XI European Meeting on Glial Cell Function in Health and Disease

Overview Session Overview Sessionprint print  

July 3, 2013 - Poster hall 6:15pm - 7:15pm
Poster Session I (even poster numbers)

Oligodendrocyte precursor cells generate astrocytes after acute cortical injury

*Xianshu Bai 1
1 University of Saarland, , Homburg, Germany
Abstract text :

Secondary injury processes after acute brain trauma involve activation of different cell types like astrocytes, oligodendrocytes and microglia cells. A complex and yet not completely understood sequence of cellular responses initiate functional recovery after the neurodegeneration process. By using double-transgenic mice GCPB (GFAP-EGFP x PLP-DsRed1) mice, we were able to identify a particular type of activated glia expressing astro- AND oligodendroglial properties simultaneously (AO cells) that transiently appeared after three types of acute cortical injuries (stab wound injury, pial vessel disruption and middle cerebral artery occlusion). AO cells could be labeled by oligodendrocyte precursor cell (OPC) markers Olig2 and Sox10, but not for markers of mature astrocytes or neurons.

Two-photon live imaging of GCPB mice revealed that AO cells originated from PLP-DsRed-positive oligodendrocyte lineage cells. Electrophysiological inspection of AO cells in acutely isolated brain slices revealed the expression of delayed rectifying, voltage gated K currents, a typical property of NG2 glia. Therefore, we classified AO cells as OPCs. To follow the fate of AO cells which disappeared about 15 days after the injury, we generated GFAP-N-Cre x PLP-C-Cre x Rosa26EYFP triple transgenic (CreC) mice. Under non-injury conditions, few recombined cells were observed. However, numerous recombined cells appeared after 1 week of stab wound injury adjacent to the lesion site, and lasted over 8 weeks. We found that 55-70 % of recombined cells were GFAP-positive, 18-29 % of them were PDGFRα-positive and 19-30 % were positive for the oligodendrocyte marker GSTπ. In line with that, about 46% of newly generated GFAP-positive astrocytes were observed from NG2-CreERT2 x Rosa26tdTomato mice after 1 week of stab wound injury. These results provide a strong evidence for OPCs giving rise to astrocytes after acute cortical injuries. To further understand the molecular mechanism, we performed intra-cerebral injection of bone morphogenetic protein 4 (BMP4, known to promote astrocyte, but blocking oligodendrocyte differentiation) into GCPB and CreC mice. BMP4, but not leukemia inhibitory factor (LIF), significantly increased the number of AO cells as well as astrocytes in the respective mice.

In conclusion, NG2/OPCs display strong potential to differentiate into astrocytes after acute cortical injury.


Enhanced human and murine oligodendrocyte differentiation in response to a selective thyroid hormone β receptor agonist.

*Emily Baxi 1 , Amanda Fairchild 1 , Carlos Pardo-Villamizar 1 , Jeffrey Rothstein 1 , Dwight Bergles 1 , Peter Calabresi 1
1 Johns Hopkins University, , Baltimore, United States
Abstract text :

Demyelinating diseases such as multiple sclerosis are characterized by loss of myelin and oligodendrocytes. Oligodendrogenesis is essential for successful remyelination and repair. Thyroid hormones are known to play an important role in developmental oligodendrogenesis and myelination and mounting evidence from rodent models of demyelination suggest that they also promote remyelination. The therapeutic application of thyroid hormone to demyelinating disorders is limited by the potential for cardiac side effects mediated by thyroid hormone receptor (THR) α signaling. Here we report that GC-1, a thyromimetic with selective THRβ1 action promotes in vitro oligodendrogenesis from both rodent and human oligodendrocyte progenitor cells. In addition, we used PDGFαR-CreER;Rosa26-eYFP double-transgenic mice to examine the effect of GC-1 on the fate of oligodendrocyte progenitor cells and find that treatment with GC-1 during developmental myelination promotes oligodendrogenesis in vivo.  These results indicate that a β receptor selective thyromimetic can enhance OL differentiation in vitro and during developmental myelination and warrants further study in demyelinating models.


Fast repopulation of microglia after ablation

*Julia Bruttger 1 , Simone Woertge 1 , Simon Yona 2 , Yochai Wolf 2 , Marco Prinz 3 , Steffen Jung 2 , Ari Waisman 1
1 University Medical Centre Mainz, , Mainz, Germany
2 Weizmann Institute of Science, , Rehovot, Israel
3 University of Freiburg, Department for Neuropathology, Freiburg, Germany
Abstract text :

Question: Unlike other glia cells, microglia originate from the myeloid cell lineage and therefore often are considered as resident brain macrophages. They act in the first response to direct injury or peripheral insults. However, their exact contribution in brain homeostasis is not yet clear. Using a novel system microglia were depleted in vivo. After optimizing depletion protocol, the role of microglia in health and disease was analyzed.

Methods: We used the new Cre-expressing mouse strain CX3CR1CreER to express a diphtheria toxin receptor (DTR) specifically on microglia/macrophages. In these mice, administration of tamoxifen leads to Cre-mediated excision of a loxP flanked transcriptional STOP element in both macrophages and microglia, thus allowing for the expression of the DTR as well as an YFP reporter. Due to their fast turnover macrophages are replaced by unaffected precursors whereas microglia persist in the modified stage. These mice were used for in vivo and ex vivo analysis of microglia by FACS and histology.

Results: Our optimized protocol with two tamoxifen injections at the age of 2 weeks resulted in 80-90% of reporter-positive microglia in adulthood, whereas all peripheral macrophages were reporter-negative. With additional three successive DT injections we could achieve a microglia ablation efficiency of 80%. Interestingly, histological as well as FACS analysis revealed 50% of microglia repopulation already 4 days after depletion. Two weeks after depletion microglia numbers were even higher compared to unaffected controls. On day 6, we found a CD45.2high Ly6C population, indicating replenishment by peripheral monocytes. In BM chimera experiments, where peripheral cells were labeled with a congenic marker, we obtained striking evidence showing that almost all of the repopulating cells are of peripheral origin.

Conclusions: After depletion microglia were rapidly repopulated, which emphasizes their critical role in brain homeostasis and function.


Enteric glia: S100B, GFAP and beyond

*David Grundmann 1 , Eva Loris 1 , Denise  Simon 1 , Franziska Markwart 1 , Wenhui Huang 2 , Frank Kirchhoff 2 , Karl-Herbert Schäfer 1
1 University of Applied Sciences, , Zweibrücken, Germany
2 University of Saarland, Molecular Physiology, Homburg, Germany
Abstract text :

Introduction: The enteric nervous system (ENS) harbours neurons, glial cells and their precursors derived from the neural crest. For years, enteric glia cells have played a “wallflower”-like role, although their importance was already recognised by various authors, including Michael Gershon, Georgio Gabella and Kristjan Jesssen, who demonstrated that enteric glia contain the glial fibrillary acidic protein (GFAP) like reactive astrocytes in the central nervous system. In recent years, the key role of enteric glia in supplying neurotrophic support, responding to inflammation or being the source for neural stem cells has become increasingly obvious. There must be more than one or two individual glial subtypes to fulfill the different tasks.

Objectives: To deliver a baseline for further studies on the role of enteric glia in different diseases and species, we tried to deliver a basic inventory which includes general (S100B) and reactive (GFAP) glial markers, as well as neural stem cell (Nestin) or markers for specific so far not identified glial cells. We studied the enteric nervous system using different transgenic mouse models for Nestin (GFP), NG2 (tdtomato) and PLP1 (DsRed) to evaluate the glial composition in the individual segments of the gastrointestinal tract. Whole-mounts were dissected from stomach, small intestine and colon of the different transgenic mice and additional immunofluorescence stainings were performed to analyze the glial phenotype.

Results: We found both S100B /Nestin and S100B /Nestin- glial cells in the myenteric plexus of the different segments of the gastrointestinal tract.In addition, we revealed that there are S100B glial cells outside the myenteric ganglia between the ganglia and in the muscle layers which are positive for the oligodendroglial or Schwann cell marker PLP1. NG2 cells (and their progeny) with different morphologies are present in the gastrointstinal tract. The NG2 proteoglycan did not co-localize with the other glial markers S100B or GFAP, but partly with PDGFRα and SK3 which are shown as fibroblast-like cell markers in the gastrointestinal tract.

Conclusion: Our data demonstrate that the enteric glial population is heterogenous. There are not only glial cells with astrocytic characteristics but also (longitutinal) glial cells with oligodendroglial or Schwann cell properties.


Thyroid hormone mediated OPC differentiation is 'Hairless'

*Matthias Hofer 1 , Alexandra Baer 2 , Moritz Rossner 3 , Matthew Trotter 1 , Mark Kotter 1
1 University of Cambridge, , Cambridge, United Kingdom
2 Medical University Vienna, Department of Neurosurgery, Vienna, Austria
3 Max Planck Institute for Experimental Medicine, , Göttingen, Germany
Abstract text :

Oligodendrocytes are the myelin forming cells of the central nervous system (CNS) that enable fast salutatory signalling transduction and mediate trophic support and protection of axons. Oligodendrocytes originate from a population of precursors cells (oligodendrocyte precursor cells, OPCs) that are formed at distinct developmental stages. The biological process leading to OPC differentiation involves a cascade of molecular events that eventually constitute the complex and multifaceted cellular program that determines the generation of mature oligodendrocytes. To identify the earliest detectable transcriptional correlates of differentiation we used a microarray approach investigating changes in gene expression occurring in primary rat OPCs within the first 12 hours after purification.

Amongst the most regulated factors that were detected was hairless (Hr), a nuclear receptor co-repressor that has been associated with thyroid hormone signalling in the neonatal CNS. Validation of the microarray results by RT-qPCR, Western blot and immunochemistry confirmed a rapid increase of Hr expression during OPC differentiation in media containing thyroid hormone. In the absence of thyroid hormone, Hr expression was suppressed and associated with impaired OPC differentiation. Similarly, RNAi mediated gene silencing of Hr induced an impairment of OPC differentiation demonstrating an important functional role of Hr in the differentiation program. Thyroid hormones regulate Hr expression via the thyroid hormone receptors THRα and THRβ as RNAi-mediated gene silencing of each of the receptors resulted in a decrease in Hr expression. Hr is known to negatively regulate RAR-related orphan receptor alpha (RORα) and Vitamin D receptor (VDR). However, RNAi-mediated gene silencing of RORα and VDR did not change the course of OPC differentiation.

Interestingly, an interaction between Hr and histone de-acetylases (HDACs), of which HDAC1 and 2 are known regulators of OPC differentiation, has been noted in the past. Using an in situ protein-protein interaction assay we were able to demonstrate a direct interaction of Hr with HDAC1 and HDAC2 in differentiating OPCs. Moreover, ChIP qPCR analysis revealed Hr-HDAC1 recruitment to the OPC differentiation inhibitor Hes5. Taken together these data show that thyroid hormone promotes OPC differentiation via Hr by modulating HDAC1 and 2 function. As iodine deficiency results in hypothyriodism our results give an example of a developmental disease that is triggered by environmental factors (the absence of iodine) causing epigenetic dysregulation.


Variable differentiation potential of NG2 glia during mouse development

*Wenhui Huang 1 , Na Zhao 2 , Alexander Cupido 2 , Xianshu Bai 2 , Anja Scheller 2 , Sandra Goebbels 3 , Frank Kirchhoff 2
1 University of Saarland, , Homburg, Germany
2 University of Saarland, Molecular Physiolgy, Homburg, Germany
3 Max-Planck-Institute of Experimental Medicine, Department of Neurogenetics, Göttingen, Germany
Abstract text :

NG2 is a type I transmembrane glycoprotein and also known as chondroitin sulphate proteoglycan 4 (CSPG4). In the central nervous system NG2-expressing cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes in the developing white matter. For temporally-controlled gene targeting of NG2 glia in vivo, we generated a mouse line in which the open reading frame of the tamoxifen-inducible form of Cre recombinase (CreERT2) was inserted into the NG2 locus by homologous recombination.

Here, we investigated the differentiation potential of NG2 glia at different developmental stages of the forebrain. Cre recombinase activity was induced at embryonic day 17.5, postnatal day 0 (P0), P3, P8, P30 and P60 by intraperitoneal tamoxifen injections into novel TgH(NG2-CreERT2) mice crossbred to Rosa26-tdTomato and Rosa26-EYFP reporter lines that helped to identify NG2 glia and its progeny. Recombination and cell identification were determined 10 to 60 days after the first tamoxifen injection.

Induction of recombination at embryonic stages revealed that embryonic NG2 cells mainly generated more NG2 glia and oligodendrocytes, however, a significant number of astrocytes could be detected as well. Recombined cells were predominantly restricted to the ventral brain. In contrast, after tamoxifen injections at P0 and P3 recombined cells were widely found in all brain regions, thereby suggesting the presence of a distinct subpopulation of NG2 cells after birth. Interestingly, only very few recombined astrocytes could be found, which are probably derived from few remaining embryonic NG2 glia. Starting from P8, NG2 glia stopped generating astrocytes, with the progeny largely restricted to the oligodendrocyte lineage. However, consistently, we found recombined NeuN cells with the morphology of neurons in the ventral cortex after administration of tamoxifen at P8. Even more of such cells were present in the whole cortex when Cre activity was induced in adult mice. The morphology, the presence of NeuN immunoreactivity and our electrophysiological characterization that demonstrated bona fide action potentials clearly identify these cells as functional neurons.

Our results suggest that NG2 cells display a broad differentiation potential that appears to be highly age-dependent during development.





Use of genetic tools to perform in vivo analysis of glia in the Enteric Nervous System

*Reena Lasrado 1 , Sarah McCallum 1 , Werend  Boesmans 2 , Pieter Vanden Berghe 2 , Vassilis Pachnis 1
1 National Institute for Medical Research, , London, United Kingdom
2 TARGID, KULeuven, , , Leuven, Belgium
Abstract text :

Neurons and glia of the Enteric Nervous System (ENS) originate from a pool of Sox10 progenitors. In addition to being a scaffold for enteric neurons, enteric glial cells (EGCs) have been suggested to function in mucosal integrity, neuroprotection, adult neurogenesis, neuro-immune interactions and synaptic transmission. Currently, diverse glial populations are known to reside within the ENS. However, whether, specific functions are carried out by specialized glial subtypes, with characteristic morphologies and molecular markers and within specific locations in the gut remains elusive.

To address this question we used a repertoire of genetic tools, immunostaining and imaging techniques. For high single-cell resolution study of EGCs, we Mosaic analysis with Double Markers (MADM) in conjunction with Sox10-Cre. We analyzed adult gut tissue to characterize the different glial populations based on morphology, position in the ENS and their relationship with the intestinal epithelial and vascular system. Moreover, we used reporter mice (Sox10-iCreERT2; R26R-YFP and GFAP-iCreERT2; R26R-YFP) to quantify the expression of three glial markers – GFAP, S100β and Sox10 in myenteric plexus (MP) preparations of adult mice.

Our investigation on MADM-MP preparations, revealed three subtypes of enteric glia in the MP of adult mice. Type I and Type II glia, present in the primary plexus of mouse ENS have been previously defined. In addition, we characterized a third subtype in the extraganglionic space of the MP of adult mice, at present termed as Type III-extraganglionic glia. Furthermore, our analyses using 3D-imaging, on MADM-gut sections allowed us to characterize the relationship of enteric glia located within the mucosa, with the intestinal epithelium and vascular system of the villi. 

Our marker expression analysis showed that while the majority of glial cells co-expressed GFAP and S100β, a large fraction of glia within the myenteric ganglia was positive for either S100β (~30%) or GFAP (~10%). We also found that S100β /GFAP- glia were abundant outside the ganglia. Most glial cells co-expressed Sox10 with GFAP and S100β yet a significant proportion (~10%) of mainly extra-ganglionic glia (Type III) expressed only Sox10. Surprisingly, ~5% of the cells that were S100β- and Sox10- displayed high GFAP expression. Use of GFAP-iCreERT2; R26R-YFP mice showed that about ~30% of YFP-labelled cells did not express GFAP, implying dynamic regulation of GFAP expression.

Overall, our high resolution studies reveal extensive heterogeneity of enteric glial cells in terms of morphology, position and expression of molecular markers suggesting differential functional roles. Our future experiments will address whether different physiological roles of EGCs can be assigned to specific subtypes.


Inhibition of endogenous Phosphodiesterase 7 promotes oligodendrocyte precursor survival and differentiation

*Eva Maria Medina-Rodriguez 1 , Francisco Javier Arenzana 1 , Jesus Pastor 2,3 , Miriam Redondo 4 , Valle Palomo 4 , Rafael Garcia de Sola 2,3 , Carmen Gil 4 , Ana Martinez 4 , Ana Bribian 1,5 , Fernando de Castro 1
1 Hospital Nacional de Paraplejicos, Grupo Neurobiologia del Desarrollo, Toledo, Spain
2 Hospital Universitario La Princesa, Neurocirugia, Madrid, Spain
3 Hospital Universitario La Princesa, Neurofisiologia clínica, Madrid, Spain
4 CSIC, Instituto de Química Médica, Madrid, Spain
5 Universidad de Barcelona, Instituto de Bioingeniería de Cataluña, Barcelona, Spain
Abstract text :

Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative demyelinating disease of the central nervous system (CNS) characterized by inflammation, which leads to formation of demyelinating areas due to loss of oligodendrocytes, astrogliosis and, finally, axonal degeneration. Different studies have suggested that 3'-5'-cyclic adenosine monophosphate (cAMP) levels might play an important role in neuroprotection and neuroinflammatory response so the modulation of this nucleotide intracellular levels might control the neuroinflammatory pathological process and, consequently, to delay the progression of MS.

 Intracellular cAMP levels depend on its synthesis by adenylyl cyclases, and on its degradation by cyclic nucleotide 3’,5’-phosphodiesterases (PDEs). Specific inhibitors for the different isoforms of PDEs family, particularly cAMP-specific PDE, emerge as putative treatments of this kind of disease. PDE7 has emerged as a new therapeutic target, not only for a variety of immunological and immunodeficiency conditions to alleviate chronic inflammation, but also for several neurodegenerative disorders, including MS, in which both immune system and CNS are implicated.

 In the present work, we have detected the expression of PDE7 in oligodendrocyte precursor cells (OPCs) isolated from cerebral cortex of P0 and P15 mice and from adult human samples derived from neurosurgery of epilepsy. OPCs are abundant in the mice and human CNS during development but they also represent 5-7% of the total number of cells in the adult CNS, where they serve as a source of new oligodendrocytes to replace those which die. However, in MS these endogenous OPCs are not able to effectively replace dead oligodendrocytes. In vitro, we have demonstrated that two newly-developed PDE7 selective inhibitors (TC 3.6 and VP 1.15) favour OPC survival and differentiation towards mature oligodendrocytes, being both more effective than the commercial PDE7 inhibitor BRL-50481. ERK intracellular pathway has been identified as a key in these cellular processes related with the cAMP/PKA/CREB pathway. Moreover, we have observed that both specific inhibitors (VP1.15 and TC3.6) increase the differentiation of human OPCs. These findings, combined with the already known anti-inflammatory effect carried out by these PDE7 inhibitors, point them as potential therapeutic agents to treat MS. The knowledge of factors that affect the biology of murine OPCs and even more, human OPCs, are critical for possible remyelination in this kind of pathologies.



High-resolution electrophysiological determination of unitary exocytic events in cultured astrocytes

*Alenka Guček 1 , Jernej Jorgačevski 1,2 , Boštjan Rituper 1 , Marko Kreft 1,2,3 , Robert Zorec 1,2
1 University of Ljubljana, , Ljubljana, Slovenia
2 Celica, Biomedical Center, , Ljubljana, Slovenia
3 University of Ljubljana, Biotechnical faculty, Ljubljana, Slovenia
Abstract text :

Bidirectional communication between neurons and astrocytes, the most abundant type of glial cells, has become a subject of numerous researches, embodying astrocytes as an important partner in synaptic modulation. Astrocytes possess Ca2 -based cytosolic excitability, which can induce release of gliotransmitters introducing (patho)physiological impact on surrounding cells. Non-vesicular and vesicular-based mechanisms appear to co-exist in astrocytes, however, the lack of understanding the basic astrocytic properties of secretion requires additional insight into regulated exocytosis. To directly monitor exocytosis of individual vesicles with the plasma membrane we employed the high-resolution cell-attached patch-clamp technique on cultured astrocytes. We monitored changes in membrane capacitance (Cm), a parameter linearly related to the surface area of the membrane. Preliminary results show, that in astrocytes discrete steps in membrane capacitance reflect unitary exo-endocytic events of single vesicles. For the first time we demonstrate direct real-time measurements of predominantly reversible capacitance steps, reflecting transient exocytosis, and irreversible capacitance steps, reflecting full-fusion exocytosis, in cultured astrocytes.


TLR-signaling induces Type I interferon responses in microglia and astrocytes and regulates leukocyte infiltration to the CNS

*Reza M. H. Khorooshi 1 , Thomas H. Holm 2 , Carsten Tue Berg 1 , Ruthe  Truong Dieu 1 , Dina Dræby 1 , Stefan Lienenklaus 3 , Trevor Owens 1
1 Institute of Molecular Medicine, , odense, Denmark
2 Medical Biochemistry, Department of Biomedicine, Århus, Denmark
3 Helmholtz Centre for Infection Research, , Braunschweig, Germany
Abstract text :

Our goal is to understand the glial signaling that controls inflammatory responses in the central nervous system (CNS). This glial response can play both a detrimental and beneficial role. Toll like receptor (TLR) signaling is implicated in responses to pathogens or endogenous signals. TLR signaling mediates immune response by inducing cytokines, including type I interferons (IFN). IFN-beta, a member of the type I IFN family, is a first-line therapeutic for multiple sclerosis. Type I IFNs signal through a common receptor, IFNAR. The aim of the present study was to investigate the in vivo response of microglia and astrocytes to CNS administration of TLR ligand/agonist, and to examine whether this response involves type I IFN signaling. Mice were administered TLR ligands/agonists by injection to the cisterna magna. We analyzed leukocyte entry to the CNS by flow cytometry, and their localization by immunostaining. The induction of IFN-beta was examined by in vivo imaging of an IFN-beta reporter mouse. Astrocytes and microglia were sorted by FACS and gene expression was measured using quantitative real time-PCR. Injection of ligands/agonists for TLR2, 3 and 4 resulted in infiltration of CD45 leukocytes after 18 hrs, most strongly by TLR2. Immunostaining showed parenchymal localization of infiltrating cells in cerebellum. FACS sorted astrocytes expressed equivalent levels of TLR3 mRNA to microglia but lower levels of TLR2 or 4. Astrocytes were induced by TLR3 signaling to express interferon regulatory factor 7, which regulates the induction of type I IFN. The induction of IFN-beta in CNS in response to TLR3 signaling was verified in IFN-beta reporter mice. TLR2, 3 and 4 signaling led to increased levels of mRNA for glial CXCL10. Together these results suggest the involvement of type I IFN signaling. However, unlike CXCL10 gene expression, that was dependent on IFNAR signaling, TLR2-induced leukocyte infiltration was not affected in IFNAR deficient mice. These studies point to a role for TLR signaling in the innate glial response that regulates CNS inflammation.


Laquinimod reduces astrocytic but not microglial NFkB activation in vitro and in vivo

*Nadine Kramann 1 , Ramona Pförtner 2 , Uwe-Karsten Hanisch 2 , Karin Hagemeier 3 , Tanja Kuhlmann 3 , Wolfgang Brück 2 , Christiane Wegner 2
1 University Medical Center Goettingen, , Göttingen, Germany
2 University Medical Center Göttingen, Department of Neuropathology, Göttingen, Germany
3 University Hospital Münster, Institute of Neuropathology, Münster, Germany
Abstract text :

Introduction: Laquinimod (LAQ) is an oral well-tolerated molecule that has been shown to reduce brain atrophy, disability progression and relapse rate in patients with relapsing-remitting multiple sclerosis. Recent experimental data indicate that LAQ minimizes demyelination, inflammation and axonal damage in mice with cuprizone challenge.

Aim: Since astrocytic NFκB activation plays a crucial role in cuprizone-induced demyelination, we investigated the effects of LAQ on CNS cells in vitro and in vivo.

Methods: In vitro experiments used primary cells to test effects of LAQ on oligodendroglial survival as well as on cytokine secretion and NFκB activation in astrocytes and microglia. Primary mouse astrocytes and microglia were pre-treated with 0, 0.25 and 2.5 µM LAQ and stimulated by pro-inflammatory cytokines. A dual-luciferase reporter assay was used to measure NFκB activity.

For in vivo experiments, 10-week-old male C57BL/6J mice were challenged with 0.25% cuprizone and treated daily with LAQ (25 mg/kg) or water. To assess astrocytic and microglial NFkB activation in vivo, nuclear translocation of NFκB p65 was assessed in astrocytes and microglia by double immunofluorescence with antibodies against p65 and Iba1 or GFAP.

Results: In vitro, astrocytic but not microglial NFκB activation was markedly reduced by LAQ as evidenced by NFκB reporter assay. In astrocytes, pre-treatment with 0.25 and 2.5 µM LAQ significantly reduced the induced NFқB activity after TNFα stimulation compared to stimulated controls. Pre-treatment with 2.5 µM LAQ also significantly reduced NFκB activation after stimulation with the combination of IL-1β and IFNγ compared to untreated stimulated controls. Oligodendroglial viability and survival were not affected by LAQ treatment. To confirm the in vivo relevance of these findings, we also examined astrocytic and microglial p65 translocation in mice with and without LAQ treatment after cuprizone challenge. The proportion of astrocytes with nuclear p65 immunoreactivity was significantly reduced in LAQ-treated mice (14.0% ± 0.9%) compared to untreated controls (25.8% ± 1.1%). Microglia did not display marked translocation of NFκB p65 after cuprizone challenge.

Conclusion: Our data indicate that LAQ prevents cuprizone-induced demyelination by attenuating astrocytic NFκB activation. In vitro, LAQ reduced the astrocytic NFκB activation by up to 46% as evidenced by NFκB reporter assay. Similar quantitative findings were obtained in vivo when LAQ treatment also led to a 46% reduction of astrocytes with NFκB activation, as evidenced by nuclear translocation of p65. These findings suggest that targeting the astrocytic NFκB pathway might have therapeutic effects in demyelinating CNS disorders.


Beta-amyloid induces apoptosis of phagocytic microglia and macrophages

*Alicia Babcock 1 , Laura Ilkjær 1 , Martin Wirenfeldt 1 , Thomas Krøigård 1 , Christa Myhre 1 , Henrik Toft-Hansen 2 , Lasse Dissing-Olesen 1 , Tomas Deierborg 3 , Sultan Darvesh 4 , Morten Jensen 5 , Mark West 5 , Bente Finsen 1
1 University of Southern Denmark, Neurobiology Research, Odense, Denmark
2 University of Southern Denmark and Odense University Hospital, Pediatrics and Clinical Immunology, Odense, Denmark
3 Lund University, Experimental Medical Science, Lund, Sweden
4 Dalhousie University , Medicine, Anatomy & Neurobiology, Halifax, Canada
5 University of Aarhus, Biomedicine, Aarhus, Denmark
Abstract text :

In Alzheimer’s disease, microglial clearance of beta-amyloid (Aβ) is considered neuroprotective. Whether Aβ uptake regulates microglial cell responsiveness, and whether macrophages participate, remains unclear. Here we investigated whether in vivo phagocytosis of endogenously-produced Aβ disturbs the turnover of microglia and macrophages by changing rates of cellular proliferation and apoptosis. Using APPswe/PS1ΔE9 Tg mice, we show that plaque-associated microglia and CD11b CD45high macrophages bind and ingest endogenously-produced Aβ in vivo. Plaque-associated microglia and CD45 leukocyte-like cells were also observed in brains from patients with Alzheimer’s disease. Phagocytic microglia and macrophages had a reduced rate of proliferation compared to Aβ- cells. Instead, high proportions of apoptotic Annexin V microglia and macrophages were found in aged APP/PS1 Tg mice. Phagocytic microglia and macrophages had greater caspase activity and increased p53 expression after in vivo uptake of Aβ, than cells that did not phagocytose Aβ. In vitro, we found that Aβ uptake induced caspase activation in microglia, whereas blocking Aβ uptake triggered apoptosis-induced cell death in macrophages. Our data demonstrate that Aβ uptake impairs microglial/macrophage viability and responsiveness. Amyloid clearance may fail as phagocytic microglia and macrophages undergo Aβ-induced apoptosis.


Olesoxime for the treatment of hereditary dysmyelinating diseases: The importance of the therapeutic window

*Mélina Begou 1 , Bérengère Depiets 2,3 , Fabrice Giraudet 4,3 , Julie Barbier 2,3 , Céline Bechon 2,3 , Magalie Michaud 5 , Rebecca Pruss 5 , Thierry Bordet 5 , Odile Boespflug-Tanguy 2,6
1 INSERM, U676, Paris, France
2 INSERM, U676, Paris, France
3 Université d'Auvergne, Faculté de Médecine, Clermont-Ferrand, France
4 INSERM, U1107, Clermont-Ferrand, France
5 TROPHOS, , Marseille, France
6 APHP, Hôpital Robert Debré, Paris, France
Abstract text :

The proteolipid protein gene (PLP1), located on the X-chromosome, undergoes alternative splicing to produce the most abundant proteins of central nervous system myelin: PLP and DM20. Related to the extent of myelin defect, mutations in the PLP1 gene in humans are associated with a spectrum of X-linked disorders, from the severe Pelizaeus-Merzbacher disease (PMD), to the mild form spastic paraplegia type 2 (SPG2). Phenotype-genotype correlation exists, large duplications of PLP1 gene are predominantly found in PMD and null mutations in SPG2.

PLP is almost 100 % conserved across mammalian species, and the large spectrum of phenotype severity is also found Plp transgenic mice. Experiments on these models have contributed to our understanding in the pathophysiology of PLP related disorders. In one hand, mice with extra copies of the Plp1 gene ( PlpTg mice) have neurological symptoms and CNS pathology similar to those found in PMD patients while mice with Plp1 gene inactivation (Plp null mice) share similarities with SPG2 patients. Then, these two mouse models represent very useful tools to evaluate the efficacy of a therapeutic strategy for the severe and the mild form of the PLP related disorders on the neuropathological and behavioral aspects.

Olesoxime (TRO19622), a cholesterol-like small molecule, has been shown to rescue motor neurons from cell death and to promote axonal regeneration both in vitro and in vivo. More recently, olesoxime has also been shown to promote central remyelination by accelerating oligodendrocyte maturation both in vitro and in vivo. Altogether, these results strongly suggest that olesoxime may be a potential drug candidate for the treatment of white matter neurodegenerative disorders and notably to PLP related disorders.

Here we treated PlpTg mice with olesoxime for 2 months starting at birth, as well as Plp null mice from 3 to 12 months of age (presymptomatic treatment) or from 9 to 15 months of age (symptomatic treatment). Behavioral and neuropathological analysis revealed that olesoxime failed to correct the development of symptoms in mice modeling the severe form of PLP related disorders. On the contrary, treatment of Plp null mice with olesoxime resulted in a 3-month delay in the onset of abnormal behaviors related to anxiety and in the slowing of central nerve conduction. These beneficial effects are observed only when the drug is administered before disease onset. Treatment with olesoxime also reduces the effect of aging on rotarod performance decline in both WT and Plp null old mice. Altogether these results suggest that olesoxime could be of interest as a treatment to delay the onset of symptoms in PLP related disorders provided that patients suffer from a mild form of the disease and are treated before the establishment of symptoms.


Lack of Microglial TREM2 Receptor Increases Susceptibility to Dopaminergic Degeneration Triggered by Systemic Inflammation

*Liviu-Gabriel Bodea 1 , Marco Colonna 2 , Harald Neumann 3
1 University of Bonn, , BONN, Germany
2 Washington University School of Medicine, Department of Pathology & Immunology, St. Louis, United States
3 University of Bonn, Insitute of Reconstructive Neurobiology, Bonn, Germany
Abstract text :

Under normal conditions, inside central nervous system the immune functions are accomplished by microglial cells. One of the receptors found on microglia is the Triggering Receptor Expressed on Myeloid cells 2 (TREM2), which signals intracellularly via an adapter molecule referred to as TYRO protein tyrosine kinase-binding protein (TYROBP, known also as DAP12). In humans, loss of function of either TREM2 or TYROBP leads to Nasu-Hakola disease, which is characterized by neuroinflammation and bone cysts.

By using a TREM2 knock-out (KO) mouse line, we provided further insights on the TREM2 function in neurodegenerative diseases.

First, we observed mild signs of dopaminergic neurodegeneration and a slight increase in microglial Iba1-immunoreactivity in different brain regions of 3 months TREM2 KO mice compared with the wild type (WT) control animals. However, the transcription levels of inflammatory cytokines (TNFα, IL1β) or inducible nitric oxide synthase (iNOS) were unaffected in mice of different ages (3, 9 or 12 months). To shorten the time required for neurodegeneration to occur, we have injected the mice intraperitoneally with lipopolysaccharides (LPS) on four consecutive days and analyzed the dopaminergic neurodegeneration in the substantia nigra after a three weeks period. While the quantification of dopaminergic neurons in the substantia nigra showed a slight decrease in number for WT animals, a higher loss was recorded in the TREM2 KO mice after systemic LPS challenge. Concomitantly, significant increase of microglia activation was detected in the LPS-treated TREM2 KO mice compared with LPS-treated WT animals.

Our results are pointing towards a neuroprotective effect of the microglial TREM2 receptor under chronic and systemic inflammatory conditions.


Emergence of tyrosine hydroxylase-positive cells in rat cortex after intraventricular injection of 6-hydroxydopamine (6-OHDA)

*Sandro Caradonna 1,2 , Britta Wachter 1 , Eva Küppers 1
1 Institute of Anatomy, Department of Cellular Neurobiology, University of Tübingen, Tübingen, Germany
2 Graduate School for Cellular and Molecular Neuroscience , Eberhard Karls University, Tübingen, Germany
Abstract text :

Intraventricular injection of 6-OHDA results in increased proliferation and de-differentiation of rat cortical astrocytes into progenitor-like cells (Wachter et al., Cell Tissue Res. 342(2):147-60 2010). The function and relevance of these cells is not yet clear. To test the hypothesis, that these cells are part of a possible compensatory mechanism to cope with the 6- OHDA-induced loss of dopaminergic neurons, we studied the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the catecholamine synthesis pathway, in the cortex of 6-OHDA-lesioned animals.

Performing immuncytochemistry at 4d after the lesion we demonstrated a 21-fold increase in the number of TH-positive (TH somata in rat cortex following 6-OHDA injection) compared to sham-lesioned animals. TH somata in the parietal cortex were restricted to the layers II-V with most of the cells being bipolar.

Combining TH immuncytochemistry with classical Nissl stain yielded complete congruency. A small fraction of TH cells co-expressed calretinin thus pointing to an interneuron affiliation. Virtually none of the TH cells expressed other markers of interneurons (calcium binding proteins, neuropeptides) or the glial markers GFAP and nestin.

In contrast, we found a co-localization of TH with markers of glial progenitor cells (Sox2 and S100β) and with PSA-NCAM, which has been shown to be expressed in immature, but not recently generated cortical neurons in cortical layer II of the cat (Varea et al.; Front. Neurosci. 5: 17 2011)

Taken together, these findings indicate that 6-OHDA lesions might induce a glial de- differentiation followed by a fate re-direction towards neuronal committed cells. Future studies have to be performed to confirm this and to find out if the TH cells are capable of dopamine synthesis.


Vanishing White Matter-mutated astrocytes impair oligodendrocyte maturation in vitro

*Stephanie Dooves 1 , Aleid van de Kreeke 1 , Niels Land 1 , Gerbren Jacobs 1 , Marjo van der Knaap 1 , Vivi Heine 1
1 VU University Medical Center, , Amsterdam, Netherlands
Abstract text :

Vanishing White Matter disease (VWM) is a severe leukoencephalopathy with mainly a childhood onset. Affected children show progressive neurological symptoms like ataxia and spasticity that eventually lead to death. Pathologically, mainly the astrocytes and oligodendrocytes of the brain white matter are affected. Previous research has shown that both are in an immature state and have an abnormal morphology.

In order to gain more knowledge about the disease pathophysiology, two mouse models for VWM are developed in our lab. Co-cultures between astrocytes (WT and VWM) and WT oligodendrocyte progenitor cells (OPCs) are done to investigate the effect of astrocytes on OPC maturation. Furthermore, induced pluripotent stem cells (iPSCs) from both VWM and WT mice are compared in their glial differentiation efficiency in vitro and in vivo.

The astrocyte-OPC co-cultures show that VWM astrocytes inhibit OPC maturation. This suggest that astrocytes might have a crucial role in the development of the oligodendrocyte and myelin pathology in VWM. Currently our research focuses on rescuing the maturation inhibition in vitro, which can aid the development of stem cell therapy for VWM.


Alpha-synuclein impairs differentiation of oligodendrocytic CG4 cells

*Benjamin Ettle 1 , Verena May 1 , Michael Wegner 2 , Eliezer Masliah 3 , Jürgen Winkler 1
1 University Hospital Erlangen, Department of Molecular Neurology, Erlangen, Germany
2 Friedrich-Alexander University Erlangen-Nuremberg, Institute of Biochemistry, Erlangen, Germany
3 University of California, Department of Neurosciences, San Diego, La Jolla, United States
Abstract text :

The atypical Parkinson syndrome multiple system atrophy (MSA) is a rare and rapidly progressive neurodegenerative disease. The major neuropathological hallmark of MSA is the presence of glial cytoplasmatic inclusions (GCIs) within oligodendrocytes consisting mainly of the primary neuronal protein alpha-synuclein (α-syn). GCI bearing oligodendrocytes become dysfunctional and thus, myelin loss accompanied by consecutive neuronal degeneration is a main characteristic of MSA observed in different regions throughout the central nervous system (CNS) including basal ganglia, pons, cerebellum, inferior olivary nuclei, and spinal cord. Although myelination is completed early after birth, oligodendrocyte precursor cells (OPCs) persist in the adult mammalian CNS. Upon injury, OPCs react with amplification and differentiate into premyelinating oligodendrocytes finally remyelinating unwrapped axons preserving them of degeneration. A recent study showed increased numbers of OPCs in MSA patients compared to control patients suggesting a disease-related alteration of this cell population in MSA (Ahmed et al., Brain Pathol., 2012). However, detailed studies analyzing oligodendrocyte differentiation in MSA are lacking. Thus, we generated human wild-type α-syn expressing CG4 cells (CG4_wt-α-syn) representing OPC-like cells with the potential to differentiate upon growth factor withdrawal and addition of triiodothyronine (T3) into premyelinating oligodendrocytes. Upon differentiation, significant fewer CG4_wt-α-syn cells express the myelin basic protein (MBP; see figure A) as a marker for terminal differentiation and additionally, mRNA levels were remarkably reduced compared to control cells (see figure B). As mRNA levels of the myelin protein proteolipid protein 1 (PLP1) as well as the major myelin-gene regulatory factor (MRF) are also reduced in differentiated CG4_wt-α-syn cells (see figure B) we hypothesized that the process of differentiation is delayed upon α-syn expression. Using various differentiation-promoting agents targeting distinct maturation-associated pathways we aimed to dissect the α-syn-mediated effect on differentiation in more detail and evaluate potential disease-modifying approaches for this devastating neurodegenerative disorder.


Glial Lazarillo protects neurons from type I Spinocerebellar Ataxia (SCA1) degeneration by a mechanism involving the control of autophagy flow and of lipid peroxide clearance.

Manuela del Caño-Espinel 1 , Diego Sanchez 1 , *Maria D. Ganfornina 1
1 Universidad de Valladolid - CSIC, , Valladolid, Spain
Abstract text :

Glial Lazarillo (GLaz) is a Drosophila homologue of Apolipoprotein D (ApoD), a lipocalin showing an increased expression with aging and neurodegeneration. GLaz/ApoD protects against oxidative stress and promotes axon regeneration after injury, but its mechanism of action is unknown. We hypothesize that GLaz/ApoD modulates membrane dynamics and oxidation. To contrast this hypothesis we study GLaz protective role on the polyQ-based Spinocerebellar Ataxia type I (SCA1) model in Drosophila.

Human polyQ-Ataxin1 expression in fly photoreceptors triggers GLaz up-regulation. Overexpressing GLaz in photoreceptors or in retinal support cells rescues neurodegeneration. When expressed in retinal support cells, the GLaz rescue is dependent on Lipocalin-receptor expression by photoreceptor neurons, and the GLaz-GFP fusion protein co-localizes with photoreceptor markers, suggesting the existence of receptor-mediated endocytosis of GLaz.

Upon neurodegeneration, oxidative stress and induction of autophagy coexist. To test whether the GLaz beneficial effects are mediated by the modulation of autophagy we quantified Atg8a expression and monitored the accumulation of ubiquitinated proteins and p62. Overexpression of GLaz reduces Atg8a induction, but concurrently reduces the accumulation of ubiquitinated proteins and p62. Upon autophagy stimulation by rapamycin treatment, the levels of SCA1-dependent accumulation of ubiquitinated proteins and p62 are further reduced by GLaz. In addition, GLaz decreases the SCA1-dependent induction of Gsts1, a SCA1 genetic modifier contributing to the clearance of lipid peroxides.

Our data support that GLaz enters the degenerating neurons by a receptor-mediated mechanism and promotes the resolution of autophagy, increasing its flow and helping to clear polyQ-induced protein aggregates. Moreover, the beneficial effects of GLaz are linked to lipid peroxide clearance, either directly or through receptor-mediated modulation of protective gene networks.

Support: MICINN(BFU2011-23978); JCyL(VA180A11-2); Fund. Rodríguez-Pascual.


Antagonizing the TGF-ß1 Receptor ALK5 reduces gliosis after Neonatal Hypoxia-Ischemia

*Mariano Guardia Clausi 1 , Zhihua Ren 1 , Dimitrios Giannakidis 1 , Steven W. Levison 2
1 NJMS-UH Cancer Center, Dept of Neurology and Neurosciences, Newark, NJ, United States
2 University of Medicine and Dentistry of New Jersey, , Newark, United States
Abstract text :

Our previous studies have shown that there is a regenerative response from the neural precursors in the subventricular zone after neonatal H-I (H-I). In vitro, neural precursors from the injured hemisphere have an increased capacity to generate oligodendrocytes and neurons. However, in vivo, after H-I there is aberrant production of astrocytes, and there is emerging evidence that reactive astrocytes inhibit the differentiation of oligodendrocyte progenitors. We have identified TGFB1 as a cytokine that is produced subsequent to H-I, that collaborates with other cytokines to stimulate the production of astrocytes from subventricular zone glial progenitors. The specific goal of this study was to evaluate gliogenesis after H-I when the TGFB1 receptor ALK5 is antagonized in the Vanucci P6 H-I rat model. By Q-PCR, the relative amount of TGFB1 mRNA peaked 7 days after H-I. Therefore, SB-505124, and ALK5 antagonist, was given intraperitoneally (i.p.) 7 days after the injury to a group of rats while another group received PBS. Animals were sacrificed at different time points and brain samples processed for Western blot analysis. Validating the importance of ALK-5 signaling, SB505124 reduced the levels of phosphorylated Smad 2/3 that increased after H-I. In another study, SB-50512 was administered i.p. from P10 to P15 and animals sacrificed at P20 for immunohistochemistry for GFAP, IBA-1, GSTpi and MBP. GFAP and IBA-1 positive cells were dramatically increased in the injured striatum and corpus callosum after H-I and MBP staining was decreased. By contrast, both the extent of injury and the degree of reactive gliosis was decreased by SB-505124 treatment. Altogether, our results indicate that SB-505124 inhibits ALK5 signaling in the damaged brain. Furthermore, SB-505124 administration decreases the extent of microgliosis and astrogliosis while preserving myelination in the damaged brain after neonatal H-I. Supported by NIH R01 HD052064 and a grant from the LeDucq Foundation awarded to SWL.


Astrocytes in the degenerating brain are primed to synthesize exaggerated levels of CXCL1 and CCL2 in response to IL-1b/TNF-a stimulation

*Edel Hennessy 1 , Colm Cunningham 1
1 Trinity College Dublin, , Dublin , Ireland
Abstract text :

Microgliosis and Astrogliosis are standard features of neurodegenerative disease. It has been shown in a number of animal models that microglia in the degenerating brain are primed to show exaggerated cytokine responses to subsequent stimulation with Toll-like receptors agonists such as LPS and poly I:C. It is not clear whether the degenerating brain shows similarly exaggerated responses to pro-inflammatory cytokines. In the current study we hypothesised that glial cells in the hippocampus of animals with chronic neurodegenerative disease (ME7 prion disease) would display abnormal responses to central cytokine challenges. In normal animals it has previously been established that intracerebral cytokine challenges elicit specific pathways, i.e. IL-1β à CXCL-1 à Neutrophil recruitment or TNFα à CCL-2 à Monocyte recruitment. Unilateral 1μl doses of TNFα (300ng/μl), IL-1β (10ng/μl) or saline were administered intrahippocampally via pulled glass microcapillary, in normal (NBH) and ME7 mice.

These animals were terminally perfused for formalin fixation and paraffin embedding at 2, 24 and 72 hours post challenge. At 2 hours post challenge ME7 microglia produced IL-1β following either IL-1b or TNFα challenge while NBH microglia did not. Furthermore, there was very robust nuclear localisation of the NFkB subunit p65 in the astrocyte population and this was associated with very marked astrocytic synthesis of the chemokines CXCL-1 and CCL-2 in response to both cytokine challenges in ME7 animals. Conversely, very limited expression of these chemokines was apparent in NBH animals similarly challenged. Thus astrocytes are the primary chemokine synthesizing brain cell and in the prion-diseased brain are they primed to produce exaggerated chemokine responses to acute stimulation with pro-inflammatory cytokines. This abnormal pattern of chemokine expression is predicted to have consequences for leukocyte infiltration and the ramifications of an altered cellular infiltration pathway for the degenerating brain will be discussed.


This work was supported by the Wellcome Trust (Grant no. WT 078300 to CC) 


Neuroprotective effects of Withaferin A in three mouse models of amyotrophic lateral             sclerosis

Priyanka Patel 1 , Vivek Swarup 1 , Daniel  Phaneuf 1 , Jasna Kriz 1 , *Jean-Pierre Julien 1
1 Laval University, , Quebec, Canada
Abstract text :

Question: Recent studies have shown that Withaferin A (WA), an inhibitor of NF-κB activity was efficient in reducing disease phenotype in TDP-43 transgenic mice model of ALS. These findings led us to test WA in three mice models of ALS, SOD1G93A mice, SOD1G37R mice and TDP-43A315T transgenic mice model.

Methods: Intraperitoneal administration of WA at a dosage of 4mg/kg of body weight was initiated from postnatal day 40 (P40) till end stage in SOD1G93A mice, from 9 months till end stage in SOD1G37R mice and from 6 months of age in TDP43A315T mice.

Results: WA was able to improve the survival by more than a week in SOD1G93A and by two weeks in SOD1G37R familial mouse model. In addition WA administration also conferred significant neuroprotection in TDP43A315T transgenic mice model. Beneficial effects of WA in SOD1G93A mice model was accompanied by reduction in loss of motor neurons and also by reduction in level of misfolded SOD1 protein in the spinal cord as detected by immunoprecipitation with antibody specific to misfolded SOD1. WA was found to be an inducer of heat shock protein 27 (HSP-27), which could possibly explain reduced level of misfolded SOD1 and increased neuroprotection in WA treated mice. Moreover real-time imaging with the use of biophotonic SOD1G93A transgenic mice carrying luc (luciferase) and gfp (green fluorescent protein) reporter genes under the control of the murine GAP-43 promoter revealed that WA was able to reduce neuronal stress at post symptomatic stage.

Conclusion: Taken together, our results suggest that WA may represent a promising therapeutic drug for treatment of ALS.


Deletion of TLR-associated signaling adaptor TRIF significantly accelerates disease progression of ALS mice.

*Okiru Komine 1 , Noriko Fujimori-Tonou 1 , Hirofumi Yamashita 2 , Yasuhiro Moriwaki 3 , Hidemi Misawa 3 , Koji Yamanaka 1,4
1 RIKEN Brain Science Institute, Laboratory for Motor Neuron Disease, Wako-shi, Saitama, Japan
2 Kyoto University Graduate school of Medicine, Department of Neurology, Kyoto, Japan
3 Keio University, Department of Pharmacology, Tokyo, Japan
4 Nagoya University, Research Institute of Environmental Medicine, Nagoya, Japan
Abstract text :

 Microglial cells are central to innate immune responses against traumatic injury and bacterial infection in the central nervous system. Recent studies suggest that innate immunity might be also involved in the pathogenesis of neurodegenerative diseases, cerebral ischemia and brain injury. Although the recent works demonstrated that adaptive immune system is involved in the motor neuron disease process, the role of innate immune system in motor neuron disease was not fully investigated.

 To assess the contribution of innate immunity in the pathogenesis of amyotrophic lateral sclerosis (ALS), the gene expression profile of lumbar spinal cord from symptomatic mutant SOD1 mice was obtained by microarray approach and subsequent pathway analysis indicated the involvement of innate immune pathway. Next, to test the role of innate immunity in the pathogenesis of ALS, SOD1G93A ALS model mice were mated with MyD88 and TRIF (TIR domain-containing adaptor inducing IFNβ) deficient mice. MyD88 and TRIF are the essential adaptor proteins for Toll-like receptor mediated signaling pathway. As compared with SOD1G93A mice, MyD88/TRIF double-deficient and TRIF-deficient SOD1G93A mice exhibited the substantially shorter survival times with accelerated disease progression. The disease duration was shortened by 50% in TRIF-deficient SOD1G93A mice. In contrast, elimination of MyD88 in SOD1G93A mice showed marginal effect in survival time. In addition, the expression levels of pro-inflammatory chemokines, CCL5 and CXCL-10 were significantly suppressed in the spinal cord of TRIF-deficient SOD1G93A mice, as compared with SOD1G93A mice. To determine the cell type in which TRIF-dependent pathway contributes to the production of these chemokines, we examined the expression of chemokines in LPS-stimulated primary microglia or astrocyte derived from TRIF-deficient or MyD88-deficient mice. TRIF-dependent induction of these chemokines was observed only in LPS-stimulated microglia. Moreover, we found that infiltration of T-lymphocytes and other immune cells were significantly decreased in the spinal cord of symptomatic TRIF-deficient SOD1G93A mice.

 These results suggest that the basal level of TRIF-dependent innate immune activation of microglia is beneficial to slow disease progression of ALS models through the maintenance of pro-inflammatory chemokines and the infiltration of the immune cells to the spinal cords. The detailed analyses to clarify the role of these infiltrating immune cells are underway.


Connexin hemichannels are activated in astrocytes of a murine model of Alzheimer's disease 

*Annette Koulakoff 1 , Chenju Yi 1 , Xin Mei 1,2 , Christian Giaume 1,2
1 Collège de France, , PARIS, France
2 Institut de la Vision, , Paris, France
Abstract text :

Accumulating evidence supports an increasing role of astrocytes in the initiation or progression of a variety of neuropathological conditions. In Alzheimer's disease (AD), numerous data indicate that astrocyte properties are modified with potential deleterious effects on neurons. Accordingly, we have described changes in the expression of astroglial connexins, the gap junction channel and hemichannel forming proteins, in the vicinity of amyloid-b (Ab) plaques in brains from AD patients and murine models of AD. Also Ab peptide was shown to trigger astroglial Cx43 hemichannel activation in culture and in acute slices leading to neuronal degeneration. Hence, we have investigated hemichannel function of astroglial connexins in 8-9 months old APPswe/PS1dE9 mice that exhibit Ab plaques in the cortex and hippocampus. Ethidium bromide (EtBr) uptake performed in acute brain hemisphere slices was used as an index of hemichannel activation and was quantified in astrocytes identified by GFAP immunostaining. In APPswe/PS1dE9 mice, compared to wild-type mice of the same age, an increased uptake of EtBr was detected in the overall population of astrocytes. This uptake was blocked by carbenoxolone and lanthanum ions, indicating connexin hemichannel involvement. Such activation may be linked to the increase in resting astroglial [Ca2 ]i described in this mouse model. Interestingly, EtBr uptake was higher in reactive astrocytes contacting Ab plaques than in non-reactive astrocytes located far from (≥ 50 µm) these deposits. We are currently investigating their respective pharmacological profiles. Moreover, in APPswe/PS1dE9 mice knock-out for Cx30 generated in our facility, EtBr uptake was comparable to that observed in APPswe/PS1dE9 mice, suggesting that Cx43 is the major contributor to the hemichannel activity observed. Altogether, these results indicate that neuroglial interactions could be affected by astroglial hemichannel activation and could account for neuronal alterations or death observed in neurodegenerative diseases.

This work was supported by the CRPCEN, FRM, LECMA and France Alzheimer.


Glia impair neuron health in Juvenile Neuronal Ceroid Lipofuscinosis

*Jenny Lange 1 , Lotta Parviainen 1 , Greg Anderson 1 , Sybille Dihanich 2 , Payam Rezaie 3 , Hannah Mitchison 2 , Brenda Williams 1 , Jonathan Cooper 1
1 Institute of Psychiatry, King's College London, , London, United Kingdom
2 University College London, , London, United Kingdom
3 Open University, , Milton Keynes, United Kingdom
Abstract text :

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of autosomal recessively inherited lysosomal storage disorders affecting children and young adults, each of which is caused by a mutation in a different gene. A common feature across all NCLs is the early, localised glial activation that occurs long before the onset of neuron loss. This glial activation appears to be an accurate predictor of which neuron populations are vulnerable and will subsequently die.

Both astrocytes and microglia express the NCL gene products and it is likely that normal glial cell function may be compromised. Given the close functional relationship between neurons and glial cells it is possible that glial activation and/or dysfunction may affect neuronal health.

We have begun to explore the biology of astrocytes and microglia in the juvenile form of NCL using primary cultures from Cln3-/- mice. Defects in both astrocytes and microglia, including altered protein secretion profiles and impaired morphological and proliferative responses were apparent. Co-culturing with mutant microglia and astrocytes influenced the survival and morphology of wild type neurons, with more profound effects upon Cln3-/- neurons. Intriguingly, these effects were largely reversed by substituting wild type glia, which rescue mutant neurons.

 A pilot study has provided similar evidence for glial dysfunction in infantile NCL, including and altered protein secretion and response to stimulation. These changes appear to be different from those observed in juvenile NCL and we are in the process of exploring whether the glial cell phenotypes discovered so far, and their impact upon neurons are specific to the different forms of the disease.


Nrf2 activators: a novel strategy to promote oligodendrocyte survival in multiple sclerosis?

*Jamie Lim 1 , Susanne  van der Pol 1 , Joost Drexhage 1 , Elga de Vries 1 , Jack van Horssen 1
1 VU University Medical Center, , Amsterdam, Netherlands
Abstract text :

To investigate the potential of different Nrf2 activators to boost antioxidant enzyme expression in oligodendrocytes and protect them from reactive oxygen species (ROS)-mediated cell death.

Oligodendrocyte damage and loss are key features of multiple sclerosis (MS) pathology and oligodendrocytes appear to be particularly vulnerable to ROS. In vitro studies showed that ROS induce cell death and prevent the differentiation of oligodendrocyte precursor cells (OPCs) into mature myelin-producing oligodendrocytes. Hence, a potential therapeutic strategy to protect these cells from ROS-mediated damage is urgently needed. Here we investigated the efficacy of several compounds that are able to boost antioxidant enzyme production, including monomethyl fumarate (MMF), tert-butylhydroquinone (tBHQ), sulforaphane (SFN) and Protandim. These compounds are thought to exert their protective function via activation of the nuclear-factor-E2-related factor-2 (Nrf2) transcriptional pathway, which is involved in the production of antioxidant enzymes necessary for oxidative stress defense.

Primary rat oligodendrocytes were treated with different concentrations of MMF, tBHQ, SFN and Protandim. Expression of antioxidant enzymes were analyzed by PCR and Western blot analyses. To study the beneficial effects of the different Nrf2 activators, oligodendrocytes were first incubated with Nrf2 activators and subsequently exposed to various concentrations of hydrogen peroxide. Oligodendrocyte cell survival was measured by a live/dead cell viability assay.

SFN, MMF and Protandim are well-tolerated and induce Nrf2-driven antioxidant enzyme production in oligodendrocytes. Protandim was the most potent compound with regard to antioxidant enzyme induction and protected oligodendrocytes against ROS-induced cytotoxicity.

Our findings indicate that several Nrf2 activators are able to induce antioxidant enzyme production in oligodendrocytes. Interestingly, Protandim, a dietary supplement consisting of herbal ingredients, was the most potent protector of primary rat oligodendrocytes.
In future experiments we will determine whether Protandim can also promote the differentiation of OPCs under oxidative stress and test the clinical efficacy of Protandim in an experimental animal model for MS.


Time-course study of NG2 glia activation in the MPTP mouse model of Parkinson’s disease 

*Yingjun Liu 1 , Jiawei Zhou 1
1 Institute of Neuroscience, SIBS, CAS, , Shanghai, China
Abstract text :

NG2 glia is the fourth type of neuroglia in vertebrate nervous system, which also known as oligodendrocyte progenitor cell (OPC) in a developmental point of view. In adult brain, NG2 glia has a small cell body with highly branched extensions and represents a new type of glial cell differing from other glial cell types such as astrocyte, microglia and mature oligodendrocytes. Published studies by others have shown that NG2 glia is able to promptly respond to brain injury and activated in several neurodegenerative disease animal models including 6-OHDA-induced rat Parkinson’s disease model. However, the pattern and function of these activated NG2 glia remain largely unknown. Here, we performed a time-course study of NG2 glia activation in MPTP mouse model of Parkinson’s disease using immunohistochemistry. It was found that NG2 glia was activated as early as 24 hours after the last MPTP injection in the substantia nigra (SN) of MPTP-treated mice. This temporal character is very similar to those of microglia, indicating NG2 glia respond to MPTP challenges very quickly. The activation of NG2 glia became stronger over time and reached the peak at about 5 days after the final MPTP injection. Then, the extent of NG2 glia activation declined gradually and diminished 10 days after MPTP injection. Interestingly, we found that prominent NG2 glia activation could only be observed in the substantia nigra. The dorsolateral striatum which is the projection target of nigral dopaminergic neurons, however, was devoid of NG2 glia activation in all the time-points examined. In summary, we characterized the NG2 glia activation in MPTP mouse model of Parkinson’s disease. These data suggest that activated NG2 glia may play a role in the degenerative process of dopaminergic neurons in PD. 


Molecular Mechanisms in Astrocytic Degradation

*Camilla Lööv 1 , Anna Erlandsson 1,2
1 Uppsala University, , Uppsala, Sweden
2 Uppsala University, Neuroscience/Neurosurgery, Uppsala, Sweden
Abstract text :

We have previously shown that astrocytes effectively engulf dead cells after trauma both in vitro and in vivo, but that they store the ingested material rather than degrade it. Compared to macrophages, which degrade engulfed, dead cells within hours, our data show that astrocytes store the ingested material for weeks before the degradation is completed. To further study the routes of degradation and the possibilities to speed up this process we have used a cell culture model where UV-treated, dead cells are added to stem cell-derived astrocytes. By labeling the dead cells with the pH-sensitive dye pHrodo prior to the engulfment, we demonstrate that the pH in the astrocytic phago-lysosomes is higher than in professional phagocytes. Interestingly, Lamp1 and Lamp2, which are involved in the phago-lysosome fusion, are both highly expressed in the astrocytes, particularly around the ingested material. Dendritic cells are known to express the inhibitory protein Rab27a that slow down the degradation in order to preserve antigen for presentation. Rab27a prolongs the actin coating around the phagosomes, which physically inhibit the phago-lysosome fusion, and interacts with Nox-2, which leads to an increased consumption of protons. Western blot analysis shows a high expression of Rab27a in our cell cultures which may explain the slow degradation. Moreover, dead cells in astrocytes are surrounded by actin rings for a long period of time after the ingestion. To counteract this, the astrocytes were treated with 0.1 or 1 µM of the actin inhibitor Latrunculin B. The number of actin rings were significantly lower in the cultures treated with 1 µM of Latrunculin B compared to controls, but the intensity of pHrodo was unaltered indicating that the prolonged degradation may be due to a higher pH in the lysosomes rather than a delayed actin coating. Next we will investigate the effect of Rab27a expression on the degradation process by performing siRNA experiments.


Energy metabolism deficits in Huntington’s disease: key role of astrocytes-neurons interactions

*Lydie Boussicault 1 , Anne-Sophie Hérard 1 , Fanny Petit 1 , Carole Malgorn 1 , Nicolas Merienne 1 , Marie Claude Gaillard 1 , Carole Escartin 1 , Thierry Delzescaux 1 , Emmanuel Brouillet 1 , Philippe Hantraye 1 , Jean Mariani 2 , Céline Vega Roiatti 2 , Gilles Bonvento 1
1 CEA, , Fontenay aux Roses, France
2 UMR CNRS 7102 UPMC, , Paris, France
Abstract text :

Huntington disease (HD) is a dominant inherited neurodegenerative disorder caused by an unstable expansion of a CAG repeat within the gene encoding for Huntingtin protein (Htt). This mutation induces formation of a poly-glutamine tract in the mutant Htt (mHtt) protein that prones its aggregation into the cells. HD is characterized by an initial and massive degeneration of the medium spiny neurons in the striatum with later neuronal loss in cortex, globus pallidus, and other structures leading to motor and cognitive symptoms. Alterations of energy metabolism contribute to HD pathogenesis but the mechanisms responsible for these alterations are not well understood. A recent PET study provided evidence for a selective impairment of striatal glycolytic and not oxidative metabolism of pre-symptomatic HD patients. In the brain, glycolysis is
predominantly an astrocytic metabolic process, whereas oxidative metabolism is primarily neuronal raising the question that metabolic defects in HD could originates from astrocytes. The purpose of our study was to characterize (1) the metabolic alterations in vivo in a transgenic mouse model of HD (BACHD mice) and (2) the respective roles of astrocytes and neurons in metabolic defects occurring in HD using an in vitro strategy. BACHD mice express the human full length Htt protein with 97 glutamine repetitions under the human Htt promoter into a Bacterial Artificial Chromosome. BACHD mice present a slow disease progression with motor deficits occurring at 6 months and progressive neuronal aggregates from 12 months, reflecting human pathology. First, we performed [14C]-2-deoxyglucose autoradiography experiments in vivo to assess energy metabolism in 14 months BACHD (n=6) and WT (n=6) mice. We performed a 3D analysis of the whole brain glucose uptake without any regional a priori. Using Statistical Parametric Mapping (SPM), a voxel-wise statistical analysis approach, we showed that compared to age-matched WT mice, 14 months BACHD mice present hypometabolism in the striatum, like pre symptomatic HD patients, and also in the hippocampus. Hypermetabolism was also detected in the hypothalamus.
Second, we performed an in vitro study to dissect out the cellular mechanisms responsible for these metabolic changes. By using cell insert, we realized neurons/astrocytes co-culture without any physical contact. This culture system allowed us to grow neurons in presence of WT or BACHD
astrocytes. We showed that both BACHD and WT neurons have a decreased [14C]-2-deoxyglucose
uptake when cultured in presence of BACHD astrocytes but not WT astrocytes. These results
collectively suggest that energy defects in BACHD mice are due to non-cell autonomous mechanisms by which astrocytes expressing mHtt induce neuronal metabolic dysfunction.


Expression and function of the late-onset Alzheimer disease associated CD33 in human microglia 

*Mona Ann Mathews 1 , Marcus Grobe-Einsler 1 , Kristin Roy 1 , Harald Neumann 1
1 University of Bonn, , Bonn, Germany
Abstract text :

Microglia are described to be involved in many neurodegenerative diseases like Alzheimer’s disease (AD). CD33 (Siglec-3) is a rapidly-evolving, highly species specific sialic-acid binding Ig-like lectin expressed primarily on cells of myeloid lineage. Recent studies have shown an association between a CD33 polymorphism and late-onset AD. The role of CD33 in human microglia was studied in induced pluripotent stem (iPS) cell-derived microglial precursor line (iPSdM).

Microglial cell lines were differentiated from iPS cells and represent in vitro models for human microglia. RT-PCR and flow cytometry analysis of iPSdM showed gene transcription and protein expression of CD33 respectively. The protein expression was increased by breaking the cis-interacting sialic acids on the microglial glycocalyx using sialidases. CD33-Fc fusion protein was designed and purified via the Fc tag to study binding patterns of CD33 to various cell types expressing different glycocalyx structures. Additionally, iPSdM exhibiting either an over expression or knockdown of CD33 will be used to determine protein functions. Furthermore, preliminary immunohistochemical analysis of control brain tissue compared to AD patients showed increased CD33 protein expression in CD68 positive cells, in the latter.

In summary, data show that CD33 is expressed by human microglial cell line enabling further characteristic and functional analysis of the protein. Furthermore, preliminary analysis on primary brain tissue corroborates the association of CD33 - expressed on microglia - to late-onset AD.


In vivo therapeutic effects and oligodendrocyte protection from excitotoxicity by the magl inhibitor JZL184

*Susana Mato 1 , Ana Bernal-Chico 1,2 , Manuel Canedo 1,2 , Maria Victoria Sánchez-Gómez 1,2,3 , Alberto Pérez-Samartín 1,2,3 , Rafael Rodríguez-Puertas 4 , Carlos Matute 1,2,3
1 University of the Basque Country-UPV/EHU, Department of Neurosciences, Leioa, Spain
2 Achucarro Basque Center for Neuroscience-UPV/EHU, , Zamudio, Spain
3 Instituto de Salud Carlos III (ISCIII; CIBERNED), , Leioa, Spain
4 University of the Basque Country-UPV/EHU, Department of Pharmacology, Leioa, Spain
Abstract text :

Multiple sclerosis (MS) is a chronic disease of the human central nervous system that is characterized by focal lesions with inflammation, infiltration of immune cells, demyelination and axonal damage. Activation of cannabinoid CB1/CB2 receptors is considered a potential therapeutic strategy for the treatment of MS, based on the evidence that exogenous cannabinoid agonists exert neuroprotective and immunosuppressive effects in experimental models of the disease. Nevertheless, the therapeutic use of synthetic and/or plant-derived agonists acting on brain cannabinoid receptors is limited by the possible adverse responses related to memory and learning impairment. An alternative approach that could avoid this limitation consists of enhancing the concentration of the main endocannabinoids (AEA, 2-AG) by increasing their synthesis or decreasing their degradation. The main objective of this study was to analyze the effects of JZL184, a selective inhibitor of 2-AG hydrolyzing enzyme monoacylglycerol lipase (MAGL), in the chronic EAE model of MS. Mice were treated daily with JZL184 (8 mg/kg and 32 mg/kg) or vehicle from onset of the motor symptoms to the end of the experiment. Comparison of the motor score curves indicated that both doses of JZL184 ameliorated the deficits observed in vehicle-treated mice during the disease course. Nevertheless, the beneficial effect of the 32 mg/kg dose was no longer evident in mice scored at 40 dpi. Chronic treatment with 8 mg/kg JZL184 reduced the conduction latency of the corticospinal tract measured at the end of the experiment, as well as the number and size of inflammatory lesions in spinal cord, whereas chronic administration of the high dose of the MAGL inhibitor had no effect on these parameters. Importantly, treatment with the 32 mg/kg dose of JZL184 reduced the coupling ability of cannabinoid receptors to Gi/o proteins, measured by [35S]GTPγS autoradiography, in all the brain areas analyzed. Finally, incubation with JZL184 prevented cytotoxicity by activation of AMPA-type glutamate receptors in oligodendrocyte precursors in vitro. This protective effect of the JZL184 was blocked by the CB1 receptor antagonist AM281. Our findings point to the involvement of CB1 receptors in the in vivo therapeutic effects of JZL184, and suggest that chronic administration of MAGL inhibitors may be a promising strategy for the treatment demyelinating disorders in which oligodendrocyte excitotoxicity plays an important role as mechanism of white matter damage.

Acknowledgements: Founded by MICINN (SAF2010-21547), CIBERNED and Gobierno Vasco. Susana Mato, Ana Bernal and Manuel Canedo are recipients of a Ramón y Cajal contract and fellowships from the University of the Basque Country-UPV/EHU and FPI, respectively.


Intracellular trafficking, matrix association and function of VEGF in astroglial cells

*Kristof Egervari 1 , Gael Potter 1 , Maria-Luisa Guzmán-Hernández 2 , Patrick Salmon 1 , Tamas Balla 2 , Bernhard Wehrle-Haller 1 , Jozsef Z Kiss 1
1 University of Geneva, , Geneva, Switzerland
2 National Institute of Child Health and Human Development, , Bethesda, United States
Abstract text :

Vascular endothelial growth factor (VEGF) is a major regulator of neurovascular remodeling following brain injury. While much have been learnt about VEGF functions on target endothelial cells, little is known about its intracellular trafficking, mode of secretion and matrix interactions in “source” cells, i.e. mainly reactive astrocytes. Here, we generated a VEGF::GFP fusion protein to follow the distribution of VEGF165 during its trafficking in primary astrocytes and COS7 cells. We found that VEGF::GFP forms dimers and gets glycosylated similarly to wild type, and as expected, the molecule follows the endoplasmic reticulum-Golgi pathway. However, its post-Golgi trafficking suggests a unique route with features that do not conform the classical constitutive secretory pathway: the secretion of VEGF165 occurs even at 19 °C and shows a Ca2 - and PKC-induced increase. We investigated the distribution of VEGF in polarized primary astrocytes in an in vitro scratch wound assay. In these cells, VEGF::GFP appeared to follow a vectorial distribution and accumulated behind the leading edge. The accumulation appeared to be on the extracellular surface, moreover, ultrastructural immunogold labeling revealed that extracellular VEGF::GFP remains associated with discrete areas of the cell membrane and is accumulated in caveolae as well as in microvesicular shedding elements. This particular localization corresponds to fibrillar adhesions (FB), where VEGF::GFP is co-localized with fibronectin. We also observed that VEGF::GFP is targeted to adherens junctions between astrocytes, however, at these sites VEGF::GFP was not co-localized with fibronectin. Finally, we show in FRAP experiments that integrin turnover is decreased in VEGF associated FBs, which raises the possibility of an autocrine/paracrine regulation of astrocytic functions. Together, these findings have strong implications for understanding focal coordination of angiogenesis and vascular remodeling by astroglia derived VEGF.


Characterization of adhesional and cytomechanical properties of single living cells and tissue slices by AFM 

*Torsten Müller 1 , Tanja Neumann 1 , Anne Hermsdörfer 1 , Carmen Pettersson 1 , Gerd Behme 1
1 JPK Instruments AG, , Berlin, Germany
Abstract text :

Topography, roughness and mechanical properties of micro environment are crucial parameters influencing cell adhesion/motility, morphology and mechanics as well as the development of cells e.g. stem/progenitor cells, and neuronal cells [1-5]. Atomic force microscopy is a powerful tool not only to study the morphology in terms of high resolution imaging and roughness measurements, but also to map mechanical and adhesive properties of the sample/cells and tissues. Combining these remarkable abilities with advanced optical microscopy allows for extensive characterization of biomaterials and tissue slices [6-8]. However, commercially existing technical solutions are either time consuming, or only limited practicable for soft, sticky, or fragile samples.  Therefore, we developed a new force curve based AFM mode - Quantitative Imaging (QI™). The novel QI tip movement algorithm prevents lateral forces and controls the vertical forces for nondestructive imaging. To demonstrate the performance and flexibility QI™ mode we investigated topography, adhesion properties, and Young’s modulus local distribution of living dorsal root ganglion cells.


A specialized platform - CellHesion® - has been developed to run single cell force spectroscopy (SCFS) with a need for long-range cell-surface and cell-cell binding experiments with up to 100 microns pulling length.  Using single cell force spectroscopy (SCFC), cell-cell adhesion can be quantified [e.g. 3], and the contribution of different components e.g. from the extra cellular matrix, can be assessed [9]. A new technical solution will be demonstrated if a cantilever attached cell can be transferred through the liquid-air interface. This approach allows to measure the adhesion of the same individual single cell on different materials within different dishes as it will presented for CHO cell adhesion on plastic as well as albumin coated surfaces.


We want to present the progress in automatic data processing and display to investigate topography, Young’s modulus and local adhesion phenomena on transparent and non-transparent biomaterials like titanium, peptide functionalized surface or cell layers, and tissue slices.



[1] Engler et al., Cell 126, 677-689 (2006)

[2] Docheva et al., Biochem. Biophys. Res. Com. 402, 361-366 (2010)

[3] M. Krieg et al.Nature Cell Biology 10(4), 429-436 (2008)

[4] P. Moshayedi et al., J. Phys.: Condens Matter 22, 194114pp. (2010)

[5]Li-Y. Chianget al., Nature Neuroscience 14, 993-1000 (2011)

[6] K. Franze et al., Biophysical J. 97, 1883-1890 (2009)

[7] A.F. Christ et al., J. Biomechanics 43, 2986-2992 (2010)

[8] Kirmse et al., J. Cell Science 124, 1857-1866 (2011)

[9] J. Friedrichs et al., Nature Protocols 5(7), 1353-1361 (2010)


The Role of FoxO3a in Oligodendrocyte Precursor Cell Differentiation

*Sara Ali Abdulla 1 , Yasir Ahmed Syed 2 , Mark Kotter 2
1 Cambridge University, , Cambridge, United Kingdom
2 Welcome Trust - MRC Center For Stem Cell Biology, , Cambrige, United Kingdom
Abstract text :

FoxO3a is a transcription factor that is highly expressed in the brain. However, little is known about the response of FoxO3a in regulating oligodendrocyte precursor cell (OPC) differentiation.  This study tested the response of FoxO3a in regulating OPC differentiation and its role in the myelination process.  A microarray analysis of early regulators of OPC differentiation showed that Foxo3a is significantly down-regulated during the onset of OPC differentiation.  In agreement, mRNA expression analysis of cell lysates collected at various differentiation time points demonstrated that the active form of FoxO3a is down-regulated during OPC differentiation. Over expression of FoxO3a through the transfection of pure OPC cultures with adenoviral vector constructs, of a constitutively active form of FoxO3a, resulted in an inhibition of OPC differentiation. On the other hand, siRNA knock downs of FoxO3a promoted OPC differentiation. In addition, ultrastructural analysis of the corpus callosum of FoxO3a null mice showed a significant increase in the number of myelinated axons in comparison to wild type. Consequently, the data suggests that FoxO3a is an essential regulator of the myelination process.


Gene Regulatory Networks Underlying Astrocyte Identity and Potential

*Angela Bithell 1 , Alessandro Michelucci 2 , Isaac Crespo 2 , Matthew Burney 1 , Caroline Johnston 1 , Kee-Yew Wong 3 , Siaw-Wei Teng 3 , Brenda Williams 1 , Lawrence Stanton 3 , Antonio Del Sol 2 , Noel Buckley 1
1 King's College London, Institute of Psychiatry,, , London, United Kingdom
2 Luxembourg Centre for Systems Biomedicine, , Luxembourg, Luxembourg
3 Genome Institute of Singapore, , Singapore, Singapore
Abstract text :

Neural stem cells (NSCs) represent a potentially valuable resource when considering repair strategies for CNS damage. Unlike their embryonic counterparts, adult neural stem cells (aNSCs) in the two neurogenic niches have an astrocyte-like identity, raising the question of what regulates this aNSC state. Some early postnatal astrocytes in the parenchyma also retain some NSC-like characteristics and in vitro display self-renewal and multipotency, though these properties are lost following in vivo maturation. Interestingly, following injury, adult astrocytes can become reactive and reacquire NSC-like properties, whilst others can generate new neurons through forced expression of specific transcription factors. We are aiming to elucidate the gene regulatory networks underlying specific NSC and astrocyte states and transitions between them using transcriptomic and epigenomic tools alongside computational methods in different NSC and astrocyte populations. We will describe our latest findings from an in vitro system that aims to model immature versus mature astrocytes derived from a common progenitor. From microarray data, we have identified candidate genes and pathways involved in regulating astrocyte potential versus differentiation, including a role for specific pro-inflammatory signalling. We will also describe the associated epigenetic signatures that accompany cell state and discuss the implications of our latest findings.


NFAT-c3 promotes calcium-dependent MMP3 expression in activated astroglial cells

*Eva Cano 1 , Fernando Neria 1 , María del Carmen Serrano-Pérez 2 , Patricia Velasco 1 , Pedro Tranque 2
1 Instituto de Salud Carlos III, , Majadahonda, Madrid, Spain
2 University of Castilla-La Mancha, Facultad de Medicina and Instituto de Investigación en Discapacidades Neurológicas, Albacete, Spain
Abstract text :

Increase in intracellular calcium ([Ca2 ]i) is a key mediator of astrocyte signalling, important for activation of the calcineurin (CN)/nuclear factor of activated T cells (NFAT) pathway, a central mediator of inflammatory events. We analysed the expression of matrix metalloproteinase 3 (Mmp3) in response to increases in [Ca2 ]i and the role of the CN/NFAT pathway in this regulation. Astrocyte Mmp3 expression was induced by overexpression of a constitutively-active form of NFATc3, whereas other MMPs and tissue inhibitor of metalloproteinases (TIMP) were unaffected. Mmp3 mRNA and protein expression was also induced by calcium ionophore (Io) and 2’(3’)-O-(4-benzoylbenzoyl) adenosine 5´-triphosphate (Bz-ATP). Mmp3 upregulation was prevented by the CN inhibitor cyclosporin A (CsA). Ca2 -dependent astrocyte Mmp3 expression was also inhibited by actinomycin D, and a Mmp3 promoter luciferase reporter was efficiently activated by increased [Ca2 ]i, indicating regulation at the transcriptional level. Furthermore, Ca2 /CN/NFAT dependent Mmp3 expression was confirmed in pure astrocyte cultures derived from neural stem cells (Ast-NSC), demonstrating that the induced Mmp3 expression occurs in astrocytes, and not microglial cells. In an in vivo stab-wound model of brain injury, MMP3 expression was detected in NFATc3-positive scar-forming astrocytes. Since [Ca2 ]i increase is an early event in most brain injuries, these data support an important role for Ca2 /CN/NFAT-induced astrocyte MMP3 expression in the early neuroinflammatory response. Understanding the molecular pathways involved in this regulation could provide novel therapeutic targets and approaches to promoting recovery of the injured brain.


Overexpression of CPEB3 leads to astrocyte dysfunction

*Tushar Deshpande 1 , Vamshidhar  Vangoor 1 , Sada Turimella 1 , Lech  Kaczmarczyk 1 , Peter  Bedner 1 , Elena  von Staden 1 , Amin  Derouiche 1 , Ronald  Jabs 1 , Gerald Seifert 1 , Christian Steinhäuser 1 , Martin  Theis 1
1 Institute of Cellular Neurosciences, , Bonn, Germany
Abstract text :

Cytoplasmic polyadenylation and element binding (CPEB) proteins are a class of translational regulators expressed in brain. CPEB1 in astrocytes regulates the translation of β-catenin and cyclin B1 mRNA. In the present study the expression and functions of other CPEB (2-4) proteins were investigated. The expression of CPEB proteins in astroglial cell types was analyzed by single cell RT-PCR, in situ hybridization, FACS analysis and immunostaining. To study the functional role of CPEB3, transgenic mice overexpressing CPEB3 in astrocytes were generated. CPEB3 overexpression without the endogenous 3’ untranslated region (UTR) led to the downregulation of astrocytic connexins (Cx43 & Cx30). Consistently, in the hippocampus of CPEB3 overexpressing mice, intercellular gap junction coupling was strongly impaired. CPEB3 overexpression also led to downregulation of Glutamate transporter 1 (GLT-1) and glutamine synthetase (GS), key players involved in extracellular glutamate clearance. We have now raised mice overexpressing CPEB3 with the endogenous 3’ UTR. In these mice, we observed the downregulation of Cx43, Cx30, GLT-1 and GS. Since interastrocytic coupling and astrocytic GLT-1 and GS activities are downregulated in epilepsy patients with hippocampal sclerosis, we hypothesize that upregulation of CPEBs in astrocytes may contribute to the pathogenesis of epilepsy by causing astrocyte dysfunction.


Transcriptional remodeling in microglia from the penumbra area in an experimental mouse stroke model

*Helene Hirbec 1 , Catherine Rey 2 , Alexis Menteyne 3 , Nicolas Nazaret 2 , Mathilde Boulpicante 1 , Joel Lachuer 2 , Etienne Audinat 3 , François Rassendren 1
1 IGF, , Montpellier, France
2 ProfileXpert, , Lyon , France
3 INSERM U603, , Paris, France
Abstract text :

Microglia are the main resident immunological cells the CNS. In the healthy brain, microglial cells are in a surveillance state whereas upon rupture of CNS homeostasis they enter activated states. In ischemic stroke, activated microglia is one of the most important cellular components of post-stroke neuroinflammation, which mainly occurs in and around the area of the infarct. Microglia activation is characterized by morphological alterations, functional and transcriptional remodeling, which account for the acquisition of immune phenotypes. Purinergic receptors are known to play important roles in microglia activation, and P2X4R have been suggested as potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain diseases. 

In the present study, we have developed an experimental approach based on laser micro-capture to isolate microglia from the penumbra area at different post-lesion times (from 4h to 7 days post-lesion). The repertoire of genes expressed by microglial cells was determined through cDNA microarray analysis. With this approach we have been able to determine clusters of genes that are co-regulated thus revealing the time course of microglia activation in this model.

In addition, we have compared the transcriptional remodeling of microglia in wild-type and P2X4-deficient mice. Our results suggest that wild-type and P2X4R KO mice present specific transcriptional profiles, which only partially overlapped. Thus our data suggest that P2X4R play significant roles in the regulation of microglial cells functions.


Promises and pitfalls of Pannexin1 transgenic mice.

Regina Hanstein 1 , Hiromitsu Negoro 2 , Naman Patel 1 , Anne Charollais 3 , Paolo Meda 3 , David Spray 1 , Sylvia Suadicani 1 , *Eliana Scemes 1
1 Albert Einstein College of Medicine, , NY, United States
2 Albert Einstein College of Medicine, Urology, New York, United States
3 University of Geneva, Cell Physiology and Metabolism, Geneva, Switzerland
Abstract text :

Gene targeting strategies have become a powerful technology for elucidating mammalian gene function. The recently generated knockout (KO)-first strategy produces a knockout at the RNA processing level and also allows for the generation of conditional KO alleles by combining FLP/FRT and Cre/loxP systems, thereby providing high flexibility in gene manipulation. However, this multipurpose KO-first cassette might produce hypomorphic rather than complete knockouts if the RNA processing module is bypassed. Moreover, the generation of a conditional phenotype is also dependent on specific activity of Cre recombinase. Here, we report the use of an efficient molecular biological approach to test pannexin1 (Panx1) mRNA expression in global and conditional Panx1 KO mice derived from the KO-first mouse line, Panx1tm1a(KOMP)Wtsi. Using qRT-PCR, we demonstrate that tissues from wild-type mice show a range of Panx1 mRNA expression levels, with highest expression in trigeminal ganglia, bladder and spleen. Unexpectedly, we found that in mice homozygous for the KO-first allele, Panx1 mRNA expression is not abolished but reduced by 70% compared to that of wild-type tissues. Thus, Panx1 KO-first mice present a hypomorphic phenotype. Crosses of Panx1 KO-first with FLP deleter mice generated Panx1f/f mice. Further crosses of the later mice with mGFAP-Cre or NFH-Cre mice were used to generate astrocyte- and neuronal-specific Panx1 deletions, respectively. A high incidence of ectopic Cre expression was found in offspring of both types of conditional Panx1 KO mice. Our study demonstrates that Panx1 expression levels in the global and conditional Panx1 KO mice derived from KO-first mouse lines must be carefully characterized to ensure modulation of Panx1 gene expression. The precise quantitation of Panx1 expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of Panx1 under physiological and pathological conditions.


Lactate modifies neuronal excitability through both NMDA and KATP receptors: importance for plasticity genes expression

*Igor Allaman 1 , Jiangyan  Yang 1 , Jean-Marie Petit 1,2 , Gabriele Grenningloh 1 , Evelyne Ruchti 1 , Pascal Jourdain 1 , Pierre Magistretti 1,2
1 EPFL, , Lausanne, Switzerland
2 Centre de Neuroscience Psychiatriques, Département de Psychiatrie, Prilly/Lausanne, Switzerland
Abstract text :

Release of glycogen-derived L-lactate from astrocytes has been shown to play an important role in the establishment of long term memory (Suzuki et al, Cell, 2011). In an accompanying poster, we present data demonstrating that in cultured neurons L-lactate induces plasticity-related genes expression through a NMDA-dependent mechanism (i.e. Arc, Zif268 and c-Fos). Here, we describe the effect of L-lactate on neuronal excitability by performing electrophysiological recordings of cultured cortical neurons. We observed that application of L-lactate (10 mM) triggers an inward current with an amplitude of -0.55 /- 0.1 nA and slow kinetics, with a peak reached at 189 /- 55s (called Ilac). The membrane current decreases gradually to reach a plateau, with a mean inward current of -0.13 /- 0.04 nA persisting up to 780s (13 minutes) after addition of L-lactate (called Ipyr).  In order to assess L-lactate specificity on the generation of these currents L-pyruvate, another monocarboxylate, as well as D-lactate, the non-metabolized enantiomer of L-lactate) were tested. In contrast to L-lactate, L-pyruvate (10 mM) induced a low-amplitude sustained current (-0.07 /- 0.03 nA) similar to the late-phase slow current evoked by L-lactate (i.e Ipyr), but no current similar to Ilac. D-lactate on its side did not elicit any specific current. Pharmacological characterization of Ilac and Ipyr currents demonstrate that Ilac is generated by NMDA receptors (as it is blocked by MK801) and relies on active NMDA receptors (as it is blocked by either glutamate or glycine binding sites inhibitors). In contrast, generation of Ipyr by both L-lactate and L-pyruvate is prevented by diazoxide, a KATP opener, whereas Ilac was unaffected by the treatment. In order to determine the importance of KATP for plasticity-related gene expression, the KATP closer glibenclamide (200 uM) was applied to neurons and mRNA gene expression of Arc and Zif268 quantified. Results obtained demonstrate that Arc and Zif268 mRNA expression are unaffected by glibenclamide. As a whole this set of data demonstrates that L-lactate generates two types of inward currents in neurons i.e. NMDA-dependent (Ilac) or KATP-dependent (Ipyr). Moreover, they highlight the NMDA-dependent Ilac current as the key mediator, in contrast to KATP, of L-lactate-induced plasticity gene expression. This is further sustained by the observation that L-pyruvate does not reproduce the effect of L-lactate on gene expression (see accompanying poster). This set of results provides novel insights into the mechanisms of action of L-lactate as an inducer of plasticity gene expression, and an important signaling molecule for neuronal plasticity.


Astrocytes in the striatum act as a reservoir of L-DOPA but less convert to dopamine

*Masato Asanuma 1 , Ikuko Miyazaki 1
1 Okayama Univ., Dept. of Brain Science, Okayama, Japan
Abstract text :

Dopamine transporter (DAT) is expressed not only in catecholaminergic neurons but also in astrocytes. We previously showed that repeated L-DOPA treatment markedly induced expression of DAT and apparent dopamine (DA)-immunoreactivity in the reactive astrocytes in the striatum of animal models of Parkinson's disease. It is thought that astrocytes can uptake both L-DOPA and DA via neutral amino acid transporter LAT and DAT, respectively. Therefore, uptake and metabolism of L-DOPA and DA in the striatal astrocytes may influence their availability in dopaminergic system of parkinsonian patients. To clarify uptake and metabolism of L-DOPA and DA in striatal astrocytes, the contents of L-DOPA, DA and their metabolites were measured after the L-DOPA/DA treatment using primary cultured astrocytes. First, we revealed the expression of neutral amino acid transporter LAT and aromatic amino acid decarboxylase (AADC) in the striatal astrocytes. The level of L-DOPA in astrocytes was markedly increased after 4-hr L-DOPA exposure, but DA was not detected in the astrocytes 4 or 8 hr after the treatment. On the other hand, the DA treatment for 4 hr increased levels of DA and its metabolites, especially DOPAC. These results indicate that uptaken DA into astrocytes is rapidly metabolized and that uptaken L-DOPA never be converted to DA in astrocytes. Furthermore, level of uptaken L-DOPA in cultured striatal astrocytes was rapidly decreased after removing extracellular L-DOPA. Taken together, the present results suggest that striatal astrocytes act as a reservoir of L-DOPA to uptake or release L-DOPA depending on the extracellular L-DOPA concentration, but have less ability to convert L-DOPA to dopamine.


Postnatal down-regulation of the γ2 subunit of GABAA receptors in NG2 cells precedes synaptic-to-extrasynaptic change in GABAergic transmission mode

*Maddalena Balia 1 , Mateo Vélez-Fort 1 , Stephan Passlick 2 , Christoph Schäfer 2 , Etienne Audinat 1 , Christian Steinhäuser 1 , Gerald Seifert 2 , Maria Cecilia Angulo 1
1 INSERM U603, , Paris, France
2 Institute of Cellular Neurosciences, University of Bonn, Bonn, Germany
Abstract text :

In neurons, synaptic and extrasynaptic GABAA receptors (GABAARs) differ in their subunit composition, conferring them distinct functional and pharmacological properties. Oligodendrocyte precursor cells, also called NG2 cells, are contacted by bona fide neuronal GABAergic synapses. However, we recently showed a synaptic to extrasynaptic switch in the mode of transmission between GABAergic interneurons and NG2 cells during postnatal development of the somatosensory cortex. We therefore hypothesized that the postnatal switch of GABAergic transmission in NG2 cells is accompanied by changes in the expression of GABAAR subunits. To test for this hypothesis, we stimulated neuronal fibers to evoke GABAAR-mediated responses in NG2 cells recorded in acute slices of the somatosensory cortex of NG2-DsRed transgenic mice. The effect of zolpidem and α5IA on evoked GABAergic responses reveals the predominance of functional α1- and α5-containing GABAA receptors, respectively, at interneuron-NG2 cell synapses early in development. However, the expression level of α5 decreases when responses rely exclusively in extrasynaptic transmission at more mature developmental stages. More importantly, specific pharmacology for the γ2 subunit, a crucial molecular component for the clustering of GABAA receptors at postsynaptic sites, demonstrated a down-regulation of this subunit in NG2 cells prior to the complete loss of GABAergic synaptic activity. In keeping with the synaptic nature of the γ2 subunit in neurons, this molecular change in NG2 cells correlates with the switch from synaptic to extrasynaptic transmission. To corroborate these functional data, we performed single cell multiplex RT-PCR of α1-5, β1-3, γ1-3 and δ subunit mRNAs in NG2 cells of the second and fourth postnatal weeks. Despite the large heterogeneity of subunit mRNA expression at both developmental stages, we found that the number of cells expressing γ2 mRNAs dramatically decreases in the fourth postnatal week. In conclusion, the expression loss of the γ2 subunit is an important molecular determinant impacting the change of transmission modes between interneurons and NG2 cells during cortical development.

Financial support: FRC, ANR blanche, ARSEP, DFG (SFB/TR3) and EU (FP7-202167 Neuroglia).


Volume coverage by microglial processes is reduced in the aging brain and occurs significantly earlier in mouse models of Alzheimer’s disease

*Rona Baron 1 , Anna Nemirovsky 1 , Alon  Monsonego 1
1 Ben-Gurion University, , Ber-Sheva, Israel
Abstract text :

Microglia integrate within the neural tissue with a distinct ramified morphology through which they scan the surrounding neuronal network, a process which appears to contribute to the integrity, maintenance and functioning of the brain. Since microglia are long-lived, they are subjected to senescence processes which may severely compromise their function with age. Here we used a digital tool for the quantitative morphometric characterization of fine cortical microglia structures in mice. We thus followed the morphological changes microglia underwent with aging and with the progression of Alzheimer’s-like disease. Compared with microglia in young mice, microglia in old mice are less ramified and possess less branches and fine processes, a phenomenon which was associated with increased expression of pro-inflammatory genes. Notably, a similar microglial pathology appeared 6-12 months earlier in mouse models of Alzheimer’s disease (AD). We thus demonstrate that in addition to promoting neurotoxic inflammation, amyloid plaques attract the microglia and modify their structure. This, in turn, causes a severe microglial process deficiency, which possibly results in compromised neuronal function and repair.


Histamine triggers microglial phagocytosis

*Liliana Bernardino 1 , Tatiana Saraiva 1 , Ana Clara Cristóvão 1 , Marta Esteves 1 , Graça Baltazar 1 , Sandra Moreira Rocha 1
1 University of Beira Interior, , Covilhã, Portugal
Abstract text :

Microglial phagocytosis is a vital phenomenon for the clearance of damaged and death cells or infectious agents in a context of brain injury or infection, respectively. In addition, microglia can boost synaptic plasticity by the phagocytosis of axon terminals and dendritic spines. Therefore, it is crucial to better understand the mechanisms involved in microglia clearance in order to devise new strategies to promote an efficient brain repair. Recently, we showed that histamine modulates microglia motility and cytokines release. In this work, we aimed to investigate the role of this molecule and its receptors in microglia-induced inflammation by evaluating microglial phagocytic activity and Reactive Oxygen Species (ROS) production. For that purpose, an IgG-opsonized latex bead assay was performed in N9 murine microglial cell lines exposed to lipopolysaccharide (LPS) or histamine 1, 10 and 100 µM. We showed that histamine significantly stimulated phagocytosis of opsonized latex beads via H1 receptor activation. This effect was accompanied by the rearrangement of cytoskeleton analyzed by phaloidin and acetylated tubulin expression and cellular distribution. The phagocytic activity was significantly reduced after pretreatment with apocynin, a putative NAPDH oxidase (Nox) inhibitor. All Nox isoforms were expressed by microglial cells, but only the expression of Nox1 was increased by treatment of histamine. Rac1, an important subunit for the activation of Nox1, was also activated by histamine. In addition, histamine induced ROS production via H1 and H4 receptor activation. In conclusion, histamine plays an important role in the regulation of microglial phagocytosis, which is mediated by Nox1 and Rac1 activation.  


Astrocytes uptake extracellular plasminogen and plasmin to control their levels.

*Aurelien Briens 1 , Matthieu Schwalm 1 , Fabian Docagne 1 , Denis Vivien 1
1 INSERM U919, , Caen, France
Abstract text :

Astrocytes are key players in the brain, cooperating with neurons to modulate crucial processes. In particular, they are able to uptake and release neurotransmitters and neuromodulators to control their synaptic level.

In our study, we hypothesize that astrocytes can regulate some effects of the serine protease plasmin in the CNS through a mechanism of uptake.

Conversion of the zymogen plasminogen to the serine protease plasmin by the activators (tPA or uPA) is the basis of fibrinolysis in the vasculature. But cerebral functions for the plasminogen activator/plasmin system have recently arisen. It has been shown that plasmin was essential to activate the Brain Derived Neurotrophic Factor (BDNF) in its mature form, promoting long-term hippocampal plasticity. In Alzheimer’s disease, plasmin plays a protective role by participating in beta-Amyloid clearance. Plasmin can also lead to neuronal injury by degrading extra-cellular matrix proteins. In animal models of multiple sclerosis, plasmin can also help removing intraparenchymal deposits of fibrin, thus limiting axonal damage. These data suggest that a system of regulation of plasmin effects in the brain is necessary.


In this study, we showed that astrocytes can actively and specifically uptake plasminogen and plasmin in a time-dependent and a dose-dependent manner.  This mechanism involves clathrin pits formation in astrocytes and the lysine-binding site of plasminogen/plasmin. Plasminogen and plasmin, following endocytosis are found in early endosomes, late endosomes, lysosomes and recycling endosomes. Finally, we noticed that astrocytes uptake plasmin more efficiently than plasminogen.


This study brings out a mechanism of glial uptake of plasminogen and plasmin. This could control their extra-cellular levels and regulate their effects within the brain. Further investigations should determine whether this mechanism can modulate plasminogen activation and synaptic plasmin activity. Besides, plasminogen activators (tPA) and cerebral substrates of plasmin (pro-BDNF) are also taken up by astrocytes. A link could thus exist between these processes to regulate the amount of synaptic mature BDNF. This would reveal a cross-talk between neurons, releasing precursors in the synapse (pro-BDNF and plasminogen) and astrocytes, regulating their activation and their clearance. Understanding and modulating plasmin uptake could be very important in pathological conditions where the plasminogen/plasmin system is involved, such as Alzheimer’s disease, multiple sclerosis or stroke.


Proteomic identification of astrocyte proteins involved in synaptic plasticity

*Karen Carney 1 , Pim van Nierop 2 , Ka Wan Li 2 , August Smit 2 , Stephane Oliet 1 , Mark Verheijen 2
1 Universite Bordeaux Segalen, Glia-Neuron Relations, Bordeaux, France
2 Vrije Universitiet, Molecular and Cellular Neurobiology, Amsterdam, Netherlands
Abstract text :

The contributions of glial cells to the modulation of adult synaptic plasticity are becoming increasingly more appreciated. Astrocytes in the supraoptic nucleus (SON) of the hypothalamus demonstrate prominent temporary structural changes during physiological events such as dehydration and lactation. Such structural remodeling is characterized by a reduction in the astrocytic coverage of oxytocin neurons and their synapses. These astrocyte morphological changes also contribute to the observed functional changes in synaptic activity during dehydration and lactation.  In order to elucidate the underlying mechanisms regulating the astrocytic contributions to synaptic plasticity we have established an astrocyte protein expression profile for structural changes in the SON during lactation and hyperosmotic challenge. For this, SON from virgin, lactating, and hyperosmotic rats were collected from acute hypothalamic slices and analyzed by mass spectrometry to identify proteins differentially regulated by the changes in synaptic structure induced by lactation and hyper-osmolarity. Our mass spectrometry data analysis has placed particular focus on regulation of astrocyte-specific proteins and the roles of these proteins in molecular pathways known to be involved in SON plasticity, such as cytoskeleton remodeling, cell adhesion, and cell signaling. Gene ontology analysis revealed over representation of pathways known to be highly active in astrocytes including metabolism and antioxidant activity. Promising target proteins for future functional studies will be discussed.


Astroglial networks set the dynamics of neuronal bursting activity  

*Oana Chever 1 , Elena Dossi 1 , Nathalie Rouach 1
1 Collège de France, , Paris, France
Abstract text :

Astrocytes play crucial roles in brain physiology by dynamic interactions with neurons. They form plastic and extensive networks mediated by gap junction channels. It has recently been shown that astroglial networks limit neuronal network activity. However, it is currently unclear how astroglial networks influence neuronal excitability and population activity. To investigate how astroglial assembly regulates neuronal network activity, we performed electrophysiological recordings in hippocampal slices (CA3 and CA1 areas) from mice with disconnected astrocytes, in which both astroglial gap junction forming proteins, connexin 30 (Cx30) and connexin 43 (Cx43), are knocked out (GFAP-Cre Cx30-/-Cx43fl/fl, dKO). Synchronized excitatory discharges were recorded in an acute pharmacological model of epileptic-like activity. In this model, spontaneous bursting discharges are characterized by a sharp and large amplitude shift in field potential, generated by a major depolarization and firing of all putative neurons. Pyramidal cells depolarized up to 40 mV during seconds and such depolarization is followed by a long-lasting undershoot of the membrane potential. We found that the frequency of bursting activity in dKO hippocampal slices is drastically increased. However, the duration of neuronal depolarizing bursts is severely reduced. Furthermore, pyramidal neurons are more depolarized due to an increase of synaptic bombardment. This suggests that increased synaptic background promoting resting membrane potential depolarization facilitates triggering of neuronal bursts, but compromises the strength of synchronized events. To investigate local dynamics of bursts generation, we performed multielectrodes arrays recordings in the CA3 area of the hippocampus. In slices from dKO mice, bursting activity in the CA3 region is generated within a more restricted area, indicating that the number of neurons to be recruited is highly decreased. Altogether, these results indicate that gap junction-mediated astroglial networks strengthen the coordination of neurons during synchronized events.


Exploring motor neuron signaling dynamics to microglia in ALS

*Joana Carolina Cunha 1 , Ana Rita Vaz 1 , Dora Brites 1,2
1 Faculty of Pharmacy, University of Lisbon, , Lisbon, Portugal
2 Faculty of Pharmacy, University of Lisbon, Department of Biochemistry and Human Biology, Lisbon, Portugal
Abstract text :

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor-neuron (MN) degeneration, mainly in motor cortex, brainstem and spinal cord. Mutations in superoxide dismutase-1 (SOD1) are commonly used to study familial forms of ALS. It has been suggested that neuroinflammation associated with microglia activation is imperative for ALS progression. Indeed, microglia may become activated and proliferate during disease onset, before MN degeneration.

This study aimed to characterize the reactivity of a microglial cell line towards LPS and their response to soluble factors released by degenerating MNs.

First, we characterized a microglial cell line obtained from CD1 mouse cortex (N9), in order to clarify the mechanisms involved in the activation pattern of these cells. For that, we incubated N9 microglia with 300 ng/ml of lipopolysaccharide (LPS) for 24 h. Morphology (anti-Iba1 staining), phagocytic ability (fluorescent latex beads) and chemotaxis to ATP (Boyden Chamber) were evaluated. We observed that N9 microglia is ramified in control conditions and after LPS treatment they change to an amoeboid morphology, which is accompanied by increased phagocytosis and decreased migratory ability, indicating that LPS induces activation of N9 cell line.

Next, we investigated the role of the released factors from a MN-like cell line expressing human SOD1 with G93A mutation (NSC-34/hSOD1G93A) on microglia functions. NSC-34 expressing human SOD1 wt were used as control. Thus, N9 microglia were incubated for 4 and 24 h with NSC-34 conditioned media that had been collected at 1 and 4 days of differentiation (DIV) and reactivity tests were performed as above. Although after 4 h of incubation no significant changes were observed, data evidenced that incubation for 24 h with NSC-34/hSOD1G93A conditioned media collected at 4 DIV changed the bipolar morphology of the N9 microglia into an active amoeboid state, decreased phagocytic ability (22%, p<0.05) and induced apoptosis (50%) (Fig.1). Moreover, microglia have shown to not be attracted by the released factors from degenerating MN, as determined by the chemotaxis assay.

Together, our results evidence that released factors from NSC-34/hSOD1G93A degenerating cells although inducing microglia morphological activation trigger a reduction in cell phagocytic ability and loss of viability, suggesting a role for microglia along ALS progression.

Funded by PTDC/SAU-FAR/118787/2010 (to DB) and PEst-OE/SAU/UI4013/2011 (iMed.UL).  from Fundação para a Ciência e Tecnologia.


The age-related hippocampal alterations of glutamatergic neurotransmission are aggravated by ω3 PUFA deficiency and reduced by fish oil supplementation in rats.

*Isabelle Denis 1 , Brigitte Potier 2 , Alizée Latour 1 , Gaelle Champeil-Potokar 1 , Marie Hennebelle 1 , Elise Maximin 1 , Benedicte Langelier 1 , Monique Lavialle 1 , Jean-Marie Billard 2 , Christine Heberden 1 , Sylvie Vancassel 1
1 INRA, , Jouy-en-Josas, France
2 INSERM , centre de psychiatrie et neuroscience, Paris, France
Abstract text :


A poor ω3 polyunsaturated fatty acids (ω3 PUFA) status, favored by the low ω3/ω6 ratio in western diets, seems to contribute to cognitive decline in the elderly, but mechanistic evidence is lacking. We therefore explored the impact of ω3 status on the evolution of glutamatergic transmission, astroglial regulation and neurogenesis in the hippocampus during ageing in rats. These processes are involved in memory formation and their dysregulation participates to the age-related brain damage leading to cognitive decline.


We have compared 6 groups of rats aged 6 to 22 months fed ω3-deficient, ω3/ω6-balanced, or ω3 (fish oil) supplemented diets: Young ω3 Balanced (YB), Deficient (YD) or Supplemented (YS), and Old ω3 Balanced (OB), Deficient (OD) or Supplemented (OS) rats. We have evaluated synaptic efficacy and plasticity (electrophysiological recording), astroglial regulations (glutamate uptake and GFAP expression), neuronal markers (glutamate transporters and receptors), neurogenesis (proliferation of neuronal precursors in the sub granular zone), and analyzed brain fatty acids composition.


Dietary modulation of ω3 intakes efficiently modified the incorporation of docosahexaenoic acid (DHA, the main ω3 in cell membranes) in brain (-50% deficient vs balanced, 10% supplemented vs balanced). Ageing induced a 35% reduction of synaptic efficacy due to decreased pre-synaptic glutamate release, and a 30% decrease in the astroglial glutamate uptake associated to a marked astrogliosis ( 100% GFAP). ω3 deficiency further decreased these hallmarks of ageing (OD vs OB rats: -35% synaptic efficacy, -15% glutamate uptake, 30% GFAP). On the opposite, ω3 supplementation increased synaptic efficacy ( 25% OS vs OD) and seems to abolish astrogliosis (OS vs YS : no change in GFAP). Neurogenesis was altered by ω3 deficiency but not by supplementation.


Our results characterize some specific age-related alterations of the glutamatergic synapse in the hippocampus that are aggravated by a dietary deficit in ω3 and attenuated by ω3 supplementation.


The role of glia cells in thyroid hormone induced regulation of neuronal  sodium current and Na+/K+-ATPase density


*Irmgard Dietzel-Meyer 1 , Birte Igelhorst 1 , Vanessa Niederkinkhaus 1 , Sivaraj Mohanasundaram 1
1 Ruhr-Universität, , Bochum, Germany
Abstract text :

Thyroid hormone has been shown to induce the secretion of proteins such as, FGF-2, EGF and neurotrophins from glial cells (see e.g. Lima et al., J. Endocrinol. 1997, 167).  On the other hand, treatment with thyroid hormone (T3) up-regulates Na -current density (NavD) in neonatal neurons (Hofmann and Dietzel, Neurosci, 2004; 125: 369). The resulting increase in neuronal excitability could explain the effects of this hormone on mental speed and some of the neurological symptoms of hyperthyroidism such as irritability and seizure susceptibility.  The effects of T3 on neuronal NavD were mimicked by incubating neuron enriched cultures with conditioned medium from T3-treated astrocytes, and were absent in neuron-enriched cultures suggesting that the factors secreted by glial cells by T3 treatment up-regulate NavD in neurons. The effect was mimicked by the addition of FGF-2 to the culture medium and blocked by antibodies against FGF-2, suggesting the involvement of this factor in the up-regulation of NavD by T3 (Niederkinkhaus et al., Mol.Endocrinol 2009; 23 :1494). Here we extend these findings showing that the neurotrophin NT-3, which also belongs to the putative factors secreted from glial cells after T3-stimulation, also increases the NavD in neuron enriched cultures. The effects of NT-3 and FGF-2 are, however, not additive. Antibodies against NT-3 potentiated the effects of FGF-2, suggesting that by converging signal cascades NT-3 could reduce the effect of FGF-2. In neuron-glia mixed cultures antibodies against NT-3 enhanced the T3-induced up-regulation of NavD, suggesting, that a co-secretion of NT-3 from glial cells could limit the effects of FGF-2. In a second series of experiments we investigated, whether the well-known effect of T3 on the expression of Na /K -ATPases, thought to contribute to the effect of thyroid hormone on metabolism, is also influenced by glial cells. We found, that the effect of T3 on 3[H]ouabain-binding and on the expression of Na /K -ATPase alpha2 subunits detected by western-blots was highest in neuron-mixed cultures, suggesting that a neuron-glia interaction is also involved in the T3 effects on Na /K -ATPase expression.


Role of the α-secretase TACE in Central Nervous System myelination

*Evelien Fredrickx 1,2 , Giorgia Dina 1 , Klaus-Armin Nave 3 , Angelo Quattrini 1 , Carla Taveggia 1
1 San Raffaele Scientific Institute, Division of Neuroscience - INSPE, Milan, Italy
2 Università Vita-Salute San Raffaele, PhD Program in Molecular Medicine - Cellular and Molecular Biology, Milan, Italy
3 Max-Planck-Institute of Experimental Medicine, , Goettingen, Germany
Abstract text :

The ADAM family of proteins belongs to the zinc family of proteases that are involved in the ectodomain shedding of several growth factors. We recently demonstrated a key role of the α-secretase TACE, also known as ADAM17, in peripheral nervous system (PNS) myelination by TACE-mediated cleavage and subsequent inhibition of Neuregulin1 (NRG1) type III activity. Unlike the PNS in which NRG1 type III is an essential instructive signal for myelination, oligodendrocyte (OL) development and myelination in the central nervous system (CNS) are likely controlled by several growth factors some of which undergo cleavage by secretases.

To study the role of TACE on OPC development, immunopanned A2B5 OPCs were cultured in proliferating or differentiating medium and treated with a soluble form of TACE (rhTACE). When OLs were scored according to their morphology, we observed enhanced OPC differentiation upon addition of rhTACE to proliferating OPCs. Addition of rhTACE to differentiating OPCs also increased the number of OLs with a complex morphology and already presenting membrane sheets, suggesting that TACE might promote OPC differentiation. To further investigate the role of neuronal TACE in CNS, we used an in vitro OL myelinating coculture system. When cocultured with TACE null DRG neurons, rat wild type OPCs never myelinate, suggesting that neuronal TACE might control OPC differentiation. In agreement, preliminary ultrastructural analyses of transgenic mice lacking TACE in CNS neurons showed hypomyelination and signs of myelin degeneration. Accordingly, conditional transgenic mice lacking TACE in OL are normally myelinated.

These studies suggest that in the CNS the α-secretase TACE promotes OPC differentiation and might regulate CNS myelination. A better understanding of the role of TACE will provide novel insights into the mechanism regulating CNS myelination.


Characterization of Schwann cells in mouse sciatic nerve slice: electrophysiological properties and neurotransmitter receptor expression.

*Nicole Fröhlich 1 , Daniela Eißler 1 , Maria Kukley 1
1 Centre for Integrative Neuroscience (CIN), , Tübingen, Germany
Abstract text :

Schwann cells (SCs) are the myelinating cells in the peripheral nervous system (PNS). Results of few immunohistochemical and pharmacological studies available to date suggest that SCs in the PNS express ionotropic and metabotropic receptors for glutamate and ACh (Dememes et al., 1995; Kinkelin et al., 2000; Loreti et al., 2006; Vrbova et al., 2009). Furthermore, neurotransmitters are able to induce changes of SC membrane potential in the giant squid axons (Villegas et al., 1987), and these changes are abolished by application of glutamate or ACh receptor antagonists (Lieberman et al., 1989; Lieberman and Sanzenbacher, 1992). These findings indicate that neurotransmitter receptors on SCs in the nerve are functional.

We are interested to understand whether SCs in the peripheral nerves of mammals express functional ionotropic glutamate receptors, and whether SCs use these receptors for communication with axons. To start addressing this question, we established a preparation of mouse live sciatic nerve slices employing “know-how” of live brain slices technique. Using this preparation, we performed whole-cell patch-clamp recordings of SCs at different stages of development (embryonic and early postnatal). We included a fluorescent dye into the pipette solution in order to perform post-recording morphological analysis of single SCs. First, we recorded current responses of SCs to a series of depolarizing voltage steps, aiming to test whether all SCs in the nerve are electrophysiologically akin. We found that mouse SCs differ in their passive membrane properties and expression of voltage-gated K channels: depending on the age, 3 to 4 cell types could be distinguished. Post-recording morphological characterization of the recorded cells showed that SCs in the mouse sciatic nerve differ in their size, as well as in the number and length of their processes. Next, we used fast pressure-induced application of glutamate to investigate whether SCs possess ionotropic glutamate receptors, and whether electrophysiologically distinct SC types differ in their expression of glutamate receptors. Application of 1 mM glutamate caused an inward current in at least two types of SCs, and preliminary analysis revealed a peak current amplitude in the range of 14-250 pA (n=18). Glutamate-induced currents in SCs were blocked by GYKI53655, indicating that mouse SCs carry ionotropic glutamate receptors of AMPA/kainate type. Currently we are studying which subunits of AMPA receptors are present in SCs, and how these receptors get activated in situ.


Neurotransmitter signaling controls exosome secretion from oligodendrocytes

*Carsten Frühbeis 1 , Dominik  Fröhlich 1 , Stefan Tenzer 2 , Wen Ping Kuo 1 , Wiebke  Möbius 3 , Aiman Saab 3 , Frank Kirchhoff 4 , Jacqueline Trotter 1 , Eva-Maria Krämer-Albers 1
1 University of Mainz, Molecular Cell Biology, Mainz, Germany
2 University of Mainz, Immunology, Mainz, Germany
3 MPI of Experimental Medicine, Neurogenetics, Göttingen, Germany
4 University of Saarland, , Homburg, Germany
Abstract text :

The biogenesis of the axon-myelin unit is controlled by reciprocal interactions between oligodendrocytes and neurons, which continue throughout life. Oligodendrocytes secrete endosome-derived microvesicles termed exosomes, implicated in intercellular communication. These exosomes carry a specific set of proteins as well as RNA species. Here, we show that the neurotransmitter glutamate stimulates exosome secretion from oligodendrocytes by provoking Ca2 entry through oligodendroglial NMDA and AMPA receptors. Furthermore, active neurons evoke exosome release from oligodendrocytes, indicating that neuronal activity controls oligodendroglial exosome release. In turn, neurons internalize oligodendroglial exosomes and functionally retrieve the exosomal cargo. Thus, oligodendrocytes may influence the neuronal metabolism by an exosome-dependent transfer of bioactive substances to neurons. Axonal degeneration resulting from lack of glial support occurs in PLP and CNP deficient mice. Intriguingly, both proteins are components of oligodendroglial exosomes. A proteomic approach revealed that amount and composition of exosomes derived from PLP-/- and CNP-/- oligodendrocytes are altered, supporting the hypothesis that disturbed intercellular transfer of substances by exosomes may contribute to axonal degeneration in these mouse models. 


Neuron-astrocyte-microglia interactions in a rat model of chronic cerebral ischemia

*Maria Grazia Giovannini 1 , Daniele Lana 1 , Alessia Melani 2 , Felicita Pedata 2
1 University, DSS, Firenze, Italy
2 University, Neurofarba, Firenze, Italy
Abstract text :

It is becoming more and more evident that proper functioning of the neuron-astrocyte-microglia triad is fundamental for the functional organization of the brain, and thus it is of the utmost  importance to better characterize their interactions in physiological and pathological processes. We recently demonstrated, in rat models of normal brain aging and LPS-induced acute inflammation, that astrocytes and microglia actively collaborate in the clearance of apoptotic neurons and neuronal debris associated with programmed cell death. Here we studied the interactions between neurons, microglia and astrocytes within the CA1 region of the hippocampus after bilateral common carotid artery occlusion (bCCAo) in the rat, a valid model of chronic cerebral hypoperfusion which leads to persistent ischemic conditions and ultimately to neuronal death. Male Wistar rats were subjected to permanent bCCAo. A group of rats was infused into the jugular vein with dipyridamole (7 days, 4 mg/kg/day by an osmotic minipump). Sham-operated animals were used as controls. Three months after bCCAo, immunohistochemical studies were performed on brain coronal slices, focussing on the hippocampal CA1 region. Using an antibody against glial fibrillary acidic protein (GFAP) no astrogliosis was detected. We found a significant increase in the number of total microglia, visualized using the IBA1 antibody, in bCCAo-treated rats in comparison to the sham group ( 18%, P < 0.01, one way ANOVA and Newman-Keuls) and this effect was completely reverted by dipyridamole (P < 0.01, one way ANOVA and Newman-Keuls). As exemplified in Figure 1, in the CA1 and Str. Radiatum of bCCAo-treated rats, many neurons (anti-NeuN antibody, red) showing signs of degeneration were closely apposed to and infiltrated by astrocyte branches (anti-GFAP antibody, green) which appeared to be bisecting the cell body into cellular debris, and microglia cells (anti-IBA1, antibody, blue) were actively phagocytosing the damaged neurons. This finding is consistent with the scavenging activity of microglia upon dying neurons or debris, a possible mechanism that prevents further injury to neighboring neurons. It will be interesting to investigate which intercellular communication mechanisms allow the recruitment and activation of different glial cells in a well-organized reciprocal interaction to scavenge the damaged neurons and to verify the mechanisms of the protective effects of dipyridamole found in this chronic cerebral ischemic model.

Partly funded by Banco S. Paolo and Boehringer Ingelheim.


Tonic suppression of synaptic and extrasynaptic inhibition in the striatum as a consequence of GLT-1 deficiency in mice carrying a mutant form of huntingtin

Anton Dvorzhak 1 , Anna Wojtowicz 1 , *Rosemarie Grantyn 1
1 University Medicine (Charité), , Berlin, Germany
Abstract text :

  • In neurodegenerative diseases, the afflicted      brain is both an important object of study and an opportunity to      characterize a given cellular interaction from a pathophysiological perspective.     

  • This dual approach is particularly advantageous      when human disease is based on a monogenetic defect and an appropriate      animal model becomes available for detailed investigation, as in case of Z_Q175_KI      (Q175), a new knock-in mouse expressing a mutant form of murine huntingtin.     

  • Electrophysiological recordings of GABAergic      unitary IPSCs from striatal output neurons (SONs) in sagittal slices from wild-type      and homozygous Q175 challenged the current viewpoint that GABAergic      transmission is enhanced in the HD striatum. Quantal analysis in      combination with high frequency stimulation and paired pulse tests revealed      that synaptic GABA release is in fact tonically suppressed resulting in      disinhibition of striatal output activity (Dvorzhak et al J Physiol 2013).     

  • The underlying mechanism involves a retrograde      endocannabinoid signalling pathway linking postsynaptic mGluR5 with      presynaptic CB1 and GABA release.  

  • In addition to this deficit, Z-Q175_KI      homozygotes exhibited a significant reduction of tonic inhibition via      extrasynaptic GABA(A) receptors.

  • Using pharmacological tools to alter the ratio of      ambient glutamate and GABA concentrations made clear that the HD-related      depression of synaptic and extrasynaptic GABAergic actions depends on the      state of both the astrocytic glutamate transporter GLT-1 and the GABA      transporter GAT-3.

  • In support of Héja and colleagues, our imaging      experiments suggest that in normal striatal astrocytes GAT-3 is in a      releasing mode and driven by the activity of GLT-1.

  • However, the latter activity is much lower in the      HD striatum. SBFI recordings of GLT-1-related Na transients and patch      clamp recordings of GLT-1 transporter currents revealed a marked reduction      (less than 50% of WT level) in SR-labelled astrocytes from symptomatic HD      mice.

  • Together, our results suggest that the deficiency      of astrocytic glutamate uptake might represent a pathophysiological key mechanism      underlying the observed disinhibition in the striatum of mice affected by Huntington's      disease.



A specific role for the Na,K-ATPase α2 isoform in the support of astrocyte glutamate uptake

Nina  Illarionova 1 , Hjalmar Brismar 1 , Anita Aperia 1 , *Eli Gunnarson 1
1 Karolinska Institutet, , Stockholm, Sweden
Abstract text :

A tightly regulated intracellular sodium (Nai) homeostasis is of fundamental importance for all cells. In astrocytes pulsative changes in the Nai concentration ([Na ]i) are regularly occurring as a result of uptake of glutamate from the synaptic space. Glutamate uptake occurs via the glutamate/Na  co-transporters GLAST and GLT-1, where one glutamate is accompanied by 3 Na  and 1 H  in exchange for 1 K . The salt pump, Na,K-ATPase (NKA), is responsible for the maintenance of the trans-membrane Na  gradient by exporting 3 Na   and importing 2 K for each ATP. NKA is dose-dependently inhibited by ouabain. Astrocytes express two isoforms of the catalytic NKA α subunit, the ubiquitous α1 and in the CNS the glia-specific α2. The isoforms differ with regard to Na  affinity, which is lower for α2 and with regard to ouabain affinity, which is higher for α2 than for α1. Although α2 mutations give rise to neurological symptoms - familial hemiplegic migraine type 2, the relative roles of the α1 and α2 isoforms in astrocytes are still incompletely understood.

We hypothesized that the low Na affinity of α2 makes it more suitable to cope with transient large increases in Nai. To test this, we compared the effect of 200µM glutamate for 10 min on real time changes of [Na ]i in primary astrocytes, which, following transient transfection,  predominantly expressed either the α1 or the α2 isoform. In α1 cells glutamate caused a significantly larger increase in [Na ]i and longer recovery time to base line [Na ]i than in cells expressing α2.  Glutamate uptake dependence on either α isoform was determined by aspartate uptake in astrocytes expressing endogenous α1 and α2. In the presence of ouabain concentrations that selectively inhibit α2, we found a modest (1.9mM) increase in [Na ]i, accompanied by a disproportionately large decrease (24%) in glutamate uptake.

It has been suggested that astrocyte glutamate transporters are clustered in microdomains where they may interact with NKA. In GST pull down assays on rat brain we found that both GLT-1 and GLAST interacted with the 1st intracellular loop of both α1 and α2, but the interaction was significantly stronger for the α2 isoform. No interaction was found with other segments of the α molecule.

The data indicate that the NKA α2 isoform, which has a more restricted expression than α1, plays a specific role for the interaction with the glutamate transporters and for sodium homeostasis in astrocytes. This specificity may be attributed to low sodium affinity of α2 and to the relatively high capacity of α2 to interact with the astrocyte glutamate transporters.



The P2X7R-Panx1 complex in glia: Role in orofacial hypersensitivity

*Regina Hanstein 1 , Maria Gulinello 2 , Menachem Hanani 3 , David C. Spray 1
1 Albert Einstein College of Medicine, Neuroscience, Bronx, United States
2 Albert Einstein College of Medicine, Rodent Behavioral Core, Bronx, United States
3 Hadassah University Hospital, Laboratory of Experimental Surgery, Jerusalem, Israel
Abstract text :

Pain is caused by aberrant neuronal responses to peripheral stimuli and glial cells are emerging as contributors to the neuronal hyperactivity. Recent work in sensory ganglia where satellite glial cells (SGCs) have close interactions with neurons has shown that SGCs can actively modulate neuronal activity. During pain, excessive amounts of ATP are released by neurons and activate purinergic receptors (P2R), in particular P2X7R that functionally interact with pannexin1 (Panx1). P2X7R activation leads to opening of Panx1 channels, providing an efflux pathway for ATP, to which neurons respond with excessive firing. To explore the involvement of this pathway in pain and analyze its impact separately in sensory neurons and SGCs, we tested pharmacological inhibitors and transgenic mice in an orofacial pain model. Transient inflammation in the submandibular region of mice was induced using complete Freund's adjuvant (CFA) and tactile sensitivity was quantified using von Frey filaments. Although inflammation was resolved by 28 days after injection, tactile hypersensitivity persisted in wildtype mice for at least 53 days, consistent with chronic post-inflammatory pain. Tactile hypersensitivity in mice was reversed by systemic injection of the Panx1/gap junction blockers mefloquine or carbenoxolone at 7 days (peak inflammation) and at 28 days after CFA-injection, suggesting the contribution of these channels to tactile hypersensitivity. In dissociated trigeminal ganglia (TG), which innervate the submandibular skin, BzATP-induced YoPro uptake into neurons and glia was prevented by mefloquine, demonstrating the functional presence of the P2X7R-Panx1 complex in TG. Moreover, TG of CFA-injected mice showed more ATP release and immunostaining of TG revealed higher expression of Panx1 at 1 week after CFA injection compared to controls. Development of hypersensitivity was prevented in P2X7R-null and in Panx1-null mice, and was attenuated in mice with glia-specific deletion of Panx1 (GFAPcre-Panx1f/f) but not with neuron-specific deletion (mNFHcre-Panx1f/f), emphasizing the importance of glial Panx1 signaling in pain. Because P2X7R and Panx1 both have a key role in the immune response by activating the inflammasome, we are currently dissecting the relative importance of neuron-glial communication compared to inflammatory responses in the development and maintenance of orofacial pain. Overall, our results show that the P2X7R-Panx1 complex likely plays a major role in signaling events contributing to tactile hypersensitivity.


Bidirectional expression of Lck-GCaMP3 and DsRed in NG2-cells as an approach for monitoring glial Ca2+-microdomains

*Bianca Verena Herl 1 , Martin Theis 1 , Christian Steinhäuser 1 , Ronald Jabs 1
1 Institute of Cellular Neurosciences, , Bonn, Germany
Abstract text :

NG2-cells are the fourth type of glial cells in the mammalian brain. They are the only non-neuronal cell type in the brain that receives synaptic input from neurons. NG2-cells exhibit a variety of Ca2 -signalling pathways, which might be triggered by pre-synaptic neuronal activity. However, no global increase in the intracellular Ca2 -concentration ([Ca2 ]i) was detected in NG2-cells employing the minimal stimulation technique at neuron-glial synapses. Therefore we assume that post-synaptic Ca2 -microdomains might be activated under physiological conditions. These Ca2 -signals are locally restricted to the plasma membrane. Membrane bound Ca2 -sensors with a high signal-to-noise ratio, such as Lck-GCaMP3, are best suited for optimal visualisation of Ca2 -microdomains. Here, we set out to express Lck-GCaMP3 in NG2-cells together with the cytosolic reporter dye DsRed.

We generated transgenic mice which express a bidirectional promoter encoding both proteins under the control of a Tet-off system. This strategy allows an exclusive expression of the transgenes only in the presence of a tet-responsive transcriptional activator (tTA).

The construct was successfully cloned and expressed in HEK293 cells. As expected, Lck-GCaMP3 was located only at the inner plasma membrane while DsRed was expressed all-over the cytosol. Functionality of the Ca2 -sensor was tested in vitro. Elevation of [Ca2 ]i reliably increased the fluorescence intensity of Lck-GCaMP3. After zygote injection seven positive founder animals could be identified and will be crossbred with a NG2-tTA mouse line.

Taken together, the presented construct appears suitable for functional imaging of Ca2 -microdomains in post-synaptic NG2-cells.


Dehydroepiandrosterone sulfate and Sulforhodamine 101 compete for active uptake by an organic anion transporting polypeptide in hippocampal astrocytes

Christian Schnell 1 , Yohannes Hagos 1 , *Swen Hülsmann 1
1 University of Göttingen, , Göttingen, Germany
Abstract text :

Sulforhodamine 101 (SR101) is widely used as a marker of astrocytes. In this study we investigated labeling of astrocytes by SR101 in acute slices from the ventro-lateral medulla and the hippocampus of transgenic mice expressing EGFP under the control of an astrocyte-specific promoter. While SR101 efficiently labeled EGFP-expressing astrocytes in hippocampus, we found that SR101-staining was very weak in the ventro-lateral medulla and rather unspecific. Thus, SR101 is not a reliable marker for brainstem astrocytes. Although carbenoxolone decreased the labeling of astrocytes in the hippocampus significantly, mefloquine which blocks pannexin and connexin hemichannels, was unable to prevent SR101 uptake in hippocampal astrocytes. Time-lapse 2-photon imaging revealed that in hippocampus both astrocytes and neurons showed temporary SR101-loading. In astrocytes the rise of SR101 fluorescence was slower as compared to EGFP-negative cells and SR101 was quickly removed from non-astrocytic cells during washout, while it was retained in astrocytes. In brainstem astrocytes, however, only a very weak and transient SR101-labeling was observed. To test if SR101 is actively removed from astrocytes in the brainstem, we applied MK-571 to block the multi-drug resistance transporters MRP-1. We did not observe any increase of SR101-labeling in brainstem astrocytes. In contrast, astrocytic SR101 labeling was significantly reduced by substrates of organic anion transport, probenecid, estron-3-sulfate and dehydroepiandrosterone sulfate suggesting that SR101 is actively transported into hippocampal astrocytes by an organic anion transporting polypeptide (OATP). Additionally, the data suggest that astrocytes modulate extracellular concentration of neurosteroids in the hippocampus.


Mapping astrocyte heterogeneity by analysis of specific cell surface marker expression

*Christina Kantzer 1 , Andreas Bosio 1 , Melanie Jungblut 1
1 Miltenyi Biotec GmbH, , Bergisch-Gladbach, Germany
Abstract text :

Astrocytes show a high level of functional and morphological heterogeneity and are involved in many aspects of neural function, e.g., formation of the blood-brain barrier, regulation of ion homeostasis, synaptogenesis, and synaptic plasticity. Despite the diversity of this cell type, the various astrocyte subpopulations are not well characterized, which is mainly due to the lack of markers that would allow for classification of astrocyte subsets.

Our study aimed at phenotyping of astrocyte subpopulations based on the expression of cell surface markers, which are associated with certain functions. We used two novel monoclonal antibodies, ACSA-1 and ACSA-2 (ACSA: astrocyte cell surface antigen), directed against extracellular epitopes of astrocyte-specific cell surface markers to identify astrocyte subtypes. The ACSA-1 antibody was generated by immunization of GLAST1 knockout mice and specifically detects the astrocyte-specific L-glutamate/L-aspartate transporter GLAST (EAAT1, SLC1A3), whereas the ACSA-2 antibody results from an immunization of rats with astrocytes isolated from GFAP-EGFP transgenic mice. A mass spectrometric approach for the ligand-based identification of the ACSA-2 antigen pointed to a number of candidates, which have to be validated by further experiments.

The antibodies ACSA-1 and ACSA-2 were carefully analyzed by co-staining experiments with commonly used intracellular astrocyte markers. We found that both antibodies specifically detect astrocytes in the developing and adult central nervous system. In contrast to antibodies against intracellular markers, such as GFAP, S100ß, or glutamine synthetase, ACSA-1 and ACSA-2 can be used to detect and isolate living astrocytes, which enables further analysis and culture of the separated cells. Flow cytometric analysis of ACSA-1 and ACSA-2 antigen expression on cells from different brain regions, as well as immunohistochemical analysis, revealed differences in the expression patterns. This allowed us to define different astrocyte subtypes especially in the cerebellum and olfactory bulb of the neonatal mouse brain.

Future work will focus on the identification of additional astrocyte-specific cell surface proteins for a comprehensive classification of astrocyte subtypes based on cell surface marker expression or expression patterns.


Glial GABA transporters downregulate enhanced neuronal activity

*Orsolya Kékesi 1 , Gabriella Nyitrai 1 , Pál  Szabó 1 , Richárd  Fiáth 2 , István  Ulbert 2 , Julianna  Kardos 1 , László  Héja 1
1 Institute of Molecular Pharmacology, , Budapest, Hungary
2 Institute of Cognitive Neuroscience and Psychology, Comparative Psychophysiology Group, Budapest, Hungary
Abstract text :

Several studies demonstrated the ability of astrocytes to sense, respond to and regulate neuronal function. Among the many functions of glial proteins, glutamate (Glu) and GABA transporters play important roles in balancing excitatory and inhibitory signals in the brain. Here we show that astrocytes regulate the tonic inhibition of neurons by the concerted action of Glu and GABA transporters, thereby protecting both neurons and glial cells from overactivation.

We demonstrate that the uptake of glutamate triggers the reverse function of glial GAT-2/3 transporters by elevating the intracellular Na concentration in astrocytes. The released GABA significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. We also describe the source of the releasing GABA that is synthesized by an alternative pathway from polyamines. Moreover, in the low-[Mg2 ] model of epilepsy, we show that blockade of the glial Glu/GABA exchange mechanism increases the duration of seizure-like events and also results in increased activity of astrocytes, demonstrating the neuroprotective impact of the mechanism. Finally, we show that the released glial GABA modulates the power of gamma range oscillation in vivo, suggesting that the Glu/GABA exchange mechanism is also functioning in the intact hippocampus under physiological conditions.

Revealing this novel molecular mechanism by which astrocytes provide an adjustable, in situ negative feedback on the excitability of neurons is expected to broaden our understanding about the regulation of neuronal activity by astrocytes and may open up new targets for the treatments of pathological conditions, such as epilepsy or ischemia.

This work was supported by grants: OTKA K 81357, TÉT NEUROGEN, TÉT MULTISCA to IU, TECH-09-AI 2009-0117 NKFP NANOSEN9, ERA-Chemistry OTKA 102166 and KMR_12-1-2012-0112 TRANSRAT.


Macrophages and microglia play distinct roles in neuropathic pain perception

*Stefanie Kraft 1 , Kelly R. Miller 1 , Christian Witzel 2 , Roland E. Kälin 1 , Gerit Pfuhl 3 , York Winter 3 , Matthias Endres 4 , Frank L. Heppner 1
1 Charité, Neuropathology, Berlin, Germany
2 Charité, Plastic Surgery, Berlin, Germany
3 Humboldt University, Cognitive Neurobiology, Berlin, Germany
4 Charité, Neurology, Berlin, Germany
Abstract text :

Question: The discovery that activation of non-neuronal CNS microglia plays a causal role in spinal processing of nociceptive signaling has shed new light on the processes underlying neuropathic pain facilitation. However, there remains much uncertainty as to the necessary contribution of microglia to enhanced pain states. We aim to define the particular role of microglia for the initiation of neuropathic pain and by answering this question also learn if the function of peripheral myeloid cells is distinct or redundant in this process.

Methods: To specifically investigate spinal microglia and peripheral macrophages in the pathogenesis of neuropathic pain, we model chronic pain by performing partial ligature of the sciatic nerve in CD11b-HSVTK mice engrafted with GFP bone marrow (GFP>CD11b-HSVTK). CD11b-HSVTK /- mice allow the exchange with peripherally-derived, GFP macrophages upon central depletion of endogenous CD11b microglia. Following this depletion/ repopulation paradigm, behavioral analyses of mechanical and thermal allodynia are conducted.

Results: We established a selective tool to exchange CNS parenchymal microglia with peripheral GFP myeloid cells. In chronic pain tests for mechanical and thermal hyperalgesia, GFP>CD11b-HSVTK /- mice show considerable decreases in paw withdrawal thresholds in response to mechanical, but not thermal stimuli ipsilateral to the injury, indicating distinct roles of microglia and macrophages in the facilitation of thermal hyperalgesia.

Conclusions: We identified a differential contribution of resident spinal microglia vs. peripheral myeloid cells in the development of neuropathic pain in GFP>CD11b-HSVTK chimeras. Future studies aim to examine the exact mechanisms underlying the distinction between these two populations.


Mechanisms of K+-clearance in the brain: The Na+/K+-ATPase as the key contributor

*Brian Roland Larsen 1 , Mette Assentoft 1 , Susan Z. Hua 2 , Kai Kaila 3 , Juha Voipio 3 , Nanna MacAulay 1
1 University of Copenhagen, , Copenhagen N, Denmark
2 University of Buffalo, , Buffalo, United States
3 University of Helsinki, , Helsinki, Finland
Abstract text :

Neuronal activity in the brain is associated with a transient increase in the extracellular K concentration. The excess K is removed from the extracellular space, primarily by the surrounding glial cells, leading to an intracellular accumulation of K via mechanisms not fully identified and/or quantified. Post-stimulus recovery of [K ]o  has been proposed to be dependent on Kir4.1-mediated spatial buffering and/or to Na /K -ATPase activity.


To resolve the molecular mechanisms involved in K clearance, we initially determined the contribution from the different K -transporting mechanisms present in primary culture of rat astrocytes and their K0.5 for K . The Na /K /2Cl- cotransporter, NKCC1, increased its activity within a physiological concentration range of K , while the Na /K -ATPase saturated at much lower concentrations. Consequently, a concentration-dependent switch occurs between the two mechanisms of K uptake in cultured astrocytes. In addition, NKCC1 was capable of mediating robust K -induced astrocytic cell swelling. Thus, NKCC1 could potentially act as a molecular mechanism responsible for clearance of the stimulus-evoked rise in [K ]o and the associated shrinkage of the extracellular space.


To determine the contribution of each of the three molecular mechanisms to K -clearance in native brain tissue, we used rat hippocampal brain slices and employed ion-sensitive microelectrodes in association with high-frequency electrical stimulation as well as focal appliances of K by ionophoresis. Inhibition of Kir4.1 (100 uM BaCl2) or NKCC1 (10 uM bumetanide) failed to show a significant effect on the rate of K removal from the extracellular space. In contrast, inhibition of the α2 and α3 isoforms of the Na /K -ATPase significantly delayed post-stimulus recovery of [K ]o and this delay was further potentiated by additional inhibition of the α1 isoform.


The Na /K ATPase emerged as the primary factor responsible for stimulus-evoked K -clearance with no evidence in favor of Kir4.1 and NKCC1 involvement. Further characterization of the different Na /K -ATPase isoforms, by heterologous expression in Xenopus laevis oocytes, revealed that the α1 isoform reached its maximal activity already at resting K concentrations, hinting at a “housekeeping” function. The α2 subunit displayed voltage-sensitivity and increased turnover rate along physiologically relevant increments in extracellular K concentration. These α2-related features may render this astrocyte-specific subunit variant specifically geared for post-stimulus recovery of [K ]o


Age-dependent activity of nitric oxide synthase during ischemic white matter injury

Jane  Zaleski 1 , Amelia Bachleda 1 , Anne Runkle 1 , *Selva Baltan 2
1 Cleveland Clinic, Neurosciences, Cleveland, United States
2 Cleveland Clinic, , Shaker Heights, United States
Abstract text :

White matter (WM) is injured in most strokes and axonal injury and dysfunction contribute to

disability associated with clinical deficits. In young WM, the damage from ischemic injury involves the sequence of energy depletion (ionic pathway), excessive glutamate release (excitotoxicity), generation of reactive oxygen species and oxidative stress (oxidative pathway). In older WM the injury is mediated by Ca2 -independent excitotoxicity due to an earlier and more robust glutamate release. Because excitotoxicity leads to oxidative stress in WM we investigated whether blocking nitric oxide synthase (NOS) activity before or after a period of oxygen glucose deprivation (OGD) promoted axon function in an age-dependent manner.


Acutely isolated optic nerves from young and old (1 and 12 month) Swiss Webster (SW) or C57BL/6 (BL6) mice were used to ascertain quantitative measurements of WM function and structure. To support a biological basis for NOS inhibitor nitro-L-arginine methyl ester (L-NAME) action in theMONpreparation, we evaluated the expression and localization of brain NOS (bNOS) using immunohistochemistry in conjunction with confocal imaging. The expression of bNOS co-localized with GFAP ( ) astrocyte nuclei, cytoplasm, end-feet as well as NF-200 ( ) axons. The pattern of bNOS expression paralleled astrocyte morphology with age and became more punctate in appearance. Evoked compound action potentials (CAPs) recovered to 21.8 ± 2.8% (n=18) after 60 min of oxygen glucose deprivation (OGD) in young BL6 MONs. Pretreatment of MONs with L-NAME (200 µM) promotedCAPrecovery to 69.4 ± 11.3 % (n=8) compared to OGD while CAPs recovered to 39.9 ± 4.4 % (n=11) when L-NAME was applied after the end of OGD. Pretreatment of MONs from 1 or 12 month old SW with L-NAME improvedCAPrecovery to 49.6 ± 3.7% (n=8, vs OGD 21.3 ± 3.7%, n=12) or to 51.1 ± 9.3 % (n=7, vs OGD 5.7 ±1.7%, n=8) respectively. L-NAME application after the end of OGD failed to promote aging axon function (8.8 ± 3.5%, n=6). Changes in NOS activity help unveil age-dependent oxidative injury mechanisms in ischemic white matter.


Neuroprotection in stroke by gonadal steroids: an active role for microglia and astroglia-microglia crosstalk

Pardes  Habib 1 , Jon Dang 1 , Daniela Dreymüller 2 , *Cordian Beyer 3
1 RWTH Aachen, Institute of Neuroanatomy, Aachen, Germany
2 RWTH Aachen, Institute of Pharmacology, Aachen, Germany
3 RWTH Aachen, , Aachen, Germany
Abstract text :

Gonadal steroid hormones reveal a potential neuroprotective role in acute brain ischemia. In rat in vivo studies using the transient occlusion of the middle cerebral artery as ischemic stroke model, we have shown that 17ß-estradiol and progesterone reduce the infarct area by more than 70% given 1h after the onset of stroke, prevent neuronal death and behavioral deficits. An intriguing aspect of this study was that the application of steroid hormones reduced the number of microglia and microglia-related markers in the penumbra during the first 24 h after the onset of stroke. Besides microglia, penumbral astroglia coevally appeared as cellular target for steroid hormones by reducing the expression of proinflammatory- and molecules necessary for the attraction and activation of microglia and lymphocytes. This suggests that protective steroid hormones influence glia cell cross-talk and activity during early damaging conditions in the lesioned brain site. By using an in vitro hypoxic approach, we have now demonstrated that microglia express steroid hormone receptors and directly respond to ischemic conditions and steroid hormone treatment by changing expression and secretion of inflammatory compounds, trans-signaling factors and by modulating phagocytotic activity. This clearly shows that microglia is an important direct target for steroid hormones but also indirectly influenced via cross-talk by adjacent astrocytes at the damaged brain site. This generally points at an extensive bidirectional crosstalk between both glial cell types to convey steroid-mediated neuroprotection in ischemic brain tissue. In summary, the balance of inflammatory astroglia-microglia interactions within the injured brain tissue during an early phase of ischemic destruction is a pivotal step for tissue repair.


The Isolectin IB4 binds RET Receptor Tyrosine Kinase in microglia

*Antonella Casamassa 1 , Laura Cerchia 2 , Carla Lucia Esposito 2 , Vittorio de Franciscis 2 , Lucio Annunziato 1 , Francesca Boscia 1
1 University "Federico II" of Naples, , Naples, Italy
2 CNR, IEOS, Naples, Italy
Abstract text :

Ret receptor tyrosine kinase is the signaling component of the receptor complex for the family ligands of the glial cell line-derived neurotrophic factor (GDNF). Ret is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons and it is necessary for the postnatal maintenance of dopaminergic neurons. Ret expression has been as well demonstrated on microglia and several evidence indicate that GDNF regulates not only neuronal survival and maturation but also certain functions of microglia in the brain.

Here we demonstrated that isolectin IB4, commonly used as a microglial marker in the brain, binds to the glycosylated extracellular domain of Ret both on the surface of living NIH3T3 fibroblasts cells stably transfected with Ret than in adult rat brain sections as revealed by immunoblotting. Further, confocal immunofluorescence analysis demonstrated a clear overlap staining between pRet and IB4 labeling in primary microglia cultures as well as in adult rat sections obtained from control or postischemic brain after permanent middle artery occlusion (pMCAO).

Interestingly, IB4 staining identified activated or amoeboid Ret-expressing microglia under ischemic conditions. Collectively, our data indicate Ret receptor as one of the IB4-reactive glycoconjugate accounting for the IB4 stain in microglia under physiological and ischemic conditions.



Post-ischemic treatment of the standardized Cordyceps militaris extract, WIB-801C reduces cerebral ischemic injury and improved long-term survival in rats.

*Geum Sil Cho 1 , Sunyoung Hwang 1 , Chung Ju 1 , Won-Ki  Kim 1
1 Korea University, College of Medicine, Department of Neuroscience, Seoul, Korea, Republic of
Abstract text :

A traditional herbal medicine, caterpillar fungus (Cordyceps militaris,) extract has been widely investigated in animal models and clinical studies for various diseases including ischemic stroke. Its major metabolite, cordycepin, has been reported to act as a selective A3 adenosine receptor agonist and to protect neurons against ischemic injury. However, their underlying mechanisms remain unclear. In the present study, we report that the standardized C. militaris extract, WIB-801C, which contains cordycepin 8% of total dry weight of the extract, markedly reduced ischemic injury by inhibiting post-ischemic inflammatory responses. Post-ischemic treatment with WIB-801C (orally administered twice at 3 and 8 h after onset of MCAO) significantly reduced infarct size and edema in rats subjected to transient middle cerebral artery occlusion (MCAO, 1.5 h) and subsequent reperfusion (22 h). WIB-801C also significantly improved integrity of glial cells in ischemic lesions, as evidenced by reduced white matter degeneration and loss of blood-brain barrier. Importantly, WIB-801C significantly ameliorated neurological deficits in not only transient but also in permanent stroke models. Moreover, WIB-801Csignificantly improved long-term survival of MCAO rats over 30 days. As we previously reported with selective A3 receptor agonists (Choi et al., 2011, Am J Pathol), WIB-801C also significantly inhibited the infiltration of ED-1 and MPO-positive inflammatory cells into ischemic lesions. The present findings indicate that a natural composite with selective A3 receptor agonist, WIB-801C confers robust neuronal/glial protection and reduced inflammatory response, leading to long-lasting improvement of post-ischemic injury in brains.

#G-S. Cho and S. Hwang contributed equally to this work



Heterogeneity of GFAP-positive glia in the cerebral cortex: from development to injury – single cell gene expression profiling

*David Dzamba 1,2 , Pavel Honsa 1,2 , Vendula Rusnakova 3 , Anders  Stahlberg 4,5 , Mikael Kubista 3,5 , Miroslava Anderova 1,2
1 Institute of Experimental Medicine, AS CR, , Prague, Czech Republic
2 Second Medical Faculty, Charles University, , Prague, Czech Republic
3 Institute of Biotechnology, AS CR, , Prague, Czech Republic
4 Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska Cancer Center, Department of Pathology, Gothenburg, Sweden
5 TATAA Biocenter, , Gothenburg, Czech Republic
Abstract text :

GFAP-positive glial cells represent a key cell type that controls and regulates a diversity of complex functions in the central nervous system. Heterogeneity among these cells has been shown previously on the level of gene expression as well as in functional studies. To unravel astrocyte heterogeneity during postnatal development and after focal cerebral ischemia, we employed single-cell gene expression profiling in acutely isolated GFAP/EGFP-positive cells from the mouse cortex. Using a microfluidic high throughput qPCR platform, we measured the expression of 47 selected genes encoding astrocytic and polydendrocytic markers, ion channels, transporters and receptors that participate in maintaining K and glutamate homeostasis in each of 292 individual GFAP/EGFP-positive glial cells. In this study we show how the expression of these selected genes changes during development (postnatal days 10, 20, 30 and 50) as well as 3, 7 and 14 days after middle cerebral artery occlusion (MCAO). Self-organizing maps and principal component analyses divided the cells according to their similarity in gene expression into three subpopulations of astrocytes present within the first 10-50 days of postnatal development (P10-P50). The first subpopulation comprises immature glia mainly from P10, characterized by the high transcriptional activity of all of the studied genes, including polydendrocytic markers. The second subpopulation is dominated by cells from P20 and displayed low transcript levels of all of the studied genes. The third subpopulation represents mature astrocytes mainly from P30 and P50. Three, seven and fourteen days after ischemia (D3, D7, D14), additional astrocytic subpopulations appear: resting astroglia (mainly from P50 and D3), transcriptionally active early reactive astroglia (mainly from D7) expressing the mRNA of polydendrocytic markers, and, presumably, permanent reactive astroglia (solely from D14). Following focal cerebral ischemia, reactive astrocytes undergo pronounced changes in their expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and markers of reactive astrocytes and polydendrocytes. From the data obtained from single cell PCR analysis, we calculated the Spearman correlation coefficients between pairs of genes, which revealed interesting positive gene expression correlations with polydendrocytic markers (Gria2-4, Grik1-5, Grin3a, Kcnj16, Kcnk2 and Kcnk10 genes). This study was further supplemented by an immunohistochemical analysis of NMDA receptor subunits and PDGFαR, which confirmed that changes in gene expressions correlate with immunodetected proteins.

GA CR 13-02154S, ASTF 110-2011, GAUK 604212


Comparative analysis of neuronal loss, glial activation and tissue degeneration in different cortical areas of adult rats following focal ischemia

*Walace Gomes-Leal 1 , Rafael Fernandes 1 , Rafael Rodrigues Lima 1 , Enio Mauricio Nery Dos Santos 1
1 Federal University of Para, , Belem, Brazil
Abstract text :

The cerebral cortex is one of the most often affected areas after stroke, but there are no studies comparing the outcome in different cortical regions after focal ischemia. The aim of this investigation was to evaluate the patterns of glial activation, tissue degeneration and neuronal loss in different survival times following cortical ischemia. Focal ischemia was induced by stereotaxic microinjections of endothelin-1 (ET-1) into the somatosensory, motor and association cortices of adult rats (N = 45). The control animals were injected with the same volume of sterile saline (N = 27). The animals were perfused at 1, 3 and 7 days after ischemia. Gross histopathology was evaluated using cresyl violet staining. . 20μm sections were submitted to immunohistochemistry for astrocytes (anti-GFAP), activated microglia/macrophages (anti-ED1) and microglia (anti-Iba1 and ED1). Tissue loss and glial activation were more intense in the somatosensory cortex than in other cortical areas. The motor cortex was the second more affected area. The association cortex was the less damaged area, which was confirmed by quantitative analysis (ANOVA-Tukey). The results suggest that an ischemic lesion of the same intensity induces a differential pattern of tissue loss and neuroinflammation, depending on the cortical area, and that the primary sensory and motor areas are more susceptible to ischemia than association areas.


A vertebrate specific Glutaredoxin affects cellular functions of oligodendrocytes

*Carsten Berndt 1 , Klaudia Lepka 1 , Katrin Volbracht 1 , Reiner Schneider 1 , Hans-Peter Hartung 1 , Tim Prozorovski 1 , Orhan Aktas 1
1 Heinrich-Heine Universität, , Düsseldorf, Germany
Abstract text :

Glutaredoxins, members of the Thioredoxin family of proteins, are general disulfide reductases maintaining and regulating cellular thiol homeostasis and signaling, respectively (1). In mammals, two cytosolic and two mainly mitochondrial located Glutaredoxins have been identified (2). We already described an essential role for the formation of a functional neuronal network during embryonic development of one of these proteins, the vertebrate specific Glutaredoxin 2 (3). Here, we will focus on the contribution of Glutaredoxin 2 on basic cellular functions of oligodendrocytes, the myelinating cells of the central nervous system, under physiological and pathophysiological situations, e.g. multiple sclerosis, which is characterized by inflammatory axonal demyelination (4). Oligodendrocyte progenitors and mature oligodendrocytes are very sensitive against oxidative stress induced by release of nitric oxide by activated microglia (5). Using A2B5 positive glia restricted progenitor cells as well as organotypic cerebellar slice cultures, we found that increased levels of Glutaredoxin 2 protect against cell death induced by either activated microglia or S-nitrosoglutathione, a physiological nitric oxide donor. Under physiological conditions elevated levels of Glutaredoxin 2 increased migration of A2B5 positive cells nearly three fold. Western Blot analyses, quantitative real time PCR, as well as immunocytochemistry revealed that Glutaredoxin 2 inhibited further differentiation of NG2 positive oligodendrocyte progenitor cells. In summary, our data indicate the importance of specific thiol redox signaling during cellular protection and function in this type of glia cells.

(1) Lillig, Berndt, Antioxid Redox Signal, accepted manuscript (2013)

(2) Lillig, Berndt, Holmgren, Biochim Biophys Acta 1780: 1304-1317 (2008)

(3) Bräutigam, Schütte, Godoy, Prozorovski, Gellert, Hauptmann, Holmgren, Lillig, Berndt, Proc Natl Acad Sci USA 108: 20532-20537 (2011)

(4) Aktas, Kieseier, Hartung, Trends Neurosci 33: 140-152 (2010)

(5) Pang, Campbell, Zheng, Fan, Cai, Rhodes, Neuroscience 168: 240-245 (2010)


Resetting translational homeostasis restores myelination in CMT1B mice

*Maurizio D'Antonio 1 , Nicolo` Musner 1 , Cristina Scapin 1 , Daniela Ungaro 1 , Ubaldo Del Carro 1 , David Ron 2 , M. Laura Feltri 1,3 , Lawrence Wrabetz 1,3
1 Ospedale San Raffaele, , Milano, United States
2 University of Cambridge, Metabolic Research Laboratories, Cambridge, United Kingdom
3 Hunter James Kelly Research Institute, , Buffalo, United States
Abstract text :

P0 glycoprotein is an abundant product of terminal differentiation in myelinating Schwann cells.  The mutant P0S63del causes Charcot-Marie-Tooth 1B neuropathy in humans, and a very similar demyelinating neuropathy in transgenic mice. P0S63del is retained in the endoplasmic reticulum of Schwann cells where it promotes unfolded protein stress and elicits an unfolded protein response (UPR) associated with translational attenuation. Ablation of Chop, a UPR mediator, from S63del mice completely rescues their motor deficit and reduces active demyelination by half. Here we show that Gadd34 is a detrimental effector of CHOP that reactivates translation too aggressively in myelinating Schwann cells.  Genetic or pharmacological limitation of Gadd34 function moderates translational reactivation, improves myelination in S63del nerves, and reduces accumulation of P0S63del in the ER.  Resetting translational homeostasis may provide a therapeutic strategy in tissues impaired by misfolded proteins that are synthesized during terminal differentiation.


Effects of TrkB expression and signalling on oligodendrocyte myelination

*Anita Ferner 1 , Junhua Xiao 1 , Lauren Giuffrida 1 , Agnes Wong 1 , Haley Peckham 1 , Trevor Kilpatrick 1 , Simon Murray 1
1 University of Melbourne, , Melbourne, Australia
Abstract text :

BDNF is known to promote central nervous system myelination both in vitro and in vivo.  Adopting in vitro myelination assays, we identified that BDNF acts through oligodendroglial-expressed TrkB receptors to promote myelination.  This was verified in vivo, as deletion of TrkB in mature oligodendrocytes (in TrkBfl/fl MBP-cre mice) resulted in a hypomyelinating phenotype during development.  Question: Here we investigate the consequence of TrkB deletion in oligodendrocyte progenitor cells (in TrkBfl/fl CNPase-cre mice) and the signalling pathways downstream of TrkB that promote myelination.

Methods and Results: TrkBfl/fl CNPase-cre mice were born in Mendelian ratios and were phenotypically indistinguishable from littermate control mice.  Surprisingly, analysis of myelinated axonal tracts in the spinal cord and optic nerve revealed normal myelin development.  In addition, the number of cells in the oligodendroglial lineage was the same as control mice during postnatal development.  These data indicate that the timing of TrkB deletion in the oligodendroglial lineage is critical, as deletion in OPCs results in no significant phenotype, whereas deletion in mature oligodendrocytes results in a hypomyelinating phenotype.  These data suggest that OPCs are able to compensate for the loss of TrkB and myelinate normally in the absence of BDNF signalling.

Our in vitro data have shown that the promyelinating influence of BDNF strongly correlates with Erk1/2 activation.  Here we show that overexpression of Erk2 in OPCs exerts a more significant promyelinating effect than Erk1.  As Erk1/2 are known to exert some of their effects through direct transcription factor phosphorylation, we have undertaken an in silico analysis of myelin-specific transcription factors and identified several that contain potential Erk1/2 binding domains and phosphorylation sites. Co-immunoprecipitation experiments show an interaction between Erk1/2 and these transcription factors. Investigation into the nature of these interactions and their functional consequences are ongoing.

Conclusions: This work suggests a novel role for Erk1/2 signalling within oligodendrocytes that regulates CNS myelination, possibly via activation of myelin-specific transcription factors.  As Erk1/2 is activated by multiple receptors, other receptors could potentially compensate for the loss of TrkB in OPCs, resulting in a normally myelinated CNS in vivo.


Role of Apolipoprotein D in macrophage recruitment and myelin phagocytosis upon peripheral nerve injury

*Nadia Garcia Mateo 1 , Diego Sánchez 1 , Concepción Lillo 2 , M Dolores Ganfornina 1
1 Instituto de Biologia Y Genetica Molecular, , Valladolid, Spain
2 Instituto de Neurociencias de Castilla y León, Universidad de Salamanca , Salamanca, Spain
Abstract text :

Apolipoprotein D (ApoD) is a Lipocalin expressed by glial cells during development, adulthood and aging of the vertebrate nervous system. In the peripheral nervous system (PNS) it is secreted by Schwann cells and fibroblasts and its expression is strongly induced upon injury.

We are interested in the function of ApoD in the molecular events that take place after a PNS injury. We have studied in vivo the process of Wallerian degeneration following sciatic nerve crush and we have assayed ex vivo the phagocytosis of labelled myelin from wt and ApoD-KO mice by flow cytometry.

The analysis of cellular processes and protein or mRNA expression of a group of signalling molecules induced by injury shows that the lack of ApoD results in an exacerbated MCP-1 and TNFa-dependent macrophage recruitment. At the injury site, free AA decreases in the wt. Lack of ApoD results in higher basal levels and a stronger injury-triggered depletion of AA. Control by ApoD of the availability of AA to produce the lipid mediators involved in macrophage recruitment is therefore a key mechanism that conditions both Wallerian degeneration and injury resolution later on.

On the other hand, the phagocytosis of ApoD-KO CNS myelin is less efficient than the phagocytosis of wt myelin. These results indicate that there are also genotype-dependent differences in myelin composition and/or in the interaction between myelin and macrophages. An electron microscopy analysis of crude myelin preparations reveals that the CNS myelin from ApoD-KO mice has abnormal periodicity and shows defective myelin compaction. Lipid analysis of ApoD-KO myelin shows altered phospholipid composition, particularly in phosphoinositid species. A study of the function of  ApoD in the myelination process is currently underway,

   Our results demonstrate that ApoD function is relevant for both, myelin membrane properties that influence myelin-macrophage interactions, and the control of the lipid-mediated signalling events controlling the extent of macrophage recruitment. 


Support:MICINN(BFU2008-01170;BFU2011-23978), JCyL(VA180A11-2).


Post-transcriptional regulation of Myelin Basic Protein during cell stress conditions

*Constantin Gonsior 1 , Jacqueline Trotter 1
1 University of Mainz, , Mainz, Germany
Abstract text :

Myelin Basic Protein (MBP), a major component of central nervous system (CNS) myelin, is essential for oligodendroglial function and myelination in the CNS and its expression is tightly regulated in time and space. The mRNA of MBP is sorted to cytoplasmic RNA granules and transported up to the distal processes of oligodendrocytes in a translationally silenced state. We and others could show that, dependent on axonal signals, activation of the non-receptor tyrosine kinase Fyn leads to a release of translational inhibition allowing synthesis of MBP protein at the axon-glial contact site. A key factor in this pathway is the Fyn target and RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) A2, that recognizes the A2 response element (A2RE) in the MBP 3’UTR and orchestrates the dynamics of the MBP mRNA granule. Moreover, we identified hnRNP F as an additional target of Fyn kinase present in these ribonucleoprotein complexes and contributing to post-transcriptional regulation of MBP.

Cell stress conditions, as present in various disease states such as multiple sclerosis, may result in the formation of cytoplasmic RNA-containing stress granules (SGs), potentially influencing the fate of myelin transcripts including their translation.MBP mRNA was shown to be sorted to these cytoplasmic foci. We here identified the hnRNPs A2 and F to be associated with oligodendroglial SGs. De-regulation of such factors involved in MBP mRNA metabolism could affect stress dependent synthesis of MBP and thus (re-)myelination and myelin maintenance.


Long-term consequences of perinatal inflammation on de- and remyelination in the central nervous system

*Viktoria Gudi 1 , Karelle  Bénardais 1,2 , Jasmin Neßler 1 , Vikramjeet Singh 1,2 , LiJie Gai 1 , Thomas Skripuletz 1 , Martin Stangel 1,2
1 Hannover Medical School, , Hannover, Germany
2 Center for Systems Neuroscience, , Hannover, Germany
Abstract text :

Perinatal inflammation is intensively discussed to have a long-term impact on various cell populations and on the development of autoimmune diseases such as multiple sclerosis (MS) in adulthood. In order to determine long-term effects of perinatal inflammation on the course of demyelination in adulthood we mimic a bacterial inflammation by exposure to lipopolysaccharide (LPS) of either pregnant or newborn mice. Demyelination as a “second hit” was induced by feeding adult mice with the oligodendrocyte toxin cuprizone (bis-cyclohexanone oxaldihydrazone). A single prenatal LPS injection at E13.5 did not affect the course of demyelination in adulthood. In contrast, serial postnatal LPS injections lead to a delayed demyelination and a reduced number of activated microglia in the corpus callosum, suggesting a long-lasting effect of perinatal inflammation on the function of microglia. Surprisingly, mice exposed to LPS after birth showed an enhancement of early remyelination accompanied by an increased number of newly differentiated oligodendrocytes. Furthermore, independent of cuprizone administration the perinatal LPS injections seem to induce a long-term impact on the blood-brain barrier (BBB) by decreasing the number of claudin-5 positive vessels. In summary, perinatal inflammation mimicked by the administration of LPS has long-lasting effects on the BBB, microglia activation, and remyelination.


Electrical activity-dependent control of myelin gene expression in vivo

*Jacob Hines 1 , Bruce Appel 2
1 University of Colorado - Denver, , Aurora, CO, United States
2 University of Colorado School of Medicine, , Aurora, CO USA, United States
Abstract text :

Question and Methods

Myelination of CNS axons is a multi-step process beginning with the production of oligodendrocyte progenitor cells (OPCs) in the ventral neural tube. Newly specified OPCs proliferate, migrate into white matter tracts, and differentiate into mature oligodendrocytes that wrap select axons with a myelin sheath. Previous studies, performed primarily in cell culture, suggest that electrical activity may regulate multiple stages of oligodendrocyte development including proliferation, migration, initial wrapping and myelination. In this study, we have examined the requirement for electrical activity in oligodendrocyte development in vivo using zebrafish as a model system.


We find that electrical activity is not required for proliferation, migration, or the initiation of axon wrapping. Rather, our data indicate a specific role for electrical activity in oligodendrocyte differentiation by regulating a subset of myelin genes, including myelin basic protein (mbp). Silencing electrical activity with tetrodotoxin reduced mRNA expression of mbp, as assessed by RNA in situ hybridization and quantitative RT-PCR. In contrast, myelin protein zero, claudin K, claudin 11a, ugt8, and 36K levels were not regulated by electrical activity. Ongoing experiments are aimed to uncover the mechanism of activity-dependent mbp mRNA expression during oligodendrocyte differentiation.


Taken together, these data indicate that electrical activity can modulate myelin gene expression in vivo but do not support the notion that electrical activity drives key axon-glia messengers that are required for the initial stages of myelination.  


Unravelling protein networks involved in peripheral nerve myelination

*Salla Kangas 1 , Steffen Ohlmeier 2 , Raija Sormunen 2 , Anthony Heape 1
1 University of Oulu, , Oulu, Finland
2 University of Oulu, Biocenter Oulu, Oulu, Finland
Abstract text :

This study aims to identify protein networks that participate in peripheral myelin formation and maintenance.

We use a cell culture system, where C57BL/6J mouse embryos (E13.5) are used for the isolation of dorsal root ganglia neuron explants. Explants are cultured in Matrigel, and endogenous Schwann cells appear in the culture within a few days. Myelin formation is induced by adding myelination-promoting substances to the culture medium. Immunocytochemistry is used to evaluate the myelination efficiency and the developmental timetable of myelination in culture. Electron microscopy is used to study structural features of myelin in culture and to compare its ultrastructural features with those of myelin in situ. RNA microarray analysis is performed to study gene expression changes during myelin development in culture. The RNA expression data indicates the changes in the RNA expression levels, but it does not give information about the amount of final protein product, their posttranslational modifications or stability. We combine the RNA expression analysis with a proteomics approach. Proteins are extracted for 2-dimensional electrophoresis analysis at the same time points, and protein spots exhibiting time-dependent changes in their expression levels are analyzed by mass spectrometry.

We show that mouse dorsal root ganglion explant cultures produce substantial amounts of mature myelin in 3 weeks after the induction of myelination. Ultrastructural analysis shows that myelin in our cell culture system has the same structural characteristics as those observed in peripheral nerves in situ. RNA microarray analysis reveals changes in myelin-, nervous system- and Schwann cell-related genes. The most significant changes in gene expression occur at the induction of myelination. At the protein level, peaks of specific proteins can be observed at the first and the last time points, which complement the results obtained using microarray data.

We have developed a reproducible and efficient method to study molecular changes in myelination at the proteomic and genomic levels. This approach will be exploited further for the characterization of the molecular networks involved in the myelination process.


Transport and translation of MBP MRNA is differentially regulated by distrinct hnRNP proteins

Julie Torvund-Jensen 1 , Jes Stensgaard 1 , Linda  Fihl 1 , Lasse  Reimer 1 , *Lisbeth Laursen 1
1 Aarhus University, , Aarhus, Denmark
Abstract text :

During the myelination process, the oligodendrocyte extends processes to and wraps around multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. This is one unique example of how cells in the central nervous system establish asymmetry. Transport of mRNA and local regulation of protein synthesis represent one mechanism by which such cellular asymmetry can be generated and the different requirements for myelin sheath at each axo-glia interaction can be controlled.  

The mRNA of a key myelin sheath protein, myelin basic protein (MBP), is known to be transported into the oligodendrocyte processes, and a tight temporal and spatial control of MBP mRNA translation is thought to be required for normal myelination. The molecular basis for the tight control of mRNA transport and translation is the assembly of large mRNA-protein complexes. Prior work has identified a number of proteins that interact with the MBP mRNA, including hnRNP-A2, hnRNP-K and hnRNP-E1. However, it is currently unknown how these protein functions together to prevent translation during transport. It is also unknown how targeting of the MBP mRNA to the myelin sheets occurs.           

To delineate the precise role of the individual binding protein in regulating MBP mRNA translation, we have identified regulatory elements within the 3’UTR of the MBP mRNA involved in translational inhibition, and we have identified binding sites in the mRNA for hnRNP-K and hnRNP-E1. Furthermore, we have analyzed the effect of siRNA-mediated knockdown and overexpression of these proteins in primary oligodendrocytes.  Together, our results allow us to propose a model, in which the individual mRNA binding proteins are assigned distinct roles for correct spatial and temporal expression of MBP.


Modulation of the proliferation and differentiative potential of adult brain subventricular zone cells by purinergic signaling in vitro and in vivo: contribution of reactive astrocytes.

*Stefania Ceruti 1 , Marta Boccazzi 1 , Chiara Rolando 2 , Maria P. Abbracchio 1 , Annalisa Buffo 2
1 Università degli Studi di Milano, , Milan, Italy
2 Università di Torino, Neuroscience NICO, Turin, Italy
Abstract text :

Evidence is accumulating that neurogenesis in the subventricular zone (SVZ) is boosted after trauma or ischemia, also through the interaction with surrounding parenchyma or niche cells. Nevertheless, the number of newborn neurons that survive and integrate in the damaged areas is negligible, suggesting a non-permissive environment. Thus, understanding the complex signaling network guiding neuroblast generation/survival could help identifying strategies to limit negative inputs and promote regeneration. Extracellular nucleotides (eNTs) are among the hypothetical modulators of SVZ cell functions, especially under pathological conditions where their concentrations raise several folds and they contribute to reactive astrogliosis. Few literature data have recently pointed for a role of the P2Y1 receptor subtype (one of the 8 known G protein-coupled nucleotide receptors) in controlling the proliferation and differentiative potential of SVZ cells. Thus, we tested the ability of ADPβS, a stable P2Y1 agonist, to modulate stem cell properties in the adult brain, with a focus on the possible modulatory effects exerted by reactive astrocytes. The administration of ADPβS in the lateral ventricle of adult mice led to reactive astrogliosis in the surrounding brain parenchima, and to a massive reaction of GFAP-expressing precursors and astrocytes in the SVZ. Also proliferation was increased, paralleled by a significant expansion of the population of Mash1 transit-amplifying cells and of doublecortin neuroblasts. Thanks to the conditional GLAST::CreERT2 YFP mouse model, we also demonstrated that ADPβS promoted the proliferation of GLAST-expressing progenitors in the neurogenic niche, and sustained their progression towards the generation of rapidly dividing transit-amplifying cells. In vitro the nucleotide analog increased the proliferation of SVZ cells grown as neurospheres, and their differentiation towards neurons, fully confirming in vivo data. Interestingly, a significant enhancement in neurosphere generation was detected when SVZ cells were initially grown in the supernatant of astrocytes exposed to ADPβS, and then shifted to normal medium. This suggests that ADPβS stimulates the release of yet-to-be identified astrocytic mediator(s) whose removal from the culture medium boosted proliferation of SVZ cells. Our results further strengthen the notion that the purinergic system is a key regulator of the neurogenic potential of SVZ cells, both directly and through the involvement of reactive astrocytes.


Hypothalamic tanycytes: a neurogenic population of radial-glial like cells in the postnatal and adult brain

Niels Haan 1 , Timothy Goodman 1 , Alaleh Nadji-Samieir 1 , Christina Stratford 1 , Ritva Rice 2 , Elie Al Agha 3 , Saverio Bellusci 3 , *Mohammad K Hajihosseini 1
1 University of East Anglia, , Norwich, United Kingdom
2 University of Helsinki, , Helsinki, Finland
3 University of Giessen, , Giessen, Germany
Abstract text :

Hypothalamus is emerging as the third neurogenic region of the postnatal and adult rodent brain. However, there is much debate about the identity of its stem/progenitor cells and very little is known about the dynamics and significance of hypothalamic neurogenesis.

To test whether hypothalamic tanycytes act as bona fide neural stem/ progenitor cells in vivo, we analysed their immumoprofile and proliferative potential, and lineage-traced them in vivo. For this, we exploited our earlier findings that in the adult hypothalamus, Fibroblast growth factor 10 (Fgf10) is expressed exclusively by tanycytes (Hajihosseini et al. 2008 MCN 37: 857-68), a radial glial-cell like population that lines the floor and ventral walls of the third ventricle. Using a line of Fgf10-lacZ reporter mice, we found that Fgf10 beta tanycytes express a panel of neural/stem progenitor markers such as Nestin, Musashi1, BLBP and Sox2. Fgf10 tanycytes also incorporated BrdU under cumulative BrdU-labelling paradigms, and formed neurospheres in vitro. To directly test the potential of Fgf10 ve tanycytes, we lineage-traced them at P28 and P70 by activating the constitutive expression of the marker gene, tomato-dsred, in these cells and their descendants.  This was achieved by applying tamoxifen to Fgf10-creERT2::Rosa26R-Tomato double transgenic mice.

After short survival time-points, tomato was detected exclusively in tanycytes, whilst with longer survival time-point, tomato cells emerged in the neighbouring parenhcyma. The majority of these were NeuN neurons with fine arborizations. We noted that the newly-generated neurons become associated mainly with hypothalamic nuclei that regulate appetite and energy-balance. Moreover, they respond to appetite/energy balance regulating signals such as acute food deprivation and leptin administration.

Our findings establish Fgf10 beta-tanycytes as a putative population of neural stem/ progenitor cells that generate appetite/energy balance regulating neurons in the postnatal and adult hypothalamus. 


Mesenchymal stem cell conditioning promotes oligodendroglial maturation

*Janusz Jadasz 1 , David Kremer 1 , Peter Göttle 1 , Nevena Tzekova 1 , Francisco J. Rivera 2 , Hans-Peter Hartung 1 , Ludwig Aigner 2 , Patrick Küry 1
1 Heinrich-Heine University, , Düsseldorf, Germany
2 Paracelsus Medical University, Institutes of Molecular Regenerative Medicine, Salzburg, Austria
Abstract text :


In demyelinating diseases such as multiple sclerosis myelin repair activities based on recruitment, activation and differentiation of resident progenitor and stem cells can be observed. However, the overall degree of successful remyelination is limited. It is therefore of considerable interest to understand oligodendroglial precursor cell (OPC) homeostasis and maturation processes in order to develop remyelination therapies. Mesenchymal stem cells (MSC) were shown to exert positive immunomodulatory effects, to reduce demyelination, to increase neuroprotection and to promote adult neural stem cell differentiation towards the oligodendroglial lineage. We here addressed whether MSC secreted factors can influence primary OPCs in a myelin non-permissive environment.


To this end we analyzed cellular morphologies, expression and regulation of key genes/proteins involved in oligodendroglial cell fate and maturation upon incubation with mesenchymal stem cell conditioned medium.


This demonstrated that MSC derived soluble factors promote and accelerate oligodendroglial differentiation, even under astrocytic endorsing conditions. Accelerated maturation featured elevated levels of 2', 3'-cyclic nucleotide 3'-phosphodiesterase and myelin basic protein expression, reduced glial fibrillary acidic protein expression and was accompanied by downregulation of prominent inhibitory differentiation factors such as ID2 and ID4.


We thus conclude that besides the previously established immunomodulatory and neuroprotective roles of MSCs these cells can also positively influence oligodendrogenesis in the adult central nervous system.


Artificial cell fate regulation of the progenitor cells in the adult spinal cord

*Masaaki Kitada 1 , Jun-ichi Suzuki 1 , Mari Dezawa 1
1 Tohoku University Graduate School of Medicine, , Sendai, Japan
Abstract text :

In mammals, regeneration capacity of the central nervous system (CNS) is considered too low to recover the neurological functions after traumatic injury. In fishes and amphibians, however, the spontaneous regeneration is vigorous enough to complete the anatomical reconstruction and functional recovery even after severe damage of CNS, in which the ependymal cells play the central role to exhibit proliferation, epithelial-mesodermal transition, cell migration, support of regenerating axons and neurogenesis to contribute to recovery from injury. These positive roles of the ependymal cells observed in fish and amphibian damaged CNS may be good candidates for treating mammalian CNS injury. In this study, we attempted to control the activity of the ependymal cells in the adult rodent spinal cord by virus-mediated gene transfer for imitating the reactions that are observed in the ependymal cells of fish and amphibian damaged CNS. We introduced the gene known to be expressed in both the ependymal cells and neural stem cells (NSCs) and found the proliferation of ependymal cells. In this case, the ependymal cells expressed glial fibrillary acific protein suggesting that this gene regulates proliferation and differentiation into astrocytes. In addition, introduction of another gene, also known to be expressed in the ependymal cells and NSCs, caused cell proliferation without differentiation tendency. These findings indicate that the cell fate of the ependymal cells in the adult rodent spinal cord can be modulated by artificial intervention. Future studies will clarify whether cell fate modification in the ependymal cells can contribute to anatomical reconstruction and functional recovery in the adult mammalian damaged spinal cord.


Endogenous retinoic acid synthesis contributes to neural stem cell differentiation

Barbara Orsolits 1 , Adrienn Borsy 1 , Emília Madarász 1 , Zsófia Mészáros 1 , Tímea Kőhidi 1 , Károly Markó 1 , Márta Jelitai 1 , Ervin Welker 1 , *Zsuzsanna Környei 1
1 Institute of Experimental Medicine, , Budapest, Hungary
Abstract text :

Retinoic acid (RA) has been widely used as a potent inducer of neuronal differentiation in various multipotent cell populations (embryonic carcinoma [EC], embryonic and neural stem [ES; NS] cells, iPS cells), in vitro. In vivo, in the central nervous system, RA action has been reported at sites of both embryonic and postnatal neurogenesis, indicating that RA signaling is involved in the regulation of neural stem cell commitment and differentiation throughout life. While NS cells respond to RA delivered to or synthetised within the brain, little is known about i; the potential of NS cells to utilize RA precursor molecules and ii; the rearrangement of the components of retinoid metabolism/signaling during NS cell development. In the presented in vitro study we monitored the changes in the retinoid machinery in differentiating NS cell populations (NE-4C NS cell line and radial glia like (RGl) cells) derived either from embryonic or adult brain tissues. We show, that early embryonic NS cells are capable to convert both vitamin A and retinyl ester to RA which serves as an auto-inductive signal to initiate the neuronal differentiation protocol. 


Selective ablation of CNS-resident microglia disturbs homeostasis within the adult hippocampal neurogenic niche

*Kelly Miller 1 , Caroline Baufeld 1 , Jochen Winterer 2 , Stefan Prokop 1 , Karen E.  Carney 3,4 , Dietmar Schmitz 2,5,6,7 , Frank L. Heppner 1,6
1 Charité - Universitätsmedizin Berlin, Department of Neuropathology, Berlin, Germany
2 Charité - Universitätsmedizin Berlin, Neuroscience Research Center, Berlin, Germany
3 VU University, Center for Neurogenomics and Cognitive Research, Amsterdam, Netherlands
4 Université de Bordeaux, Neurocentre Magendie, Iserm Unité 862, Bordeaux, Netherlands
5 Bernstein Center for Computational Neuroscience Berlin, , Berlin, Germany
6 Charité - Universitätsmedizin Berlin, Cluster of Excellence 'NeuroCure', Berlin, Germany
7 DZNE - German Center for Neurodegenerative Diseases, , Berlin, Germany
Abstract text :

Adult neurogenesis plays a critical role in the overall health of, and repair processes occurring within the nervous system.  While a great deal of information has been gained about the elements of the neurogenic niche and the factors that mediate neurogenesis, many unanswered questions remain.  Specifically, studies suggest that microglia have a significant impact on adult neurogenesis, but no unifying theory exists to explain their exact role in this process.  In order to determine if microglia play a pivotal role in adult hippocampal neurogenesis and to examine the mechanisms through which microglia exert their effects on the hippocampal neurogenic niche, we employed a transgenic mouse model that allows for the selective ablation of microglia in the central nervous system (CNS), the CD11b-herpes simplex virus thymidine kinase (CD11b-HSVTK) mouse.  Using this system, we removed microglia from the adult hippocampal neurogenic niche and evaluated the effects of this manipulation on neurogenesis and hippocampal homeostasis, including immunohistochemical analyses of stem cell survival and proliferation, as well as analyses of neuroblast development, migration and electrophysiological activity.  Taken together, these studies help to provide a better understanding of the factors governing adult neurogenesis and further our knowledge about the increasingly diverse role of microglia in the CNS.


Characterization of neural precursors derived from mouse iPS cells: in vitro and in vivo after transplantation into the central nervous system

*Sabah Mozafari 1 , Antoine  Marteyn 1 , Cecilia  Laterza 2 , Cyrille  Deboux 1 , Gianvito  Martino 2 , Anne  Baron-Van Evercooren 1
1 Université Pierre et Marie Curie, Institut du Cerveau et de la Moelle épinière, Paris, France
2 San Raffaele Scientific Institute, Institute of Experimental Neurology–DIBIT 2, Division of Neuroscience, Milan, Italy
Abstract text :

   Experimental studies have shown that loss of myelin results in axonal loss and disability. So, finding of an expandable and autologous source of myelin-forming cells to enhance remyelination is required. Induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) have been developed recently. The remyelination potential and safety of these cells still remained to be well-addressed. The main goal of this study is to characterize mouse iPS-NPCs in vitro and in vivo after transplantation.

   We used embryonic mouse neural progenitor cells (mNPCs) as control and characterized mouse iPS-NPCs for their expression of major markers of immature or mature cells by immunostaining. RT-PCR was performed to analyze expression of Nestin, Olig2, β3-tubulin, Olig1, Sox10, and NKX2.2 mRNAs. Furthermore, to investigate the fate of these cells in vivo, we injected the lysolecithin to induce focal demyelination in the spinal cord of adult nude or Shiverer mice. We transplanted cells in the lesion site 2 days after demyelination. Animals were sacrificed 2 days, 1, 2, 6 and 8 weeks post transplantation to assess survival and differentiation of the grafted cells.

   Our preliminary results showed that miPS-NPCs similar to mNPCs were Nestin , Ki67 , Olig2 , β3-tubulin but O4- and MAP5-. miPS-NPCs, were more proliferative than mNPC. Moreover, miPS-NPC expressed all mRNA transcripts except Sox10. A first line of miPS-NPCs, was transplanted in the newborn Shiverer:Rag forebrain or the demyelinated spinal cord of immunosuppressed Shiverer mice with a constant failure in terms of cell survival beyond 2 days of transplantation, highlighting the fragility of some of the reprogrammed cell lines. Cells of a second line of miPS-NPCs were transplanted in nude mice to be sacrificed at 1, 2 and 6 weeks. An additional serie of transplantation was performed in the demyelinated Shi:Rag spinal cord. Data at 1 and 2 weeks indicate excellent survival and integration of the miPS-NPC at both time points. Some of the grafted cells expressed GFAP, Olig2 and Ki67 (few only) and so far, no tumors were observed at these time-points. Immunohistochemistry with other markers and analysis of 6 weeks post transplantation animals, are in progress.

   Our preliminary results show that although miPS-NPCs have very similar immature phenotype of mNPCs in vitro, they may have different survival capacities in vivo. Future studies will reveal whether transplanted cells which showed short-term survival after grafting are capable of differentiation into myelin-forming cells. Sponsored by MSIF and ELA.


Oligodendrogliogenic and neurogenic adult subependymal zone neural stem cells constitute distinct lineages and exhibit differential responsiveness to Wnt signaling

*Felipe Ortega 1 , Sergio Gascon 2 , Giacomo  Masserdotti 2 , Aditi Deshpande 2 , Christiane  Simon 2 , Judith Fischer 3 , Leda Dimou 2 , Dieter Chichung Lie 4 , Timm Schroeder 3 , Benedikt Berninger 1
1 Johannes Gutenberg University Mainz, , Mainz, Germany
2 Ludwig Maximilians Universität, Physiological Genomics, München, Germany
3 Institute of Stem Cell Research, Helmholtz Zentrum München, ISF, München, Germany
4 University Erlangen-Nürnberg, Erlangen, , Erlangen, Germany
Abstract text :

The adult mouse subependymal zone (SEZ) harbors adult neural stem cells (aNSCs)that give rise to neuronal and oligodendroglial progeny. However it is not known whether the same aNSC can give rise to neuronal and oligodendroglial progeny or whether these distinct progenies constitute entirely separate lineages. Continuous live imaging and single cell tracking of aNSCs and their progeny isolated from the mouse SEZ revealed that aNSCs exclusively generate oligodendroglia or neurons, but never both within a single lineage. Moreover, activation of canonical Wnt signaling selectively stimulated proliferation within the oligodendrogliogenic lineage, resulting in a massive increase in oligodendrogliogenesis without changing lineage choice or proliferation within neurogenic clones. In vivo activation or inhibition of canonical Wnt signaling respectively increased or decreased the number of Olig2 and PDGFR-a positive cells, suggesting that this pathway contributes to the fine tuning of oligodendrogenesis in the adult SEZ. 


CCL-1 in the spinal cord contributes to neuropathic pain induced by nerve injury.

*Nozomi Akimoto 1 , Kenji  Honda 2 , Daisuke  Uta 3 , Hidemasa  Furue 3 , Mizuho  Kido 4 , Keiji  Imoto 3 , Yukio Takano 2 , Mami Noda 1
1 Kyushu University, Pathophysiology, Fukuoka, Japan
2 Fykuoka University, Physiology and Pharmacology, Fukuoka, Japan
3 National Institute for Physiological Sciences, , Okazaki, Japan
4 Kyushu University, Molecular Cell Biology and Oral Anatomy, Fukuoka, Japan
Abstract text :

Cytokines such as interleukins are known to be involved in the development of neuropathic pain through activation of neuroglia. However the role of chemokine (C-C motif) ligand 1 (CCL-1), a well-characterized chemokine secreted by activated T cells, in the nociceptive transmission remains unclear. Here we examined actions of CCL-1 on synaptic transmission, glial cells and cytokines in the spinal dorsal horn, and found that CCL-1 is one of the key mediators involved in the development of neuropathic pain. After a unilateral and partial sciatic nerve ligation (PSNL), expression of CCL-1 mRNA was transiently detected from the ipsilateral dorsal root ganglion (DRG) and spinal cord, and CCL-1 receptor CCR-8 protein was expressed in the superficial dorsal horn. Recombinant CCL-1 injected intrathecally to naïve mice induced hyperalgesia which was prevented by the supplemental addition of N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801. Patch-clamp recordings from spinal cord slices revealed that CCL-1 enhanced excitatory synaptic transmission in the substantia gelatinosa (lamina II). We showed that CCL-1 induced phosphorylation of NMDA subunit, NR1 and NR2B, and increased expression of glial cells activation markers (Iba-1, CD11b and GFAP) and cytokines (IL-1beta, TNF-alpha and IL-6) in the spinal cord. The tactile allodynia induced by nerve ligation was attenuated by prophylactic and chronic administration of neutralizing antibody against CCL-1 and in CCR-8 knock-down mice. Our results indicate that CCL-1 is one of the key molecules in pathogenesis and CCL-1/CCR-8 signaling system can be a potential target for drug development in the treatment for neuropathic pain.


Therapeutic Efficacy of Suppressing the JAK/STAT Pathway in Multiple Models of Neuroinflammation

*Etty (Tika) Benveniste 1
1 Univ. of Alabama at Birmingham, , Birmingham, United States
Abstract text :

Increasing evidence indicates that pathogenic T helper cells and myeloid cells, including dendritic cells, macrophages and microglia, are involved in the pathogenesis of Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis, an animal model of Multiple Sclerosis. The Janus Kinase/Signal Transducer and Activator of Transcription  (JAK/STAT) pathway is utilized by cytokines for signaling, and is critical for development, regulation and termination of immune responses. Dysregulation of the JAK/STAT pathway has pathological implications, especially in autoimmune and neuroinflammatory diseases. Many of the key immunoregulatory cytokines involved in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis, including IL-6, IL-12, IL-23, IFN-γ and GM-CSF, use the JAK/STAT pathway to induce biological responses. As such, targeting JAKs has implications for treating autoimmune inflammation of the brain. In this study, we use a novel JAK1/2 inhibitor, AZD1480, to investigate the therapeutic potential of inhibiting the JAK/STAT pathway in five different models of Experimental Autoimmune Encephalomyelitis. AZD1480 treatment effectively inhibits disease severity in MOG-induced classical and atypical Experimental Autoimmune Encephalomyelitis models by preventing entry of immune cells into the brain, suppressing differentiation of Th1 and Th17 cells, deactivating myeloid cells such as macrophages and dendritic cells, inhibiting STAT activation in the brain, and reducing expression of pro-inflammatory cytokines and chemokines. Furthermore, treatment of SJL/J mice with AZD1480 delays disease onset of PLP-induced relapsing-remitting disease, and reduces clinical severity. Suppression of the JAK/STAT pathway by AZD1480 treatment was also effective in reducing ongoing paralysis induced by adoptive transfer of either path­­­­­ogenic Th1 or Th17 cells.  In vivo AZD1480 treatment impairs both the priming and expansion of T-cells in Experimental Autoimmune Encephalomyelitis development. Immune modulation induced by AZD1480 involves attenuation of antigen-presentation function by downregulation of MHC class II and CD40 expression, as well as a direct inhibitory effect on Th1 cell polarization. These results collectively indicate that inhibition of the JAK/STAT pathway has striking clinical efficacy in multiple pre-clinical models of Multiple Sclerosis, and suggest the feasibility of the JAK/STAT pathway as a target for immunomodulatory therapy in Multiple Sclerosis.



Primary microglia lack strict regulation of inflammasome-mediated activation as compared to myeloid macrophages

*Saskia Burm 1 , Ella  Zuiderwijk-Sick 1 , Anke 't Jong 1 , Celine van der Putten 1 , Linda van Straalen 1 , Sandra Amor 2 , Paul van der Valk 2 , Jack van Horssen 3 , *Jeffrey Bajramovic 1
1 Biomedical Primate Research Centre, , Rijswijk, Netherlands
2 VU Medical Centre, Department of Pathology, Amsterdam, Netherlands
3 VU Medical Centre, Department of Molecular Cell Biology, Amsterdam, Netherlands
Abstract text :

Myeloid cells can respond to intra- and extracellular danger/stress signals by inflammasome-mediated activation. This process consists of two steps: Toll-like receptor-induced production of pro-IL-1b followed by inflammasome-mediated cleavage and secretion of bioactive IL-1b. Inflammasome-mediated activation is strictly regulated by expression of regulatory proteins and by inhibitory processes like autophagy. Recent studies describe that microglia are markedly different from other myeloid cells. They originate from a separate progenitor, are chronically exposed to the neural microenvironment and their activation may be regulated differently. The objective of this study was to determine the expression profile of inflammasome components in primary microglia and to compare inflammasome-mediated microglial activation and its regulation with other myeloid cells.

Primary microglia, bone marrow- and blood-derived macrophages (BMDMs) of adult rhesus macaques of the same donor were profiled for all NOD-like receptors (NLRs), inflammasome-associated adaptor proteins, caspases, and regulatory proteins by qPCR. Inflammasome function and kinetics were assessed by exposing LPS-primed microglia to ATP, Silica or MSU (danger-associated molecular patterns: DAMPs) followed by measuring the transcription of pro-IL-1b-encoding mRNA and the secretion of IL-1b.

Primary microglia expressed NLRs (NALP1-3, NOD1/3/4, AIM2, IPAF, NAIP), adaptor proteins (ASC), caspases (1/3-5/7/8), and regulatory proteins (A20), and the expression profile closely resembled that of BMDMs. Priming of microglia with LPS induced high levels of pro-IL-1b-encoding mRNA, but IL-1b protein was only secreted in response to subsequent stimulation with various DAMPs. Interestingly, in LPS-primed BMDMs the window for inflammasome-mediated activation was 4-8 hours, while LPS-primed microglia remained sensitive for inflammasome-mediated activation for at least 20 hours.

In conclusion, primary microglia express multiple inflammasome components closely resembling the expression profile of BMDMs and they can be induced to form functional inflammasomes. Importantly, primed microglia remain sensitive to inflammasome-mediated activation for much longer than other macrophages. We will present data on whether this reflects a difference in negative regulation of the inflammasome, or differences in autophagy or apoptosis sensitivity.


Lipopolysaccharide-induced brain activation of the indoleamine 2,3-dioxygenase and depressive-like behavior are impaired in obese and diabetic db/db mice

*Nathalie Castanon 1 , Anne-Laure Dinel 1 , Caroline André 1 , Agnès Aubert 1 , Sophie  Layé 1
1 Nutrition and Integrative Neurobiology, , Bordeaux, France
Abstract text :

Converging clinical studies report an increased prevalence of comorbid neuropsychiatric symptoms, particularly major depressive disorders, in a number of conditions (e.g., aging, obesity) and diseases (e.g., atherosclerosis, congestive heart failure, rheumatoid arthritis) sharing inflammation as a common denominator. By using an experimental approach in mice exposed to innate immune system (ISS) stimulation, we demonstrated that induction of depressive-like behavior is mediated by cytokine-induced brain activation of a tryptophan-catabolizing enzyme, the indoleamine 2,3-dioxygenase (IDO). In the metabolic syndrome (MetS), a widely accepted concept that identifies a cluster of individual risk factors for type 2 diabetes and cardiovascular disease (including obesity, metabolic dysregulations and basal low-grade inflammation), neuropsychiatric symptoms emerge as significant factors for aggravation of the disease and related outcomes. Recently, we demonstrated in a model of MetS, the diabetic and obese db/db mice that cognitive alterations and increased anxiety-like behavior are related to hippocampal inflammation. On the other hand, depressive-like behavior is not affected by basal inflammation displayed by db/db mice. Given the data linking increased depressive-like behavior and IDO activation by cytokines in conditions of ISS stimulation, the question arises as to whether depressive-like behavior is increased in those conditions in db/db mice.

To answer this question, we measured in db/db mice and in their healthy db/ littermates the effect of a lipopolysaccharide (LPS) challenge (5 µg/mouse, ip) on behavioral reactivity in a depression test (the forced swim test, FST), plasma levels and hippocampus expression of inflammatory cytokines and related neuronal targets, and brain IDO activity.

Plasma levels and hippocampus expression of IL-1β, IL-6, TNFα and IL-10 are similarly increased 2h after LPS in both db/ and db/db mice. As expected, brain IDO activity and duration of immobility in the FST are increased in LPS-treated db/ mice 24h after LPS. On the contrary, induction of brain IDO activity is significantly blunted in db/db mice compared to their db/ counterparts and no increase of depressive-like behavior is observed. Moreover, some data suggest an impairment of the neuroimmune interactions in db/db mice.

We need now to understand how obesity and related neurobiological alterations impair IDO activation by cytokines and their consequences in terms of vulnerability to infections in MetS.


Does tissue Transglutaminase play a role in leukocyte/monocyte infiltration during experimental multiple sclerosis?

*Navina Chrobok 1 , Claudia Sestito 1 , Erik NTP Bakker 2 , Susanne  van der Pol 3 , Elga HE de Vries 3 , Keith K Fenrich 4 , Franck Debarbieux 4 , Micha MM Wilhelmus 1 , Benjamin  Drukarch 1 , Anne-Marie van Dam 1
1 VU university medical center, Department of Anatomy and Neurosciences, Amsterdam, Netherlands
2 Academic Medical Center, Department of Biomedical Engineering and Physics, Amsterdam, Netherlands
3 VU university medical center, Department of Molecular Cell Biology and Immunology, Amsterdam, Netherlands
4 Université de la Méditerranée, Institut de Biologie du Developpement de Marseille-Luminy, Marseille, France
Abstract text :


Multiple sclerosis (MS) is a chronic neuroinflammatory disease which manifests in neurological deficits caused by inflammation, demyelination and axonal damage. A pathological hallmark is the infiltration of lymphocytes and monocytes into the central nervous system, which subsequently serve as a source of inflammatory mediators contributing to local activation of glial cells and further production of inflammatory mediators.

Tissue Transglutaminase (TG2) is a multifunctional enzyme whose expression and activity is enhanced during inflammatory processes. We previously observed that the expression of TG2 is increased in monocytes in lesions in post-mortem material of MS patients. Moreover, TG2 activity and expression is enhanced in chronic-relapsing experimental autoimmune encephalomyelitis (cr-EAE) in rats. In this same MS model, pharmacological inhibition of TG2 activity dramatically reduced clinical symptoms and attenuated the influx of monocytes. In the present study we question whether TG2 plays a role in mouse EAE, as transgenic mice have to be used for subsequent in vivo imaging of leukocytes/monocytes during EAE.

Methods and results

EAE was induced in TG2-/- mice and littermate wildtype mice (C57Bl/6) using MOG35-55. During the early phase of disease (day 14-15), the clinical symptoms observed were significantly less severe in TG2-/- compared to the wildtype mice. Moreover, the maximal clinical score was significantly lower in the knockout mice. There was no difference in the day of onset of disease symptoms between the two groups.

Secondly, we aimed at visualizing leukocytes/monocytes in the spinal cord of LysM-GFP and CD11c-YFP mice suffering from MOG35-55-induced EAE using intravital video microscopy. A permanent window was fixed on top of the spinal cord of mice to allow reimaging of the same field of view in the same mouse over the disease course. In a pilot experiment we observed numerous GFP positive cells in the white matter around the imaged blood vessel in the spinal cord of a LysM-GFP EAE mouse. Although equally present, this was less prominent in a CD11c-YFP mouse suffering from EAE. Subsequent immunohistochemical analysis revealed that various cell types had infiltrated the spinal of cord of both mice.


To further address the role of TG2 in the infiltration of leukocytes/monocytes into the spinal cord of EAE mice in vivo, specific inhibitors for TG2 activity will be administered to the transgenic mice and leukocytes/monocytes will be visualized using intravital video microscopy.



The modification of myeloid-derived suppressor cell population by the synthetic retinoid AM80 abolishes symptom recovery in a murine model of multiple sclerosis

Verónica Moliné-Velázquez 1 , Fernando de Castro 1 , *Diego Clemente 1
1 National Hospital for Paraplegics, , Toledo, Spain
Abstract text :

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. The Relapsing-Remitting (RR-MS) is the most frequent clinical variant, which is characterized by a relapsing phase with inflammatory cell infiltrates and a remitting period, where patients partially recover. Among the different cell types involved in the necessary immunomodulation to allow the relapsing-to-remitting transition, the role of myeloid-derived suppressor cells (MDSCs) gains importance. MDSCs form a heterogenic population of immature myeloid cells that is able to suppress the inflammatory response. This cells act, among other mechanisms, through Arginase-I (Arg-I) activity on T-cells. A previous study of our group showed that Arg-I -MDSCs transiently enter the spinal cord of EAE mice and takes part in the immune response control by inducing T cell apoptosis around the peak of the clinical score. Therefore, changes in the MDSC population during EAE should modify the evolution of the disease.

The retinoid acid family molecules are used for the treatment of different leukaemia due to their role as MDSC differentiation factors into diverse cell populations, abolishing T cell immunosuppression. AM80, a synthetic analogue of the retinoic acid with a higher bioavailability and less side effects than the natural ones, has controversial actions on EAE depending on its administration period. In this work, we administered AM80 specifically in the critical moment for immune modulation (around the maximum clinical score). Drug administration affected MDSC population and clearly worsened the EAE course. Our results point to endogenous strengthening of MDSC population as a new and promising therapeutic strategy to treat MS by speeding up the transition from the relapsing to the remitting period.


This work was supported by the Spanish Ministerio Economía y Competitividad (SAF2009-07842; SAF2012-40023; RD07-0060-2007 and RD12-0032/0012/F.E.D.E.R., European Union, “Una manera de hacer Europa”), Gobierno de Castilla-La Mancha (PI2009/26) and ARSEP Foundation (France). DC and FdC are hired by SESCAM.


Cox-2 inhibitors reduce microglia inflammation in vivo

*Alexander Cupido 1 , Bogdan Catalin 1,2 , Frank Kirchhoff 1
1 University of Saarland, , Homburg, Germany
2 University of Medicine and Pharmacology, Department of Physiology, Craiova, Romania
Abstract text :

Question - Methods

In vivo imaging of transgenic fluorescent mice in the CNS by two-photon laser-scanning microscopy (2P-LSM) has become a powerful tool in neuroscience. For in vivo imaging of the cortex a craniotomy of the skull has to be made. In addition to anaesthesia and analgesia treatment, we occasionally apply anti-inflammatory drugs to prevent immunological activity that can obscure the images. As anti-inflammatory drug we used Carprofen ((RS)-2-(6-chloro-9H-carbazol-2-yl)propanoic acid), a known cyclooxygenase-2 (COX-2) inhibitor.

Adult CX3CR1EGFP mice in which microglia are labeled by expression of the green fluorescent protein EGFP were treated with a single dose of Carprofen or with vehicle 12 hours before the craniotomy. In all mice we exposed the right somato-sensory cortex, and quantified the microglial response to a laser-induced micro-lesion. This micro-lesion was caused by increasing the power of the laser for 1 second in the middle of the region of interest.

Results - Conclusions

Unexpectedly, we observed that Carprofen reduced the microglial process motility significantly. The speed with which the processes approached the lesion dropped from 0.5 µm per minute in the untreated mice to 0.2 µm per minute in the treated mice.

A molecular understanding of microglia response in inflammatory processes and how anti-inflammatory drugs modify normal microglia response will provide a strong impact in developing treatment strategies for diseases with strong inflammatory components.


IOP Induces upregulation of MHC-II and GFAP in the glia of Contralateral Mice Retina To Experimental Glaucoma

*Rosa De Hoz Montañana 1 , Beatriz I Gallego 1,2 , Blanca Rojas 1,3 , Juan J Salazar 1,2 , Ana I Ramirez 1,2 , Alberto Triviño 1,3 , Francisco J Valiente-Soriano 4 , Manuel Vidal-Sanz 4 , Jose Manuel Ramirez 1,3
1 Universidad Complutense (UCM), Inst Invest Oftalmol Ramón Castroviejo, Madrid, Spain
2 Universidad Complutense (UCM), Facultad Optica y Optometria, Madrid, Spain
3 Universidad Complutense (UCM), Departamento Oftalmologia. Facultad Medicina, Madrid, Spain
4 Universidad de Murcia, Facultad de Medicina, Murcia, Spain
Abstract text :

Purpose: To study the effects of laser-induced ocular hypertension (OHT) in the macro- and microglia of eyes with OHT (OHT-eyes) and contralateral eyes two weeks after lasering. 

Methods: Adult Swiss mice were divided into two groups: naïve (n=6) and lasered (n=6). Retinal wholemounts were immunostained with antibodies against GFAP, Iba-1 and MHC-II.

Results: In the naïve retinas, weak constitutive MHC-II expression was scarcely found in some Iba-1 microglial cells and rarely in GFAP astrocytes. Only a small dendritiform subpopulation of Iba-1 cells, located in the juxtapapillary area and in the marginal region of the retina, had a strong MHC-II immunoreaction.  In comparison with naïve both, in contralateral and in OHT-eyes: i) GFAP was upregulated in Müller cells and microglia was activated; ii) MHC-II was upregulated on macroglia and microglia. In microglia, it was similarly expressed in contralateral and OHT-eyes. By contrast, in macroglia, MHC-II upregulation was observed mainly in astrocytes in contralateral eyes and in Müller cells in OHT-eyes

 Conclusions: Both, contralateral and OHT-eyes had macro- and microglial retinal changes in MHC-II expression after two weeks of laser-induced OHT. Our results suggest that the gliotic reaction in contralateral untreated eyes could be related to the immune response. On the basis of the glial changes observed, the use of the contralateral eye as a control in experimental unilateral OHT should be reconsidered.


Quantitative and phenotypic analysis of mesenchymal stromal cell graft survival and recognition by microglia and astrocytes in mouse brain.

Nathalie De Vocht 1 , Dan Lin 2 , Jelle  Praet 3 , Chloe Hoornaert 4,5 , *Kristien Reekmans 4,5 , Debbie Le Blon 4,5 , Jasmijn Daans 4,5 , Patrick Pauwels 6 , Herman Goossens 5 , Niel Hens 2 , Zwi Berneman 4,5 , Annemie Van der Linden 3 , Peter Ponsaerts 4,5
1 University of Antwerp, , Wilrijk, Belgium
2 University of Hasselt, Center for Statistics, Hasselt, Belgium
3 University of Antwerp, Bio-Imaging Lab, Antwerp, Belgium
4 University of Antwerp, Laboratory of Experimental Hematology, Antwerp, Belgium
5 University of Antwerp, Vaccine and Infectious Disease Institute, Antwerp, Belgium
6 University of Antwerp, Laboratory of Pathology, Antwerp, Belgium
Abstract text :

Question: Although cell transplantation is increasingly suggested to be beneficial for the treatment of various neurodegenerative diseases, the therapeutic application of such intervention is currently hindered by the limited knowledge regarding central nervous system (CNS) transplantation immunology. In this study, we aimed to investigate the early post transplantation innate immune events following grafting of autologous mesenchymal stromal cells (MSC) in the CNS of immune competent mice.

Methods and results: First, the survival of grafted Luciferase/eGFP-expressing MSC (MSC-Luc/eGFP) was demonstrated to be stable from on day 3 post implantation using in vivo bioluminescence imaging (BLI), which was further confirmed by quantitative histological analysis of MSC-Luc/eGFP graft survival. Additional histological analyses at week 1 and week 2 post grafting revealed the appearance of (i) graft-surrounding/-invading Iba1 microglia and (ii) graft-surrounding GFAP astrocytes, as compared to day 0 post grafting. While the density of graft-surrounding astrocytes and microglia did not change between week 1 and week 2 post grafting, the density of graft-invading microglia significantly decreased between week 1 and week 2 post implantation. However, despite the observed decrease in microglial density within the graft site, additional phenotypic analysis of graft-invading microglia, based on CD11b- and MHCII-expression, revealed > 50% of graft-invading microglia at week 2 post implantation to display an activated status. Although microglial expression of CD11b and MHCII is already suggestive for a pro-inflammatory M1-oriented phenotype, the latter was further confirmed by: (i) the expression of NOS2 by microglia within the graft site, and (ii) the absence of arginase 1-expression, an enzyme known to suppress NO activity in M2-oriented microglia, on graft-surrounding and -invading microglia.

Conclusions: In summary, we here provide a detailed phenotypic analysis of post transplantation innate immune events in the CNS of mice, and warrant that such intervention is associated with an M1-oriented microglia response and severe astrogliosis.


The neuroprotective effect of a PPAR-γ agonist in microglia-induced neuronal death is abrogated if CD200-CD200R1 interaction is disrupted

*Guido Dentesano 1,2 , Joan Serratosa 1 , Josep M. Tusell 1 , Unai Perpiñà 1 , Josep Saura 2 , Carme Solà 1
1 IIBB-CSIC, IDIBAPS, Cerebral Ischemia and Neurodegeneration, Barcelona, Spain
2 University of Barcelona, IDIBAPS, Biochemistry and Molecular Biology Unit. School of Medicine, Barcelona, Spain
Abstract text :

Microglial cells are involved in inflammatory and immune responses of the CNS. In the presence of pathological insults, they acquire reactive phenotypes aimed at re-establishing brain homeostasis and minimizing neuronal damage. However, reactive microglia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect. Consequently, the progression and resolution of microglial activation have to be tightly controlled to avoid detrimental secondary effects. Signals arising from neuronal cells play an important role in the activation state of microglial cells. Among them, inhibitory mechanisms, such as neuronal CD200 and microglial CD200R1 interaction, keep the pro-inflammatory phenotype of microglia under control. Alterations in the expression of CD200 and CD200R1 have been described in pathological conditions, and the modulation of CD200-CD200R signalling could be an interesting target to be considered in therapeutic approaches against neuroinflammation occurring in neurodegenerative diseases.

Little is known on the molecular mechanisms involved in the regulation of CD200 and CD200R1 expression. The aim of the present work is to study the possible modulation of CD200 and CD200R1 by PPAR-γ agonists, and the involvement of CD200-CD200R1 in the anti-inflammatory and neuroprotective effects of PPAR-γ activation. We have used mouse microglial, mixed glial and neuronal cell cultures, as well as neuron-microglia co-cultures. CD200R1 expression was detected in microglial cells, and it was decreased in response to pro-inflammatory stimuli such as LPS/IFN-γ. The PPAR-γ agonist 15-deoxy-D12, 14-prostaglandin J2 (15d-PGJ2) abrogated the inflammatory response in LPS/IFN-γ-treated microglial cells and prevented CD200R1 expression inhibition. CD200 expression was detected in neuronal cultures and, at a lesser extent, in astrocytes in mixed glial cultures. LPS/IFN-γ-treatment did not modify CD200 expression in neuronal cultures, but it induced an increase in mixed glial cultures. This increase was inhibited by 15d-PGJ2 pre-treatment. 15d-PGJ2 also protected against LPS/IFN-γ-induced neurotoxicity in neuron-microglia co-cultures, but this effect was abolished when CD200-CD200R1 interaction was interrupted using an anti-CD200R1 blocking antibody. These results show that PPAR-γ agonists modulate CD200 and CD200R1 expression in reactive glial cells, and that CD200-CD200R1 interaction is necessary for the neuroprotective effect of PPAR-γ agonists.

Supported by La Marató de TV3 Foundation and grants PI10/378 and PI12/00709 (Instituto de Salud Carlos III, Spain).


Characterization and modulation of HmIba1 as an activation marker for microglia in the invertebrate model, the leech Hirudo medicinalis.

*Francesco Drago 1 , Alice Accorsi 2 , Pierre-Eric Sautière 1 , Françoise Croq 1 , Christophe Lefebvre 1 , Christelle Van Camp 1 , Jacopo Vizioli 1
1 University Lille 1, , Villeneuve d'Ascq, France
2 Universita di Modena e Reggio emilia, Dipartimento di Biologia, Modena, Italy
Abstract text :

Question: Ionized Calcium-binding Adapter molecule 1 (Iba1), is a 17 kDa cytokine-inducible protein, produced by activated macrophages during chronic transplant rejection and in inflammatory reactions. In central nervous system (CNS), Iba1 is a sensitive marker associated to activated microglia and is upregulated following neuronal death or brain lesions. Iba1-like factors have been described in several metazoan and share a well conserved amino acid primary structure throughout evolution suggesting a common, functional role.

The medicinal leech Hirudo medicinalis is able to regenerate its CNS after injury, leading to a complete functional repair. Similarly to Vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. We investigated the expression of Hirudo Iba1 to track the activation state of leech microglial cells involved in nerve repair events.

Results: We recently identified a gene, named HmIba1, coding a 17.5 kDa protein showing high similarity with Vertebrate Iba1 factor. Quantitative RT-PCR analyses showed that HmIba1 is constitutively expressed in cultured nerve chains. A weak down regulation was observed in the days following experimental injury. Gene transcripts rise back to basal level one week later. Cultured nerve chains stimulated with ATP shows a significant increase of HmIba1 transcript 6 hours after treatment. Immunoblot analysis, performed with anti-HmIba1 polyclonal antibodies, revealed an immunopositive band at the predicted size. The presence of HmIba1 protein in naïve and experimentally challenged tissues was evaluated by immunohistochemistry. The protein is constitutively present in spread, stellar shaped microglial cells, distributed in connective fibers and in segmental ganglia. A few hours after experimental injury of CNS, the amount of immunopositive microglial cells increases at the lesion site and at the cut end of nerve fibers until. The amount of HmIba1 cells in connectives rapidly increases in ATP treated nerve chains. This augmentation is visible in ganglia microglia and in connective fibers, where cells located between axon fibers display an elongated and stretched shape.

 Conclusion: HmIba1 is a good marker of activated microglia. Like in vertebrates, the ATP induces its expression in leech CNS. Also if the functional role of HmIba1 has to be further elucidated, this molecule appears as a good activation marker of microglia and an interesting tool to study and follow the activity of such cells during nerve repair in leech.


Acidosis affects interleukin-1β processing in glial cells

*Michelle Edye 1 , Stuart Allan 1 , David Brough 1
1 University of Manchester, , Manchester, United Kingdom
Abstract text :

Acidosis is a clinical consequence of many major diseases. Non-infectious diseases are increasing in prevalence and many have been shown to have an inflammatory component, which in the absence of infection, will be sterile. The NLRP3 inflammasome, a multimeric protein complex which induces processing of IL-1β into its active form, is a well established mediator of sterile inflammation and is known to drive the worsening of brain injury in numerous experimental disease paradigms. We sought to investigate whether acidic conditions, typical of a disease environment, affected IL-1β processing in primary mouse glial cells.


Mixed glial cultures were grown from wild type and NLRP3 knock-out mice. Culture media was reduced to pH6.2 and IL-1β release was measured following addition of activators of the NLRP3 inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, ATP) with or without pre-treatment with caspase-1 or cathepsin D inhibitors (YVAD-CHO or pepstatin A respectively). Subsequently, IL-1β release was measured following addition of lactic acid with or without pre-treatment with the above inhibitors.


At pH6.2, activators of the NLRP3 inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, ATP) induced the release of IL-1β from mixed glial cultures. The IL-1β released at this low pH was 20kDa in size in addition to the mature 17kDa IL-1β. This 20kDa IL-1β release was maintained in NLRP3 deficient cells and was not significantly altered by pre-treatment with the caspase-1 inhibitor YVAD-CHO. Lactic acid, itself released during disease and a common cause of acidosis, also induced the release of 20kDa IL-1β from mouse glial cultures. As with the NLRP3 activators under acidic conditions, the lactic acid-induced 20kDa IL-1β release was maintained in NLRP3 knock-out cultures and with pre-treatment with YVAD-CHO. Pre-treatment with the cathepsin D inhibitor pepstatin A, however, significantly reduced the 20kDa IL-1β released with the NLRP3 activators and lactic acid.


Here we show that under disease relevant conditions (low pH), 20kDa IL-1β is released from mouse glial cultures and this 20kDa IL-1β is independent of the classical NLRP3 inflammasome/caspase-1 pathway and likely mediated by cathepsin D. Further investigation of this caspase-1-independent IL-1β pathway may in future provide novel targets for the treatment of inflammatory disease.


Astrocytary IL-6 involment in EAE and traumatic lesions

*Maria Erta 1 , Mercedes Giralt 1 , Silvia Jimenez 1 , Gemma Comes 1 , Juan Hidalgo 1
1 Universitat Autònoma Barcelona, , Bellaterra/ Barcelona, Spain
Abstract text :

Interleukin-6 (IL-6) is a highly plurifunctional cytokine, with many pleitropic actions, considered one of the main cytokines controlling the immune system and coordinating it with the nervous and endocrine systems.

IL-6 is produced in multiple cell types in the CNS, and in turn many cells do respond to it. It is therefore important to ascertain which the contribution of each cell type is in the overall role of IL-6 during both physiological and pathological conditions. Astrocytes are major responders to IL-6 as well as one of the main CNS producers of IL-6. For this work we used astrocytary IL-6 KO (Ast-IL6 KO) mice, which we already proved to have an important role in physiological conditions (like body weight control and exploratory/ locomotion behavior), in order to test astrocytary IL-6 role during a neuroinflammation situation. For this purpose, we induced either an extensively used animal model of Multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), or a traumatic brain injury (cryolesion) in our mice.

Regarding EAE, results indicate that lack of astrocytary IL-6 delays clinical course of EAE and ameliorate EAE symptomatology in Ast-IL6 KO respect littermate controls in a gender-dependent way. Further immunohistochemistry analyses confirm a decreased number of cellular and lymphocytes infiltrates and lesser demyelination, angiogenesis and gliosis in spinal cord of Ast-IL6 KO animals. Regarding traumatic injury, astrocytary lack of IL-6 facilitates microgliosis, lymphocytes infiltration and a faster decrease of the injured area.

All these results are likely to bring some answers to astrocyte-secreted IL-6 involvement in neuroinflammation pathways and EAE pathogenesis. 


Cross-talk between neurons and non-neuronal cells within sensory neurons : effects on ATP-gated P2X3 receptors

Alessia Franceschini 1 , Sandra Vilotti 1 , Tanja Bele 2 , Arn M.J.M. Van Den Maagdenberg 3 , Andrea Nistri 1 , *Elsa Fabbretti 2
1 International School for Advanced Studies (SISSA), , Trieste , Italy
2 University of Nova Gorica, , Vipava, Slovenia
3 University Medical Centre, Dept Human Genetics, , Leiden, Netherlands
Abstract text :

Enhanced activity of trigeminal ganglion neurons is thought to underlie neuronal sensitization facilitating the onset of chronic pain attacks. Chronicity might establish a neuroinflammatory ganglion profile with inflammatory cells contributing to the hypersensitive phenotype. We first investigated whether, in trigeminal sensory ganglia, cytokines such as TNFa might contribute to a local inflammatory phenotype of a transgenic mouse model of familial hemiplegic migraine type-1 (FHM-1, Cacna1a R192Q knock-in mice). With respect to wild-type, R192Q KI trigeminal ganglia were enriched in activated macrophages and expressed higher mRNA levels of IL1b, IL6, IL10 and TNFa cytokines and the MCP-1. Functional consequences of crosstalk between macrophages and sensory neurons were studied in primary ganglia cultures, where larger release of soluble factors and larger currents mediated by pain-transducing ATP-gated P2X3 receptors were found. Consistently, we observed that, following LPS injection, TNFa expression and macrophage occurrence were significantly higher in R192Q knock-in ganglia with respect to wild-type ganglia. Our data suggest that, complex cellular and molecular environment of sensory ganglia could support a new tissue phenotype compatible with a neuroinflammatory profile. We propose that, in selected patients, this condition might contribute to pain pathophysiology through release of soluble mediators, including TNFa and ATP that may modulate the crosstalk between sensory neurons and resident glia, underlying the sensitisation process. Supported by Telethon (GGP10082), Cariplo Foundations, ARRS grant (1000-11-310161) and Crossborder Cooperation Programme Italia – Slovenia grant MINA.


Lack of astrocytic interleukin-6 enhances high-fat diet-induced obesity in mice

*Olaya Fernández Gayol 1 , Mercedes Giralt 1 , Amalia Molinero 1 , Beatriz Ferrer 1 , Gemma Comes 1 , Juan Hidalgo 1
1 Universitat Autonòma de Barcelona, , Bellaterra, Spain
Abstract text :

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in inflammatory and non-inflammatory responses. Among the latter, it participates in the regulation of body weight and metabolism. IL-6-deficient mice develop mature onset obesity and have higher blood glucose, as well as impaired glucose tolerance and elevated blood leptin levels, suggesting that IL-6 participates in suppressing adiposity in mice. Moreover, transgenic mice with astrocyte-targeted production of IL-6 challenged with a high-fat diet are resistant to high-fat diet-induced obesity, highlighting the role of centrally produced IL-6 in the regulation of body weight.

In this context, we hypothesize that mice lacking IL-6 produced by astrocytes (ast-IL6KO) will be more prone to develop obesity. We therefore challenged ast-IL6KO mice (obtained with the Cre-lox technology) with a high fat diet (58% kcal from fat) for 17 weeks and compared their body weight and food intake with those of mice fed a standard diet (18% kcal from fat). On weeks 14 and 15, the metabolic status was evaluated by an insulin tolerance test (ITT) and an oral glucose tolerance test (OGTT), respectively.

Regarding body weight gain, we see a clear increase in ast-IL6KO females on a high-fat diet in comparison to their controls with no apparent difference in food intake, which is also already apparent in the control diet. In males a similar tendency is observed.

Part of this difference can be attributed to the heavier subcutaneous white adipose tissue depots (relative to body weight) in ast-IL6KO mice (Fig 1).

Insulin and oral glucose tolerance are compromised in mice fed a high-fat diet, but no differences between genotypes are observed.

Taken together, these results indicate that centrally produced IL-6 has indeed a major role in the regulation of body weight, affecting subcutaneous white adipose tissue, but without significantly altering peripheral glucose metabolism.

We are currently working on assessing hypothalamic neuropeptides with in situ hybridization to study the effects of astrocytic IL-6 deficiency at the central level.


Expression of Calreticulin and Other Endoplasmic Reticulum Stress Molecules in a rat model of inflammatory demyelination

*Una FitzGerald 1 , Mary  Ní Fhlathartaigh 1 , J.  McMahon 1 , R.  Reynolds 2
1 National University of Ireland, Galway, , Galway, Ireland
2 Imperial College London , , London, United Kingdom
Abstract text :

Endoplasmic reticulum (ER) stress is a signalling pathway, linked to many neurological diseases including multiple sclerosis (MS). Studies of human tissue in our lab have shown ER stress-associated molecules at increased levels, in early biopsy MS samples and in cortical lesions. Increasing evidence suggests that calreticulin (CRT), a multifunctional ER-resident protein and ER stress responder, plays a role in cell clearance and it has been implicated in autoimmunity. To extend our human tissue work, we set up an EAE model of inflammatory demyelination in Dark Agouti (DA) rats and report the profile of expression of CRT and other ER stress signalling molecules in demyelinated spinal cord lesions and control samples. To induce spinal cord demyelination, the DA rats were injected with recombinant murine myelin oligodendrocyte (rmMOG) protein mixed with incomplete Freund’s adjuvant (IFA). Control rats were injected with a PBS/IFA or with 100 ml of PBS only. Animals were then monitored over a 43-day period and clinical scores assigned. Molecular analyses was carried out by real-time PCR to determine transcriptional expression profiles of Grp78/BiP, CHOP, CRT and XBP-1. BiP and CHOP showed a trend towards up-regulation whereas spliced XBP1 displayed a downward trend in EAE, compared to controls, but this was not statistically significant. In contrast, immunohistochemical staining of tissue sections allowed semi-quantitative comparison of protein expression in the lesion (L), lesion edge (LE), normal-appearing white matter (NAWM), central canal (CC) and grey matter (GM). Statistical analyses revealed that CHOP (p<0.001), p-EIF2a (p<0.01) and CRT (p<0.05) were significantly increased in EAE spinal cord white matter lesions and in the normal-appearing white matter compared to healthy control tissue. Interestingly, there was also an up-regulation of CHOP (p<0.05), and p-eIF2a (p<0.01) in the grey matter and central canal of diseased spinal cord tissue. Morphological criteria and dual immunofluorescent labelling confirmed expression of ER stress protein in neurons, astrocytes, microglia or oligodendrocytes. An intriguing finding was localisation of CRT to the rim of ORO-positive myelin fragments and the ‘patchy’ nature of CRT staining seen when tissue was dual-labelled with CRT and GFAP or IBA1. These results are the first demonstration of significantly higher levels of CRT in rodent EAE. CHOP and p-eIF2a data has also not been reported in rat EAE. This study highlights the potential importance of ER stress in inflammatory demyelination.

The authors acknowledge support from the Irish Research Council and from the Foundation Office of NUI, Galway.


Are microglia just macrophages? Analysis of both cell types functions after brain injury.

*Sylvie Girard 1 , David Brough 1 , Stuart Allan 1
1 University of Manchester, , Manchester, United Kingdom
Abstract text :

Objectives: Microglia are a brain resident population of immune cells, involved in homeostatic surveillance of the brain parenchyma, with high importance in the regulation of inflammatory processes after injury. There are unresolved questions regarding microglia origin, and their similarities to peripheral macrophage populations. Our objective was to compare the capacity of microglia and bone marrow derived macrophages (BMDMs) to adopt different phenotypes, and how this influenced cell death after brain injury.

Methods: We compared the phenotype of BMDMs and microglia (both BV2 microglial cell line and primary microglia) after treatment with well known polarising agents. Lipopolysaccharide (LPS) was used as an inducer of the classical M1 phenotype and IL-4 was used to induce an alternative M2 phenotype. Microglia or BMDMs, of different phenotypes, were added to hippocampal organotypic slices (HOSC) subjected to oxygen glucose deprivation (OGD) as an in vitro model of brain injury.

Results: BMDMs and microglia (BV2 and primary microglia) both adopt M1 or M2 phenotypes after treatment with LPS or IL-4 respectively. However, addition of polarised microglia or BMDMs onto HOSC resulted in different outcomes. Addition of activated BMDMs, of either M1 or M2 phenotypes, led to cell death in control HOSC and increased death when combined with OGD. On the other hand, addition of BV2-microglia did not induce any cell death in control HOSC, and were protective after OGD, except for M1-microglia which were toxic. Endogenous microglia within the HOSC were also able to adopt different phenotypes and exert neuroprotection after IL-4 treatment. These ex vivo data correlated with the in vivo situation where we observed increases in microglial activation early after experimental stroke, although the vast majority of these activated cells did not express markers of the M1 phenotype. 

Conclusions: This study highlights functional differences between macrophages and microglia, especially in response to brain injury. Although microglia, like peripheral macrophages, can adopt different phenotypes, their functions are fundamentally different in a model of brain injury. These data provide new insights into the protective role of microglia after brain injury.


Altered microglial response in pre-symtomatic SOD1 mutant mediated disease

*Mathieu Gravel 1 , Jasna Kriz 1 , Essam Abdelhamid 1
1 Universite Laval - CHUQ, , Quebec, Canada
Abstract text :

Although major clinical symptoms in Amyotrophic lateral sclerosis (ALS) disease arise from the degeneration and death of motor neurons, excitotoxicity from glial cells dysfunction and inflammatory processes are very likely to be involved in the neurodegenerative process. Microglial activation is associated with a significant induction of several Toll-like receptors (TLRs). To study the TLR2 response in-vivo, SOD1G93A mice were crossed with the TLR2-Luc-GFP reporter mice, a transgenic mouse bearing the dual reporter system luciferase and green fluorescent protein under the transcriptional control of the murine TLR2 promoter.  Double transgenic mice and littermates controls were monitored longitudinally using in-vivo biophotonic/bioluminescent imaging to measure microglial activation over the course of the disease.

Surprisingly, this analysis did not reveal an increase of activation in SOD1G93A mice as the disease progressed, with only weak signal coming from olfactory bulb and brain area. However, quantification of this signal suggested a lower level of TLR2 activation in the SOD1G93A mice compared to wild-type littermates. To further study the microglial impairment observed in the SOD1G93A mice, these transgenic mice were given LPS with i.p. injections at 5mg/kg and were followed for 48h by bioluminescence imaging, both at a pre-symptomatic age (60 days) and closer to paralysis (115 days). The level of TLR2 activation was lower in the SOD1G93A mice than the wild-type littermates, both at 60 days and 115 days. Immunostaining of olfactory bulbs reveal the same phenomenon, which is less Iba1 and less TLR2-positive cells in the SOD1G93A tissue. mRNA analysis by in-situ hybridization confirms a similar pattern of microglial response. In a parallel study, primary microglial cell culture, derived from adult mice (60days) were similarly stimulated with LPS to further understand the altered SOD1 mutant microglia response. Here again, SOD1 cells appears less responsive to LPS stimulation as observed in our mice studies. These combined results suggest that SOD1G93A microglial cells might have a pre-symptomatic suboptimal immune response.


Upregulation of c-Jun in the 3xTrg AD model regulates miR-155 expression in glial cells and contributes to neuroinflammation. 

*Joana Guedes 1,2,3 , Luís Pereira de Almeida 4,3 , Maria Conceição Pedroso de Lima 5,3 , Ana Luísa Cardoso 3
1 University of Coimbra, Center for Neuroscience and Cell Biology, PDBEB - PhD Programme in Experimental Biology and Biomedicine, Coimbra, Portugal
2 University of Coimbra, IIIUC - Institute for Interdisciplinary Research, Coimbra, Portugal
3 University of Coimbra, CNC - Center for Neuroscience and Cell Biology, Coimbra, Portugal
4 University of Coimbra, Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, Coimbra, Portugal
5 University of Coimbra, Department of Life Sciences, Faculty of Science and Technology, Coimbra, Portugal
Abstract text :

MiRNAs are small non-coding RNA molecules that modulate gene expression at a post-transcriptional level and their role in the regulation of biological processes makes them an emerging class of therapeutic targets. Dysregulation of miRNA networks has been linked to neurodegeneration and immune dysfunctions and has become a research focus in the context of Alzheimer’s disease (AD).

In this work, we demonstrate that miR-155 expression is increased in the brain of 3×Trg AD transgenic mice, prior to senile plaque formation, and co-localized with intraneuronal APP accumulation in the cortex and hippocampus. In view of the pro-inflammatory functions of miR-155 and aiming at clarifying its role in AD, we evaluated the levels of miR-155 in Aβ-stressed astrocytes and microglia cultures, two brain-related cell types involved in neuroinflammation. We show that miR-155 expression levels are increased in astrocytes and microglia following activation with Aβ fibrils but not with Aβ oligomers. These findings correlate with the observed decrease in SOCS-1 expression, a miR-155 validated target, and with the increase in the production of TNF-α, IL-1β and IL-6 by these cells. Importantly, we show that miR-155 expression is regulated by c-Jun, since silencing of this transcription factor led to the reduction of miR-155 levels following astrocyte and microglia exposure to Aβ fibrils or LPS. c-Jun was also found to be upregulated in the 3×Trg AD transgenic model since early ages, which can help to explain the observed early increase in miR-155 expression.

Overall, our results demonstrate the important role of miR-155 in AD and show that silencing of c-Jun in glial cells may constitute an interesting and promising anti-inflammatory therapeutic strategy towards this disease, by targeting miR-155-mediated inflammatory responses.


Peripheral poly I:C-induced neuroinflammation: role of Toll-like receptor 3 (TLR3) in microglia


*Masataka Ifuku 1 , Shamim  Hossain 1 , Mami Noda 2 , Toshihiko  Katafuchi 1
1 Kyushu University, , Fukuoka, Japan
2 Kyushu University, Laboratory of Pathophysiology, Fukuoka, Japan
Abstract text :

It has been suggested that peripheral infection/inflammation may play a role in the onset or development of not only neurodegenerative diseases but also depression, autism and chronic fatigue syndrome through inflammatory responses in the brain. To investigate the role of microglia in this hypothesis we used a model of neuroinflammation induced by an intraperitoneal (i.p.) injection of the Toll-like receptor 3 (TLR3) agonist, polyinosinic:polycytidylic acid (poly I:C) in rats. As we reported previously an injection of poly I:C (i.p) decreased the daily amounts of spontaneous running wheel activity to ~60% of the preinjection levels until day 7. Microglia were morphologically activated in the prefrontal cortex (PFC) until 72 hrs after the injection of poly I:C. Pretreatment with minocycline for the 3 consecutive days (40 mg/kg/day) blocked the poly I:C-induced decrease in the running wheel activity as well as microglial activation. Quantitative analysis of mRNA levels demonstrated that interferon-α (IFN-α) increased in the PFC on both days 1 and 7. We found in the present study that poly I:C injection induced increases in mRNAs relevant to TLR-signaling such as TLR3, interferon regulatory factor 3 (IRF3) and IRF7 in the PFC. Furthermore, direct application of poly I:C to the primary cultured microglia also induced an enhanced expression of IFN-α, TLR3, IRF3 and IRF7. It has been reported that permeability of blood-brain barrier is increased after poly I:C injection (Wang et al., 2004). Therefore, it is possible that the peripheral poly I:C enters into the brain to induce neuroinflammation by activating microglia. Interestingly, intracerebroventricular (i.c.v.) injection of primary cultured microglia activated by poly I:C, but not by lipopolysaccharide, effectively produced a significant decrease in spontaneous activity in rats. Taken together, these findings suggest that microglial activation is important for poly I:C-induced neuroinflammation through inducing TLR3-related genes. Roles of each gene in the behavioral changes will be further studied.


Phenotype and function of CD11c (dendritic) cells within the CNS

*Kerstin Immig 1 , Martin Gericke 1 , Andreas Lösche 2 , Jäger Kathrin 2 , Lena Wendeburg 3 , Uwe-Karsten Hanisch 4 , Knut Biber 3 , Ingo Bechmann 1
1 Institute of Anatomy, University of Leipzig, Leipzig, Germany
2 IZKF-FACS-Core Unit, University of Leipzig, Leipzig, Germany
3 Department of Psychiatry and Psychotherapy, Section of Molecular Psychiatry, University of Freiburg, Freiburg, Germany
4 Institute of Neuropathology, University of Göttingen, Göttingen, Germany
Abstract text :

The brain’s immune privilege has been attributed to a lack of dendritic cells (DC) within its parenchyma and the adjacent meninges implying maintenance of antigens rather than their presentation in lymphoid organs (LO). Using CD11c-gfp mice, we have recently reported the existence of a CD11c/IBA-1/CD11b -population in the juxtavascular parenchyma which extent their processes into the glia limitans, an ideal position for antigen-presentation. Therefore, we phenotypically compared them to the CD11c /CD45 population (all isolated with the same protocol used for brain CD11c cells) from lung, liver and spleen in healthy mice using 7-color flow cytometry. We found unique, site-specific expression patterns of F4/80, CD80, CD86, CX3CR1, CCR2, FLT3 and MHC-II. As described before, two different CD45 -populations (CD45high and CD45int) in the brain can be separated, whereas liver, lung and spleen exhibit a more homogeneous CD45high population. A higher percentage of the brain`s CD45 /CD11c -cells were F4/80-positive compared to spleen, liver and lung, but expressed it at lower levels. Within the CD45int /CD11c -population most cells expressed the microglia marker CX3CR1 and CCR2low (marker for inflammatory, motile cells). Most importantly, compared to spleen and liver, CD45int/CD11c -cells from the brain almost completely lacked MHC-II expression and CD45high/CD11c -cells from the brain have a lower percentage of MHC-II -cells. Since the CD45int cells are widely regarded as the resident, intraparenchymal microglial population and CD45high as DCs and perivascular macrophages, our data confirm the view that a small subpopulation of microglia (CX3CR1high, F4/80 ,CCR2low) share established immune phenotypical characteristics of DCs (CD11c ). Additionally we showed that intraparenchymal CD11c microglia are unique in their low expression of MHC-II. Their weak expression of CD80 is in line with a tolerogenic phenotype.


Supported by DFG (FOR 1336/B2)


CD14 as a key regulator of TLR-mediated responses of microglia

*Hana Janova 1 , Tommy Regen 2 , Denise van Rossum 1 , Sandra Ribes 1 , Alexander Götz 1 , Roland Nau 1 , Wolfgang Brück 1 , Uwe-Karsten Hanisch 1
1 Universitätmedizin Göttingen, , Göttingen, Germany
2 Universitätsmedizin der Johannes Gutenberg-Universität, , Mainz, Germany
Abstract text :

Every year, hundred thousands of people die of meningitis, an infectious disease of CNS that can be caused by Gram-negative bacteria E.coli. The survival of these people is dependent on the early recognition of the rising danger followed by a swift response by innate immune cells of the CNS, the microglia. These cells express a large arsenal of pattern-recognition receptors (PRR), with the best-known Toll-like receptor 4 (TLR4) as a main sensor of lipopolysacharide (LPS), a cell wall component of Gram-negative bacteria. This receptor triggers the response via both MyD88- and TRIF-dependent signalling pathways, leading to production of cyto- and chemokines and thereby alarming and attracting the immune cells of the periphery to invade the brain. However, TLR4 is only fully functional in complex with several co/receptors, such as CD14. Even though CD14 has been traditionally considered simply as a LPS affinity provider, recent data indicate that CD14 is also necessary for the endocytosis of TLR4, which links TLR4 to intracellular signalling via the adapter protein TRIF. Our latest data reveal the critical role of this receptor for the profile as well as the magnitude of microglial cyto/chemokine production in response to diverse LPS variants. CD14 increases the sensitivity towards LPS in a cell type-specific manner, making microglia far more sensitive to LPS than bone marrow and peritoneal macrophages. While striatal applications of low LPS doses reveal less incoming neutrophils into a brain of CD14ko mice, as compared to wildtypes, high doses LPS lead to an excessive neutrophil infiltration.  This phenomenon is well correlating with the observations in vitro, where CD14 absence in microglia stimulated with high doses LPS causes an excessive production of selected chemokines, with the highest impact on the neutrophil chemoatractant CXCL1. These regulatory activities of CD14 require its membrane insertion and prolonged functionality, pointing to an involvement of signalling.  In order to reveal candidates for such signalling mechanisms, we tested the role of Syk and PLC. Even though these enzymes were described to play a role in CD14-dependent signalling in dendritic cells, they have no contribution in microglia, further pointing to a cell type-specific organization of the CD14/TLR4 complex in microglia. Importantly, we identified receptor systems that impose influences on CD14 expression itself, which would thereby determine the extent and impact of a CD14-mediated regulation of TLR4 functions. Supported by the DFG (FOR1336).


Role of anti-inflammatory lipid mediators in the resolution of inflammation in microglial cells

*Corinne Joffre 1 , Charlotte Rey 1 , Agnès Aubert 1 , Sophie Layé 1
1 INRA, , Bordeaux, France
Abstract text :

Question: Microglial cells are the cellular components of the brain immune system. In response to systemic bacterial infection they become activated and are strongly involved in the local inflammatory response. They produce pro- and anti-inflammatory cytokines, in particular IL-1b, IL-6 and TNFalpha, and IL-10, respectively. Dietary polyunsaturated fatty acids (PUFA) are able to regulate neuroinflammation. N-6 and n-3 PUFA are precursors of anti-inflammatory mediators such as lipoxin A4 (LXA4) synthesized from arachidonic acid and resolvin E1 (RvE1) and D1 (RvD1) produced from eicosapentaenoic acid and docosahexaenoic acid, respectively. These mediators are synthesized by 5- and 15-lipoxygenases and exert their anti-inflammatory actions by acting on specific G-protein coupled receptors in particular on ALX and ChemR23. The aim of this study was to determine the effects of lipoxin and resolvins on the resolution of inflammation in microglial cells stimulated with lipopolysaccharide (LPS).

Methods: We used BV-2 cells, a murine microglial cell line. In a first experiment aimed at determining the time course of the induction of expression of the lipoxygenases and receptors, they were incubated with LPS. In a second experiment aimed at determining the effects of the resolvins and lipoxin on the production of IL-10, IL-1b, IL-6 and TNFalpha, they were firstly incubated with LXA4, RvD1 and RvE1 and then incubated with LPS.

Results: Our results indicated that LPS only enhanced the expression of ALX, the receptor for RvE1 and LXA4 (Í2) (p&lt;0.05). The expression of the 15-lipoxygenase was also affected by LPS (p&lt;0.01) and varied during the time course, reaching a maximum after 6h of incubation (p&lt;0.05). The production of the proinflammatory cytokines decreased after 18h of incubation: IL-1b with RvD1 (100 nM, p&lt;0.001) and RvE1 (10 nM, p&lt;0.01); IL-6 with RvE1 (1 and 10 nM, p&lt;0.05 and p&lt;0.001); TNFalpha with RvE1 (1 and 10 nM, p&lt;0.001 and p&lt;0.01). The production of the anti-inflammatory cytokine IL-10 increased only when cells were incubated with LXA4 after 6h (1 and 10 nM, p&lt;0.01).

Conclusions: These results suggested a potential role of PUFA-derivates in the resolution of inflammation.


Microglial P2Y receptor-mediated motility in situ

*Renaud Jolivet 1 , Christian Madry 1 , David Attwell 1
1 University College London, , London, United Kingdom
Abstract text :

Microglia are the resident macrophages of the brain and the first responders to disturbances of brain homeostasis. They exist in one of two broadly defined states, a rather inaptly named “resting state” in which they exhibit a ramified morphology and, upon tissue disturbance, an “activated state” in which they exhibit an amoeboid morphology. The characterization as “resting” is in stark contrast with the frantic morphological changes that ramified microglia undergo in brain slices and in vivo, constantly extending and retracting processes. While the purinergic receptor P2Y12 has been identified as a key receptor via which microglial processes are guided to a source of ATP or to the site of an injury, the signals and pathways guiding microglial processes in the healthy tissue remain elusive.

Here, by imaging microglia in acute hippocampal brain slices, we assessed the baseline and targeted motility of microglial processes in the presence of different pharmacological agents targeting purinergic signaling. We found that while bath application of a P2Y12 specific blocker alone (PSB-0739 at 500 nM) or of a P2Y1 specific blocker alone (MRS2179 at 25 µM) did not affect the baseline motility of microglial processes, bath application of a P2Y1/12/13 blocker (MRS2211 at 25 µM) strongly reduced the baseline motility of microglia, leading them to retract most of their processes. Moreover, a strong rebound of motility was observed after washing out MRS2211 that did not lead to microglial activation within the time window of the experiment (up to 90 minutes). In experiments measuring the chemotactic response of microglia to a pipette containing 1 mM of ATP inserted into the slice, we found that chemotaxis was completely abolished by PSB-0739 (bath applied at 2 µM) but not by MRS2211 (20 µM). Finally, no chemotaxis was observed in response to a pipette containing UDP-glucose (5 mM), an agonist of the P2Y14 receptor. These results confirm that P2Y12 alone controls the targeted chemotactic response of microglial processes to ATP, with no discernable involvement of P2Y1, 13 or 14, but suggest that P2Y1, 12 and 13 might collectively play a role in controlling the baseline motility of microglial processes in the healthy tissue. How the different P2Y receptors interact with each other to control microglial movements remains to be determined.

Supported by an EU Marie Curie Fellowship, the Wellcome Trust and the ERC.


Transcriptional and epigenetic control of microglia polarization into inflammatory or alternative phenotype

*Bozena Kaminska 1 , Michal  Dabrowski 1 , Marta Maleszewska 1 , Aleksandra Steranka 1 , Magdalena Smiech 1 , Aleksandra Ellert-Miklaszewska 1
1 Nencki Institute of Experimental Biology, , Warsaw, Poland
Abstract text :

Microglia are innate immune cells residing in the central nervous system and their activation is observed in virtually all neurological diseases. These cells accumulate at the sites of infection or injury of the central nervous system and initiate cascades of inflammation that can turn pathological when is chronic or uncontrolled. Microglia infiltrate malignant gliomas where they switch to a cytoprotective, pro-invasive phenotype and support tumor progression. Primary rat microglia cultures stimulated with lipopolysaccharide (LPS) or glioma-secreted factors become activated but adapt different fates and use distinct signaling pathways. Global gene expression profiling followed by functional analysis revealed activation of distinct transcriptional, signaling and metabolic pathways. Microglia stimulated with LPS up-regulate innate/immune and inflammatory genes encoding cytokines, chemokines, immune mediators and neurotoxic factors. Many genes characteristic for classical inflammatory response failed to be induced or were down-regulated in glioma-activated microglia. Specific responses include induction of genes coding for transcription factors: inhibitors of DNA binding 1/3 and c-Myc, markers of the cytoprotective phenotype and immunosuppressive cytokines/chemokines. Such global, complex and stable changes in gene expression could involve epigenetic changes such as histone modifications, DNA methylation and remodeling of chromatin structure. To identify epigenetic mechanisms associated with establishment of different phenotypes of microglia, we investigated changes in the profile of chromatin modifications of differentially expressed, candidate genes. DNA methylation was studied by methyl-specific PCR followed by sequencing. A profile of four covalent histone modifications, characteristic for transcriptionaly active or silenced chromatin, was studied by chromatin immunoprecipitation followed by qPCR. The results suggest that in untreated microglia most of the genes, which become transcribed in inflammatory or alternatively activated cells are in open chromatin conformation that allows quick expression directly after stimulation. Most of the analyzed genes did not show DNA methylation. Changes in histone modifications were delayed and associated with gene repression that suggests their role consolidation of the polarized phenotype. Inflammatory activation of microglia seems to be "default response" that has to actively suppressed to reveal/restore cytoprotective phenotype.


Progesterone attenuates astro and microgliosis and decreases inflammatory reaction following spinal cord injury

*Florencia Labombarda 1 , Susana Gonzalez 1 , Ignacio Jure 2 , Alejandro De Nicola 1
1 IBYME/CONICET/UBA, , Buenos Aires, Argentina
2 IBYME/CONICET, , Buenos Aires, Argentina
Abstract text :

Reactive gliosis and inflammatory mediators are implicated in demyelination and secondary damage after spinal cord injury (SCI). We have previously reported that after SCI, short-term progesterone treatment (3 days) stimulates oligodendrocyte precursor cells proliferation and decreases reactive gliosis, whereas chronic treatment (21 days) differenciates oligodendrocytes precursor cells into mature oligodendrocytes and enhances remyelination. Presently, we further studied whether progesterone was able to modulate the inflammatory reaction and cytokine production by astrocytes and microglial cells. Thus, the time-course mRNA expression of pro-inflammatory cytokines (IL1β, TNFα and IL-6) and pro-inflammatory enzymes (COX-2 and iNOS) was studied by PCR in Real Time.  Results showed that the highest increase in cytokine and pro-inflammatory enzymes mRNA production occurred in rat spinal cord 6 h after SCI. Progesterone treatment significantly decreased the early rise of proinflammatory mediators mRNAs at 6h. As progesterone action in spinal cord involves multiple mechanisms, the role played by the classical progesterone receptor (PR) on the progesterone inhibitory effects on cytokines, was assessed in PRKO mice. In agreement with data obtained in the rat model, SCI strongly stimulated TNFα, IL1β and IL-6 mRNA levels in spinal cord of both wild type and PRKO mice. However, whereas progesterone treatment inhibited the mRNAs of cytokines in wild type mice 6h after SCI, it was ineffective in PRKO mice, involving PR in the inhibition of cytokines production. Finally, we measured astrocyte and microglial cells density by immunohistochemsitry after 6 h of progesterone treatment. The steroid administration significantly decreases GFAP and Ox-42 cells, which correlated with cytokine and pro-inflammatory enzymes inhibition. We conclude that progesterone attenuates reactive gliosis and inflammatory reaction, probably reducing the secondary spinal cord damage and favouring remyelination. Supported by the University of Buenos Aires, Ubacyt 20020100200053



Microglial CX3CR1-CX3CL1 signaling contributes to astroglial scarring following mesenchymal stem cell grafting in mouse brain

*Debbie Le Blon 1 , Chloé Hoornaert 1,2 , Nathalie De Vocht 1 , Jasmijn Daans 1,2 , Kristien Reekmans 1,2 , Zwi Berneman 1,2 , Peter Ponsaerts 1,2
1 University of Antwerp, , Antwerp, Belgium
2 Vaccine and Infectious Disease Institute, , Antwerp, Belgium
Abstract text :


Mesenchymal stem cell (MSC) transplantation is widely suggested to become a promising strategy for future cell-based therapeutic intervention following injury or disease of the central nervous system (CNS). However, preceding data by our laboratory has demonstrated the initiation of strong glial cell responses following transplantation of MSC in mouse brain. Hereby, activated microglia will invade and surround the MSC implant, whereas astrocytes will form a dense glial scar around the MSC implant (De Vocht et al., 2011 & 2012; Praet et al., 2012).

Aims of the study:

To investigate whether CX3CL1-CX3CR1 signaling contributes to microglia migration/activation and subsequent astrogliosis following MSC transplantation in the CNS. 


First, in order to allow localization of grafted MSC in vivo, wt C57BL/6 MSC were transduced with a lentiviral vector encoding the blue fluorescent protein (BFP). Next, BFP MSC were injected into the CNS (striatum) of CX3CR1 /- (n=10) and CX3CR1-/- (n=7) transgenic mice. These mice have respectively one or both copies of the CX3CR1 gene replaced by the eGFP reporter gene. At 10 days post-transplantation, histological analyses were performed to determine Iba1 microglia and S100β astrocytes within and surrounding the BFP MSC graft site. Astrogliosis was determined based on staining for GFAP. CX3CR1 expression was evaluated based on eGFP expression. All histological evaluations were quantified using TissueQuest and/or ImageJ cell analysis software.


(i) BFP MSC graft survival is observed in both CX3CR1-/- and CX3CR1 /- mice; (ii) microglia migration towards grafted MSC occurs independent of functional CX3CR1-CX3CL1 signaling; (iii) down-regulation of eGFP gene-expression (and thus also CX3CR1 gene expression) is observed in both CX3CR1-/- and CX3CR1 /- mice on MSC graft-invading microglia, but not on MSC graft-surrounding microglia; (iv) the absence of functional CX3CR1-CX3CL1 signaling in CX3CR1-/- mice results in significantly less astrogliosis around the MSC graft site, as compared to the MSC graft site in CX3CR1 /-  mice .


Here we identified CX3CR1-CX3CL1 signaling as a potential target to modulate and/or control astroglial scarring following (stem) cell grafting in the CNS. The latter is of significant importance as grafted cells can only functionally interact with (injured) brain tissue in the absence of a graft-surrounding astroglial scar.


Expression of UNC-93B1 in the CNS

*Seija Lehnardt 1 , Dorothea Gaessler 1
1 Charité-Universitätsmedizin, , Berlin, Germany
Abstract text :

Toll-like receptors (TLRs) are key molecules of the innate and adaptive immune response in vertebrates. The original protein Toll in Drosophila melanogaster regulates both host defense and morphogenesis during development. TLRs recognize host- and pathogen-derived stimuli. Single-stranded RNA is sensed by TLR7 localized to the endolysosomal compartment of immune cells. We found that both extracellular viral RNA and host-derived microRNAs induce cell-autonomous and microglia-mediated neuronal cell death through the endosomal receptor TLR7 in vitro and in vivo. However, the exact intracellular signalling cascade and the cellular mechanisms through which TLR7 leads to tissue injury in this context remained unclear. UNC93B1 is a molecule specifically involved in trafficking of nucleotide-sensing TLRs, such as TLR7, in immune cells of both humans and mice. UNC93B1 physically interacts with this receptor in the endoplasmic reticulum (ER), and the function of the membrane protein UNC93B1 is to deliver the nucleotide-sensing receptors from the ER to endolysosomes. Making use of real-time PCR, immunocytochemistry, and histochemistry we systematically examined the expression of UNC93B1 in the murine CNS. Whereas a distinct expression for this molecule was observed in microglia and astrocytes, expression of UNC93B1 in cultured neurons was negligible. However, expression of this molecule was detected in cortical neurons of brain sections. Moreover, UNC93B1 was strongly regulated during different embryonic, postnatal, and adult stages of the developing mouse brain. Neurons of various brain regions were identified as the main cell type expressing UNC93B1 in the developing brain.

Taken together, our data reveal a specific expression pattern of UNC93B1 in the CNS, in particular in a developmental context, and lay foundation for further investigation of the pathophysiological significance of this molecule for both injurious and developmental processes in the central nervous system of vertebrates.


Myosin-dependent functions in microglia

*Stefanie Janßen 1 , Viktoria Gudi 1 , Corinna Trebst 1 , Martin Stangel 1
1 Medical School Hannover, , Hannover, Germany
Abstract text :

Microglial cells were shown to play an important role in many diseases of the central nervous system, not least due to their capability to become activated upon signs of brain injury. They migrate to lesion sites, proliferate, release cytokines and chemokines, and phagocytose cells or cellular debris. Therefore, the microglial cytoskeleton has to be highly rearrangeable. One major element of the contractile system in nonmuscle cells is nonmuscle myosin II (NM II). As the role of NM II in glial cells is poorly characterized, it is an aim of this project to study NM II-dependent functions in microglia. We found that inhibition of NM II by blebbistatin, which is a highly selective inhibitor of NM II adenosine phosphatase, prevents any morphological shaping in freshly plated microglia and leads to functional deficits during migration and phagocytosis of fluorescence-labeled beads. Using confocal microscopy, we could find diffuse expression of NM II in resting microglia in vitro, whereas activation with bacterial lipopolysaccharide (LPS) led to perinuclear accumulation of NM II protein. In an activated state, microglial cells release pro-inflammatory cytokines and reactive oxygen species; interestingly, in the presence of blebbistatin the release of nitric oxide (NO) as well as rearrangement of NM II was prevented. Taken together, these results illustrate a key role for NM II in microglial shaping and functioning. Ongoing studies will improve our knowledge on spatial and functional aspects of the actomyosin complex during demyelinating processes and open targets for development of therapeutic strategies towards remyelination in the CNS.


Deciphering the role of astroglial Cx30 at the gliovascular interface

*Anne-Cécile Boulay 1 , C. Giaume 1 , M. Cohen-Salmon 1
1 CIRB, , Paris, France
Abstract text :

Astrocytic endfeet are specialized processes, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. One of the most striking properties of astrocyte endfeet is their enrichment in the gap junction proteins connexin (Cx) 43 and Cx30 allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. However, the role of these proteins at the gliovascular interface is not fully understood. We have recently demonstrated that astroglial perivascular Cxs expression starts after birth during blood-brain barrier maturation and controls its integrity (Ezan et al. 2012). The aim of the present study was to decipher the role of Cx30 at the gliovascular interface, by analyzing the brain vascular transcriptome in a new mouse model of Cx30 inactivation (Cx30) (Boulay et al., 2013). Total RNAs of purified brain vessels from adult Cx30and wild-type littermates (Yousif et al., 2007) were analyzed by high throughput sequencing. Results were further validated by quantitative RT-PCR.

Cx30 purified vessels showed essentially a strong upregulation (fold change > 12) of Sgcg. This gene encodes gamma-sarcoglycan, a membrane glycoprotein associated with the dystrophin-dystroglycan complex which is essential for muscle integrity and is impaired in Duchenne muscular dystrophy (Petrof et al., 1993). At the gliovascular interface, the dystrophin-dystroglycan complex anchors astrocyte endfeet to the basal lamina (Nico et al., 2010), and is crucial to the BBB integrity. However, expression of Sgcg and its role in the brain has until now only been poorly documented. Moreover, we found an upregulation of S100a8 and S100a9 (fold change 6,3 and 5,5, respectively) encoding the calgranulin A and B, two proteins present in neutrophils and monocytes, and involved in their migration to inflammatory sites, attachment to endothelial cells and extravasation (Ryckman et al., 2003).

Altogether, our results suggest that Cx30 might modify the dystrophin-dystroglycan complex thereby influencing blood-brain barrier properties, and control the attachement as well as the transmigration of leukocytes through the endothelium, thus contributing to the regulation of inflammatory processes in the brain.


Comparative study of human mesenchymal stem cells isolated from the olfactory system and bone marrow: Effects on myelination.

*Sue Barnett 1 , Steven Johnstone 1 , Susan Lindsay 1
1 University of Glasgow, , Glasgow, United Kingdom
Abstract text :

Mesenchymal stem cells (MSCs) have been proposed as candidates for transplant mediated repair in multiple sclerosis (MS).  Clinical trials are ongoing using autologous systemic transplantation of bone marrow derived MSCs into relapsing remitting, secondary progressive and primary progressive MS.  Previously we have shown that rat and adult human olfactory mucosa (OM) contain a mesenchymal-like stem that resides in the lamina proporia (LP-MSCs).  Since the olfactory system is one of the only CNS tissues that promote neurogenesis throughout life, cells from olfactory biopsies have been proposed as excellent candidates for CNS repair. Thus, we have made a detailed comparison of MSCs isolated from bone marrow with MSCs isolated from the olfactory mucosa. Using immunocytochemistry and q-PCR purified human LP-MSCs expressed typical bone marrow derived (BM-) MSC markers including CD90, CD105, CD73, CD166, CD271, NG2, fibronectin and STRO-1, however they expressed more nestin mRNA. LP-MSCs formed spheres, were clonogenic and differentiated into bone and fat. Comparative micro(mi) RNA-based fingerprinting (SistemQCtm from Sistemics) demonstrated 64% homology between the two MSCs, with only 26 differentially expressed miRNAs. LP-MSC conditioned medium (CM) promoted oligodendrocyte precursor cell proliferation and induced a highly branched morphology. Both LP and BM-MSCs promoted OPC proliferation and differentiation, but only myelinating cultures treated with CM from LP and not BM-MSCs had a significant increase in myelination. Comparison with fibroblasts and contaminating OM fibroblast like-cells showed the promyelination effect was LP-MSC specific. Preliminary in vivo cell transplantation studies suggest LP-MSCs can promote many neurites in to the lesion and myelination around the lesion may be increased. Thus LP-MSCs harvested from human OM biopsies may be an important candidate for cell transplantation by promoting CMS myelination.


ROCK inhibitors decrease actin stress fibres and GFAP while elevating stellation, L-glutamate uptake and AHNAK expression in astrocytes on 3D nanoscaffolds

*Philip Beart 1 , Natasha Zulaziz 2 , Chew Ling Lau 1 , David Nisbet 3 , Malcolm Horne 1 , Ross O'Shea 2
1 University of Melbourne, The Florey Institutes of Neuroscience and Mental Health, Parkville, Australia
2 La Trobe University, Department of Human Biosciences, Bundoora , Australia
3 Australian National University, Research School of Engineering, Canberra, Australia
Abstract text :

Astrocytes are dynamic cells performing vital roles in neuronal survival, and failure of these processes contributes to neuropathologies. Manipulation of Rho kinase (ROCK) has beneficial effects on glia, including astrocytes, where ROCK inhibitors produce a pro-survival phenotype. Question - How such results translate to clinical actions in vivo needs further attention and we explored actions of ROCK inhibitors on astrocytes maintained under more physiological 3D primary culture. Methods - Random and aligned nanoscaffolds were engineered from poly-ε-caprolactone. Primary astrocytes (PND 1.5 C57Bl6 mice) were subcultured and plated at 10 days(div) in 96-well plates, on random or aligned scaffolds in 96-well plates (8,000 cells/well), or on glass coverslips in 24-well plates (20,000 cells/well). Astrocytes were treated 8 div later with vehicle, dbcAMP (100 μM), or Rho kinase inhibitors Y27632 (30 μM) or Fasudil (100 μM) for another 3 div when biochemical and morphological analyses were undertaken. Results - Astrocytes possessed cobblestoned morphology in 2D culture, but were dramatically different in phenotype on scaffolds: tight clusters formed on random scaffolds, with elongated processes on aligned scaffolds. Immunoblotting showed GFAP expression was always reduced on scaffolds relative to 2D cultures. Drug treatments decreased intensity of F-actin staining, increasing that of G-actin (disassembly actin stress fibres) notably on aligned scaffolds, where AHNAK expression was elevated by all drugs indicative of extensive enlargesome activity. AHNAK labelling was similar to GFAP but more widespread with Y27632 and Fasudil, which produced extensive process outgrowth. Fasudil and Y27632, unlike dbcAMP, had less effect on GFAP immunolabelling in 3D cultures. Processes infiltrated both types of scaffolds, showing more growth along aligned nanofibres. On random scaffolds, Y27632 and Fasudil elevated L-glutamate transport. Conclusions - Astrocytes flourished on biomatrices and the combination of bioengineering and ROCK inhibition produced synergism in decreasing cytoskeletal stress, elevating EAAT activity and process outgrowth, beneficial effects for brain repair.


Sip1 expression by Schwann cells plays a role in peripheral nerve regeneration

*Bastian Brinkmann 1 , Susanne Quintes 1 , Madlen Ebert 1 , Victor  Tarabykin 2 , Danny Huylebroeck 3 , Ueli Suter 4 , Klaus-Armin Nave 1 , Michael Sereda 1,5
1 Max Planck Institute of Experimental Medicine, Department of Neurogenetics, Goettingen, Germany
2 Charité Universitätsmedizin Berlin, Neurocure Excellence Cluster, Institute of Cell and Neurobiology, Berlin, Germany
3 University of Leuven, Laboratory of Molecular Biology (Celgen), Department of Development and Regeneration, Leuven, Belgium
4 ETH Hönggerberg, Institute of Cell Biology, Department of Biology, Zürich, Switzerland
5 University of Göttingen (UMG), Department of Clinical Neurophysiology, Goettingen, Germany
Abstract text :

Smad-interacting protein-1 (Sip1, Zeb2, Zfhx1b) has been shown to be required for CNS myelination. In the present study, we show that Sip1 is also indespensible for myelination by Schwann cells, the glial cells of the peripheral nervous system. After nerve injury, Schwann cells undergo a dramatic phenotype transition that resembles a transdifferentiation process known from tumorigenesis and tissue repair. While Sip1 expression is low in mature cells, that have completed myelination, it is upregulated several days after acute nerve trauma. We have generated mutant mice that lack Sip1 specifically in mature Schwann cells by using tamoxifen inducible Cre recombination. In these conditional mutants, regeneration of crushed sciatic nerve is impaired. Functional recovery as assessed by footprint analysis is slow and remyelination is severely delayed. Our results suggest an essential role of Sip1 in Schwann cell transdifferentiation after nerve trauma.


Combination of growth factor treatment and scaffold deposition following experimental traumatic brain injury show a temporary effect on cellular regeneration

*Fredrik Clausen 1 , Anna Erlandsson 1
1 Uppsala University, , Uppsala, Sweden
Abstract text :

Due to the restrictions in regenerative capacity of the brain the tissue most severely damaged after traumatic brain injury (TBI) will end up as a fluid filled cavity. Presently, it is an impossibility to effectively restore lost brain tissue, but ongoing research on the stimulation of endogenous neural stem cells has increased the hope to viably and functionally repopulate the injured parenchyma. However, it is crucial that possible therapies can show a long-term effect on both regeneration and functional recovery to be of clinical interest.

In this study the enhanced induction of neurogenesis in rats after experimental TBI was evaluated three or six weeks after injury. Severe focal TBI was performed using the controlled cortical impact model after which a combination therapy of intracerebroventricular administration of epidermal growth factor (EGF) for seven days followed by deposition of extracellular matrix scaffold, containing vascular endothelial growth factor, into the cortical cavity. The treatment was devised to accomplish an optimal effect on the stem cell regeneration. The animals with six weeks survival time were functionally evaluated using the Morris water maze (MWM). Before sacrifice the animals were injected with bromodeoxyuridine (BrdU) to identify newly generate cells. Sections were made and immunohistochemically double stained for BrdU and cell type markers for neurons, astrocytes and blood vessels. The sections were micrographed and analyzed for hemispheric tissue loss as well as number of new astrocytes, neurons and blood vessels.

Three weeks after injury there was a significant treatment effect as shown by an increase in neuronal and astrocytic regeneration. However, after six weeks there was no difference in the number of newly generated neurons and astrocytes. Evaluation of tissue loss and spatial learning in the MWM corroborated that the treatment had no effect at the later time point. The results clearly show the importance of long-term studies in rodents to ensure that a promising effect on tissue regeneration and functional outcome is not merely temporary.



Role of Tumor Necrosis Factor-Alpha Inhibition following Spinal Cord Injury

*Ditte Gry Ellman 1 , Valerie Bracchi-Ricard 2 , Hans Gram Novrup 1 , Anjana Jain 2 , Lise Lyck 1 , Louise Lykkemark 1 , David E. Szymkowski 3 , Damien E Pearse 2 , John R Bethea 2 , Kate Lykke Lambertsen 1,2
1 University of Southern Denmark, , Odense C, Denmark
2 University of Miami Miller School of Medicine, , Miami, Denmark
3 Xencor Inc, , Monrovia, United States
Abstract text :

Tumor necrosis factor (TNF) is a pleotrophic cytokine involved in normal brain function and inflammation, apoptosis, and other responses that occur following injury and disease. There is a considerable amount of confusion as to whether or not TNF activation is beneficial or detrimental following injury to the CNS. Genetic studies in mice have suggested that inflammation in disease models involves soluble TNF (solTNF) and that maintenance of innate immune function involves transmembrane TNF (tmTNF). These findings suggest that the outcome of selective pharmacologic inhibition of TNF may depend on whether the pharmacologic intervention is targeted towards solTNF or tmTNF.

Therefore, we took advantage of a dominant-negative inhibitor of solTNF, XPro195, that selectively inhibits solTNF and compared the effect of this inhibitor to a more widely used IgG1 Fc-TNF receptor (TNFR) 2 fusion protein Etanercept, an inhibitor of both solTNF and tmTNF, that has been a successful treatment strategy for several autoimmune diseases. However, Etanercept has been shown to display severe side effects by increasing the risk of infections.

Female C57BL/6 mice were subjected to a spinal cord injury at T9 level. Mice were divided into three groups, receiving Etanercept, XPro1595, or saline. The drug was delivered directly onto the lesion site using Alzet mini osmotic pumps for 3 days starting immediately after introduction of the lesion. Mice were allowed to survive 35 days. Functional evaluation was performed using the Basso Mouse Scale, Rung Walk, Open Field test and Hargreaves test. In contrast to Etanercept, XPro1595 significantly ameliorated recovery of hind limb function (evaluated by motor recovery score) compared to saline when administered continuously to the lesioned cord, whereas s.c. injections every 3 days for 8 weeks following SCI had no effect.

Our study suggests that selective inhibition of solTNF is beneficial following SCI when administered directly to the contused spinal cord and raises the possibility that selective inhibition of solTNF may represent a new therapeutic strategy for the treatment of SCI without compromising the innate immune response.


Neuregulin-1 type I enhances functional recovery after acute peripheral nerve injury and rescues axonal loss in a mouse model for Charcot Marie Tooth disease 1A

*Robert Fledrich 1 , Ruth M. Stassart 1 , Lauren M. Haag 2 , Peter Veselcic 1 , Dirk Czesnik 3 , Klaus-A. Nave 1 , Michael W. Sereda 1,3
1 Max Planck Institute of Experimental Medicine, , Göttingen, Germany
2 Ruhr-Universität , , Bochum, Germany
3 Universitätsklinik, Neurophysiologie, Göttingen, Germany
Abstract text :

After peripheral nerve injury, axons rapidly degenerate and start regrowing only after a few days. Full functional recovery, however, is rare. A progressive loss of intact axons also determines the clinical phenotype of chronic demyelinating peripheral neuropathies, such as Charcot Marie Tooth disease type 1A (CMT1A). CMT1A is the commonest inherited neuropathy, caused by a duplication of the peripheral myelin protein 22kDa (PMP22) gene. We demonstrate that the transgenic neuronal overexpression of the growth factor neuregulin-1 type I enhances axonal regeneration and functional recovery after experimental acute peripheral nerve injury. Similarly, neuregulin-1 type I overexpression preserves axonal loss and improves nerve function in a mouse model for CMT1A. Importantly, neuregulin-1 type I induced axonal preservation in CMT1A mice was independent of myelination, as myelin sheath thickness was not altered.

We conclude that axonal recovery and preservation relies on common supportive factors and that paracrine neuregulin-1 type I constitutes a beneficial signal in peripheral nerve disorders.


Olfactory ensheathing cells promote neurite outgrowth from organotypic spinal cord co-cultures

*Karen Gladwin 1 , Stuart Law 1 , Daqing Li 1 , David Choi 1
1 University College London, , London, United Kingdom
Abstract text :

Olfactory ensheathing cells (OECs) are unique glia with potential for therapeutic transplantation for the repair of spinal cord injuries (SCI).  OECs possess many characteristics favorable for the repair of SCI including the secretion of growth and survival factors, the expression of cell adhesion molecules and the ability to integrate with astrocytes.  OECs have also been reported to promote exceptional outgrowth of severed axons across the inhibitory environment of the lesion scar.  The unique combination of regenerative properties suggests OECs would be beneficial for the repair of ventral spinal root avulsion injuries.  After ventral root avulsion anterior horn neurons often fail to regenerate axons back into the surgically re-implanted nerve root resulting in poor functional recovery. 

To evaluate the efficacy of OEC transplants for the repair of ventral root avulsions, the ability of OECs to promote neurite outgrowth from spinal cord neurons was investigated in a spinal cord organotypic co-culture.  The integration of OECs with the spinal cord tissue was also examined by confocal fluorescence microscopy.  OECs were harvested from adult Sprauge Dawley (SD) rats, cultured for 14 days and transfected with GFP.  Slices of cervical spinal cord were prepared from P8 SD pups and cultured on collagen coated membrane inserts.  Each slice was surrounded by a collagen gel with or without the addition of GFP OECs and Glial derived neurotrophic factor (GDNF).  Our results indicate that OECs alone and in combination with GDNF have a positive effect on neurite outgrowth and morphology.


This work was supported by the European Research Council and the International Spinal Research Trust.


Tamoxifen promotes CNS remyelination by modulation of the PKC signalling pathway in oligodendrocyte precursor cells (OPCs)

*Ginez Gonzalez 1 , Alexandra Baer 2 , John Rundle 1 , Yasir Syed 3,4 , Matthias Hofer 3,4 , Robin Franklin 3,5 , Chao Zhao 5 , Mark Kotter 3,4
1 University of Cambridge, , Cambridge, United Kingdom
2 University of Vienna, , Vienna, Austria
3 University of Cambridge, Clinical Neuroscience/BRC, Cambridge, United Kingdom
4 Laboratory for Regenerative Medicine (LRM), , Cambridge, United Kingdom
5 University of Cambridge, School of Veterinary Medicine, Cambridge, United Kingdom
Abstract text :

Remyelination is a regenerative process during which demyelinated axons are enwrapped by newly formed myelin sheaths. In the central nervous system (CNS), this process takes place in two stages: first, during the recruitment stage, oligodendrocyte precursor cells (OPCs) divide, migrate and engage the demyelinated axons. Then, during the differentiation stage, OPCs differentiate to myelin-forming oligodendrocytes. Under pathological conditions such as multiple sclerosis (MS), remyelination often fails and as a consequence chronic demyelinated areas accumulate in MS. Post mortem evidence suggests that OPC differentiation is often impaired in MS patients. However, the reason why OPC differentiation fails in MS is not completely understood. It has been proposed that MS lesions contain factors that act as specific inhibitors of OPC differentiation. Amongst them, myelin associated inhibitors play an important role. We have demonstrated that the inhibitory effect of myelin protein extracts (MPE) is mediated by activation of signalling cascades including PKCα-MARCKS in vitro. Furthermore, modulation of PKCα has been shown to rescue OPC differentiation in the presence of inhibitory MPE. Here we demonstrate that tamoxifen, a drug that is used in various clinical settings because of its PKC-inhibitory activity, is able to promote OPC differentiation in vitro

In a series of in vivo experiments, we tested the hypothesis that inhibition of PKCα by tamoxifen promotes CNS remyelination. Demyelination was induced in the caudal cerebellar peduncle (ccp) of young-adult Sprague-Dawley rats by the administration of ethidium bromide. Animals received daily doses of tamoxifen. Although tamoxifen treatment did not induce changes in the number of OPCs expressing immature markers (NG2 and PDGFR-α), a significant increase in the number of cells expressing mature marker of oligodendrocytes (CC1/Olig2 and PLP) was detected. Furthermore, analysis of resin-embedded tissue demonstrated that tamoxifen significantly promotes remyelination in the CNS when compared to controls (Mann-Whitney’s test, p<0.05). Although further investigations are required to elucidate the mechanisms by which tamoxifen exerts CNS remyelination, our results demonstrate that administration of tamoxifen represents a potent and clinically accessible strategy to promote endogenous myelin regeneration.


HDACs in control of maintenance and regeneration in Schwann cells

Valérie Brügger 1 , Céline Pattaroni 1 , Patrick Matthias 2 , Ueli Suter 3 , *Claire Jacob 1
1 University of Fribourg, , Fribourg, Switzerland
2 FMI, , Basel, Switzerland
3 ETH Zurich, , Zurich, Switzerland
Abstract text :

Histone deacetylases (HDACs) are key transcriptional regulators that control gene expression by remodeling chromatin and modifying the activity of transcription factors. There are 18 known mammalian HDACs, subdivided into four classes. Using mouse genetics, we have shown that the two class I HDACs HDAC1 and HDAC2 are crucial for survival and myelination of Schwann cells, the myelinating glia of the peripheral nervous system (PNS), and for maintenance of peripheral nerve integrity in adults. Our current aim is to understand whether and how HDACs can influence the regeneration process in Schwann cells. Our data suggest important functions of HDACs in PNS regeneration.


Bone marrow-derived mesenchymal stem cell intraventricular injection in a chronically demyelinated mouse model

*Jonathan Jones 1 , Pablo Cruz Martínez 1 , Salvador Martinez 1
1 Neuroscience Institute, Univ. Miguel Hernandez, , San Juan, Alicante, Spain
Abstract text :

Mesenchymal stem cell transplantation has been proven to have beneficial effects in various degenerative diseases, including demyelinating models. Both in our lab as well as others have shown that they express a number of trophic factors that are capable of inducing remyelination, mainly by activating nearby oligodendrocyte progenitors towards mature oligodendrocytes that in turn remyelinate the damaged area. However, this effect was only observed locally, in the area surrounding the graft, thus in order to achieve general remyelination in various brain structures simultaneously, we decided to perform bone marrow-derived mesenchymal stem cell injections into the lateral ventricles. In this manner, the cells may attach to various areas such as the hippocampus, corpus callosum and fornix, among others, all of which are demyelinated. Previous to the graft, the cells were incubated with iron nanoparticles. This way, it is possible to track in vivo the grafted cells by magnetic resonance imaging (MRI). As a result, the cells were observed at different time points (0-15-30-60-90 days) by MRI. Also, the demyelinated areas can also be visualized and myelin quantified using image analysis software. This allows the quantification myelin density in the same individual mice at different moments before and after transplantation.


CD300f immunoreceptor contributes to peripheral nerve regeneration after crush injury

Patricia Solari 1 , Natalia Puig 1 , María Luciana Negro 1 , Laia Acarin 2 , Joan Sayós 3 , Hugo Peluffo 4 , *Natalia Lago 5
1 Institut Pasteur de Montevideo, Neurodegeneration Laboratory, Montevideo, Uruguay
2 Faculty of Medicine, Autonomous University of Barcelona, Unit of Histology, Barcelona, Spain
3 CIBBIM-Nanomedicine Program, Hospital Universitari Vall d’Hebrón, Institut de Recerca (VHIR), Universitat Autònoma de Barcelona, Immunobiology Group, Barcelona, Spain
4 Faculty of Medicine, UDELAR and Institut Pasteur de Montevideo, Department of Histology and Embryology and Neurodegeneration Laboratory, Montevideo, Uruguay
5 Institut Pasteur de Montevideo, , Montevideo, Uruguay
Abstract text :

It is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (CNS). After injury of the peripheral nervous system (PNS), Schwann cells and macrophages phagocyte myelin debris in Wallerian degeneration in order to regenerate axons to distal targets. The immunoreceptor CD300f is normally expressed in the myeloid line and in nervous system in microglia, oligodendrocytes and neurons under certain conditions. However, little is known about the CD300f ligands. By using a CD300f-Fc fusion protein we have analyzed the expression of the CD300f ligands in the PNS. Moreover, we have also analyzed the possible role of the CD300f immunoreceptor in peripheral nerve regeneration by blocking the interaction between CD300f and its ligands with the same fusion protein. Thy1-YFP-H mice sciatic nerves were injected with CD300f-Fc, control mIgG2a or PBS immediately before a crush injury. The results show that CD300f ligand is expressed in Schwann cells. Moreover, after a sciatic nerve injury, animals injected with the CD300f-Fc protein show a lower number of YFP-positive fibers growing into the tibial nerve after 10 days post-lesion (dpl) than control groups. Moreover, the CD300f-Fc group shows a higher number of macrophages and CD206-positive cells at 10 dpl when compared to control groups. We do not see these differences in axon regeneration and macrophage infiltration after 28 dpl. We have also evaluated the mRNA expression of the pro-inflammatory cytokine IL-1β at 24 hours after crush and injection of the fusion protein. Q-PCR shows an up-regulation of the mRNA in control groups and a lower mRNA expression level in the CD300f-Fc protein group. Together these results show that the pair CD300f receptor and ligand is implicated in some aspects of Wallerian degeneration and nerve regeneration such as the modulation of both the influx and phenotype of macrophages.


Phosphorylation of aquaporin-4 at Ser111 is not required for channel gating

*Mette Assentoft 1 , Shreyas  Kaptan 2 , Robert  Fenton 3 , Susan Z.  Hua 4 , Bert  de Groot 2 , Nanna MacAulay 1
1 University of Copenhagen, Department of Cellular and Molecular Medicine, Copenhagen, Denmark
2 Max Planck Institute for Biophysical Chemistry, Computational Biomolecular Dynamics Group, Gottingen, Germany
3 Aarhus University, Department of Biomedicine and InterPrET Center, Aarhus, Denmark
4 State University of New York in Buffalo, Department of Physiology and Biophysics, Buffalo, United States
Abstract text :

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 has been described as an important entry and exit site for water during formation of brain edema and regulation of AQP4 is therefore of broad interest. Phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. Protein kinase (PK)-dependent phosphorylation of Ser111 has been reported to increase the water permeability of AQP4 expressed in an astrocytic cell line. This possibility was, however, questioned based on the crystal structure of the human AQP4. Our study aimed to resolve if Ser111 was indeed a site involved in phosphorylation-mediated gating of AQP4. The water permeability of AQP4-expressing Xenopus oocytes was not altered by a range of activators and inhibitors of PKG and PKA. Mutation of Ser111 to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of AQP4. PKG activation had no effect on the water permeability of AQP4 in primary cultures of rat astrocytes. Molecular dynamics simulations of a phosphorylation of AQP4.Ser111 recorded no phosphorylation-induced change in water permeability. A phospho-specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser111, failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. Thus, our data indicate a lack of phosphorylation of Ser111 and of phosphorylation-dependent gating of AQP4. 


Astrocyte volume regulation in the cortex of alpha-syntrophin-negative GFAP/EGFP mice

*Olena Butenko 1 , Jana Benesova 1 , Michaela Mikesova 1 , Pavel Honsa 1 , David Dzamba 1 , Jan Kriska 1 , Vendula Rusnakova 2 , Mikael Kubista 2,3 , Miroslava Anderova 1
1 Institute of Experimental Medicine, , Prague, Czech Republic
2 Institute of Biotechnology, , Prague, Sweden
3 TATA Biocenter, , Gothenburg, Sweden
Abstract text :

The formation of brain edema, which accompanies ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to extracellular K elevation and neurotransmitter accumulation in the extracellular space. An increased uptake of these osmotically active substances, predominantly provided by astrocytes, is accompanied by intracellular water accumulation via aquaporin-4 (AQP4). Previously, it has been shown that the removal of perivascular AQP4 via the deletion of alpha-syntrophin, a protein responsible for anchoring AQP4 on the astrocytic membrane, delays edema formation and K clearance (Amiry-Moghaddam et al.,2003, PNAS 11;100(23):13615-20). Therefore, we aimed to elucidate the impact of alpha-syntrophin deletion on astrocyte volume changes in the cortex during pathological states, such as hypoosmotic stress or oxygen-glucose deprivation (OGD), using three-dimensional (3D) confocal morphometry in situ. In addition, single cell RT-qPCR profiling was carried out to reveal possible differences in the expression profiles of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. In order to visualize individual astrocytes that lack alpha-syntrophin, double transgenic mice were generated by crossbreeding GFAP/EGFP mice (Nolte et al., 2001, Glia. 33(1):72-86) with alpha-syntrophin knockout mice (Amiry-Moghaddam et al., 2003, PNAS 11; 100(23):13615-20). 3D-confocal morphometry revealed that alpha-syntrophin deletion results in significantly smaller/slower astrocyte swelling when induced by 20 min hypoosmotic stress (210 mOsm), 30 min OGD or by high extracellular K (50 mM), while alpha-syntrophin deletion had no effect on the astrocytic shrinkage evoked by hyperosmotic stress (350 mOsm). The volume recovery of cortical astrocytes from GFAP/EGFP/alpha-syntrophin knockout mice was significantly slower following their exposure to hypo- or hyperosmotic stress, whereas no differences were found in astrocyte volume recovery following OGD or after their exposure to high extracellular K . Compared to the cortical astrocytes of GFAP/EGFP mice, single cell RT-qPCR analyses revealed that astrocytes from GFAP/EGFP/alpha-syntrophin knockout mice express higher mRNA levels for two-pore domain K channels (TWIK-1, TASK-2, TASK-3), inwardly or outwardly rectifying K channels (Kir3.1, Kir5.1, Kv1.3, Kv1.6) and chloride channels (ClC1,ClC4), while mRNA expression for the glutamate transporter GLT-1 is lower. In summary, the deletion of alpha-syntrophin slowed down astrocyte swelling during hypoosmotic stress, OGD or high K ; however, it also resulted in alterations in astrocytic gene expression profiles.

Supported by grants GA CR 13-02154S and P304/12/G069 from the GACR.



MLC1 dysfunction causes brain oedema with white matter vacuolation in murine brain.

*Mohit Dubey 1 , Marianna Bugiani 2 , M Ridder 2 , J.C Lodder 1 , M Verheijen 1 , HD Mansvelder 1 , I Boor 1,2 , MS van der Knaap 1
1 VU-University, Amsterdam, , Amsterdam, Netherlands
2 VU-Univ Medical Center, Department of Child Neurology, Amsterdam, Netherlands
Abstract text :

MLC1 is an eight transmembrane domain protein highly expressed in brain. Human mutations in MLC1 lead to the rare genetic disorder “megalencephalic leukoencephalopathy with subcortical cyst” (MLC). Characteristic for the disease are the onset of macrocephaly within the first year of life, and a slowly progressive loss of motor skills with epilepsy and cognitive decline. At the cellular level, countless fluid-filled vacuoles occur within myelin sheaths surrounding axons and in astrocytic endfeet. In cell culture models, MLC1 mutations are associated with defects in chloride currents and cell volume regulation. Thus far, the expression and function of MLC1 in intact murine and human brain are still controversial.

To understand the role and developmental expression of MCL1 in the brain, we have developed a MLC1 mutant null mouse model carrying an eGFP reporter gene under the MLC1 promotor. We now show the exclusive expression of MLC1 in astrocytes throughout the brain with specifically higher expression around blood vessels, at the sub-ventricular zone and at the glia limitans. The functional loss of MLC1 in mutant mice recapitulates in part the human MLC disease. KO mice show macrocephaly with high brain wet weight. Water filled vacuoles develop in the white matter of the cerebrum and in large fiber tracts of the brainstem. By contrast, the heterozygous loss of MLC1 has no consequences on myelin structural integrity. Both heterozygous and homozygous MLC1 KO mice have dysmorphic peri-vascular and peri-ventricular astrocytes. In contrast to humans, KO mice have no motor deficits, but are hyperactive and show an anxiety-like behavior.

In the MLC1 KO mouse, a dysfunction of an astrocytic protein causes loss of myelin structural integrity leading to vacuolating myelinopathy. Therefore, the MLC mutant mouse could be a key model to study the astrocytic involvement in brain water and ion homeostasis.


Connexin mediated glial networks are heterogenous among brain regions

*Stephanie Griemsmann 1 , Lena Claus 1 , Simon Höft 1 , Peter Bedner 1 , Jiong Zhang 1 , Ronald Jabs 1 , Martin Theis 1 , David W Cope 2 , Vincenzo Crunelli 2 , Gerald Seifert 1 , Christian Steinhäuser 1
1 University of Bonn, , Bonn, Germany
2 University of Cardiff, , Cardiff, United Kingdom
Abstract text :

Astrocytes are connected with each other via gap junction channels. These astroglial networks fulfil a variety of functions in the brain, e.g. potassium buffering and metabolite transport. In this study we compared glial networks in different brain regions to investigate site specific effects on network formation. We combined electrophysiology and immunohistochemstry with semi-quantitative RT-PCR and Western blot analysis to investigate connexin expression, gap junction coupling and antigen profiles. Experiments were performed in wild type and transgenic mice with glia specific fluorescence labelling as well as in Cx30ko mice. Astrocytes were investigated between postnatal days 12-60.

Gap junction networks in the CA1 region of the hippocampus and the ventrobasal thalamus show abundant coupling in hGFAP-EGFP mice. Intriguingly, we found significant coupling between oligodendrocytes and astrocytes in the thalamus, while in the hippocampus panglial coupling was less abundant. Other glial cells did not participate in the networks. We also found that a fluorescent glucose analog, 2-NBDG, propagates through the thalamic panglial network. The function of these panglial networks remains largely unclear.

In heterozygous Cx43-ECFPki mice, deletion of one allele of the major hippocampal Cx43 significantly reduced the number of coupled astrocytes only in the hippocampus, while the thalamic networks remained unchanged.

SR101 labelling of astrocytes and subsequent 2P microscopy identified a significant subset of thalamic SR101 cells lacking Cx43-ECFP expression. SR101 did not label oligodendrocytes as analysed in PLP-GFP mice. Semi-quantitative RT-PCR and Western blot analysis revealed stronger expression of Cx30 in thalamic nuclei while Cx43 levels were higher in the hippocampus. This indicates a minor role for Cx43 in gap junction coupling of astrocytes in the thalamus. Consistent with these findings, the thalamus of Cx30ko mice displayed a strong decrease in astrocytic cell coupling compared to wild type littermates.

Together, these results indicate that thalamic astrocytes differ in various aspects from their counterpart in other brain regions and support the emerging concept of astrocyte heterogeneity.

Supported by DFG (SFB-TR3) and EU (FP7-202167 Neuroglia).


Temporally controlled ablation of astroglial P2Y1 receptors

*Hannah Maria Jahn 1 , Aiman Samir Saab 1 , Alexej Verkhratsky 2 , Frank Kirchhoff 1
1 University of Saarland, , Homburg, Germany
2 University of Manchester, Faculty of Life Science, Manchester, United Kingdom
Abstract text :

Purinergic signaling is the most diverse system in astrocytes to communicate with other glial cells and neurons. Astrocytes express a variety of P2X (ionotropic) and P2Y (metabotropic) receptors. Especially in case of brain injury, ATP levels and receptor expression on astrocytes are increased. ATP is immediately degraded to ADP, AMP and adenosine. These nucleotides/nucleosides act back on the preferred purinoreceptor expressed on glial and neuronal cells. In the last decade researchers tried to evaluate the functional impact of astrocytic purinoreceptors on the complex information flux at the tripartite synapse. A prominent role has been suggested for the P2Y1 receptor subtype. We generated conditional mouse mutants to investigate the function of P2Y1 receptors in astrocytes in vivo and crossbred mice carrying the floxed P2Y1 gene with mice that express the inducible DNA recombinase (CreERT2) under the control of the GLAST (L-glutamate/L-aspartate transporter) locus.

Ablation of the P2Y1 receptor is induced in development and adulthood by intraperitoneal tamoxifen injections. Successful recombination of the targeted P2Y1 receptor is evaluated by qRT-PCR on genomic DNA and mRNA usually 21 days after tamoxifen application. At the genomic level we find ~25% and ~50% recombination in astrocytes of the cerebellum and hippocampus, respectively. Similar recombination frequencies are observed in young (p11) or adult mice (6-10 weeks). When looking at the transcript level, the mRNA is reduced to 57% in the cerebellum of adult mice and to 76% in the hippocampus. In young mice we determined a reduction to 40% and 42% mRNA expression in the cerebellum and hippocampus, respectively. Given the high level of P2Y1 expression on other cells of the brain these findings indicate a successful astrocyte-specific knockout. Further fluorescence-activated cell sorting (FACS) of recombined cells expressing the red fluorescent protein td-tomato will be used to verify the knockout. The potential function of these astroglial receptors in neuronal networks will be as well investigated using histological (EM and light microscopy), two-photon Ca2 imaging in situ and in vivo and behavioral approaches.


Oligodendrocyte-targeted gene therapy to treat leukodystrophy

*Alexia Kagiava 1 , Irene Sargiannidou 1 , Stavros Bashardes 2 , Jan Richter 2 , Natasa Schiza 1 , Christina Christodoulou 2 , Angela Gritti 3 , Kleopas Kleopa 4
1 The Cyprus Institute of Neurology and Genetics, Neuroscience Laboratory, Nicosia, Cyprus
2 The Cyprus Institute of Neurology and Genetics, Department of Molecular Virology , Nicosia, Cyprus
3 Fondazione Centro San Raffaele del Monte Tabor, , Milan, Italy
4 The Cyprus Institute of Neurology and Genetics, Neuroscience Laboratory and Neurology Clinics, Nicosia, Cyprus
Abstract text :

Question: Pelizaeus-Merzbacher-like disease (PMLD) is a hypomyelinating leukodystrophy caused by mutations in the GJC2 gene encoding the gap junction (GJ) protein connexin47 (Cx47). Cx47 is expressed in oligodendrocytes and forms most GJ channels to astrocytes and to other oligodendrocytes. Since PMLD-associated GJC2 mutations cause loss of Cx47 function, gene replacement strategies may be promising for developing future treatments. A mouse model for PLMD, the Cx32/Cx47 double knockout (KO), is characterized by early onset of severe leukodystrophy at 4 weeks of age leading to death by 6 weeks, offering the possibility to test therapies. The aim of the present study was to generate a lentiviral vector to allow gene delivery specifically to oligodendrocytes, and to examine the transduction efficacy, distribution, duration and levels of EGFP reporter gene and Cx47 expression throughout the CNS, in order to establish a method for oligodendrocyte-targeted gene therapy.

Methods: The expression cassette with the Cx47 gene along with the IRES-EGFP as a reporter gene under the control of oligodendrocyte-specific CNP promoter was cloned into the lentiviral vector pCCLsin.PPT.hPGK.GFP.pre. Mock vectors lacking the Cx47 gene were also generated as controls. Viral particles were produced to high titers and purified, before injection. Lentiviral vectors were delivered into the brain of wild type (WT) and Cx47KO mice at postnatal day 1 (P1), intraventricularly and in the stratum radiatum of the dorsal hippocampus. EGFP and Cx47 expression was assessed using immunochemistry and immunoblot analysis at different time points post injection.

Results: We found widespread expression of virally delivered EGFP in different brain regions colocalizing with oligodendrocyte markers (CC1 and Olig2). The expression of EGFP was detected from P15 until 3 months post-injection. On average 20.3±2.56 % of oligodendrocytes were EGFP positive, with highest rates in the subventricular zone (29.3±6.31%, n=6) and olfactory bulb (19.9±4.36%, n=8) and lower rates in the cortex (16.1±2.60 %, n=6) and corpus callosum (12.3±3.40%, n=3). In Cx47KO brain expression of virally delivered Cx47 was also found after P21 with formation of GJ plaques in a subpopulation of oligodendrocytes.

Conclusions: Our results show that neonatal lentiviral gene delivery may result in stable and widespread CNS expression targeted to oligodendrocytes by using cell specific promoters. Thus, gene therapy approaches using lentiviral vectors may be feasible for future treatment of leukodystrophy and should be further studied.


Acknowledgement: This project is funded by the European Leukodystrophy Association, ELA (Grant 2011-025I2 to KAK).


Identification of epigenetic modifications associated with inhibition of the stem and tumorigenic properties of glioblastoma stem cell.

*Alexandra Bogeas 1 , Klaudia  Kuranda 2 , Luis Gustavo Dubois 1 , Elias A. El-Habr 1 , Salwa  Sayd 1 , Francois-Xavier  Lejeune 1 , Amelia  Dias-Morais 1 , Thierry  Virolle 3 , Michele  Goodhardt 2 , Marie-Pierre Junier 1 , Hervé Chneiweiss 1
1 Inserm U894, , Paris, France
2 Inserm UMRS 940, , Paris, France
3 Inserm U898, , Nice, France
Abstract text :

A growing body of evidence indicates that glioblastoma stem-like cells (GSCs) play a central role in human glioblastoma development and resistance to current therapies.  We recently described a cluster of micro-RNAs, the miR-302-367, which induces the exit of GSCs from their stem state and suppresses their tumorigenic properties in an irreversible manner (Fareh et al, 2012). To elucidate the gene networks that govern GSC exit from their stem state, we used ChIP-seq (chromatin immunoprecipitation followed by next generation DNA sequencing), to identify gene loci showing enrichment of the active (H3K4me3) and repressive (H3K27me3) epigenetic marks in GSC-miR-302-367 as compared to naïve GSCs. Functional significance of the ChIP-seq results was evaluated with confrontation of the ChiP data with the transcriptomes of the corresponding cells derived from exon array hybridization.


Western blot analysis showed that global H3K4me3 and H3K27me3 expression levels were similar in GSC and GSC-miR-302-367. ChIP-seq analysis revealed similar gross number of gene loci associated with H3K4me3 or H3K27me3 (13000 and 5000 genes, respectively) in either cell type. Interestingly, 16% (2381 genes) of the analyzed genes exhibited a change in either H3K4me3 or H3K27me3, or both in miR-302-367-GSCs as compared to naïve GSCs. These changes resulted into a transcript variation in 14% of the cases (332/2381) considering as significant an increase or a decrease of at least 2-fold and the 77% of these genes translated into congruent alterations in the corresponding transcript levels.


Analysis of the functional significance of the changes observed using functional annotation databases identified novel gene networks, likely to participate in the regulation of GSC properties. Studies under way focus on members of these networks specifically activated in miR-302-367 GSCs that might alter GSC dialog with their microenvironment.


Pro- versus anti-tumor activities of microglia/macrophages: experimental and mathematical modeling approaches

*Liliana del Rocío Cisneros Castillo 1 , Alina  Toma 2 , Thorsten M.  Buzug 3 , Anne Régnier-Vigouroux 1,4
1 German Cancer Research Center, , Heidelberg, Germany
2 University of Lübeck, Institute of Medical Engineering, Centre of Excellence for Technology and Engineering in Medicine (TANDEM), Lübeck, Germany
3 University of Lübeck, Institute of Medical Engineering, Lübeck, Germany
4 Johannes-Gutenberg University Mainz, , Mainz, Germany
Abstract text :

Tumor infiltrating microglia/macrophages (TIMs) constitute the largest population of infiltrating cells in glioblastoma (GBM), the most aggressive brain tumor. Data from the clinic and from experimental work performed in murine and human models indicate that TIMs play a significant role in GBM biology as they support proliferation, migration and invasion of tumor cells. Evidence is amounting that tumor cells actually polarize TIMs towards this M2-like, tumor-supportive phenotype. We showed that toll-like receptor 3 ligand reverses this M2- into a M1-like phenotype. Pre-activated, M1-like polarized TIMs incubated with spheroids of GBM cells reduced migration, killed and phagocytosed tumor cells over a 15 day-period, indicating a sustained M1 activation of TIMs in absence of exogenously added stimuli. In order to analyse in more detail TIMs-GBM cells interactions, we have undertaken two approaches. We use spheroids of cells (tumor with/without TIMs) embedded in collagen matrix, in which TIMs can be implanted, as an experimental model. This three-dimensional in-vitro system is well suited for a qualitative and quantitative monitoring of cell proliferation, death, migration and invasion. Data experimentally generated are then implemented in a mathematical model that is, to our knowledge, the first one proposed to take into account tumor cells and TIMs in order to simulate GBM progressive behaviour. In a first step, the capacity of spheroids made of murine glioma cells to invade collagen matrices was evaluated in absence and presence of microglia. Invasion was monitored by photography of the spheroids and images were processed with Photoshop CS5 software. In order to achieve a precise determination of invasion, we chose to measure the diameter of the core plus the invasive rim as a read out of expansion rate. Untreated microglia promoted growth and invasion of tumor cells. Data generated through the proposed in-vitro system were comparable to and reproduced the in-silico simulations obtained with the mathematical model, hence validating our mathematical and experimental approaches. We currently evaluate the rate of proliferation and death of tumor cells in various settings that modulate TIMs polarization, using flow cytometry and confocal (live) imaging. Data contributed by these two approaches should facilitate the delineation of a predictive model for tumor progression in a TIMs-enriched microenvironment with possible therapeutic fallouts.


Regulation of glioblastoma stem-like cells properties by GABA-related metabolites.

*Luiz Gustavo Dubois 1,2 , Elias A. El-Habr 2 , Joanna Lipecka 2 , Mohamed Fareh 3 , Vivaldo Moura-Neto 1 , Thierry Virolle 3 , Hervé Chneiweiss 2 , Marie-Pierre Junier 2
1 Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, Rio de Janeiro, Brazil
2 1. Inserm U894, , Paris, France
3 2.INSERM U898, , Nice, France
Abstract text :

A growing body of evidence indicates that Glioblastoma stem-like cells (GSCs)
play a central role in glioblastoma development and resistance to current therapies.
We recently described a cluster of micro-RNAs, the miR-302-367, which induces the
exit of GSC from their stem state and suppresses their tumorigenic properties in an
irreversible manner (Fareh et al, 2012). We postulated that such a drastic change in
the cell phenotype should be accompanied with metabolic alterations that could be
instrumental in the loss of functional properties of GSCs induced by the micro-RNA

Metabolome profiling by mass-spectrometry of GSCs and GSC-miR-302-
367 pointed to changes in the GABA synthesis pathway (see abstract El-Habr et al).
Remarkably, exposure of naïve-GSC to metabolites of the GABA synthesis pathway
found to be overproduced in GSC-miR-302-367 reproduced at least in part the
effects of miR-302-367, including inhibition of clonality, down-regulated expression
of self-renewal markers, and loss of self-renewal properties. The metabolites studied
acted by interfering with progression of the cell cycle and the nuclear localization of
transcription factors crucial for maintenance of GSC stem-like properties. Studies
assaying the effects of the metabolites on GSC tumor-initiating properties are under

These results demonstrate that metabolic regulations can participate in
the control of GSC properties, and opens novel paths in therapeutic targeting of


*LGD and EAE contributed equally. 



An allograft glioma model reveals the dependence of aquaporin-4 expression on the brain microenvironment

*Petra Fallier-Becker 1 , Susan Noell 1 , Marcos Tatagiba 1 , Rainer Ritz 1 , Hartwig Woburg 1 , Karen Wolburg-Buchholz 1
1 University of Tuebingen, , Tuebingen, Germany
Abstract text :

Aquaporin-4 (AQP4), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. In contrast, most cultivated glioma cell lines do not express AQP4, and primary cell cultures of human glioblastoma lose it during the first passages. Accordingly, in two glioma cell lines of the rat (C6 cells and RG2 cells) and in SMA mouse glioma cell lines we found no AQP4 expression. This let us consider the possibility that AQP4 expression depends on brain microenvironment. AQP4 negative rat glioma cells were implanted into rat brain. Within two weeks, a tumor developed. AQP4 staining of the tumor cells was positive. However, if the identical cells were implanted into the rat’s flank, they did not express AQP4. In contrast to the normal brain, where AQP4 staining is polarized in the astrocytic endfoot membranes, the AQP4 staining in C6 and RG2 tumors was distributed over the whole glioma cell as in human glioblastoma. We conclude that the micro-environment is crucial for AQP4-expression in brain and brain-tumor.


CDH5 is specifically activated in glioblastoma stem like cells and contributes to vasculogenic mimicry induced by hypoxia

*Xiang Zhang 1 , Xing-gang  Mao 1 , Xiao-yan  Xue 1 , Wei Zhang 1
1 Xijing Hospital, Fourth Military Medical University, , Xi'an, China
Abstract text :

Background: A proportion of glioblastoma (GBM) stem like cells (GSCs) expressing
endothelial cell (EC) marker CDH5 (VE-cadherin or CD144) can trans-differentiate into
ECs and form blood vessels. However, the implication of CDH5 expression in gliomas,
and how its expression is regulated in GSCs remain to be clarified.
Methods: The mRNA and protein levels of CDH5 were detected in glioma samples and
cultured cell lines, and the prognostic value of CDH5 expression level for GBM patients
was evaluated. Bioinformatics analysis was performed to reveal the potential functional
roles of CDH5 in GBM. Gene knockdown induced by shRNA, chromatin
immunoprecipitation analysis and vasculogenic tube formation assay were performed
to investigate the relationships between hypoxia, CDH5 expression level and
Results: CDH5 was overexpressed in gliomas, correlated with tumor grades, and an
independent adverse prognostic predictor for GBM patients. CDH5 was specifically
activated in GSCs but not in non-GSCs or neural stem cells (NSCs), and CDH5 cells
could produce xenogafts in immunocompromised mice. Bioinformatics analysisdemonstrated that CDH5 might interact directly with hypoxia inducible factor 2α
(HIF2α). CDH5 expression was significantly upregulated in GSCs, but not in non-GSCs
or normal NSCs, under a 1% O2 condition. Both HIF1α and HIF2α positively regulated
CDH5 level in GSCs, and could bound to the promoter of CDH5. Furthermore, CDH5
contributed to the vasculogenic mimicry of GSCs, especially under hypoxic conditions.
Conclusions: The specific expression of CDH5 in GSCs may contribute to GSC-derived
neovasculogenesis in GBM, especially under hypoxic conditions, revealing novel
tumorigenic mechanisms contributed by GSCs.