Targeting of CH25H to boost p62-dependent autophagic degradation of α-synuclein in cell and mouse models of Parkinson's disease.

Abstract

Insufficient understanding of α-synuclein turnover mechanisms has impeded successful clinical translation for Parkinson's disease (PD). Here, we pinpointed cholesterol 25-hydroxylase (CH25H) as a pivotal regulator of α-synuclein degradation. Through bulk RNA sequencing of substantia nigra tissue from the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, along with reanalysis of published datasets from induced pluripotent stem cell-derived astrocytes of patients with PD, we observed an elevated CH25H expression in PD-associated astrocytes. This finding was validated by combined fluorescence in situ hybridization for <i>Ch25h</i> and immunofluorescence staining for GFAP in mouse substantia nigra sections. Conditional knockout or knockdown of astrocytic <i>Ch25h</i> alleviated PD-like motor deficits and reduced dopaminergic neuronal loss in MPTP and α-synuclein preformed fibril (PFF) mouse models. Using 4D label-free proteomics and molecular docking approaches, we uncovered a shared binding domain on p62 where both CH25H and α-synuclein interact. Proximity ligation assays in cultured astrocytes showed that <i>Ch25h</i> overexpression promoted formation of p62/CH25H complex, whereas it inhibited p62/α-synuclein interaction. Conversely, <i>Ch25h</i> knockdown enhanced p62/α-synuclein complex formation and facilitated α-synuclein degradation. 25-Hydroxycholesterol, the enzymatic by-product of CH25H, did not affect the expression of α-synuclein in astrocytes, suggesting an activity-independent influence of CH25H on α-synuclein clearance. In addition, treatment with a p62 polypeptide (60 to 90 amino acids) effectively facilitated α-synuclein clearance by sequestering free CH25H in both cultured astrocytes and mice in the PFF model. Collectively, our study provides insights into the mechanisms underlying α-synuclein turnover and suggests promising avenues for disease-modifying interventions in synucleinopathies.