Pharmaceutical integrated stress response enhancement protects oligodendrocytes and provides a potential multiple sclerosis therapeutic

Abstract

Oligodendrocyte death contributes to the pathogenesis of the inflammatory demyelinating disease multiple sclerosis (MS). Nevertheless, current MS therapies are mainly immunomodulatory and have demonstrated limited ability to inhibit MS progression. Protection of oligodendrocytes is therefore a desirable strategy for alleviating disease. Here we demonstrate that enhancement of the integrated stress response using the FDA-approved drug guanabenz increases oligodendrocyte survival in culture and prevents hypomyelination in cerebellar explants in the presence of interferon-γ, a pro-inflammatory cytokine implicated in MS pathogenesis. In vivo, guanabenz treatment protects against oligodendrocyte loss caused by CNS-specific expression of interferon-γ. In a mouse model of MS, experimental autoimmune encephalomyelitis, guanabenz alleviates clinical symptoms, which correlates with increased oligodendrocyte survival and diminished CNS CD4+ T cell accumulation. Moreover, guanabenz ameliorates relapse in relapsing-remitting experimental autoimmune encephalomyelitis. Our results provide support for a MS therapy that enhances the integrated stress response to protect oligodendrocytes against the inflammatory CNS environment.

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.

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.

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.

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.

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).

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.

Figure 7. Guanabenz treatment alleviates clinical severity of relapse in mice with relapsing-remitting EAE.

Six- to eight-week old female SJL mice were immunized with proteolipid protein 139–151 to induce R-EAE. R-EAE mice that displayed an acute phase of disease were treated at the beginning of remission (on average 5 days after onset of disease) with vehicle (n=13) or 8 mg kg⁻¹ of guanabenz (n=14) daily and monitored for clinical symptoms. All R-EAE mice that displayed no relapse (9 out of 22 vehicle-treated and 9 out of 23 guanabenz-treated) were removed from the analysis. Unpaired t-test, *P<0.05. Data are presented as mean±s.e.m. Clinical scores of each treatment group are aligned by day of onset of disease (defined as the day the mouse reaches a clinical score of 1) and averaged by day. One representative experiment of two is presented.