Spatial Transcriptomics Analysis Uncovers ER stress in MANF-deficient Purkinje Cells Underlying Alcohol-induced Cerebellar Vulnerability in Mice

Cerebellar Purkinje cells (PCs) are among the most vulnerable neurons to alcohol neurotoxicity. Alcohol can induce endoplasmic reticulum (ER) stress and alter the structure and function of PCs. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress inducible protein highly expressed in PCs. It is neuroprotective in various pathological conditions where ER stress is induced. However, it is unknown whether MANF plays a role in protecting PCs from alcohol induced ER stress. In this study, we generated PC-specific MANF knockout (KO) mouse model to test the hypothesis that MANF deficient PCs are more susceptible to binge alcohol exposure induced ER stress and neurodegeneration in the adult brain. We found that PC-specific MANF KO animals show moderate motor function deficit, which was exacerbated by alcohol exposure. Interestingly, female KOs were more sensitive than male KOs to alcohol-induced motor function impairments. In accordance with the behavior changes, alcohol exposure also caused UPR activation, increased intranuclear expression of calcium binding protein Calbindin, and PC degeneration in female but not male MANF KO mice. Spatial transcriptomics and high throughput in situ analyses demonstrated that MANF deficiency altered the transcriptomic landscape in PCs in a sex-specific manner and triggered the expression of genes involved in protein folding and response to ER stress. These results suggests that MANF KO PCs may be predisposed with a higher risk to UPR activation and ER stress in a sex dependent manner, contributing to their vulnerability to alcohol neurotoxicity.