CHARACTERIZATION OF CNS STEM CELLS USING SOMATIC MOSAICS

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The existence of multipotent stem cells in the primarily post-mitotic cellular environment of the adult brain has sparked intense research into identification of putative cell type(s) and potential functions. Elucidation of stem cell identity could have an immediate impact regarding its potential use in therapies for neurodegenerative diseases or age-related degeneration. Controversy has arisen regarding the identity of the CNS stem cell and its step-wise lineage progression from stem cell to post- mitotic neuron and/or glia. Recent suggestive studies concerning stem cells located in the subventricular zone (SVZ) have shown that astrocyte- like cells found in the SVZ have the potential to self-renew and/or give rise to cells downstream in the rostral migratory pathway. The development of a novel somatic mosaic approach is described in this application to definitively assess the role of SVZ astrocytes as stem cells, where the overall hypothesis is as follows: Selective removal of GFAP- positive SVZ astrocytes, purported to be multipotent stem cells, will result in the concomitant loss of neuroblastic cells and mature neurons that normally arise from the SVZ. Specifically, a germline-transmitted transgene construct will be used that carries cis recombination elements, loxP, flanking strong transcriptional termination sequences. When activated by the somatic delivery of cre recombinase via a defective herpes amplicon vector a permanent alteration in transgene structure will be catalyzed specifically in the genome of SVZ astrocytes, thereby effecting focal expression of a pro-drug enzyme that sensitizes these cells to a cell death-promoting pro-drug. Cells in which recombination induces transgene expression will also be tagged via co-induction of a histochemical marker, allowing for determination of cell fate in the absence of pro-drug. Experiments described in this proposal constitute the analysis of the properties of the somatic mosaic system that are anticipated to allow for appropriate design, execution, and interpretation of future experiments where the system will be applied to address the function of SVZ astrocytes in the adult rodent.