Olig1/2 overexpression improve functional outcome

III. RESULTS

5. Olig1/2 overexpression improve functional outcome

Locomotor recovery was evaluated using BBB score. The hind-limbs of all the animals in the two groups (GFP group vs Olig/1/2 group) were completely paralyzed one day after contusion injury, and locomotor function gradually improved thereafter. At 3 weeks after injury, the mean score of Olig1/2 injected rats were significantly higher than GFP injected rats, and remained higher thereafter (Fig. 17A). Repeated measures two-way ANOVA revealed a significant treatment effect on locomotor recovery (p<0.01), and one-way ANOVA at each time point showed significant differences between Olig1/2 and GFP group at 3,4,5, and 6 weeks after SCI.

As a result of the injury paradigm used in this study, I predicted that differences between Olig1/2 and GFP groups would be limited to the fine details of locomotion. The most accurate test to assess the fine details of locomotion after SCI is CatWalk gait analysis (Hamers et al., 2001). The CatWalk measurements were taken while the animal walked on a flat runway. All animals (GFP group n=9, Olig1/2 group n=8) were trained on the behavioral test, and baseline measurements were obtained before injury. At 4 weeks after injury, fore-paw interlimb distance was not significantly affected by injury (Fig. 17B). Hindlimb interlimb distance, in contrast, significantly increased following contusion injury by about 40% in GFP injected rats (Fig. 17C). Olig1/2 injected rats almost completely normalized the interlimb distance. The GFP injected rats with contusion injury also exhibited shorter stride length in both limbs than intact operated rats (Fig. 17D). Olig1/2 injected rats tended to restore normal stride length, although the difference was not statistically significant. The sign of hindlimb angle is determined by whether the limb stands inwards or outwards relative to

horizontal line. For the right limb if the limb is placed outward, the value is positive, if inward, the value is negative; for the left paw it is the other way around. In intact rats, the angle of the LF limb was negative and that of the RF limb was positive, with an absolute value ranging from 10° to 20° in the forelimbs (data not shown). In contrast, the absolute values of the hindlimbs were near zero (Fig. 17E), suggesting that the intact rats usually walked with outward bilateral fore-limbs and almost straight bilateral hindlimbs. Hindlimb angles were affected differentially by the contusion injury (42.2°±8.1° and 17.1°±7.8° for GFP and Olig1/2 groups, respectively). I next determined the absolute number of CC1-positive mature oligodendrocytes at 6 weeks after contused injury (Fig. 18). In Olig1/2 group, the absolute number of CC1 positive cells rose up to 4745385±399613.5, and the compared to GFP group (2950256±688207.9) were statistically significant (p<0.001). These result suggest that Olig1/2 overexpression into the contused spinal cord enhances recovery of open field locomotion, improves quality of the hindlimb movement during locomotion, and promotes differentiation from GPCs to mature oligodendrocyte.

Fig. 17. Olig1/2 improves locomotor recovery. (A) Comparison of BBB locomotor scale.

Hind limb locomotor function was scored as from 0 to 21. Olig1/2 group showed significantly inproved locomotor behavior uo to 6 weeks after SCI. ** p<0.01, *** p<0.001

compared to sham by repeated measures two-way ANOVA followed by Tukey's post hoc analysis. (B-E) Gait parameters were assessed at 4 weeks after SCI using CatWalk task.

Interlimb distance (ILD) or base of support of (B) Fore limb and (C) Hind limb. (D) Stride langth (SL) between fore limb and hind limb. (E) Note that the angle of Hind limbs placement, as determined by the angle between the second toe and the horizontal line. * p<0.05, ** p<0.01, *** p<0.001 by one-way ANOVA followed by Tukey’s post hoc analysis. White, orange, and violet bars represent sham (n=17), GFP (n=9), and Olig1/2 (n=8) groups, respectively.

Fig. 18. olig1/2 increased mature oligodendrocyte. (A-D) Confocal images of mature oligodendrocyte marker CC1+/DAPI+ double stained cells were sections from GFP (A, B) and Oig1/2 (C, DF) groups. Scale bars; 100 um (A, C), 50 um (B, D). (E) The number of CC1+ cells of spinal cord tissue was stereologically counted and compared between the two groups at 6 weeks after SCI. *** p<0.001 by t-test analysis. Orange and violet bars represent GFP (n=9) and Olig1/2 (n=8) groups, respectively.

Part C. Mechanisms in the regulation of the glioma formation by OLIG genes

1. Olig genes regulate tumorous transformation of GPC in vitro and in vivo.

In the previous experiment, injection of olig2-retrovirus after contusive SCI led to a glioma formation. To verify that Olig2 induces tumorous transformation in GPCs, primary GPC culture was established and treated with olig2-retrovirus. Relevant parameter for tumor formation was cell clonogenic efficiency, which measures the ability of sparsely seeded cells (1x10⁴ cells/6-cm dish) to form colonies. A2B5 positive GPCs were purified with the immunopanning method and then infected with GFP-only, olig1-GFP, olig2-GFP, and olig1/2-GFP retroviruses at 1x10⁶ cfu dosage. The numbers of colonies formed in soft agar were counted at 14 days after retroviruses infection. In Olig2 infected GPCs, colony formation was dramatically increased to about 27 fold compared with GFP-only (7.7±3.5 and 207±42.6 for GFP-only and Olig2 groups, respectively; n=3 for each group).

Morphological examination of the colonies revealed that the Olig2 infected GPCs became rounded, tightly packed, and grew to a much higher density than other group colonies. The colony forming activity was not affected by Olig1 retrovirus infection (15.3±5.5). Treatment of retrovirus coexpressing Olig1 and Olig2 (Olig1/2) resulted in a much smaller number of colonies (45.3±12.1) than Olig2 alone (p<0.001; Fig. 19). Olig1/2 infected GPCs formed slightly more colonies in soft agar than GFP-only or Olig1 group, but the difference was not statistically significant. These results indicated that Olig2 overexpression in proliferating GPCs induces the tumorous transformation, but the coexpression of Olig1 could counteract the Olig2-induced tumorous transformation of GPCs.

To evaluate the tumorous transformation of Olig infected GPCs in vivo, GFP-only, Olig1, Olig2, or Olig1/2 infected GPCs (number of transplanted cells; 3 × 10⁵) were transplanted into the right striatum of nude mice. At 4 weeks after the stereotaxic transplantation of Olig infected GPCs (3x10), animals were sacrificed and stained with cresyl violet. Two out of 4 animals with transplantation of Olig2 transduced GPCs demonstrated hyperplasia and focal necrosis, histologic features typical of malignant glioma (3 of 7) (Fig. 20C). In contrast, none of the nude mice transplanted with GFP-only or Olig1 transduced GPCs developed tumors (0 of 4 for each group) (Fig. 20). Furthermore, coexpression of Olig1 with Olig2 did not result in tumor in any of 4 animals, indicating that Olig1 prevented Olig2-induced tumor formation. These in vivo results confirmed that overexpression of Olig2 induces tumorous transformation in GPCs and coepxression of Olig1 counteract the Olig2-induced transformation.

Fig. 19. The clonogenic efficiency of olig genes-infected GPCs. (A-D) Representative images of Soft agar colony formation assay with olig genes infected GPCs. GPCs (1x10⁴ cells/6-cm dish) were infected 3days after seeding with GFP-only retrovirus (A), olig1 retrovirus (B), olig2 retrovirus (C), and olig1/2 retrovirus (D) (1x10⁶ cfu). 14 days after infection, the cells were stained Cresyl Violet for macroscopic observation of colonies. (E) The Olig2 group was significantly increased the number of colonies but Olig1/2 group prevented number of colonies by Olig2. *** p<0.001 by one-way ANOVA followed by Tukey’s post hoc analysis. Orange, sky blue, dark blue, and violet bars represent GFP, Olig1, Olig2, and Olig1/2 groups, respectively. n=3 for each group.

Fig. 20. Nude mice bearing established GPCs xenograft infected with Olig2 retrovirus.

(A-D) Representative images of retroviruses infected GPCs xenograft into the right striatum of nude mice. 4 weeks after transplantation of retroviruses infected GPCs (3x10⁵), brain sections were stained Cresyl Violet. (C) The hyperchromatic cells formed a tumor-like mass.

High power image is tumor mass. (A) GFP-only. (B) Olig1. (D) Olig1/2. Scale bars; 2mm.

2. Olig genes regulate expression of tumor suppressor p21

Olig genes are important transcription factors for the development of oligodendrocyte lineage cell. As was shown in the previous experiments, however, Olig2 overexpression in proliferating GPCs induces the tumor formation but the coepxression of olig1 and olig2 prevents tumor formation by olig2. Next, I sought to elucidate the molecular mechanisms in the regulation of the tumor formation by olig1 and olig2 gene. I focused my subsequent studies on the relationship between Olig2 and p21 because targeted disruption of p21 has been shown to enhance the proliferation of adult neural progenitor cells (Kippin et al., 2005). Lysates of GFP-only infected GPCs, olig1-infected GPCs, olig2-infected GPCs, and olig1/2-infected GPCs were subjected to immunoblot analysis. GPCs transduced with Olig1 alone did not show changes in p21 level. Intriguingly, protein level of p21 was significantly lower in olig2-infected GPCs. However, when Olig1 was coexpressed with Olig2, p21 level expression was maintained as similar to control values. (Fig. 21A). The protein level of tumor suppressor p53, an upstream regulating factor for p21, did not change markedly among the four groups (Fig. 21B). These results suggest that regulation of tumorous transformation by olig genes may be mediated by tumor suppressor p21.

The activity of p21 is regulated primarily at the transcriptional level (Gartel and Radhakrishnan, 2005). p21 contains canonical E box DNA-binding elements in the upstream promoter/enhancer region of the gene (Ligon et al., 2007), leading to the hypothesis that Olig1 and Olig2 might directly regulate p21 transcriptional activity through such sites (Fig.

21A). Therefore, I tested regulation of p21 transcriptional activity by olig genes using luciferase assay. In HEK293 cell line, ectopic Olig2 expression resulted in a marked

suppression of p21 promotoer driven luciferase acitivty expression. However, cotransfection Olig1 cDNA restored luciferase activity in a dose dependent manner (0.01-1ug) (***

p<0.01;Fig. 22B, C). Collectively, these data showed that Olig2 interacts directly with upstream regulatory elements of p21 and functionally can repress its expression and Olig1 may compete with olig2 to upstream regulatory elements of p21. Olig1 upregulated expression of the tumor suppressor p21 while Olig2 downregulated.

Fig. 21. Olig1 and Olig2 regulated the levels of p21. Whole-cell lysates from cultures of GPCs infected for 3 days with GFP-only, olig1, olig2, and olig1/2 retroviruese were analyzed immunoblot analysis for protein levels of the tumor suppressor p21 (A) and p53 (B). β-actin was used as loading control. A representative experiment from among four is shown. * p<0.05, ** p<0.01 by one-way ANOVA followed by Tukey’s post hoc analysis.

Orange, sky blue, dark blue, and violet bars represent GFP, Olig1, Olig2, and Olig1/2 groups, respectively. n=4 for each group.

Fig. 22. Olig genes regulate transcriptional activity of p21 promoter region. (A) Diagram of p21 promoter region. E-boxes (red) are shown within the region that is bound by Olig1 and Olig2 (red box). (B) p21 luciferase reporter is repressed by Olig2 in HEK293 cell line.

*** p<0.001 by one-way ANOVA followed by Tukey’s post hoc analysis. n=3 for each experiment. (C) Olig1 dose dependent manner increase p21 luciferase reporter activity repressed by Olig2. *** p<0.001 by one-way ANOVA followed by Tukey’s post hoc analysis. n=4 for each experiment.

IV. DISCUSSION

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

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

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

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.

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

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

문서에서 Regulation of Oligodendrogenesis in a Rat Contusive Spinal Cord Injury (페이지 84-0)