Regulation of Oligodendrogenesis in a Rat Contusive Spinal Cord Injury

Abstract

"Loss of oligodendrocytes (OLs) and ensuing demyelination significantly hamper functional recovery following traumatic spinal cord injury (SCI). Although proliferating glial progenitor cells (GPCs) are present in the lesioned spinal cord, spontaneous remyelination is extremely limited. The failure of remyelination after SCI may be due to inadequate signaling to generate sufficient OLs from GPCs. In this thesis research, I tested a hypothesis that manipulation of post-injury microenvironment or intrinsic transcriptional machinery could enhance the extent of oligodendrogenesis in the lesioned spinal cord. Specifically, ex vivo delivery of vascular endothelial growth factor (VEGF) was employed to increase oligodendrogenesis in rat spinal cord contusion model. I also delivered Olig genes to the proliferating GPCs to examine if activation of transcription factors which are required for proper development oligodendrocytes can promote oligodendrogenesis in injured adult spinal cord.

VEGF exerts various trophic effects for neural cells including neural stem and/or progenitors in addition to stimulating angiogenesis. The first part of my thesis study delivered VEGF gene carried by immortalized human neural stem cells one week after contusion injury and examined whether the ex vivo VEGF delivery promoted the proliferation and differentiation of GPCs. The ex vivo approach resulted in a marked elevation of VEGF in the injured spinal cord tissue and a concomitant increase in phosphorylation of VEGF receptor flk-1. Stereological counting of BrdU+ cells revealed that the ex vivo VEGF delivery significantly enhanced cellular proliferation at 2 weeks after SCI. The number of proliferating NG2+ glial progenitor cells (NG2+/BrdU+) was also increased. Furthermore, the VEGF delivery increased the number of early proliferating cells that differentiated into mature oligodendrocytes, but not astrocytes, at 6 weeks after SCI. F3.VEGF treatment also increased the density of blood vessels in the injured spinal cord and enhanced tissue sparing. These anatomical results were accompanied by improved BBB locomotor scores, suggesting that VEGF can be used as an effective therapeutic reagent to improve functional outcomes after SCI.

The second part of my thesis research was to test a hypothesis that introduction of Olig genes into proliferating GPCs could increase the OL generation after SCI. To deliver Olig genes selectively to proliferating cells, recombinant retroviruses encoding Olig1 or Olig2 with enhanced green fluorescent proteins (eGFPs) were directly injected into the injured rat spinal cord immediately after contusive injury. Surprisingly, introduction of Olig2 led to a marked hyperplasia of GFP+ cells at 1 week after SCI. Fluorescence activated cell sorting and subsequent culture of GFP+ cells revealed Olig2-induced tumorous transformation of GPCs. In contrast, Olig1 did not alter the number of GFP+ proliferating cells. Simultaneous introduction of Olig1 and 2 (Olig1/2) led to a more than two-fold increase in the number of GFP+ cells without tumor formation. Olig1/2 significantly increased the proportion of NG2+ GPCs and CC1+ mature oligodendrocytes compared to GFP only or Olig1-GFP retrovirus injection groups, and this was accompanied by an increase in the expression of myelin proteins. Introduction of Olig1/2 enhanced the expression of transcription factors involved in diverse stages of oligodendrocyte development, especially Sox10 that drives terminal OL differentiation. Finally, retroviral introduction of Olig1/2 significantly improved a quality of hindlimbs locomotion and increased the total number of oligodendrocytes at 6 weeks after SCI. Simultaneous activation of both Olig1 and Olig2 genes may be highly beneficial for SCI by both enhancing oligodendrocytic differentiation (and/or specification) and increasing the proliferation of glial progenitor cells.

In the last part, I explored the mechanism by which Olig genes regulates glioma formation which was unexpectedly observed during the second part of thesis research. Introduction of Olig2 gene into proliferating GPCs induced dramatic and transplantation of GPCs transduced with Olig2 into the brains of nude mice resulted in brain tumor, supporting a notion that overactivation of Olig2 gene carries a risk of tumor formation. Combined introduction of Olig1 with Olig2 (Olig1/2) prevented Olig2-induced hyperplasia of GPCs. I found that p21, a tumor suppressor and inhibitor of stem cell proliferation, was directly repressed by Olig2. However, p21 protein level was not decreased by Olig1/2. The Olig genes regulated p21 expression by altering transcriptional activity on the p21 promoter site.

In the present thesis research, I demonstrated that manipulation of microenvironment or activating intrinsic transcriptional machinery can promote endogenous oligodendrogenesis following SCI. The results indicated that providing VEGF to the lesioned spinal cord can increase the GPC proliferation and the number of newly born oligodendrocytes. Furthermore, activation of both Olig1 and Olig2 transcription factors in proliferating GPCs may enhance the GPC proliferation and their differentiation into mature OLs. I also found that activation of Olig2 alone may lead to a formation of glioma by a p21-dependent mechanism. These studies suggest that endogenous oligodendrogenesis can be stimulated in the lesioned spinal cord and may be utilized as a therapeutic intervention to improve functional outcome after SCI."