Spatial transcriptomics analysis uncovers ER stress in MANF-deficient Purkinje cells underlying alcohol-induced cerebellar neurodegeneration in mice

Alcohol use disorders (AUD) is one of the most prevalent mental disorders in the United States affecting more than 10% of the adult population. Cerebellar atrophy and Purkinje cell (PC) degeneration are frequently observed in patients with AUD. Alcohol can cause endoplasmic reticulum (ER) stress in PCs and alter PC structure and function. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress inducible protein highly expressed in PCs. It is neuroprotective in various ER stress-related pathological conditions. However, it is unknown whether MANF plays a role in protecting PCs from alcohol-induced ER stress and neurodegeneration. In this study, we generated PC-specific MANF knockout (KO) mouse model to test the hypothesis that MANF-deficient PCs are more susceptible to alcohol-induced ER stress and neurodegeneration in the adult brain. We employed a binge alcohol exposure paradigm and tested the effect of alcohol and MANF deficiency on molecular, cellular, and behavioral outcomes in the adult animals. We also performed spatial transcriptomics and high throughput in situ analyses to profile gene expression changes in response to MANF deficiency. We found that alcohol exacerbated the motor function deficits in PC-specific MANF KO animals. Interestingly, female KOs were more sensitive to alcohol-induced motor function impairments than male KOs. In accordance with the behavior changes, alcohol exposure activated the unfolded protein response (UPR), increased intranuclear expression of calcium binding protein, and caused PC degeneration in female but not male MANF KO mice. Spatial transcriptomics and high throughput Xenium in situ analyses revealed 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 transportation, and response to ER stress. Our study reveals that MANF-deficient PCs are predisposed with a higher risk to UPR activation and disrupted calcium homeostasis in a sex-dependent manner, which may underline their sex-specific vulnerability to alcohol-induced neurodegeneration. These findings suggest that ER stress plays a significant role in alcohol-triggered neurodegenerative process in the cerebellum, and MANF may possess therapeutic potentials in AUD via its capacity in restoring ER and calcium homeostasis.