Комбинированная терапия резко поощряет воспоминание.

Introduction

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS) that has an as-yet unclear etiology and is currently incurable ( Plemel et al., 2017 ). MS is typically characterized by demyelination and axonal degeneration, which contribute to progressive neurological disability ( Thompson et al., 2018 ). The pathogenesis of MS involves an aberrant autoimmune response and a progressive failure of remyelination, both of which are complex processes ( Healy et al., 2022 ). Major characteristics of MS include acute exacerbations of multifocal inflammation mediated by the activation of B and T lymphocytes that act against CNS antigens. Concurrently, there is a progressive failure of remyelination due to an intrinsic deficiency in myelin production by oligodendrocyte precursor cells (OPCs) ( Faissner et al., 2019 ; Healy et al., 2022 ; Ma et al., 2024 ). Among the immune cells implicated in MS pathogenesis, B cells play a crucial role in activating cytokine responses and proinflammatory T cells ( Weber et al., 2010 ; Sabatino Jr et al., 2019 ). Current therapeutic strategies primarily focus on immunomodulation, such as depleting B cells with anti-CD20 monoclonal antibodies (αCD20) ( Hauser et al., 2008 ; Li et al., 2018 ). These treatments effectively suppress acute inflammatory activity, particularly during the early stages of the disease, and slow disease progression ( Zhang et al., 2024 ). However, they fail to address the chronic progression of MS and the associated disability due to their inability to directly promote OPC differentiation and remyelination ( Yi et al., 2023 ). Recent studies have highlighted the potential of small molecules to promote myelin regeneration ( Mei et al., 2014 ; Najm et al., 2015 ; Hubler et al., 2018 ; Niu et al., 2021 ). Although promising, these compounds might not be able to sufficiently modulate the immune environments, limiting their effectiveness. Therefore, an integrative approach capable of modulating the immune response while simultaneously enhancing remyelination is highly desired.

Combination therapies (CTs), which involve the use of two or more agents with synergistic or additive effects, have been extensively employed to achieve precise and effective treatments ( Crunkhorn, 2016 ; Brown et al., 2021 ; Kirchhammer et al., 2022 ; Mugarza et al., 2022 ). In the context of autoimmune diseases, CTs often incorporate immunosuppressants, biologics, and small-molecule inhibitors to better regulate inflammation, reduce disease activity, and minimize organ damage, ultimately enhancing patient outcomes. Additionally, CTs can lower the risk of drug toxicity by reducing doses for each agent while maintaining therapeutic efficacy, highlighting the potential of CTs as a powerful and versatile strategy for addressing complex conditions, such as MS.

Herein, we investigated a CT for MS that integrates an immunomodulator αCD20 with a small-molecule inhibitor Clemastine (CLM, an inhibitor of muscarinic acetylcholine receptor 1) ( Mei et al., 2014 ) to concurrently modulate immune responses and promote OPC differentiation and in situ remyelination. Compared to monotherapies with either αCD20 or CLM alone, the CT dramatically activated signaling pathways and genes associated with myelin development and markedly promoted OPC differentiation and remyelination. Consequently, CT treatment led to substantial improvements in locomotion in the experimental autoimmune encephalomyelitis (EAE) model, indicating functional recovery. Furthermore, a reduced dose of αCD20 or a shorter duration of CLM treatment achieved a comparable reversal of disease symptoms. These findings underscore the potential of CTs to address the multifaceted pathobiology of MS and lay the foundation for the development of novel therapeutic approaches.

Results

CT efficiently depletes B cells and suppresses T cell activation

To investigate the CT for MS, EAE mice were treated with saline (Control), αCD20 (one dose), CLM (daily injections for 10 consecutive days), or a combination of αCD20 and CLM (named Combo hereafter), and comprehensive assessments were performed to evaluate the efficacy of these treatments in immune modulation and remyelination ( Figure 1 A). One of the primary causes of MS is an aberrant autoimmune response mediated by B and T lymphocytes. To evaluate the effects of CT on modulating immune responses, peripheral blood cells were analyzed using flow cytometry. As a result, a significant decrease in B cell populations was observed immediately after treatment in both the αCD20 and Combo groups, but not the CLM and Control groups, supporting an efficient depletion of B cells by αCD20 ( Figure 1 B and D; Supplementary Figures S1 B, S2 B, and S4 B). Concomitantly, a reduction in CD4 + and CD8 + T cells was observed in the αCD20 and Combo groups, indicating suppressed T cell activation and polarization ( Figure 1 C, E, and F; Supplementary Figures S1 C, S2 C, and S4 C). By performing quantitative real-time PCR (qRT-PCR), we analyzed the expression of the master transcription factors (TFs) ( Figure 1 G) and inflammatory cytokines ( Figure 1 H) associated with Th1, Th2, Th17, and Treg cells, respectively, in the lumbar spinal cord, the major segment of MS pathogenesis in EAE mice. Remarkably, both the Combo and αCD20 groups exhibited substantial reductions in the expression of Tbx21 and Rorc , the typical TFs for Th1 and Th17, respectively, supporting suppression of T cells ( Figure 1 G). Typically, the proinflammatory responses in EAE mice peak at
16–20 days post-induction (dpi) and gradually decline by 30 dpi in the Control group ( Figure 1 I–K; Supplementary Figures S1 D, S2 D, and S4 D). We observed lower expression of corresponding proinflammatory cytokines, including IFN -γ, TNF-α , and IL-17 , in the Combo and αCD20 groups at 25 dpi, demonstrating a pronounced alleviation of T cell-mediated inflammation ( Figure 1 H–K). Furthermore, both Combo and αCD20 significantly reduced Th1 and Th17 proinflammatory cytokines over time ( Figure 1 I–K). Of note, although a slight decrease in the expression of TFs and proinflammatory cytokines was detected in the Combo group compared to the αCD20 group, it was not statistically significant ( Figure 1 I–K; Supplementary Figure S3 B and C). Similarly, the other proinflammatory factors, such as Cxcl1, Cxcl10, Ccl20, IL-1β, IL-6, Mmp3 , and Mmp9 , were downregulated slightly or comparable in the Combo group compared to the αCD20 group ( Figure 1 L). In addition to the lumber spinal cord, the suppressed Th1 and Th17 activity was also observed in B cell-depleted groups in the spleen, with a slight decrease in the Combo group compared to the αCD20 group ( Figure 1 M and N). In contrast, the CLM group showed limited impact on modulating inflammatory responses ( Figure 1 H–N). These findings demonstrate that CT can efficiently deplete B cells, suppress T cell activation, and repress proinflammatory responses.

CT efficiently depletes B cells and suppresses T cell activation. ( A ) Diagram describes the strategy for CT. CLM was administered daily at 15–24 dpi, and αCD20 was administered at 15 dpi. ( B – F ) Flow cytometry analysis of CD3 + T/B220 + B cells ( B ) and CD4 + T/CD8 + T cells ( C ) from peripheral blood cells. The percentages of B220 + B cells ( D ), CD4 + T cells ( E ), and CD8 + T cells ( F ) were quantified. n  = 4 mice per group. ( G and H ) qRT-PCR analysis of the expression of TFs ( Tbx21, Gata3, Rorc , and Foxp3 ) and cytokines ( IFN-γ, IL-4, IL-17 , and IL-10 ) in the lumbar spinal cord, normalized to the Control group. n  = 4 independent biological experiments. ( I – K ) The mRNA expression of IFN-γ ( I ),
TNF-α ( J ), and IL-17 ( K ) in the lumbar spinal cord at the indicated time points. n  = 4 independent biological experiments. ( L – N ) qRT-PCR analysis of the expression of proinflammatory factors ( Cxcl1, Cxcl10, Ccl20, IL-1β, IL-6, Mmp3 , and Mmp9 ) in the lumbar spinal cord ( L ), TFs ( Tbx21, Gata3, Rorc , and Foxp3 ) in the spleen ( M ), and cytokines ( IFN-γ, IL-4, IL-17 , and IL-10 ) in the spleen ( N ), normalized to the Control group. n  = 4 independent biological experiments. Data are presented as mean ± SEM. P -values were calculated using one-way ANOVA test. * P  < 0.05, ** P  < 0.01, and *** P  < 0.001 compared with the Control group; # P  < 0.05, ## P  < 0.01, and ### P  < 0.001 compared with the Combo group ( D – K , M , N ). * P  < 0.05, ** P  < 0.01, and *** P  < 0.001 ( L ).

CT facilitates OPC repopulation and remyelination

Robust remyelination is mediated by OPC repopulation and subsequent differentiation and maturation to form myelin ( Yi et al., 2023 ). To assess whether CT could facilitate OPC repopulation, we analyzed OPC density (Sox10 + ) within lesions (Iba1 + ) in EAE mice ( Figure 2 A and D; Supplementary Figures S1 E, S2 E, and S4 E). OPCs usually repopulate in the lesion area beginning at 16 dpi and gradually decrease alongside remyelination from ∼20 dpi to 30 dpi, as observed in the Control group ( Figure 2 A and D; Supplementary Figures S1 E, S2 E, and S4 E). During the early stage
(16–20 dpi), the CLM and Combo groups showed more Sox10 + OPCs in the lesion among the four groups ( Figure 2 D; Supplementary Figures S1 E and S2 E). In the late stage (20–30 dpi), the density of OPCs gradually decreased, particularly in the Combo group ( Figure 2 A and D; Supplementary Figures S2 E and S4 E). Next, to evaluate OPC differentiation, we assessed the area of the lesion (Iba1 + area), as well as the extent of myelin formation (mean fluorescence intensity of myelin basic protein, MFI of Mbp) in the focal lesion, in the lumbar spinal cord over time ( Figure 2 B, E, and F; Supplementary Figures S1 F, S2 F, and S4 F). In the Control group, the lesion area increased from 16 dpi to 25 dpi, with partial recovery by ∼25–30 dpi. In the αCD20 group, the lesion area decreased by ∼20 dpi and, subsequently, the myelin formed in the lesion and increased over time. The CLM group showed notably reduced lesion area compared to the Control group, particularly during 25–30 dpi. Of note, compared to the αCD20 group, the CLM group exhibited a slight delay in myelin formation (evidenced by Iba1 + area and MFI of Mbp) at the early stage (16–20 dpi) and an even smaller lesion area but robust expression of Mbp by 25 dpi. The Combo group exhibited a remarkable reduction in lesion area as early as 20 dpi, which showed the smallest lesion area and the most dramatic formation of myelin in the lesion among all groups at 25 dpi. These observations were further confirmed by myelin gold staining, showing a more significantly reduced lesion area and robust formation of myelin in the Combo group at 25 dpi ( Figure 2 C, G, and H; Supplementary Figures S1 G, S2 G, and S4 G). Furthermore, we analyzed the expression of myelin-related genes and found that as remyelination progressed, the expression of OPC-related ( Olig2, Pdgfrα , and Sox10 ) and oligodendrocyte-related ( Mbp, Mag , and Mog ) genes was dramatically increased in the treatment groups ( Supplementary Figures S1 H, S2 H, S3 D, and S4 H). In brief, in the αCD20 group, these genes showed the increased expression from 25 dpi to 30 dpi, whereas in the CLM and Combo groups, an earlier upregulation was observed from 20 dpi to 30 dpi. Notably, the Combo group demonstrated the highest expression of these myelin-related genes among all groups at 25 dpi ( Supplementary Figures S1 H, S2 H, S3 D, and S4 H). These findings demonstrate that CT can activate myelin genes and dramatically promote OPC repopulation and remyelination.

CT greatly facilitates OPC repopulation and remyelination. ( A and B ) Immunofluorescence analysis of OPCs (Sox10 + ) and myelin (Mbp + ) in the lumbar spinal cord lesions (Iba1 + ). ( C ) Myelin gold staining shows myelin in the spinal cord. Scale bar, 100 μm (left, original image for each group) or 50 μm (right, zoomed-in image for the boxed area). ( D – H ) The density of OPCs ( D ), Iba1 + area ( E ), MFI of Mbp ( F ), demyelination area ( G ), and myelin abundance ( H ) in white matter (WM) were quantified. n  = 4 mice per group. Data are presented as mean ± SEM. P -values were calculated using one-way ANOVA test. * P  < 0.05, ** P  < 0.01, and *** P  < 0.001.

CT significantly promotes functional recovery in EAE mice

Remyelination contributes to the recovery of neuronal function. To evaluate the effects of CT, disease severity was monitored in EAE mice by systematically assessing Basso, Beatlie, Bresnahan (BBB) locomotor rating scale scores. As shown in  Figure 3 A, progressive paralysis in EAE mice began at 10–15 dpi, with disease progression peaking at 15–30 dpi, in the Control group. Treating mice with αCD20 resulted in rapid hindlimb motor function recovery; however, this effect diminished in the late phase (∼25–30 dpi). In contrast, the CLM group demonstrated notable motor recovery by 25–30 dpi, a time window correlated with its dynamics in promoting OPC repopulation and remyelination. Notably, the Combo group recovered earlier and faster while maintaining a more sustained therapeutic effect than the other groups. To understand the dynamics of recovery, we analyzed the distribution of BBB scores for each group ( Figure 3 B). Briefly, at 20 dpi, the αCD20 and Combo groups outperformed the CLM group (75.0%, 75.0%, and 66.0% of mice scoring in the 2.0–3.0 range and 25.0%, 0.0%, and 34.0% in the 0.0–2.0 range for the αCD20, CLM, and Combo groups, respectively). With the progress of recovery, the CLM group gradually exhibited a comparable or even superior effect over the αCD20 group. At 25 dpi, 26.0%, 20.0%, and 75% of mice in the CLM, αCD20, and Combo groups, respectively, showed BBB scores in the 0.0–2.0 range. Intriguingly, at 30 dpi, the CLM and Combo groups exhibited better recovery than the αCD20 group. Particularly, the majority of mice (82.0%) in the Combo group showed BBB scores within the 0.0–2.0 range. These findings underscore the superior efficacy of CT in reducing disease severity and promoting functional recovery in EAE mice. Furthermore, we analyzed the locomotion of mice by rotating rod tests with both fixed ( Figure 3 C and D) and accelerating ( Figure 3 E and F) speeds, respectively. As a result, in both tests, the αCD20 and CLM groups outperformed the Control group, whereas the Combo group showed the most pronounced improvement at 25 dpi ( Figure 3 C–F). Collectively, these results demonstrate that CT can significantly promote the functional recovery of EAE mice.

CT significantly promotes functional recovery in EAE mice. ( A ) BBB scores for the indicated groups over time. ( B ) The distribution of BBB scores at 20, 25, and 30 dpi, respectively. ( C – F ) Rotating rod tests with fixed speed ( C and D ) and accelerating speed ( E and F ) were performed at the indicated time points. n  = 4 mice per group. Data are presented as mean ± SEM. P -values were calculated using one-way ANOVA test. * P  < 0.05, ** P  < 0.01, and *** P  < 0.001.

CT exhibits synergistic effects in promoting remyelination

The superior recovery observed in the Combo group than monotherapies suggests synergistic or additive effects of the immunomodulator and CLM on remyelination. To investigate these effects, we analyzed the expression of key genes of the pathways directing OPC differentiation and myelin development. Intriguingly, the CLM and Combo groups showed significantly higher expression of Shh, Fgf17, and Egf at 25 dpi ( Figure 4 A). Shh is a morphogen that plays a vital role in myelin development ( Fuccillo et al., 2006 ). Fgf17 has been reported to induce OPC proliferation and differentiation ( Iram et al., 2022 ). EGF possesses the ability to promote the survival and self-renewal of OPCs and helps them differentiate into oligodendrocytes ( Fortin et al., 2005 ). The activation of these genes strongly supports the ways through which CLM promotes OPC differentiation and remyelination. Meanwhile, although no significant changes in the expression of these genes were observed in the αCD20 group, immunomodulation may indirectly facilitate myelin formation. As a support, the activation of the myelin genes in the Combo group gradually increased and peaked at 25 dpi ( Figure 4 A). Overall, the Combo group remarkably activated these genes to much more extent than the CLM group across the main time window for remyelination, indicating synergistic effects executed by αCD20 in Combo ( Figure 4 B–D). Together, these results suggest that the immunomodulators in Combo may synergistically support small molecules to induce OPC differentiation and remyelination.

CT activates myelin-related genes. ( A ) qRT-PCR analysis of the expression of Shh, Egf , and Fgf17 in the lumbar spinal cord, normalized to the Control group. n  = 4 independent biological experiments. ( B – D ) The mRNA expression of Shh ( B ), Egf ( C ), and Fgf17 ( D ) in the lumbar spinal cord at the indicated time points. n  = 4 independent biological experiments. Data are presented as mean ± SEM. P -values were calculated using one-way ANOVA test. * P  < 0.05, ** P  < 0.01, and *** P  < 0.001.

CT provides an effective therapeutic strategy for MS

Frequent injections in clinical therapy would lead to infusion- or injection-related reactions and are not cost-effective. Reducing the dosage or shortening the treatment duration may help minimize risks and adverse effects while maximizing therapeutic benefits, thus achieving a more targeted and efficient therapy. To optimize CT, we tested whether a half dose of αCD20 (from 10 mg to 5 mg per kg body weight) or a half course of CLM (from 10 days to 5 days) would also reverse the disease symptoms ( Figure 5 A and B). As a result, even with a half dose of αCD20, this optimized CT condition considerably suppressed T cell activation, evidenced by the reduced expression of Tbx21 and Rorc , the key TFs for Th1 and Th17, respectively ( Figure 5 C and D). In addition, the half-dose CT inhibited proinflammatory factors and reduced inflammation to a similar level as those in the Combo and αCD20 groups, supporting a satisfactory efficacy ( Figure 5 E–G). On the other hand, the half-course CT exhibited comparable effects as the Combo group, but much more pronounced than the CLM group, in suppressing proinflammatory responses, suggesting a limited role of CLM in immunomodulation ( Figure 5 C–G).

CT provides an effective therapeutic strategy for MS. ( A ) Diagram describes the strategy for optimized CT. ( B ) The table shows detailed information of individual treatments. ( C – G ) qRT-PCR analysis of the expression of Tbx21, Rorc, IFN-γ, TNF-α , and IL-17 in the lumbar spinal cord, normalized to the Control group. n  = 4 independent biological experiments. ( H – J ) Immunofluorescence analysis of myelin (Mbp + ) in spinal cord lesions (Iba1 + ) at 30 dpi. The Iba1 + area ( I ) and MFI of Mbp ( J ) were quantified. ( K – M ) Myelin gold staining shows myelin in the spinal cord. The demyelination area ( L ) and myelin abundance ( M ) were quantified. Scale bar, 100 μm. n  = 4 mice per group. ( N and O ) Rotating rod tests with fixed speed ( N ) and accelerating speed ( O ) were performed at the indicated time points. Data are presented as mean ± SEM. P -values were calculated using two-tailed unpaired Student's t -test * P  < 0.05, ** P  < 0.01, and *** P  < 0.001 compared with the Half dose group; # P  < 0.05, ## P  < 0.01, and ### P  < 0.001 compared with the Half course group.

Next, we assessed their efficiency in remyelination. In brief, both optimized CTs (half-dose and half-course) significantly reduced the demyelination area (Iba1 + area) and resulted in a robust myelin formation, as evidenced by Mbp staining ( Figure 5 H–J) and myelin gold staining ( Figure 5 K–M). Intriguingly, although the optimized CT regimens did not surpass the original CT (Combo), they still outperformed the corresponding monotherapies, suggesting that they may be sufficient for immunomodulation and myelin repair and thus retain significant therapeutic efficacy. Accordingly, the behavioral assessment by rotating rod tests with fixed speed and accelerating speed revealed superior motor coordination and endurance by optimized CTs compared to monotherapies ( Figure 5 N and O), further demonstrating their effectiveness in promoting functional recovery. In summary, our findings suggest that a CT regimen that simultaneously targets immunomodulation and OPC differentiation represents an effective therapeutic strategy for MS ( Figure 6 ).

Working model for CT strategy. CT possesses superior efficacy than monotherapies in promoting remyelination and functional recovery of EAE mice by modulating the immune responses (extrinsic factors) and activating OPC differentiation (intrinsic factors) simultaneously.

Discussion

In this study, we established and optimized the CT strategy for treating demyelinating diseases. By combining the use of an immunomodulator (αCD20) and a small-molecule inhibitor (CLM), the CT not only refines the inflammatory cytokine milieu but also activates the pathways for myelin development and induces the expression of myelin genes, thus significantly promoting remyelination and recovering neuronal function. Therefore, our study provides strong evidence that a CT strategy achieves a more rapid, effective, and sustained recovery over monotherapies in EAE mice, offering a promising way for addressing complex pathobiological conditions of MS.

Current therapeutic strategies for MS primarily focus on immunomodulation. B cell depletion, achieved mainly through targeting CD20, changed the therapeutic landscape of MS. However, despite that a few monoclonal antibodies are clinically available and only one was regularly approved for primary progressive MS, safety issues, such as the development of hypogammaglobulinemia, are the major concerns. Thus, the exploration of alternative dose schedules might be effective in mitigating these risks. CT has been widely used for treating complex pathobiological conditions, including MS. The current CTs available for MS are predominantly immune-targeted. The drawbacks of these strategies include that more profound suppression of the immune system may result in possible detrimental effects on protective inflammation or natural repairing brain mechanisms. Ideal combinations could include agents that have distinct and complementary modes of action, which may show additive or synergistic effects. In our study, we used αCD20, an immunomodulator, and CLM, a small-molecule chemical inducing OPC differentiation, simultaneously, each of which targets distinct mechanisms. Intriguingly, αCD20 depleted B cells and suppressed proinflammatory responses immediately, which may establish an immune environment that is favorable for subsequent OPC repopulation and differentiation and remyelination. As a support, the Combo group exhibited more profound improvement in remyelination than using αCD20 or CLM alone, further suggesting synergistic effects. This multifaceted approach is particularly advantageous in MS, a condition characterized by immune dysregulation and demyelination. Furthermore, the dual action of CT enables a reduced need for prolonged and/or high-dose medications, thereby potentially minimizing adverse side effects. The rapid and sustained recovery achieved with CT underscores its potential as a transformative therapeutic strategy for MS.

To elucidate the underlying mechanisms of CT, we explored the function of immunomodulators and small-molecule drugs in demyelination and remyelination. While the function of αCD20 is mainly to suppress proinflammatory responses, CLM is more potent in triggering OPC differentiation, probably through activating the myelin development-related signaling. Additionally, we refined the CT protocols with optimal doses and treatment durations. Further efforts are required to focus on expanding the repertoire of immunomodulators and small-molecule drugs for CT. By integrating diverse immunomodulators with a broad spectrum of small molecules—each with distinct and potentially complementary mechanisms of action—it could be possible to create more targeted and personalized therapeutic strategies for MS.

Materials and methods

Mouse experiments

Mouse experiments were performed in accordance with protocols approved by the Department of Laboratory Animal Science, Shanghai Jiao Tong University School of Medicine. Mice were housed in the specific pathogen-free facility and were allowed free feeding.

EAE model

To generate EAE model, female C57BL/6 mice (8 weeks old) were subcutaneously immunized with 100 μl myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide (ChinaPeptides, 163913-87-9) in a completed Freund's adjuvant (Sigma, F5506) emulsion with Mycobacterium tuberculosis H37Ra (BD, 231141). One hour after immunization, mice were intraperitoneally injected with 100 ng of pertussis toxin (List Labs, 181) in 100 μl 1× phosphate buffered saline (PBS). A second dose of pertussis toxin was administrated on the next day. EAE model mice were monitored daily for clinical scores. Once the mice reached the disease peak (around Day 15), they were randomly divided into groups and were intraperitoneally injected with CLM (10 mg/kg, TargetMol, T19966 ) and/or αCD20 (10 mg/kg, Biocell, BP0356) in the treatment groups or solvents (saline) in the Control group.

EAE was scored as follows: 0, no abnormality; 1, mild limp tail weakness (floppy tail); 2, tail paralysis; 3, tail paralysis and hind leg paresis; 4, hind leg paralysis or mild forelimb weakness; and 5, quadriplegia or moribund state.

qRT-PCR

Total RNA from animal tissues or cells was isolated using the TRNzol Kit (TIANGEN, DP424) and was reverse-transcribed into cDNA with the Reverse Transcription Kit (Vazyme, R223) according to the manufacturer's instruction. The cDNA was amplified by qRT-PCR using SYBR green reagent (Vazyme, Q411) with a real-time PCR instrument (Bio-Rad) and normalized to the internal control Gapdh . The primer sequences are listed in Supplementary Table S1 .

Flow cytometry

Cells (2 × 10 6 ) were incubated with 5 ml acidic buffer (50 mM glycine–HCl, 5 mM KCl, and 130 mM NaCl, pH 3.0) for 4 min at room temperature and subsequently washed and used for flow cytometry. The flow cytometry protocol used to
phenotype mononuclear cells involved staining of dead cells using violet viability stain (AF700, BD, 564997) for 25 min at room temperature. Subsequently, cells were incubated with antibodies against CD3 (BioLegend, 100274), B220 (BioLegend, 103251), CD45 (BD, 553079), CD11b (BD, 612800), CD4 (BD, 561830) or CD8 (BD, 552877) for 30 min at 4°C. CD3 + or B220 + cells and CD4 + or CD8 + cells were visualized by incubation with streptavidin-PerCP (BD Biosciences) for 30 min at 4°C. Finally, cells were fixed in 1% Cytofix. Flow cytometric analysis was performed on a FACS LSRII using FACSDiva software (BD Biosciences).

Immunostaining

Anesthetized mice were perfused through the left cardiac ventricle with ice-cold 0.9% saline followed by 4% paraformaldehyde (PFA), and the tissues were removed. The spinal cord was dissected, fixed with 4% PFA overnight, therewith dehydrated in 30% sucrose in saline overnight at 4°C. After that, the tissues were embedded in O.C.T. (SAKURA, 4583) and frozen at −80°C. Frozen sections were cut at a thickness of 20 μm. Cryo-sections were washed with 1× PBS and permeabilized with 0.2% Triton X-100 for 10 min, then blocked with 5% normal donkey serum containing 0.1% Triton X-100 for 1 h, and overlaid with diluted primary antibodies (1:200, listed in Supplementary Table S2 ) at 4°C overnight. The next day, the sections were washed and incubated in Alexa Fluor-conjugated secondary antibodies with
4′,6-diamidino-2-phenylindole (DAPI) for 2 h at room temperature. The coverslips were washed three times in PBS and then mounted with a Mounting medium. Immunofluorescence images were visualized with a laser scanning confocal microscope (Leica TCS SP8).

Myelin gold staining

TrueGold Myelin Stain Kit (Oasisbiofarm, BK-AC001) was used for myelin staining. Briefly, frozen sections derived from the spinal cords were washed with 1× PBS and distilled water three times. Then, slides were dried for 30 min at 37°C, incubated with the staining reagent for 30 min at 45°C, rinsed in distilled water, and incubated with terminating reagent for 30 min at 45°C. After rinsing slices, the sections were mounted with a Mounting medium.

Rotating rod tests

Before the experiment, several training sessions on the rotarod were required for mice to acclimatize to the device and to ensure their comfort during the test. Upon initiating the experiment, the mouse was gently placed on the rotarod, and the rod rotated at a slow speed, allowing the animal to gradually adapt to the movement. The mouse must maintain its balance on the rod to prevent falling off. For the fixed-speed experiment, the rotarod was set to a constant speed of 20 rounds per minute (rpm/min). In the accelerating experiment, the rotarod started at 4 rpm/min and accelerated to a rate of 40 rpm/min. The speed at which the rodent fell off the rod and the total time were recorded.

Statistics

Statistical analyses were carried out using GraphPad Prism 8 (GraphPad Software). Values are presented as mean ± standard error of the mean (SEM) and quantified from at least three independent biological replicates unless otherwise stated. No statistical methods were used to predetermine the sample size. For two-group comparisons, a two-tailed unpaired Student's t -test was used to determine statistical significance. For comparisons of multiple groups, a one-way analysis of variance (ANOVA) with a post-hoc Tukey's test was conducted. The specific statistical approach is described in each figure legend. No marking was made for not significant.

Supplementary Material