Introduction of Olig2-expressing retrovirus after contusive SCI induced

III. RESULTS

1. Introduction of Olig2-expressing retrovirus after contusive SCI induced

To determine the effects of OLIG2 overexpression on proliferating glial progenitor cells after SCI, I injected a total of 6 µl of the Olig2-GFP retrovirus solution into the spinal cord at 3 mm rostral and 3 mm caudal (3 μl each) to the lesion epicenter 30 minutes after contusive SCI. Unexpectedly, histological examination at 7 days after SCI revealed massive proliferation of hyperchromatic cells in all the animals (6 out of 6) injected with Olig2-GFP retrovirus (Olig2 group) (Fig. 8). The hyperchromatic cells frequently packed together and formed a tumor-like mass (Fig. 8B, arrowheads). At the same time, they showed diffuse infiltrative behavior at the margin of the tumor-like mass, often encroaching the gray-white matter junction. In contrast, none of animals (0 out of 6) injected with control retrovirus (GFP only, GFP group) developed such a tumor-like mass composed of hyperchromatic cells (Fig. 8A), and the obliteration of the gray-white matter junction by infiltrating cells was not observed. In order to test if the Olig2-expressing retrovirus-induces hyperplasia specifically in an injury setting, I also injected Olig2-GFP retrovirus in sham operated animals (Fig. 8C).

There was no animal that showed the massive hyperplasia (0 out of 4), indicating that the Olig2-retrovirus induced formation of a tumor-like mass only in the injured spinal cord. In transverse spinal cord sections, the hypercellular regions were filled with GFP positive cells that often clustered together (Fig. 8E). In GFP group, much fewer numbers of GFP positive cells were scattered in the spinal cord parenchyma (Fig. 8D). Most of the GFP cells in Olig2

group expressed a proliferation marker Ki67 (Fig. 8G), indicating that these cell were highly proliferative. The majority of GFP cells in Olig2 group were positive with OPC marker NG2.

In contrast, only about 30% of GFP cells in GFP group were NG2 positive cells (Fig. 8H,I).

The GFP+ cells in Olig2 group were not positive for neural stem cell marker Sox2, astrocytic lineage marker GFAP, or mature oligodendrocyte marker CC1. I evaluated GFP+ cells timorous character by CD133 tumor marker. Olig2 group increased CD133+/GFP+ cells (Fig.

9). These data indicated that Olig2 gene introduction to proliferating cells induced massive hyperplasia of NG2 positive OPCs.

Fig. 8. Olig2 overexpression induced the formation of glioma after SCI. (A-C) Representative images of eriochrome / Cresyl Violet stained sections at the 1.8 mm rostral to the epicenter, epicenter, and 1.8 mm caudal to the epicenter. (B) The hyperchromatic cells formed a tumor-like mass (arrowheads). Scale bars; 1mm. (D-I) Confocal images of GFP+

cells colocalized with proliferating cell marker ki67 (F,G) and OPCs marker NG2 (H,I) from GFP group (D,F,H) and Olig2 group (E,G,I). Scale bars; 100um.

Fig. 9. GFP+ cells in Olig2 group is oligdendrocyte lineage cell. (A-F) Confocal images of GFP+ cells colocalized with astrosytic lineage marker GFAP (A,B), neural stem cell marker Sox2 (C,D), and tumor cell marker CD133 (E,F) from GFP group (A,C,E) and Olig2 group (B,D,F). Scale bars; 100um.

To characterize the hyperplastic GFP cells more thoroughly, retrovirus-injected spinal cords were dissected and dissociated, and subjected FACS to isolate GFP positive cells. As expected, the number of GFP cells isolated from Olig2 group was 3 times higher than that from GFP group (11.7±3.7%, and 3.3±0.8% for GFP, and Olig2 groups, respectively). Consistent with the phenotypic properties in vivo, 83.3±3.0% of GFP cells isolated from Olig2 group were positive for NG2, whereas 33.2±6.1% of GFP cells from GFP group were colocalized with NG2 (Fig. 10A,B). 2.2±1.1% of GFP cells isolated from Olig2 group were positive for GFAP (30.9±3.6% for GFP group) (Fig. 10C,D) or 4.1±1.3%

of GFP cells isolated from Olig2 group were positive for Sox2 (11.5±4.7% for GFP group) (Fig. 10E,F). To determine whether Olig2 overexpressing GFP cells underwent tumorous transformation, soft agar colony forming assay was performed. At 14 days after inoculation inside soft agar, GFP cells isolated from Olig2 group formed numerous colonies numerous colonies were found (Fig. 10G-I). In contrast, only few colonies were observed in the dishes where GFP cells isolated from GFP group were inoculated. Collectively, these results suggest that Olig2 overexpression in proliferating cells after SCI induces the formation of glioma composed of oligodendrocyte lineage cells.

Fig. 10. Olig2 overexpression is lead to glioma formation in vitro. (A-F) Confocal images of GFP+ FACS sorted cells colocalized with OPC marker NG2 (A,B), astrocytic lineage marker GFAP (C,D), and neural stem cell marker (E,F) from GFP+ group (A,C,E) and olig2 group (B,D,F). Scale bars; 200um. (G,H) Soft agar colony formation assay of GFP+ FACS sorted cells from GFP and olig2 group. Cresyl Violet stained colonies. (I) The number of colonies was significantly increased olig2 group. *** p<0.001 by t-test analysis. Orange and dark blue bars represent GFP (n=3) and Olig2 (n=3) groups, respectively.

52 2. Effects of Olig genes on proliferation of GPCs

Olig1 gene was introduced into proliferating cells after SCI using the same method.

The animals with Olig1-GFP retrovirus (Olig1 group) did not show the proliferation of hyperchromatic cells on Cresyl Violet stained tissue sections (Fig. 11A). Given the findings that Olig2 led to glioma formation, I generated retrovirus expressing both Olig1 and Olig2 (Olig1/2-GFP retrovirus) and examined whether simultaneous overexpression of Olig1 and Olig2 genes might result in different outcomes. Overexpression of both Olig1 and Olig2 did not result in tumor-like hyperplasia (Fig. 11B), suggesting that simultaneous Olig1 activation prevented the Olig2-induced glioma formation. However, the number of GFP cells in animals with Olig1/2-GFP retrovirus (Olig1/2 group) was significantly increased compared to GFP or Olig1 group (Fig. 11C-I) along the rostrocaudal extent (p < 0.001 by repeated measures two-way ANOVA). The total number of GFP cells estimated by the stereological method was almost three-fold higher in Olig1/2 group than that in GFP group (GFP, 456615;

Olig1/2, 1212615). The number of GFP cells in Olig1 group (76400) tended to be higher than that in GFP group, but the difference was not statistically significant. To compare proliferative activities, the percentage of GFP positive cells colocalized with a proliferation marker Ki67 was obtained (Fig. 11J-M). The percent Ki67 positive GFP cells were significantly higher in Olig1/2 than GFP and Olig1 groups (p<0.01 and p<0.5, respectively).

Fig. 11. Introduction of olig genes enhanced cellular proliferation. (A,B) Representative images of eriochome / Cresyl Violet stained sections at the 1.8 mm rostral to the epicenter, epicenter, and 1.8 mm caudal to the epicenter from olig1 group (A) and olig1/2 group (B).

Scale bars; 1 mm. (C-H) Confocal images of GFP+ cells were sections from GFP (C,D), olig1 (E,F), and olig1/2 (G,H) groups. Scale bars; 200 um (C,E,G), 100 um (D,F,H). (I) The number of GFP+ cells of spinal cord tissue was stereologically counted and compared between the four groups at 1 week after SCI and injection with retroviruses. ** p<0.01, ***

p<0.001 by one-way ANOVA followed by Tukey’s post hoc analysis. Orange, sky blue, violet, and dark blue bars represent GFP, olig1, olig1/2, and olig2 groups, respectively. n=5 for each group. (J-L) Confocal images of GFP+ cell colocalized with proliferating cell maker ki67 from GFP (J), olig1 (K), and olig1/2 (L) groups. Arrows indicated Ki67+/GFP+ cells.

Scale bars; 100 um. (M) The percentage of Ki67+/GFP+ cells of spinal cord tissue was counted and compared between the three groups at 1 week after SCI. * p<0.05, ** p<0.01 by one-way ANOVA followed by Tukey’s post hoc analysis. Orange, sky blue, and violet bars represent GFP, Olig1, and Olig1/2 groups, respectively. n=3 for each group.

3. Influence of Olig gene overexpression on OL differentiation

I next determined the phenotypic characteristics of GFP positive cells. About 30%

of GFP cells were positive for OPC marker NG2 and PDGFRα in GFP group (Fig. 12A, D, J, K). Although Olig1 overexpression did not significantly increase the percent NG2 positive cells (Fig. 12B). Simultaneous overexpression of Olig1 and Olig2 robustly increase the proportion of NG2 positive GFP cells (50.7±5.1%; p<0.001 vs. GFP and Olig1 group) (Fig.

12C, J). I also analyzed the colocalization with another OPC marker PDGFRα, and obtained very similar results (Fig. 12D-F, K). These results suggested that introduction of both olig1 and olig2 genes into proliferating glial progenitor cells promoted their specification for the oligodendrocytic lineage cell. In GFP group, only 11.4±2.3% of GFP cells differentiated into CC1 positive mature oligodendrocytes by 7 days after SCI (Fig. 12G, L). However, introduction of Olig1 gene significantly increased the percent CC1 positive cells (20.4±3.1%, p<0.05 vs. GFP group) (Fig. 12H, L). In Olig1/2 group, the mean percent CC1 positive cells rose up to 29.8±6.3%, and the differences compared to GFP and Olig1 groups were statistically significant (p<0.001 and p<0.05, respectively) (Fig. 12I, L). The percentage of GFP cells differentiating into GFAP positive astrocytes tended to be reduced by olig genes (22.7±5.1%, 20.6±4.8%, and 16.4±5.3% for GFP, Olig1, and Olig1/2 groups, respectively), but the differences between groups were not statistically significant. I also examined neuronal differentiation by NeuN and Tuj-1 staining, but could not find any GFP cells that expressed either of the neuronal markers (data not shown). In addition, very few GFP cells were positive for microglial marker Iba1 regardless of experimental groups (data not shown).

Fig. 12. Differentiation of oligodendrocyte lineage cells by Olig genes. (A-I) Confocal images of spinal cord tissue sections doubly stained with NG2+/GFP+ (A-C), PDGFRα+/GFP+ (D-F), and CC1+/GFP+ (G-I). Arrows indicated NG2+/GFP+, PDGFRα+/GFP+, and CC1+/GFP+ cells. Scale bars; 100 um. (inset panels; 20um confocal

montage with orthogonal views taken at the center of one cell). The percentage of NG2+/GFP+ (J), PDGFRα+/GFP+ (K), and CC1+/GFP+ (L) cells of spinal cord tissue was stereologically counted and compared between the three groups at 1 week after SCI. * p<0.05, *** p<0.001 by one-way ANOVA followed by Tukey’s post hoc analysis. Orange, sky blue, and violet bars represent GFP , Olig1, and Olig1/2 groups, respectively. n=5 for each group.

Next, I examined whether newly proliferating glial progenitor cells (GFP positive) after SCI could differentiate into myelinating oligodendrocytes. I found evidence that GFP cells expressing myelin basic protein (MBP) were closely associated with neurofilament positive axons, suggesting that the GFP cells enwrapped the axons (Fig. 13A-D). To determine quantitatively the effects of olig genes on the production of myelinating oligodendrocytes, spinal cord tissues from different groups were homogenized and expression of myelination-related proteins such as proteolipid protein (PLP) and MBP was analyzed by western blot (Fig. 13E,F). Introduction Olig1 slightly increase the expression of both PLP and MBP. The expressions of PLP and MBP were markedly enhanced in Olig1/2 group compared to GFP and Olig1 group. These results suggest that Olig1 may tend to promote differentiation of glial progenitor cells into myelinating oligodendrocytes and introduction of both Olig1 and Olig2 may greatly potentiate the production of myelinating cells.

Fig. 13. Olig1/2 enhanced differentiation for oligodendrocyte lineage cells. (A-D) GFP+

cell of Olig1/2 injected group harboring an oligodendrocytic morphology are found both in the grey and white matter, and some GPF+ cells warp around myelinated axon. Arrows indicated GFP+/MBP+/NF+ cells. Scale bar; 100 um. (E, F) The western blot analysis showed that Olig1/2 infected GPCs increased mature oligodendrocyte marker MBP and PLP, and Quantitative protein expression of MBP and PLP by olig genes. Data presented as the proportion of normal protein expression level after normalization with β-actin. * p<0.05, **

p<0.01, *** p<0.001 by one-way ANOVA followed by Tukey’s post hoc analysis. Orange,

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

4. Alteration of transcription factor expression by olig genes

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 primary GPC cultures. A2B5 positive GPCs were purified with the immunopanning method and then infected with GFP only, olig1-GFP, and olig1/2-GFP retroviruses. I verified the overexpression of Olig1 in the GPCs infected by olig1-GFP retrovirus compared to GFP only (Fig. 14A). The overexpression of both Olig1 and Olig2 was also confirmed in the GPCs infected by olig1/2-GFP retrovirus. I initially performed immunoblot analysis on cultured GPCs using antibodie. 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). Expression of Nkx2.2 increased by Olig1 and Olig1/2 but not increased by GFP (Fig.14B). 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). Olig1/2 increased 1.5 fold of Sox9 expression (1.0±0.30, 0.84±0.33, and 1.5±0.22 for GFP, Olig1, and Olig1/2 groups, respectively) and significally increased Sox10 compared to GFP and olig1 group (1.06±0.24, 1.57±0.66, and 3.81±0.86 for GFP, Olig1, and Olig1/2 groups, respectively).Finally, I also examined whether the expression of inhibitory HLH transcription factor Id2, which functions to inhibit OL differentiation (Wang et al., 2001; Samanta and Kessler, 2004), could not affected by introduction of olig genes. I observed that the progressive increase in Olig1 or

Olig1/2 overexpression was accompanied by corresponding changes in protein expression patterns of specific transcription factors. At the Olig1/2 overexpressed OPCs were increase of express Nkx2.2, Sox9, and Sox10 but not increase Id2 compared with only eGFP or olig1-eGFP infected OPCs (Fig. 14). To further examine the in vivo expression of specific transcription factors include Nkx2.2, Sox9, and Sox10 by Olig1/2 overexpression after SCI (Fig. 15). These results suggests that olig1/2 increase expression level of transcription factor Sox10 (Fig 16), transcription factor of oligodendrocyte terminal differentiation, but not changed expression level of myelin inhibitory transcription factor Id2 following SCI.

Fig. 14. Olig1/2 increased expression of oligodendrocyte lineage specific transcription factors in cultured GPCs. (A) Expression of Olig 1 and Olig2 into GPCs induced olig genes. (B-E) western blot analysis of early development stage specific transcription activator, Sox9 (C) and inhibitor, Id2 (E), and late development stage specific transcription activators,

Nkx2.2 (B) and Sox10 (D) of oligodenodrocyte lineage cells. Data presented as the proportion of normal protein expression level after normalization with β-actin. * p<0.05, **

p<0.01, *** p<0.001 by one-way ANOVA followed by Tukey’s post hoc analysis. Orange (n=4), sky blue (n=4), and violet (n=4) bars represent GFP, Olig1, and Olig1/2 groups, respectively.

Fig. 15. Expression of oligodendrocyte lineage transcription factors by Olig1/2 after SCI. (A-C) Confocal image of GFP cells colocalized with Nkx2.2 (A-C), Sox9 (D-F), and Sox10 (G-I) at 1 week after SCI. Arrows indicate GFP+/Nkx2.2 (C), Sox9 (F), and Sox10 (I).

Scale bars; 50 um.

Fig. 16. Expression of oligodendrocyte terminal differentiation transcription factor, Sox10. (A-C) Confocal image of spinal cord sections triple stained with GFP (green), Sox10 (red), and DAPI (blue) cells from GFP (A), Olig1 (B), and Olig1/2 (C) groups. Arrows indicate Sox10+/GFP+/DAPI+ cells. Scale bars; 100um. (D) The percentage of Sox10+/GFP+ cells of spinal cord tissue was stereologically counted and compared between the three groups at 1 week after SCI. * p<0.05, *** p<0.001 by one-way ANOVA followed Tukey’s post hoc analysis. Orange, sky blue, and violet bars represent GFP, Olig1, and Olig1/2 groups, respectively. n=4 for each group.

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

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