Promoting Oligodendrogenesis and Myelin Repair Using the Multiple Sclerosis Medication Glatiramer Acetate

Promoting oligodendrogenesis and myelin repair using the multiple sclerosis medication glatiramer acetate

Viktor Skihara, Claudia Silvaa, Andrew Chojnackia, Axinia Do¨ringa, William B. Stallcupb, Samuel Weissa, and V. Wee Yonga,1

aHotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada and bThe Burnham Institute for Medical Research, La Jolla, CA 92037

Communicated by Michael Sela, Weizmann Institute of Science, Rehovot, Israel, August 30, 2009 (received for review May 31, 2009) The formation of oligodendrocytes (oligodendrogenesis) and my- are immunoreactive for neurotrophic factors such as BDNF (7). elin is regulated by several neurotrophic factors. Strategies to However, any approach to use leukocytes to deliver neurotro- increase the level of these trophic molecules may facilitate repair phic factors for repair must balance their potential detriments in in demyelinating conditions, such as multiple sclerosis (MS). Be- exacerbating the pathology of MS. cause leukocytes are a source of neurotrophic factors, and as Glatiramer acetate (GA), a medication used in relapsing- glatiramer acetate (GA) generates T helper 2 (Th2) lymphocytes remitting MS (8), generates GA-reactive T lymphocytes that are that are not known to be harmful, we tested the hypothesis that of the T helper 2 (Th2) anti-inflammatory bias (9, 10). GA- GA regulates oligodendrogenesis and myelin formation. First, we reactive Th2 cells accumulate in the CNS, where they have been generated GA-reactive Th2 cells and determined that they pro- shown to produce not only anti-inflammatory cytokines but also duced transcripts for neurotrophic factors, including insulin-like neurotrophic factors (11). Importantly, Th2 cells have not been growth factor-1 (IGF-1). The conditioned medium from GA-reactive found to be neurotoxic, unlike the proinflammatory Th1 or Th17 T cells elevated IGF-1 protein and promoted the formation of lymphocytes that destroy neurons in tissue culture (12). More oligodendrocyte precursor cells (OPCs) from embryonic brain- recently, GA has also been found to modulate monocytoid cells

derived forebrain cells in culture. We next subjected mice to into those that express anti-inflammatory cytokines (13). NEUROSCIENCE lysolecithin-induced demyelination of the spinal cord. At 7 days Here, we have tested the hypothesis that T cells exposed to GA after the insult, the number of OPCs in the demyelinated dorsal elevate neurotrophic factors that are important for oligoden- column was higher than that in uninjured controls, and was further drogenesis in culture. Furthermore, we have used mice with increased by the daily s.c. injection with GA. Increased OPC gen- lysolecithin-induced demyelination to address whether treat- eration by GA was associated temporally with the elevation of ment with GA would increase OPC numbers and remyelination IGF-1 and brain-derived neurotrophic factor (BDNF) in the spinal within the lesioned spinal cord. These studies have relevance to cord. Finally, the resultant remyelination at 28 days was higher in the biology of harnessing the benefits of inflammation to evoke mice treated with GA during the first 7 days of injury compared repair. with vehicle controls. These results indicate that GA promotes oligodendrogenesis and remyelination through mechanisms that Results involve the elevation of growth factors conducive for repair. T Cells Exposed to GA Elevate Their Production of Growth Factors in Vitro. Cells obtained from the lymph nodes of GA-pretreated beneficial inflammation ͉ neurotrophic factors ͉ oligodendrocyte ͉ mice (Fig. S1) were incubated with APCs and GA, resulting in regeneration ͉ remyelination a proliferative response (Fig. S2) that suggested the presence of T cells reactive to GA. Cell conditioned medium from these ultiple sclerosis (MS) is characterized by inflammation GA-reactive cells was then examined for the accumulation of IFN-␥, a Th1 cytokine, and for IL-5, a Th2 cytokine. Increasing Mand demyelination in the CNS. The demyelinated lesions ␥ can be repaired and, indeed, the extent of remyelination is culture periods with GA resulted in loss of detectable IFN- and substantial in some patients (1). There is increasing interest in steady accumulation of IL-5 (Fig. S2), indicating that the GA- facilitating remyelination as its benefits extend beyond restora- reactive T cells were of the Th2 phenotype, as reported by others tion of nerve impulse conduction to preventing axonal degen- (9–11). With this confirmation, we examined the capacity of eration, since axon and myelin units have dynamic interactions GA-reactive T cells to produce growth factors. From several involving survival signaling. mouse donors previously treated with GA, lymph node cells were Much has been learned about the process of remyelination (2) restimulated with GA and RNA was harvested. Fig. 1 shows that and key steps include the proliferation and maturation of increases in transcripts encoding IGF-1 and PDGFaa were oligodendrocyte precursor cells (OPCs) and the appropriate evident after 3 days of GA treatment. Transcripts for BDNF was interactions of oligodendrocyte processes with axons to form variable, with three cultures elevating this trophic factor in myelin. A large number of molecules are critically involved in the response to GA whereas three did not. proliferation, maturation and survival of OPCs, and among these The cell-conditioned medium collected from T cells over 3 are neurotrophic factors such as platelet-derived growth factor days of culture was analyzed for IGF-1 protein content. IGF-1 (PDGF), insulin-like growth factor-1 (IGF-1) and brain-derived

neurotrophic factor (BDNF) (3–6). Accordingly, strategies that Author contributions: V.S. and V.W.Y. designed research; V.S., C.S., A.C., and A.D. performed have been used to induce remyelination in animals have included research; W.B.S. and S.W. contributed new reagents/analytic tools; C.S., A.C., A.D., and V.W.Y. the provision of neurotrophic factors through infusion or gene analyzed data; and V.S., C.S., A.C., A.D., W.B.S., S.W., and V.W.Y. wrote the paper. therapy. The authors declare no conﬂict of interest. Another means to deliver growth factors to the nervous system Freely available online through the PNAS open access option. takes advantage of the observation that leukocytes are rich 1To whom correspondence should be addressed. E-mail: [email protected]. sources of neurotrophic factors. Indeed, even the disease- This article contains supporting information online at www.pnas.org/cgi/content/full/ promoting inflammatory cells of perivascular cuffs in MS lesions 0909607106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909607106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 24, 2021 Fig. 1. GA-reactive T cells in tissue culture produce growth factors. Real-time PCR analyses and GADPH-normalized levels show that compared with non-GA exposed cells (control), treatment of T cells with GA resulted in increase in levels of IGF-1 and PDGFaa. Each circle, square, or triangle within a given growth factor dataset represents a separate culture. *, P Ͻ 0.05; **, P Ͻ 0.01, compared with non-GA exposed controls (t test).

protein was significantly elevated in T-cell cultures treated with GA, either collected at first in vitro restimulation with GA (day 3), or after restimulation a week after (day 10) (Fig. S2).

Conditioned Medium from GA-Reactive T Cells Increases the Number of OPCs in Culture. We evaluated whether growth factors produced Fig. 3. GA treatment increases injury-induced rise of OPC numbers. (A) The by GA-reactive T cells (Fig. 1) were sufficient to regulate the number of PDGR␣-positive OPCs were counted in the dorsal column of 10 formation of OPCs from neural precursor cells. Single cells from sections, each spaced 100 ␮m apart and centered around the lesion epicenter. the anterior entopenduncular area (AEP) of embryonic day 15 The sum per mouse was then plotted in A, where each value is from a separate mouse. There were 7 uninjured mice, 8 lysolecithin-vehicle mice, and 11 mice were exposed to conditioned medium collected from lysolecithin-GA animals. **, P Ͻ 0.01; ***, P Ͻ 0.001 (one way ANOVA with GA-reactive T cells for 72 h. Fig. 2 and Fig. S3 show that there Bonferroni multiple comparison test). (B) Data of OPC numbers (mean Ϯ SEM) were significantly more OPCs and Ki67-positive cells when the in discrete sections around the lesion epicenter are displayed, to indicate that embryonic brain cells were incubated with medium from GA- the increase in OPCs induced by GA is widespread across several sections reactive T cells, compared with medium from control T cells (not containing demyelination. *, P Ͻ 0.05, one way ANOVA with Bonferroni restimulated with GA). These results show that GA-reactive T multiple comparison test. All analyses were blinded. cells secrete factors conducive for oligodendrogenesis. day 7. In the area of demyelination, a dense accumulation of GA Treatment of Mice with Lysolecithin Injury Increases the Number Iba1ϩ microglia/macrophages was readily observed. Moreover, of OPCs in the Lesioned Spinal Cord. We next investigated whether OPCs identified by PDGFR␣ immuno-labeling and as discrete GA could increase OPC numbers in vivo. To better simulate cells could be discerned (Fig. S4). We blindly enumerated conditions in patients, we injected mice daily with s.c. GA. the number of OPCs in the dorsal column across 10 sections Previous studies have shown that significant demyelination (14, of the spinal cord, each spaced 100 ␮m apart, and centered 15) and newly generated OPCs (16) could be found by day 7 after around the epicenter of injury. Fig. 3 shows that in mice lysolecithin administration, so we chose this time point for subjected to lysolecithin injury and treated s.c. with PBS vehicle, analysis. Fig. S4 of MBP immunoreactivity shows that demyeli- the average total number of OPCs in the dorsal column summed nation of the dorsal column of the spinal cord was extensive at across 10 sections of individual mice averaged 257. This represents an increase from uninjured mice (average sum of 141 OPCs across 10 sections). Impressively, daily treatment with s.c. GA for 7 days after lysolecithin injury resulted in a further increase of OPC numbers, where an average of 390 OPCs across 10 sections was found per mouse (Fig. 3). The increase of OPC numbers in the dorsal column induced by GA compared with vehicle occurred not only at the lesion epicenter, but also rostrally or caudally across 300–400 ␮mon either side (Fig. 3) containing a demyelinating lesion. Overall, these results demonstrate that the daily s.c. injection of GA for 7 days after lysolecithin injury results in an elevation of the injury-induced increase of OPCs in the spinal cord and that this increase occurs locally in the dorsal column that contains a demyelinating injury.

Alterations of Cytokine and Growth Factor Levels in Spinal Cords of Fig. 2. Conditioned medium (CM) from GA-reactive T cells promotes the GA-Treated Mice. development of OPCs in vitro. OPCs were identiﬁed by PDGFR␣ labeling We determined whether GA treatment of lysolecithin-injured mice led to alterations of inflammatory whereas cycling cells were tagged by Ki67. Values are mean Ϯ SEM. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001, compared with control non-T-cell-exposed cytokines or growth factors in vivo. Coronal spinal cord blocks medium. a, P Ͻ 0.05; b, P Ͻ 0.01, comparing GA T-cell medium data to control from around the injury site were harvested from mice treated s.c. T-cell CM (one-way ANOVA with Bonferroni multiple comparisons). with GA or vehicle daily for 7 days after lysolecithin injury, and

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909607106 Skihar et al. Downloaded by guest on September 24, 2021 Fig. 4. Inflammatory and growth factor profiles at day 7 after lysolecithin injury. (A) Displays data from real-time PCR for IL-5 and IL-12; the IL-5:IL-12 ratio of individual mice shows that GA biased the cytokine profile within the spinal cord toward one that is less proinflammatory. Each histogram is of 12–15 mice.(B) GA treatment is found to increase IGF-1 and BDNF transcripts. Each histogram represents 9–12 mice. All values are mean Ϯ SEM. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001, compared with uninjured controls, one way ANOVA with Bonferroni multiple comparison test.

RNA was collected and subjected to real-time PCR. Fig. 4 shows (14–16), we found that the extent of eriochrome cyanine staining that lysolecithin injury led to rise of levels of the Th2 cytokine, was greater in the dorsal column at 28 days of vehicle-treated IL-5; and IL-12, a proinflammatory cytokine expressed by APCs mice compared with that at 7 days after lysolecithin injury (Fig. including macrophages and important in the generation of Th1 5). An initial extensive demyelination at 7 days, which resulted cells (IFN-␥ was not detected). Analysis of the ratio of IL-5 to in a large area of the dorsal column being devoid of intact myelin, NEUROSCIENCE IL-12 from individual mice showed that this ratio was highly was progressively repaired by day 28. Impressively, in mice elevated by GA compared with vehicle-treatment. treated with daily s.c. GA for the first 7 days of injury, erio- For growth factors (Fig. 4), we found that IGF-1 transcript chrome cyanine staining at 28 days indicated that an even greater increased after lysolecithin injury in vehicle treated mice com- degree of remyelination had occurred compared with the profile pared with uninjured controls, and this was elevated further after in vehicle-treated mice (Fig. 5). Much of the initially demyeli- injury by GA treatment. BDNF did not rise after injury from nated area of the dorsal column in the GA-treatment group uninjured controls, but GA treatment significantly raised BDNF contained patches of blue profiles (Fig. S5), reminiscent of levels. ‘‘shadow plaques’’ that have been used to describe remyelination in the human CNS. GA Treatment Increases Indices of Remyelination 28 Days After We used the pixel intensity of eriochrome cyanine staining to Lysolecithin Injury. In correspondence with the documented re- document the area of each dorsal funiculus that was devoid of myelination that occurs after lysolecithin-induced demyelination myelin. Such analyses were conducted across 10 sections span-

Fig. 5. Evolution of de- and remyelination and impact of GA. (A) Displays representative eriochrome cyanine staining whereby demyelination is very prominent and comparable in both the vehicle and GA-treated mice at 7 days after lysolecithin injury. Progressive remyelination indicated by increased eriochrome cyanine proﬁles in the dorsal column is evident at day 28 after injury, and this is enhanced further in mice treated with GA. Ten times original magniﬁcation. (B) The area of demyelination (mean Ϯ SEM) within the dorsal column of sections caudal and rostral to the lesion epicenter is displayed; demyelination is calculated based on pixel intensity devoid of eriochrome cyanine staining, and was determined using Image-Pro Plus software. (C) The volume of remaining demyelination encompassing 1 mm of the spinal cord shows a smaller remaining lesion (t test) in GA-treated mice compared with that in the vehicle group.

Skihar et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 24, 2021 ning 1 mm and centered around the lesion epicenter. Fig. 5 Specific growth factors have been infused or genetically demonstrates that the remaining demyelination at day 28 was expressed in animal models of demyelination in attempts to more extensive in vehicle-treated mice compared with that in the promote remyelination. IGF-1 treatment of rats subjected to GA-treated group, and this was evident not only around the EAE increased the numbers of proliferating oligodendrocyte lesion epicenter, but also rostrally and caudally. precursors (21). For BDNF, a single dose in rabbits subjected to Finally, we documented the area of demyelination per section cervical spinal cord ventral root avulsion and replantation and summed these across 10 sections spanning 1 mm per mice promoted remyelination of axons that regenerated from the to obtain an index of the lesion volume. Fig. 5 reveals that the replantation (22). volume of remaining demyelination at day 28 was significantly The provision of a particular growth factor may sometimes less in the GA-treatment group (average of 0.26 Ϯ 0.03 mm3 ϫ result in proliferation of cells but not their subsequent matu- 10Ϫ3) (mean Ϯ SEM) compared with vehicle controls (average ration. This is highlighted by Woodruff et al. (23) where the of 0.55 Ϯ 0.10 mm3 ϫ 10Ϫ3)(P Ͻ 0.05, Mann–Whitney unpaired transgenic increase of PDGFaa led to a rise in numbers of T test). This was not the result of differing lesion volumes at day OPCs after lysolecithin demyelination; however, there was a 7 after the lysolecithin insult, which was found to be similar in trend toward lower remyelination in the PDGF transgenics, both groups (average of 0.98 Ϯ 0.21 mm3 ϫ 10Ϫ3 vs. 0.72 Ϯ 0.20, which the authors speculated to be due to the prolonged vehicle vs. GA, mean Ϯ SEM, n of 7 each, P Ͼ 0.05). Comparing mitogenic stimulus. across time points, the lesion volume at day 28 in the vehicle These results indicate that although single growth factor group was 56% of that at day 7 whereas that in the GA group at treatment may be adequate to affect repair, this may be subop- day 28 was 36% of that at day 7. timal or counterproductive in some cases. The concomitant Overall, studies of indices of remyelination suggest that GA provision of multiple growth factors, to include those with treatment in the first 7 days after lysolecithin administration proliferative and/or differentiating capacity, would appear to be favors a more pronounced repair over the ensuing 28 days. required to enhance repair.

Discussion Beneficial Aspects of Neuroinflammation. One means to deliver Summary of Results. We have determined that in culture, GA- multiple growth factors into the CNS takes advantage of the reactive T cells produced growth factors described for oligoden- observation that leukocytes have the capacity to generate a drogenesis, and that the conditioned medium from GA-reactive range of growth factors (7). For example, activated T cells, B T cells increased the number of OPCs that were formed from cells, and macrophages express nerve growth factor and BDNF embryonic brain cells. After demyelination of the spinal cord in in vitro, and inflammatory infiltrates of T cells, NK cells, and mice, the daily treatment for 7 days with s.c. GA elevated the macrophages in the CNS of animals afflicted with experimental injury-induced rise of OPCs correspondent with an mRNA autoimmune encephalomyelitis (EAE) were immunoreactive for profile in the injured tissue that was anti-inflammatory and neurotrophin-3 and BDNF (24). These reports highlight that proreparative. Although the size of the demyelination at day 7 there are beneficial aspects to neuroinflammation, which should was comparable between the two groups, suggesting that GA did not be unexpected since a main role for an inflammatory not modulate grossly the initial demyelinating events, determi- response in other tissues is to enhance repair. In accordance, nations at day 28 show increased remyelination when mice were Schwartz and colleagues (25) have noted that even autoreactive exposed to GA during the first 7 days of injury. T cells could prevent neuronal death in neurotrauma. There is increasing evidence that inflammation aids remy- Growth Factors Regulate the Generation of OPCs in Culture, in elination. The depletion of macrophages (26) or T cells (27) Development, and in Remyelination. Recovery after demyelination impairs remyelination after lysolecithin-induced demyelina- requires the presence of OPCs and the ability of these cells to tion. Conversely, the promotion of an inflammatory response divide, migrate, mature, and enwrap axons in response to signals by creating a stab injury to the spinal cord (28) or by injecting in demyelinated lesions. The generation and maturation of OPCs zymosan along with OPC transplants into the retina (29) are dependent on several growth factors. In tissue culture increased remyelination. studies, the proliferation of OPCs is stimulated by PDGF, among The benefits of inflammatory cells are attributed in part to others. BDNF increases the proliferation of OPCs and their their production of neurotrophic factors described above but maturation into oligodendrocytes (3, 4). IGF-1 has strong ca- other aspects of an inflammatory response may also help. For pacity to facilitate both OPC proliferation and maturation (5), instance, proteases released by inflammatory cells may help and is a survival factor for oligodendrocytes. remove nonpermissive molecules, such as proteoglycans that Developmental studies have also highlighted the importance hinder recovery (14). The challenge of harnessing beneficial of growth factors. Transgenic mice overexpressing PGDF pre- neuroinflammation is how to avoid its potential detriments, sented with hyperproliferation of precursor cells whereas PDGF given the extensive literature that persistent neuroinflammation null mice had severely reduced number of OPCs (6). IGF-1 can harm the CNS. transgenic mice have larger brain and higher myelin content than controls (17). Conversely, IGF-1 null mice have lower number of Harnessing the Benefits of Inflammation with GA. GA has been in OPCs at 1–3 weeks of age, and mice with deficient IGF receptor clinical use to treat MS for over 15 years where it has been shown 1 signaling have retarded developmental myelination (18). to be safe, to reduce relapse rate and to produce longterm Growth factors are also critical during remyelination. The stabilization of disease in a significant proportion of patients (8). increase of several growth factors within the demyelinated CNS The mechanisms of GA in MS are multiple, and include the has been reported (19). IGF-1 transgenic mice remyelinated generation of Th2 cells and Type 2 monocytes (9, 13). These cells more readily compared with wildtype after demyelination of the are not known to harm the CNS and may elicit CNS outcomes corpus callosum; moreover, type 1 IGF receptor null mice did differently from other inflammatory cell types. When Th2 cells not remyelinate adequately after insult, corresponding with the were contrasted with proinflammatory Th1 cells, it was found failure of OPCs to accumulate, proliferate or survive in mutants that Th2 cells interacted with glia to elevate neurotrophins; in (20). It must be noted that although we have focused discussions contrast, the interaction of Th1 cells with glia did not affect to PDGF, BDNF, and IGF-1, a myriad of molecules has been neurotrophins but enhanced the levels of proinflammatory described to influence myelin formation, including the chemo- molecules (30). In our study, we were unable to reproducibly kine GRO-␣, hormones, notch signaling and LINGO. detect T cells at sites of demyelination by immunohistochemistry

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909607106 Skihar et al. Downloaded by guest on September 24, 2021 or flow cytometry (data not shown), even though the PCR data presence of 10 ␮g/mL GA. Three days after, total RNA was extracted from cells (Fig. 4) suggested a Th2:Th1 bias in GA-treated mice. Future with 1 mL of TRIzol solution (Life Technologies BRL). The mRNA expression of studies will need to examine representation of Th2 versus Th1 T IGF-1, BDNF, PDGFaa, and the housekeeping gene, 18s rRNA, was analyzed by cells, or Type 2 monocytes, in detail in the demyelinated spinal real-time PCR. All PCR primer sets were purchased from SA Biosciences (IGF-1, cord after GA-treatment. PPM03387A; BDNF, PPM03006A; and PDGFaa, PPM03103A). For 18s rRNA the In accordance with potentially useful outcomes of GA for following forward (F) and reverse (R) primers were used, F; GTAACCCGTT- the CNS, GA has been found to confer neuroprotection in GAACCCCATT, R; CCATCCAATCGGTAGTAGCG. animal models of optic nerve injury, EAE, ALS, Parkinson’s, Value of growth factor expression from each PCR was normalized to 18s rRNA value from the same RNA extract. By comparing the ratio of normalized and Alzheimer’s disease (31). GA has also been studied in the growth factor value for a given T-cell culture exposed to GA versus a sister context of neural repair. Immunoglobulins harvested from culture that is not, the relative fold change in transcript was thus obtained for GA-primed mice and injected into mice with viral-induced that culture. demyelination of the spinal cord promoted remyelination (32); To verify that protein for growth factors was also elevated in GA-reactive whether similar outcome could result from daily s.c. injections T cells, conditioned media collected for cytokine analyses described earlier to mimic the mode of treatment of MS patients with GA was were subjected to the measurement of a selected growth factor, IGF-1, using not addressed. Treatment of EAE mice using GA has been ELISA (Biosource). Furthermore, the cell conditioned medium was analyzed reported to increase the extent of neurogenesis (33) and the for their capacity to promote OPC in culture as described in SI Text. number of OPCs (34) in the CNS. Overall, these studies support the repair potential of GA that is suggested by our Lysolecithin-Induced Demyelination in the Spinal Cord of Mice and Treatment study. with GA. C57BL/6 adult 2–3-month-old male mice were anesthetized i.p. with a mixture of ketamine (85 mg/kg) and xylazine (15 mg/kg). A 1.5 ␮L solution Conclusion of 1% D-lysophosphatidylcholine (lysolecithin; Sigma) was injected over 1.5 We have determined that the MS medication, GA, promotes min into the dorsal column of the T3 spinal cord segment using a 32 G needle, oligodendrogenesis and indices of remyelination in mice sub- attached to a 5 ␮L Hamilton syringe (Hamilton). The needle was left in the jected to lysolecithin-induced demyelination. This demonstra- spinal cord for an additional 2 min to prevent backﬂow of the lysolecithin. tion of the myelination capacity of GA injections in a nonin- Muscles, connective tissue and skin were then sutured above the injection site. flammatory animal model, suggests the direct effect on myelin Animals received daily injections of 2 mg per mouse of GA dissolved in formation is not solely due to the reduction of a harmful saline s.c. for 7 days starting from the day of lysolecithin administration. This dose was used by Aharoni et al. in mice to increase neurogenesis (33) or inflammatory milieu. The results herein indicate that it is document OPC numbers (34) in EAE. Control animals were treated with daily possible to employ GA to harness the benefits of neuroin- s.c. injections of vehicle (PBS) after the lysolecithin insult. NEUROSCIENCE flammation for repair. The data also suggest that the long term Mice were killed for the evaluation of OPC numbers and cytokine/growth benefits observed in patients on GA treatment (8) may in part factor content in the spinal cord at day 7 after demyelination, and for the be due to remyelination, and this deserves further evaluation. evaluation of myelin reformation at day 28, as described in detail in SI Text.

Materials and Methods ACKNOWLEDGMENTS. This work was supported by grants from Teva Phar- Assessment of Neurotrophic Factors Produced by GA-reactive T Cells in Vitro. maceutical Industries, Israel, the Multiple Sclerosis Society of Canada, and Responder and APC populations (see SI Text) freshly harvested from mice were NeuroScience Canada. Dr. Skihar was supported by a fellowship from the cocultured (3 million each, in 3 mL medium) in 6-well plates in the absence or Multiple Sclerosis Society of Canada.

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