Guanabenz protects oligodendrocytes against IFN-γ

We have previously reported that addition of IFN-γ to differentiating oligodendrocyte precursor cells (dOPCs) in vitro causes significant apoptotic cell death that is associated with ER stress14. We therefore sought to determine whether guanabenz could protect oligodendrocytes from IFN-γ-mediated death. We began by treating dOPCs with IFN-γ alone, guanabenz alone or IFN-γ plus guanabenz concomitantly for 48 h, at which point the ratio of live and dead cells in each group was measured. Whereas guanabenz treatment alone had no effect on cell survival, IFN-γ treatment resulted in a 22.5% decrease in dOPC survival compared with control untreated cells (Fig. 1a). Treatment with IFN-γ+2.5 μM guanabenz, however, significantly increased the number of surviving cells, and treatment with IFN-γ+5.0 μM guanabenz restored cell survival to control levels, demonstrating that guanabenz protected cells from IFN-γ-mediated death. TdT-mediated dUTP nick end labeling (TUNEL) staining confirmed that guanabenz protected the dOPCs from apoptotic death (Fig. 1b).

Figure 1: Guanabenz protects differentiated rat oligodendrocyte progenitor cells from IFN-γ-mediated death and prolongs the integrated stress response. (a) Quantification of dOPCs stained with PI/FDA to identify dead/live cells after 48 h of differentiation. Data represent three individual dOPC isolations, each with three replicates per group. (b) IFN-γ-mediated apoptotic death was confirmed via TUNEL staining of dOPCs 48 h after differentiation. N=3. (c) Western blot analysis of dOPCs treated continuously with IFN-γ alone or IFN-γ+5.0 μM guanabenz and probed with p-eIF2α, a marker of ISR activity, and eIF2α. (d) Quantification of extended time points in a western blot. Blot represents one of four individual isolations; graph represents the average of four isolations. Mixed results ANOVA (a), unpaired t-test (b,d), *P<0.05, **P<0.005, ***P<0.0005. Data are presented as mean±s.e.m. Full size image

A recent study28 demonstrated that guanabenz protected HeLa cells from ER stress by inhibiting the dephosphorylation of eIF2α. We therefore examined p-eIF2α levels in dOPCs treated continuously with IFN-γ or IFN-γ+5.0 μM guanabenz over time by immunoblot. Expression of p-eIF2α increased steadily in both treatment groups as predicted, given that the ISR is activated in oligodendrocytes in response to the presence of IFN-γ (ref. 14). At 4 and 12 h, both groups showed similar levels of p-eIF2α expression (Fig. 1c,d). By 20 h, however, p-eIF2α expression had significantly decreased in dOPCs treated only with IFN-γ but remained high in cells treated with IFN-γ+5.0 μM guanabenz (P=0.043, unpaired t-test). This result suggests that guanabenz protected oligodendrocytes from IFN-γ-mediated apoptosis by driving persistence of the ISR. Interestingly, by 28 h after treatment (Fig. 1d), expression of p-eIF2α decreased in both groups and remained steady until 48 h, demonstrating that guanabenz’s enhancement of the ISR is transient in vitro.

Guanabenz protects myelinating cerebellar slices from IFN-γ

Having found that guanabenz is able to protect dOPCs from IFN-γ-mediated death, we next investigated whether the surviving dOPCs were able to successfully myelinate axons. We treated rat cerebellar slice cultures sectioned at postnatal day 11 (P11) for 7 days with IFN-γ alone or IFN-γ and increasing doses of guanabenz. In this experimental time frame, dOPCs are able to mature and begin to myelinate axons, as demonstrated by the finding that myelin basic protein (MBP) immunostaining in normal explants at P20 show distinct and well-organized myelinated fibres (Fig. 2a, arrowheads). When IFN-γ was added to these slice cultures, however, myelin organization was severely disrupted (Fig. 2b). Co-culture with 2.5, 5.0 or 10.0 μM guanabenz resulted in restored myelination to IFN-γ-treated explants (Fig. 2c–e).

Figure 2: Guanabenz decreases IFN-γ-induced hypomyelination in rat cerebellar slice cultures. (a–e) Anti-MBP staining of myelinated fibres (a, arrowheads) in slice cultures that were (a) untreated, (b) treated with IFN-γ or (c–e) concomitantly treated with IFN-γ and 2.5, 5.0 or 10.0 μM guanabenz. Images representative of two or three slices per treatment; the experiment was performed twice. (f) Electron microscopy analysis of slice cultures. Note the significant increase in the number of myelinated axons (arrowheads) when guanabenz and IFN-γ were concomitantly added to slices. Myelinated axons per field were determined by analysis of a minimum of 200 axons per condition. (g) Toluidine blue staining of slices left untreated, treated with IFN-γ alone or treated with IFN-γ and guanabenz. Images represent a minimum of three sections per treatment. Unpaired t-test, *P<0.05. Data are presented as mean±s.e.m. Scale bars, 100 μm (a–e), 2 μm (f) and 20 μm (g). WM: white matter, GL: granule cell layer, PCL: Purkinje cell layer. Full size image

Electron microscopy analysis to examine the extent of myelination in the explants revealed that, compared with sections treated only with IFN-γ, sections treated concomitantly with guanabenz had 57.5% more myelinated axons (P=0.022, unpaired t-test; Fig. 2f, arrowheads). Indeed, toluidine blue staining revealed that the IFN-γ treatment alone appeared to destroy the general cytoarchitecture of the slice tissue (Fig. 2g), whereas addition of 10.0 μM of guanabenz along with IFN-γ protected the tissue such that myelination and the cellular architecture were preserved. Therefore, guanabenz treatment appears not only to reduce IFN-γ-mediated hypomyelination but also to have neuroprotective properties as well.

Guanabenz protects oligodendrocytes from IFN-γ in vivo

We have previously described a transgenic mouse model system in which the ectopic expression of IFN-γ by astrocytes is regulated by tetracycline14. In these mice, the transcriptional control region of the glial fibrillary acidic protein (GFAP) gene drives expression of the tTA protein, which is the transcriptional activator of the tetracycline regulatory element (TRE) that drives IFN-γ expression. Transcriptional activation of the TRE/IFN-γ transgene by tTA is prevented by the presence of doxycycline, a tetracycline derivative. This inducible system allows for regulated expression of IFN-γ specifically in the CNS and independently from the adaptive immune response. Using these transgenic mice, we have shown that CNS-specific expression of IFN-γ results in significant oligodendrocyte loss and hypomyelination in the absence of an adaptive immune response14. Moreover, studies of GFAP/tTA;TRE/IFN-γ transgenic mice revealed that IFN-γ-mediated insults in the CNS became more severe when ISR capabilities were diminished14, and less severe when the ISR was enhanced26. We hence used these mice to investigate whether guanabenz could protect oligodendrocytes from inflammation-induced cell death and prevent myelin loss in vivo.

In wild type littermates, immunostaining at P18 for MBP and aspartoacylase (ASPA), a mature oligodendrocyte marker34,35, revealed distinct myelin tracts and a large population of mature oligodendrocytes in the corpus callosum (Fig. 3a), whereas in vehicle-treated mice with CNS-specific expression of IFN-γ, a 60.6% (P=0.011, unpaired t-test) loss of mature oligodendrocytes and hypomyelination were observed (Fig. 3b). IFN-γ-expressing mice that were treated daily from P7 with 4 mg kg−1 of guanabenz showed a 51.3% (P=0.008, unpaired t-test) increase in the number of mature oligodendrocytes compared with vehicle-treated mice and a restoration of myelination to levels observed in wild type littermates (Fig. 3c,d). These findings demonstrate that guanabenz is able to protect oligodendrocytes and myelin from the detrimental effects of IFN-γ in vivo.

Figure 3: Guanabenz protects oligodendrocytes and myelin from IFN-γ-mediated loss in vivo. (a–c) Immunofluorescent staining for MBP and ASPA, a mature oligodendrocyte marker, in the medial corpus callosum of (a) vehicle-treated wild-type littermates, (b) vehicle-treated GFAP/tTA;TRE-IFN-γ transgenic mice and (c) guanabenz-treated GFAP/tTA;TRE-IFN-γ mice at P18. Mice were treated with vehicle or 4 mg kg−1 of guanabenz daily from P7 to P18. (d) Quantification of ASPA+ cells in the medial corpus callosum of wild type littermates and GFAP/tTA;TRE-IFN-γ mice treated with vehicle or guanabenz. Images represent four to six mice per group; graph represents the average of values from four to six mice per group. Unpaired t-test, **P<0.005, #P<0.05 as compared with vehicle-treated control. Scale bar, 200 μm. Data are presented as mean±s.e.m. Full size image

Guanabenz ameliorates chronic EAE disease symptoms in mice

EAE is a CD4+ T cell-mediated model of MS in which adult mice are immunized with a component of myelin in adjuvant (typically complete Freund’s adjuvant, CFA). The resulting myelin peptide-activated CD4+ T cells infiltrate the CNS, resulting in lesioned areas of inflammation, oligodendrocyte apoptosis, demyelination and axonal degeneration predominantly in the spinal cord. Inflammatory insults in mice with EAE manifest as MS-like clinical symptoms such as ataxia and paralysis36. As we clearly observed the ability of guanabenz to protect developing oligodendrocytes and myelination from IFN-γ-induced loss in vitro and in vivo, we next sought to explore whether guanabenz treatment could protect mature oligodendrocytes and myelin to provide therapeutic benefit to mice with chronic EAE.

We began by determining a dose of guanabenz that could potentially enhance ISR activity in EAE mice. Data from our dOPC cultures (Fig. 1) and cerebellar explants (Fig. 2) demonstrated that 2.5–10.0 μM guanabenz was sufficient to protect oligodendrocytes and myelin from inflammatory loss. Pharmacokinetic analysis of serum and brain tissue from wild type EAE mice treated with 4, 8 or 16 mg kg−1 of guanabenz daily for >20 days revealed a striking concordance between these efficacious in vitro concentrations and the EAE brain exposures (Supplementary Table 1): brain tissue levels of drug were above 2 μM at 2 h in all dosage groups, and were above 2 μM at 4 h for the 8 and 16 mg kg−1 groups. Guanabenz levels were also substantially lower in serum than in the brain, with the brain:serum ratio of guanabenz in our EAE mice similar to those reported in rats37 and rhesus monkeys38. While guanabenz was eliminated much more rapidly in our mice than as reported in humans, the serum exposure in mice 4 h post injection was similar to that previously reported in human volunteers at doses that have been approved by the FDA for therapeutic use39. These findings indicated that 4–16 mg kg−1 treatment with guanabenz in mice would likely achieve CNS levels sufficient to modulate the ISR.

Indeed, while vehicle-treated EAE mice typically develop clinical symptoms about 10 days after immunization with disease severity peaking roughly a week later (Fig. 4a), daily treatment of EAE mice with 4, 8 or 16 mg kg−1 of guanabenz beginning at post-immunization day 7 (PID7) significantly delayed the onset of clinical symptoms (defined as a clinical score of 1.0; Fig. 4a,b). Notably, the average peak of clinical disease in EAE mice treated with 8 mg kg−1 guanabenz (2.8) was also significantly lower than in vehicle-treated EAE mice (3.8, P=0.032, unpaired t-test), such that the average vehicle-treated EAE mouse developed hindlimb paralysis and even forelimb paresis, whereas the average guanabenz-treated EAE mouse experienced only hindlimb paresis (Fig. 4c). In addition, the incidence of disease in all guanabenz-treated EAE mice was lower than in vehicle-treated EAE mice (Fig. 4d). These results demonstrate that guanabenz treatment significantly delays disease onset at all doses tested, while treatment with 8 mg/kg guanabenz also diminishes severity of disease.

Figure 4: Guanabenz treatment delays and alleviates clinical symptoms in mice with chronic EAE. (a) Clinical scores of wild type C57BL/6J female mice immunized with CFA and MOG 35–55 to induce chronic EAE, treated with vehicle (n=15) or 4 mg kg−1 (n=15), 8 mg kg−1 (n=15) or 16 mg kg−1 (n=14) guanabenz daily from PID7 to the end of the study. (b–d) Average onset of disease, defined as the day a clinical score of 1.0 was first reached in each mouse (b), peak of disease (c) and incidence of disease (d) of all treatment groups. Data in (a–d) represent one of two studies conducted with similar results and presented as mean±s.e.m. Unpaired t-test, *P<0.05, ***P<0.0005 compared with vehicle. Full size image

Guanabenz alters the molecular and cellular response to EAE

Given the ability of guanabenz treatment to significantly ameliorate EAE, we next sought to determine the underlying mechanism of this protection. Lumbar spinal cords were isolated from PID15 EAE mice (age corresponding to the average peak of disease in vehicle-treated EAE mice) that were treated daily with vehicle or 8 mg kg−1 of guanabenz beginning PID7 for immunohistochemical and biochemical analyses. Since EAE and MS are characterized by focal inflammatory lesions of demyelination and oligodendrocyte loss, we analysed the ability of guanabenz treatment to decrease the number of spinal cord lesions as well as the degree of oligodendrocyte loss within the lesions. Histological analysis of lumbar spinal cord cross-sections revealed inflammatory foci in the vehicle-treated EAE sections as expected (Fig. 5a, arrowhead), but surprisingly none in the guanabenz-treated EAE tissue. Staining with luxol fast blue (LFB) also revealed demyelination around these areas of cell infiltration (Fig. 5b, dotted areas) in vehicle-treated EAE tissue only. Both findings were further emphasized in higher magnification images of toluidine blue-stained sections (Fig. 5c, arrows). To identify and analyse these focal areas of infiltration, we used CD3 as a general T cell marker and confirmed that mice immunized with adjuvant only, which did not develop any clinical symptoms, also did not have any detectable CD3+ cells in the spinal cord as expected (Fig. 5d, Supplementary Fig. 1). In vehicle-treated EAE mice, however, we identified large focal areas in the white matter that contained on average 800 CD3+ cells per mm2. Designating these highly concentrated areas of CD3+ cells as ‘lesion’ sites (Fig. 5e), we found that the vehicle-treated EAE mice had 65.7% (P=0.026, unpaired t-test) of the mature oligodendrocytes found in regions of matched size and location in mice immunized with adjuvant only (Fig. 5f). Anti-CD3 immunostaining in the guanabenz-treated EAE sections, however, revealed no detectable CD3+ cells and hence no lesion sites, and indeed counts of mature oligodendrocytes in regions of matched size and location to vehicle-treated EAE mice revealed cell numbers comparable to those in mice treated with adjuvant only (Fig. 5d–f). Thus, at PID15, the typical EAE characteristics of infiltrating T cells within the spinal cord, oligodendrocyte loss and regions of demyelination are present in vehicle-treated but not guanabenz-treated mice.

Figure 5: Guanabenz treatment alters T cell distribution and upregulates ISR activity while protecting oligodendrocytes in chronic EAE. All analyses conducted using lumbar spinal cords of PID15 mice immunized with adjuvant only (CFA) or adjuvant with MOG 35–55 (EAE), then treated with vehicle or guanabenz from PID7 to 15. (a) Haematoxylin and eosine (H&E) staining. Note only the vehicle-treated EAE sample displays cellular infiltrates (arrowhead). (b) LFB staining. Note the demyelinated focal areas (dotted areas) in vehicle-treated EAE mice only. (c) Higher magnification of sections stained with toluidine blue. Note the demyelination (arrows) in areas of cellular infiltration in vehicle-treated EAE mice. (d) Quantification of cells positive for CD3, a T cell marker. (e) Isolated pockets of these CD3+ areas were considered ‘lesion’ areas and quantified. (f) Quantification of cells positive for ASPA, a mature oligodendrocyte marker, in ‘lesion’ areas. (g) Quantification of cells positive for both p-eIF2α and tubulin polymerization promoting protein (TPPP), a mature oligodendrocyte marker. Unpaired t-test, *P<0.05, **P<0.005, ***P<0.0005. Data in d–g represent an average of five to six mice per group, presented as mean±s.e.m. (h) Immunoblot analysis of the pro-apoptotic ISR protein CHOP in lumbar spinal cord lysates. GAPDH is presented as a loading control. Blot representative of n=4 per group. Scale bars, 200 μm (a,b), 50 μm (c). Full size image

The ongoing presence of T cells in the spinal cord at this EAE time point is heavily dependent on debris resulting from oligodendrocyte and myelin loss40. As our earlier in vitro and in vivo experiments had demonstrated that enhancement of the ISR with guanabenz treatment can protect oligodendrocytes from death, we next examined whether the ISR could be playing a role in delaying and alleviating EAE by protecting mature oligodendrocytes from inflammatory loss, thereby limiting detectable T cell presence at PID15. We therefore stained PID15 EAE lumbar spinal cord tissue sections with markers for p-eIF2α and mature oligodendrocytes. These data showed that, while roughly 50% of the mature oligodendrocytes in adjuvant-only-treated mice expressed p-eIF2α, more than 80% of mature oligodendrocytes expressed p-eIF2α in both the vehicle- and guanabenz-treated EAE mice (Fig. 5g). As MS and EAE lesions19,20,21,22 have previously been shown to have high ISR activity, the finding that vehicle-treated EAE tissue had high expression of the ISR pathway was expected. Since sections from guanabenz-treated EAE mice appeared similarly lesion-free and intact as those from adjuvant-only-treated mice, however, the finding that the ISR was also highly upregulated in the guanabenz-treated sections suggests that the ISR may have a role in enhancing oligodendrocyte survival and subsequently delaying EAE onset and alleviating disease severity.

In PID15 EAE lumbar spinal cord lysates, immunoblots probing the protein CHOP revealed further evidence of ISR activity (Fig. 5h). CHOP is a pro-apoptotic protein that is downstream of p-eIF2α in the ISR pathway, and its high expression is indicative of cell loss as a result of an overwhelmed ISR. Interestingly, Tsaytler et al.28 found that as guanabenz merely prolonged the phosphorylation of eIF2α, treatment with guanabenz did not induce higher CHOP expression. We found that CHOP was highly expressed in vehicle-treated EAE tissue, but not in guanabenz-treated EAE tissue or samples from mice immunized with adjuvant only (Fig. 5h). Taken together, these findings of the key markers of ISR activity, p-eIF2α and CHOP, indicate that guanabenz treatment enhanced the ISR in EAE mice, potentially resulting in ISR-mediated protection of oligodendrocytes from EAE inflammatory loss and subsequently inhibiting continuation of the activated immune response within the CNS.

Guanabenz alters the number of activated CD4+ cells in EAE

While our earlier experiments clearly show that enhancement of ISR activity can protect oligodendrocytes and myelin, upregulation of this pathway has been found to lead to suppression of the inflammatory response16,41,42. Studies have also shown that agonism of the α2-adrenergic receptor, another known function of guanabenz, may also modulate inflammation43,44,45. We therefore tested whether guanabenz treatment had any immunomodulatory effects in PID15 EAE mice. Compared with vehicle-treated EAE mice, EAE mice treated daily from PID7 to PID15 with 8 mg kg−1 guanabenz showed no difference in the numbers of CD4+ T cells, CD8+ T cells, B cells, macrophages or dendritic cells in the lymph node as determined by flow cytometric analyses (Fig. 6a). In addition, guanabenz treatment had no effect on lymph node T cell proliferation or Th1 (IFN-γ) or Th17 (interleukin (IL)-17 and granulocyte–macrophage colony-stimulating factor (GM-CSF)) cytokine production in response to anti-CD3 (1 μg ml−1), OVA 323–339 or myelin oligodendrocyte glycoprotein (MOG) 35–55 (20 μg ml−1; Supplementary Fig. 2a–d). By contrast, guanabenz-treated EAE mice showed a significant increase in the number of splenic CD4+ T cells and B cells (Fig. 6b). When equal numbers of total splenocytes were reactivated ex vivo, however, again no significant differences in recall responses were found (Supplementary Fig. 2e–h). Together, the above findings suggest that guanabenz treatment during EAE has no effect on peripheral T cell proliferation or cytokine production, but may cause retention of CD4+ T cells within the spleen.

Figure 6: Guanabenz treatment protects oligodendrocytes and alters CD4+ T cell populations in two EAE mouse models. Flow cytometry analysis of immune cell populations in the (a) inguinal lymph nodes, (b) spleen and (c) CNS, taken from PID15 mice with actively induced chronic EAE treated daily with vehicle or guanabenz beginning PID7. Data are representative of four mice per group; experiment performed twice. (d,e) Analysis of IFN-γ and IL-17 protein expression in the lumbar spinal cord of PID9 and PID15 chronic EAE mice. PID9, n=4–5 mice per group, PID15, n=6–9 mice per group. (f–o) Flow cytometry analysis of adoptive transfer EAE mice. Recipient mice were treated daily with vehicle or guanabenz beginning PCT day 0. C57BL/6 mice that received no blast cells and only two pertussis toxin treatments (Ptx only) were included as flow analysis controls. (f) All mice were followed for EAE disease severity until euthanized, with five mice in each group remaining on day 16. The last guanabenz treatment was on day 9, and the subsequent rapid clinical decline of these animals demonstrated that they were the recipients of active T cells and the protective nature of guanabenz. On days 3, 6 and 10 the CNS was collected from four representative mice in each treatment group, and the number of (g) total live CD4+ T cells, (h) dead CD4+ T cells, (i) AnnexinV+ CD4+ T cells, (j) Ki67+ CD4+ T cells, (k) CD44hi CD4+ T cells, (l) IFN-γ+ CD4+ Th1 cells and (m) IL-17+ CD4+ Th17 cells was assessed via flow cytometry. The number of (n) live mature GALC+ MOG+ oligodendrocytes present within the CNS and the number of (o) A2B5+ PDGFRα+ early progenitor OPCs were also assessed via flow cytometry. The data are presented as the average number of cells over time. *P<0.05, **P<0.005, ***P<0.0005, as compared with vehicle-treated mice. Data represents average of four mice per group, presented as mean±s.e.m. Full size image

In the CNS, in line with our histological findings (Fig. 5), significantly fewer CD4+ T cells and microglia were found in the guanabenz-treated EAE mice (Fig. 6c). While ISR-mediated protection of oligodendrocytes may limit subsequent T cell responses, it is also possible that guanabenz treatment directly affects the ability of CD4+ T cells to traffic into the CNS and/or become fully activated. We began our examination of these possibilities by evaluating cytokine expression at the initiation (PID9) and peak (PID14–15) of clinical disease in the lumbar spinal cord tissue. Interestingly, at both PID9 and PID15, protein levels of GM-CSF in the vehicle- and guanabenz-treated EAE mice were significantly higher than those in mice treated with adjuvant only (Fig. 6d,e). Similarly, vehicle- and guanabenz-treated EAE mice yielded higher protein levels of IFN-γ than adjuvant mice at both time points, which was reflected in higher IFN-γ mRNA levels (Supplementary Fig. 3). While this also held true for IL-17 protein and mRNA levels at PID9, both protein and mRNA levels of IL-17 were similar in all samples by PID15 (Fig. 6d,e, Supplementary Fig. 3). These findings indicate that guanabenz treatment is not inhibiting the presence of the key encephalitogenic factors needed for EAE development within the CNS.

We explored the effect of guanabenz on T cell trafficking into the CNS further using the MOG 35–55 adoptive transfer model of EAE in C57BL/6 mice. MOG 35–55 blast cells were transferred into naïve recipient mice that then received daily treatment of either vehicle or guananbenz (8 mg kg−1). The mice were monitored for disease over a 16-day-time course, and the data show that clinical disease severity peaked at a clinical score of 2 in the vehicle-treated mice and was strikingly reduced in the guanabenz-treated mice (Fig. 6f).

The use of the adoptive transfer model guaranteed that guanabenz treatment could not affect initial activation or expansion of T cells while allowing modulation of the in vivo reactivation and subsequent proliferation of the myelin peptide-specific CD4+ T cells required for transfer EAE46. To determine how guanabenz treatment might alter CD4+ T cell activation in vivo, spleen and CNS samples were collected 3, 6 and 10 days post-cell transfer (PCT). Vehicle- and guanabenz-treated recipient mice both had significantly higher numbers of live CD4+ T cells within the CNS on PCT3 and PCT6 compared with non-recipient mice injected with pertussis only, indicating that the drug does not prevent initial T cell infiltration into the CNS. On PCT10, however, vehicle-treated recipients displayed a significant increase in the number of live CD4+ T cells compared with earlier time points, while guanabenz-treated recipient mice only displayed a minimal increase (Fig. 6g). These data correlate with our findings in the actively-induced chronic EAE model, in which guanabenz-treated mice had significantly lower CD4+ T cell numbers compared with vehicle-treated mice (Fig. 6c). Vehicle-treated mice on PCT10 also displayed significantly greater numbers of proliferating (Ki67+), activated (CD44hi) Th1 (IFN-γ+) and Th17 (IL-17+) CD4+ T cells within the CNS as compared with guanabenz-treated mice (Fig. 6j–m and Supplementary Figs 5–8). Meanwhile, in the spleen, vehicle-treated mice showed an increase in numbers of Th1 and Th17 cells from PCT3 to PCT10 (Supplementary Figs 4–8). Conversely, the guanabenz-treated PCT10 mice had significantly higher numbers of dead CD4+ T cells in the CNS as compared with vehicle-treated PCT10 mice (Fig. 6h,i) and significantly decreased numbers of live CD4+ T cells (Fig. 6g), resulting in significantly lower numbers of Ki67+, CD44hi, IFN-γ+ and IL-17+ CD4+ T cells (Fig. 6j–m). Guanabenz treatment also resulted in an increase in the number of dead CD4+ T cells within the spleen on PCT3 compared with vehicle treatment (Supplementary Figs. 4-8).

We also used flow cytometry47 to characterize oligodendrocyte lineage cells in the adoptive transfer recipients. Consistent with our histological findings (Fig. 5f), there was not an observed loss of mature (GALC+, MOG+) oligodendrocytes in the guanabenz-treated mice over the 10-day-time course, while this was readily observed in the vehicle-treated recipients (Fig. 6n, Supplementary Figs 9 and 10). The loss of oligodendrocytes in the vehicle-treated recipients correlated with an increase in oligodendrocyte progenitor cells (A2B5+, PDGFRα+), which was not observed in the guanabenz-treated recipients (Fig. 6o).

Collectively, these data demonstrate that guanabenz treatment results in both a decrease in the number of activated CD4+ T cells and an increase in the number of dead CD4+ T cells in the CNS while protecting mature oligodendrocytes. Whether the decreased T cell activation occurs directly by a T cell- or CNS antigen-presenting cell (APC)-intrinsic mechanism and/or indirectly by limiting the amount of myelin antigen available secondary to guanabenz-induced oligodendrocyte protection requires additional investigation.

Guanabenz alleviates relapse in relapsing-remitting EAE

The ability of guanabenz to protect oligodendrocytes and myelin both in vitro and in vivo, in addition to delaying and alleviating disease in chronic EAE, suggests that it has potential as a therapeutic agent for MS. In patients with relapsing-remitting MS, the predominant human disease presentation, treatment is initiated only when clinical symptoms are already present. To investigate whether guanabenz has therapeutic potential for this clinical setting, we used the SJL/J mouse relapsing-remitting EAE (R-EAE) model48,49. Similar to human patients with MS, these mice develop symptoms, undergo remission and experience subsequent clinical relapses. We thus treated SJL/J mice with R-EAE with 8 mg kg−1 of guanabenz daily beginning at the onset of remission. Encouragingly, guanabenz-treated R-EAE mice displayed a 47.9% reduction in clinical severity at peak of relapse (clinical score of 0.9) compared with vehicle-treated R-EAE mice (1.8, P=0.038, unpaired t-test, Fig. 7), such that guanabenz-treated R-EAE mice experienced tail limpness, the initial sign of the clinical onset of EAE, whereas vehicle-treated R-EAE mice showed hindlimb ataxia. These results indicate that treatment with guanabenz can be effective following the onset of disease.