XI European Meeting on Glial Cell Function in Health and Disease

Overview Session Overview Sessionprint print  

July 3, 2013 - Poster hall 5:15pm - 6:15pm
Poster Session I (uneven poster numbers)

Function of the Nkx2.2 transcription factor in oligodendrocytes and their progenitor cells

C. Fenger 1 , M. Thomassen 2 , B. Emery 3 , T. Kuhlmann 4 , T. Kruse 2 , *Bente Finsen 1
1 University of Southern Denmark, Department of Neurobiology Research, Odense C, Denmark
2 Odense University Hospital, Human Microarray Centre, Odense C, Denmark
3 University of Melbourne, Centre of Neuroscience, Melbourne, Australia
4 University Hospital of Münster, Neuropathology, Münster, Germany
Abstract text :

Background: The importance of the Nkx2.2 transcription factor during oligodendrogenesis has been documented by findings of impaired oligodendrogenesis in perinatal Nkx2.2 deficient mice. However, only four Nkx2.2 target genes have been identified so far in oligodendrocyte (Ol)-lineage cells. In concordance with its content of activator and repressor domains, Nkx2.2 promotes the expression of Ermn and Plp1 and represses the expression of Mbp and Sirt2, although they are all essential for generation of functional myelin.

Hypothesis: Nkx2.2 promotes oligodendrogenesis and myelination of CNS by acting as activator and repressor of several genes depending on the presence of other transcription factor/co-factors at different stages of the oligodendrogenesis.

Method: To further elucidate the function of Nkx2.2 during oligodendrogenesis, primary Nkx2.2 target genes were identified in murine Ol progenitor cells (OPC) and in vitro differentiated Ols by chromatin immunoprecipitation combined with DNA sequencing. Furthermore, the transcriptome were investigated in OPC/Ol containing CNS structures of postnatal Nkx2.2 deficient and wild type mice by microarray, qPCR, and in situ hybridization.

Results: In total, 14.470 putative primary Nkx2.2 target genes were identified in OPCs and/or Ols of which 51 were significantly lower expressed in Nkx2.2 deficient mice. These genes included Nkx2.2, the known target genes; Mbp, Plp1, and Sirt2, and several genes with known or possible roles in cell division, myelination, myelin compaction, and axonal protection suggesting that Nkx2.2 activates the expression of these genes in OPCs and/or Ols. Since many of these genes appear to be target genes in OPCs and Ols, although they are not expressed by both cell types, the results of this study open the possibility that Nkx2.2 works as activator and repressor at different stages of the oligodendrogenesis. Higher NG2 and PDGFαR mRNA levels and an apparently higher number of NG2 and PDGFαR expressing OPCs in the Nkx2.2 deficient mice suggest that Nkx2.2 is important for the terminal Ol differentiation including myelination, but not for the cell fate specification of OPCs in the developing murine CNS.

Conclusion: Nkx2.2 has impact on the expression of several genes with known or possible roles in cell division, myelination, myelin compaction, and axonal protection.


Glial differentiation is delayed in the hypothalamus of rats malnourished during a restrict period in early life

Michael  Rocha 1 , Priscilla Fernandes 1 , Alex C. Manhães 1 , *Penha C. Barradas 1 , Frank Tenorio 1
1 Universidade do Estado Rio de Janeiro, Instituto de Biologia, Rio de Janeiro, Brazil
Abstract text :

Nutrient restriction during the perinatal period exerts a profound influence on brain development. Previous studies using an undernutrition protocol (0% protein diet) during the first half of the lactation period in rats showed that the offspring presented an altered feeding pattern, which reflected a metabolic imprinting effect on feeding behavior. There is a delay in the pattern of NPY expression in the arcuate-paraventricular (ARC-PVN) pathway, reflecting an effect on the development of the hypothalamic circuitry, leading to metabolic imprinting. Here we studied the effects of undernutrition on glial differentiation using the same model. We used rats from 5 to 30 postnatal (P) days of age whose dams were either fed a 0% protein diet (DG) or a normoprotein diet (CG) from P1 to P10. We assessed Ki67, vimentin, GFAP, ED-1 and CNPase distribution in hypothalamic nuclei using immunohistochemistry. Our results showed impairment in cell proliferation, more evident in the PVN and in the lateral hypothalamus (LH), where DG animals showed a lower number of Ki-67positive (Ki67 ) cells at P5 when compared to CG. However, at P10 and P15, the number of Ki67 cells was significantly increased in DG. From P20 onwards, staining intensity remained relatively stable and similar in all groups. DG animals showed a delay in astroglial differentiation, presenting a decrease in GFAP expression and a peak of vimentin expression at P10 and P15 accompanied by an increase in vimentin/Ki67 cells. An increase in the number of ED1 positive cells next to the third ventricle was also shown at the same ages in DG. CNPase staining was very faint in most nuclei and did not show any obvious difference in DG animals. Our results suggest that glial differentiation is delayed in DG, which may contribute to the changes in hypothalamic circuitry that causes metabolic imprinting.



Intrinsic mechanisms regulating oligodendrocyte progenitor cell division: the role of Citron-kinase

*Enrica Boda 1 , Silvia Piretta 2 , Federico Bianchi 3 , Ferdinando Di Cunto 3 , Annalisa Buffo 2
1 University of Turin, , Orbassano (Turin), Italy
2 University of Turin, Dept. of Neuroscience, Neuroscience Institute Cavalieri Ottolenghi, Orbassano (Turin), Italy
3 University of Turin, Molecular Biotechnology Center, Dept. of Genetics, Biology and Biochemistry, Turin, Italy
Abstract text :

Oligodendrocyte progenitor cells (OPCs) comprise the main cycling cell population of the CNS parenchyma during the early postnatal period and at adult stages. However, the molecular mechanisms implicated in OPC divisions are still by large obscure. With the aims to unveil i) whether OPC divisions exploit the same molecular machinery of neuronal precursors, and ii) whether distinct OPC subsets exist with different cell division machineries, we focused on a mutant mouse where Citron-kinase, a crucial regulator of cytokinesis in neuronal precursors, is germinally ablated (CIT-K KO). These mice have severe CNS developmental abnormalities, reduction of selected neuronal populations due to apoptosis triggered by defective divisions, display ataxia, epileptic seizures and die by the third postnatal week. Interestingly, they were reported to have myelin defects, suggesting that the CIT-KO affects oligodendroglial cells. Notably, already early after birth we found a 2-fold decrease in OPC density in both the cortical grey (GM) and white matter (WM), compared to wild-type mice (WT). Here, most OPCs were hypertrophic and multinucleated, indicating a cytokinesis failure after nuclear division. At later ages, OPCs progressively disappeared from the cortical GM, while in both WM and ventral areas of the telencephalon (i.e. striatum, hypothalamus), uni-and multi-nucleated OPCs could be detected, although at lower densities compared to WT. These data suggests OPC heterogeneity in both CIT-K requirement for cytokinesis and susceptibility to death. However, even where normal uninucleated OPCs persisted, we could not find pre-myelinating or myelinating cells, in line with additional differentiation defects. To discriminate the contribution of cell-autonomous vs. environmental factors in differentiation impairment, we performed homochronic grafts of WT fluorescently tagged (GFP ) cells into perinatal CIT-KO mice. Strikingly, GFP OPCs invaded the whole CIT-K KO brain and actively divided. However, they hardly ever differentiated into more mature cells at difference with cells grafted in the WT, indicating that altered environmental signals contribute to myelination defects in CIT-K KO. In conclusion, our results show that CIT-K is involved also in glial cell divisions and suggest distinct CIT-K requirement and proneness to death of dorsal and ventral OPCs. Additionally, CIT-K ablation results in pervasive nervous tissue alterations affecting OPC maturation. Further studies will clarify the molecular mechanisms underlying these alterations.


Proliferation of reactive astrocytes is enriched in juxtavascular positions

*Jesica Frik 1,2 , Sophia Bardehle 1,3 , Martin  Krüger 4 , Ingo  Bechmann 4 , Leda Dimou 1,3 , Nikolaus Plesnila 5,6 , Magdalena Götz 1,3,6
1 Institute of Physiology, LMU, Physiological Genomics, Munich, Germany
2 University of La Plata, Institute for Biotechnology and Molecular Biology, La Plata, Germany
3 Institute for Stem Cell Research, Helmholtz Zentrum, , Munich, Germany
4 Institute for Anatomy, University of Leipzig, , Leipzig, Germany
5 Institute for Stroke and Dementia Research (ISD), University of Munich Medical School, Experimental Stroke Research, Munich, Germany
6 Munich Cluster for Systems Neurology (Synergy), , Munich, Germany
Abstract text :

Astrocytes fulfill essential functions under physiological conditions and are involved in various beneficial and adverse functions after brain injury (reviewed by Sofroniew 2009). In order to improve functional recovery after injury, it is essential to delineate the beneficial from adverse effects. However, little is yet to know which extent distinct sets of astrocytes perform distinct functions or whether all astrocytes behave in a uniform manner. Live imaging of astrocytes after stab wound injury in the adult cerebral cortex in vivo revealed a striking heterogeneity of astrocyte behaviour with distinct subsets extending long processes towards the site of injury, others proliferating and yet other subtypes undergoing only hypertrophy. Most strikingly, the vast majority of astrocytes proliferating were located with their somata directly at blood vessels (capillaries or post-capillary vessels). This close proximity was confirmed at ultrastructural level and identified as juxtavascular positions, sometimes adjacent to pericytes located in the perivascular space.

This novel population of reactive astrocytes at juxtavascular positions is of particular interest, as the capacity of astrocytes to reactivate proliferation (Buffo et al. 2008; Simon et al. 2011) and stem cell potential (reviewed by Robel et al. 2011.) seems to be tightly linked (Sirko et al., Cell Stem Cell, in press). Therefore we examined whether this population of juxtavascular astrocytes would also preferentially proliferate in even more severe injury conditions, such as 1 hour of middle cerebral artery occlusion (MCAO). The proliferation of juxtavasular astrocyte was analyzed immunohistochemically in fixed brain sections co-stained for S100b/GFAP/Ki67/CD31 at different time points after injury. At 3 days post injury, 18% of all astrocytes were actively dividing (Ki67 ). Strikingly the majority of these was again located at juxtavascular positions (68%), suggesting a general bias of astrocytes at this position to proliferate after injury, while astrocytes further distant from blood vessels virtually fail to divide. We shall further present data from conditional knock-out mice with reduced proliferative activity of juxtavascular astrocytes in order to elucidate the function of this highly specific astrocyte population and their proliferative reaction after injury.


Continuous live imaging of reactive astrocyte divisions in vitro

*Gabor Heimann 1 , Magdalena Götz 1,2,3 , Swetlana Sirko 1,2
1 Institute of Physiology, Ludwig-Maximilians University Munich, Physiological Genomics, München, Germany
2 Institute for Stem Cell Research, Helmholtz Zentrum Munich, German Research Center for Health, , München, Germany
3 Munich Cluster for Systems Neurology (SyNergy), , München, Germany
Abstract text :

Astrocytes are multifaceted cells in the adult central nervous system and react to pathology

of the adult brain with a finely graded continuum of morphological and behavioral changes.

Given that astrocytes in healthy adult brain rarely divide outside the stem cell niches, but

activate both proliferation as well as a stem cell potential after injury (for review see Robel et

al., 2011), it is important to understand their exact mode of cell division. Indeed, a defining

hallmark of a stem cell is the capacity to self-renew – often by asymmetric cell division.

Therefore we set out to image reactive astrocyte divisions in vitro by developing a primary

culture system for reactive astrocytes obtained from the somatosensory cortex of adult mouse

at 5 days after stab wound injury. Continuous live imaging and single cell tracking (Costa et

al., 2008, 2011) allowed us observing the mode of cell division mode as well the cell fate of

their immediate progeny. Results of our study reveal (i) that reactive astrocytes can undergo

multiple rounds of cell division, (ii) the SHH signaling has a profound influence on this

behavior, (iii) reactive astrocytes divide with distinct modes of cell division, which can be

influenced by the local microenvironment. Taken together, this new culture system allows

close examination of signals on the mode and multitude of reactive astrocyte cell divisions

and provides a great tool to identify signals derived from the extracellular matrix, other cell

types or as diffusible signals.


Mouse Schwann cell culture and the expression of L-MAG in Schwann cells and in myelinating cocultures

*Henrika Honkanen 1 , Anthony Heape 1
1 University of Oulu, , Oulu, Finland
Abstract text :

The knowledge of the control of SC proliferation is essential for understanding how the PNS nerve develops, how the myelin sheath is formed by SCs, and what happens during the degeneration of myelin sheath. Here we investigated the effects of several growth factors with the aim of improving the efficiency of our published protocol for expanding mouse SCs (Honkanen et al, 2007). We have studied the expression of L-MAG in Schwann cells alone, and during early phases of myelination in an attempt to better understand the role of L-MAG during PNS myelination.                  

Studies of Mouse Schwann cell culture. The proliferation of SCs is regulated by axonal signals and several growth factors.  Growth factors that are essential for rodent SC survival in culture include heregulin-β1 and forskolin. In the case of rat SCs, these factors are also sufficient to allow a robust proliferation. In contrast, the proliferation of mouse-derived SCs in culture requires the presence of other growth factors. We studied effect of several factors known to be expressed in SCs after nerve injury on mouse SC proliferation. These included fibroblast growth factor type 2 (FGF), pituitary extract (PE), epidermal growth factor, platelet-derived growth factor BB, and transforming growth factor β1. We found that heregulin-β1 and forskolin are essential for mouse SC to survive in culture, FGF and PE were the best combination of growth factors promoting the mouse SC proliferation, and that neuronal axons provide effective mitogenic environment for mouse SCs.

The expression of the Large Myelin-Associated Glycoprotein in PNS was studied in SCs cultured alone and in cocultures of SCs and DRGNs. When Schwann cells are alone without axons, expression of the cytoplasmic domain of L-MAG is seen in the nucleus of Schwann cells. When Schwann cells make contact with neuronal axons, the location of the expression  shifts out of the nucleus to the perinuclear and cytoplasmic regions; the extracellular domain is not visible. until the Schwann cells initiate the myelination programme. These results support the hypothesis that L-MAG might have role in the regulation of the myelination programme in the PNS.


The effect of microglia and secreted factors on cell density in oligodendrocyte precursor cell culture

*Karolina Kleinsimlinghaus 1 , Meray Serdar 2 , Romy Marx 2 , Irmgard D. Dietzel 2
1 Ruhr-Universität , , Essen, Germany
2 Ruhr-Universität Bochum, Biochemie II - Neurobiochemie, Bochum, Germany
Abstract text :

In order to develop strategies to expand olidodendrocyte progenitor cultures (OPCs), leading to as high yields of OPCs as possible, we investigated the influence of different culture conditions on OPC proliferation rate (using a BrdU-assay) and the number of surviving cells (by counting DAPI-stained nuclei). After we had observed, that the choice of the basal medium (neurobasal, RPMI or DMEM) exerts a pronounced effect on OPC proliferation we here investigated the effect of cell density on OPC proliferation.

Starting from a small seeding density (10.000 cells/cm2) a tenfold increase in seeding density only resulted in a fourfold augmentation of the density of surviving cells after 7 days in culture. Using plating densities of 5.000; 20.000 and 80.000 cells/cm2 we observed, that an increase in seeding density led to a decrease in the proliferation rate, as tested with a BrdU-assay and monitored after 3 days in culture.  Similarly, the percentage of A2B5-positive, immature, cells was lower, the higher the cell density.

We then tested whether this finding is due to proliferation inhibiting factors secreted in cultures with high seeding densities and whether the remaining microglia population could be involved. To this aim we seeded OPCs at low density (5.000 cells/cm2) and cultivated them with the medium supernatant of cultures with high density (80.000 cells/cm2). Furthermore we seeded OPCs in low density (5.000 cells/cm2 ) and co-cultivated them with an added density of microglia as counted in cultures of high density (80.000 cells/cm2).

The resulting cell counts after 3 days showed, that the percentage of BrdU positive cells after cultivation with the supernatant was significantly reduced compared with control cultures, although the number of A2B5-positive cells was not influenced by this treatment. The addition of microglia to the cultures lead to a strong reduction of surviving cells, even though the percentage of BrdU and A2B5 positive cells were not altered. So apoptosis, necrosis or phagocytosis might be responsible for the reduction of surviving cells.

Our findings suggest that the reduction of surviving OPCs in high density cultures is due to secreted factors inhibiting OPC proliferation and that microglia could be involved in this effect.



ROCK inhibition with Y27632 promotes the proliferation and cell cycle progression
of cultured astrocyte from spinal cord

*Xiang Luo 1
1 Huazhong University of Science and Technology, , Wuhan, China
Abstract text :

   Rho-associated Kinase (ROCK) has been identified as an important regulator of proliferation and cell cycle progression in a number of cell types. Although its effects on astrocyte proliferation have not been well characterized, ROCK has been reported to play important roles in gap junction formation, morphology, and migration of astrocytes. In the present study, our aim was to investigate the effect of ROCK inhibition by [( )-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] (Y27632) on proliferation and DNA synthesis in cultured astrocytes from rat spinal cord and the possible mechanism involved. Western blots showed that treatment of astrocytes with Y27632 increased their expression of cyclin D1, CDK4, and cyclin E, thereby causing cell cycle progression. Furthermore, Y27632-induced astrocyte proliferation was mediated through the extracellular-signalregulated kinase signaling cascade. These results indicate the importance of ROCK in astrocyte proliferation.


Specification and maintenance of oligodendrocyte precursor cells from neural progenitor cells: involvement of microRNA-7a

*Xianghui Zhao 1
1 the Fourth Military Medical University, , Xi'an, China
Abstract text :

The generation of myelinating cells from multipotential neural stem cells in the CNS requires the initiation of specific gene expression programs in oligodendrocytes (OLs). We reasoned that microRNAs (miRNAs) could play an important role in this process by regulating genes crucial for OL development. Here we identified miR-7a as one of the highly enriched miRNAs in oligodendrocyte precursor cells (OPCs), overexpression of which in either neural progenitor cells (NPCs) or embryonic mouse cortex promoted the generation of OL lineage cells. Blocking the function of miR-7a in differentiating NPCs led to a reduction in OL number and an expansion of neuronal populations simultaneously. We also found that overexpression of this miRNA in purified OPC cultures promoted cell proliferation and inhibited further maturation. In addition, miR-7a might exert the effects just mentioned partially by directly repressing proneuronal differentiation factors including Pax6 and NeuroD4, or proOL genes involved in oligodendrocyte maturation. These results suggest that miRNA pathway is essential in determining cell fate commitment for OLs and thus providing a new strategy for modulating this process in OL loss diseases.


Cell-specific receptor expression defines the differential response of astrocytes versus microglia to oncostatin M

*Iain Campbell 1 , Meng-Ping Hsu 1
1 University of Sydney, , Sydney, Australia
Abstract text :

The IL-6/gp130 family of cytokines including IL-6, IL-11, leukemia inhibitory factor (LIF) and oncostatin M (OSM), are pivotal mediators in a wide range of processes in the CNS ranging from neurodevelopment to neuroinflammation. Surprisingly little is known about the relative responses of glial cells to these cytokines. In addressing this problem we first examined the expression of the receptors for IL-6, IL-11, LIF and OSM in cultured murine astrocytes versus microglia. In astrocytes, the OSMR mRNA was most abundant while low levels of the IL-6R, IL-11R and LIFR mRNAs were also detectable. In comparison, in microglia, neither the OSMR nor LIFR mRNAs were detectable while the IL-11R mRNA and IL-6R mRNA were present at similar and higher levels, respectively. Immunoblotting for the IL-6R and OSMR proteins revealed that both astrocytes and microglia produced IL-6R however, the OSMR was abundant in astrocytes but not detectable in microglia. Consistent with the pattern of receptor expression, astrocytes and microglia both responded to hyper-IL6 with strong activation of STAT3. However, in response to OSM, STAT3 activation was induced in astrocytes but not microglia. Moreover, in contrast to a previous report, OSM failed to activate NFkB or induce NOS2 in microglia. In conclusion, these findings, (1) identify notable differences in the expression of some key IL-6/gp130 family cytokine receptors in astrocytes and microglia, and (2) provide the molecular basis for the differential response of astrocytes versus microglia to OSM. Supported by NHMRC project grant 632754.


RXR Signalling and Regulation in Oligodendrocyte Lineage Cells

*Alerie Guzman de la Fuente 1 , Jeffrey  Huang 1 , Brahim  Nait Oumesmar 2 , Charles ffrench-Constant 3 , Robin Franklin 1
1 Wellcome Trust/MRC Stem Cell Institute, , Cambridge, United Kingdom
2 Centre de Recherche de l'Institut du Cerveau et de la Moelle Epinière - CRICM, , Paris, France
3 MRC Centre for Regenerative Medicine, Queen's Medical Research Institute , , Edinburgh, United Kingdom
Abstract text :

Myelin sheaths are lost from CNS axons in demyelinating diseases. New sheaths can be regenerated following the recruitment of adult neural progenitor cells (OPC) into areas of injury and their differentiation into myelinating oligodendrocytes. Although this regenerative process (called remyelination) occurs efficiently in the early stages of Multiple Sclerosis, its efficiency gradually declines leaving axons demyelinated and vulnerable to degeneration. Several signalling pathways are involved in regulating OPC differentiation and subsequent myelination. Understanding their mechanisms is necessary to design regenerative therapies to overcome remyelination failure.

We have identified RXRγ as a key positive regulator of OPC differentiation and remyelination (Huang et al., 2011). RXRγ is a nuclear receptor forms a heterodimer with other nuclear receptors to activate its downstream signalling cascades.

Here we describe RXRγ binding partners expressed in both OPC and oligodendrocytes, and the binding of VDR, RARβ and PPARγ to RXRγ within oligodendrocyte lineage cells by coimmunoprecipitation. VDR- RXRγ heterodimer is necessary for the oligodendrocyte differentiation. Treatment of oligodendrocytes with VDR antagonist inhibits OPC differentiation and abrogates the effect of 9-cis retinoic acid, an RXRγ agonist, in OPC differentiation.

Our data suggests a model in which RXRγ is an essential anchor for different nuclear receptors, including VDR, with a shifting and complex RXRγ signaling in oligodendrocyte lineage cells.


The involvement of NO-mediated signaling in PDT-induced injury of neurons and glial cells

*Vera Kovaleva 1 , Elena Berezhnaya 1 , Mikhail Rudkovskii 1 , Anatoly Uzdensky 1
1 Southern Federal University, , Rostov-on-Don, Russian Federation
Abstract text :

Photodynamic therapy (PDT) is currently used in oncology, particularly, in treatment of brain tumors. The possible role of NO-mediated signaling in photodynamic injury and protection of neurons and surrounding glial cells (GC) was studied. Crayfish stretch receptor consisting of a single neuron enveloped by GC was photosensitized with alumophthalocyanine Photosens (10 nM, 30 min incubation) and irradiated with laser diode (670 nm, 0.4 W/cm2). Application of NO generators notably 10 mM sodium nitroprusside and 10 mM NONOate reduced PDT-induced necrosis of GC and showed the same tendency in neuronal necrosis. NONOate significantly increased PDT-induced apoptosis of GC. Inhibitor of neuronal NO-synthase L-NAME (1mM) significantly increased the percentage of necrotic glial cells after PDT but did not influence neuronal necrosis. This confirmed the anti-necrotic effect of NO in glial cells and involvement of neuronal NO synthase in protection of glial cells. 1 mM L-NAME and 1 mM L-NNA (another inhibitor of neuronal NO-synthase) as well as 50 μM S-Methhylisothioharnstoff Sulfat (inhibitor of inducible NO synthase) protected GC from PDT-induced apoptosis. Therefore NO may be involved in PDT-induced apoptosis of glial cells. Inhibition of NO-activated protein kinase G with 10 μM KT5823 decreased the percentage of necrotic glial cells but not neurons. Therefore protein kinase G appeared to be involved in PDT-induced necrosis of GC, possibly independently on NO. KT5823 also increased the level of apoptosis of GC indicating the anti-apoptotic role of protein kinase G. Thus NO is involved in regulation of PDT-induced necrosis of neurons and glial cells as well as in apoptosis of glial cells.


In vivo imaging of inflammation, de-and remyelination using Fluorescence Molecular Tomography (FMT)

*Stefanie Albrecht 1 , Christiane Geyer 2 , Lydia Wachsmuth 3 , Thomas Vogl 4 , Cornelius Faber 3 , Michel Eisenblätter 2 , Tanja  Kuhlmann 1
1 University Hospital Münster, Institute of Neuropathology, Münster, Germany
2 University Hospital Münster, IZKF Münster Core Unit Optical Imaging (OPTI), Clinical Radiology, Münster, Germany
3 University Hospital Münster, Experimental NMR, Clinical Radiology, Münster, Germany
4 University Münster, Institute of Immunology, Münster, Germany
Abstract text :

Multiple sclerosis (MS) is a chronic disease characterized by inflammation, demyelination, gliosis and axonal degeneration. Until now, no imaging techniques exist that allow the specific detection of these different pathologies in vivo. Therefore our aim was to establish imaging techniques to detect specifically inflammation as well as remyelination in vivo. Feeding mice with cuprizone induces demyelination in many brain regions and is a frequently used animal model to study remyelination. Using Fluorescent Molecular Tomography (FMT) we were able to follow inflammation as well as de-and remyelination in the cuprizone model. Cuprizone induced inflammation was imaged using a fluorescent conjugated antibody against the proinflammatory macrophage marker myeloid-related protein 14 (MRP-14). The reduction as well as progression of myelination after cuprizone withdrawal was followed via the fluorescent myelin binding compound 3,3ß-diethylthiatricarbocyanine iodide (DBT). Due to different fluorescence characteristics of the antibody and the myelin binding compound, we are able to longitudinally and simultaneously image the degree of inflammation and de- and remyelination. The FMT studies were accompanied by Diffusion Tensor Imaging (DTI) studies to visualise the changes in radial diffusivity of the corpus callosum in mice fed with cuprizone during de- and remyelination. Additionally, extensive histological and electronmicroscopical analyses were applied to verify the FMT and DTI results.


Decreased expression of alpha1 subunit of Na+/K+-ATPase in the ALS rat brain

*Danijela Bataveljic 1 , Ljiljana Nikolic 2 , Pavle Andjus 1
1 Faculty of Biology, University of Belgrade, Center for Laser Microscopy, Belgrade, Serbia
2 Institute for Biological Research “Siniša Stanković”, University of Belgrade, Department of Neurophysiology, Belgrade, Serbia
Abstract text :

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting predominantly motoneurons but with a particular pathological role of non-neuronal cells. Previous research suggested that increased concentration of extracellular potassium could lead to motoneuron dysfunction. The Na /K -ATPase should regulate the homeostasis of potassium ions and thus help maintain regular neuronal activity. This homeostasis may be regulated by Na /K -ATPase in both neurons and astrocytes. In order to reveal the possible role of the Na /K -ATPase in ALS pathology, we explored the expression of the alpha1 catalytic subunit of Na /K -ATPase in neurons and astrocytes from different brain regions of the SOD1G93A rat model of ALS. Labeling immunofluorescently the alpha1 isoform of the Na /K -ATPase revealed a decreased signal in the trigeminal nucleus and cerebellum of the ALS rat. Double immunofluorecence with NeuN for neurons showed that the reduced alpha1 was on the neuronal surface in the trigeminal nucleus. In addition, the alpha1 ring-like staining of cerebellar granule neurons was lower in ALS as compared to the wild type rat. In comparison to neurons, astrocytes from the wild type rat were characterized with a lower expression of alpha1. In the examined brain regions we did not observe a prominent difference in alpha1 expression between astrocytes of wild type and ALS rats. Based on the findings of this study the prominent reduction of Na /K -ATPase level observed in neurons and not in astrocytes could contribute to further understaning of impaired ion homeostasis affecting proper neuronal physiology in ALS. The role of glial Na /K -ATPase needs to be further studied with markers of other alpha subunit isoforms.


CD38 deficiency inhibits Alzheimer's disease pathology in a mouse model

*Eran Blacher 1
1 Tel-Aviv University, , Tel Aviv, Israel
Abstract text :

Alzheimer’s disease (AD) accounts for the majority of cases of dementia worldwide, affecting over 50% of the population over 85. The disease is characterized by a progressive memory loss, cognitive deterioration, behavioral disorders, deposit of beta-amyloid (Ab) peptides, neurofibrillary tangles, reactive astrocytosis and activation of microglia cells. Mutations in the genes that encode APP (Amyloid Precursor Protein) and components of the proteases that generate amyloid beta cause familial forms of AD.

Microglia, the resident immune cells of the central nervous system (CNS) were suggested to play both beneficial and harmful roles in the course of the disease. Therefore, modulating the microglial response is being considered as a promising approach for the treatment of this disease.

We have recently shown using in vitro and in vivo systems that the ectoenzyme CD38, regulates microglial activation. In view of the significant role of activated microglia in AD and the important role played by CD38 in microglial activation, we hypothesized that CD38 deficiency would affect the course of the disease.

In order to test this hypothesis, we implicated the APPswePS1dE9 transgenic mouse model for AD, and compared the cognitive performance (as evident by behavioral studies) of aged APPswePS1dE9 mice, to APPswePS1dE9 Cd38-/- mice. In addition, Abload, accumulation of microglia and astrocytes was assessed in the brains of these mice. Our preliminary results show that CD38 deficiency has a neuroprotective role in this AD mouse model as indicated by improvement in cognitive performance and dramatic reduction in Aβ load


Pericytes are preserved in Alzheimer’s Disease

*Lasse Brandt 1 , Francisco Fernández-Klett 1 , Laura W. Harris 2 , Sabine Bahn 2 , Josef Priller 1
1 Charité-Universitätsmedizin Berlin, Department of Neuropsychiatry and Laboratory of Molecular Psychiatry, Berlin, Germany
2 University of Cambridge, Centre for Neuropsychiatric Research, Institute of Biotechnology, Cambridge, United Kingdom
Abstract text :

Pericytes are vascular mural cells which are enriched within the capillary basement membranes of the brain. Recent studies have provided evidence that pericytes regulate the blood-brain barrier (BBB) and modulate cerebral blood flow. Neurovascular dysfunction and BBB damage are hallmarks of Alzheimer’s disease (AD), but the role of pericytes in AD pathogenesis is still unclear.

We have examined post-mortem samples of middle frontal gyrus in collaboration with the University of Cambridge after ethical approval by the local IRB. Ten cases with neither history of neurological disorder nor evidence of neuropathology were compared with 10 AD cases provided by the Neurological Foundation of the New Zealand Human Brain Bank. We performed immunostainings for laminin and platelet-derived growth factor receptor (PDGFR)-β which mark vessels and pericytes, respectively. We also stained for Aβ1-42 for evidence of amyloid pathology. Quantification of pericyte and vessel density was performed using stereological methods, which are considered as gold standard in morphometric quantification. The Spaceballs method was used to measure the density of capillaries. Pericyte density was assessed using the Optical Fractionator.

The age of the subjects did not differ between the control and AD groups (76,2±4,0 vs. 75,4±5,5 years). The cortex of AD cases contained more capillary vessels than controls (mean±SD: 308±40 vs. 365±41 mm/mm3; p<0,01; Fig. 1A). The capillary density increased in correlation to the total plaque load (R=0,58; p<0,001). In contrast, we could not observe a significant difference in the number of pericytes per mm capillary length between control and AD cases (8,56±1,21 vs. 8,17±0,94 cells/mm; p=0,44; Fig. 1B). Relative to tissue volume, the cortex of AD individuals showed a tendency to contain more pericytes than controls (2624±406 vs. 2985±588 cells/mm3; p=0,13; Fig. 1C).

This is the first study using state-of-the-art techniques to investigate the relationship between capillaries and pericytes in AD brains. Impaired clearance of β amyloid across the pericyte-regulated BBB and deficient microvascular regulation of blood flow may contribute causally to AD pathogenesis. In consequence, loss of pericytes could contribute to disease progression. However, our results show that AD cortical pathology is characterized by an increase in the density of capillary vessels without deficit in pericyte coverage. Thus, they do not substantiate the notion that pericyte loss is a significative feature of AD.


Changes of the extracellular space diffusion parameters during aging in a triple transgenic animal model of Alzheimer’s disease

*Michal Cicanic 1 , Lydia Vargova 1,2 , Magdalena Kulijewicz-Nawrot 2 , Jose Julio Rodriguez 2,3,4 , Eva Sykova 1,2
1 Charles University, 2nd Faculty of Medicine, Department of Neuroscience, Prague, Czech Republic
2 Institute of Experimental Medicine AS CR, Department of Neuroscience, Prague, Czech Republic
3 IKERBASQUE, Basque Foundation for Science, , Bilbao, Spain
4 University of the Basque Country UPV ⁄ EHU, Department of Neuroscience, Leioa, Spain
Abstract text :

Amyloid-β (Aβ) deposits in the brain extracellular space (ECS) and neurofibrillary tangles are typical features of Alzheimer’s disease (AD). Both affections are expressed in triple transgenic (3xTg-AD) mice, making them a useful model for studying the pathophysiology of AD. In these mice, significant changes in astroglial morphology appear in the limbic brain structures [1, 2], which may affect the diffusion of neuroactive substances through the ECS.

Using the real-time iontophoretic method, we determined the ECS volume fraction α (α = ECS volume / total tissue volume) and the geometrical factor tortuosity λ (λ2 = free / apparent diffusion coefficient) in the CA1 region of the hippocampus, dentate gyrus (DG) and prelimbic cortex (PLC) in brain slices obtained from 10-month-old (10m) and 20-month-old (20m) 3xTg-AD mice and age-matched wild-type (wt) controls. To study potential diffusion anisotropy, the measurements were performed along 3 orthogonal axes: medio-lateral, rostro-caudal and ventro-dorsal. Astroglial morphology and the presence of Aβ were assessed using immunohistochemical staining for GFAP and Aβ.

We found no anisotropy in the CA1 region, DG, or PLC, thus the data for each structure along the three orthogonal axes were pooled. The ECS diffusion parameters measured in the CA1 are shown in table 1. In the CA1 of 10m mice, there was no significant difference in the ECS diffusion parameters between wt and 3xTg-AD mice. In 20m mice, α decreased by 9 % in wt but increased by 27 % in 3xTg-AD mice in comparison with the values found in the younger animals. During aging, there was also a significant decrease in λ in wt but not in the 3xTg-AD mice. A similar pattern of changes in the ECS diffusion parameters during aging was also observed in the DG and PLC. Unlike in the CA1, we found significant differences in young animals: α was lower in the DG and λ was higher in the DG and PLC of 3xTg-AD mice than in wt mice. In 20m 3xTg-AD mice, Aβ deposits accompanied with astroglial atrophy or hypertrophy were observed.  

We suggest that the amyloid load in 3xTg-AD mice and the subsequent prevailing astroglial atrophy affect the normal aging process by inducing an increase in the ECS volume during aging instead of the normal decrease. This leads to a larger ECS space in aged 3xTg-AD mice than in age-matched wt mice, which may alter the efficacy of extrasynaptic as well as synaptic transmission and contribute to the impaired cognitive functions in AD.

The study was supported by the grants: GACR P304/11/0184, GACR P304/12/G069.

1. Olabarria et al. Glia 2010. 58(7): 831-8.

2. Kulijewicz-Nawrot et al. J Anat 2012. 221(3): 252-62.


Intravital characterization of microglia in Alzheimer’s Disease

*Natalia H. Drost 1 , Jan-Leo Rinnenthal 1 , Debora Pehl 1 , Frank L. Heppner 1
1 Charité - Universitätsmedizin Berlin, , Berlin, Germany
Abstract text :

Question: Microglia are attracted to and surround β-amyloid deposits, the main hallmark of Alzheimer’s disease (AD), suggesting a role for these cells in disease pathogenesis. However, recent in vivo data using the CD11b-HSVTK system for microglial depletion suggests that resident microglia are not sufficiently capable of restricting and clearing β-amyloid plaques. To underpin the CD11b-HSVTK system and shed more light into the biological mechanistics of microglial depletion mediated by Ganciclovir (GCV), we aim to intravitally monitor the depletion process.

Methods: We are using CD11b-HSVTK transgenic mice crossed to Fractalkine-GFP /- mice harboring green fluorescent microglia. A chronic cranial window is implanted onto the head of the offsprings followed by depletion of microglia and subsequent in vivo two-photon (2P) imaging. As 2P imaging is able to visualize the upper 300 µm of the cortex, a new topical depletion approach of CD11b-HSVTK microglia was established. Here, GCV is applied directly onto the brain surface in the area of the cranial window through a catheter.

Results: Manipulation of the brain skull by installation of the cranial window and placing a catheter for topic GCV application resulted in strong depletion of microglia up to 85% in Fractalkine-GFP /- mice crossed to CD11b-HSVTK mice.

Conclusions: Taken together, we established a minimal invasive technique for visualization of the microglia depletion process in CD11b-HSVTK mice using intravital 2P microscopy. These tools will allow the underpinning of the CD11b-HSVTK system as well as the study of relevant biological questions involving resident and peripheral derived microglia in AD.


Treatment early in life with the lectin ConA decelerates spreading depression in well-nourished and early-malnourished adult rats

*Geórgia S. Ferreira Soares 1 , Liliane Cabral Cavalcanti 1 , Rubem Carlos Araújo Guedes 1
1 Universidade Federal de Pernambuco, , Recife, Brazil
Abstract text :

Background and Purpose: The lectin Concanavalin A (ConA), obtained from the seeds of Canavalia ensiformis, stands out as a biochemically, biophysically and structurally well characterized lectin, commonly used as histochemical marker, due the its high selectivity to microglial cells. Early nutritional insults have important effects on neural and glial electrophysiological activity because nutrients regulate brain development during early postnatal life, when neural and glial cells present higher degree of plasticity. Herein we characterized in adult rats the electrophysiological changes caused by the systemic treatment early in life with the lectin ConA by recording the electrophysiological phenomenon of cortical spreading depression (CSD). Additionally, we evaluated the impact of early nutritional deficiency on the CSD effects of ConA. Methods: Two nutritionally distinct groups of male newborn rats – well-nourished (W; n=26) and malnourished (M; n=33) were treated from the 5th to the 24th day of postnatal life with daily intraperitoneal injections of saline (Sal) or ConA (Sigma-Aldrich) at doses of 1mg/kg (group W-L1 and M-L1) or 10 mg/kg (Groups W-L10 and M-L10). Two groups of “naive” (non-injected) pups were used as additional controls. When the pups reached adult age (90-120 days), under ip anesthesia (1g/kg urethane 40mg/kg chloralose) they were submitted to the CSD recording (ECoG and slow potential change) in two points of the parietal cortical surface. CSD was triggered every 20 minutes by applying 2% KCl for 1 min at a point in the frontal cortex. This study was approved by the ethics committee of our University, Case No.: 23076.031272/2010-53. Results: Compared to W-Sal controls (mean±sd velocity in mm/min=3.41±0.10), M-Sal rats presented significantly higher velocities (4.26±0.16; p<0.05). In both nutritional conditions L1 and L10 treated groups presented significantly lower CSD velocities (3.12±0.14 and 2.82±0.18 for the W-L1 and W-L10, respectively, and 3.74±0.13 and 3.25±0.16 for the M-L1 and M-L10). This effect was dose-dependent, and was greater in the M-condition. The naïve- and Sal-groups had similar CSD velocities. Conclusions: The lectin ConA administered early in life to rats decelerated CSD in a dose-dependent manner at adulthood, suggesting a long-lasting effect. The largest relative reduction was observed in the M group, suggesting modulation of the effect by the nutritional status. The involvement of glial cells in this effect is discussed.


N-Arachidonoyl – Dopamine (NADA) is a novel neuroprotective Endocannabinoid.


*Urszula Grabiec 1 , Marco Koch 2 , Robert Kraft 3 , Kerstin Hill 4 , Claudia Merkwitz 2 , Chalid Ghadban 1 , Beat Lutz 5 , Alex Straiker 6 , Faramarz Dehghani 1
1 Martin Luther Universität Halle Wittenberg, Institut für Anatomie und Zellbiologie, Halle (Saale), Germany
2 Universität Leipzig, Germany, Institut für Anatomie, Leipzig, Germany
3 Universität Leipzig, Carl-Ludwig Institut für Physiologie, Leipzig, Germany
4 Rudolf-Boehm Institut für Pharmakologie und Toxikologie, Universität Leipzig, Leipzig, Germany
5 Johannes Gutenberg-Universität, Institut für Physiologische Chemie und Pathobiochemie, Mainz, Germany
6 Gill Center for Biomolecular Medicine , Department of Psychological and Brain Sciences, Bloomington, USA, United States
Abstract text :

Endocannabinoids exert numerous effects in the CNS under physiological and pathological conditions. The aim of the present study was to examine whether the novel endocannabinoid N-arachidonoyldopamine (NADA) may protect neurons in excitotoxically lesioned organotypic hippocampal slice cultures (OHSC). OHSC were excitotoxically lesioned by application of N–methyl–D–aspartate (NMDA, 50 µM) for 4 h and subsequently treated with different NADA concentrations (0.1 pM – 50 µM) alone or in combination with cannabinoid receptor antagonists. NADA protected dentate gyrus granule cells and caused a faint reduction in the number of microglial cells. The number of degenerated neurons significantly decreased between 100 pM and 10 µM NADA. O-1918, 6-iodonordihydrocapsaicin and HC-030031 showed no effects at all used NADA concentrations. In next step we looked at the MAP kinase signal transduction pathway activation after stimulation with NADA. For this purpose we used neuronal hippocampal cell line HT22, microglial cell line BV2 and astrocytes, because of their appearance in OHSC. NADA did not influence the activation p38 and p44/42 MAPK in analyzed cells (after 30 min, 2 h, 6 h, 24 h). Our findings demonstrate that NADA protects dentate gyrus granule cells by acting upon CB1. NADA reduced the number of microglial cells at distinct concentrations. TRPV1 was not involved in NADA mediated neuroprotection. Thus, our data implicate that NADA-mediated activation of neuronal CB1 may serve as a novel pharmacological target to mitigate symptoms of neuronal damage.


Ammonium induces an astroglial calcium dysbalance in different brain regions

*Nicole Haack 1 , Christine R. Rose 1
1 Heinrich Heine University Düsseldorf, , Duesseldorf, Germany
Abstract text :

Hepatic encephalopathy (HE) is a serious neurological disorder caused by liver failure. The prime candidate responsible for HE pathology is an increased ammonium concentration; and following acute liver failure, ammonium can reach levels of up to 5 mM in the brain. Astrocytes are a major target of ammonium toxicity and earlier studies suggested that this toxicity includes a disturbance in intracellular calcium signaling.

In the present study, we analyzed the effect of acute hyperammonia on the intracellular calcium concentration of astrocytes in tissue slices of mouse brain, using quantitative intracellular calcium imaging with the indicator dye Fura-2. Astrocytes were identified by staining with the vital dye SR101; cerebellar Bergmann glial cells were identified based on their morphology. In all brain regions studied, the hippocampal CA1 area, somatosensoric cortex, and cerebellar cortex, bath perfusion with 5 mM ammonium for 30 min induced a small, but persistent elevation in intracellular calcium, averaging about 50 nM. In addition, a fast and transient increase by on average 100 nM that lasted about 10 minutes was seen in a small percentage of astrocytes in hippocampal and cortical slices at the onset of ammonium perfusion. This transient increase was never observed in cerebellar Bergmann glial cells. Both the transient, as well as the plateau phase of ammonium-induced calcium changes were unaltered in the presence of TTX, indicating that they are independent from action potential generation by neurons. Furthermore, ammonium-induced calcium increases largely persisted upon removal of extracellular calcium, indicating that they are caused by release from intracellular stores.

Taken together, our experiments demonstrate that ammonium evokes complex changes in the intracellular calcium concentration of astrocytes in the intact tissue, which differ both between cells in one preparation as well as between different brain regions. Because of the central role of astrocyte calcium in gliotransmission, the observed ammonium-induced calcium dysbalance might result in disturbed neuron-glia interaction and contribute to the pathology of HE.


The L-type voltage-gated calcium channel subunit alpha1C (CaV1.2) is expressed in astrocytes around beta-amyloid plaques in an Alzheimer mouse model

*Christian Humpel 1 , Nina Daschil 1 , Gerald J. Obermair 2 , Bernhard E. Flucher 2 , Birgit Hutter-Paier 3 , Manfred Windisch 3 , Josef Marksteiner 1
1 Lab. Psychiatry and Exp. Alzheimers Res., , Innsbruck, Austria
2 Div. Physiology, , Innsbruck, Austria
3 JSW Lifesciences, , Grambach, Austria
Abstract text :

Growing evidence suggests that amyloid-β peptides (Aβ) may affect the modulation of L-type voltage-gated calcium channels (LTCC) in Alzheimer’s disease (AD). We have recently shown that the LTCC alpha1C subunit (CaV1.2, a1C) is expressed in activated astrocytes around plaques in transgenic mice expressing the human AβPP751 with the London (V717I) and Swedish (K670M/N671L) mutations (Willis et al., J Alzheimers Disease 20: 1167-1180. 2010). The aim of the present study using this Alzheimer mouse model is (1) to investigate in more detail the cellular expression of the a1C subunit in the brains in a time- and region specific pattern, (2) to determine quantitative mRNA expression profiles of all LTCC subunits in the hippocampus and (3) to perform a co-localization study with the auxiliary calcium channel beta4 subunit. (4) Further we aim to culture primary astrocytes and stimulate with recombinant Aβ(42) to induce and study a1C expression in vitro.

            Our data show that 11 month old tg mice displayed a significant increase of Aβ-plaques in the brain, compared to wt mice. Furthermore, we show that the a1C subunit colocalizes with 90% of the Aβ-positive plaques.  The cellular expression of a1C was predominantly found in activated GFAP astroglia around plaques. qPCR profiles of the hippocampus revealed expression of the majority of calcium channel subunits, which was stable during aging. The auxiliary beta4 subunit was expressed in these mice but did not co-localize with a1C astroglia around plaques. In cultures of primary astrocytes, Aβ(42) slightly enhanced the a1C mRNA expression after 3 days. We are currently underway to characterize this expression in more detail.

            In summary, our data provide evidence for a largely stable expression of most LTCC subunits in Alzheimer tg mice during aging. Finally, the expression of a1C but not beta4 is upregulated in activated astrocytes located around Aβ-plaques and Aβ(42) may directly induce astroglial CaV1.2 calcium channels.


This study was supported by the Sonderforschungsbereich SFB F4405-B19 and F4406-B19

of the Austrian Science Funds.


Lysosomal enzymes in the hippocampal glial cells of kainic acid treated rats: potential implication in temporal lobe epilepsy

*Satyabrata Kar 1 , Mayukh Banerjee 1 , Anna Sasse 1 , Yanlin Wang 1 , Mahua Maulik 1
1 University of Alberta, , Edmonton, Canada
Abstract text :

The temporal lobe epilepsy (TLE) is the most common form of epilepsy that originates from the hippocampus and then propagates to other limbic areas such as the amygdala and entorhinal cortex. The pathological feature associated with TLE is hippocampal sclerosis which is characterized by atrophy, induration, gliosis and loss of neurons in CA1, CA3 and the dentate hilar regions. Some animal models, albeit do not exactly match the complex etiologies identified in humans, are found to recapitulate most of the pathological features observed in TLE. There is evidence that administration of kainic acid can cause seizures in the CA3 region of the hippocampus that can lead to loss of neurons and astrogliosis characteristic of TLE, but the underlying mechanisms associated with the degeneration of neurons remain unclear. Since lysosomal enzymes, cathepsins B and D, can have important roles in the loss of neurons in a variety of experimental conditions, we evaluated their potential roles along with the insulin-like growth factor-II (IGF-II) receptor, which is involved in the intracellular transport of these enzymes, in the kainic acid treated rats. Our results clearly showed that systemic administration of kainic acid evoked severe loss of neurons along with hypertrophy of astrocytes and microglia in the hippocampal region of the adult rat brain. The levels and expression of cathepsins B and D as well as IGF-II receptor increased progressively with time in the hippocampus of kainic acid treated rats compared to control rats. The activity of both cathepsins B and D was also found to be enhanced in the hippocampus of treated rats. Our double labelling studies revealed that expression of both cathepsins were initially increased in the pyramidal neurons and then decreased with time. This was accompanied by the expression of immunoreactive IGF-II receptors as well as cathepsins B and D in a subset of GFAP-labelled activated astrocytes and Iba1-labelled microglia in kainic acid treated rats. These results, taken together, suggest that enhanced levels/expression and activity of lysosomal enzymes may have a role in the loss of neurons observed in kainic acid treated rats.


Early activation of microglia has a central role in the disease pathogenesis of progressive myoclonus epilepsy, EPM1

*Inken Körber 1 , Tarja Joensuu 1 , Anna-Elina Lehesjoki 1 , Outi Kopra 1
1 Folkhälsan Institute of Genetics and University of Helsinki, Research Programs Unit, Molecular Neurology and Neuroscience Center, Helsinki, Finland
Abstract text :

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1, OMIM 254800) is an autosomal recessively inherited neurodegenerative disorder. It is caused by loss-of-function mutations in the cystatin B gene (CSTB, OMIM 601145), encoding an inhibitor of lysosomal cysteine proteases. The precise role of CSTB and the mechanisms by which its loss leads to EPM1 remain poorly understood. The mouse model for EPM1, cystatin B-deficient (Cstb-/-) mouse, recapitulates the key clinical features of EPM1 including myoclonic seizures and progressive ataxia. It also shows widespread neuron loss and gliosis in the brain. Interestingly, in Cstb-/- mice pronounced microglial activation has been detected in selected brain areas already in presymptomatic mice, preceding astrocytosis and neuronal death.

In this study, we examine the microglial contribution to the neuronal dysfunction and death in EPM1. We aim to characterize the functional properties of Cstb-/- microglia and their effects on the survival of Cstb-/- neurons.

Our results show that the Cstb mRNA level in cultured wild type mouse microglia analyzed by real time quantitative PCR is high in relation to primary astrocytes or neurons. A gene-expression profiling of microglia from Cstb-/- mice and from wild type mice has been obtained by using the Affymetrix Mouse Exon 1.0 ST Array and reveals a down-regulation of interferon-regulated pathways in Cstb-/- microglia. The stimulation of primary wild type mouse microglia with the endotoxin lipopolysaccharide (LPS) up-regulates Cstb mRNA expression measured by real time quantitative PCR. Cstb-/- microglia show an altered inflammatory response to the stimulation with LPS by increased secretion of chemokines demonstrated by cytokine array analyses. Moreover, the release of nitric oxide from Cstb-/- microglia measured by Griess assay is elevated.

Our data indicate an altered response of primary Cstb-/- microglia to inflammatory stimuli, which may contribute to neurodegeneration in EPM1. The data provide a basis for further detailed studies on the pathophysiology and therapy of this devastating disease.


GDNF induces secretion of Cyr61 from retinal Müller glial cells – a novel neuroprotective factor in retinal degeneration

*Joanna Kucharska 1 , Patricia del Rio 1 , Blanca Arango-Gonzalez 2 , Matteo Gorza 1 , Annette  Feuchtinger 3 , Marius Ueffing 1,2 , Steffanie Hauck 1
1 Helmholtz Zentrum Munchen, Research Unit Protein Science, Munchen, Germany
2 University of Tuebingen , Division of Experimental Ophthalmology , Tuebingen, Germany
3 Helmholtz Zentrum Munchen, Institute of Pathology, Munchen, Germany
Abstract text :

Retinitis pigmentosa is a group of inherited disorders affecting photoreceptors or retinal pigment epithelium (RPE) that leads to progressive loss of vision. The Pde6brd1 mouse model is a very well-known animal model for retinal degeneration, in which rods carry a mutation in β subunit of rod cGMP-phosphodiesterease. Glial cell line-derived neurotrophic factor (GDNF) has already been shown to rescue morphology as well as function of rod cells in Pde6brd1 mouse. This effect was indirect, through stimulation of Müller glial cells (RMG). To better understand the neuroprotective effect of GDNF, primary retinal RMG were stimulated in vitro with GDNF and the secreted proteome was analyzed by proteome profiler arrays. Among others, Cyr61/CCN1, a member of CCN family, was found strongly induced upon RMG stimulation with GDNF. When applied directly to medium, Cyr61 significantly reduced photoreceptor death in organotypic ex vivo cultures of Pde6brd1 retinas. In order to identify the target cells of Cyr61 we treated, ARPE19 and MIO-M1 cell lines with Cyr61, and observed an increase in phosphorylation of Akt and Erk1/2 signaling molecules. These results suggest that stimulation of RPE and RMG cells may have a protective influence on photoreceptor survival in organotypic ex vivo conditions. We postulate Cyr61 as a novel potential candidate for future therapeutic approaches in neurodegenerative retinal disorders.


Axonal degeneration is limited in the optic nerve of EAE-induced mice by AAV2 transduction of Retinal ganglion cells (RGCs) with a site-specific phospho-mutant CRMP-2

*Jae young Lee 1 , Rachel Kenny 1 , Micheal F Azari 1 , Pei Mun Aui 1 , Kylie Magee 1 , Alan Harvey 2 , Steven Petratos 1
1 Monash University, , Clayton, Australia
2 The University of Western Australia, School of Anatomy, Physiology and Human Biology, Crawley, Australia
Abstract text :

Question: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterised by demyelination and axonal degeneration. The molecular mechanisms that underpin axonal degeneration are relatively unexplored in MS. Studies using the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), have shown that the collapsin response mediator protein 2 (CRMP-2), which play a significant physiological role in neuronal cell bodies and axons within the CNS, is phosphorylated during the neurodegenerative phase of the inflammatory disease.


Methods: We investigated the limitation of axonal degeneration by transducing retinal ganglion neurons with a phosphorylation mutant of CRMP-2 utilising an intraocular adeno-associated virus 2 (AAV2) delivery system in EAE-induced mice. The other group of mice injected with AAV2 consisting of the green flourescent protein reporter only (AAV2-GFP) with EAE was used as a control (n=8 per each group).


Results: We showed substantial preservation of axons of the optic nerve in the EAE-induced mice injected with the AAV2 carrying the CRMP-2 phospho-mutant compared (~10-fold difference) with those mice injected with AAV2-GFP. The AAV2-GFP transduced axons showed significant degeneration during the peak stage of EAE.


Conclusion: Our data suggest that phosphorylation of CRMP-2 may be a central mechanism that governs axonal degeneration during inflammatory demyelination of the CNS (as occurs in MS) and inhibition of phosphorylation of CRMP-2 may be of therapeutic potential for the progressive phase of the disease in MS patients.


Glial pathology in the prefrontal cortex affects the cognitive function of the rat

*Ana Lima 1 , Magda Reis 1 , Ana Filipa Oliveira 1 , Vanessa Morais Sardinha 1 , Cristina Mota 1 , Luísa Pinto 1 , Fernanda Marques 1 , João José Cerqueira 1 , Nuno Sousa 1 , João Filipe Oliveira 1
1 Life and Health Sciences Research Institute - ICVS, , Braga, Portugal
Abstract text :

The arising of the “tripartite synapse” concept and the discovery of the astrocytic excitability have been highlighting astrocytes as active elements concerning information flow in the brain. However, the impact of such remarkable features in complex brain function is still under-explored mostly due to the difficulty of studying neuron-astrocyte interactions in vivo.

The aim of this work was to use an in vivo model of astrocytic dysfunction and investigate the impact of this treatment in complex cognitive functions. The rationale consisted in studying behavioral performance that relies on the prefrontal cortex, such as behavioral flexibility and working memory in an animal model of astrocytic dysfunction. For that purpose, we used a pharmacological model in which Wistar-Han rats were subjected to bilateral intracranial injections of aminoadipate in the prelimbic portion of the medial prefrontal cortex to cause astrocyte depletion specifically in this region, mimicking pathological states such as depression, in which marked decreases of GFAP-positive cells are observed. This animal model was tested for its cognitive abilities by the attentional set-shifting task (ASST) and water maze-based tests.

A clear impairment of cognitive function was observed in the animals treated with aminoadipate. A detailed morphological and histological analysis showed that along with the astrocytic lesion, neurons were also affected at the site of lesion. This seems to contribute to the cognitive decline observed in this model and may explain similar observations under pathological states.


Assessment of Cell Toxicity and Matrix Metalloproteinase-9 expression by Antiretroviral Drugs in Cultured Primary Astrocytes. 

Tiziana Latronico 1 , Emilia Raimondo 1 , Fabio Mengoni 2 , Miriam Lichtner 3 , Claudio Maria Mastroianni 3 , *Grazia Maria Liuzzi 1 , *Grazia Maria Liuzzi 1
1 University of Bari, , Bari, Italy
2 Sapienza University, , rome, Italy
3 Sapienza University, Polo Pontino, , Rome, Italy
Abstract text :

Background: The toxic effects of new antiretroviral drugs on the central nervous system (CNS) are unclear. Because these drugs penetrate the brain even at low concentrations, it becomes crucial to determine the doses which can be toxic for the CNS resident cells. Moreover, after the recent introduction into clinical practice, it is unclear whether the efficacy of the antiretroviral drugs of new generation may also derive from their ability to exert extravirological effects on factors responsible for the development of HIV brain injury, e.g. matrix metalloproteinases (MMPs). Objective: To investigate on the toxicity of four different antiretroviral drugs and their ability to modulate the expression of gelatinase B (MMP-9) in astrocyte cultures.Methods: Primary cultures of rat astrocytes were activated by exposure to 10 mg/ml lipopolysaccaride (LPS) (positive control) and simultaneously treated for 20 h with increasing doses (1-5-10-25. 50 mM) of: Efavirenz (EFV); Darunavir (DRV); Maraviroc (MVC) or Raltegravir (RAL). MMP-9 mRNA expression was assessed by RT-PCR. Quantitative determination of MMP-9 expression was done by computerized scanning densitometry. Single drug toxicity was assessed by the MTT test. Each drug was considered toxic at the concentration able to induce a percentage of cell survival above 60%. Results: The treatment with antiretroviral drugs inhibited MMP-9 mRNA expression in LPS-activated astrocytes in a dose-dependent manner. In particular, a statistically significant inhibition of MMP-9 expression was observed when astrocytes were treated with 25mM EFV (54% of inhibition) or with 50 mM RAL (43% of inhibition). As assessed by the MTT test, the toxicity of the antiretrovirals ranges from 10 and 50 mM. In particular, EFV was toxic for astrocytes at the concentration of 25 mM, MVC at 10 mM, while DRV and RAL were toxic at the concentration of 50mM.Conclusions: The present results indicate that EFV and RAL directly inhibit MMP-9 expression in LPS-activated astrocytes with mechanisms that are independent from their antiviral activity. The toxic doses of antiretrovirals are much higher than those found in the CSF of HIV-positive patients. Our results highlight some beneficial/deleterious extra-viral effects of the antiretroviral drugs that may be useful to improve the development of new therapeutic strategies for the management of HIV infection.

Supported by a grant of Programma Nazionale AIDS, ISS,Italy(N. 40H8).


Effects of prenatal drinking on astrocytes parameters and behavior.


*Paula Lunardi 1 , Giovana Brolese 1,2 , Nubia B. Cunha 1,2 , Franciane L. Pedroso 1 , Carlos Alberto Gonçalves 1,2 , D. Engelke 3 , P.  Nardin 1 , C.  Batassini 1 , A.C.  Tramontina 1
1 Universidade Federal do Rio Grande do Sul, Instituto de Ciências Básicas da Saúde, Programas de Pós-Graduação em Bioquímica, Porto Alegre, Brazil
2 Universidade Federal do Rio Grande do Sul, Instituto de Ciências Básicas da Saúde, Programas de Pós-Graduação em Neurociências , Porto Alegre, Brazil
3 Universidade Federal de São Paulo – UNIFESP, Programas de Pós-Graduação em Neurociências, São Paulo, Brazil
Abstract text :

Background: Although it is widely accepted that prenatal ethanol exposure (PNEE) to high doses has long-lasting detrimental effects on brain development, the case for moderate exposure remains controversial. Recent evidence demonstrates that astrocytes are affected by PNEE, suggesting that alterations in these cells may participate in CNS disturbance associated with ethanol. The aim of this study was to investigate the effects of PNEE with moderate doses by evaluating offspring’s astrocyte parameters in acute hippocampal slices. In addition, we checked anxiety-like behavior and memory of the offspring on the thirtieth postnatal day. Methods: The dams were divided in four groups and had received 50 mL of nonalcoholic or alcoholic beer solution - Control, Vehicle (nonalcoholic solution), EtOH 5% (nonalcoholic solution 5%v.v ethanol) or EtOH 10% (nonalcoholic solution 10%v.v ethanol) during gestational day 1 up to weaning (postnatal day 22). Male offspring (30 – 35 days old) were sacrificed and hippocampal acute slices were prepared. We analyzed GFAP content, S100B and glutamate uptake as astrocyte parameters. We tested the animals in the plus-maze discriminative avoidance task to check anxiety and memory. Results: GFAP content decreased after PNEE for EtOH 5% and EtOH 10% treatment (F3,59 = 17.75, p < 0.0001). Interestingly, we found that only the EtOH 10% treatment showed an increase of hippocampal S100B content (t = 2.38, p = 0.02) and in the CSF (F3,18 = 3.249, p = 0.0462), but no difference in secretion. Glutamate uptake was significantly decreased for EtOH 10% group (F3,20 = 4.069, p =0.0208). To perform the plus-maze discriminative avoidance task we selected pups from control, vehicle and EtOH 10% treatment on PND 30. Two-way ANOVA revealed significant effects of treatment (F2,46 = 4.32, p = 0.019), arm type (F1,46 = 103.9, p < 0.0001) and treatment x arm type interaction (F2,46 = 6.66, p < 0.01). In the test session, only a significant prenatal treatment group effect (F2,46 = 6.44 p < 0.01). Bonferroni’s post-hoc test revealed that only control group presented significantly less time in the enclosed aversive arm than nonaversive one. Conclusions: In this study we showed that PNEE with moderate doses could alter the structure and functional astrocytes balance. Thus, high levels of S100B are indicated in some neurodegenerative disorders. Taken this data together we could demonstrate that moderate ethanol exposure can be harmful to fetal brain.



Oligodendrocytes, small heat shock protein HSPB5 (alpha-B-crystallin) and Huntington's disease: What is the link?

*Nash Matinyarare 1 , Ursula Puentener 1 , Jessica Teeling 1 , Jenny Morton 2 , Andreas Wyttenbach 1 , V Hugh Perry 1 , Vincent O'Connor 1 , Shmma Quraishe 1
1 University of Southampton, , Southampton, United Kingdom
2 University of Cambridge, Department of Pharmacology, Cambridge, United Kingdom
Abstract text :

Introduction Huntington’s disease (HD) is a neurodegenerative disease characterised by huntingtin protein misfolding and the intra-cellular accumulation of mutant huntingtin (mHtt). Accumulation of mHtt is most pronounced in neurons and is thought to underpin neuronal dysfunction. Oligodendrocytes have been shown to accumulate mHtt aggregates, which may contribute to cellular dysfunction in these cells. Evidence from diffusion tensor MRI (DT-MRI) studies shows that white-matter changes are amongst the earliest pre-symptomatic changes observed in HD. These findings have been taken to implicate axonal and or glial aberrations prior to disease onset.

Methods We have used a variety of techniques, which include, emulsion in situ hybridisation, whole brain sub-fractionation, immunohistochemistry, meso scale cytokine assay, and western blots.

Results Our investigations using the R6/2 mouse model show that mHtt aggregates result in a specific down-regulation of the small heat shock protein (sHSP) HSPB5 (alpha-B-crystallin). Localisation of HSPB5 by whole brain sub-fractionation shows that HSPB5 is enriched in the myelin fraction. Emulsion in situ hybridisation further shows localises HSPB5 in oligodendrocytes. Detergent extraction of myelin fractions show differential HSPB5 partitioning between soluble and insoluble fractions. Cumulatively, these findings suggest that HSPB5 is constitutively expressed in oligodendrocytes and is present in two distinct pools. Recent studies implicate HSPB5 as being a negative down-regulator of inflammation. As such down-regulation of the sHSP in R6/2 mice may promote increased oligodendrocyte vulnerability to mHtt cytotoxicity. To investigate the role of HSPB5 as a negative regulator of inflammation, we obtained transgenic HSPB5 knockout mice. The knockout mice are viable and show very little phenotypic difference against littermate controls. We have analysed the inflammatory profiles of these mice using the meso scale. To validate our findings in the R6/2 mice, we are currently investigating human tissue from distinct Vonsattel stages of disease, to see if HSPB5 downregulation is also observed in the human disease.

Conclusion As HSPB5 has several critical cellular roles, its down-regulation may compromise oligodendrocyte structure and function, which ultimately has negative consequences for neurons. It is therefore worthwhile to investigate it’s specific role in the CNS.


The role of Connexin43 gap junctions and hemichannels in the pathophysiology of Amyotrophic Lateral Sclerosis (ALS). 

*Akshata Almad 1 , Nicholas Maragakis 1
1 Johns Hopkins University, , Baltimore, United States
Abstract text :

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder that results in the loss of motor neurons (MN) leading to paralysis and eventual death of patients. Genetic studies using animal models and in vitro studies show that in addition to neurons, astrocytes are key contributors to the disease progression. Astrocytes form a highly coupled intercellular network in the central nervous system through gap junctions (GJs). GJs are composed of connexin subunits with connexin 43 (Cx43) being the predominant astrocyte connexin, which conducts crucial homeostatic functions of regulating ions and metabolites. However, under pathological conditions GJs can perform aberrant functions such as excessive propagation of excitatory molecules such as Ca2 , glutamate and other cell death signals along the astrocyte networks. Under disease conditions, connexins can also release these signaling molecules into the extracellular milieu through hemichannels. Since astrocytes are involved in the disease progression of ALS, we are exploring if abnormal GJ activity can serve as a potential mechanism for the reported astrocyte-mediated toxicity.

We used the SOD1G93A mutation as a ALS mouse model to address the role of connexins in ALS. We observed that at the end stage of the disease, spinal cords of SOD1G93A mice exhibited a significant increase in expression of connexin 43 protein compared to their spinal cords of control mice. This increase in Cx43 co-localizes primarily to astrocytes in the gray matter of the spinal cord. Importantly, human post-mortem tissue from ALS patients compared to control patients exhibited a significant increase in Cx43 RNA and protein levels in the motor cortex and a trend for increase in Cx43 in the cervical spinal cord. This is an important finding in pursuing Cx43 as a potential candidate contributing to astrocyte-mediated toxicity in ALS. In addition, when astrocytes were isolated from WT and SOD1G93A mice, endogenous levels of Cx43 RNA and protein were elevated in SOD1G93A mice compared to the WT derived astrocytes. We are currently investigating if functional properties of astrocytes from SOD1G93A mice are altered in addition to biochemical changes. In vitro studies using Cx43 specific blockers to assess neuroprotection are also being conducted in co-cultures of neurons and astrocytes. These findings will be novel and an important step towards harnessing therapeutic strategies for ALS.



Roles of anosmin-1 and FGF-2 in the biology of adult oligodendrocyte precursor cells.

*Ana Bribian 1,2 , Eva Medina-Rodriguez 1 , F. Javier Arenzana 1 , Verónica Murcia-Belmonte 1 , Pedro F Esteban 1 , Fernando de Castro 1
1 Hospital Nacional de Parapléjicos, , Toledo, Spain
2 Insitut de Bioenginyeria de Catalunya, , Barcelona, Spain
Abstract text :

During development, oligodendrocyte precursor cells (OPCs) migrate until their final destination and then differentiate to myelin-forming oligodendrocytes. OPCs exist in mature rodent and human central nervous system (CNS; around 5-7% of the total cells) and constitute an interesting source regenerative therapies in demyelinating diseases, like multiple sclerosis (MS). Different molecules are involved in OPC morphofunctional development during embryogenesis and postnatal stages, and some of them are upregulated in MS lesions, suggesting their involvement in the pathogenesis of the disease.

This is de case of anosmin-1, an extracellular matrix glycoprotein coded by the KAL1 gene and responsible for the X-linked form of Kallmann syndrome. The best known mechanism of action of anosmin-1 seems to be mediated through the interaction of this protein with fibroblast growth factor receptor 1 (FGFR1) and the modulation of the activation of this receptor by FGF2. This protein participates in the adhesion, migration and differentiation of various cell types in the CNS; among others, anosmin-1 promotes the adhesion of neurons, neurite outgrowth, axonal guidance and branching of different CNS projection neurons, as well as having a role in the migration of different types of neuronal precursors, immortalized GnRH-producing neurons and embryonic OPCs.  In addition, previous results of our group also suggest a role of anosmin-1 in demyelinated lesions from MS patients and point to the feasible pharmacological and genetic manipulation of the FGF2/FGFR-1/anosmin-1 system on endogenous and/or exogenous OPCs in demyelinating lesions.

In the present work we studied the functional implications of anosmin-1 and FGF-2 on neurobiology (cell death, proliferation, motility, migration) of postnatal/adult murine OPCs isolated from cerebral cortex (P0, P15, P60) and of OPCs isolated from adult human biopsies, using both in vivo (immunohistochemistry) and in vitro (chemotaxis chambers, migration, BrdU uptake, TUNEL, immunocytochemistry) techniques. These results would be useful for the design of effective neuroreparative therapies in MS and other demyelinating diseases.



Funded: Ministerio Economía y Competitividad (SAF2009-07842; SAF2012-40023, RD07-00606-2007, RD12-0032-12), partially by F.E.D.E.R.; European Union, “Una manera de hacer Europa”, Gobierno de Castilla-La-Mancha (PI2009-26; PAI2010/012), Fundación Eugenio Rodríguez Pascual, Red Española de Esclerosis Múltiple (RETICS  RD07/0060/2007, RD07/0060/0015). AB has a postdoctoral Sara Borrell program grant ISCIII/MINECO. EMMR has a pre-doctoral fellowship from Ministerio Economía y Competitividad (SAF2009-07842). PFE and VM are currently hired by ADE10/0010 (MINECO). FdC is hired by Sescam.


Astrocyte-derived TG2 contributes to ECM production and aggregation, and cell adhesion

*Nathaly Espitia Pinzon 1 , Wia Baron 2 , John JP Breve 1 , Benjamin Drukarch 1 , Anne-Marie van Dam 1
1 VU University Medical Center, , Amsterdam, Netherlands
2 University Medical Center Groningen, Cell Biology, Groningen, Netherlands
Abstract text :

Multiple Sclerosis (MS) is a chronic demyelinating disease of the central nervous system in which astroglial cells become hypertrophic, produce various extracellular matrix (ECM) proteins which become aggregated when secreted. These aggregated ECM proteins contribute to a non-permissive environment for repair. Tissue Transglutaminase (TG2) expression is enhanced in astrocytes in MS lesions. This enzyme is well-known for protein cross-linking capacities. Moreover, TG2 can act as a co-receptor for β-integrins to bind to ECM proteins, thereby mediating cell adhesion processes.


In this study we questioned whether astrocyte-derived TG2 affects ECM protein production and/or aggregation, and if astrocyte-derived TG2 mediates cell adhesion onto various ECM proteins. Primary rat astrocytes were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype TG2. Alternatively, primary rat astrocytes were transduced with a lentiviral vector expressing scrambled shRNA or TG2 shRNA to knockdown endogenous rat TG2. The rat primary astrocytes overexpressing human TG2 showed an increased production of fibronectin and collagen V. Conversely, knocking-down TG2 showed a decrease in fibronectin and collagen production. Interestingly, overexpression of TG2 resulted in enhanced aggregation of extracellular laminin.  Also, human astrocytoma cells (U373) were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype TG2 and allowed to adhere onto ECM-coated wells. Astrocytoma cells overexpressing human TG2 showed increased adhesion onto laminin-, and fibronectin-coated wells.


We conclude that astrocyte-derived TG2 affects production/aggregation of fibronectin and laminin. Moreover, the astrocyte-derived TG2-mediated elevated production/aggregation of fibronectin and laminin coincides with more adherence of astrocytes onto these ECM proteins. We hypothesize that TG2-mediated enhanced production/aggregation of fibronectin and laminin is related to increased astrocyte adhesion which together could contribute to the non-permissive environment for repair in the central nervous system of MS patients. 


Cyclic AMP signaling promotes differentiation of astrocyte transcriptome

Sonia Paco 1 , Manuela Hummel 2 , Virginia Pla 1 , Lauro Sumoy 3 , *Fernando Aguado 1
1 University of Barcelona, , Barcelona, Spain
2 Centre for Genomic Regulation, , Barcelona, Spain
3 Institute for Predictive and Personalized Medicine of Cancer, , Badalona, Spain
Abstract text :

cAMP signaling produces dramatic changes in astrocyte morphology and physiology. However, its involvement in phenotype acquisition and the transcriptionally mediated mechanisms of action are largely unknown. Here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of cAMP pathways. A bulk of astroglial transcripts were differentially regulated by cAMP signaling. cAMP analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. Moreover, genes typically activated in reactive cells, such as immunological mediators and scar components, were repressed by cAMP. Gene Set Enrichment Analysis and evaluation in situ of gene expression in astrocytes in different states showed that cAMP signaling conferred a mature and in vivo–like transcriptional profile to cultured astrocytes. These results indicate that cAMP signaling is a key pathway restricting developmental and activation features of astrocytes and promoting their maturation. A positive modulation of cAMP signaling is suggested to suppress the mechanisms of activation driven by pathological situations and to promote the physiologically normal state of differentiated astrocytes.


PGC-1α expression in neurons and glia cells

*Hanna Bayer 1 , David Pasche 1 , Irma Merdian 1 , Judith Eschbach 1 , Patrick Weydt 1 , Anke Witting 1
1 Experimental Neurology, , Ulm, Germany
Abstract text :

Neurodegenerative diseases are characterized by mitochondrial and metabolic abnormalities that can be detected in neurons and non-neuronal cells. Recent evidence from Huntington disease, Parkinson disease and amyotrophic lateral sclerosis research shows that peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1-α (PGC-1α), a master regulator of mitochondrial biogenesis, plays a crucial role in the mitochondrial dysfunction occurring during neurodegeneration. Of note, we recently reported specific PGC-1α isoforms which are under the control of a brain-specific promoter (Soyal et al., Hum Mol Gen 2012).

Here we examine the expression of these brain-specific PGC-1α isoforms in neurons and glia cells. We characterize the abundance of the different isoforms of PGC-1α in primary murine cell cultures of neurons, glia cells, including oligodendrocytes, astrocytes and microglia under control conditions and metabolic stress.

We show that PGC-1α is differentially expressed in the different cell types, e.g. neurons and oligodendrocytes express much higher levels of the novel brain-specific isoforms of PGC-1α. The different expression levels might reflect the different metabolic needs of the different cell types.


Analysis of astrocyte-specific gene recombination in the brain

*Carmen Bohn 1 , Hannah Jahn 1 , Frank Kirchhoff 1
1 University of Saarland, , Homburg, Germany
Abstract text :

Astrocytes are the most abundant glial cell type. They express a range of neurotransmitter receptors localized along their processes that cover synapses and constitute “microdomains” for signalling pathways. To investigate astroglial purinergic P2Y1 and glutamatergic NMDA receptors we generated knockout mice in which gene recombination of “floxed” receptors was controlled by an astrocyte-specific and tamoxifen-inducible Cre recombinase (GLAST-CreERT2).

Here, we present data on the DNA recombination efficiencies of GLAST-CreERT2 x floxed P2Y1 mice and GLAST-CreERT2 x floxed GluN1 mice in different brain regions (cerebellum, hippocampus, brainstem, optic nerve and cortex). Recombination is determined by quantitative real-time PCR of genomic DNA using primers across the loxP sequences flanking exon 1 of the p2ry1 gene and exon 11-21 of the grin1 gene, respectively. DNA recombination was induced in young adult mice by intraperitoneal tamoxifen injection for three consecutive days and analyzed three weeks later. Primers were designed such that reduction of the PCR signal reveals increasing recombination (i.e. gene excision and knockout) compared to control. The degree of reduction in the investigated brain regions is expected to be similar in both mouse lines; allowing conclusions (1) about the reliability of the GLASTCreERT2/loxP system in general (i.e. recombination efficiency at different chromosomal locations), and (2) gives a percentage of GLAST-positive cells (predominantly astrocytes) in the given brain area. First data demonstrate 30 % recombination in the cortex, which is well in line with the percentage of astrocytes in this brain region. These results could be confirmed by using a reporter mouse line expressing the red fluorescent protein tdtomato (R26-tdtomato) and using immunohistochemistry with astrocyte markers.

In conclusion, the combination of quantitative real-time PCR analysis of gene recombination and cell-specific Cre mouse lines represents a reliable approach to reveal the percentage of a given cell type in a given tissue region.


Identification and characterization of novel subtypes of astrocytes

*Zhuoxun Chen 1 , Mausam Ghosh 2 , Rita Sattler 1 , Michael Robinson 2 , Jeffrey Rothstein 1
1 Johns Hopkins University, , Baltimore, United States
2 University of Pennsylvania, , Philadelphia, United States
Abstract text :

Astrocytes, the most abundant cell type in the central nervous system (CNS), are a heterogeneous cell population that is despite its functional importance still not well defined. While astrocytes are no longer just considered the supporting cells in the CNS and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. Studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter 1 (GLT-1) with significantly increased levels of GLT-1 in the spinal cord compared to brain. The mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. To study GLT-1 regulation in more detail, we generated a family of transgenic mice using increasing lengths of the 5’ non-coding region of the GLT-1 gene controlling expression of tdTomato. These mice were crossed with full length BAC-GLT-1-EGFP mice to compare astrocytic expression of the reporter genes. Interestingly, only a subpopulation of GFP-positive astrocytes was found to have strong tdTomato fluorescence signal, when we crossed an 8.3 kb EAAT2 promoter fragment mouse with the BAC-GLT-1 mouse, suggesting the existence of astroglial subtypes that are differentially regulated for EAAT2 expression. Such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. To this end, we purified both tdTomato /GFP and tdTomato-/GFP astrocytes from multiple CNS regions by fluorescence-assisted cell sorting (FACS) and analyzed their transcriptomic profiles using microarray analysis. A list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative RT-PCR and immunofluorescence. Pax6 and Kir4.1 were found to be highly expressed in astrocytes, consistent with previous findings. More importantly, Kir4.1 mRNA was noted to be selectively enriched in cortical tdTomato /GFP cells, compared to GFP only cells. It suggests Kir4.1 may serve as an astroglial subtype marker.


Understanding the role of microRNAs in microglia-mediated neuroinflammation

Shweta Jadhav 1 , Mahesh Choolani 1 , Eng-Ang Ling 1 , Jia Lu 2 , *Thameem Dheen 1
1 National University of Singapore, , Singapore, Singapore
2 DSO National Laboratories, , Singapore, Singapore
Abstract text :

Microglia are the resident immune cells of the central nervous system (CNS) and respond to detrimental signals such as neuronal injury and infection by releasing proinflammatory cytokines and chemokines. However, neuronal damage caused due to excessive microglial activation is a well known phenomenon in neurodegenerative diseases.

MiRNAs are novel regulators of gene function, controlling several biological processes. Recent reports show altered miRNA expression in immune-mediated pathologies, suggesting that miRNAs have roles in modulating immune responses. Thus, the present study was initiated to identify microRNAs and their target mRNAs which can modulate microglial inflammatory responses. 

A pilot study was designed to understand the role of miRNA-200b in modulating microglia-mediated immune response as this was found to be localized in the microglia.  Recently, miRNA 200b has been shown to target several proteins including c-Jun, the substrate of JNK MAPK (mitogen-activated protein kinase) which mediates the release of proinflammatory cytokines in activated microglia. qRT-PCR revealed the decrease of miR-200b  expression level in activated BV2 microglia. Loss-of-function and gain-of-function studies confirmed c-Jun to be the target of miR-200b in microglia. Overexpression of miR-200b in activated microglia resulted in a decrease in c-Jun and JNK expression and activity, thereby dysregulating the MAPK-JNK pathway and proinflammatory cytokines. Further, treatment of neuronal cells, MN9D with conditioned medium obtained from activated microglial cells, resulted in increased inflammatory-mediated cell death upon knockdown of miR-200b. Overexpression of miRNA-200b also reduced the phagocytic ability in activated microglia.

Taken together, these results demonstrate the important role of miR-200b in modulating the MAPK pathway via c-Jun which in turn affects different aspects of the inflammatory process accompanying microglia activation including cytokine response, NO production, phagocytosis and neuronal cell death. Thus, miR-200b may prove to be a useful target for developing therapeutic strategies to control microglial mediated inflammation in neurodegenerative diseases.

Further in order to understand the global miRNA changes contributing to microglial activation, a global miRNA microarray was carried out using control, LPS-activated and Amyloid β-activated primary microglia. This screen identified several families of miRNAs differentially expressed between the different treatment groups enabling us to analyze the miRNA signature in activated microglia.


The RNA helicase DDX5 binds MBP mRNA and regulates MBP expression on a posttranscriptional level

*Peter Hoch-Kraft 1 , Constantin Gonsior 1 , Robin White 1 , Eva-Maria Kraemer-Albers 1 , Jaqueline Trotter 1
1 Johannes Gutenberg-University Mainz, , Mainz, Germany
Abstract text :

Myelin Basic Protein (MBP) is one of the major components of the myelin sheath and has been shown to be essential for the proper assembly of compact myelin. Consequently, animal models, lacking MBP, like the shiverer mutant mouse, show a severe CNS dysmyelination phenotype. The regulation of MBP synthesis is thereby an important step during the maturation of oligodendrocyte progenitor cells (OPCs) to myelinating oligodendrocytes (OL). In the polarized OL, MBP mRNA is translocated to the myelin compartment and its translation en route is precisely regulated in time and space mediated by the hnRNP A2-dependent RNA-trafficking pathway. hnRNP A2 binds the A2 response element (A2RE) in the MBP 3’UTR and recruits RNP complexes that mediate the fate of the mRNA, according to extracellular stimuli. This results in the localised translation of MBP at the axonal contact site. Analysis of proteins co-precipitating with hnRNP A2 yielded the RNA binding protein Dead Box RNA Helicase 5 (DDX5), as a novel potential component of MBPmRNA-associated protein complexes. Our observations show that DDX5 is associated with MBP mRNA and is capable of regulating the levels of MBP Protein on a posttranscriptional level. In line with its decreasing expression levels during OL development in vitro, DDX5 may act as an inhibitor of MBP expression and subsequently initiation of OL myelination or remyelination in the adult brain.


Novel conditional GCaMP3 mouse lines for imaging Ca2+ signals in astrocytes

*Amit Agarwal 1 , Ethan Hughes 1 , Dwight Bergles 1
1 Johns Hopkins University, , Baltimore, United States
Abstract text :

Astrocytes elevate intracellular Ca2 in response to stimulation of metabotropic receptors. These Ca2 transients are linked to processes as diverse as synaptic plasticity and functional hyperemia, but the relevance of this signaling remains uncertain. Although most studies of Ca2 signaling in astrocytes have focused on somatic events, anatomical and functional studies indicate that Ca2 signaling in astrocytes may be compartmentalized into functionally isolated “microdomains.” To facilitate analysis of Ca2 signaling, we generated transgenic mice in which the cytosolic and membrane anchored variant of genetically encoded calcium indicator GCaMP3, referred as cGCaMP3 and mGCaMP3 respectively, can be expressed conditionally in a cell specific manner. A ubiquitous CAG promoter is used to control cGCaMP3 and mGCaMP3 expression, and a “stopper” sequence flanked by loxP sites was placed upstream of the coding sequence, preventing expression until Cre excises this DNA. These constructs were targeted to ROSA26 locus to ensure widespread expression. To evaluate whether cGCaMP3 and mGCaMP3 were expressed at levels sufficient to resolve Ca2 transients, we bred R26-lsl-cGCaMP3 and R26-lsl-mGCaMP3 mice to GLAST-CreER mice. Double transgenic offspring were injected with tamoxifen at 3 weeks of age and analyzed 2-3 weeks later. Histology revealed that cGCaMP3 and mGCaMP3 were expressed in astrocytes throughout the brain. Astrocytes in acute cortical slices exhibited large amplitude, spontaneous increases in both cGCaMP3 and mGCaMP3 fluorescence. The signal to noise ratio of the fluorescence intensity was greatly improved in astyrocytes expressing mGCaMP3, partially due to the ability of mGCaMP3 to better resolve near membrane Ca2 flux. Ca2 transients were typically restricted to small regions of an individual astrocyte, consistent with Ca2 elevations within microdomains. Spontaneous Ca2 transients were occasionally observed in the soma of astrocytes expressing cGCaMP3 but not mGCaMP3, and were not coincident with activity in the processes, suggesting that these regions are functionally uncoupled. To determine if GCaMP3 expression is sufficient to visualize Ca2 signaling in vivo, we made cranial windows over the somatosensory cortex and performed 2-photon imaging. Microdomain Ca2 transients were prominent in cortical astrocytes, indicating that these mice provide new opportunities for understanding the significance of this form of signaling.


Contribution of different carbonic anhydrase isforms to proton dynamics in mouse cerebellar glial cells and neurons

*Marco D Alt 1 , Joachim W Deitmer 1
1 TU Kaiserslautern, , Kaiserslautern, Germany
Abstract text :

Cell survival and proper enzyme function require a strictly regulated intracellular pH. The intracellular buffering of protons is accomplished by (1) intrinsic buffers and (2) the CO2/HCO3- buffer system. The latter being an open buffer system, because biomembranes are usually permeable to CO2, and, in addition, most cells express HCO3- transporting membrane proteins. The enzyme family of carbonic anhydrases (CAs) catalyses the reversible reaction from CO2 and H2O to HCO3- and H , and thereby modulates the intracellular H buffer dynamics.We have performed calibrated in situ live-cell imaging with the proton-sensitive fluophore BCECF in glial cells and neurons of acute cerebellar slices of wild-type and knockout mice. The studies were used to quantify the intracellular buffer capacity and to dissect the contribution of the intracellular CAII and the extracelluar CAIV by comparing intracellular H shifts in glial cells and neurons from wild-type mice and mice deficient in their CAII or CAIV gene. We found that only one proton in 400000 is unbound and thereby chemically active, and that about 50% of this buffer capacity is mediated by the CO2/HCO3- buffer system and the other 50% by intrinsic buffers. The rate of CO2-induced change in intracellular H concentration was increased by intracellular CAII in both glial cells and neurons. The extracellular CAIV on the other hand affected primarily neurons, but also showed unexpected intracellular activity (Schneider et al. 2013, PNAS 110). The rates of acidification and alkalinisation in wild-type and knockout mice were significantly reduced in the presence of the CA blocker 6-Ethoxy-2-benzothiazolsulfonamid (10 µM). We confirmed CA protein expression by Western blot and could also show that the expression of the remaining CA is not upregulated. These results provide new insights into cellular proton-coupled processes in acute neural tissue, which shows some new complex roles of carbonic anhydrases in shaping proton dynamics in cells and tissues.

Supported by the Deutsche Forschungsgemeinschaft De 231/24-1.


Axon Initial Segment Associated Microglia

*Kelli Baalman 1 , Matthew Rasband 1
1 Baylor College of Medicine, , Houston, United States
Abstract text :

Microglia have long been known to respond to virtually any insult to the central nervous system. They quickly migrate to the site of the insult, and play many important roles, such as barricading the injury site, phagocytosing debris, and releasing cytokines. The role of microglia in the normal brain, however, has only recently begun to be appreciated. With the advent of in vivo imaging, microglia have been shown to be constantly surveying their environment with rapid extensions and retractions of their processes. Subsequent studies have shown that these ‘resting’ microglia (now known as ‘surveying microglia’) are indeed active throughout development and adulthood.  During development, microglia have been shown to be involved in synaptic monitoring and pruning as well as synaptic stripping after injury. Thus far, many studies have focused on the role of microglia at the synapse. However, we report here, for the first time, that in the cortex of normal adult rodents, a small percentage of microglia are specifically associated with the axon initial segment (AIS). The AIS is characterized by a high density of voltage-gated ion channels and plays an important role in initiation of the action potential and maintenance of neuronal polarity. This interaction seems to be limited to the ‘surveying’ phenotype of microglia and much less frequent in ‘activated’ microglia. This overlap of processes appears early in development and continues throughout adulthood although the function of microglia at the AIS is still currently unknown.


Role of neuronal injury in the control of microglia reactivity exerted by GDNF

*Graça Baltazar 1 , Julieta Oliveira 1 , Tiago Roxo 1 , Carla Fonseca 1
1 Faculty of Health Sciences, CICS-UBI, , Covilhã, Portugal
Abstract text :

Neuroinflammation is a pathological hallmark in patients and experimental models of Parkinson’s disease. Both present the classical features of inflammation, with evidence of an uncontrolled process. Moreover, microglia may become activated early in the disease process and remain primed, responding strongly to subsequent stimuli, and thereby enhancing inflammation-induced oxidative stress and cytokine-dependent toxicity in vulnerable neuronal populations.

We have previously demonstrated that glial cell line-derived neurotrophic factor (GDNF), released by ventral midbrain astrocyte cultures, potently prevents microglial activation induced by a pro-inflammatory agent. Therefore, GDNF is an important mediator in the astrocyte-microglia crosstalk keeping microglia in a resting state. However, in the brain, microglia and astrocytes are not alone and other cell types, e.g. neurons, may interfere and influence this astrocytic control of inflammation. Therefore, it is important to investigate if the presence of neurons alters GDNF-mediated astrocytic control of microglial activity. In this work we aimed at investigating whether the presence of neurons, injured or not, changes the ability of astrocyte-derived GDNF to prevent microglial activation. For that purpose, we used primary cultures of ventral midbrain astrocytes and microglia, as well as neuron-astrocyte mixed cultures from the same brain region.

Neuron-astrocyte cocultures were exposed to vehicle (control) or to the DA neurotoxin MPP . The media conditioned by control and MPP -challenged neuron-astrocyte mixed cultures, or by astrocyte cultures, was collected and transferred to ventral midbrain microglia cultures, which were then exposed to the pro-inflammatory agent lipopolysaccharide. The effect of conditioned media obtained from the different cell culture systems on microglial activity was determined by analyzing the production of nitric oxide by the Griess reaction and of the phagocytic activity by determining the engulfment of fluorescent microspheres by fluorescence microscopy.


Synaptically evoked calcium signals in astrocytic processes enhance the stability of excitatory synapses

*Yann Bernardinelli 1 , Elia Janett 2 , Irina Nikonenko 1 , Emma V. Jones 3 , Carmen E. Flores 1 , Bernadett  Boda 1 , Keith K. Murai 3 , Christian Bochet 2 , Dominique Muller 1
1 University of Geneva, Basic Neurosciences, Geneva, Switzerland
2 University of Fribourg, Chemistry, Fribourg, Switzerland
3 McGill University, Center for Research in Neurosciences, Montreal, Canada
Abstract text :

Neuron-glial communication in the CNS is fundamentally important for many brain processes including synaptic transmission and plasticity. Perisynaptic astrocytic processes (PAP) contact excitatory synapses, forming tripartite structures with neurons. In the hippocampus, the morphology of PAP has been shown to remodel rapidly and continuously but the mechanisms and roles of this form of structural plasticity remain unknown. This study investigated the physiological mechanisms driving PAP movements and their role during long-term potentiation (LTP). PAP and spines contacts were labeled by viral gene delivery of farnesylated fluorescent proteins in hippocampal slice cultures and imaged by confocal microscopy. Electron-microscopy of infected slices confirmed that PAP-synapse morphology is preserved in organotypic cultures. PAP movements adjacent to dendritic spines were evaluated with an index of motility (MI). Increasing neuronal activity by Schaffer collaterals (SC) stimulation elevated PAP MI and this was prevented by both TTX and mGluR inhibition, suggesting that neuronal activity modulates PAP movements. We next investigated whether intracellular calcium (Ca2 i) in astrocytes influenced PAP motility. BAPTA-AM bulk-loading specifically chelated Ca2 i in astrocytes and reduced PAP movements. When exogenous Gq-coupled receptors MrgA1 or MrgC11 were specifically targeted to astrocytes, their agonist FMRFa induced Ca2 i increases and accelerated PAP motility. Moreover, FMRFa delivery at the synaptic level by two-photons flash photolysis was sufficient to elevate PAP MI. We then investigated PAP dynamics in relation to LTP. Application of theta-burst SC stimulation initially increased PAP motility, but then resulted 30min later in a decrease of PAP motility. Interestingly, the reduction of PAP movements correlated with both spine enlargement and increased PAP’s spine coverage. To assess the consequences of these changes, we then specifically triggered elevation of PAP movements at the synaptic level by flash photolysis. Spine stability analysis 24 hour after uncaging revealed that spines in contact with activated PAPs were more stable than others. This study suggests that excitatory synapses control the motility of surrounding PAPs through the triggering of neurotransmitter-evoked astrocytic Ca2 i elevations. Increased PAP motility seems to be necessary to elevate their coverage of the synapse during LTP, leading to higher synapse stability.


Strongly reduced density of gray matter glutamine synthetase expressing astroglial cells in major depression but not bipolar disorder

*Hans-Gert Bernstein 1 , Gabriele Meyer-Lotz 1 , Martin Walter 1 , Henrik Dobrowolny 1 , Johann Steiner 1 , Bernhard Bogerts 1
1 University of Magdeburg, , Magdeburg, Germany
Abstract text :


Glutamate and GABA are the chief excitatory and inhibitory neurotransmitters in the adult CNS.  The mainly astroglia-located enzyme glutamine synthetase is required to synthesize  glutamine from the re-uptake of either glutamate or GABA. Thereby, this enzyme significantly contributes to the control of brain glutamate and GABA levels. There is good evidence that both neurotransmitter systems are dysregulated in depression. Hence, we decided to study the cellular expression of this enzyme in subjects with major depression and bipolar disorder. 

 Material and Methods

We investigated the numerical density of glutamine synthetase immunoreactive astroglial cells in different brain areas  (prefrontal cortex, anterior insula) of  14 subjects with major depression, 15 subjects with bipolar disorder and 19 matched control cases.

 Results and Discussion

We found that compared with controls in cases with major depression there was a significant reduction of the density of immunostained glial cells in all brain regions studied, whereas bipolar cases showed a normal expression of the enzyme.

 Our findings point to a profound disturbance of the glutamate-glutamine-GABA cycle in major depression, which is in good agreement with neuroimaging observations and molecular biologic data of others.


Microglia reactivity to amyloid-β oligomers (AβO) changes according to experimental ageing

*Cláudia Caldeira 1 , André Frederico 1 , Ana Rita Vaz 1 , Adelaide Fernandes 1,2 , Dora Brites 1,2
1 Faculty of Pharmacy-University of Lisbon, , Lisbon, Portugal
2 Faculty of Pharmacy-University of Lisbon, Biochemistry and Human Biology, Lisbon, Portugal
Abstract text :

Healthy brain aging is characterized by neuronal loss and cognitive decline being inflammation a major causative factor. Microglial activation is considered to be a major driver for the neuropathological findings in Alzheimer’s disease (AD). We evaluated whether young vs. aged microglia responded differently to Aβ soluble oligomers (AβO) and to conditioned media from AβO-treated hippocampal neurons.

Microglia from 1 day CD1 pups were incubated for 24 h at 2 (young) and 16 (old) days in vitro (DIV)   with AβO (Aβ1-42 at 50 and 1000 nM). In parallel, E16 mice hippocampal neurons were treated with AβO at 4 (young) and 18 (old) DIV for 24 h, and the incubation media used as conditioned media to treat microglia, at respective DIV, for further 24h. Cell migration (Boyden chamber), extracellular content in glutamate (commercial kit), and activity of matrix metalloproteinases (MMP)-2 and -9 (gelatin zymography) were assessed.

An increased migration towards AβO and ATP was observed in young microglia but almost undetectable in aged cells (p<0.01). Interestingly, microglia exposure to conditioned media from 1000 nM AβO-treated neurons markedly decreased both young and old microglia migration, while treatment with media from 50 nM AβO-treated neurons enhanced migration, mainly in the young microglia (p<0.05). Concerning glutamate, aged cells released less than young ones upon AβO treatment (p<0.01). In addition, when exposed to neuronal conditioned media, only the young microglia were able to exert a neuroprotective role by reducing the media glutamate content. Curiously, while AβO promoted a microglia release of MMP-9, independently of cell age, it only induced MMP-2 secretion in old cells in an AβO concentration-dependent manner (p<0.05 for 1000 nM AβO). In contrast, conditioned media from AβO-treated neurons elicited MMP-2 release only from young microglia.

Together, our data point to a loss of microglia function towards AβO with ageing and are unique in suggesting that senescent microglia may release high levels of MMP-2 in AD. In addition, our results indicate that AβO-treated neurons enhance the release of MMP-2 even by young microglia. Whether this finding may contribute to enhance inflammatory stress and the onset of AD will be the aim of further studies.

Funded by Edgar Cruz e Silva–2012 from GEECD and Pest-OE/SAU/UI4013/2011 from FCT (to DB) and Amadeus Dias from the University of Lisbon (to AF).


Axonal degeneration is assisted cell non-autonomously by glial cells

*Alejandra Catenaccio 1 , Pablo Silva 1 , Felipe Court 1
1 Pontificia Universidad Católica de Chile, , Santiago, Chile
Abstract text :

Axonal degeneration is an active process involved in a variety of neurodegenerative conditions triggered by diverse stimuli. This degenerative process can cause permanent loss of function, so it represents a focus for neuroprotective strategies. We have recently shown that axonal degeneration triggered by distinct mechanical and toxic damage is dependent on the activation of the mitochondrial permeability transition pore (mPTP), which probably represents a central execution program of axonal degeneration, upon which several pathways converge (Barrientos et. al., Journal of Neuroscience 2011). Nevertheless, the participation of other cellular types in this degenerative process has been largely overlooked.

After axonal damage in the peripheral nervous system (PNS), Schwann cells enveloping axons have been regarded as responsible for clearing degenerating axonal debris and to mount an inflammatory response for tissue clearance by invading macrophages. Nevertheless, a possible function of glial cells in modulating axonal degeneration has not been addressed so far. Here we explored the possibility that glial cells actively participates not only in the clearance of degenerating axons, but directly in the axonal degeneration program during Wallerian degeneration.

We found that axonal injury activates an early response of Schwann cells that results in the fragmentation of their associated axons by actin-rich cytoplasmic domains of Schwann cells known as Schmidt-Lanterman incisures (SLI). This first step of Schwann cell fragmentation of axons is dependent on the ERK signaling pathway, wich control the dedifferentiation program initiated in  Schwann cells after nerve injury (Napoli et. al., Neuron 2012). In the axonal compartment, injury triggers a parallel cell autonomous degenerating program dependent on mitochondrial mPTP activation, which is cell non-autonomously assisted by Schwann cells through the activation of a cell extrinsic pathway of axonal destruction by TNF-alpha secretion.

We showed for first that time that axonal degeneration in the PNS proceeds by axonal autonomous mechanisms, as well as cell non-autonomous ones dependent on SCs. Therefore, effective targets for neuroprotection should consider not only the axonal compartment, but also the associated glial cell.


Microglial modulation of synaptic strength at the first synapse in the nociceptive pathway

*Anna K Clark 1,2 , Doris Gruber-Schoffnegger 3 , Jürgen  Sandkühler 3
1 Medical University of Vienna, Centre for Brain Research, Vienna, Austria
2 King's College London, Wolfson Centre for Age Related Diseases, London , Austria
3 Medical University of Vienna, , Vienna, Austria
Abstract text :

Long-term potentiation of synaptic strength (LTP) in nociceptive pathways represents a cellular model for some forms of pain amplification. Extensive evidence indicates that microglia contribute to hypersensitivity in chronic pain models. Recent evidence also suggests that glial cell activity is required for spinal LTP (Gruber-Schoffnegger et al., 2013). Here we investigated whether stimulation of microglia is sufficient to induce an amplification of synaptic strength at the first synapse in the nociceptive pathway. We utilised the chemokine Fractalkine (FKN) in order to specifically stimulate a pro-nociceptive spinal microglial phenotype via the CX3CR1 receptor (Clark et al., 2007), and examined whether FKN was sufficient to modulate synaptic transmission between primary afferent C-fibres and spinal lamina I neurons in vitro.


Monosynaptic C-fibre evoked excitatory postsynaptic currents (EPSCs) were recorded in lamina I neurons using whole-cell patch-clamp in rat transverse spinal cord dorsal-root slice preparations. Spontaneous EPSCs were recorded simultaneously. In all experiments Bicuculline and Strychnine were added to the bath solution to block GABAA- and glycine-mediated synaptic currents.


The superfusion of spinal cord slices in vitro with FKN results in a rapid facilitation of evoked EPSCs in spinal lamina I neurons receiving monosynaptic input from primary afferent C-fibres. Both the microglial cell inhibitor Minocycline and a FKN neutralising antibody, completely prevented FKN-induced changes in synaptic transmission. In addition, the inclusion of the Ca2 chelator BAPTA or the NMDA receptor antagonist MK801 in the pipette solution completely prevented FKN-induced enhancement of synaptic strength, suggesting a post-synaptic Ca2 -dependent mechanism of LTP induction. Analysis of paired-pulse ratio (PPR) indicated that FKN-induced facilitation of synaptic strength is accompanied by a decrease in PPR suggesting a pre-synaptic mechanism of LTP expression. In support of this finding, FKN increases the number of spontaneous EPSCs recorded from lamina I neurons.


These data indicate that stimulation of microglial cells in order to induce a pro-nociceptive activity state is sufficient for facilitation of synaptic strength within the dorsal horn. Both pre- and post-synaptic mechanisms contribute to FKN-induced facilitation of synaptic strength.


This work was funded by a Wellcome Trust Flexible Travel Fellowship to AKC.


Clark AK, Yip PK, Grist J, Gentry C, Staniland AA, Marchand F, Dehvari M, Wotherspoon G, Winter J, Ullah J, Bevan S, Malcangio M (2007). Proc Natl Acad Sci U S A 19;104:10655-10660.

Gruber-Schoffnegger D, Drdla-Schutting R, Hönigsperger C, Wunderbaldinger G, Gassner M, Sandkühler J (2013). J. Neurosci., In Press.


Tailoring substrates for long-term organotypic culture of adult neuronal tissue

*Valentina Dallacasagrande 1 , Mareike Zink 2 , Steven Huth 3 , Alexander Jacob 3,4 , Marcus Müller 3 , Josef Käs 2 , Stefan Mayr 3,4 , Andreas Reichenbach 1
1 Paul Flechsig Institute, , Leipzig, Germany
2 University, Physics, Leipzig, Germany
3 Leibniz Institute for Surface Modification , , Leipzig, Germany
4 Translational Centre for Regenerative Medicine , , Leipzig, Germany
Abstract text :

Organotypic explants culture has a pivotal role in studying the complex structure of neuronal tissue. Although embryonic tissue explants and slices are used to obtain long term culture, most applications such as drug testing require adult tissue for reliable results. In this study, we show that nanostructured metal-oxide arrays can be employed to yield long-term organotypic culture of adult neuronal tissue explants as demonstrated for the adult guinea pig retina and adult murine brain slices. Even after 14 days of culture, the thickness and the layered structure of the retina were well maintained. Quantitatively, the number of nuclei within the inner and outer nuclear layers was comparable to freshly isolated retinae and no significant change in the densities of the nuclei within the layers was observed. The outer plexiform layer, as the site of synaptic connection between photoreceptors and neurons, was still well preserved. Moreover, the thickness of the photoreceptor layer was conserved, indicating that the inner and the outer segments of the photoreceptors survived well during two weeks culture. This is hardly observed in long-term cultures since the outer segments were cultured without retinal pigment epithelium. Concerning the adult murine brain slices from neo-cortex, after 9 days of culture, the neurofilaments and cell nuclei were still well preserved and the neuronal network displayed the typical arrangement of long axons. However, preservation of the tissue depends strongly on the geometry of the nanostructured array surface such as roughness and spacing, whereby organotypic brain slice culture requires different geometries than retina culture. Since organotypic culture of adult tissue could only be obtained for about 7 days with conventional techniques, our new substrate material could be the breakthrough for in vitro studies on tissue regeneration, neuroplasticity, as well as drug testing.


The “Yin and Yang” in depression: how astrocytes and neurons differently respond to antidepressants to remodel neuronal synaptic contacts

*Barbara Di Benedetto 1 , Sebastian Giusti 1 , Annette Vogl 1 , Elisabeth Butz 1 , Theo Rein 1 , Damian Refojo 1 , Rainer Rupprecht 1
1 Max Planck Institute of Psychiatry, , Munich, Germany
Abstract text :

Functional alterations in synaptic contacts have often been described as a hallmark of major depressive disorder (MDD). Antidepressants (ADs) have been shown to restore neuronal circuits through an enhancement of synaptogenesis in some regions of the brain. Nevertheless, the underlying mechanisms are still unclear. Glia cells have been since long acknowledged as active partners of neurons in orchestrating molecular signals crucial for the proper arrangement of neuronal circuits in the developing and adult brain. Therefore, understanding how both neurons and astrocytes respond to antidepressants (AD) is of high interest.

Using rat C6 glioma cells and primary cultures of astrocytes and neurons, we showed a time-dependent cell-autonomous modulation of the ERK/MAPK signalling pathway after acute treatment with various classes of ADs in both cell types. Specifically, glia cells responded to ADs with the simultaneous and fast activation (after 10 min treatment) of both ERK1/2, in contrast to treatment with antipsychotics or mood stabilizers. This activation induced 48 hrs later an increased release of GDNF, a factor involved in synapse formation and axonal wiring, which was MAPK-dependent. On the contrary, hippocampal neurons showed a reduction in ERK activity that correlated with neuronal activity inhibition after short term (10 min) ADs administration, as demonstrated by quantification of c-Fos expression after KCl stimulation. Interestingly, this inhibitory effect was reversible after long term (48 hrs) ADs treatment.

To further identify whether GDNF released from astrocytes treated with ADs might be responsible for long term changes in neuronal synapses, we examined how ADs influenced synaptic densities in neurons alone or co-cultured with astrocytes. Strikingly, we found that the number of synapses was reduced at 48hrs after AD treatments, but only in the presence of intact astrocytes and not in cultures of neurons alone or neurons treated with astrocyte-conditioned media. This effect was reversed after 120 hrs treatment and rescued by the soluble form of the specific GDNF receptor GFRalpha1, but not by GDNF alone. Moreover, live imaging of astrocytes showed dramatic morphologic changes of their processes in response to ADs that might underlie the observed synaptic remodelling. Our analysis of changes occurring at the neuron-glia interface upon AD treatments might elucidate novel mechanisms of ADs action that may open the avenue for unravelling the role of astrocytes in psychiatric diseases and implement pharmacologic treatment regimens for MDD patients.


D-Serine released by astrocyte can modulate the respiratory rhythm in neonatal mice

*Jaime Eugenin 1 , Sebastian Beltran-Castillo 1 , Isabel Llona 1
1 USACH, , Santiago, Chile
Abstract text :


Activation of NMDA receptors increase the respiratory frequency in mammals. Astrocytes are in intimate contact with neurons, specially in glutamatergic synapses, and are able to sense neuronal activity, react to, and even influence nearby neurons. Furthermore, they can sense changes in H or PCO2, and in response to these stimuli, they can release molecules like ATP and D-serine, which will activate neuronal circuits. Then D-serine, an agonist of the glycine site of the NMDA receptor, can affect the respiratory rhythm during hypercapnia. Our aim was to study the probable role of D-serine in the modulation of the respiratory rhythm in mice neonates.



Fictive respiration was recorded with suction electrodes from C4-C5 ventral roots in “en bloc” (brainstem-spinal cord) preparations from P0-P3 CF1 mice. Superfusion was done with artificial cerebrospinal fluid equilibrated with O2:CO2 = 95%: 5%, (pH 7.4,28ºC) containing different D-serine concentrations (0.1-100 µM) in presence or absence of D-amino acid oxidase (DAAO), which degrades D-serine. In addition, hypercarbic acidosis (switching equilibration from 5 to 10% CO2, changing pH from 7.4 to 7.2) was done in absence or presence of DAAO.



Application of D-serine increased the frequency of the respiratory rhythm in a concentration-dependent way. Application of DAO attenuated the effects of D-serine application. Likewise, DAAO reduced slightly the effect of hypercarbia on the respiratory rhythm.



Our preliminary experiments indicate that D-serine is a potent agent increasing the respiratory rhythm frequency in in vitro neonatal preparations, and likely, in association with ATP, is mediating the respiratory response to hypercarbia. 


Neurofilaments protect oligodendrocytes from lysolecithin toxicity in vitro

*Catherine Fressinaud 1 , Joël Eyer 1
1 University Hospital, , Angers, France
Abstract text :

Neurofilaments (NF) and co-isolated proteins increase the proliferation and differentiation of oligodendrocytes (OL) in vitro (Fressinaud et al., 2012). Since NF are released in the cerebrospinal fluid in MS, and their concentration correlates with disease severity, they might then regulate remyelination by OL. Thus, NF effects were determined in a model of OL cultures treated with lysophosphatidyl choline (LPC) (Fressinaud, 2005). LPC decreases the proliferation of OL progenitors (OLP) and the differentiation of surviving cells, and it destroys myelin-like membranes. Recovery was significantly improved by NF fractions, as well as tubulin (TUB), added after LPC removal: compared to cultures treated with LPC alone the number of OLP (A2B5 cells), and their proliferation increased significantly, as well as the number of differentiated (CNP ) and mature (MBP ) OL. Moreover, NF and TUB protected OL from LPC toxicity when added at the time of LPC treatment: they increased OLP proliferation, as well as the number of CNP and MBP OL significantly, compared to cultures treated only with LPC. On the contrary irrelevant proteins (actin, and skin proteins) were ineffective, demonstrating the specificity of the cytoskeleton proteins effects. Importantly, NF and TUB increase OLP survival and proliferation when challenged with LPC, without delaying differentiation and maturation. We hypothesize that release of NF and TUB following axonal damage in MS could participate in the regulation of remyelination through this process. This putative phenomenon might be complexified by alterations of axon protein expression, or of proteolysis, known to occur in MS models.


Transfer of Exosomes from Oligodendrocytes to Neurons

*Dominik Fröhlich 1 , Wen Ping Kuo 1 , Carsten Frühbeis 1 , Wiebke Möbius 2 , Klaus-Armin Nave 2 , Anja Schneider 2 , Mikael Simons 2 , Matthias Klugmann 3 , Sheena Pinto 4 , Bruno Kyewski 4 , Jacqueline Trotter 1 , Eva-Maria Krämer-Albers 1
1 University of Mainz, Molecular Cell Biology, Mainz, Germany
2 Max Planck Institute of Experimental Medicine, Department of Neurogenetics, Göttingen, Germany
3 University of New South Wales, Department of Physiology, Sydney, Australia
4 German Cancer Research Center, Division of Developmental Immunology, Heidelberg, Germany
Abstract text :


Exosomes are small membranous vesicles of endocytic origin which are released by almost every cell type and have been implicated to play important roles in intercellular communication.  We have recently discovered that oligodendrocytes secrete exosomes containing a distinct set of proteins as well as mRNA and microRNA. Intriguingly, oligodendroglial exosome release is stimulated by the neurotransmitter glutamate indicating that neuronal electrical activity controls glial exosome release. In this study we examined the role of exosomes in neuron-glia communication and its implications in glial support.


To analyze the transfer of oligodendroglial exosomes to neurons, we exposed cultured cortical neurons to fluorescently labeled oligodendroglial exosomes. Indeed, cultured cortical neurons internalized and accumulated oligodendroglial exosomes in the neuronal cell soma in a time-dependent manner. Addition of endocytosis inhibitors or expression of dominant negative dynamin interfered with neuronal exosome internalization indicating that exosome uptake is mediated by clathrin-dependent endocytosis. Furthermore, neuronal internalization of exosomes resulted in functional retrieval of exosomal cargo in vitro and in vivo upon stereotactic injection of exosomes. To investigate the influence of oligodendroglial exosomes on neuronal gene expression we performed a microarray screen of neuronal mRNAs after exosome exposure. Interesting candidates were further validated by qRT-PCR.


Taken together, our results represent a proof of principle of exosome transmission from oligodendrocytes to neurons suggesting a new route of horizontal transfer in the CNS.


Characterization of glial cells in organotypic cultures of rat retina

*Beatriz I. Gallego 1 , Ana I Ramírez 1,2 , Michelle Dierstein 3 , Rosa de Hoz 1,2 , Blanca Rojas 1,4 , Alberto Triviño 1,4 , José M Ramírez 1,4 , Marius Ueffing 3 , Juan J Salazar 1,2 , Blanca Arango-Gonzalez 3
1 Universidad Complutense (UCM), Inst Invest Oftalmol Ramón Castroviejo, Madrid, Spain
2 Universidad Complutense (UCM), Facultad Optica y Optometria, Madrid, Spain
3 Eberhard Karls Universität Tübingen, Institute for Ophthalmic Research , Tübingen, Germany
4 Universidad Complutense (UCM), Departamento Oftalmologia. Facultad Medicina, Madrid, Spain
Abstract text :


Organotypic retinal culture is a useful tool to perform preclinical drug testing, in which cellular interactions as well as tissue three-dimensionality are preserved. The present study aimed to provide a detailed characterization of the morphologic changes of macro and microglial cells in this culture system.


Retinas were isolated from 7 days old Crl: CD(SD) rats with the retinal pigment epithelium attached as described previously (Arango-Gonzalez et al. 2010). Explants were cultured for 4, 10 and 14 days (DIV4, DIV10 and DIV14). Glial cell populations were characterized by immunostaining cryosections and wholemounts of age-matched control and cultured retinas using specific antibodies against GFAP, vimentin and CD11-b.


In DIV4 and DIV10 cultures, GFAP-positive astrocytes were more robust and not homogeneously distributed as observed in vivo: in some retinal areas the astrocytic network was thicker and in other regions astrocytes were sparsely distributed. At DIV14 only few thinned GFAP-positive astrocytes remained. GFAP immunoreactivity of Müller cells was up-regulated in culture. However, no major difference was observed in vimentin staining between both, in vivo and in vitro retinas. Müller cells extended processes from the inner to the outer limiting membrane were observed for all examined retinas. Microglial cells stained with CD11-b at DIV4 and DIV10 had somas more robust than in vivo and cell processes were thicker and retracted. Same cells at DIV14 exhibited mainly a rounded morphology.


Progressive morphological changes were observed in glial cell populations in retina explants. These changes include reactive macrogliosis, rearranged astrocytic distribution and microglial activation. Since glial response is a hallmark of several retinal diseases, our organotypic retinal culture is a valuable resource for future investigations in retinal degenerative processes and therapy.


Epigenetic induction of the Ink4a/Arf locus prevents Schwann cell overproliferation during nerve regeneration and after tumorigenic challenge

*Clara Gomis Coloma 1 , Jose Antonio Gomez-Sanchez 2,1 , Cruz Morenilla-Palao 1 , Gloria Peiro 2 , Eduard Serra 3 , Manuel Serrano 4 , Hugo  Cabedo Marti 2,1
1 Instituto Neurociencias Alicante UMH-CSIC, , San Juan de Alicante, Spain
2 Hospital General Universitario Alicante, , Alicante, Spain
3 Institut de Medicina Predictiva i Personalitzada del Cancer (IMPPC), , Barcelona, Spain
4 Centro Nacional de Investigaciones Oncologicas (CNIO), , Madrid, Spain
Abstract text :

The number of Schwann cells is fitted to the axonal length in the peripheral nerves. This setting is lost when tumorigenic stimuli induce uncontrolled Schwann cell proliferation, generating tumours such us neurofibromas and schwannomas. Schwann cells proliferate as well during Wallerian degeneration. In both cases proliferation is finally arrested. We show that in neurofibroma, the induction of Jmjd3 removes trimethyl groups on lysine-27 of histone-H3 and epigenetically activates the Ink4a/Arf-locus, forcing Schwann cells towards replicative senescence. Remarkably, loss of function of this mechanism allows unrestricted proliferation, inducing malignant transformation of neurofibromas. Interestingly, our data suggest that in injured nerves, Schwann cells epigenetically activate the same locus and go as well into the senescence program. Indeed, when this pathway is genetically blocked, cell proliferation results increased after nerve injury. We postulate that the Ink4a/Arf-locus is expressed as part of a physiological response that prevents uncontrolled proliferation of the de-differentiated Schwann cells generated during nerve regeneration, a response that is also activated to avoid overproliferation after tumorigenic stimuli in the peripheral nervous system.


Basal forebrain lesions reduce acetylcholinergic tone, induce microglial priming and predispose mice to inflammation-induced cognitive deficits

*Éadaoin W. Griffin 1 , Robert Field 1 , Colm Cunningham 1
1 Trinity College Dublin, , Dublin, Ireland
Abstract text :

Microglia in the degenerating or aging brain are primed by aspects of pathology to produce exaggerated pro-inflammatory responses to subsequent central and systemic inflammatory insults. However, the mechanisms by which microglia become primed, rather than adopting an M1 phenotype, are not clear. Triggers for priming may include altered expression of complement, recognition of amyloid or neuronal/synaptic debris for phagocytosis, decreased engagement of microglial regulatory molecules such as CD200R, TREM2 or fractalkine receptor. There is also evidence that loss of basal neurotransmitter tone may contribute to this state. Microglia are known to express the nicotinic cholinergic a7 receptor, which has been shown to influence macrophage and microglial reactivity. In the current study we performed limited lesions of the basal forebrain cholinergic system using the murine-p75-saporin immunotoxin (mu-p75-sap), to investigate the degree to which loss of cholinergic tone predisposes the microglia to subsequent inflammatory stimulation and to assess the consequences of this for cognitive function in the vulnerable brain. Intracerebroventricular injection of mu-p75-sap (0.08μg) depleted cholinergic neurons in the basal forebrain and decreased cholinergic innervation of the hippocampus, but left performance on hippocampal-dependent reference and working memory tasks relatively intact. However, decreased cholinergic innervation of the hippocampus conferred increased susceptibility to cognitive deficits induced by systemic LPS (100μg/kg) 40 days after lesioning, but microglia were not primed 40 days after 20% lesions. To investigate the regional, temporal and neurochemical basis of this we induced more severe lesions of the basal forebrain (using 1.2μg mu-p-75 sap) and assessed microglial priming at 14 or 40 days post-lesion. These data demonstrate that microglia are primed by low dose challenge at 14 but not 40 days and, when more robust lesions are induced, priming remains even at 40 days. These responses are observed at the site of injury (medial septum) but more profoundly at the site of denervation, the hippocampus. Indicating that neuronal injury and, particularly, loss of cholinergic tone have a profound effect on the reactivity of the microglia to subsequent inflammatory challenge. These data expand our knowledge of microglial priming and have clear implications for the interaction of cholinergic tone and inflammation in cognitive dysfunction such as dementia and delirium in which cholinergic dysfunction is implicated. 


Astrocyte TNFα-dependent alteration of hippocampal excitatory synaptic transmission in a mouse model of Multiple Sclerosis

*Samia Habbas 1 , Mirko Santello 1 , Hiltrud Stubbe 1 , Giovanna Zappia 1 , Nicolas Liaudet 1 , George Kollias 2 , Adriano Fontana 3 , Tobias Suter 3 , Andrea Volterra 1
1 University of Lausanne, , Lausanne, Switzerland
2 Institute for Immunology, , Vari, Greece
3 University Hospital Zurich, , Zurich, Switzerland
Abstract text :

Astrocytic signalling via purinergic P2Y1R reversibly potentiates excitatory transmission at perforant path-granule cell (PP-GC) hippocampal synapses. The effect, mediated via glutamate release and activation of presynaptic NR2B-NMDAR, requires constitutive levels of TNFα as permissive factor (Jourdain et al., 2007 and Santello et al., 2011).  Here we show that when TNFα levels increase the cytokine becomes instructive and directly evokes glutamatergic gliotransmission. Thus, brief application of high TNFα causes persistent potentiation of excitatory transmission, measured as increased mEPSC frequency in GCs. This effect is both TNFR1- and NR2B-dependent because it is blocked in TNFR1-/- mice and by ifenprodil, a selective NR2B antagonist. In order to establish if the effect depends on TNFR1 signaling in astrocytes, we created mice where TNFR1, knocked out in all cells, can be conditionally re-expressed only in astrocytes. With this model, we observed that astrocyte TNFR1 expression is sufficient to reestablish the synaptic effect of TNFα. Next, we examined whether a similar TNFα-dependent effect could be observed in defined pathological conditions. We selected Multiple Sclerosis (MS) because: 1) >50% of the MS population suffers from cognitive impairment possibly due to hippocampal dysfunction (Benedict and Zivadinov, 2011), and 2) TNFα and TNFR1 are key players in both the human pathology (Gregory et al., 2012) and in its murine model, Experimental Auto-immune Encephalomyelitis (EAE) (Probert et al., 2000).  We recorded PP-GC synaptic activity after EAE induction via Adoptive Transfert (AT-EAE) and found that mEPSC frequency was increased compared to control animals. Moreover synaptic alteration in AT-EAE mice depended on astrocytic TNFR1 because the effect was absent in TNFR1-/- mice but reappeared upon TNFR1 re-expression in astrocytes. NR2B receptors are also necessary because in vivo ifenprodil injection prevented synaptic alteration. Overall, our study reveals a specific mechanism responsible for hippocampal synaptic dysfunction possibly relevant for cognitive impairment in MS. Research supported by SNSF grant 31003A-140999 and NCCR “Synapsy” to AV.


Quantitative profiling of retinal Müller glial cell surface proteome changes in response to LPS treatment

*Stefanie Hauck 1 , Christine von Toerne 1 , Jennifer Behler 1 , Juliane Merl 1 , Marius Ueffing 1,2
1 Helmholtz Zentrum München, , Neuherberg, Germany
2 University Medical Centre, Centre of Ophthalmology, Tübingen, Germany
Abstract text :

Retinal Müller glial cells (RMG) are the main glial cell type in the retina and are essential players providing neurotrophic, metabolic and structural support for retinal neurons as well as mediating inflammatory responses in the retina. While some key neuroprotective molecules in the context of retinal degeneration have been demonstrated to be of RMG origin, there is yet no comprehensive understanding on the multifaceted role of RMG in orchestrating diverse intercellular functions. In order to systematically as well as functionally analyse RMG reactivity to retinal degeneration and thus explore the RMG-derived signalling molecules in depth, we aim at comprehensively profiling proteome-wide cellular responses to stimuli. We thus developed a proteomics approach screening specifically for cell surface and membrane proteins as well as secreted protein expression profiles and in addition monitor quantitative changes induced by prototype inflammatory inducer lipopolysaccharide LPS.

This workflow utilises sugar residues of cell surface proteins on intact RMG which are mildly oxidized and then covalently coupled to biotin. Biotinylated proteins are affinity purified and glycosylated peptides are specifically released by PNGaseF followed by protein identification and quantification by label-free LC-MSMS. This workflow resulted in the identification of more than 500 proteins on cell surfaces complemented by more than 700 proteins identified in the RMG secretome. Bioinformatic analysis allocates 75% of the cell surface proteins to be truly membrane or extracellular matrix proteins. This comprehensive cell surfaceome includes transmembrane receptors, transporters, adhesion molecules, signalling molecules and proteases, including 18 CD markers, 18 integrins, 41 solute carriers, two ephrins, five ephrin receptors and six plexins. Treatment of cells with LPS results in a highly reproducible significant shift of cell surface proteome with upregulation of 36 proteins and downregulation of 13 proteins. Among those LPS-induced cell surface expression changes are proteins that suggest an active role of these glial cells in inflammatory processes. The specific changes will be discussed in detail.

Cell surface biotinylation on glycosyl-residues in combination with label-free LC-MSMS is a sensitive and reproducible method to profile cell surface proteomics and sheds light on the biological properties of cells.


Neuroprotection of Retinal Ganglion Cells by Müller Glia and Astrocytes

*Jennifer Higginson 1 , Daniel Piso 2 , Patricia Veiga-Crespo 1 , Sansar Sharma 3 , Elena Vecino 1
1 Universidad del Pais Vasco, , Leioa, Spain
2 ESS Bilbao, Research in Control and Diagnostics, Leioa, Spain
3 New York Medical College, Department of Cell Biology and Anatomy, Valhalla, NY, United States
Abstract text :

Following retinal disease or injury, for example in glaucoma or retinal ischemia, axonal degeneration and death of retinal ganglion cells (RGC) results in irreversible blindness. The retinal glial cells, astrocytes and Müller glia, provide structural and trophic support to the RGCs in the healthy retina and may also have a function in promoting cell survival after injury. In rat models of glaucoma and ischemia, Müller glia were found to adopt a branched morphology, suggesting that there exists a degree of cell plasticity, which may have functional significance. In this study, we have investigated the relationship between glia and RGCs in the healthy and damaged retina both in vivo and in vitro. In damaged retinas, Müller glia significantly modify their branches enveloping RGCs in vivo. Moreover, in vitro Müller cells promote significant neurite elongation when they are in close contact with RGCs. In addition to this, the organization of astrocytes in the retina of a rat model of glaucoma was found to be significantly different to those of a healthy retina. These alterations in glial cell morphology may reflect changes in their relationship with RGCs and could signify profound changes in their secretome, which may influence RGC survival.  


Neuron-astrocyte interactions during the development of the somatosensory cortex in a genetic model of absence epilepsy: from morphology to in vivo calcium imaging

*Guillaume Jarre 1 , Séverine Stamboulian 1 , Antoine Depaulis 1 , Jean-Claude Platel 1 , Isabelle Guillemain 1
1 Grenoble-Institut des neurosciences Inserm U 836 -, , Grenoble cedex 9, France
Abstract text :

Epilepsy is one of the most frequent neurological diseases that affects up to 1% of the world population and which is associated with a hyper-synchronicity and hyper-excitability of neuronal networks. Absence epilepsy is a non-convulsive epileptic syndrome characterized by spike-and-wave discharges (SWD) on the electroencephalogram (EEG) associated with a behavioral arrest. It occurs during childhood and, in most of the case, disappears before adulthood, suggesting a dysfunction of brain development and maturation. We have recently shown in the GAERS, a genetic model of absence epilepsy, that spontaneous seizures are initiated in the barrel region of the somatosensory cortex (SSC) and then spread to the rest of the cortex and the thalamus. These seizures are characterized by SWD which occur spontaneously around 25th day after birth (P25) but are preceded by EEG abnormalities as soon as P15. To understand what makes this SSC network more synchronous and excitable, we performed a morphological study of neurons and astrocytes by estimating densities of NeuN and S100-positive cells respectively. Although we found similar densities of neurons and astrocytes between GAERS and non-epileptic controls, the thickness of the SSC was 30% smaller in GAERS. Western blotting quantification of GFAP, another astrocytic marker, was significantly higher at P15-P17, as compared with non-epileptic control. Moreover, GFAP-immunolabeling suggested that these astrocytes were reactive. We hypothesized that these modifications are linked with an alteration of astrocytic and/or neuronal physiological calcium excitability that could lead to the occurrence of epileptic seizures. To better evaluate the role of astrocytes and neurons networks during the development of SSC between P15 and P25, we used two-photon microscopy imaging coupled with EEG recording in animal under anesthesia and neuroleptanalgesia. We first analyzed neuropile, representing ascendant projection of deeper cortical layers, astrocytes and neurons calcium activities. Preliminary results obtained from animals under isoflurane anesthesia showed similar neuropile activities. Astrocytic and neuronal activities were too weak to highlight differences between GAERS and non-epileptic control. This is likely due to the mode of anesthesia known to strongly decrease astrocyte calcium signaling and neuronal synchronization. Current experiments are therefore performed using neuroleptanalgesia know to allow the occurrence of SWD. The expected data should lead to a better understanding of neuron-astrocyte interactions during brain development and the mechanism underlying epileptogenesis in a genetic model of epilepsy.


Misfolded truncated tau protein influences neuron-glial interaction via regulation of the “On” and “Off” signalling molecules

*Zuzana Kazmerova 1 , Norbert Zilka 1,2 , Monika Zilkova 1,2 , Tomas Smolek 1,2 , Michal Novak 1,2
1 Institute of Neuroimmunology, , Bratislava, Slovakia
2 AXON Neuroscience SE, Grӧsslingova 45, Bratislava, Slovakia
Abstract text :

The reciprocal communication between neuronal and glial cells represents the key component of the immunosurveillance system of the brain.  It has been proposed that the neuro-glial interaction is impaired in human Alzheimer’s disease. In this study we focus on neuronal “On” and “Off” signalling molecules in the transgenic rat model for Alzheimer’s disease. Using enzyme-linked immunosorbent assay we determined the level of CD47, CX3CL1 (“Off” signalling molecules) and MMP3, TREM2 (“On” signalling molecules) in brain homogenates. Our results demonstrated significantly up-regulated level of CD47 (p < 0,001) in our AD rat transgenic animal model. On the other hand, quantification of MMP3 level revealed significant decrease of MMP3 expression (p < 0,001) in tested transgenic animals. Previous studies showed that CD47 and MMP3 are predominantly expressed on neuronal cells in CNS where CD47 functions normally as a marker of “self” to protect intact body component or “don’t eat me” molecule which protect CD47-expresing cells from phagocytosis. Our results indicate that overexpression of CD47 on neuronal cells expressing pathologically modified truncated tau protein can be used as a neuroprotective mechanism potentiating spread and accumulation of pathological modified tau protein in neurons affected by AD pathology which in final stage support progression of the developing disease. In conclusion, we showed, that pathologically modified Tau protein can modulate specific “On and Off” signalling molecules and thus affects neuron-glia interaction in AD.

 Acknowledgement: This work was supported by Axon Neuroscience and research grants VEGA 2/0161/11, 2/0205/11, 2/0193/11, APVV 0200-11 and structural fund 26240220046.


Protection effect of glial cell line-derived neurotrophic factor on neurons and glial cells under photodynamic injury

*Maxim Komandirov 1 , Evgenya Knyazeva 1 , Mihail Rudkovsky 1 , Uliya Fedorenko 1 , Grigory Fedorenko 1 , Anatoly Uzdensky 1
1 Southern Federal University, , Rostov-on-Don, Russian Federation
Abstract text :

Tissue reactions on external injuries depend on intercellular interactions that are provided in particular by neurotrophic factors. Neurotrophic factors are mediators that transmit “survival signal” in nervous system. The influence of the glial cell line-derived neurotrophic factor (GDNF, 10 ng/ml) on photo-induced changes in neuronal activity and ultrastructure and death of neurons and glial cells (GC) was studied in this work to investigate the role of neurotrophic intercellular interactions in cell survival under the photodynamic injury. Photodynamic injury is a very effective inducer of the oxidative stress. Crayfish stretch receptor (SR) was the model object in this research. Cells were photosensitized with aluminophthalocyanine “Photosens” and illuminated with laser (640 nm). We used the method of dual fluorochroming of specimens with propidium iodide and Hoechst 33342 to visualize morphologic features of photo-induced apoptosis of GC and necrosis of neurons and GC. Photodynamic injury caused the necrosis of neurons and GC and apoptosis of GC. GDNF significantly reduced the level of photo-induced cell death both necrosis and apoptosis. In presence of GDNF after illumination ultrastructure of cells were more saved then without one. There was the segregation in the Nissl bodies abundant in ribosomes, cisterns of endoplasmic reticulum (ER) and mitochondria. Large number of ribosomes and polysomes means that protein synthesis was saved and proteins were stored and packed in dictyosomes to be transported to the periphery of neurite. Hereby, GDNF protected neurons and GC of SR from photo-induced damages


Impact of oligodendrocyte-derived exosomes on neuronal metabolism: a role in neuroprotection? 

*Wen Ping Kuo 1 , Dominik  Fröhlich 1 , Carsten  Frühbeis 1 , Christoph Zehendner 2 , Heiko Luhmann 2 , Jacqueline Trotter 3 , Eva-Maria Krämer-Albers 3
1 University of Mainz, , Mainz, Germany
2 University of Mainz, Physiology and Pathophysiology, Mainz, Germany
3 University of Mainz, Molecular Cell Biology, Mainz, Germany
Abstract text :

Question: Myelination in the CNS requires an intimate communication between oligodendrocytes and neurons. Since oligodendrocytes maintain axonal integrity, we examined the role of oligodendroglial exosomes in axon-glia communication and their potential impact on the neuronal metabolism. Exosomes are nano-sized vesicles carrying a specific set of lipids, proteins, and RNAs to target cells to functionally impact their behavior. Our previous studies indicate that neuronal electrical activity controls glial exosome release resulting in subsequent neuronal uptake and functional retrieval of the exosomal content. Proteomic analysis of oligodendroglial exosomes revealed a list of candidates with potential neurotrophic action in neurons. To elucidate the impact of oligodendroglial exosomes on the neuronal metabolism, we analyzed the metabolic activity of neurons after co-culture with oligodendrocytes or direct treatment with exosomes.

Methods: Neurons were subjected to different stress paradigms such as oxidative stress, hypoxia, and nutrient deprivation. Neuronal vitality was assessed by MTT assay and the mitochondrial membrane potential was visualized by staining with MitoCapture

Results: Neurons grown under optimal conditions were not affected by the presence of exosomes. Intriguingly, when neurons were subjected to stress (oxidative stress, nutrient deprivation, oxygen-glucose deprivation) their metabolic activity was significantly increased in the presence of exosomes. When challenged with oxidative stress prior to exosome treatment, neurons were not able to recover. MitoCapture staining demonstrated that oligodendroglial exosomes prevent the breakdown of the mitochondrial membrane in nutrient-deprived neurons.        

Conclusions: Our results indicate that exosome supply is protective for neurons but is unlikely to support their recovery. We suggest that oligodendroglial exosomes carry neuroprotective substances, which protect neurons from stress. 


The fate of mDach1-expressing cells in the dorsal part of the lateral ventricles following focal cerebral ischemia

*Miroslava Anderova 1,2 , Helena Pivonkova 1,2 , Pavel Honsa 1,2
1 Institute of Experimental Medicine, AS CR, , Prague, Czech Republic
2 Second Medical Faculty, Charles University, , Prague, Czech Republic
Abstract text :

The mDach1 gene, involved in the development of the neocortex and the hippocampus, is expressed by neural stem cells (NSCs) during early neurogenesis, and in the adult mouse brain, mDach1 gene activity persists in particular subpopulations of neurons and astrocytes of the cortical plate, in the CA1 layer of the hippocampus and in certain cell subpopulations in the dorsal part of the lateral ventricles (LV). In this study we aimed to elucidate the role of mDach1-expressing cells in adult neurogenesis and gliogenesis under physiological as well as post-ischemic conditions, employing transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the D6 promotor of the mDach1 gene. Middle cerebral artery occlusion (MCAo) was used as a model of ischemic injury, and sham-operated mice were used as controls. We isolated GFP cells from the dorsal part of the LV of sham-operated- and post-ischemic brains and employed a neurosphere-forming assay in order to estimate their ability to proliferate and self-renew. Furthermore, we followed their immunocytochemical and electrophysiological properties during in vitro differentiation and compared the differentiation potential of GFP cells isolated from the controls to those isolated from post-ischemic brains. The GFP cells isolated from the dorsal part of the LV of controls formed neurospheres and differentiated only into a glial phenotype. The GFP cells isolated from the dorsal part of the LV of post-ischemic brains were able to form neurospheres as well; however, besides their differentiation into a glial phenotype, they also gave rise to cells with the properties of neuronal precursors. We also performed in situ immunohistochemical/electrophysiological analyses of GFP cells in the adult brain of controls and those after MCAo. In situ analyses revealed that GFP cells expressed the phenotype of adult NSCs or neuroblasts in controls or following ischemia and that their number was increased in both hemispheres following MCAo. Compared to controls, we found that the number of GFP/doublecortin-positive cells significantly increased in the dorsal part of the LV as well as the number of GFP cells in the olfactory bulb, where they probably differentiated into calretinin interneurons. Our results indicate that GFP cells with an active mDach1 gene in the dorsal part of the LV play an important role in the increased production of neuroblasts after injury and that this process is also enhanced in the contralateral hemisphere. Collectively, our results reveal the involvement of the mDach1 gene in adult neurogenesis. Cells expressing this gene exhibit the properties of adult NSCs or neuroblasts and respond to MCAo by enhanced neurogenesis.

Supported by GA CR P303/12/0855, GAUK 383711.


Lactate increases TREK channel activity in CA1 stratum radiatum Astrocytes

*Aditi Banerjee 1 , Swagata Ghatak 1 , Sujit Sikdar 1
1 Indian Institute of Science, , Bangalore, India
Abstract text :

Background: Astrocytes play a major role in the pathology of cerebral ischemia. In order to minimize post-ischemic neuronal damage, astrocytes increase glutamate uptake, to reduce excitotoxicity. CA1 stratum radiatum astrocytes in the hippocampus express two pore domain potassium channel TREK1, which contributes to their characteristic linear current-voltage profile (Zhou M et al., 2009). Inhibition of TREK1 channel in astrocytes decreases glutamate clearance capacity and increases ischemia-evoked neuronal apoptosis (Wu X et al., 2012). Global cerebral ischemia increases expression of TREK1 channel in astrocytes (Pivonkova H et al., 2010). Astrocytes meet the energy requirements of neurons by releasing lactate. Lactate concentration reaches upto 25 – 30 mM in the ischemic hemisphere of the brain, when intravenously injected and reduces neuronal lesions (Berthet C et al., 2012).

Objective: Though lactate is a known neuroprotective agent, its mechanism of action is not known. We hypothesized that lactate can provide neuroprotection by activating TREK channels. The effect of lactate on the electrophysiology of TREK channels was studied both in an in vitro brain slice model, after blocking the activity of other ion channels and in a heterologous expression system.

Results: Whole cell patch clamp experiments, on CA1 stratum radiatum astrocytes, in acute hippocampal slices, significantly increased TREK channel activity by 23.3 ± 6.4% and hyperpolarized their resting membrane potential by 5 ± 0.6 mV, on bath application of 30 mM lactate. Comparison of rectification index at negative potentials between control and 30 mM lactate treatment showed no significant difference, suggesting that the conductance activated by lactate is outward rectifying. Lactate-evoked increase in TREK channel activity was reversibly inhibited by 200 µM quinine, a potent inhibitor of TREK channels. Further, lactate was unable to increase TREK channel activity after disruption of intracellular lactate uptake with 2 mM α-cyano-4-hydroxy cinnamate, a blocker of monocarboxylate transporter. This was confirmed by inside-out patch clamp experiments on human TREK1 channel expressed in HEK293 cells.

Conclusion: The experimental observations suggest uptake of lactate by astrocytes to activate TREK channels intracellularly.

References:       Berthet C et al., 2012. Cerebrovasc Dis 34:329.

                        Pivonkova H et al., 2010. Neurochem Int 57:783.

                        Wu X et al., 2012. J Mol Neurosci DOI 10.1007/s12031-012-9875-5.

                        Zhou M et al., 2009. J Neurosci 29:8551.


Pyruvate carboxylation in astrocytes and the pentose phosphate pathway are affected after neonatal hypoxic-ischemic brain injury - a 13C NMR spectroscopy study.

*Eva Brekke 1 , Tora Morken 2 , Marius Widerøe 3 , Asta Håberg 4 , Ann-Mari Brubakk 2 , Ursula Sonnewald 4
1 Norwegian University of Science and Technology, , Trondheim, Norway
2 Norwegian University of Science and Technology, Department of Laboratory Medicine, Women and Children's health, Trondheim, Norway
3 Norwegian University of Science and Technology, Department of Circulation and Medical Imaging, Trondheim, Norway
4 Norwegian University of Science and Technology, Department of Neuroscience, Trondheim, Norway
Abstract text :


Pyruvate carboxylase (PC), the main anaplerotic enzyme in the brain, is predominantly located in astrocytes. In the adult brain, ischemia is known to reduce pyruvate carboxylation. Moreover, reperfusion after ischemia leads to production of reactive oxygen species (ROS). The pentose phosphate pathway (PPP) is, by making NADPH necessary for the regeneration of reduced glutathione, a major pathway in the protection against ROS. However, astrocytic and neuronal metabolic function in the neonatal brain after hypoxic-ischemic brain injury (HI) remains to be explored.



HI was induced in 7-day-old rats by unilateral severing of the carotid artery followed by 2 hours recovery and subsequent exposure to hypoxia (8%O2) for 90 min. 30 min after end of hypoxia (the early reperfusion phase), rats were injected with [1,2‑13C]glucose and decapitated 30 min later. One group was sham-operated, and not exposed to hypoxia, thus reflecting normal metabolism in the neonate. Extracts of ipsilateral hemispheres from HI and sham animals were analysed with 1H- and 13C-NMR spectroscopy.



The sham animals had similar glutamine content, but lower amounts of 13C labelled glutamine compared to corresponding values from adult rats, reflecting a generally lower rate of glucose metabolism in the neonatal astrocytes. However, approximately equal amounts of 13C labelled isotopomers of glutamine were derived from PC and pyruvate dehydrogenase (PDH), resulting in a relatively high PC/PDH-ratio compared the same ratio in adults. Following HI and reperfusion, a reduction in glucose metabolism and mitochondrial metabolism was seen by increased glucose and reduced incorporation of 13C labelling in glutamate, glutamine and aspartate via PC and PDH. However, the PC/PDH-ratio in glutamine was similar in HI and sham. Total mean values of glutamate, glutamine and aspartate were decreased, but only the latter to a significant degree. Labelling in lactate via the PPP was reduced following HI.



The low labelling of lactate via the PPP indicates that the flux through the PPP was in fact reduced in the early reperfusion phase after HI. Moreover, mitochondrial metabolism of pyruvate from glucose was reduced in both astrocytes and neurons. Impaired anaplerosis in astrocytes was confirmed by lower amounts of aspartate. However, the proportionally similar decrease in 13C labelling in glutamate and glutamine via PC, and the maintained PC/PDH-ratio implies that astrocytes continue to provide metabolic support for the neurons during the early reperfusion phase.


Hypoxia/ischemia increases the expression of TREM2 in gray and white matter of neonatal mice brain

*Mariela Chertoff 1 , Kalpana  Shrivastava 1 , Lydia Gimenez Llort 2
1 Universidad Autonoma de Barcelona , Neuroscience Institute, Department of Cell Biology, Physiology & Immunology, Medical Histology, Barcelona, Spain
2 Universidad Autonoma de Barcelona, Neuroscience Institute, Department of Psychiatry and Forensic Medicine, Barcelona, Spain
Abstract text :

Recently, rare variants or mutations of Triggering Receptor Expressed on Myeloid cells 2 (TREM2) had been associated with increased risk of Alzheimer´s Disease, Frontotemporal Dementia-like Syndrome and Early onset Dementia and. The loss-of-function of TREM2 or its coreceptor DAP12 is responsible for the recessively inherited Nasu-Hakola disease (also known as PLOSL). The brains of PLOSL affected patients show strong microglial activation in the cerebral white matter. Reports demonstrate that TREM2-mediated phagocytic function of microglia is required for debris clearance and CNS tissue homeostasis. Perinatal brain injury is the underlying etiology for a host of developmental disabilities that includes spastic motor deficits and cognitive, behavioral and learning difficulties. Hence, our aim is to characterize the expression of TREM2 in the control of neuroinflammation following hypoxia/ischemia (HI) in the newborn brain.

We performed HI brain damage in postnatal day 7 (P7) C57/BL6 mice by permanent left carotid occlusion and litters were exposed to 8% of oxygen balanced with nitrogen  for 50 minutes in a hypoxic chamber with controlled humidity and temperature maintained at 37ºC. Pups were then returned to their dam until sacrifice. The age-matched controls and samples from 3 hours to 7 days after hypoxia were collected and processed for immunohistochemistry.

In control animals, TREM2 expression was observed in the corpus callosum and in the subventricular zone at P7 that almost disappeared at P10. Following HI, an increase in TREM2 staining was observed in the corpus callosum, hippocampus, caudate-putamen, fimbria, cortex and thalamus in the ipsilateral (IL) hemisphere, following a similar regional pattern of microglia activation. The increased expression of TREM2 was observed from 24 hours to 7 days post-hypoxia. TREM2 colocalized mainly with microglia markers, such as Iba-1 and CD68. Oligodendrocyte expression of TREM2 was also analyzed.

In conclusion, HI produced an increase in TREM2 expression on the IL damaged regions. These results suggest that the modulation of TREM2 might be a possible target for damage control in neonatal brain.


Supported by Fundación La Maratò de TV3(2011-110531). M.C. holds a Marie Curie International Incoming fellowship (2009-IIF-253110).


Keywords: microglia, TREM2, Hypoxia/ischemia, white matter, M1/M2 microglia


Distinct subsets of interleukin-1 receptor antagonist producing cells are neuroprotective after focal cerebral ischemia in mice


*Bettina Hjelm Clausen 1 , Kate Lykke Lambertsen 1 , Alicia Babcock 1 , Christian von Linstow 1 , Tomas Deierborg 1 , Bente Finsen 1
1 Institute for Molecular Medicine, Neurobiology Research, Odense, Denmark
Abstract text :

Objective: The cytokine interleukin-1 (IL-1) and its naturally occurring receptor antagonist (IL-1Ra) play a key role in determining neuronal cell death and survival in focal cerebral ischemia. The objective of this study was to determine the cellular production of IL-1/IL-1Ra, and to test the neuroprotective potential of post-surgically injected IL-1Ra overexpressing bone marrow (BM) cells in a mouse model of focal cerebral ischemia.

Materials and methods: C57BL/6 mice were injected i.v. with BM cells isolated from sIL-1Ra overexpressing mice, 30 min after permanent middle cerebral artery occlusion (pMCAo). Physiological parameters and behaviour were recorded post-surgically, infarct sizes were estimated, and microglial-leukocyte expression of IL-1/IL-1Ra was analyzed by flowcytometry, in situ hybridization and immunohistochemistry.

Results: We identify microglia, and not recruited leukocytes as the major producers of IL-1Ra after pMCAo in mice, and we show by using IL-1Ra knock out mice that microglial-produced IL-1Ra is neuroprotective. We report that the sIL-1Ra produced by the post-surgically injected BM cells, potentiates the neuroprotective effect of microglial-derived IL-1Ra, at both 24 h and 5 days, which is consistent with behavioural improvement at 5 days, and detection of recruited BM cells in the ischemic area 1.5 h after pMCAo. Interestingly, we also find that the sIL-1Ra overexpressing BM cells stimulate microglial production of IL-1Ra 6 h after pMCAo, at which time both IL-1α and IL-1β is upregulated.

Conclusion: Our results provide proof of principle that increasing the production of IL-1Ra by recruited BM cells can counteract the effect of IL-1a/b, increase neuronal survival and improve motor function alone or through induction of microglial-produced IL-1Ra after pMCAo in mice.



Attenuated Inflammatory Response in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Knock-Out Mice following Stroke

*Christiane Frahm 1 , Matthias W. Sieber 1 , Nadine Jaenisch 1 , Martin Brehm 1 , Madlen Guenther 1 , Bettina Linnartz-Gerlach 2 , Harald Neumann 2 , Otto W. Witte 1
1 University Hospital Jena, , Jena, Germany
2 University Hospital Bonn, , Bonn, Germany
Abstract text :


Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial surface receptor involved in phagocytosis. Clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. The role of TREM2 following stroke is currently unclear.

Methods and Results

As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). Quantitative PCR (qPCR) revealed a greatly increased transcription of TREM2 after stroke (~10fold). We subsequently analyzed the expression of pro-inflammatory cytokines, chemokines and their receptors in TREM2-knockout (TREM2-KO) mice via qPCR. Microglial activation (CD68, Iba1) and CD3-positive T-cell invasion were analyzed via qPCR and immunohistochemistry. Functional consequences of TREM2 knockout were assessed by infarct volumetry. The acute inflammatory response (12 h reperfusion) was very similar between TREM2-KO mice and their littermate controls. However, in the sub-acute phase (7 d reperfusion) following stroke, TREM2-KO mice showed a decreased transcription of pro-inflammatory cytokines TNFα, IL-1α and IL-1β, associated with a reduced microglial activity (CD68, Iba1). Furthermore, TREM2-KO mice showed a reduced transcription of chemokines CCL2 (MCP1), CCL3 (MIP1α) and the chemokine receptor CX3CR1, followed by a diminished invasion of CD3-positive T-cells. No effect on the lesion size was observed.


Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice. We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke.


Increased expression of hyperpolarization-activated cyclic nucleotide-gated channels in reactive astrocytes after ischemia 

*Pavel Honsa 1,2 , Lenka Harantova 1,2 , Helena Pivonkova 1 , Vendula Rusnakova 3 , David Dzamba 1,2 , Mikael Kubista 3,4 , Miroslava Anderova 3,2
1 Institute of Experimental Medicine, Department of Cellular Neurophysiology, Prague, Czech Republic
2 2nd Faculty of Medicine, Charles University, , Prague, Czech Republic
3 Institute of Biotechnology, Laboratory of Gene Expression, Prague, Czech Republic
4 TATA Biocenter, , Gothenburg, Czech Republic
Abstract text :

Astrocytes respond to central nervous system (CNS) injury by the formation of a glial scar that develops within few days after insult and is characterized by astrocyte proliferation and cellular hypertrophy. Reactive astrocytes change their immunohistochemical profile, such as increasing expression of GFAP, nestin and vimentin; however, they also markedly alter the expression of ion channels, receptors and transporters, which participate in the maintenance of ionic-, water- and neurotransmitter homeostasis, such as K channels, aquaporins or glutamate transporters. Here, we aimed to characterize the gene expression profile and functional properties of reactive astrocytes 5 weeks after global cerebral ischemia (GCI) or focal cerebral ischemia (FCI) with a specific focus on K channels or non-specific cationic channels. GCI was induced in adult rats by bilateral, 15-minute common carotid artery occlusion combined with low oxygen, while FCI was induced in adult EGFP/GFAP mice by permanent middle cerebral artery occlusion. Using the patch-clamp, we investigated the membrane properties of astrocytes in situ 5 weeks after GCI in the rat hippocampal CA1 region and 5 weeks after FCI in the mouse cortex. Regardless of the type of ischemic injury, astrocytes from both CNS regions depolarized their membrane potential by ~14 mV 5 weeks after ischemia. When compared to astrocytes from the non-injured CA1 region or the cortex, the post-ischemic astrocytes displayed large hyperpolarization-activated time- and voltage-dependent, non-inactivating inward currents, the current density of which increased 3-fold in response to GCI or FCI. In addition, these non-specific cationic channels were sensitive to ZD7288 and to a low extracellular Na concentration, suggesting that they may belong to the family of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We have taken advantage of fluorescently labeled astrocytes in EGFP/GFAP mice and isolated EGFP cells by FACS from non-injured as well as ischemic regions and performed gene expression profiling using single-cell RT-qPCR. Our data revealed that cortical astrocytes after FCI express, besides the increased expression of Gfap, Gfapδ, Aqp1 and 9, extremely high levels of Hcn1-3 transcripts that encode HCN 1-3 channels. Moreover, our immunohistochemical analyses confirmed the presence of HCN1-3 in reactive astrocytes, especially HCN1, 2 and 3 channels. In summary, our results show that reactive astrocytes from post-ischemic tissue express HCN channels and that their activity might significantly contribute to Na influx, possibly maintaining ATPase function and/or contributing to intracellular Na -dependent events, such as glutamate transporter or Na /Ca2 exchanger reversal.

GA CR 13-02154S, P304/12/G069 and GAUK 383711.



[1,6-13C]glucose metabolism in immature and in differentiated oligodendrocytes in vitro

*Ana Amaral 1 , Ursula Sonnewald 2 , Mark Kotter 1
1 University of Cambridge, , Cambridge, United Kingdom
2 Norwegian University of Science and Technology, , Trondheim, Norway, Norway
Abstract text :

Oligodendrocytes are the myelinating cells of the central nervous system (CNS). They are responsible for ensheathing myelin layers around axons, which contributes to improve conduction of neuronal impulses and additionally provides axonal support. Oligodendrocytes are generated by oligodendrocyte precursor cells (OPCs), a self-renewing and proliferating adult stem/precursor cell population in the CNS. This process occurs not only during developmental myelination but also mediates CNS remyelination, a regenerative process that restores myelin sheaths to demyelinated axons.

Although energy metabolism in the oligodendrocyte lineage is thought to be very active to support the lipid production specific to this cell type, it is still poorly characterized. As a first step towards a detailed characterization of the energy metabolism associated with specific lineage stages we investigated how glucose - the main cerebral energy substrate - is metabolized at an immature OPC stage and in differentiated oligodendrocytes. Primary cultures of OPCs and oligodendrocytes were incubated with medium containing [1,6-13C]glucose for 4h and metabolites in both cell extracts and culture medium were collected and analysed for excess % 13C enrichment using Gas-Chromatography-Mass Spectrometry (GC-MS) as well as for total amounts using HPLC. The significant 13C-enrichment in citrate, malate, and aspartate in cell extracts indicate that oligodendrocytes oxidize glucose-derived metabolites extensively in the Tricarboxylic Acid (TCA) Cycle. Moreover, they release 13C-labelled aspartate and glutamate to the extracellular medium, although a significant consumption of aspartate, glutamate and glutamine from the medium was also observed. Enrichment of extracellular lactate and increased alanine synthesis in oligodendrocytes as compared to OPCs suggests that the rate of aerobic glycolysis is increased in immature as compared to differentiated cells.

In conclusion, these data confirm that both OPCs and oligodendrocytes are highly metabolically active and, in addition to extensively oxidizing glucose, synthesize and release distinct metabolites. We anticipate that further studies on this subject will contribute to a better understanding of the physiological role of oligodendrocytes in the CNS and the role of glycolysis and mitochondrial metabolism during their maturation. 


Silencing or knocking-out of the Na+/Ca2+ exchanger 3 (NCX3) impairs oligodendrocyte differentiation 

*Francesca Boscia 1 , Carla D'Avanzo 1 , Anna Pannaccione 1 , Agnese Secondo 1 , Antonella Casamassa 1 , Luigi Formisano 1 , Natascia Guida 1 , Lucio Annunziato 1
1 University of Naples "Federico ", Neuroscience, Napoli, Italy
Abstract text :

Changes in intracellular [Ca2 ]i levels have been shown to influence the developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of myelination and remyelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and NCX3, play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte phenotype. In fact, while NCX1 was down-regulated, NCX3 was strongly up-regulated during the oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas the knocking down of the NCX3 isoform in OPCs prevented the up-regulation of the myelin protein markers CNPase and MBP, its overexpression induced an up-regulation of CNPase and MBP. Furthermore, NCX3 knock-out mice exhibited not only a reduced size of spinal cord but also a marked hypomyelination, as revealed by the decrease in MBP expression and by the accompanying increase in OPCs number. Collectively, our findings indicate that calcium signaling mediated by NCX3 plays a crucial role in oligodendrocyte maturation and myelin formation.


The growth factor NRG induces NMDA receptor dependent myelination by oligodendrocytes, employing Akt and CREB signalling

*Kimberley Evans 1 , Iben Lundgaard 2 , Aryna Luzhynskaya 2 , John Stockley 2 , Zhen Wang 3 , Charles ffrench-Constant 4 , David Attwell 5 , Ragnhildur Karadottir 2
1 University of Cambridge, , Cambridge, United Kingdom
2 University of Cambridge, Wellcome Trust - MRC Cambridge Stem Cell Institute, John van Geest Centre for Brain Repair & Dept. of Veterinary Medicine, Cambridge, United Kingdom
3 CNRS, Institut de Biologie de l'Ecole Normale Supérieure Génomique Fonctionnelle, Paris, France
4 University of Edinburgh, MRC Centre for Regenerative Medicine, Centre for Multiple Sclerosis Research, Edinburgh, United Kingdom
5 University College London, Department of Neuroscience, Physiology & Pharmacology, London, United Kingdom
Abstract text :

The growth factor neuregulin (NRG) can regulate myelination by signalling to ErbB receptors, respectively, on myelinating cells. Furthermore, in CNS neurons, NRG  increase the expression of NMDA receptors. Oligodendrocytes at all developmental stages in the white matter exhibit NMDA evoked currents, mediated by receptors with very weak magnesium block which are expressed in the myelin1,2, indicating that they might play a role in myelination.

In an assay in which cortical oligodendrocytes ensheath dorsal root ganglion cells3 we found that NRG and NMDA receptors interact to regulate myelination. Without NRG, blocking NMDA receptors had no effect on myelination. Adding NRG increased myelination at all time-points analysed (2-5 weeks), suggesting that NRG did not merely accelerate myelination. Strikingly, NRG led to the majority of myelination becoming dependent on activation of NMDA receptors and action potential activity. NRG’s effect was associated with a 4-fold increase in NMDA receptor current in oligodendrocyte lineage cells. Thus, NRG switches myelination from a default programme, which is independent of neuronal activity, to a mechanism that is regulated by glutamate released from active axons. The effects of NRG were associated with an increase in the phosphorylation of Akt and the transcription factor CREB, but not associated with changes in the phosphorylation of ERK.


These data reveal a function for oligodendrocyte NMDA receptors. The absence of NRG in multiple sclerosis lesions, and enhanced remyelination by added NRG, suggest a role for neuregulin/NMDA receptor dependent remyelination after pathology.


1. Káradóttir et al., (2005) Nature 438, 1162-1166.

2. Káradóttir et al., (2008) Nature Neuroscience 11, 450-456

3. Wang et al., (2006) Glia 55, 537-45


Supported by the Wellcome Trust, MRC, EU FP7 Leukotreat, FP7 Marie Curie Training Network, Action Medical Research, and the Royal Society.


Dysregulation of GPR17, a key receptor involved in oligodendrocyte maturation, as a novel potential pathogenetic mechanism in demyelinating diseases

*Marta Fumagalli 1 , Elisabetta Bonfanti 2 , Simona Daniele 3 , Davide Lecca 2 , Giusy T Coppolino 2 , Maria Letizia Trincavelli 3 , Claudia Martini 3 , Maria P Abbracchio 2
1 Università degli Studi di Milano, , Milano, Italy
2 Università degli studi di Milano, Dep. of Pharmacological and Biomolecular Sciences, Milan, Italy
3 Università di Pisa, Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, Pisa, Italy
Abstract text :

The P2Y-like Gi-protein-coupled receptor GPR17 is activated by both uracil nucleotides and cysteinyl-leukotrienes, two families of mediators involved in trophic responses and in the repair of central nervous system lesions (Ciana et al., EMBO J, 2006). In both brain and spinal cord, GPR17 is transiently expressed by a subpopulation of neural progenitors expressing the proteoglycan NG2 (NG2 cells, also known as Oligodendrocyte Precursor Cells, OPCs) in transition from precursors to premyelinating phenotypes, whereas it is not present on mature oligodendrocytes. Previous data in cultured OPCs showed that early GPR17 activation by its endogenous ligands promotes, while inhibition by antagonists or silencing RNAs impairs, OPC differentiation (Lecca et al., Plos One, 2008; Fumagalli et al., J Biol Chem, 2011). Altogether, these data point at GPR17 as a key functional modulator of oligodendrocyte maturation.

Here, we used primary OPC cultures to investigate the mechanisms underlying GPR17 endogenous regulation. This is quite important since GPR17 is upregulated at injury sites in both a demyelinating in vivo model (Boda et al., Glia, 2011) and in multiple sclerosis (MS) patients (Chen et al., Nat Neurosci 2009), which, could, in turn, cause defective myelination. In line with these findings, we have recently shown that GPR17 forced over-expression at late differentiation stages, obtained by transfecting cultured OPCs with a GFP-GPR17 fusion vector, indeed impairs terminal cell maturation. This suggests that the receptor needs to be down-regulated/desensitized to allow OPC terminal maturation. Physiologically, GPR17 down­regulation may occur through agonist-induced receptor phosphorylation via G-protein coupled receptor kinases (GRKs) (Daniele et al., J Pharm Exp Ther, 2011; Frantangeli et al., J Biol Chem, 2013), which are, in turn, controlled by mTOR kinases, and are altered in MS. Our in vitro data show that rapamycin, an inhibitor of mTOR kinase, reduces GRK2 levels, with parallel increases in GPR17 expression and strong impairment of OPC maturation. Globally, these data suggest that dysregulation of these interconnected pathways leading to aberrant GPR17 overexpression may prematurely block OPC maturation. Analysis of GPR17 in vivo in MS models will confirm if the receptor is dysregulated during disease development and will help finding ways to re-establish myelin repair in demyelinating diseases.

Sponsored by Fondazione Italiana Sclerosi Multipla, grant 2010/R/2 to MPA




Role of Jun activating binding protein 1 (Jab1) in Central Nervous System (CNS) myelination

*Cristina Rivellini 1 , Emanuela Porrello 1 , Giorgia Dina 1 , Klaus-Armin Nave 2 , Corinna Lappe-Siefke 2 , Ruggero Pardi 3 , Angelo Quattrini 1 , Stefano Carlo Previtali 1
1 San Raffaele Scientific Institute, , Milan, Italy
2 Max Planck Institute of Experimental Medicine, Neurogenetics, Goettingen, Germany, Germany
3 San Raffaele scientific Institute, Division of Immunology, Transplantation and Infectious Disease , Milan, Italy
Abstract text :

Proper control of cell cycle, proliferation and differentiation is fundamental to regulate oligodendrocyte (OL) development and recruitment of oligodendrocyte precursors (OPC) after brain damage. Failure in this process results in severe dysmyelinating disorders and defective brain repair. The molecular machinery that couples differentiation to proliferation withdrawal plays therefore a critical role in brain development and repair and is regulated by multiple extracellular cues including extracellular matrix (ECM) and growth factor derived signals. However, how these signals are integrated into OL to control cell cycle progression and differentiation is only partially understood.

We are investigating the role of Jun activating binding protein 1 (Jab1), an intracellular signalling molecule, shuttling between nucleus and cytoplasm, involved in the control of cell proliferation, survival and gene transcription. Recent evidence showed that Jab1 function is regulated by ECM and growth factors. We demonstrated that Jab1 is expressed in glial cells in peripheral and central nervous system (CNS), and conditional inactivation of Jab1 in Schwann cells results in dysmyelinating neuropathy.

Here we present preliminary data showing that Jab1 plays a role in CNS development. Mice with conditional inactivation of Jab1 in OL, by CNP-Cre tansgene, show CNS hypomyelination, including corpus callosum, optic nerve and spinal cord. Concomitant fiber degeneration was observed. Our preliminary data suggest that Jab1 expression in OL plays a role in CNS myelination and possibility axonal survival. 


A3 adenosina receptor triggers oligodendrocyte death and myelin loss 

*Estibaliz González-Fernández 1 , Rogelio Arellano 1,2,3 , Alberto Pérez-Samartín 1,2,3 , María Victoria Sánchez-Gómez 1,2,3 , Carlos Matute 1,2,3
1 University of Basque Country-UPV/EHU, , Leioa, Spain
2 Achucarro Basque Center For Neuroscience, , Zamudio, Spain
3 Instituto de Salud Carlos III (ISCIII; CIBERNED), , Leioa, Spain
Abstract text :

Adenosine is a potent neuromodulator which can be released from most cells and its extracellular concentration increases dramatically after brain injury. It acts on four G protein-coupled receptors, A1, A2a, A2b and A3, and their activation is involved in myelination as well as in apoptosis linked to neurodegenerative diseases. In this study, we initially found that rat oligodendrocytes in vitro express all four adenosine receptor subtypes, adenosine transporters ENT1 and ENT2 and the adenosine degrading enzymes adenosine deaminase and adenosine kinase. Activation of adenosine receptors caused mitochondrial dysfunction and oligodendroglial apoptosis. Notably, selective activation of adenosine receptors revealed that A3 receptors are the main subtype mediating oligodendrocyte death. Thus, A3 receptor agonist 2-CI-IB-MECA caused a robust increase in ROS levels, mitochondrial membrane depolarization and caspase-dependent apoptosis. In turn,  selective A3 receptor activation induced MAPK modifications compatible with cellular damage, which was prevented by the selective antagonist MRS1220. The modulation of G protein and adenylate cyclase suggested that 2-CI-IB-MECA acts via cAMP-PKA. Moreover, selective activation of A3 receptor triggered intracellular calcium mobilization via PLC pathway. Finally, we used cerebellar organotypic slices and optic nerve ex vivo to investigate adenosine-mediated oligodendroglial death in more integral preparations. Consistent with data in dissociated cultures, A3 receptor activation induced a significant damage in the optic nerve as well as in the cerebellum white matter, an effect that was prevented by caffeine, an adenosine receptor blocker. Together, these results indicate that adenosine can trigger oligodendrocyte death via activation of A3 receptors, and suggest that this mechanism may contribute to the etiology of demyelinating diseases.


Histone Methyltransferase Enhancer of Zeste Homolog 2 regulates Schwann cell differentiation

*André Heinen 1 , Nevena Tzekova 1 , Nina Graffmann 1 , Klintsy Julieta Torres 1 , Markus Uhrberg 1 , Hans Peter Hartung 1 , Patrick Küry 1
1 Heinrich Heine University Düsseldorf, , Düsseldorf, Germany
Abstract text :


Epigenetic control is crucial for the differentiation of a variety of cells including oligodendrocytes, the myelinating glial cells of the central nervous system. However, studies about the implication of epigenetic factors in peripheral nervous system maturation are just emerging and we were wondering whether methylation of histones is involved in this process.


We describe the function of EZH2 in primary Schwann cells and DRG cocultures using immunohistochemistry, transfection and lentiviral transduction, shRNA, qRT-PCR analysis, morphological analysis, Western blotting and chromatin immunoprecipitation.


Here, we demonstrate for the first time the impact of a histone methyltransferase, encoded by the enhancer of zeste homolog 2 (EZH2) gene, on Schwann cell differentiation. In sciatic nerves, EZH2 expression was found in Schwann cells and to peak perinatally. Suppression of EZH2 expression in cultured primary rat Schwann cells reduced the length of cell processes. These morphological changes were accompanied by widespread alterations in the gene expression pattern, including downregulation of myelin genes and induction of p57kip2, which we have recently identified as an intrinsic inhibitory regulator of Schwann cell maturation. In addition, we show that EZH2 suppression in dorsal root ganglion cocultures interferes with in vitro myelination. Chromatin
immunoprecipitation analysis revealed binding of EZH2 at the p57kip2 promoter and reduction of histone H3K27 trimethylation upon gene suppression. EZH2 suppression-dependent effects on morphology and myelin genes could be reversed by concomitant suppression of p57kip2,
indicating that p57kip2 is a downstream effector of EZH2. Furthermore, we describe Hes5 as transcriptional repressor of myelin genes in Schwann cells, which was induced upon EZH2 suppression and downregulated in p57kip2-suppressed Schwann cells.


We have identified a molecular link between histone methylation and control of Schwann cell differentiation and demonstrate that this epigenetic mechanism is crucial for glial differentiation to proceed.


Remyelination after Cuprizone treatment: Galectin-3 involvement.

*Hernan Hoyos 1 , Mariel Marder 1 , Gabriel Rabinovich 2 , Laura Pasquini 1 , Juana Pasquini 1
1 FFYB-UBA IQUIFIB-CONICET, Biological Chemistry, Buenos Aires, Argentina
2 FCEN-UBA IBYME-CONICET, Cell Biology, Buenos Aires, Argentina
Abstract text :

Cuprizone (CPZ) is a copper chelator which induces reproducible demyelination
in the mouse brain (Matsushima and Morell, 2001). CPZ-induced demyelination
is characterized by oligodendroglial cell loss, myelin sheath degeneration
and astrocyte and microglia recruitment to the lesioned area. The CPZ model
causes demyelination without participation of the immunological system.
Galectin-3 (Gal-3) is a 31 kDa lectin which binds to β-galactosides and is widely
spread among different cell types and tissues. We have previously found
that Gal-3 is involved in the control of myelin integrity and function and drives
oligodendroglial cell differentiation (Pasquini et al., 2011). Remyelination is
a regenerative process during which new myelin sheaths wrap axons after
pathological demyelination (Fancy et al., 2011). Eight-week-old Lgals3-/- and
wild type (WT) mice were fed a diet containing 0.2% CPZ w/w during 6 weeks,
after which CPZ was withdrawn in order to evaluate remyelination 2 weeks
after. According to our results, CPZ-induced demyelination in Lgals3-/- mice
showed an exacerbated astrocytic and microglial response as compared to WT
littermates. Electron microscopy showed a significant lack of myelinated axons
in Lgals3-/- mice as compared to controls. Furthermore, the few myelinated
axons present were nearly 50% less myelinated than those of controls and
were found to be collapsed. Remarkably, the remyelination process seemed to
be faster in Lgals3-/- mice than in WT. Remyelinated Lgals3-/- mice showed a
higher Myelin Basic Protein (MBP) recovery rate as compared to their controls.
Flow cytometry assays showed a sharper microglial response in Lgals3-/- mice,
which was supported by an exacerbated number of CD11b and CD45 cells.
However, electron microscopy images from remyelinated Lgals3-/- animals
showed, again, collapsed axons with a defective myelin wrap, as compared to
WT mice showing normal axons without any relevant myelin wrap disruption.
Behavioral performance observed during CPZ treatment recovery correlates
with alterations in the morphological studies, which show that neither Lgals3-/-
nor WT mice reach basal myelination levels.


2‘,3‘-cyclic nucleotide 3‘-phosphodiesterase (CNP) deficiency causes axonal loss and hypermyelination in the sensory peripheral nervous system

*Theresa Kungl 1 , Tobias Nientiedt 1 , Kim J. Neufeld 1 , Michael W. Sereda 1,2 , Klaus-Armin Nave 1
1 MPI of Experimental Medicine Göttingen, , Göttingen, Germany
2 University of Göttingen, Neuropathology, Göttingen, Germany
Abstract text :

CNP is expressed in myelin forming glial cells of the central (CNS) and peripheral nervous system (PNS). It is present in cytoplasmic regions like Schmidt-Lanterman incisures and the paranodal compartments, but is excluded from compact myelin. Mice deficient of CNP display axonal loss without an obvious myelination defect in the CNS. So far the role of CNP in the PNS remains unclear. Here, we demonstrate that CNP deficient mice exhibit alterations in mechanical and thermal sensation. Histological analyses of CNP deficient mutants reveal loss of sensory axons when quantified in the dorsal root and the predominantly sensory saphenous nerve. In contrast ventral roots remain unaltered. Moreover, electron microscopical assessments of saphenous nerves display a hypermyelination of small to mid-sized caliber axons and an overrepresentation of the smallest fibers. The myelin pathology of sensory axons is reflected by alterations of sensory nerve conduction velocities. Thus, our data demonstrate that, contrary to findings in the central nervous system, CNP has an impact on myelination and is especially important for the integrity of small to mid-sized caliber axons within the sensory peripheral nervous system.


Transferrin and Thyroid hormone converge in the control of myelinogenesis

*Leandro Marziali 1 , Paula Franco 1 , Juana  Pasquini 1
1 University of Buenos Aires, , Caba, Argentina
Abstract text :

Promyelinating effects of both Apo-Transferrin (aTf) and Thyroid Hormone (TH) on rat brain are well documented. TH effects are mediated by nuclear receptors which act as transcription factors and regulate different cell processes.  Previous results from our laboratory showed that TH promotes the commitment of oligodendrocyte progenitors (OPCs) to mature myelinating oligodendrocytes (OLG). On the other hand, aTf is able to promote the commitment of neural stem cells (NSC) to cells of the oligodendroglial linage and favors OLG maturation after an intracranial injection. Our previous demonstration that TH administration is able to up-regulate Tf mRNA expression leads us to hypothesize that both factors converge in the control of oligodendrogenesis. To test if the combined effects of both aTf and TH are required for proper myelination in the rat brain, studies were done at P10 and P20 for each experimental condition. A hyperthyroid state was rendered by daily subcutaneous TH administration from the day of birth (Hyper). Hypothyroidism was achieved by giving propylthiouracil to the mother from gestational day 18 to the end of the experiment (Hypo). Half of the hypo pups received an intracranial injection of aTf at postnatal day 3 (Hypo aTf). A set of euthyroid animals received an intracranial injection of aTf at postnatal day 3 (aTf). Control groups received an intracranial injection or subcutaneous saline solution (C).

At P10, Hyper animals showed an up-regulation of 55% in Tf mRNA expression and 87% in protein levels as compared to controls. Hypo showed a decrease of 25% in Tf mRNA levels. This effect was not affected by exogenous administration of aTf.

At P20, Hyper showed no differences in the expression of Tf mRNA or protein levels as compared to controls. Hypo showed a decrease of 25% in Tf mRNA and a 40% decrease in protein levels. This effect was not affected by exogenous administration of aTf. No differences were observed in the expression of Insulin Growth Factor I (IGF-I) or IGF-I Receptor between groups at P10 or P20.

Immunohistochemical analyses were performed at P20 in all groups usingspecific markers anti CAII, anti MBP, anti RIP and PDGFRα.

Our conclusion is that, in a hypothyroid state, Tf is not able to produce OLG and myelin maturation at the corpus callosum, which indicates that TH is necessary for aTf action. However, the finding that hyperthyroid animals showed a significant increase in Tf mRNA strongly suggests that Tf could be involved in TH effects.

New experiments are carrying out in order to knock down the Tf gen to probe this last piece of evidence.


Wnt-signalling regulates oligodendrogenesis in the dorsal postnatal subventricular zone

*Kaz Azim 1,2 , Bruno Fischer 1 , Konrad Basler 3 , Lukas Sommer 4 , Arthur Butt 2 , Olivier Raineteau 1
1 University of Zürich/ETHZ, Brain Research Institute, Zürich, Switzerland
2 University of Portsmouth, IBBS, School of Pharmacy and Biomedical Sciences, Portsmouth, United Kingdom
3 University of Zürich, IMLS, Zürich, Switzerland
4 University of Zürich, Institute of Anatomy, Zürich, Switzerland
Abstract text :

In the developing postnatal forebrain, the myelin forming cells, oligodendrocyte (OL) lineage cells originate from neural stem cells (NSCs) of the dorsal subventricular zone (SVZ) of the lateral ventricle. The mechanisms that regulate OL progenitor (OP) differentiation from postnatal SVZ sources are relatively complex but the early NSC-intrinsic signals are unknown.

Gene expression profiling of the microdissected SVZ regions revealed that Wnt-responsive genes were enriched in the postnatal dorsal SVZ compared to the lateral SVZ. We performed inhibition of GSK3β by intraventricular infusion of ARA to initiate Wnt-signalling. Several approaches confirmed activation of the Wnt-signalling pathway in the dorsal SVZ as well as within OPs. Thus, gene expression profiles of Wnt-target genes of the dorsal SVZ indicated that Axin2, Lef1, Fzdl1 and Tcf4, but not those other pathways, were dramatically up-regulated. Furthermore, nuclear β-catenin was transiently up-regulated in the dorsal SVZ including in a large population of Sox10-EGFP expressing cells following pharmacological Wnt-activation. Gene expression profiling and immunostainings revealed that the densities of NSCs, cycling progenitors and subsequent OP differentiation in the dorsal SVZ were augmented.

In parallel, we genetically manipulated Wnt-signalling in stem and progenitor cells of the dorsal wall of the lateral ventricle. Targeted dorsal SVZ electroporation of Cre-GFP plasmids in floxed stabilised β-catenin (gain-of-function) and floxed β-catenin (loss-of-function) transgenic mouse lines were performed and resulted in a phenocopy of the results observed following ARA-intraventricular infusion.

In summary, our findings illustrate that Wnt-signalling endogenously persists in the dorsal SVZ after birth. Pharmacological as well as genetic interventions reveal an important role of this signalling pathway in the regulation of OP genesis during the period of myelination.


Enteric stem cell niche in Hirschsprung's disease


*Cornelia Irene Hagl 1 , Theisen Marie 1 , Heumüller-Klug Sabine 1 , Wink Elvira 1 , Schäfer Karl-Herbert 1
1 Medical Faculty Mannheim, University of Heidelberg, , Mannheim, Germany
Abstract text :

The transplantation of neural crest derived stem cells (NCSC) is a potent alternative for the treatment of Hirschsprung’s disease (HSCR), especially as the microenvironment in the aganglionic segment is suitable for enteric stem cell survival and differentiation. Ideally, the cells transplanted are derived from progenitors within the intestine. To gain further insight in possible stem cell niches the compelling evidence that mouse enteric glia can also be neuronal precursors was related to human tissue. Human colon from infants with Hirschsprung’s disease was investigated with a panel of glial and stem cell markers.

Tissue samples from infants with Hirschsprung’s disease were investigated with glial and stem cell markers along the gut axis. The tissue samples from ganglionic, aganglionic and transient segments were immuonstained either for S100/nestin, S100/p75, GFAP/nestin and GFAP/p75.

In all samples investigated, nestin positive ganglia could be found, even in the distal parts with a severe hypo- or aganglionosis. Beside nestin positive cells in all segments, there were also different expression pattern glial markers within the ganglia, indicating that distinct phenotypes of glia cells could be found.

Neural and glial precursor cells are present in the ganglionic as well as in the hypoganglionic segments of Hirschsprung’s colon, suggesting that these cells might be suitable for NCSC generation and further transplantation.


microRNA regulation of neural precursor maintenance and specification

*Laura Hudish 1 , Bruce Appel 1
1 University of Colorado Denver School of Medicine, , Aurora, United States
Abstract text :

During development neural precursors (NPs) both divide, to expand the cell population, and produce many different kinds of neurons and glia. This balance appears to be regulated by Par complex proteins, which polarize neural precursors and can thereby direct daughter cells for different fates. How Par complex proteins are regulated to appropriately polarize NPs remains unknown. In recent years, regulation of gene function by microRNAs has emerged as an important mechanism during development. Using bioinformatics we identified the polarity gene pard3 as a candidate target for microRNA219 (miR-219). miR-219 is specifically expressed in the developing central nervous system (CNS) of vertebrates and miR-219-deficient zebrafish embryos have a deficit of oligodendrocytes, the myelinating glial cells of the CNS. Because a disruption in polarity could affect the types of cell divisions that NPs undergo, thus altering the balance of cell types that arise, we hypothesize that neural precursor maintenance is regulated by modulation of polarity cues through miR-219. By using an in vitro reporter assay we found that miR-219 can downregulate expression of a luciferase gene fused to the pard3 3’UTR. Reduction of pard3 function in zebrafish embryos suppressed the oligodendrocyte phenotype resulting from loss of miR-219 function and injection of a morpholino oligonucleotide designed to block binding of miR-219 to its pard3 target sequence phenocopied miR-219 knock down. Together, these data provide strong evidence that pard3 is a functionally relevant in vivo target of miR-219. To further investigate the role of miR-219 function we tested expression of NP, radial glial and neuronal markers in miR-219-deficient embryos. The number of cells expressing Sox2, a marker of NPs and neural stem cells, was significantly increased upon miR-219 knock-down. Concomitantly, miR-219-deficient embryos had a dramatic deficit of radial glia and late born neurons. These data provide evidence for a new mechanism of NP regulation, in which miR-219 regulates Pard3 levels, thereby regulating the transition of dividing neural precursors to differentiated neurons and glia.


Adult neural stem cells generate waves of oligodenrocyte progenitor cells that populate transiently the corpus callosum but do not contribute to its pool of oligodendrocytes.

*Ilias Kazanis 1 , Robin Franklin 1
1 University of Cambridge, , Cambridge, United Kingdom
Abstract text :

In the subependymal zone (SEZ) cytogenic niche of the adult mouse brain neural stem cells drive the continuous generation of new cells, mostly of olfactory bulb interneurons, but also of cells of the oligodendroglial lineage. Previous reports have shown that newly-born cells exit the SEZ and migrate to the adjacent corpus callosum (cc) where they differentiate into oligodendroglial cells. However, the real contribution of these niche-derived oligodendrocyte progenitor cells and oligodendrocytes (nOPCs and nOligos) to the oligodendroglial population of the cc has not been assessed so far. In this study we used the hGFAP-CreERT2 x Rosa26-EYFP double transgenic mice in order to label specifically the progeny of SEZ-located adult neural stem cells. We found that cells expressing markers of OPCs, such as PDGFRα and Olig2, are constantly generated in the SEZ and migrate to the proximal fraction of the cc. Interestingly though, these cells do not remain in the cc for more than 15 days with their contribution to the total population of oligodedroglial cells remaining stably below 2% even in the ageing brain. Moreover, immunostaining for markers of cell cycle revealed that a higher fraction of nOPCs undergoes mitosis, when compared with local OPCs; hence, they constitute almost 25% of proliferating OPCs of the cc. Notably, during their transient presence in the cc, nOPCs express markers of maturing oligodendrocytes and are incorporated in established local cell structures. When the cc is challenged with a focal demyelinating insult the local and the niche-derived oligodendroglial machineries exhibit differential cell kinetics, nOPCs increase their contribution to the total pool of oligodendroglial cells; however, their presence remains transient.  


Isolation of radial glia-like neural stem cells from fetal and adult mouse brain via selective adhesion to a novel adhesive peptide-conjugate

*Timea Kohidi 1 , Károly Markó 1 , Nóra  Hádinger 1 , Tibor Andrási 1 , Gábor Mező 2 , Emilia Madarász 1
1 IEM-HAS, , Budapest, Hungary
2 Eötvös Lóránd University, HAS, , Budapest, Hungary
Abstract text :

Rapid procedure for isolating radial glia-like cells from fetal, perinatal or adult rodent brain was established by employing the preferential adhesion of neural stem cells to a novel synthetic adhesive polypeptide (AK-cyclo[RGDfC]). Radial glia-like (RGl) neural stem/progenitor cells grew readily on the peptide-covered surfaces under serum-free culture conditions in the presence of EGF as the only growth factor supplement. Proliferating cells derived either from fetal (E 14.5) forebrain or from different regions of perinatal or adult brain maintained several radial glia-specific features including nestin and RC2 immunoreactivity, Pax6, Sox2, Blbp and Glast gene expression. Proliferating RGl cells were obtained also from non-neurogenic zones including the parenchyma of the adult cerebral cortex and dorsal midbrain. Regardless of origin in space and age, all RGl populations expressed positional genes (Pax6, Olig2, Dlx2, Emx2) characteristic to developing forebrain regions. Upon appropriate induction, cells cloned from different fetal or adult brain areas could generate astrocytes, oligodendrocytes and neurons with different phenotypes. All RGl lines gave rise to GABAergic neurons. Significant differences however, were found among the cell lines in generation of glutamatergic and cathecolamine-synthesizing neurons and in the production of oligodendrocytes. Despite of uniform expression of positional genes, mature derivatives of RGl cells displayed characteristics resembling the region of their origin.  Sub-clones of channelrhodopsin-expressing RGl cells have been also established in order to investigate stem cell responses and pretended developmental shifts in response to light-evoked bioelectric stimuli.


The Wnt signaling pathway affects the differentiation potential of neonatal neural stem cells in vitro

*Jan Kriska 1,2 , Pavel Honsa 1,2 , David Dzamba 1,2 , Lucie Tumova 1 , Vladimir Korinek 1 , Miroslava Anderova 1,2
1 Institute of Experimental Medicine, , Prague, Czech Republic
2 2nd Faculty of Medicine, Charles University in Prague, , Prague, Czech Republic
Abstract text :

Wnt proteins regulate many processes during embryonic development and play an important role in the proliferation/differentiation of neuronal progenitors and in the establishment of neurogenic niches. Here we aimed to clarify the effect of the Wnt signaling pathway on the proliferation and differentiation of neonatal neural stem cells (NSCs) isolated from the subventricular zone (SVZ) and their membrane properties during differentiation in vitro. To suppress the Wnt signaling pathway, the Rosa26-DN-TCF4 mouse strain was used, allowing the manipulation of Wnt signaling in the nucleus via the Cre/lox system. Crossbreeding Rosa26-DN-TCF4 mice with the Rosa26-CreERT2 strain enabled Cre-mediated excision triggered by tamoxifen (4OHT), after which EGFP-DN-Tcf4 protein is constitutively produced, thus inhibiting the expression of the target genes driven by the Wnt signaling pathway. In vitro experiments were performed in proliferation medium in order to estimate the impact of Wnt signaling inhibition on the ability of NSCs to form neurospheres or during their differentiation using 4OHT untreated- (control) and treated cultures. To follow the fate of NSCs, the expression of neuronal/glial markers was analyzed 8 days after the onset of in vitro differentiation. The patch-clamp and Ca2 imaging were employed to characterize the membrane properties of differentiated NSCs. The cells were divided into 3 groups based on their electrophysiological and immunocytochemical properties. GFAP cells displayed passive time- and voltage-independent K currents, and their average membrane potential (Vm) was -77 mV and input resistance (IR) was 92 MΩ, while DCX or MAP2 cells expressing fast activating outwardly rectifying K currents (KA), and delayed outwardly rectifying K currents (KDR) were defined by a Vm of -72 mV and high values of IR (1943 MΩ). NG2 cells with a complex current pattern expressed inwardly rectifying K (KIR) currents, in addition to KDR and KA currents, and their Vm was -76 mV and IR was 383 MΩ. The electrophysiological analysis revealed that the inhibition of the Wnt signaling pathway significantly increased the incidence of cells with a complex current pattern, while the number of cells with the marked expression of outwardly rectifying K currents declined. Wnt signaling inhibition also resulted in increased KIR and decreased KA current amplitudes. 4OHT-treated cells were also hyperpolarized when compared to controls. Immunocytochemistry using antibodies against MAP2 and DCX showed that neuronal progenitors in 4OHT-treated cultures were less developed, having fewer and less branched processes. In summary, our data imply that the canonical Wnt pathway in the neonatal SVZ promotes NSC differentiation into cells with neuronal characteristics. GACR P303/12/0855; P304/12/G069


Influence of demyelination and aging on adult oligodendrocyte precursor cells RNA profil: towards an identification of new molecular cues for myelin repair

*Sarah Moyon 1 , Marie-Stephane Aigrot 1 , Luce Dauphinot 1 , Marie-Claude Potier 1 , Matthew Trotter 2 , Jeffrey Huang 3 , Robin Franklin 2 , Catherine Lubetzki 1
1 CRICM, , Paris, France
2 University of Cambridge, , Cambridge, United Kingdom
3 Georgetown University, , Washington, United States
Abstract text :

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous
system (CNS). Spontaneous remyelination can occur in MS, but it is most often insufficient,
mainly linked to defect of oligodendrocyte progenitor cells (OPCs). In experimental
murine model of demyelination, the repair capacity is robust and sufficient, even if it might
decrease with aging. One major therapeutic goal in MS is to favour endogenous myelin
repair, to prevent neurodegeneration and disability progression. Using a transcriptomic
approach on pure populations of OPCs, we try to identify mechanisms specifically involved
in OPCs activation.
We set up a method to isolate a purified population of OPCs, by fluorescent-
activated cell sorting from PDGFαR::GFP mouse brains. We created the gene expression
profile of neonatal OPCs, adult OPCs and adult oligodendrocytes (OLs) in control
conditions and of adult OPCs in cuprizone-induced demyelinating conditions.
We analyzed the transcriptomic profile of adult OPCs, neo-natal OPCs and OLs, and
identified more than 1000 differentially expressed genes. After demyelination, we
identified more than 2000 adult-OPC-specific genes that are either up- or down-regulated
compared to control conditions. Preliminary analysis suggests that adult OPCs are more
“mature” than neonatal OPCs and partially revert to a more immature profile after
Bio-informatic analysis is ongoing, with the perspective of identifying key
candidates for myelin repair capacity, then develop functional experiments to validate their
involvement in remyelination. In parallel, we plan to compare the transcriptomic profile of
adult OPCS from aged versus young mice, to get insight into age-related decrease of repair


Regulation of ischemia-induced progenitor cell proliferation in the adult mouse hippocampus by the ERK/MAPK effector ribosomal S6 kinase.

Kate Karelina 1 , Diego Alzate-Correa 1 , *Karl Obrietan 1
1 Ohio State University, , Columbus, United States
Abstract text :

Ischemia-induced progenitor cell proliferation is a prominent example of the adult mammalian brain’s ability to regenerate injured tissue resulting from pathophysiological processes.  A key locus of regeneration is the neurogenic niche located in the subgranular zone of the dentate gyrus (SGZ).  Within the SGZ, radial glia-like progenitor cells generate neural precursor cells which migrate into the granule cell layer (GCL) of the dentate gyrus, mature, and integrate into the hippocampal synaptic network.  In order to better understand and ultimately exploit the cell signaling mechanisms that regulate ischemia-induced proliferation in the SGZ, we tested the role of the p42/44 mitogen-activated protein kinase (MAPK) cascade effector ribosomal S6 kinase (RSK) in this process.  Using the endothelin-1 ischemia model in C57Bl/6 mice, we report that intrahippocampal cerebral ischemia triggered RSK activation in SGZ progenitor cells.  Further, microinjection of a RSK inhibitor significantly reduced ischemia-induced SGZ progenitor cell proliferation.  Using the neurosphere assay, we also show that SGZ- and subventricular zone (SVZ)-derived adult neural stem cells (NSC) exhibit a significant reduction in proliferation in the presence of RSK and MAPK inhibitors.  Taken together, these data indicate that RSK functions as a cell-autonomous regulator of ischemia-induced progenitor cell proliferation.


Astrocyte-Tissue inhibitor of metalloproteinases-1: The TIMP-ed balance of neuroinflammation: Relevance to HIV-1-associated neurocognitive disorders

*Anuja Ghorpade 1 , Jerel Adam Fields 1 , Clara Chao 1 , Kathleen Borgmann 1 , Lin Tang 1
1 University of North Texas, Health Science Center, Fort Worth, United States
Abstract text :

The pathogenesis of neurodegenerative disorders, including Human Immunodeficiency Virus (HIV)-1 associated neurocognitive disorders (HAND), is exacerbated by an imbalance between metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). As the TIMPs exhibit diverse non-classical functions including anti-apoptotic effects, the induction of TIMP-1 in neuroinflammatory conditions likely serves multiple roles in addition to modulating MMP activity. Our work demonstrates differential TIMP-1 expression in acute versus chronic activation of astrocytes and HIV-1 associated dementia (HAD) brain tissues. The TIMP-1 promoter harbors five consensus CCAAT boxes. CCAAT enhancer binding protein (C/EBP)b levels were elevated in brain specimens from HIV-1 patients and this transcription factor regulated astrocyte TIMP-1 expression. HIV-relevant stimuli increased C/EBPb expression in human astrocytes and localized the factor to the nucleus. Overexpressing C/EBPb in astrocytes increased TIMP-1 promoter activity, mRNA and protein expression, while knockdown of C/EBPb decreased TIMP-1 mRNA and protein expression. ERK1/2 activation is critical for IL-1b-mediated astrocyte TIMP-1 expression and p38K activation contributes to IL-1b-induced astrocyte TIMP-1 and C/EBPb expression. These data suggest that ERK1/2 signals downstream of C/EBPb to facilitate IL-1b-induced astrocyte TIMP-1 expression. The role of astrocyte-TIMP-1 as a neurotrophic factor was examined. Interestingly, cotreatment with TIMP-1 protected neurons from apoptosis and reversed neuronal morphological changes induced by staurosporine (STS) and HIV-1ADA virus. Further, the anti-apoptotic effect was specific to TIMP-1 and partially independent of MMP-inhibition. Additionally, TIMP-1 modulated the Bcl-2 family of proteins and inhibited opening of mitochondrial permeability transition pores induced by HIV-1 or STS. Together, these findings describe a novel function, regulatory mechanism and direct role of astrocyte-TIMP-1 in neuroprotection suggesting its therapeutic potential in HAD and possibly in other neurodegenerative diseases.


Impact of microglia-mediated inflammation on hypothalamic homeostasis

*Caroline Baufeld 1 , Kelly Miller 1 , Frank L. Heppner 1
1 Charité - Universitätsmedizin Berlin, Department of Neuropathology, Berlin, Germany
Abstract text :

Diets high in fat are known to cause inflammation in the periphery as well as the central nervous system. In peripheral adipose tissue, it has been shown that inflammation is primarily mediated by macrophages that are recruited to the tissue. Similarly, a recent study demonstrated reactive microgliosis in the hypothalamus of mice fed a high-fat diet. In the CNS, however, the relevance of microglial activation in response to a high fat diet remains unclear. We aim to determine if microglia activation occurring in response to overnutrition is a cause or consequence of dysregulation of energy homeostasis and obesity.

To determine if the absence of microglia (and therefore microglia-mediated inflammation) may influence the metabolic response to a high fat diet, we used a transgenic mouse model, the CD11b-HSVTK mouse (TK), which allows for an inducible, specific ablation of microglia in response to treatment with the nucleoside analog, gancilovir (gcv). Mice were treated with gcv for 4 weeks during which time they were maintained on a diet high in fat (60%) or a respective control diet. Body weight, food intake, locomotor activity and energy expenditure were measured and compared to wildtype mice.

Analyses revealed alterations in the metabolic phenotype of mice fed both HFD and control diet in the absence of microglia cells. Our findings point towards a physiological role of microglia in energy homeostasis, the study of which will be the focus of further experiments.


Immune complexes of beta amyloid with specific monoclonal antibodies induce neuronal loss via microglial activation

*Vilmante Borutaite 1 , Ramune Morkuniene 1 , Aurelija Zvirbliene 2 , Indre Dalgediene 2 , Paulius Cizas 1 , Silvija Jankeviciute 1 , Gintaras Valincius 3
1 Lithuanian University of Health Sciences, , Kaunas, Lithuania
2 Vilnius University, Institute of Biotechnology, Vilnius, Lithuania
3 Vilnius University, Institute of Biochemistry, Vilnius, Lithuania
Abstract text :

Oligomeric forms of beta amyloid (Aβ) peptides are considered as the main toxic factors in pathogenesis of Alzheimer’s disease (AD). In recent years, one of the most promising therapeutic means for AD was thought to be immunization with specific antibodies against Aβ. Several therapeutic antibodies were developed, however, most of the clinical trials were canceled due to unexpected deaths and neuroinflammatory responses in some patients. The causes and mechanisms of such responses are currently only partially understood.  We have recently raised monoclonal antibodies against oligomeric forms of Aβ and were investigating whether these antibodies would prevent the neurotoxicity of Aβ oligomers in primary neuronal-glial cerebellar granule cell (CGC) cultures. Antibodies were not directly toxic to CGCs when applied at concentrations relevant to concentrations reported to be in blood serum. However, surprisingly, the antibodies when in complexes with Aβ oligomers or firbrils dramatically increased the neurotoxicity of Aβ. Similar effects were also observed with antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein VP1, human metapneumovirus nucleocapsid protein and measles virus nucleocapsid protein strongly potentiated the neurotoxicity of their antigens. The neurotoxicity of antibody-oligomeric antigen complexes was abolished by removal of the Fc region from the monoclonal antibodies or by the removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. In conclusion, we find that immune complexes formed by Aβ oligomers or other oligomeric antigens and their specific monoclonal antibodies can cause neuronal death in primary neuronal-glial cultures via Fc-dependent microglial activation. The results suggest that therapies resulting in antibodies to oligomeric Aβ or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation.  Additionally, if endogenous antibodies contribute to neuronal loss in AD or viral encephalitis, suppression of microglial activation may be therapeutic. 


Cannabigerol quinone exerts therapeutic effects in experimental autoimmune encephalomyelitis

*Francisco Javier Carrillo-Salinas 1 , Miriam Mecha 1 , Ana Feliu 1 , Leyre Mestre 1 , Irene Cantarero 2 , Carmen  Navarrete 3 , Eduardo Muñoz 2 , Carmen Guaza 1
1 Cajal Institute (CSIC), , Madrid, Spain
2 University, Medicine, Córdoba, Spain
3 VivaCell Biotechnology España, , Córdoba, Spain
Abstract text :

Phytocannabinoids (pCBs) without psychotropic effects are considered of special interest as novel therapeutic agents in CNS diseases. These pCBs include cannabidiol (CBD), cannabigerol (CBG), D9 tetrahydrocannabivarin (D9THCV) and cannabidivarin (CBDV). We have developed a series of new cannabinoid quinones, among them the CBG quinone (VCE-003) that shows PPARg and CB2 receptors agonism. In addition we have found that VCE-003 activates the Nrf2/ARE pathway in neuronal cell lines. In the present study, we investigated the therapeutic potential of VCE-003 in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) by immunization con MOG 33-55. VCE-003 (5mg/Kg ip, daily) was administered to susceptible C57/BL6 mice at the onset of symptomatology. Clinical score and weights of mice were daily recorded until the day of sacrifice (28 days post-immunization). VCE-003 treatment delayed the onset of disease and ameliorated the symptomatology. Histological analysis of spinal cord of EAE mice treated with VCE-003 showed decreased microglia reactivity and reduced cellular infiltrates, in particular CD4 T lymphocytes. Double labeling with Neurofilament and the myelin protein, RIP indicated that VCE-003 diminished the axonal damage. Demyelination was evaluated by Luxol fast blue labeling. Changes in the expression of several cytokines, adhesion molecules and Nrf2-dependent genes were determined by qRT-PCR. The implication of PPARg and CB2 receptors in the beneficial effects of VCE-003 in the EAE model of MS is being investigated by using specific receptors antagonists. Taken together our results support the potential of VCE-003 for the treatment of MS and other chronic inflammatory diseases.


This work was supported by the MINECO grants IPT-2011-0861-900000 (VivaCell, EM and CG), SAF2010-17501 (CG) and SAF2010-19292 (EM).


Complement-independent modulation of chemokine expression by antibodies in myelinated cultures.

*Katie Jean Chapple 1 , Eva-Maria Oesau 1 , Maren Lindner 1 , Christopher  Linington 1
1 University of Glasgow, , Glasgow, United Kingdom
Abstract text :

A significant proportion of children with multiple sclerosis (MS) or acute disseminated encephalomyelitis (ADEM) develop autoantibody responses directed against myelin oligodendrocyte glycoprotein (MOG). The pathological relevance of this autoantibody response is unknown, but it is generally assumed that it exacerbates demyelination via activation of complement and/or cell meditated effector mechanisms. However in a majority of cases the MOG-specific autoantibody titre is far lower than that required to induce widespread demyelination and exacerbate disease severity in animal models of MS. To explore the possibility MOG-specific antibodies may play other more subtle roles in disease pathogenesis we investigated possible effects in myelinating cultures derived from embryonic rat spinal cord; a model system that allows us to explore antibody-dependent effects in the absence of exogenous complement and effector cells. Myelinating cultures were treated continuously with monoclonal antibodies specific for either MOG (clone Z2, IgG2a), sulphatide (clone O4, IgM), proteolipid protein (PLP) (clone O10, IgM), or an appropriate isotype control from 18 days in vitro onwards.

In the absence of an exogenous source of complement none of these antibodies induced demyelination, but by 24 DIV had all had a significant inhibitory effect on myelination compared to cultures treated with appropriate isotype control antibodies. To investigate possible mechanisms contributing to this inhibitory effect qPCR arrays were used to determine if this complement-independent effect on myelination was associated changes in expression of immune mediators. Unexpectedly we found recognition of antigens exposed at the surface of the myelin sheath induced a rapid increase in expression of three chemokines known to be involved in recruitment of effector T cells into the CNS. Within 24 hours of adding antibody to the cultures expression of CCL5, CCL20 and CXCL11 increased by at least three orders of magnitude and then declined to baseline over the following five days despite continuous presence of antibody. This transient increase in mRNA transcripts for these cytokines resulted in sustained protein synthesis and secretion of biological active products as demonstrated by analysis of culture supernatants.

These results challenge the traditional view that myelin-specific autoantibodies contribute to the pathogenesis of diseases such as MS and ADEM by virtue of their ability to initiate immune-mediated demyelination. We now demonstrate that even in the absence of recruited immune effector mechanisms myelin-specific autoantibodies not only inhibit myelination but trigger secretion of chemokines predicted to trigger or exacerbate inflammation within the CNS.


Neuroprotective function of microglial Siglec-E

*Janine Claude 1 , Bettina Linnartz-Gerlach 1 , Harald Neumann 1
1 Reconstructive Neurobiology, , Bonn, Germany
Abstract text :

Microglia have innate immune receptors recognizing pathogens and disease-associated molecular patterns but also molecules that could sense the intact tissue. A subfamily of these receptors is the inhibitory signaling sialic acid-binding immunoglobulin-like lectin (Siglec) group including Siglec-E that has an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail to suppress activatory microglial signals.

In this study, we used primary and stem cell-derived microglia that were modified by lentiviral vectors. Here we show that Siglec-E is expressed on microglia and is up-regulated following interferon-γ (IFN-γ) treatment. We performed lentiviral knock-down and overexpression of Siglec-E. Lentiviral overexpression of Siglec-E decreased, while knock-down increased the phagocytosis of neural debris and its associated reactive oxygen burst. The extracellular domain of Siglec-E linked to a Fc-part of immunoglobulin bound to the sialic acid residues of the neuronal glycocalyx. Therefore, we co-cultured these modified microglia with primary hippocampal neurons. Overexpression and knock-down of Siglec-E showed an increase and decrease in relative neurite-length, respectively. The neuroprotective effect of Siglec-E was abrogated after removal of the sialic acid residues on the neuronal glycocalyx. Treatment with the anti-oxidant Trolox abolished the neurotoxic effect of the Siglec-E knock-down on neurite length.

In summary, our data suggests an immunomodulatory function of Siglec-E on microglia which leads to a neuroprotective phenotype by decreasing the production of reactive oxygen species and a reduced phagocytosis rate of neural debris.


Characterization of the role of the Metallothionein-1 in an animal mouse model of Alzheimer’s disease.

*Gemma Comes 1 , Yasmina Manso 1 , Monica Belfiore 1 , Javier  Carrasco 1 , Juan Hidalgo 1
1 Universitat Autònoma de Barcelona, , Bellaterra. Barcelona, Spain
Abstract text :

 Metallothioneins (MTs) are low molecular weight, zinc- and copper-binding proteins which are involved in antioxidant, anti-inflamatory and anti-apoptotic processes.

MT-1&2 levels have been found to be increased in several human neurodegenerative diseases including Alzheimer’s disease (AD). Moreover, MT1 2 are also upregulated in different AD mouse models, for example Tg2576 mice, which show a significant up-regulation of these proteins in the vicinity of the amyloid plaques.

In the present study we generated a double transgenic mouse line that develops AD-like pathology in addition to having an overexpresion of MT1, in order to determine the role of MTs in different aspects of AD pathology.

The results show that the overexpression of MT1 does affect the mortality rate of the Tg2576 and control mice in a gender-dependent manner and partially reverses the behavioural phenotype of young (4-5 months) Tg2576 mice, reducing the exploratory activity and improving the learning process; in contrast, MT1 does not cause relevant changes in deambulations and anxiety.

On the other hand, the amyloid cascade and neuroinflammation is increased in the hippocampus of old (15-16 months) APPTgMT mice, but the lower level of gliosis in the hippocampus of young male APPTgMT mice suggests that the overexpression of MT1 is capable of reducing inflamatory response well before amyloid plaques are formed but not afterwards.

Further molecular and immunohistochemistry analyses are underway to give further insight into the role of MTs in amyloidosis and neuroinflammation in this AD mouse line.


Anti-epileptic drugs (AEDs) alter the microglial in-/ activation state in astroglia/microglia cocultures

*Hannes Dambach 1 , Daniel Hinkerohe 2 , Zahra Moinfar 1,3 , Nora Prochnow 1,3 , Andreas Hufnagel 4 , Pedro Michael Faustmann 1,3
1 Ruhr-Universität Bochum, Neuroanatomie und Molekulare Hirnforschung, Bochum, Germany
2 Knappschafts-Krankenhaus, Neurologie, Bochum, Germany
3 Ruhr-Universität Bochum, International Graduate School of Neuroscience (IGSN), Bochum, Germany
4 Neurologische Privatpraxis, , Düsseldorf, Germany
Abstract text :

Background:The contribution of glial cells in the pathophysiology of epilepsy is increasingly valued. Furthermore, clinical and experimental evidence suggests a direct relationship between epileptic activity and CNS inflammation, which is characterized by accumulation, activation and proliferation of microglia and astrocytes. Concomitant glia-mediated mechanisms of action of several AEDs have been proposed. However, their direct effects on glial cells, especially microglia, have jet been hardly investigated. We aimed to investigate the influence of commonly used AEDs on the glial viability and microglial in-/ activation state in a physiological and inflammatory modified in-vitro astroglia/microglia coculture model.

Methods:Primary astrocytic cultures were prepared from brains of postnatal (P0–P2) Wistar rats and cocultured with a physiological amount of 5% (M5), as well as 30% (M30) microglia in order to mimic inflammatory conditions. Cocultures were treated for 24 hours with valproic acid (VPA), carbamazepine (CBZ), phenytoin (PHE) and gabapentine (GBT) with a concentration of 10, 25, 50 and 100 µg/ml. Viability and proliferation was measured using the tetrazolium (MTT) assay. The microglial activation state was determined by immunocytochemical labeling using a monoclonal antibody to the ED1 marker.

Results:M5 and M30 cocultures showed a dose-dependent, significant reduction in glial viability after incubation with PHE and CBZ. Furthermore, low doses of VPA led to highly significant microglial activation in M5 cocultures. However, CBZ significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia in the inflammatory modified M30 cocultures. 

Conclusion: CNS inflammation is characterized by a disturbance of glial cell functions. Strong microglial activation, a typical hallmark of inflammation, was induced by VPA in M5 and continued in M30 cocultures. With regard to the direct relation between CNS inflammation and seizures, VPA seems to be unsuitable to reduce inflammatory condition. The reverse effect was achieved after CBZ. We noticed significant microglial inactivation, after incubation of the M30 cocultures. As it has been demonstrated for levetiracetam before, we assume a beneficial therapeutic effect of anti-epileptic drugs (AED) with an anti-inflammatory glial potential in epileptic patients with persistent inflammation.


Peroxisome Proliferator-Activated Receptor-γ agonists protect oligodendrocyte from mitochondrial stress 

*Roberta De Simone 1 , Antonietta Bernardo 1 , Chiara De Nuccio 1 , Sergio Visentin 1 , Luisa Minghetti 1
1 Istituto Superiore di Sanità, , Rome, Italy
Abstract text :

We have previously shown that natural (15-deoxy-D12,14 prostaglandin J2, 15d) and synthetic (pioglitazone) agonists of peroxisome proliferator activated receptor-γ (PPAR) potentiate intrinsic cellular mechanisms protecting oligodendrocyte (OL) progenitors (OPs) from oxidative insults and promote their differentiation to OLs. In addition, PPAR-γ agonists potentiate mitochondrial activities, as the mitochondrial respiratory chain activity and the regulation of cytoplasmic Ca2 waves, which are known to be crucial for OL differentiation.

In the present study we sought to investigate whether PPAR-γ agonists can protect OL cultures from conditions causing mitochondrial stress.

First, to specifically induce a mitochondrial impairment, we used the complex I inhibitor rotenone. As expected rotenone, at concentration not affecting cell viability, significantly inhibited OL differentiation, as indicated by the reduced number of cells expressing specific markers of differentiation (O4 and O1). In PPAR-γ agonist–treated OLs the inhibitory effects of rotenone were significantly attenuated, suggesting a protective effect of the agonists against the mitochondrial toxin.

We next examined a condition mimicking inflammatory stress by challenging OP cultures with TNF-a, an inflammatory cytokine known to retard the differentiating program of OPs. In parallel with the expected reduction of the percentage of O4 and O1 positive cells, the cytokine induced a significant reduction of mitochondrial membrane potential (mMP), suggesting an impairment of the mitochondrial functions. The simultaneous treatment with TNF-a and PPAR-γ agonists (15d or pioglitazone) significantly reverted both TNF-a dependent reduction of OL differentiation and mMP. At the molecular level, we found that in OP cultures, PPAR-γ agonists increased the expression of the uncoupling protein-2 (UCP-2), a mitochondrial protein known to contribute to the protection of mitochondria against oxidative stress.

These findings suggest that PPAR-γ agonists protect OLs and promote myelination through several mechanisms, including those involving mitochondrial functions. Our studies support the therapeutic potential of PPAR-γ agonists in brain diseases in which mitochondrial alteration, oxidative stress and demyelination occur and point to the need to better understand the role of PPAR-γ and its agonists in OL biology. 


N-3 polyunsaturated fatty acids protect against the cognitive effects of a peripheral inflammation by targeting microglia morphofunctional activity

*Jean-Christophe Delpech 1 , Charlotte Madore 1 , Agnès Aubert 1 , Corinne Joffre 1 , Sophie Layé 1 , Agnès Nadjar 1
1 NutriNeuro, UMR INRA 1286, Université Bordeaux, , Bordeaux, France
Abstract text :

Microglia is the main cellular component of the cerebral innate immune system. During a peripheral immune challenge, microglial cells are activated, leading to alteration of hippocampal memory. This modulation of cognitive processes is exerted by signals produced by immune-like processes and in particular the proinflammatory cytokine Interleukin-1 beta (IL-1β). Depending on the type of signals produced after an inflammatory event, microglia develops specific activities, including cytotoxic, neuroprotective or phagocytic activities in order to come back to brain homeostasis. As described for macrophages, microglia can adopt M1 (proinflammatory) or M2 (phagocytic) phenotypes distinguishable through their pattern of factors expression and specific cell surface markers.  Interestingly, polyunsaturated fatty acids (PUFAs) are precursors of bioactive lipid messengers involved in the regulation of inflammation and in particular, docosahexanoic acid (DHA, 22:6n-3) an n-3 PUFA, presents anti-inflammatory properties. The aim of our study was thus to investigate the effect of an increase of n-3 PUFAs on the inflammatory response and cognitive abilities after a peripheral immune challenge. To increase n-3 PUFAs, we took advantage of transgenic mice carrying the fat-1 gene from the roundworm Caenorhabditis elegans. This fat-1 transgenic mouse is capable of producing n-3 fatty acids from the n-6 type endogenously, eliminating confounding factors of the diet. This conversion leads to abundant n-3 fatty acids with reduced levels of n-6 fatty acids in tissues, including the brain. To induce a neuroinflammation, we injected mice intraperitoneally with lipopolysaccharide (LPS), components of gram negative bacteria walls. We first studied the microglial phenotype 24 h after LPS injection and found that microglia in Fat-1 mice presented a significant increase of M2 phenotype markers. We then measured cytokine expression in the hippocampus after a LPS challenge and found a significant decrease in IL-1β mRNA in Fat-1mice compared to wild-type littermates. In terms of hippocampal memories abilities, only control mice presented a deficit in the Y-maze task whereas Fat-1 mice were able to perform the test correctly. Our results indicate that 24 h after a LPS injection, Fat-1 mice presented a decreased of the inflammatory response and normal hippocampal memory abilities compared to wild-type littermates.


Microglial activation beyond the Substantia Nigra in Parkinson’s Disease

*Karlijn Doorn 1 , Paul J. Lucassen 1 , John G.J.M. Bol 2 , Benjamin Drukarch 2 , Wilma D.J. van de Berg 2 , Anne-Marie van Dam 2
1 University of Amsterdam, Swammerdam Institute for Life Sciences, , Amsterdam, Netherlands
2 VU Medical Center, Dept. Anatomy and Neurosciences, Amsterdam, Netherlands
Abstract text :

It has become clear that the characterization of Parkinson’s disease (PD) as an isolated disorder of the dopaminergic system is an oversimplification of the complex pathogenesis of the disease. Various brain areas are affected in PD, showing α-synuclein (α-syn) accumulation, a major hallmark of PD pathology. For instance, the olfactory bulb (OB) reveals α-syn aggregates in an early stage of the disease, and the hippocampus (HC) is affected in a rather late stage of the disease (Braak et al., 2003). Furthermore, neuroinflammation, i.e. activation of microglial cells in the substantia nigra (SN), has been widely implicated in PD progression (McGeer et al., 1988). In the present study, we question whether microglial activation occurs in brain regions beside the SN which are affected in PD patients. 

To this end, we studied microglial activation and protein pathology in the OB and HC of clinically and neuropathologically verified PD patients (Braak 4-6) and control subjects (Braak 0). Human post-mortem formaldehyde-fixed material of PD patients included SN (n=13), OB (n=9) and HC (n=12), and material from age-matched control subjects without neurological deficits included SN (n=10),OB(n=6), and HC (n= 8).

The presence of α-syn pathology concentrated in the anterior olfactory nucleus of the OB and in the CA1-2 region of the HC. Furthermore, using CD68 as a microglial marker, we observed a significant increase in the number of immunopositive microglial cells, with an activated, amoeboid morphology, in the SN as well as in the OB and HC of PD patients compared to control subjects. Co-localization studies indicated that these activated microglia were found in the proximity of α-syn inclusion bodies and neurites, but did not co-localize suggesting that the microglial cells do not actively phagocytose α-syn.

We conclude that microglial activation occurs in other brain regions beside the substantia nigra of PD patients. The functional role of the microglial cells in those brain regions and their contribution to PD pathology remains to be established. 



Braak, H., Tredici, K. D., Rüb, U., de Vos, R. A. I., Jansen Steur, E. N. H., and Braak, E. (2003) Staging of brain pathology related to sporadic Parkinson's disease. Neurobiology of aging 24, 197-211.McGeer, P. L., Itagaki, S.,

Boyes, B. E., and McGeer, E. G. (1988) Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology 38, 1285-6.



Inflammatory profiling of the satellite glial cells in the dorsal root ganglia of rat experimental neuropathic pain models

*Petr Dubovy 1 , Vaclav Brazda 2 , Ivana Hradilova-Svizenska 1 , Ilona Klusakova 1 , Lucie Strejckova 1
1 CEITEC and Medical Faculty, Masaryk University, , Brno, Czech Republic
2 CEITEC Masaryk University, Cellular and Molecular Neurobiology, Brno, Czech Republic
Abstract text :

The satellite glial cells (SGC) envelope the primary sensory neurons of the dorsal root ganglia (DRG) and react intensely to various types of nerve injury that induce neuropathic pain. Unilateral chronic constriction injury (CCI, n=64) and spare nerve injury (SNI, n=32) of the rat sciatic nerve well as i.p. application of paclitaxel (PAC, n=12) were used as experimental models of neuropathic pain (NPP). NPP induction was tested by withdrawal threshold of mechanoallodynia and thermal hyperalgesia. Expression of TNFa, IL-6 and their receptor and signaling (TNFR1, TNFR2, IL-6R, gp130, STAT3) proteins and mRNA were investigated bilaterally by immunohistochemistry and in situ hybridization in both lumbar (L4-5) and cervical (C7-8) DRG 1, 3, 7 and 14 days from treatment. The SGC were identified by colocalization with GFAP or GS.

Our results demonstrate significant inflammatory activation of the neurons and their SGC not only in DRG associated but also non-associated with injured nerve. A distinct expression of cytokines and their receptors was identified in SGC surrounding large-sized DRG neurons. Significant inflammatory reactions of SGC were found in all DRG of PAC-treated rats. Moreover, inflammatory activation SGC was also observed in DRG of sham-operated rats indicating other kinds of trigger than traumatic nerve injury. The nerve injury and PAC-treatment also triggered SOCS3 expression in SGC to control STAT3 activation. Inflammatory activation of SGC significantly contributes to ectopic activation of the DRG neurons not only associated with injured nerve, and is involved in NPP induction.

This work was supported by the project “CEITEC - Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund and the EU 7th FP under the "Capacities" specific programme (Contract No. 286154 – SYLICA)".


I-TAC signalling in primary rodent astrocytes and human glioma cells requires CXCR4

*Jürgen Engele 1 , Malte Puchert 1 , Veysel Ödemis 1
1 University of Leipzig, , Leipzig, Germany
Abstract text :

It is currently believed that CXCL12 predominantly signals through CXCR4. In addition, it is assumed that the alternate CXCL12 receptor, CXCR7, represents a non-classical G protein-coupled receptor which primarily acts as a modulator of the function of CXCR4. Discrepant from this view, we demonstrated recently that in primary rodent astrocytes and human glioma cell lines, SDF-1 exclusively signals through CXCR7 by a G protein-dependent mechanism. We now provide evidence that CXCR4 is essential for CXCL11/I-TAC signalling in primary rodent astrocytes and some human glioma cells. Treatment of cultured rat astrocytes with CXCL11 for 10 min resulted in the dose-dependent activation (phosphorylation) of Erk1/2 and Akt with maximum activation in the presence of 100 ng/ml of the chemokine. CXCL-11-dependent activation of both signalling molecules persisted in astrocytes in which expression of the established receptors for CXCL11, CXCR3 and CXCR7, were inhibited by RNAi. However, CXCL11-dependent activation of Erk and Akt was abrogated following siRNA-mediated inhibition of CXCR4. Likewise, CXCL11 failed to activate Erk and Akt in cortical astrocytes cultured from a CXCR4-/- transgenic mouse line. Moreover, similar to primary astrocytes CXCL11 activated ERK and Akt in the human glioma cell line, A767. Again activation of both signalling molecules was abrogated following RNAi-mediated inhibition of CXCR4. CXCL11-dependent activation of Erk and Akt further remained undetectable in a non CXCR4-expressing subpopulation of A767 cells, previously isolated by flow cytometry. Together, these findings unravel a unique processing of CXCL11 and CXCL12 signalling in primary astrocytes which is preserved at least in some malignant astroglial cells.


Imaging reactive astrocytes in vivo by positron emission tomography with TSPO radioligands

Sonia Lavisse 1 , Martine  Guillermier 1 , Anne-Sophie Hérard 1 , Fanny Petit 1 , Marion Delahaye 1 , Nadja Van Camp 1 , Lucile Ben Haim 1 , Vincent Lebon 1 , Frédéric Dollé 2 , Thierry Delzescaux 1 , Gilles Bonvento 1 , Philippe Hantraye 1 , *Carole Escartin 1
1 MIRCen, , Fontenay-aux-Roses, France
2 SHFJ, , Orsay, France
Abstract text :

Astrocyte and microglia become reactive under many brain pathological conditions, making this process of neuroinflammation a surrogate marker of neuronal dysfunction.

Several studies have reported that reactive microglia overexpress the translocator protein 18kDa (TSPO, formerly known as peripheral benzodiazepine receptor). Positron emission tomography (PET) using radioligands of TSPO is thus considered as a potent technique to detect reactive microglia in situ. However, it is still controversial whether TSPO PET imaging is also able to monitor reactive astrocytes in situ. This is an important question as reactive astrocytes and reactive microglia play very different roles in brain physiology and could impact disease progression in opposite ways.

To address this question, we used a model of  selective astrocyte activation through unilateral lentiviral gene transfer of the cytokine ciliary neurotrophic factor (lenti-CNTF) in the rat striatum. CNTF induced an extensive activation of astrocytes, which overexpressed GFAP and became hypertrophic. Microglia, on the contrary, displayed a minimal increase in the expression of markers of reactivity.

CNTF-activated astrocytes overexpressed TSPO at the mRNA and protein levels. PET imaging experiments demonstrated a significant and specific binding of two TSPO radioligands [18F]DPA-714 and [11C]SSR180575 in the lenti-CNTF-injected striatum.

We show that reactive astrocytes can be monitored by TSPO-PET imaging in the rat brain. This technique is thus well suited to monitor reactive microglia as well as reactive astrocytes, the two cell types involved in neuroinflammation, but would not allow their discrimination in situ


Cannabidiol provides long-lasting protection against the deleterious effects of inflammation in a viral model of multiple sclerosis: a role for A2A receptors

*Ana Feliu 1 , Miriam Mecha 1 , Francisco Javier Carrillo-Salinas 1 , Carmen Guaza 1
1 Cajal Institute, CSIC, , Madrid, Spain
Abstract text :

Inflammation in the central nervous system (CNS) is a complex process that involves a multitude of molecules and effectors, and it requires the transmigration of blood leukocytes across the blood-brain barrier (BBB) and the activation of resident immune cells. Cannabidiol (CBD), a non-psychotropic cannabinoid constituent of Cannabis sativa, has potent anti-inflammatory and immunosuppressive properties. Yet, how this compound modifies the deleterious effects of inflammation in TMEV-induced demyelinating disease (TMEV-IDD) remains unknown. Using this viral model of multiple sclerosis (MS), we demonstrate that CBD decreases the transmigration of blood leukocytes by downregulating the expression of vascular cell adhesion molecule-1 (VCAM-1), chemokines (CCL2 and CCL5) and the proinflammatory cytokine IL-1β, as well as by attenuating the activation of microglia. Moreover, CBD administration at the time of viral infection exerts long-lasting effects, ameliorating motor deficits in the chronic phase of the disease in conjunction with reduced microglial activation and pro-inflammatory cytokine production. Adenosine A2A receptors participate in some of the anti-inflammatory effects of CBD, as the A2A antagonist ZM241385 partially blocks the protective effects of CBD in the initial stages of inflammation. Together, our findings highlight the anti-inflammatory effects of CBD in this viral model of MS, and demonstrate the significant therapeutic potential of this compound for the treatment of pathologies with an inflammatory component.


A novel human in vitro microglia model  

*Luis Filgueira 1 , Samar Etemad 2 , Marc Ruitenberg 3
1 University of Fribourg, , Fribourg, Switzerland
2 University of Western Australia, , Perth, Australia
3 University of Queensland, Biomedical Sciences, Brisbane, Australia
Abstract text :

Microglia are the resident innate immune cells of the brain. As the brain macrophages, microglia share common characteristics with the monocyte-macrophage lineage such as expression of surface markers,  chemokine and cytokine receptors, phagocytosis and antigen presentation. Microglia responds with activation to injury, pathogen, as well as endogenous and exogenous toxins. This activation is usually followed by secretion of pro inflammatory cytokines and chemokines. Activated microglia may contribute to pathological processes, but they play also a key role in the development of the central system and in neurogenesis through secretion of neurotrophic factors.

To date, there is no standardized simple model available to investigate the biology of human microglia. The aim of this study was to establish a new in vitro microglia model using blood-derived precursor cells. For that purpose, human peripheral blood monocytes were cultured in serum free medium in the presence of a mixture of cytokines and chemokines (M-CSF, GM-CSF, NGF and CCL2) to generate monocyte-derived microglia (M-MG). Monocyte-derived dendritic cells (M-DC) were also generated as a control population using GM-CSF and IL-4. The human microglia cell line HMC3 was used as control.

M-MG were clearly different in morphology, phenotype and function from M-DC, but shared many properties with HMC3 cells. M-MG acquired a ramified morphology with primary and secondary processes, comparable to HMC3. They expressed very low levels of CD45, CD14 and HLA-DR, CD11b and CD11c; but a distinct pattern of chemokine receptors, including CCR1, CCR2, CCR3, CCR4, CCR5, CXCR1, CXCR3, CX3CR1. Similar to HMC3, under non-activated condition, the M-MG secreted of IL-8 and IL-6. In comparison with M-DC, M-MG displayed lower T-lymphocyte stimulatory capacity, as well as lower phagocytosis activity.

In summary, we have established a new protocol for the generation of human monocyte-derived microglia, which is is feasible, well standardized and reliable, as it uses well defined culture medium and recombinant cytokines, but no serum or conditioned medium. This model will certainly be very helpful for future studies investigating the biology and pathology of human microglia.


Brain response to traumatic brain injury: interleukin-6 relevance

*Mercedes Giralt 1 , Olaya Fernandez 1 , Raquel Ramos 1 , Amalia Molinero 1 , Juan Hidalgo 1
1 Universitat Autònoma de Barcelona, , Bellaterra, Barcelona, Spain
Abstract text :


Interleukin-6 (IL-6) is a cytokine with major regulating effects of the inflammatory response. Moreover, IL-6 is a neuropoietin that has neurotrophic effects related to neuronal survival and protection. To establish the importance of IL-6 produced only in the central nervous system, we have generated mice producing IL-6 essentially only in the brain by crossing GFAP-IL6 mice (transgenic mice with astrocyte-targeted production of IL-6) with IL-6 KO mice (IL-6-deficient).  We studied the inflammatory response in a traumatic brain injury model, cryolesion, after 3 and 10 days post-lesion GFAP-IL6-IL6 KO mice, comparing them with appropriate controls.

In basal conditions WT and  IL-6 KO mice showed a similar phenotype. This was also the case for GFAP-IL6 and GFAP-IL6-IL-6 KO mice, which showed prominent astrogliosis, microgliosis, increased recruitment of T lymphocytes and vascularisation compared with the other two groups. 

In response to cryolesion of the cortex an increased astrogliosis, microgliosis, recruitment of T lymphocytes and vascularisation was observed.  IL-6 deficiency produced an altered inflammatory response, in a time-and gender-dependent manner.

This study with IL-6 KO and GFAP-IL6 transgenic mice indicates that during an acute neuropathological insult such as traumatic brain injury IL-6, from either the brain or the periphery, has an important role on the inflammatory response.


Regulation of microglial proliferation in chronic neurodegeneration

*Diego Gomez-Nicola 1 , Nina L. Fransen 1 , Stefano Suzzi 1 , V. Hugh Perry 1
1 University of Southampton, , Southampton, United Kingdom
Abstract text :

An important aspect of chronic neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, Huntington’s and prion disease, is the generation of an innate inflammatory response within the central nervous system (CNS). Microglial and astroglial cells play a key role in the development and maintenance of this inflammatory response, showing enhanced proliferation and morphological activation. Using a laboratory model of chronic neurodegeneration (ME7 murine model of prion disease), we studied the time-course and regulation of microglial proliferation. Our results show that resident microglial cells have an increased proliferation rate during the development of the disease, leading to a significant increase in the population, without a contribution from circulating cells. Microglial proliferation is differentially regulated in diverse regions of the CNS, pointing to a heterogeneous development of the pathology. We have identified novel molecular regulators of the proliferative response, and addressed the significance of the contribution of microglial cells to the pathological course of the disease by modifying their proliferation. We also found a correlation of our results with the scenario present in chronic human neurodegenerative conditions variant Creutzfeldt-Jakob Disease (vCJD) and Alzheimer’s disease. Our results demonstrate that microglial proliferation is an important feature of the evolution of chronic neurodegenerative disease, with direct implications for understanding the contribution of the CNS innate immune response to disease progression.


Stimulation of the IL-1 Signaling Pathway by CNS-Infiltrating Myelin Reactive T Cells in Zones of Axonal Degeneration

*Manuela Grebing 1 , Helle Hvilsted Nielsen 2 , Christina Fenger 1 , Martin  Söderman 1 , Katrine Tækker Jensen 1 , Bettina Hjelm Clausen 1 , Kate Lykke Lambertsen 1 , Mads Thomassen 3 , Torben A. Kruse 3 , Bente Finsen 1
1 University of Southern Denmark, , Odense C, Denmark
2 Odense University Hospital, Department of Neurology, Odense, Denmark
3 Odense University Hospital, Department of Human Genetics, Odense, Denmark
Abstract text :

Recently, we showed a beneficial effect of myelin reactive T cells on oligodendrocyte precursor cell differentiation in zones of axonal degeneration in the hippocampal dentate gyrus, which mirrors grey matter injury in multiple sclerosis. This effect was associated with a marked expression of T-cell cytokines, such as Interferon-γ and interleukin-17, enhanced microglial clearance of myelin debris, and an enhanced sprouting of calretinergic axons. To gain better insight in which cytokines and signaling pathways are elevated in response to the T cell enhanced regenerative responses, we performed a mRNA microarray study, in which the transcript profile in hippocampi from mice with adoptively transferred proteolipid protein specific T cells combined with an axonal lesion were compared to the transcript profile from mice with axonal lesion. Thereby we identified 1446 transcripts, which were differently expressed. We identified the interleukin-1 (IL-1) signaling pathway as a potential target by performing pathway analysis using DAVID and AmiGO databases. We discovered that IL-1α, IL-1β and IL-1 receptor antagonist (RA) mRNA as well as the IL-1 signaling pathway were highly upregulated in response to myelin reactive T cells. In order to determine if IL-1β mRNA was translated into protein, we performed immunohistochemical stainings on tissues from mice with 2 days post lesion survival. Expression of IL-1β was observed in the molecular layer of T cell infiltrated, but not in T cell naïve mice with axonal lesion, suggesting that myelin reactive T cells stimulate IL-1β protein expression. Ongoing studies will determine the expression of IL-1α and IL-1RA protein and determine the cellular expression of the IL-1 signaling pathway in T-cell infiltrated versus non-T cell infiltrated mice with axonal lesion. Understanding the regulation of the expression of IL-1α, IL-1β and IL-1RA in T cell compared to non-T cell infiltrated CNS may lead to a better understanding of the functional consequences of inflammatory responses in the CNS.


Supported by the Danish Multiple Sclerosis Society, the Augustinus Foundation and the University of Southern Denmark


Key words:

Myelin reactive T cells, IL-1 signaling pathway, myelin debris


Ageing augments the behavioural response to systemic Salmonella Typhimurium infection in mice – do microglia play a role?

*Adam Hart 1 , Steven G. Booth 1 , Ursula Püntener 1 , Hugh Perry 1 , Jessica Teeling 1
1 University of Southampton, , Southampton, United Kingdom
Abstract text :

Infections often induce sickness behaviour, such as lethargy, social withdrawal and anhedonia. This behavioural response is exaggerated during ageing as a consequence of microglial priming1. In this study we extended our previous work2 to examine whether S. typhimurium infection causes a prolonged behavioural response, whether ageing modulates the sickness behaviours induced by S. typhimurium infection and what involvement microglia may have in these changes.

Young (3m) and aged (18m) C57/Bl6 mice were administered 3 x 105 CFU of attenuated S. typhimurium (SL3261) via intraperitoneal injection and tested in several behavioural assays at 3 different timepoints – before infection, 1 week after infection or 3 weeks after infection. The behavioural assays used were the novel object recognition test, open field test, static rod test and the forced swim test. Body weights were recorded daily. Tissue was collected from saline treated and mice infected for 1, 8, 15, 22 or 61 days and analysed by qPCR.

We found that young mice lost weight only in the first 24h after infection, whereas aged mice exhibited a biphasic pattern of weight loss, with the second phase of weight loss beginning 1 week after infection and continuing for approximately 2½ weeks. Aged mice also demonstrated a co-ordination and balance deficit in the static rod test 1 week after infection compared to uninfected or 3 week post infection aged mice, whereas young mice were unimpaired at any timepoint. S. typhimurium infection caused a significant, progressive reduction in open field rearing activity at 1 week and 3 weeks after infection in both young and aged mice. A similar but not significant trend was apparent in novel object performance. Forced swim test data showed a trend towards depressive behaviour at 1 week after infection, but not at 3 weeks.  Analysis of pro-inflammatory mediators in the hippocampus did not indicate significantly upregulated IL-1β, TNF-α, COX-1 or COX-2 transcript at any timepoint, suggesting that microglia might not play a significant role in mediating these behavioural effects, or that the changes in these molecules were too subtle to reliably detect using qPCR in the hippocampus. The effects of ageing on S. Typhimurium induced weight and co-ordination changes may be a result of regional differences in the sensitivity of microglia to peripheral infection within the CNS or increased sensitivity to activation of neuronal circuits involved in weight loss or co-ordination with age.

1. Godbout et al, FASEB, 2005, 19:1329

2. Püntener et al, J Neuroinflammation, 2012, 9:146.


Is an inflammation caused by injury necessary for the brain?

*Hiroko Ikeshima-Kataoka 1,2 , Sayaka Inui 2 , Masato Yasui 2 , Yutaka  Matsui 3 , Toshimitsu  Uede 4
1 Waseda Univ., Facul. Sci. & Engin., Tokyo, Japan
2 Keio Univ. Sch. Med. , Dept. Pharmacol. & Neurosci. , Tokyo, Japan
3 Hokko Memorial Hosp. , Div. Cardiovasc. Med., Hokkaido, Japan
4 Hokkaido Univ., Dept. Matrix Med., Inst. Genet. Med. , Hokkaido, Japan
Abstract text :

 At pathological condition such as injury or ischemia to the central nervous system (CNS), microglial cells and astrocytes become activate and inflammation occurs like proliferate, migrate and secrete some pro-inflammatory cytokines, IL-1ß, TNF-α or IL-6. However, it is unclear that the reason why inflammation is induced in the CNS, and whether or not it is necessary for the brain during recovery from pathological situation. 

 To know the molecular mechanisms for inflammation occurs in activated glial cells, we made a stab wound mouse model to the brain. We performed microarray analysis for RNA extracted from the brain tissue around stab wound site compared among day after 0, 1, 3 and 7. It revealed that most of the genes from top 20 genes at high expression level around lesion site were concerned in immunological or inflammatory functions. We successfully identified and focused on to osteopontin (OPN), which is an inducer of pro-inflammatory cytokine production expressed not only in reactive microglial cells but also in reactive astrocytes around the injured CNS. Furthermore, we also found receptors against OPN functioning in the injured brain. However, functional role of OPN in reactive astrocytes is not yet clarified. To analyze the central role of OPN in reactive astrocytes, we examined primary culture of astrocytes from OPN-deficient (OPN/KO) mice and found that the morphology of these cells was unusual. By the stimulation with lipopolysaccharide to the primary culture of astrocytes from OPN/KO mice, some of the pro-inflammatory cytokine expression was altered. Moreover, reactivity of astrocytes caused by stab wound to the brain was examined using analogue of nucleic acid, bromo-deoxy-uridine (BrdU), and clarified that it was decreased in OPN/KO mice.

  From these results, we conclude that OPN might play a major role in the activation of astrocytes under inflammation occurred to the CNS.


Does systemic inflammation contribute to the progression of age-related hearing loss?

*Bethan Impey 1 , Andrew E Causon 2 , Akosua Agyemang-Prempeh 2 , Joanne L Bailey 3 , Carl Verschuur 2 , Tracey A Newman 3
1 University of Southampton, , Southampton, United Kingdom
2 University of Southampton, Hearing and Balance Centre, Faculty of the Environment and Engineering, Southampton, United Kingdom
3 University of Southampton, CES, IfLS, Faculty of Medicine, Southampton, United Kingdom
Abstract text :

Background: Age-related hearing loss (presbycusis) affects half of people by the age of 75. It has a large impact on quality of life and is associated with accelerated cognitive decline. However, presbycusis is not an inevitable process of aging, suggesting that its progression could be minimised.

Hearing relies on sound vibrations from the middle ear to elicit movement of the basilar membrane in the cochlea, resulting in excitation of hair cells.  Spiral ganglion neurons relay the inner hair cell signals to the central auditory system via the vestibulocochlear nerve. Sounds are integrated in the central auditory pathway and then processed by cognitive areas of the brain.

Degeneration of cochlear structures, including hair cells and spiral ganglion cells occurs in presbycusis. In the central auditory system there are alterations in neuronal plasticity and neurodegeneration may occur.

Chronic inflammation has previously been correlated with age-related hearing loss in humans, implicating immune cells in the progression of presbycusis. Microglia within the central nervous system are susceptible to priming by neurodegeneration or aging, both factors in presbycusis. It is feasible that microglia in the cochlea, an organ with similar immune privilege as the brain, could also become primed. Priming increases the chance of microglia becoming activated by subsequent inflammatory events, such as ‘flu, which may cause them to contribute to neurodegeneration in the cochlea and central auditory system.

Hypothesis: Our working hypothesis is that as age-related hearing loss progresses, microglia in the cochlea and central auditory pathway will become primed. We predict that systemic inflammation will cause primed microglia to be activated to a pro-inflammatory damaging phenotype, which will increase the rate of cochlea and auditory pathway degeneration and hence hearing loss progression.

Methods: C57BL/6J mice are genetically predisposed to develop age-related hearing loss.  Young and middle-aged mice will be challenged with LPS or saline (control) and microglial phenotype assessed in the cochlea and central auditory system. Neurodegeneration will be evaluated at corresponding points in the cochlea and auditory pathway using molecular techniques.

Conclusion: If our hypothesis is proved this would suggest that rigorous control of infection in older individuals would reduce progression of hearing loss by minimising degeneration in the auditory system.


Immunomodulatory properties of LIF and OSM in multiple sclerosis

*Kris Janssens 1 , Helena Slaets 1 , Bart Vanwijmeersch 1 , Piet Stinissen 1 , Jerome Hendriks 1 , Niels Hellings 1
1 Biomedical Research Institute, , Diepenbeek, Belgium
Abstract text :

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), characterized by multifocal inflammatory infiltrates, demyelination, and axonal loss and degeneration. In the cerebrospinal fluid and lesions of MS patients leukemia inhibitory factor (LIF) and oncostatin M (OSM), members of the IL-6 cytokine family, are upregulated. In humans, LIF and OSM both signal through the LIF receptor (LIFR), however OSM can also activate its specific receptor, OSMR. LIFR signaling promotes the survival of glial cells and neurons, and is thought to limit the development of pathogenic T helper 17 cells while promoting differentiation of protective regulatory T cells. OSMR signaling is also neuroprotective, however the effects on immune cells are not yet elucidated.

In this study, the immunomodulatory effects of LIFR and OSMR signaling in humans are investigated. We determined which immune cells express the receptors for LIF and OSM in healthy donors and compared this to the expression levels in MS patients. We found that in blood of healthy donors approximately half of the monocytes express the LIFR and OSMR, while 5-10% of the T cells and B cells express the receptors. More importantly, in untreated MS patients higher numbers of T cells and B cells express both the LIFR and OSMR as compared to healthy controls and treated MS patients. Available treatments suppress the immune system, resulting in less activated T and B cells, which could explain the lower receptor expression. Indeed, we measured the receptor expression on T cells and B cells after activation and demonstrated this strongly induces expression of both receptors.

Currently, we are investigating the effect of LIF and OSM on proliferation, cytokine production and antigen presentation of the different immune cell subsets. As blood circulating immune subsets of MS patients have a higher expression of LIF and OSM receptors, patients show increased susceptibility for modulation by these cytokines. Thus, their immunoregulating properties together with the protection of glial cells and neurons indicates that these cytokines are promising candidates for the treatment of MS and other neuroinflammatory diseases.


Correlation of binding of  [3H]PK11195 to lesion type and presence of activated microglia in human multiple sclerosis tissues

*Saga Johansson 1 , Naghmeh Fouladi 1 , Julian Goggi 1 , Paul Jones 1 , Will Trigg 1
1 GE Healthcare, Medical Diagnostics, Amersham, United Kingdom
Abstract text :


Multiple sclerosis (MS) is a progressive neuroinflammatory disorder. During the time course of disease, there is a widespread microglial activation associated with extensive and chronic neurodegeneration and progressive loss of motoric and cognitive functions. Translocator protein 18kDa (TSPO, also known as peripheral benzodiazepine receptor) is highly expressed in vivo by activated microglia, and is an attractive target for diagnostic imaging of microglia in MS.

Results and conclusions

The aim of the present study was to evaluate the specific binding of [3H]PK11195, a compound targeting TSPO, to human tissue sections from patients with MS, and to correlate these findings with presence of TSPO microglia in different types of lesions. Specific binding of [3H]PK11195 was evaluated using autoradiography, and histological analysis was performed on tissue sections to evaluate lesion type, TSPO expression and presence of microglia. There was a clear correlation between level of specific binding of [3H]PK11195 and lesion type, such that there was an increased binding to tissue with active or chronic-active lesions compared to control tissue or tissue with chronic lesions. This was concomitant with an increased TSPO expression and increased presence of microglia in tissue sections with active or chronic-active lesions. In conclusion, specific binding of [3H]PK11195 is correlated with lesion type, TSPO expression and presence of microglia in tissue sections from MS patients.


Mesenchymal stem cells require to be in close vicinity with microglia to arrest cell cycle

*Shinsmon Jose 1 , Sharmili Vidyadaran 1
1 Universiti Putra Malaysia, Department of Pathology, Selangor, Malaysia
Abstract text :


We are exploring use of mesenchymal stem cells (MSC) as a tool to modulate microglia inflammatory responses and have previously demonstrated that MSC reduce BV2 microglia proliferation and increase nitric oxide (NO) secretion following lipopolysaccharide (LPS) stimulation. Here we describe that MSC modulate proliferation of BV2 microglia through a mechanism independent of NO. MSC reduce proliferation of stimulated BV2 cells by 30% (p<.05) and the reduction was unaffected even when NO were reduced to levels comparable to that of unstimulated BV2 cells (6.07 ± 0.6µM) using the iNOS inhibitor L-NAME. Viability of BV2 microglia determined by Annexin V/PI staining remained unaffected by MSC (≥ 94.02 ± 1.72%), ruling out apoptosis as a mode for inhibiting microglia proliferation. To then determine whether MSC arrests BV2 microglia cell cycle at any particular phase, propidium iodide (PI) staining revealed that the percentage of BV2 microglia in the S-phase upon LPS stimulation (54.77 ± 1.5) is reduced to 48.54 ± 1.0% following direct coculture with MSC. This modulation of microglia cell cycle by MSC is dependent on cell-cell contact. It is therefore probable that MSC and microglia require being in close vicinity to exert an anti-proliferative effect. In an effort to examine this, migration of both cells across a semipermeable membrane in the presence of 1µg/mL LPS was analysed. Although MSC migrate marginally to an LPS stimuli (67.0 ± 15.1), they are chemoattracted towards microglia (213.3 ± 19.8 cells). This demonstrates that MSC respond to chemotactic cues by microglia, more so if the microglia are activated (272.3 ± 18.8; p<.05). Migration of cells within tissue requires digestion of extracellular matrix by proteases, and accordingly, expression of MMP2 and MMP9 increased in LPS-stimulated cocultures. This increased expression was accompanied with increased activity, as assessed by gelatin zymography. This work suggests that MSC and microglia migrate towards each other which may enable MSC to modulate microglia proliferation in a contact-dependent manner through cell cycle modulation. 


Differential roles for the small heat shock protein Alpha B-Crystallin in de- & remyelination

*Hedwich Kuipers 1 , Jane Yoon 1 , Johannes Winderl 1 , Jack van Horssen 2 , Theo Palmer 3 , Lawrence Steinman 1
1 Stanford University, Neurology and Neurological Science, Stanford, CA, United States
2 VU University Medical Center, Molecular Cell Biology and Immunology, Amsterdam, United States
3 Stanford University, Neurosurgery, Stanford, CA, United States
Abstract text :

The small heat shock protein alpha B-crystallin (cryab) is expressed by oligodendrocytes (OL) and astrocytes at several stages of multiple sclerosis (MS) lesions. Cryab has been shown to be protective and therapeutic in the EAE model of MS, possibly due to its anti-apoptotic functions and regulation of inflammatory pathways. In this study, we further investigated the role of cryab in de- and remyelination in vivo, applying the cuprizone model of demyelination to cryab-/- mice.

Surprisingly, we found that cuprizone-induced lesions are significantly smaller, less severe and less inflammatory in cryab-/- mice, compared to wild type (wt) controls. Staining for microglia and astrocytes revealed that astrocytes are the main inflammatory cell type that is affected by cryab expression: lesions in cryab-/- mice display less reactive astrogliosis than wt controls. Conversely, although less severe, lesions in cryab-/- mice also contain fewer oligodendrocyte progenitor cells (OPC) than in wt mice. In addition, our data suggest that remyelination is less efficient in cryab-/- mice than in wt controls and that remyelinated areas in cryab-/- mice contain fewer OL than remyelinated areas in wt mice.

Together, we hypothesize that cryab, due to its anti-apoptotic actions, mediates protection in both astrocytes and OL, enabling on one hand reactive astrogliosis, which contributes to demyelination, and on the other hand OL survival, facilitating remyelination. Additional in vitro experiments support this hypothesis, revealing that cryab-/- astrocytes are less reactive than wt astrocytes and that siRNA-mediated knockdown of cryab affects OL survival. Interestingly, analyzing phosphorylation patterns of cryab in cuprizone lesions revealed that cryab is differentially phosphorylated in astrocytes and OL. Furthermore, we found that cryab is differentially phosphorylated in astrocytes in active demyelinating MS lesions, indicating that phosphorylation of the protein might underlie its (pathogenic) role in active demyelination.

These paradoxical findings not only suggest a role for cryab as a potential target in a regenerative approach for MS treatment, but also point to a possible pivotal role for reactive astrogliosis in cuprizone-induced demyelination and, more importantly, in early MS pathology.


MC4R activation induces BDNF expression through ERK and PI3K in rat astrocytes.

*Mercedes Lasaga 1 , Lila Carniglia 1 , Daniela Durand 1 , Carla Caruso 1
1 School of Medicine - Univesrity of Buenos Aires, , Buenos Aires, Argentina
Abstract text :

Melanocortin 4 receptor (MC4R) is predominantly expressed in the brain and it is the only MCR expressed in astrocytes. Our previous results showed that a-melanocyte-stimulating hormone (a-MSH) the anti-inflammatory action is mediated by MC4R in astrocytes and in the hypothalamus of male rats and that these effects may lead to neuroprotection. We have already demonstrated that NDP-MSH (an a-MSH analogue) increased brain-derived neurotrophic factor (BDNF) expression through the cAMP-PKA-CREB pathway in astrocytes. In the present study we examined the participation of mitogen activated protein kinases (MAPK) and phosphatidylinosotol-3 kinase (PI3K)-Akt pathways in MC4R signaling in astrocytes. We also investigated the effect of a-MSH on BDNF expression in vivo in brains of male rats.We preincubated cultured rat primary astrocytes with MAPK (p38, JNK and ERK) and PI3K-PDK1-Akt inhibitors 15 min before addition of 1 mM NDP-MSH for 1 h. We found that NDP-MSH-stimulating effect on BDNF expression assayed by qRT-PCR was abolished only in the presence of ERK and PI3K inhibitors. Accordingly, levels of phospho-ERK1/2 determined by western blot were increased by NDP-MSH whereas phospho-Akt levels were not modified at 30 min. NDP-MSH-induced ERK1/2 activation was decreased by adenylate cyclase and PI3K inhibitors but not by a PKA inhibitor, suggesting a cAMP and PI3K involvement in this effect. We also investigated if a-MSH was able to induced BDNF expression in vivo. For that purpose we injected male rats with a-MSH (0.5 mg/kg, ip) or vehicle (saline) once and sacrificed them after 3 h. Rats were also injected twice daily and for two days and killed 48 h after the first injection. We observed that BDNF mRNA levels increased after a-MSH treatment at 3h in the hypothalamus whereas in the cortex BDNF expression was not modified. At 48 h BDNF expression was not modified by a-MSH. We showed by immunohistochemistry that MC4R and BDNF co-localizes with neurons and astrocytes in the brain. Since melanocortins have anti-inflammatory and neuroprotective effects in the brain, the mechanisms described in astroglia help understand MC4R action. Our results indicate that melanocortins induce BDNF expression in astrocytes through ERK and PI3K, suggesting that this effect could be involved in the neuroprotective actions of melanocortins. The fact that BDNF expression also increases in vivo in the hypothalamus reinforce this idea.



Microglia activation in the leech Hirudo medicinalis: HmC1q promotes the microglial accumulation through the distinct recognition of gC1qR and cC1qR receptors.

*Christophe Lefebvre 1 , Annelise Bocquet-Garcon 1 , Jacopo Vizioli 1 , Christelle Van Camp 1 , Pierre-Eric Sautière 1 , Francesco Drago 1 , Michel Salzet 1 , Françoise Croq 1
1 University Lille 1, , Villeneuve d'Ascq, France
Abstract text :

Question: Unlike vertebrates, the medicinal leech, Hirudo medicinalis, is able to functionally repair its central nervous system after an injury. Of interest, when the CNS is injured in H. medicinalis, microglial cells migrate to the site of lesion. This accumulation is known to be essential for the usual sprouting of injured axons and leads to a functional nerve repair. Because of the insignificant infiltration of macrophages in the injured leech CNS, we investigate the chemotactic mechanisms of the recruitment of only resident microglial cells in order to explore in fine the crosstalk between damaged neurons and activated microglia leading to the leech CNS repair.

Methods: In vitro and ex vivo chemotactic assays were performed by using recombinant form of HmC1q on microglial cells which were pre-incubated or not with anti-cC1qR or anti-gC1qR antibodies. Then, affinity purification analyses using biotinylated C1q were performed from leech microglial cell protein extracts. Finally, immunohistochemistry analyses were located the production of newly characterized C1q receptors in microglial cells.

Results: We demonstrated that rHmC1q contributes to the recruitment of some leech microglial cells. Chemotaxis assays showed that the blocking of respective receptors, homologous to human gC1qR and cC1qR, inhibits the HmC1q-dependent recruitment. Importantly, affinity purification analyses demonstrated the interaction between both receptors and HmC1q. Finally, those receptors were located on distinct microglial subsets.

Conclusion: This work is used to specify the activation processes of only resident microglial cells. The results show the importance of HmC1q in the microglial accumulation leading to the nerve repair. In H. medicinalis, HmC1q activity is driven through two different receptors which are not present on the same cells suggesting that HmC1q activates two distinct microglial cell subpopulations.


Periplaques form extensive lesions in multiple sclerosis spinal cords

*Alice Lhuillier 1 , Marie Chanal 1 , Géraldine Androdias 1 , Richard Reynolds 2 , Christian Confavreux 1 , Serge Nataf 1
1 Lyon Neurosciences Research Center, , Lyon, France
2 UK MS Tissue Bank, Imperial College of London, , London, United Kingdom
Abstract text :

QUESTION: Our knowledge of multiple sclerosis (MS) neuropathology has benefited from a number of studies that precisely depicted different types of plaques in white or grey matter areas. Also, both inflammation and diffuse axonal loss in the normal appearing white matter (NAWM) were extensively described. However, little attention was given to the so-called periplaque, which is usually defined as a partially demyelinated ribbon of tissue surrounding the plaque. Whether periplaques correspond to expanding lesions, sites of ongoing remyelination or areas of tract degeneration remains uncertain. METHODS: In this context, our study aimed to bring quantitative insights to the neuropathology of MS periplaques in the spinal cord of primary or secondary progressive MS patients. A neuropathological quantitative assessment of inflammation, axonal loss and myelin loss was performed concurrently in the periplaques, plaques and normal-appearing white matter (NAWM) of cervical spinal cord samples from 16 patients with progressive MS. RESULTS: Periplaques formed large areas of incomplete myelin loss that extended distant away from the border of plaques. Axonal loss was quantitatively similar in plaques and periplaques but signs of axonal dystrophy were predominantly observed in plaques. Surprisingly, axons that remained myelinated in periplaques presented a thicker myelin sheath than myelinated axons of the NAWM. Inflammation in the periplaque was mainly characterized by an accumulation of macrophages/microglia that were closely apposed to myelin sheaths but exerted poor phagocytic activity. Finally, we found that neuropathological features of periplaques were overall disconnected from that of plaques with regard to size, shape, inflammation and axonal integrity. CONCLUSIONS: Our work indicates that in MS spinal cords, periplaques correspond to demyelinating rather than remyelinating lesions. It further suggests that in progressive forms of MS, periplaques are likely to impact significantly on neurological disability. Finally, we propose that tract degeneration might not be the only cause of periplaque extension and that slowly-expanding demyelination, temporally remote from plaque formation, might occur in periplaques. Molecular results will be presented that support these hypotheses.


The Hedgehog pathway promotes optimal Blood Brain Barrier functioning

*Jorge Ivan Alvarez 1 , Aurore Dodelet-Devillers 1 , Hania Kebir 1 , Pierre Fabre 2 , Igal Ifergan 1 , Simone Terouz 1 , Karolina Wosik 1 , Mike Sabbagh 1 , Monique Bernard 1 , Elga de Vries 3 , Frederic Charron 2 , Alexandre Prat 1
1 Université de Montréal, Faculty of Medicine, CHUM Research Center, Neuroimmunology Unit, Montréal, Canada
2 Institut de Recherches Cliniques de Montréal (IRCM), Laboratory of Molecular Biology of Neuronal Development, Montréal, Canada
3 VU Medical Center, Department of Molecular Cell Biology and Immunology, Amsterdam, Netherlands
Abstract text :

Blood brain barrier-endothelial cells (BBB-ECs) are surrounded by astrocyte endfeet known to regulate BBB permeability. Recent studies indicate that components of the Hedgehog (Hh) pathway play an important role in vascular proliferation, differentiation and tissue repair in adult tissues. As BBB disruption is early observed in Multiple Sclerosis (MS), this study aimed to determine the role of astrocyte-secreted Sonic Hh (Shh) in the maintenance of BBB functions. We found that human astrocytes express and secrete Shh and conversely, that human BBB-ECs bear the Hh receptor Patched-1, the signal transducer Smoothened (Smo), as well as transcription factors of the Gli family. Transendothelial resistance and permeability experiments using primary cultures of human BBB-ECs showed that activation of the Hh pathway stimulated expression of junctional proteins and promoted a BBB phenotype. In vivo permeability assays showed that blocking the Hh pathway increased barrier permeability and lack of Shh significantly compromised the expression of junctional proteins and the BBB phenotype. To distinctively demonstrate the role of the Hh pathway in inducing barrier properties at the CNS endothelium, we generated a conditional knockout specifically deleting the signal transducer Smo from ECs (Tie2-Cre; Smoc/c). Analysis of these mice at E14, E18, P5, P19 and P55 showed a significant increase in BBB permeability to endogenous and exogenous tracers and these alterations correlated with a decrease in the expression of junctional proteins, altered expression of basement membrane proteins and reduced association of astrocyte endfeet with the vasculature. Our findings show that while activation of the Hh pathway restricts BBB permeability, Hh neutralization impacts on BBB formation and stability during fetal development and adulthood.


Understanding and manipulating the astrocyte response to spinal cord injury using diblock copolypeptide hydrogels for depot delivery of bioactive molecules

*Mark Anderson 1 , Shanshan  Zhang 2 , Yan Ao 1 , Timothy J. Deming 2,3 , Michael V. Sofroniew 1
1 University of California, Los Angeles (UCLA), Neurobiology, Los Angeles, United States
2 University of California, Los Angeles (UCLA), Chemistry, Los Angeles, United States
3 University of California, Los Angeles (UCLA), Bioengineering, Los Angeles, United States
Abstract text :

One goal for improving outcome after spinal cord injury (SCI) is to promote regrowth of injured nerve fibers across areas of tissue damage surrounded by astroglial scars that inhibit such growth. Little is known about how astrocytes assemble to form this scar, and a deeper understanding of the cellular and molecular mechanisms that control this process is needed if this goal is to be achieved. In vitro data suggest that certain bioactive molecules can alter astrocyte morphology into a more growth supporting state. Other molecules have been shown to promote axon growth. Clinical application of many of these molecules would require neurosurgical delivery directly into the spinal cord. Injectable biomaterials have the potential to serve as depots and scaffolds for in vivo delivery of bioactive molecules. We are developing di-block co-polypeptide hydrogels (DCH) as fully synthetic biomaterials that could safely and easily be injected into specific sites after SCI to deliver molecules in order (i) to understand better, and (ii) to manipulate, the mechanisms that control glial scar formation. We have previously shown that DCH are biocompatible after injection into brain or spinal cord and self-assemble into structures with finely controllable properties. Our current work shows that DCH depots can deliver diffusible bioactive growth factors that influence local neurons or astrocytes in predictable ways and form gradients that are effective up to several mm away from depots. We are now testing the ability of DCH depots to deliver specific growth factors that will manipulate scar-forming cells when DCH are injected at clinically realistic sub-acute times after SCI. Supported by Wings for Life, CA
State Roman Reed Fund, and Adelson Medical Research Foundation.


Grey Matter Astrocytes Activated by Remote Axonal Transection Mediate Structural Plasticity

*Daniel Barson 1 , Giulia Tyzack 1 , Sergey Sitnikov 1 , Chao Zhao 1 , Robin Franklin 1 , Ragnhildur Thora Karadottir 1 , Andras Lakatos 1
1 University of Cambridge, , Cambridge, United Kingdom
Abstract text :

The role of increased activity of astrocyte networks in structural synaptic plasticity following remote axonal injury is unexplored. We set out to investigate the efficiency of synaptic rearrangements in models where astrocyte reactivity is selectively diminished by the inhibition of the main cytokine pathway in transgenic mice (TG) in comparison to that observed in wild type mice (WT). First, synapse densities were compared in the facial nuclei two weeks after unilateral facial nerve transection by synaptotagmin-1/PSD-95 immunolabelling and ultrastructural analysis. We found that the recovery of synapse density was decreased by 40% in TG mice compared to WT mice, and that the reduction in synaptic density correlates with a relative loss of neuronal coverage by reactive astrocyte end-feet in the same conditions. Specifically, we observed a 30% reduction in excitatory synapse density in the TG mice. Second, we developed an organotypic entorinho-hippocampal slice culture system in which astrocyte activation and synaptic plasticity could be more closely monitored in real time. This was assisted by two-photon microscopy, using glial Ca2 -imaging and dendritic spine motility analysis after perforant pathway transection. In summary, we demonstrated that full astrocyte activation is necessary for partial structural recovery of synapses after axonal injury. Our work, using simplified models, may help experimental approaches aimed at optimizing adaptive plasticity in CNS injuries.


Propentofylline improves oligodendrocyte remyelination following gliotoxic injury in the rat brainstem

*Eduardo Bondan 1 , Maria de Fátima Monteiro Martins 1,2 , Deborah Eillen Menezes Baliellas 2 , Caio Fernando Monteiro Menezes 2 , Sandra Castro Poppe 2
1 Universidade Paulista, , São Paulo, Brazil
2 Universidade Cruzeiro do Sul, , São Paulo, Brazil
Abstract text :

Propentofylline (PROP) is a xanthine derivative with pharmacological effects distinct from those of the classical methhylxanthines theophylline and caffeine. It depresses activation of microglial cells and astrocytes which is associated with neuronal damage during inflammation and hipoxia. It is largely known that ethidium bromide (EB) injection into the CNS induces local oligodendroglial and astrocytic death, resulting in primary demyelination, blood-brain barrier disruption and Schwann cell invasion, and thus serving as an experimental demyelinating model in animals. The aim of this study was to evaluate if PROP had the capacity of affecting glial cell behaviour during the process of demyelination and remyelination following gliotoxic injury with EB. Wistar rats were divided into 2 groups: I- rats injected with 10 microlitres of 0.1% EB into the cisterna pontina and treated with PROP; II- rats injected with EB and not treated with the xanthine. PROP (Agener) treatment was done using 12.5 mg/kg/day by intraperitonial route during all experimental period. The rats were euthanized from 7 to 31 days after EB injection and brainstem sections were collected and processed for light and transmission electron microscopy studies. Results from both groups were compared by using a semi-quantitative method developed for documenting in semithin sections the extent and nature of remyelination in gliotoxic lesions. In general terms, by 7-11 days following gliotoxic lesion, the center of the lesion was expanded and filled with huge amounts of myelin debris among foamy macrophages and demyelinated axons. No astrocytic processes were found in this site and some lymphocytes were seen in the neuropil and around blood vessels. The most prominent feature of the 15th day was the initial association at peripheral locations between naked axons and remyelinating cells. Schwann cells were associated with one or multiple demyelinated axons or already forming thin myelin lamellae around single axons in astrocytic-free areas. Oligodendrocytes began to form thin myelin sheaths in areas completely or partially filled with astrocytic prolongations. By 21-31 days, it became evident that rats treated with PROP presented a small improve in the repair process when compared to untreated animals, the latter presenting greater amounts of myelin-derived membranes in the central area and a lesser extension of oligodendrocyte remyelination, but without any apparent difference regarding to Schwann cells. By 31 days results showed that PROP administration following EB injection seemed to stimulate oligodendroglial remyelination (mean remyelination scores of 3.61±0.21 for oligodendrocytes and 1.20±0.41 for Schwann cells) in comparison to untreated animals (scores of 2.98±0.44 and 0.83±0.31, respectively).


S1PR1-modulation in the convalescence period improves functional recovery and reduces reactive astrogliosis in experimental stroke

*Robert Brunkhorst 1 , Nathalie Kanaan 1 , Alexander  Koch 2 , Helmuth Steinmetz 1 , Josef  Pfeilschifter 2 , Waltraud Pfeilschifter 1,2
1 Goethe University Hospital Frankfurt, Department of Neurology, Frankfurt, Germany
2 Goethe University Hospital Frankfurt, Department of General Pharmacology and Toxicology, Frankfurt, Germany
Abstract text :

The sphingosine 1-phosphate (S1P) signaling pathway is known to influence astroglial reactivity in experimental autoimmune encephalomyelitis and the synthetic S1P analog FTY720 has been shown to provide neuroprotection in experimental models of acute stroke. However, the effects of a manipulation of S1P-S1P1 signalling at later time points after experimental stroke have not yet been investigated. We examined whether a relatively late initiation of a FTY720 treatment has a positive effect on long-term neurological outcome with a focus on reactive astrogliosis, synapses and neurotrophic factors. 

We induced photothrombotic stroke (PT) in adult C57BL/6J mice and allowed them to recover for three days. Starting on post-stroke day 3, mice were treated with FTY720 (1 mg/kg b.i.d.) for 5 days. Behavioral outcome was observed until day 31 after photothrombosis and periinfarct cortical tissue was analyzed using tandem mass-spectrometry, TaqMan®analysis and immunofluorescence with especial emphasis on markers of astroglial reactivity. 

FTY720 treatment results in a significantly better functional outcome persisting up to day 31 after PT. This is accompanied by a significant decrease in GFAP-immunoreactivity (-ir) and an increase in PSD-size. However no change in GFAP-mRNA, GS-ir or CS56-ir was observed.  While FTY720-treatment leads to a decrease in S1P concentration, it increases VEGF-mRNA in the periinfarct cortex.

The initiation of FTY720 treatment in the convalescence period has a positive impact on long-term functional outcome, probably mediated through reduced astrogliosis, a modulation in synaptic morphology and an increased expression of neurotrophic factors.



Does the developmental heterogeneity of oligodendrocyte origin influence remyelination of the adult central nervous system?

*Abbe Crawford 1 , R. Tripathi 2 , W. D. Richardson 2 , R. J. M. Franklin 1
1 University of Cambridge, Department of Veterinary Medicine, Neurosciences, Cambridge, United Kingdom
2 University College London, Wolfson Institute for Biomedical Research, London, United Kingdom
Abstract text :

The oligodendrocyte precursor cell (OPC) is the major cell type responsible for remyelination, a regenerative process vital to the repair of areas of demyelination in the central nervous system (CNS).  OPCs originate from two discrete locations, ventral and dorsal, within the developing nervous system, raising the question of whether these may represent two functionally distinct OPC populations. The purpose of this project is to study and compare the roles of the two populations in remyelination.  Using a transgenic mouse line in which ventral OPCs express green fluorescent protein, and dorsal OPCs express TdTomato, lysolecithin injection into the spinal cord was performed to create focal white matter demyelinating lesions.  The remyelination process was then monitored to 21 days post lysolecithin injection.  The results obtained so far have shown that both OPC populations take part in remyelination, but that the dorsal population is the most active, showing a greater proliferative response and a significant increase in cell number within the lesioned tissue.  Identifying the factors that enable the dorsal population to become the major cellular player in remyelination may reveal new targets for its therapeutic enhancement.


Three Ca2+ channel inhibitors in combination reduce chronic secondary degeneration following neurotrauma

Donna Savigni 1 , Ryan O'Hare Doig 1 , Charis Szymanski 1 , Carole Bartlett 1 , Ivan Lozic 1 , Nicole Smith 1 , *Melinda Fitzgerald 1
1 The University of Western Australia, , Crawley, Australia
Abstract text :

Following neurotrauma, cells beyond the initial trauma site undergo secondary degeneration, with excess Ca2 a likely trigger for loss of neurons, compact myelin and function. Treatment of secondary degeneration by limiting excess Ca2 entry into cells using inhibitors of specific Ca2 channels showed promise in preclinical studies, but clinical trials were disappointing and combinatorial approaches are acknowledged as necessary. We assessed efficacy of every possible combination of three Ca2 channel inhibitors at reducing secondary degeneration 3 months after partial optic nerve (ON) transection in rat. We used lomerizine to inhibit voltage gated Ca2 channels (for 3 months); oxidised adenosine-triphosphate (oxATP) to inhibit purinergic P2X7 receptors and/ or 2-[7-(1H-imidazol-1-yl)-6-nitro-2,3-dioxo-1,2,3,4-tetrahydro quinoxalin-1-yl]acetic acid (INQ) to inhibit Ca2 permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (both for the first 2 weeks after injury). Only the three Ca2 channel inhibitors delivered in combination completely preserved visual function to levels not different from normal animals.

 In order to determine the mechanism/s by which the three inhibitors delivered in combination preserved function, we assessed neuroprotection, nerve swelling, myelin compaction and node/paranode complexes. Preservation of retinal ganglion cell (RGC) somata and axons is unlikely to have accounted for the full recovery of function as only partial neuroprotection of RGCs vulnerable to secondary degeneration was achieved. A range of the Ca2 channel inhibitor combinations prevented swelling of optic nerve vulnerable to secondary degeneration, with no one agent selectively effective. Each of the treatments involving lomerizine significantly increased the proportion of axons with normal compact myelin and decreased the proportion of axons with partially decompacted myelin, indicating the importance of sustained control of Ca2 flux via voltage gated Ca2 channels for prevention of chronic myelin decompaction. Nevertheless, limiting decompaction of myelin or formation of atypical node/ paranode complexes was not sufficient for preservation of function in our model. It is likely that the additional prevention of the lengthening of the paranodal gap that was only achieved by treatment with the three Ca2 channel inhibitors in combination was an important effect that contributed to the associated preservation of visual function by this combinatorial treatment strategy.


Demyelinated axons regulate their own remyelination via glutamate signalling to oligodendrocyte precursor cells

*Helene Gautier 1 , Kimberley Evans 1 , Iben Lundgaard 1 , Cristina Lao-Peregrín 1 , Robin J M Franklin 1 , Ragnhildur Thora Káradóttir 1
1 Wellcome Trust/MRC Cambridge Stem Cell Institute, , Cambridge, United Kingdom
Abstract text :

During development, some OPCs differentiate into myelinating oligodendrocytes while others remain into adult life, where they comprise approximatley 5% of CNS cells and are the main proliferating cells present.  In demyelinating disease such as multiple sclerosis, adult OPCs differentiate into new oligodendrocytes that remyelinate the axons, although this process eventually fails or is incomplete, leading to sustained clinical deficits.

Glutamate and electrical activity influence OPC differentiation and myelination in normal development. OPCs receive synaptic input from unmyelinated axons, possibly to initiate myelination. Both OPCs and mature oligodendrocytes respond to glutamate via AMPA and NMDA receptors. Activation of glutamate receptors in vitro increase OPC migration but decreases proliferation.

Here we examine the role of glutamate signalling in remyelination following experimental demyelination in the adult rodent  CNS. We voltage-clamped OPCs in brain-slices of adult rat cerebellar peduncle containing focal areas of primary demyelination and post-identified these by NG2-immunolabelling. Recruited OPCs mainly expressed AMPA receptors at the peak of the OPCs proliferation (5-10 days post-lesion), as over 90% of the glutamate evoked current was blocked with NBQX (AMPA/Kainate receptor antagonist) and unaffected by AP5 (NMDA receptor antagonist).  

The demyelinated axons continued to propagate action potentials with latencies similar to those in unmyelinated axons during development.  Critically, the demyelinated axons established synapses with OPCs expressing voltage-gated sodium currents.  These synaptic inputs had identical decay times to those recorded during developmental myelination. Pharmacological inhibition of neuronal activity or glutamate signalling decreased remyelination efficiency by impairing differentiation.

These results indicate that 1) glutamate currents are mainly mediated via AMPA and kainate receptors during the OPC recruitment 2) OPCs establish synaptic contact with demyelinated axons and 3) the neuronal activity is essential for remyelination. Together, these data suggest that demyelinated axons remain active and communicate with OPCs through glutamate release during the regenerative process to guide their remyelination.


Hydrogel as a tool for vascular endothelial growth factor gradual release in peripheral nerve regeneration.

*Sara  Gnavi 1 , Chiara Tonda Turo 2 , Laura di Blasio 3 , Francesca Ruini 2 , Luca  Primo 3 , Stefano Geuna 1,4 , Giovanna Gambarotta 4 , Isabelle Perroteau 4
1 University of Torino, Neuroscience Institute Cavalieri Ottolenghi, Orbassano, Italy
2 Polytechnic University of Torino, Department of Mechanics, Torino, Italy
3 University of Torino, Institute for Cancer Research and Treatment, Candiolo, Italy
4 University of Torino, Department of Clinical and Biological Science, Orbassano, Italy
Abstract text :

The aim of this study was to obtain an hydrogel for the controlled delivery of Vascular Endothelial Growth Factor (VEGF-A165).

Hydrogels are a class of liquid-gel biomaterials with high water content, typically composed of cross-linked, three-dimensional hydrophilic water-soluble polymer networks. The advantages of injectable hydrogel systems are: biocompatibility, biodegradability, adjustable shape, low cost and similarity to the extracellular matrix; moreover, they can be used as growth factor and cell delivery systems for tissue engineering applications.

Agarose/Gelatin (A/GL) hydrogel was cross-linked with genepin. Rheological measurements show that A/GL hydrogel can be injected through a syringe into the inner cavity of a nerve guide. In vitro assays performed to evaluate adhesion, proliferation, viability and migration – using a fibroblast cell line (NIH3T3), Neonatal Olfactory Bulb Ensheathing Cells (NOBEC) and a Schwann cell line (RT4-D6P2T) - show that hydrogel allows cell adhesion, proliferation, viability and migration.

VEGF-A165 is a potent angiogenic factor and angiogenesis has long been recognized as an important and necessary step during tissue repair. VEGF is known to have a positive effect on Schwann cell proliferation and migration, neuron survival and outgrowth of regenerating nerve fibers.

The hydrogel preparation protocol was optimized to be functionally efficient at physiological conditions, allowing the loading of VEGF without the risk of its denaturation.

Different amounts of VEGF (50-100-200 ng/ml) were incorporated in the A/GL hydrogel and the releasing rate in vitro up to 65 days was quantified by ELISA . Released VEGF bioactivity was validated in Human Umbilical Vein Endothelial Cells (HUVEC) and in NOBEC by evaluating  phosphorylation of VEGF-Receptor 2 (VEGFR2), akt and erk1-2. Moreover, dorsal root ganglia explants were cultured on hydrogel containing different amounts of VEGF, and an increased neurite outgrowth was quantitatively assessed.

These results demonstrate that VEGF can be successfully incorporated and bioactive released from A/GL hydrogel inducing VEGFR2 activation and neurite outgrowth.

In vivo test are ongoing on adult rats: one centimetre lesions on medial nerve were performed, followed by implantation of Porous Poly-e-caprolactone tubes filled with A/GL hydrogel containing VEGF-A165. Functional tests and histological analysis will be carried out to evaluate peripheral nerve regeneration.


Human pluripotent stem cell-derived oligodendrocyte precursor cells for spinal cord injury repair

*Anu Hyysalo 1,2 , Meeri Mäkinen 1,2 , Laura Ylä-Outinen 1,2 , Tiina Joki 1,2 , Susanna Narkilahti 1,2
1 University of Tampere, Institute of Biomedical Technology, Tampere, Finland
2 BioMediTech, , Tampere, Finland
Abstract text :

Introduction: Loss of neural connections, degradation of myelin sheath, and death of oligodendrocytes arise as secondary damage in spinal cord injury (Emery et al., 1998). Currently, no effective treatment for spinal cord injury exists. However, functional recovery through remyelination by oligodendrocyte transplantation could provide a possible treatment. For that aim, human oligodendrocytes are needed in large amounts, and they can be produced from human pluripotent stem cells. Studies with animal cells suggest that electrophysiological properties of oligodendrocyte precursor cells (OPCs) can have an effect in the myelination capacity of the cells (Karadottir et al. 2008). Characteristics of developing human oligodendrocytes are not excessively studied but several studies indicate heterogeneity among cells expressing generally accepted OPC marker chondroitin sulphate proteoglycan (NG2) (Polito and Reynolds, 2005). Thus, thorough characterization of OPCs is critical for determination of safety and efficacy of these cells.

Materials and Methods: Previously, our group has developed and published protocols for differentiation of oligodendrocytes and OPCs from human embryonic stem cells (Sundberg et al. 2010, Sundberg et al. 2011). Published protocols have been modified for production of OPCs for research purposes, and produced cells are used for characterization of OPCs. Ultimately the aim is to define an OPC population with optimal myelination capacity.   

Results and Discussion: Currently, human oligodendrocytes and OPCs can be produced from pluripotent stem cells. These cells are routinely characterized in protein expression level, and methods for more comprehensive characterization are under development. Produced OPCs can be purified with fluorescence-activated cell sorting based on NG2 protein expression in order to study if this cell population is heterogeneous in terms of myelination capacity.

Conclusions: Oligodendrocytes and OPCs can be produced from human pluripotent stem cells. Studies are ongoing to determine the molecular characteristics and myelination capacity of the differentiated and purified OPC- population.



Living art – method for labeling living human derived neural cells with fluorescent probes

*Tiina Joki 1 , Meeri Mäkinen 1 , Laura Ylä-Outinen 1 , Heli Skottman 2 , Riikka Äänismaa 1 , Susanna Narkilahti 1
1 University of Tampere, Institute of Biomedical Technology, Tampere, Finland
2 University of Tampere, Eye group, Tampere, Finland
Abstract text :

Background: Fluorescent probes as such are not new but their use as tracking living cell populations in vitro is a tool that offers novel possibilities. Fluorescent probes attaching to living cells can be used e.g. in long term follow up studies with possibility to identify the labeled cell population or to enhance cell visibility in opaque cultures. These features are useful in many research areas in the field of applied sciences e.g. development of controlled neurotoxicity screening platforms, in vitro neural disease modeling, and biomaterial research for craft development.

The aim of this study was to create a non-invasive tool for cell visualization and cell population tracking that can be used in long term studies.

Methods: This study was performed using human pluripotent stem cell lines derived in Institute of Biomedical Technology (former Regea), University of Tampere, Finland. Cells were differentiated to neural cells using neural differentiation protocol described earlier by Lappalainen and colleagues. Fluorescent probes used in this study were 1) 5-chloromethylfluorescein diacetate (CellTracker Green CMFDA, CT) and 2) 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD). Cell viability and proliferation of the labeled populations was briefly studied. Also, the labeled cells were characterized using immunocytochemistry and neuronal functionality was studied using micro electrode array (MEA).

Results: During labeling optimization a wide range of different labeling parameters was tested for both probes to obtain long term labeling. Most suitable parameters for human neural cells were 10 µM CT with 72 hours incubation time and 10 µM DiD with 2 hours incubation time. With these concentrations the labeling was visible up to 4 weeks and had no statistical effect on cell viability. CT labeling was not affecting to cell proliferation but with DiD slight decrease in proliferation was seen in long term culture. Labeled populations contained both MAP-2 positive neurons and GFAP-positive astrocytes. Also, the CT or DiD labeled population and their mixed-cultures expressed normal spontaneous network activity when cultured on MEA-platform.

Conclusions: This study indicated that fluorescent probes CT and DiD are suitable for long-term labeling of human derived neural cultures in vitro. The labeling had no negative effects on cell behavior with the exception of DiD labeled cells having slight decrease on cell proliferation. Importantly, the labeled neural networks were electrically functional.


Perineurial Glia: First Repsonders After a Peripheral Nerve Injury

*Sarah Kucenas 1 , Wendy Lewis 1
1 University of Virginia, , Charlottesville, United States
Abstract text :

Although often described as a hard-wired component of the vertebrate body, the nervous system is a plastic and considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity of its core components. Unlike the CNS, the PNS is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that underlie this phenomenon. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. They are ensheathed by myelinating or non-myelintaing Schwann cell glia, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. This structure forms from centrally-derived glial cells and serves as a protective barrier that is essential for nerve function. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris, regenerate and re-innervate targets. Schwann cells have been shown to play an important role in this process by trans-differentiating, proliferating, clearing debris, and guiding re-growing axons, but less is known about the potential role of perineurial glia. To investigate the role of perineurial cells in PNS regeneration, we have developed an injury response assay that uses a Micropoint laser to create injuries along the motor nerves in live transgenic zebrafish. Time-lapse imaging of injured nerves reveals that perineurial glia rapidly respond to nerve injury and extend processes toward the injury zone. This is in contrast to Schwann cells, which we observe orienting towards the distal stump where they engulf and clear axonal debris. These data demonstrate that perineurial glia respond immediately to motor nerve injuries in a manner distinct from Schwann cells, and future work is aimed at defining the molecular mechanisms that mediate the cellular responses of perineurial glia and Schwann cells, as well as determining if developmental paradigms are recapitulated in these glial populations during the regenerative process.


Canonical Wnt signaling and formation of the glial scar

*Joel Levine 1 , Justin P. Rodriguez 1
1 Stonybrook University, Dept. of Neurobiology and Behavior, Stony Brook, United States
Abstract text :

When the brain or spinal cord is injured, glial cells in the damaged area undergo complex changes resulting in the formation of the glial scar. Whether the scar is beneficial and detrimental to recovery remains controversial. The signals that initiate the formation of the glial scar are unknown. Because both canonical and non-canonical Wnts are increased after spinal cord injury (SCI), we examined the role of canonical Wnt signaling in the glial reactions to CNS injury. To disrupt -catenin-dependent Wnt signaling specifically in OPCs, we created transgenic mice carrying an OPC-specific conditionally deleted β-catenin gene. After moderate contusion injuries to the thoracic spinal cord of control mice, OPCs proliferate and accumulate in the penumbra region surrounding the injury epicenter. In the -catenin-depleted mice, there is reduced proliferation and accumulation of OPCs after SCI, reduced accumulation of activated microglia/macrophages and reduced astrocyte hypertrophy. Using a crushed optic nerve model, we show that these reduced glial reactions create an environment that is permissive for axonal regeneration. These results suggest that canonical Wnt signaling in glia after CNS injury is necessary for the formation of the glial scar and they identify Wnt signaling as a new therapeutic target for promoting axon regeneration.


In search of the satellite glia role: insights from their membrane properties

*Sylvia Agathou 1 , Iben Lundgaard 2 , Kim Evans 2 , Katrin Volbracht 2 , Ragnhildur Thora  Káradóttir 2
1 University of Cambridge, , Cambridge, United Kingdom
2 University of Cambridge, Wellcome Trust/MRC Cambridge Stem Cell Institute, John van Geest Centre for Brain Repair & Dept. of Veterinary Medicine, , Cambridge, United Kingdom
Abstract text :

The crucial role of central nervous system (CNS) glial cells in the integrity and physiology of neuronal networks, is well known. However, little is known about glial cells in the peripheral nervous system (PNS). One of the main glial cell types in the PNS is the perineuronal satellite glial cells (SGCs), that surround DRG neurons in an envelope-like fashion. Despite their abundance in peripheral nerves, having stem cells properties and playing a role in neuropathic pain, there is  limited information on their  physiology.  Although SGCs have  similarities to astrocytes in terms of purinergic- and gap junction- mediated signaling, their membrane properties and ionotrophic glutamate receptor expression are more or less unknown.

Our aim was therefore to characterize the membrane properties and glutamatergic ion channel expression of SGCs. We performed patch-clamp experiments on SGCs wrapped around sensory neurons in the rat dorsal root ganglion (DRG) in vitro. Whole-cell recordings revealed that SGCs are slightly hyperpolarized in comparison to the neurons they ensheath, with a resting membrane potential of approximately ~80mV and a high input resistance (&gt;1GΩ). Moreover, upon membrane depolarization no detectable voltage-gated Na , Ca2 or K currents  were detected. Extracellular application of Glutamate agonist,  Kainic acid, N-Methyl-D-aspartic acid (NMDA) and 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) during the recording, did not evoke any response from the cells, indicating their lack of ionotropic glutamatergic receptor (iGluR) expression. Double immunocytochemistry on lucifer yellow (LY) filled SCGs revealed that they express the SCIP/Oct6 transcription factor, which is also expressed by promyelinating Schwann cells.

DRG ganglion SGCs differ from CNS perineuronal cells as they lack glutamatergic receptors, but are similar to astrocytes as they have no voltage-gated ion channels, are gap-junctionally coupled and express glutamine synthetase. Thus, we are currently addressing if SGCs respond to neuronal activity in a similar manner to astrocytes in the CNS with the use of calcium imaging. These studies will provide further insights into the physiological role of SGCs in the PNS.


Inhibition of P2X4 function by P2Y6 UDP receptors in microglia

*Louis-Philippe Bernier 1,2 , Ariel Ase 2 , Eric Boue-Grabot 3 , Philippe Seguela 2
1 University of British Columbia, , Vancouver, Canada
2 McGill University, , Montreal, Canada
3 Université de Bordeaux, , Bordeaux, France
Abstract text :

ATP-gated P2X4 receptor channels expressed in spinal microglia actively participate in central sensitization, making their functional regulation a key process in chronic pain pathologies. P2Y6 metabotropic Gq-coupled receptors, also expressed in microglia, are involved in the initial response to nerve injury, triggering phagocytosis upon activation by UDP. It has been reported recently that expression of both P2X4 and P2Y6 is upregulated in activated microglia following nerve injury. We show here, in resting as well as LPS-activated primary microglia, that P2Y6 decreases P2X4-mediated calcium entry and inhibits the dilation of P2X4 channels into a large-conductance pore measured with a YO-PRO-1 uptake assay. Furthermore, P2Y6 activation modulates the ATP-dependent migration of microglia, a process likely involved in their shift from migratory to phagocytic phenotype. Reconstituting the P2X4-P2Y6 interaction in recombinant systems shows that P2Y6 activation decreases P2X4 current amplitude, activation and desensitization rates, and reduces P2X4 channel permeability to the large cation NMDG . Phospholipase C-mediated hydrolysis of the phosphoinositide PI(4,5)P2, a necessary cofactor for P2X4 channel function, underlies this inhibitory crosstalk. As extracellular levels of both ATP and UDP are increased in the spinal cord following nerve injury, the control of P2X4 activity by P2Y6 might play a critical role in regulating neuropathic pain-inducing microglial responses.


Astrocytes and S1P Receptors.

*Kumlesh Dev 1
1 Trinity College Dublin, , Dublin, Ireland
Abstract text :

The family of sphingosine 1‐phosphate receptors (S1PRs) are G protein‐coupled comprising five subtypes (S1P1R–S1P5R). These receptors are expressed in cells of the immune, cardiovascular, and central nervous systems (CNS), in addition to others. S1PRs play important roles in celular proliferation, differentiation, survival and migration. Recently, the immunomodulatory drug, Gilenya® has been approved as the first oral therapy for Multiple Sclerosis (MS), after proving efficacious in clinical trials. The active ingredient of Gilenya® is the phosphorylated compound FTY720 (pFTY720), which is a potent agonist on all S1PRs, except S1P2Rs. pFTY720 has been suggested to work as a ‘functional antagonist’ causing S1P1R internalisation in lymphocytes, thus limiting T cell auto‐immunity. In addition to regulating the immune system, the lipophilic nature of the pro‐drug FTY720 allows it to readily cross the blood‐brain‐barrier (BBB) where it may also activate S1PRs expressed on both neurons and glia. Here, the role of S1PRs in the CNS was investigated, by specifically investigating their role in astrocyte function. A range of methods were used to examine the effects of S1PR activation and their roles in astrocytes, including: (i) S1P1R subtype trafficking, (ii) transient and continued intracelular signalling, (iii) astrocyte cell migration, (iv) release of pro-inflammatory cytokines, (v) oligodendrocyte cell differentiation and survival and (vi) myelination state in brain slice cultures. The data showed that activation of the S1P1R subtype leads to (i) its internalisation and (ii) continued signalling in astrocytes, and that S1P1Rs were found to (iii) promote astrocyte migration, (iv) limit release of pro-inflammatory cytokines, (v) promote oligodendrocyte survival and (vi) limit demyelination. These studies demonstrate a role for S1P1Rs in regulating astrocyte function and suggest their use a drug targets for neuroinflammtory and neurodegenerative disorders.


Purinergic receptors in adipose-derived stem cells differentiated to Schwann cells

*Alessandro Faroni 1 , Ambra Grolla 2 , Giorgio Terenghi 1 , Valerio Magnaghi 3 , Alexei Verkhratsky 2
1 The University of Manchester, Faculty of Medical and Human Sciences, Manchester, United Kingdom
2 The University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom
3 The University of Milan, Department of Pharmacological and Biomolecular Sciences, Milan, United Kingdom
Abstract text :

Schwann cells (SC) play important roles in the development and regeneration of the peripheral nervous system (PNS) following injury. Several molecules such as neurosteroids and neurotransmitters have been suggested as potential pharmacological targets in regulating SC physiology and regenerative potential. Nevertheless, the slow growth rate and difficulties in harvesting limit SC applications in regenerative medicine.

Adipose-derived stem cells (ASC) can be differentiated into a SC-like phenotype (dASC) sharing morphological and functional properties with SC, thus representing a valid SC alternative. We have previously shown that dASC express γ-aminobutyric-acid (GABA) receptors (i.e. GABA-A and GABA-B receptors), which modulate their proliferation and neurotrophic potential, although little is known about the role of other neurotransmitter systems in ASC.

In this study we investigated the expression of purinergic receptors in dASC. Using RT-PCR, Western blot analyses and immunohistochemistry we have demonstrated that ASC express P2X3, P2X4 and P2X7 purinoceptors. Interestingly, differentiation of ASC towards glial phenotype was accompanied by up-regulation of P2X4 and P2X7 receptors. Using Ca2 -imaging techniques, we have shown that stimulation of purinoceptors with adenosine-5'-triphosphate (ATP) results in intracellular Ca2 signals, indication functional activity of these receptors.. Moreover, we have shown that the increase of intracellular Ca2 leads to SC death, an effect that can be prevented using specific P2X4 or P2X7 antagonists.

Altogether, these results show, for the first time, the presence of functional purinergic receptors in SC-like derived from ASC and their link with critical physiological processes such as cellular death and survival. The presence of these novel pharmacological targets in dASC might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches using dASC for peripheral nerve repair.


K+ effects on oligodendrocytes in ischaemia

*Nicola Hamilton 1 , Karolina Kolodziejczyk 1 , David Attwell 1
1 UCL, , London, United Kingdom
Abstract text :

The myelin sheaths wrapped around axons by oligodendrocytes are crucial for brain function. In ischaemia the myelin is damaged, partly as a result of glutamate release. Glutamate is thought to activate oligodendrocyte AMPA/kainate and NMDA receptors, causing a Ca2 influx that leads to myelin disruption and a loss of action potential propagation. We now show that, although ischaemia-evoked membrane current changes in oligodendrocytes are triggered by glutamate release, they are dominated by a K influx caused by a rise of extracellular [K ]. This influx is opposed by a reduction of the membrane K conductance. Unexpectedly, activation of NMDA and AMPA/kainate receptors does not raise [Ca2 ]i or [Na ]i in oligodendrocytes, but leads to a rise of [K ]i. Thus, myelin disruption and loss of action potential propagation may result from the activation of ionotropic glutamate receptors on cells other than oligodendrocytes, triggering an excessive influx of K that leads to osmotic swelling in the tiny intra-myelin spaces. Ischaemic damage to oligodendrocytes therefore resembles the swelling occurring in astrocytes rather than the excitotoxic damage occurring in neurons

Acknowledgements Supported by the Wellcome Trust, ERC and EU (Leukotreat).


Scratch-wound induced alterations in glial glutamate transporter distribution and function.

*W. Karl Kafitz 1 , Alexandra E. Schreiner 1 , Julia Langer 1 , Martin C. Stock 1 , Christine R. Rose 1
1 Heinrich Heine University Düsseldorf, , Düsseldorf, Germany
Abstract text :

Rapid extracellular removal of glutamate, the major excitatory neurotransmitter in the CNS, is essential for normal brain function. This task is primarily accomplished by the action of the sodium-dependent, high-affinity transporters GLAST and GLT-1 (rodent analogous of EAAT1 and EAAT2), which are mainly expressed by astrocytes. Impairment or failure of GLAST and GLT-1 plays an important role in many pathological conditions.

In the present study, we analysed changes in glutamate transporter expression and function following a mechanical lesion in organotypic slice cultures of the mouse hippocampus using immunohistochemistry, western blots and dynamic imaging. The lesion was positioned perpendicular to the stratum pyramidale in the CA1 area and comprised the entire hippocampus proper. After three-six days, a glial scar had formed along the lesion site. Activated astrocytes in close proximity (100-150 µm) to the lesion (“scar cells”) directed long, palisading GFAP-positive processes towards the lesion, had significantly swollen somata and lost their ability to take up SR101. Furthermore, some exhibited distinct clustering of GLAST and GLT-1 immunoreactivity. Scar cells showed greatly diminished increases in intracellular sodium in response to application of D-aspartate, an agonist for glutamate transporters. Astrocytes in the periphery to the lesion, in contrast, maintained their ability to take up SR101 and showed only slight up-regulation of GFAP, as well as less swollen cell bodies. Cells in the periphery displayed only marginal changes in glutamate transporter immunoreactivity and unaltered amplitudes of sodium changes in response to D-aspartate.

Taken together, our data show that mild astrogliosis in the periphery of a mechanical lesion is not accompanied by a significant change in glial glutamate uptake capacity. At the scar itself, a strong clustering of glutamate transporters is observed that apparently goes along with a severe functional reduction in glutamate uptake.

Supported by the DFG (Ro2327/4-3).


Astrocytic CX43 hemichannels and gap junctions play a crucial role in development of chronic neuropathic pain following spinal cord injury

*Michael Chen 1 , Benjamin Kress 1 , Xioaning Han 1 , Katherine Moll 1 , Weiguo Peng 1 , Ru-Rong Ji 1 , Maiken Nedergaard 1
1 University of Rochester School of Medicine and Dentistry, Center for Translational Neuromedicine and Department of Neurosurgery and Neurology, Rochester, NY, United States
Abstract text :

Chronic neuropathic pain is a frequent consequence of spinal cord injury (SCI). Yet despite recent advances, upstream releasing mechanisms and effective therapeutic options remain elusive. Previous studies have demonstrated that SCI results in excessive ATP release to the peritraumatic regions and that purinergic signaling, among glial cells, likely plays an essential role in facilitating inflammatory responses and nociceptive sensitization. We sought to assess the role of connexin 43 (Cx43) as a mediator of CNS inflammation and chronic pain. To determine the extent of Cx43 involvement in chronic pain, a weight-drop SCI was performed on transgenic mice with Cx43/Cx30 deletions. SCI induced robust and persistent neuropathic pain including heat hyperalgesia and mechanical allodynia in wild-type control mice, which developed after 4 weeks and was maintained after 8 weeks. Notably, SCI-induced heat hyperalgesia and mechanical allodynia were prevented in transgenic mice with Cx43/Cx30 deletions, but fully developed in transgenic mice with only Cx30 deletion. SCI-induced gliosis, detected as upregulation of glial fibrillary acidic protein in the spinal cord astrocytes at different stages of the injury, was also reduced in the knockout mice with Cx43/Cx30 deletions, when compared with littermate controls. In comparison, a standard regimen of post-SCI treatment of minocycline attenuated neuropathic pain to a significantly lesser degree than Cx43 deletion. These findings suggest Cx43 is critically linked to the development of central neuropathic pain following acute SCI. Since Cx43/Cx30 is expressed by astrocytes, these findings also support an important role of astrocytes in the development of chronic pain.


Metabolic reprogrammation following mouse cortical astrocytes transformation in vitro: a proteomic study

*Azeddine Bentaib 1 , Pascal  De Tullio 2 , Marie Pierre  Junier 3 , Pierre  Leprince 1
1 University of Liège, Giga Neurosciences, Liège, Belgium
2 university of Liège, Drug Research Center, Liège, Belgium
3 University of Paris V, Équipe Plasticité gliale, Paris , France
Abstract text :

Gliomas are the most frequent primitive CNS tumors and are thought to derive from astrocytes or from neural progenitors/stem cells. However, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified.  TGF alpha is frequently over-expressed in the early stages of glioma progression. Sharif & al (Oncogene. 2007 (19):2695-706) previously demonstrated that prolonged exposure of normal astrocytes to TGF alpha is sufficient to trigger their reversion to a neural progenitor-like state. When astrocytes de-differentiated with TGF alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting.

Our study aims to identify and characterize the protein signature of those in vitro transformed cells in an attempt to understand their neoplastic behavior and the effect of transformation on metabolic processes. This involves a global proteomic analysis using the 2D-DIGE methodology and a study of the metabolism of these cells (carbon source, lactate, glucose and glutamine use, ROS metabolism…) by 1H- and 13C-NMR NMR quantification of metabolites. Such approaches are expected to provide information allowing understanding the metabolic reprogrammation that occurred during transformation.

The comparative 2D-DIGE proteomic analysis of normal and transformed astrocytes shows that during transformation, the cells increase their expression of glycolytic enzymes, thus acquiring the ability to use aerobic glycolysis (Warburg effect).  Moreover, the transformed cells reduce their capacity for tricarboxylic acid oxidation and for neurotransmitters (glutamate and GABA) metabolism. Ingenuity Pathway Analysis indicates major effects on carbohydrates, amino acids and nucleotides metabolic components. Using enzymatic activity measurements and the detection of protein isoforms by 2D-Western blot and zymography, we document a change in expression and activity of various isoenzymes that may be responsible for those metabolic reprogrammations.


Proteomic profiling of human induced pluripotent stem cell-derived microglia upon exposure to glioblastoma cells

*Judy Choi 1,2 , Kristin Roy 3 , Tore Kempf 4 , Martina Schnölzer 4 , Agnes Hotz-Wagenblatt 4 , Harald Neumann 3 , Anne Régnier-Vigouroux 1,2
1 German Cancer Research Center, Program Infection and Cancer, Heidelberg, Germany
2 Johannes Gutenberg University of Mainz, Faculty of Biology, Mainz, Germany
3 University Hospital Bonn, University of Bonn, Neural Regeneration, Institute of Reconstructive Neurobiology, Bonn, Germany
4 German Cancer Research Center, Protein Analysis Facility, Heidelberg, Germany
Abstract text :

Question: Glioma-associated microglia/macrophages contribute to at least 1/3 of the total tumor mass and are known to support glioma growth and invasion. However, there have been several studies demonstrating the potential of modulating microglia/macrophages response to reduce glioma growth or invasion (Kees et al. 2012; Synowitz et al. 2006), warranting the need to better understand the interaction between glioma and microglia. While it is the gold standard to study glioma using human specimens, research involving human microglia is often challenged with the problem of low cell yield.

Method: Recently, human induced pluripotent stem cell-derived microglia (iPSdM) cell lines were developed as a substitute for primary microglia according to an established protocol (Beutner et al. 2010). Two iPSdMs were used to determine their responses in the presence of glioblastoma cells. Using LC-MS/MS method, proteomic profiles were generated and compared between iPSdMs exposed to human glioblastoma cells and iPSdMs exposed to normal human astrocytes as control. 

Results: Immunolabeling of the two iPSdM cell lines showed specific expression of microglial markers such as Iba-1, CD11b, and CD68. The comparative proteomic analyses indicated that microglia exposed to glioblastoma cells showed differential protein expressions relevant for cytoskeletal activity, energy production, and cell survival compared to control.

Conclusion: This is the first study showing the proteomes of human induced pluripotent stem cell-derived microglia exposed to glioblastoma cells, and the findings could provide further insight of the interaction between microglia and glioblastoma.


Cytostatic and cytotoxic effects induced by M2 muscarinic receptor activation on human glioblastoma cells.

*Maria Di Bari 1 , Vanessa Tombolillo 1 , Francesco Alessandrini 1 , Claudia Conte 1 , Francesca  De Grassi 2 , Ruggero Ricordy 2 , Ada Maria Tata 1
1 University of Rome Sapienza, , Rome, Italy
2 CNR, , Rome, Italy
Abstract text :

Glioblastomas (GBM) are the most common brain tumors in humans. Although advances in the chemotherapeutic management of glioblastoma have been made, almost 80% of the patients die within the first 2 years after diagnosis. On the basis of these observations, the identification of  new molecules that can counteract the GBM growth and invasiveness appears relevant.

Previously, we have demostrated that M2 muscarinic receptor agonist arecaidine inhibited cell proliferation in a time and dose-dependent manner and induced a severe apoptosis both in two glioma stable cell lines (U251 and U87) and in primary cultures derived from human biopsies. In order to clarify the mechanisms causing the decreased cell proliferation, we have evaluated the ability of M2 receptor activation to counteract the Notch-1 and EGFR pathways. The analysis by real time PCR and western blot have demonstrated that, in both cell lines, M2 receptor caused a decreased expression of EGFR and Notch-1 and its ligands (e.g. delta-1, jagged-1 and -2), suggesting that the decreased cell proliferation may dependent on altered expression and activity of these pathways. Although FACS analysis have showed that arecaidine induced an arrest of cell cycle progression, we observed, in both cell lines, a significant increase of dividing cells already after 24 hours of treatment. In particular, the number of metaphases increased significantly, while the percentage of anaphases diminished.                                                                                                            

Furthermore we observed in both cell lines, a dis-regulation of mitotic spindle assembly, misalignment of chromosomes and the presence of multipolar spindles. Moreover, due to prolonged activation of the promethaphase/methaphase mitotic checkpoint, chromosomes appeared more condensed in cells treated with arecaidine. FACS analysis and immunocitochemistry using anti-histone γ-H2AX, a marker of double strand breaks, have also demonstrated that arecaidine treatment induced DNA damage. On the other hand, M2 agonist induced also oxidative stress, followed by an increased expression of the enzyme superoxide dismutase and sirtuins (SIRT1 and SIRT2).

In conclusion, the data obtained suggest that the activation of M2 receptor induces cytostatic and cytotoxic effects in glioblastoma cell lines, opening new therapeutic perspectives for this receptor in glioblastoma therapy.


Tumor suppression effects of miRNA-302-367 on glioblastoma stem cells are correlated with altered GABA metabolism. 

*Elias A.  El-Habr 1 , Luiz Gustavo Dubois 1 , Mohamed  Fareh 2 , Alexandra  Bogeas 1 , François-Xavier  Lejeune 1 , Joanna  Lipecka 1 , Thierry  Virolle 2 , Marie-Pierre Junier 1 , Hervé  Hervé Chneiweiss 1
1 INSERM U894, , Paris, France
2 INSERM U898, , Nice, France
Abstract text :

Glioblastomas are the most frequent and aggressive form of adult primary brain tumors. Glioblastoma stem cells (GSCs) are thought to play key roles in the development and resistance of the tumor to existing radio- and chemotherapies. Our previous results showed that the cluster of microRNAs, miRNA-302-367, induces loss of GSC stem-like and tumor-inducing properties (Fareh et al. 2012). To further understand the molecular pathways controlling the peculiar properties of GSCs, we sought for metabolic changes likely to accompany the drastic change in cell phenotype induced by miR-302-367 expression.

We measured the intracellular and secreted levels of 271 metabolites by mass spectrometry. Reconstitution of the metabolomes revealed significant and coordinated changes in components of the Krebs cycle, and the glutamine/glutamate neuropeptide metabolic pathway that suggested altered turnover of the GABA synthesis pathway. Exon array hybridization and Western blot analysis, revealed changes in expression of several enzymes of the GABA synthesis pathway in miR-302-367-GSCs. Of note, none of the analyzed enzyme transcripts appeared as a direct target of miR-302-367.

Our results could be integrated in a complex multistep schema compatible with enhanced turnover of the GABA synthesis pathways, resulting in decreased cell GABA levels and increased levels of GABA by-products, as observed in miR-302-367 GSCs. Further studies showed that these metabolic changes participate in the induced loss of GSC properties (see abstract by Dubois et al).

* EAE and LGD contributed equally