Decreased mitochondrial activity in the demyelinating cerebellum of progressive multiple sclerosis and chronic EAE contributes to Purkinje cell loss

Abstract

In multiple sclerosis (MS), cerebellar gray matter atrophy, white matter demyelination, and Purkinje cell (PC) loss have been linked to tremors, impaired motor control, and loss of coordination. Similar pathologies have been observed in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). This study hypothesized that inflammatory demyelination of the cerebellum alters overall mitochondrial function and is a contributor to axon degeneration and PC loss. Postmortem cerebellar tissue from MS patients, particularly those with secondary progressive MS, showed decreased mitochondrial complex IV (COXIV) activity and significant PC loss. Inflammation, PC axon demyelination, axon degeneration, and parallel fiber loss were also evident. These findings were mirrored in late-stage EAE mice, which also showed increased inflammation and demyelination, reduced PC COXIV activity, and overall PC loss. Further analysis of EAE mice revealed altered mitochondrial structure, modified mitochondrial respiration, and reduced levels of mitochondrial genes involved in energy production. These findings indicate that both human MS and mouse EAE share similar cerebellar changes linked to mitochondrial dysfunction. Thus, late-stage EAE is a valuable model for studying MS-related cerebellar pathology, and mitochondria may be a potential therapeutic target for MS treatment.

Keywords: COXIV; axon damage; cerebellar pathology; experimental autoimmune encephalomyelitis; mitochondria respiration.

Fig. 1.

Neuropathological analysis of the human postmortem cerebellum. (A, i) Table of MS brain samples used for IHC. (ii) Confocal image of normal cerebellum stained for NF200 (red) and PLP (green); dashed rectangle marks PLP WM analysis region. (iii) PLP staining in normal and MS cerebellum. (iv) PLP expression significantly decreases in MS WM. (B, i) Confocal image of MS cerebellum stained for CD45 (red) and GFAP (green); dashed area marks lesion regions. (ii) High-magnification images of GFAP+ and CD45+ cells in lesions. (iii) GFAP and CD45 expression significantly increase in ML, GCL, and WM of MS tissue. (C, i) NF200 (red) and DAPI (blue) staining show NF200 variability in MS and normal sections. (ii) Representative parallel fibers in ML. (iii) NF200+ parallel fiber expression significantly decreases in MS cerebellum. n = 4 normal, n = 11 MS tissue.

Fig. 2.

(A, i) Representative normal (NM) and MS cerebellar sections showing Calbindin (green), in the PCL and ML of the cerebellum. (ii) There was a significant decrease in Calbindin cell counts in the MS PCL and ML as compared to the normal. (iii) There was a significant decrease in PC counts in both the MS female and MS male samples. (B, i) Representative images from postmortem normal and MS ML and PC layers showing COXIV (red), Calbindin (green), and DAPI (blue) staining. (ii) There was a significant decrease in COXIV puncta in the PC somas of MS cerebellum. (iii) There was a significant decrease in COXIV puncta in both females and males compared to the normal group. n = 4 normal tissue, n = 11 MS tissue. *P < 0.05, **P < 0.01 with Student’s t test.

Fig. 3.

Experimental design and EAE scores. (A, i) Schematic of EAE induction and longitudinal assessment of cerebellar and PC mitochondrial pathology. (ii) Three representative EAE experiments are shown. EAE mice (red) show disease onset around day 10. (B, i) MBP (green) and DAPI (blue) staining in a normal cerebellum. (ii) MBP analysis in WM and GCL across groups. (iii and iv) MBP expressions significantly decrease in WM and GCL at EAE D21, D40, and D60. (C, i and ii) Thy1 (green) and Iba-1 (red) staining with DAPI (blue); analysis regions marked. (iii and iv) Iba-1 expression significantly increases in WM and GM at EAE D21, D40, and D60. n = 8 mice/group for IHC.

Fig. 4.

WM axon damage and parallel fiber loss in EAE. (A, i) Thy1-YFP cerebellum image highlighting the analyzed axon blebbing region. (ii) Axon blebbing increases at EAE D21 and persists, with swollen, discontinuous axons at EAE D40 and D60. (B, i) NF200 (red) and DAPI (blue) staining show PC bodies and parallel fibers. (ii) NF200+ parallel fibers significantly decrease at EAE D40 and D60. n = 6 mice/group.

Fig. 5.

EAE-associated PC loss and mitochondrial pathology. (A, i) GFAP (green) and COXIV (red) staining show COXIV reduction in the GCL at EAE D21, later decreasing in the PCL and increasing in the GCL at EAE D40 and D60. (ii) Higher magnification highlights COXIV loss in the PCL at EAE D60. (iii) Quantification confirms a significant COXIV decrease at EAE D21. (B, i) Calbindin+ PCs (green) exhibit loss and altered dendritic arborization at EAE D60. (ii) Calbindin and COXIV costaining show COXIV reduction in PCs. (iii) PC counts significantly decline at EAE D40 and D60. (iv) COXIV puncta in PC somas significantly decrease at EAE D60. (C, i) EM high magnification images of cerebellar WM, PC bodies, and mitochondria (mito) at EAE D40 compared to normal. (ii) Axon diameter and G-ratio comparison. (iii and iv) EAE D40 exhibits increased axon diameter and G-ratio. (v) Mitochondrial size vs. count in EAE D40 and normal groups. n = 4 to 5 mice/group for EM analysis.

Fig. 6.

Decrease in coupled respiration at peak EAE disease. (A) Seahorse assay of cerebellar mitochondrial OCR. (i) EAE D21 showed reduced coupled respiration compared to normal after ADP, Oligomycin, FCCP, and Rotenone/Antimycin A addition. (ii) No significant OCR differences were observed between normal and EAE D60. (B, i) Western blot of mitochondrial homogenates. (ii) No differences in COXI, COXII, COXIII, or ATP Synthase across groups, but CIV-MTCO1 (COXIV) and syntaphilin were significantly reduced in EAE D40 and D60. n = 4 to 6 mice/group.

Fig. 7.

Impaired mitochondrial oxidative phosphorylation gene expression in late-stage EAE cerebellum. (A, i) NanoString analysis of gene expression in EAE D40 and normal mouse cerebellum (n = 3-4/group). Image credit: Created in BioRender. Feri, M. (2025) https://BioRender.com/9669j9h, (ii) Heatmaps show expression levels (low: red, high: green), and (iii) a volcano plot highlights differentially expressed genes (fold change, P-value < 0.50). (B) Heatmaps display downregulated genes in EAE D40 related to oxidative phosphorylation: (i) Complex I (Ndufa12, Ndufa3, Ndufa6, Ndufb10, Ndufb8, Ndufs8), (ii) Complex IV (Cox4i1, Cox5a, Cox5b, Cox6a1, Cox6b1, Cox7c, Cox8a), and (iii) Complex V (Atp5k, Atp6v1f). (C) Schematic summarizing affected OXPHOS genes. Image credit: Created in Biorender. Feri, M. (2025) https://BioRender.com/a87e2gt.

Fig. 8.

Summary of PC Loss in MS/EAE: (A) Under normal conditions, myelinated PCs receive excitatory and inhibitory inputs. In peak EAE, demyelination, inflammation, and astrogliosis induce PC stress, leading to axon damage and initial PC loss. In chronic EAE, persistent damage results in increased axon blebbing, swelling, and further PC and parallel fiber loss. (B) Mitochondria redistribute to meet rising energy demands during demyelination, but sustained stress overwhelms them. Prolonged disease leads to COXIV loss, reduced energy production, and PC degeneration. Image credit: Created in BioRender. Feri, M. (2025) https://BioRender.com/t12njs8.