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XI European Meeting on Glial Cell Function in Health and Disease

Overview Session Overview Sessionprint print  
 

July 3, 2013 - Room 2 11:10am - 12:55pm
Workshop III (continued): Astrocyte Heterogeneity and Transcriptome Analysis
11:10am
W03-04

Endothelin-1: An astrocyte-derived signal that regulates oligodendrocyte development and regeneration

*Vittorio Gallo 1,2 , Tim Hammond 1,2 , Ana Gadea 1 , Jeff Dupree 3
1 Children's National Medical Center, Center for Neuroscience Research, Washington, DC, United States
2 The George Washington University, Institute for Biomedical Sciences, Washington, DC, United States
3 Virginia Commonwealth University, Department of Anatomy and Neurobiology, Richmond, VA, United States
Abstract text :

We have previously demonstrated that Endothelin-1 (ET-1) induces a reactive phenotype in astrocytes in vitro and in vivo. After 48 h ET-1 exposure, microarray analysis of cultured primary astrocytes showed that ET-1 promoted expression of Notch ligands Jagged1, Delta1, and Contactin1, but not Jagged2 or Delta3. Our microarray analysis also found significant increases in Stat-3, Nestin, AdamTS,  Laminin Alpha-5, Laminin Beta-1, and Laminin Gamma-1 expression. Western blot analysis confirmed that ET-1 induced Notch ligand expression in astrocytes. ET-1-treated astrocytes inhibited oligodendrocyte progenitor cell (OPC) differentiation in vitro through Notch activation in astrocyte-OPC co-cultures. ET-1 was expressed at high levels by astrocytes in the corpus callosum following lysolecithin-induced demyelination. Infusion of ET-1 into remyelinating lesions during the OPC differentiation phase prevented OPC differentiation and limited remyelination efficiency. Conversely, infusion of the ET-receptor pan-antagonist PD142,893 into transgenic Notch reporter mice drastically reduced Notch activation, and accelerated the rate of OPC differentiation and remyelination. We ablated ET-1 production specifically from astrocytes using an inducible Cre system (GFAPCreERT2;ET-1flox/flox) (ET-1CKO), and found that the rate of remyelination after lysolecithin injection was drastically increased. Our findings indicate that ET-1 is a remyelination inhibitory signal that promotes astrocyte reactivity and activation of Notch in OPCs after demyelination. Blocking ET-1 activity could constitute a new strategy to promote repair following demyelination.



11:45am
W03-05

Identification and genomic analysis of human neural and oligodendrocyte progenitor cells

*Fraser J. Sim 1
1 SUNY University at Buffalo, Department of Pharmacology and Toxicology, Buffalo, United States
Abstract text :

The successful development of therapies for demyelinating disease is reliant on a thorough understanding of the molecular signaling cascades that regulate neural and oligodendrocyte progenitor recruitment and differentiation. As the extent and nature of species-specific differences are largely unknown, the question remains whether effective strategies to augment rodent repair will be effectively translated into human patients. This workshop is focused on techniques used for the molecular study of defined cell progenitor populations from the adult and fetal human brain. Cell type-specific antigenic as well as promoter/enhancer-based approaches for isolation of distinct stages of lineage differentiation will be reviewed. We will discuss the use of multicolor FACS and phenotypic characterization to determine the heterogeneity and overlap between progenitor populations. Microarray analysis techniques using R/Bioconductor for the analysis of gene expression, data exploration, pathway analysis and the comparative analysis of human and rodent gene expression will be introduced. We will discuss specific advanced genomic methods such as receiver operating characteristic (ROC), gene set enrichment analysis and (GSEA) and weighted gene co-expression network analysis (WGCNA) and their limitations and interpretation. Data from primary human CD133/CD140a FACS-sorted neural stem and oligodendrocyte progenitor cells will be presented and example code provided for participants. Finally, the utility and future directions of genomic and epigenetic analysis of human precursors will be discussed.



12:20pm
W03-06

Fluorescent labelling of astroglial vesicular compartments in cultures and in situ.

V. Kasimov 1 , A. Gourine 1 , K. Naibova 2 , J. Hewinson 2 , B.H. Liu 2 , A.G. Teschemacher 2 , *S. Kasparov 2
1 University College, , London, United Kingdom
2 University of Bristol, , Bristol, United Kingdom
Abstract text :

The role and even the very existence of vesicular exocytosis from astrocytes remains a hotly debated matter. Previous studies used a variety of methods to demonstrate vesicles which could potentially store and release putative “glio-transmitters” in cultured astrocytes, but the evidence for such vesicles in astrocytes in situ is limited. We have generated adenoviral vectors which drive expression of three fluorescently tagged vesicular proteins in astrocytes in order directly visualise such vesicles. We used vesicular glutamate transporter (VGLUT2) fusion with eGFP to visualised putative glutamate containing vesicles, lysosomal tetraspanin CD63 fusion with a red monomeric protein mKate to target lysosomes and a vesicular ATP transporter (VNUT) fusion with eGFP to visualise putative ATP-containing vesicles. Confocal images of cultured astrocytes unequivocally demonstrate that the three tags label different pools of vesicular organelles which can be distinguished based on their size, predominant distribution within the cell and motility. The average size of VGLUT2- positive vesicles was 0.3±0.05 µm, VNUT-positive 0.4±0.05 µm and 1±0.1 µm for CD63 positive lysosomes (~100 vesicles of each type measured in at least 5 different cells). The same viral vectors were also injected into the brain of rats and 7-8 days later acute slices were prepared and imaged immediately using a confocal microscope. Numerous motile EGFP-fluorescent vesicles were found in live astrocytes of various brain regions. Although these results do not directly demonstrate the role of these vesicular organelles in release of “glio-transmitters” they suggest that such vesicles are not an artefact of culturing and that glutamate and ATP are likely to be stored in different classes of vesicles which to not seem to overlap with lysosomes.