Autophagy Modulation in a Murine Model of Cuprizone-Induced Demyelination

Abstract Multiple sclerosis (MS) is a chronic immune-mediated disorder affecting the central nervous system (CNS), characterized by demyelination and neuronal loss. Optic neuritis is the first clinical manifestation of MS in about 40% of patients and it occurs in up to 70% over the course of the disease (Tong et al., 2025). Although inflammation is recognized as the driver of lesion formation, neurodegenerative mechanisms are responsible for the axonal loss and the resulting neurological disability (Haki et al., 2024). Autophagy is a key homeostatic process involved in degradation and recycling of cellular components, essential for neuronal integrity (Palmer et al., 2025). Alterations in this process have been associated with several neuroinflammatory and neurodegenerative conditions; however, its contribution to MS pathology remains unclear. The present study aims to evaluate the regulation of autophagy in the optic nerve and corpus callosum in a cuprizone-induced mouse model of MS. Demyelination was induced by feeding 8-week-old male C57BL/6J mice a diet containing 0.2% (w/w) cuprizone for 8 weeks. Protein expression levels were analyzed by western blotting and immunofluorescence. Proteomic profiling was performed using mass spectrometry. Cuprizone exposure induced a marked reduction in myelin basic protein (MBP) levels in both the corpus callosum and the optic nerve, accompanied by increased glial fibrillary acidic protein (GFAP) expression. In the corpus callosum, the levels of autophagy markers (i.e. p62 and LC3-I/II) were increased. PINK1 and Parkin expression was significantly upregulated, whereas optineurin levels remained unchanged. In the optic nerve, Parkin expression was increased, while no changes were observed in retinal tissue. Proteomic analysis of the corpus callosum identified 5,987 proteins, of which 139 were significantly modulated followed cuprizone treatment. These preliminary findings indicate a dysregulation of autophagy and mitophagy in the corpus callosum and optic nerve during cuprizone-induced demyelination and support further investigation of these pathways as potential modulators of neurodegeneration in MS. Bibliography: • Haki, M., Al-Biati, H. A., Al-Tameemi, Z. S., Ali, I. S., & Al-Hussaniy, H. A. (2024). Review of multiple sclerosis: Epidemiology, etiology, pathophysiology, and treatment. Medicine, 103(8), e37297. • Palmer, J.E., Wilson, N., Son, S.M., Obrocki, P., Wrobel, L., Rob., M, Takla, M., Korolchul, V.I., Rubinsztein, D.C. (2025) Autophagy, aging, and age-related neurodegeneration. Neuron,113(1), 29 • Tong, B., Zhang, X., Hu, H., Yang, H., Wang, X., Zhong, M., Yang, F., & Hua, F. (2025). From diagnosis to treatment: exploring the mechanisms underlying optic neuritis in multiple sclerosis. Journal of translational medicine, 23(1), 87.