Date on Master's Thesis/Doctoral Dissertation

12-2011

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Anatomical Sciences and Neurobiology

Committee Chair

Whittemore, Scott R.

Author's Keywords

Oligodendrocytes; OPC; Histone deacetylase; HDAC inhibition; Cell culture; DNA damage

Subject

Oligodendroglia; Neurochemistry

Abstract

Recent studies indicate post-translational deacetylation by members of the superfamily of histone deacetylase complexes (HDACs) is necessary for oligodendrocyte precursor cell (OPC) differentiation into mature oligodendrocytes (OLs). However, it remains unknown if transient HDAC inhibition may promote OPC survival as has been shown in other cell types and neurological disease models. Chapter 2 demonstrates the development of a novel primary culture of OPCs from mouse pup cortices enriching the O4+ OPC population using Magnetic Activated Cell Sorting (MACS) technology. Once plated, OPCs were glial fibrillary acidic protein- (GFAP-), A2B5+, NG2+, and O4+. When induced to differentiate, mouse OPCs became either more complex O4+ and O1 + oligodendrocytes (OLs) or GFAP+ astrocytes. Bi-potentiality is lost in co-culture, however, with rat embryonically derived dorsal root ganglia (DRG) as mature OLs aligned with DRG neurites. In Chapter 3, following OPC treatment with the FDA approved pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), approximately 65% reduction in cell survival relative to vehicle-treated OPCs was observed. SAHA-mediated death of OPCs induces apoptosis partly by caspase activation with improvement of OPC survival when SAHA is accompanied by treatment with the general caspase inhibitor q-vd-oph. SAHA treatment of CNP-EGFP transgenic mouse pups increased the number of caspase activated apoptotic cortical OPCs compared to vehicle treated pups (p < 0.05). In Chapter 4, we extended this study to spinal cord-derived rat primary OPCs to determine putative mechanism(s). Rat OPCs were similarly susceptible to SAHA-mediated HDACi. SAHA treatment of rat OPCs increased phosphorylated histone H2A.X+ (yH2AX) indicating the presence of HDACimediated DNA double-strand breaks. SAHA treatment also increased phosphorylated p53 and cleaved caspase 3 levels suggesting HDACi P53 mediated, caspase-dependant rat OPC apoptosis. These results provide strong evidence of HDACi-mediated apoptosis of normal mouse pup and adult rat OPCs. SAHA is currently involved in more than 180 clinical trials, some of which include a pediatric patient population, and is in consideration for use in the treatment of psychiatric and neurodegenerative conditions. These results strongly suggest that such clinical use of SAHA may negatively impact OPC survival and potentially be detrimental to the myelinating brain and spinal cord.

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