Therapeutic potential of blocking GAPDH nitrosylation with CGP3466b in experimental autoimmune encephalomyelitis

Abstract

Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system (CNS). Although classically considered a demyelinating disease, neuroaxonal injury occurs in both the acute and chronic phases and represents a pathologic substrate of disability not targeted by current therapies. Nitric oxide (NO) generated by CNS macrophages and microglia contributes to neuroaxonal injury in all phases of MS, but candidate therapies that prevent NO-mediated injury have not been identified. Here, we demonstrate that the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is robustly nitrosylated in the CNS in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. GAPDH nitrosylation is blocked in vivo with daily administration of CGP3466b, a CNS-penetrant compound with an established safety profile in humans. Consistent with the known role of nitrosylated GAPDH (SNO-GAPDH) in neuronal cell death, blockade of SNO-GAPDH with CGP3466b attenuates neurologic disability and reduces axonal injury in EAE independent of effects on the immune system. Our findings suggest that SNO-GAPDH contributes to neuroaxonal injury during neuroinflammation and identify CGP3466b as a candidate neuroprotective therapy in MS.

Keywords: GAPDH; experimental autoimmune encephalomyelitis; multiple sclerosis; neuroinflammation; neuroprotection; nitric oxide; nitrosylation.

Figure 1

CGP3466b prevents GAPDH nitrosylation within the CNS in the MOG_{35 − 55} EAE mouse model of MS. (A) Nitrosylated GAPDH (SNO-GAPDH) was assayed by biotin switch from spinal cord lysates in control (CFA alone) mice at day 15 and at onset (post-immunization day 10), peak (post-immunization day 15), and chronic (post-immunization day 28) stages of EAE. Total GAPDH in tissue lysate was included as loading control. (Left) Representative immunoblot. (Right) Quantification of SNO-GAPDH at peak EAE. The ratio of SNO-GAPDH to total GAPDH band intensity was used for quantification, and results were normalized to CFA alone control. Data represent mean ± SEM of three mice per group. (B) CGP3466b was administered at indicated doses by daily i.p. injection beginning on post-immunization day 0, and nitrosylated GAPDH and β-tubulin were assayed by biotin switch on post-immunization daiy 15. (Left) Representative immunoblot. “No asc” = no ascorbate control. (Right) Quantification of SNO-GAPDH levels following treatment with vehicle or CGP3466b 4 mg/kg. SNO-GAPDH levels were quantified as described in (A). Data represent mean ± SEM of three mice per group. **p < 0.01 by student's t-test (A) or one-way ANOVA with Tukey's multiple comparisons test (B). Full blots are shown in Supplementary Figure 1.

Figure 2

Blocking SNO-GAPDH with CGP3466b is neuroprotective in MOG_{35 − 55} EAE. (A, B) CGP3466b 4 mg/kg was administered by daily i.p. injection beginning on post-immunization day 0 (prophylactic paradigm). Clinical scoring was performed by a blinded observer. Prophylactic treatment with CGP3466b attenuated neurologic deficits (A) without impacting weight loss (B). Data represent n = 17 (vehicle) and n = 10 (CGP3466b) mice per group. (C) CGP3466b 4 mg/kg was administered by daily i.p. injection beginning on post-immunization day 10 (therapeutic paradigm), and clinical scoring was performed by a blinded observer. Data represent n = 20 (vehicle) and n = 26 (CGP3466b) mice per group. (D) To quantify axonal injury, mice were treated with vehicle or CGP3466b 4 mg/kg by daily i.p. injection beginning on post-immunization day 0, and SMI-32+ axonal spheroids were examined via immunofluorescence staining of proximal optic nerve at post-immunization day 28. A representative image is shown (left), along with quantification performed from n = 3 mice per group (right). (E) Mice were treated with vehicle or CGP3466b 4 mg/kg by daily i.p. injection beginning on post-immunization day 10, and SMI-32+ axonal spheroids were examined via immunofluorescence staining of proximal optic nerve at post-immunization day 28. A representative image is shown (left), along with quantification performed from n = 3 mice per group (right). *p < 0.05, **p < 0.01 by Mann-Whitney U-test (A, C) or student's t-test (D, E). Data shown as mean ± SEM.

Figure 3

CGP3466b has minimal direct effects on myeloid and lymphoid cells in vitro. (A) Bone marrow derived macrophages (BMMs) were treated with LPS (100 ng/mL) plus vehicle or the indicated concentrations of CGP3466b for 24 h. Cytokine concentrations in media were determined by ELISA. Data represent mean ± SEM of n = 3 or n = 4 independent experiments performed in triplicate. (B) BMMs and (C) Bone marrow derived dendritic cells (BMDCs) were treated with LPS (100 ng/mL) plus vehicle or the indicated concentrations of CGP3466b, and expression of activation markers was measured by flow cytometry. Data are a representative example of n = 3 independent experiments. (D) CD4+ lymphocytes were stimulated with anti-CD3/CD28 antibodies (3 ug/mL) in the presence of vehicle or the indicated concentrations of CGP3466b. Percentage of CD4+ lymphocytes expressing the given activation markers was measured by flow cytometry. Data represent mean ± SEM of three biological replicates.

Figure 4

CGP3466b does not impact CNS immune infiltration in MOG_{35 − 55} EAE. (A) CD45+ infiltrates in lumbar spinal cord at post-immunization day 18. Mice were treated with vehicle or 4 mg/kg CGP3466b daily starting on post-immunization day 0. A representative image is shown (left), along with quantification performed from n = 3 mice per group (right). (B) Iba1+ infiltrates in optic nerve at post-immunization day 28. Mice (the same cohort depicted in Figure 2D) were treated with vehicle or 4 mg/kg CGP3466b daily starting on post-immunization day 0. A representative image is shown (left), along with quantification performed from n = 3 mice per group (right). (C) Infiltrating macrophages and (D) resident microglia in the brain and spinal cord were examined via flow cytometry on post-immunization day 18 for expression of arginase-1 as a percentage of total macrophages or microglia, respectively. Data represent mean ± SEM of 3 mice per group. (E) Infiltrating macrophages and (F) resident microglia were examined via flow cytometry on post-immunization day 18 for expression of MHC II, shown as mean fluorescence intensity. Data shown from 3 mice per group. (G) Th1 cells and (H) Th17 cells were quantified via flow cytometry on post-immunization day 18 in the brain and spinal cord as a percentage of total CD4+ cells. Data represent mean ± SEM of 3 mice per group. All statistical analyses performed with student's t-test.