Spontaneous remyelination after contusive SCI is very limited in the adult spinal cord. Many lines of recent studies have demonstrated that such limitation is attributable to, at least in part, restricted differentiation of endogenous GPCs in vivo. In this thesis, I describe strategies to overcome such restriction by cell extrinsic and intrinsic molecules.
Part A. Ex vivo VEGF delivery following contusion spinal cord injury
The present experiment adopted an ex vivo approach for stable and robust grafts. These findings indicate that the ex vivo approach using immortalized NSCs ensured a stable and effective increase of the ambient concentration of VEGF in the injured spinal cord, which would be highly demanding or very costly to achieve by direct infusion of VEGF. As gene delivery vehicles, NSCs exhibit inherent long-distance migratory capabilities and a remarkable capacity to integrate with host neural tissue (Jandial et al., 2008). Especially, immortalized human NSCs have shown exceptional capability to find pathological regions (Muller et al., 2006). The majority of F3.VEGF NSCs in this study were also found around
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the lesion cavities, even though they were injected at 2 mm rostral and caudal to the epicenter. Thus, it is highly likely that F3.VEGF grafts functioned as localized and sustained cellular sources providing VEGF directly to the lesion site.
The major finding of this study was that F3.VEGF grafts markedly increased the number of BrdU+ proliferating cells. Approximately 40% of all the proliferating cells were NG2+ cells in all the groups. This percentage is comparable to the data of the previous report that almost half of the acutely dividing cells were NG2 immunoreactive (Zai and Wrathall, 2005). Other proliferatingcells after SCI are thought to encompass macrophages / microglial cells, Schwann cells, mature glial cells, ependymal cells, fibroblasts, and enthothelial cells (Namiki and Tator, 1999; McTigue et al., 2001; Yang et al., 2006). It is likely that the ex vivo delivery of VEGF promoted proliferation of these potentially proliferating cells to a similar extent. Indeed, the mitogenic role of VEGF has been demonstrated for very different kinds neural cells. For examples, application of VEGF increased the number of neuronal cells in developing retina (Yourey et al., 2000), and VEGF promoted proliferation of astrocytes (Rosenstein et al., 1998), Schwann cells (Sondell et al., 1999), and neural stem or progenitor cells (Jin et al., 2002). In contrast to my present results, a previous study reported that a single injection of VEGF immediately after injury did not increase the number of BrdU+
cells (Widenfalk et al., 2003). The discrepancy might be explained by more effective and stable expression of VEGF by the ex vivo approach taken in this study.
Transplantation of F3.VEGF cells markedly increased the extent of proliferation of NG2+ glial progenitor cells. NG2 and PDGFα are coexpressed in O2A glial progenitor cells
(Nishiyama et al., 1996), and there is evidence that NG2+ cells differentiate into mature oligodendrocytes in vivo (Nishiyama et al., 1999). NG2 positive cells increase the extent of
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proliferation after SCI (McTigue et al., 2001), and proliferating NG2 positive cells are thought to differentiate into mature oligodendrocytes replacing the ones that are lost secondary to injury (Zai and Wrathall, 2005). I found that ex vivo delivery of VEGF increased the number of early proliferating cells that differentiated into mature oligodendrocytes at 6 weeks after SCI. These results suggest that VEGF expanded a pool of proliferating NG2 positive cells after SCI that eventually differentiated into mature oligodendrocytes. Notably, the number of newly born astrocytes at 6 weeks after SCI was not affected by F3.VEGF grafts. NG2+ cells could generate at least a portion of GFAP+
astrocytes composing the glial scar. my data suggest that the increase in proliferating NG2+
cells by F3.VEGF grafts did not contribute to the generation of new astrocytes in the glail scar (Alonso, 2005). It is possible that proliferating NG2+ cells which are destined to take the astrocytic fate did not respond to VEGF. Alternatively, the effects of VEGF on genesis of new astrocytes might be antagonized by transplanted stem or progenitor cells as cellular vehicles that were shown to suppress activation of astrocytic scars (Li et al., 2005; Davies et al., 2006). I did not find evidence that new neurons were generated by any of the experimental intervention. The molecular environment of the spinal cord is predominantly gliogenic, but not conducive for the generation of new neurons (Horner et al., 2000;
Shihabuddin et al., 2000). I assumed that VEGF provided by F3.VEGF grafts could not overcome the limitation imposed by the molecular niches in the spinal cord.
Increases in the number of proliferating NG2+ glial progenitor cells and newly born oligodendrocytes can have important implications in the recovery of neuroglical function after SCI. Demyelinating lesions in the white matter are at least partially responsible for functional deficits after SCI (Waxman, 1992; Cao et al., 2005). It has been demonstrated that
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newly generated oligodendrocytes are capable of remyelinating axons (Yang et al., 2006).
Therefore, it is conceivable that the expanded pool of proliferating glial progenitor cells by F3.VEGF could induce remyelination and lead to functional recovery. I showed that F3.VEGF grafts markedly enhanced tissue sparing. NG2 glial progenitor cells stimulated by VEGF might differentiate into myelinating oligodendrocytes and enhance remyelination, eventually leading to the increase in the volume of myelinated white matter. It is also possible that neuroprotective effects and/or angiogenic activity of VEGF played a more major role in enhanced tissue sparing. A single injection of VEGF immediately after SCI increased the density of blood vessels and decreased apoptosis of neural cells (Widenfalk et al., 2003). Moreover, VEGF also prevented retraction and promoted regeneration of the corticospinal axons after spinal cord transaction (Facchiano et al., 2002). Therefore, enhanced tissue sparing and the resulting improvement of locomotor function by F3.VEGF could be ascribed to a combination of the multifaceted trophic effects of VEGF on glial progenitor cells, endothelial cells, neural cells, and injured axons.
In summary, I showed a successful delivery of VEGF to the injured spinal cord tissue by genetically modified human NSCs as cellular vehicles. VEGF overexpressing NSC graft exerted a previously unreported effect of VEGF on glial progenitor cells following SCI:
transplantation of F3.VEGF increased the proliferation of NG2+ glial progenitor cells and the number of newly born oligodendrocytes. Therefore, VEGF can be therapeutically utilized to repair white matter pathology in SCI. VEGF also improved angiogenesis and tissue sparing, indicating multifaceted actions for repair of injured spinal cord. The strategy of human NSC-based VEGF delivery may have potential to be clinically translated for the victims of SCI.
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Part B. Regulation of GPCs by Olig genes following contusion spinal cord injury
My results provide the first direct evidence that olig1 and olig2 plays an important role in the oligodendrogenesis from OPCs to myelinating oligodendrocytes after SCI. I used eGFP-expressing retroviruses to genetically manipulate proliferative cells in the injuried spinal cord. In this study, I injected olig1 or/and olig2 expressing eGFP+ retrovirus into the contused spinal cord. I describes that differential and cooperative actions of olig1 and olig2 on proliferating glial progenitor cells after SCI.
This thesis showed that overexpression of olig2 can give rise to a oligodendroglioma of proliferating cells that closely resemble immature oligodendrocyte progenitor cells after SCI. Molecular and cytogenetic analysis has shown that human glioma often contain multiple genetic lesions that underlie glioma in humans include activating mutations in the EGFR and PI-3 kinase pathways and loss-of-function mutations in tumor suppressors such as p53 and p16/p19 (Barber et al., 2004; Samuels et al., 2004; Takei et al., 2008; Kawamura et al., 2009). These findings have led to the assumption that glioma formation requires the accumulation of multiple genetic lesions. However, studies using molecular and cytogenetic analysis of human tumors are correlative in nature, and it is not known how many genetic lesions are actually required to glioma formation. Moreover, these mutations are not unique to tumors of the CNS. Lineage-restricted pathways that regulate CNS tumor behavior may represent more specific therapeutic targets with little potential to affect off-target cell types. One very important implication to my study is that the
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histological hallmarks of glioma can be driven by overexpression of a single gene. By 7 dpi, 100% of the olig2-driven tumors have shown features of oligodendroglioma, including marked NG2+ OPCs proliferation, massive growth, and CD133+ cells (Fig. 8-10).
I propose that olig2-expressing retroviruses drive the formation of glioma so quickly because they not only transform the infected cells but they also transform the environment by SCI. In both normal and malignant neural stem cells, olig2 functions as a direct repressor of cell cycle inhibitor gene, p21. Furthermore, Olig2 regulated lineage-restricted pathway that is critical for proliferation of normal and tumorigenic CNS stem cells (Ligon et al., 2007).
Transcription regulation orchestrates oligodendrogenesis during CNS development (Miller, 2002; Ross et al., 2003). Olig1 and olig2 play important roles in generating oligodendrocytes during embryogenesis (Zhou et al., 2000; Lu et al., 2001). Olig2 is required for oligodendrocyte lineage specification during CNS development, whereas olig1 contributes more to oligodendrocyte differentiation and maturation (Lu et al., 2001;
Takebayashi et al., 2002). Their coexpression during SCI in the adult was unknown. This study showed that overexpression of Olig1 or Olig2 alone fails to promote oligodendrocyte differentiation compared to GFP alone group. However, Olig1/2 is enhanced proliferation and differentiation of proliferating OPCs following contusive SCI. These data indicate that olig1 is required for the differentiation and maturation of adult OPCs following contusive SCI. Whether Olig2 plays an important role in the proliferation and differentiation of oligodendrocyte following SCI is not known, since olig2 overexpression induce glioma formation. I found that olig1/2 increased the percentage of early proliferating NG2+ or PDGFRα+ OPCs that differentiated into CC1+ mature oligodendrocytes at 1 week after
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injured. These results suggest that Olig1/2 expanded a pool of proliferating NG2+ cells after SCI that eventually differentiated into mature oligodendrocytes (Fig. 12). Although I cannot formally rule out the possibility that Olig1/2 might regulate oligodendrocyte differentiation through an unidentified intercellular signaling molecules. I propose that Olig1/2 has a direct role in the control of expression of myelin structure genes (MBP and PLP) and oligodendrocyte differentiation. It is plausible that Olig1/2 bHLH transcription factor may directly bind to the promoters of structural myelin genes and subsequently regulate their expression (Fig. 13).
To determine potential mechanisms for the effects of olig genes on the GPC differentiation. I examined the expression of transcription factors involved in oligodendrocyte lineage differentiation in vivo and in vitro. I first looked at the expression of homeodomain transcription factor Nkx2.2. Nkx2.2 plays an important role during the entire course of oligodendrocyte differentiation (Nicolay et al., 2007). Sox9 and Sox10, two highly related group E Sox proteins, are both implicated in oligodendrocyte development, but at different stages. Sox9 exerts a strong influence on the initial specification of OLs (Stolt et al., 2003). In contrast, Sox10 plays a key role in promoting terminal OL differentiation (Stolt et al., 2002; Li et al., 2007). I also examined whether the expression of HLH transcription factor Id2, which functions to inhibit OL differentiation (Wang et al., 2001; Samanta and Kessler, 2004), could be affected by introduction of olig genes. I observed that the progressive increased in Olig1/2 group was accompanied by corresponding changes in protein expression patterns of specific transcription factor, Sox10 but not changed expression of myelin inhibitory transcription factor, Id2. Olig1/2 in vivo and in vitro causes ectopic expression of Sox10. Sox10 contains a DNA binding domain of the high mobility group
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(HMG) and is predominantly expressed in mature oligodendrocytes (Stolt et al., 2002). It is also known to directly bind to and activate the myelin basic protein promoter, thus suggesting a role in differentiation of oligodendrocytes.
Increase in the number of oligodendrocytes can have important implications in the recovery of neurological function after SCI. Demyelination lesions in the white matter are at least partially responsible for functional deficits after SCI (Waxman, 1992; Cao et al., 2005).
Olig1/2 improved quality of hind limb locomotion as assessed by BBB score and footprint analysis using CatWalk task at 6 weeks after SCI. They exhibited better coordination between hind- and forelimbs. The smaller distance between the two hindlimb (interlimbs distance) in olig1/2 group suggests an improved balance during locomotion but hindlimb angles did not completely improve by olig1/2 (Fig. 17). However, Olig1/2 have been shown to increase the number of CC1+ mature oligodendrocytes (Fig. 18). These results suggests that the expanded pool of mature oligodendrocytes by olig1/2 could attribute to behavioral improvement.
In summary, I showed that introduction of olig2 gene into proliferating GPCs induced dramatic hyperplasia resulting in glioma formation. In contrast, Olig1 did not affect proliferation of GPCs, but increased the proportion of mature oligodendrocytes, indicating that olig1 promotes differentiation. Introduction of olig1/2 gene into proliferating GPCs following SCI increased the number of proliferating cells without tumor formation, promoted differentiation of GPCs into mature oligodendrocytes even further than olig1 alone, were increase of specific transcription factor, Sox10, and improved quality of hind limb locomotor behavior. Therefore, Olig1 and Olig2 may differentially regulate GPCs following SCI. Olig2 may preferentially promote proliferation even leading to a tumor formation.
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Olig1 may be primarily involved in differentiation of GPCs and/or initial specification of oligodendrocytic lineage cells. Olig1 may counteract propensity of timorous transformation by olig2. Olig2 may synergistically potentiate the effects of olig1 on differentiation and/or specification of oligodendrocytic lineage cells. Balanced activation of both Olig1 and Olig2 may be highly beneficial for SCI by both enhancing oligodendrocyte differentiation (and/or specification) and increasing the proliferation of GPCs. Modulation of olig gene functions could be utilized as a novel target for spinal cord repair.
Part C. Mechanisms in the regulation of the glioma formation by OLIG genes
The results in the third part of thesis provided the detailed mechanism of tumor regulation by olig1 and olig2 genes. During the course of the experiments to test whether overexpression of Olig2 promotes endogenous oligodendrogenesis, I unexpectedly observed tumors that consist of NG2 positive glial progenitor cells (Fig. 8, 9). To verify the tumorous transformation of GPCs transduced with Olig2, Olig2 retrovirus was treated to primary GPC culture, and the dramatic increase of colonies in soft agar formation assay was found. Only the animals (nude mice) with transplanted cells transduced with Olig2 developed tumors in the brain. So the above pieces of evidence support my argument that overexpression of olig2 gene may be sufficient to induce tumorous transformation of GPCs. Olig2 gene might be a therapeutic target for the diseases with oligodendrocyte and myelin pathology (Maire et al., 2009). Several studies tried to manipulation olig2 gene to direct neural stem and/or progenitors (Copray et al., 2006; Hwang et al., 2009; Maire et al., 2009). The tumorigentic
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potential of Olig2, which was discovered in my thesis study, should be taken in consideration when designing a therapeutic strategy target olig2 gene.
Previous literatures have noticed the association between Olig2 and glioma.
Although the cellular origin for glioma remains obscure (Sanai et al., 2005). Recent studies have highlighted similarities with multipotent neural stem cell and progenitor cells found in the developing and adult mammalian CNS (Jackson et al., 2006). Highly tumorigenic subpopulations of cells in gliomas have been identified that express the hematopoietic stem cell marker CD133. In analogy to neural progenitor cells of the adult CNS (Sanai et al., 2005), glioma might be viewed as populations of developmentally blocked stem cell marked by CD133, eGFP+ proliferating cells marked by Ki67, and noncycling progeny cells make up the bulk of the tumor mass. Olig2 transcription factor is expressed in 100% of adult gliomas, irrespective of grade (Ligon et al., 2004). In adult glioma, Olig2 expression marks essentially 100% of the CD133-positive putative glioma stem cell. Olig2 is also required for tumor formation in the same p16/p19 double null murine model of adult gliomas (Bruggeman et al., 2007). A recent study showed that Olig2-mediated repression of tumor suppressor p21 is critical in the development of glioma (Ligon et al., 2007). Therefore, I also examined the expression level of p21 in GPCs transduced with Olig2, and found a significant reduction. In contrast, Olig2 transduction did not change the level of p53, an upstream factor regulating p21, indicating that p21 is a direct target of Olig2.
I also found that coexpression of Olig1 completely block the Olig2-induce tumorous transformation of GPCs. Olig1 coexpression also prevented Olig2-mediated reduction of p21 level. Since the level of p21 is regulated primarily at the transcriptional level (Gartel and Radhakrishnan, 2005), and p21 promoter contains canonical E box
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binding elements that allow binding of bHLH transcription factors, I hypothesized that olig genes regulates p21 transcriptional level through p21 promoter region. Indeed, the luciferase experiment showed that co-transfection of olig1 cDNA restored transcriptional activity of p21 promotor region in a dose dependent manner. These findings suggest a possibility that Olig1 and Olig2 may bind to the same promoter sequence, and they may compete with each other for binding. Further studies employing EMSA or chip assay would address this question. I found that Olig1 alone did not produce any change in p21 expression level, indicating that Olig1 may function to inhibit transcriptional repression of Olig2 in a dominant negative fashion. Therefore, a balance between Olig1 and Olig2 transcriptional factors may be important to keep GPCs from uncontrolled tumor formation. A recent study also reported the imbalance expression of Olig1 and Olig2 in glioma (Ligon et al., 2007).
The results from the current thesis experiments may provide an insight into a mechanism of glioma formation.
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V. CONCLUSION
The current thesis study provides strong evidence that enhancing remyelination by endogenous OPCs could be an important component for SCI repair strategies to improve functional outcomes. A limited number of OPCs, the presence of inhibitory factors for OL differentiation, and/or the lack of growth factors for OL survival and myelination in the injured spinal cord may all contribute to this failure of endogenous remyelination. Therefore, strategies to promote significant functional recovery after SCI by remyelination from endogenous OPCs may be proven feasible once mechanisms regulating their proliferation, differentiation, and maturation are better understood. The current study proved that the ex vivo delivery of VEGF and introduction of olig genes into proliferating OPCs are effective approaches to enhance remyelination and locomotion functional recovery after SCI. Further studies may find more effective extracellular factors that could regulate proliferation and differentiation of endogenous oligodendrogenesis after injury. In addition, combination of different extracellular factors at different time points after SCI may result in a more optimal regulation of post-injury oligodendrogenesis. In a similar vein, complex transcriptional networks to finely adjust differentiation of OLs are being uncovered and recent studies also showed novel intrinsic mechanism of OL differentiation such as micro RNA and epigenetic control (Liu et al., 2009; Dugas et al., 2010; Liu and Casaccia, 2010; Zhao et al., 2010).
Therefore, there will be more therapeutic targets to manipulate intrinsic OL differentiation mechanisms. As the current thesis revealed, possibility of tumor formation should be taken into consideration.
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The modification of intrinsic properties of GPCs, incorporating therapeutic genes relevant for specific disease conditions, modulation of disease-specific microenvironment, and appropriate combination of these approaches will potentiate therapeutic efficacy of cell-based therapy for the diseases in the spinal cord. The diversity of mechanisms that might promote recovery from SCI could increase the options for developing novel therapies, but it also makes it more important to identify the mechanisms that are activated by any one treatment. A better understanding of molecular events characterizing each transition could provide an important framework for future therapeutic studies aimed at repair of demyelination after SIC.
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