Inhibition of miR-148a-3p Ameliorates Prion Accumulation by Restoring ATP6AP2-Mediated Lysosomal Acidification

Prion diseases (PrDs) are fatal neurodegenerative disorders characterized by the accumulation of misfolded prion protein (PrPSc) and progressive impairment of protein quality-control systems. Lysosomal dysfunction and defective autophagic flux contribute to PrPSc accumulation, but the upstream regulatory mechanisms remain incompletely defined. In this study, we investigated the regulatory role of miR-148a-3p during prion infection. We found that miR-148a-3p expression is markedly increased in ME7- and 139A-infected mouse brains and prion-infected SMB-S15 cells. Dual-luciferase assays confirmed that miR-148a-3p directly targets the PRNP 3′UTR, negatively regulating cellular PrP expression. In prion-infected cells, inhibition of miR-148a-3p reduced proteinase K (PK)-resistant PrP (PrPres), delayed RT-QuIC conversion, and decreased PMCA-amplified PrPres. Through bioinformatic prediction and experimental validation, we identified ATP6AP2, a V-ATPase-associated regulator of lysosomal acidification, as a high-confidence target of miR-148a-3p. ATP6AP2 expression was significantly decreased in sCJD transcriptomic data, scrapie-infected mouse brains, and prion-infected cells. Importantly, inhibition of miR-148a-3p restored ATP6AP2 expression, which improved lysosomal acidification and alleviated impaired autophagic flux. Furthermore, overexpression of ATP6AP2 directly reduced PrPSc accumulation in SMB-S15 cells and diminished prion seeding capacities in RT-QuIC and PMCA assays. These findings identify miR-148a-3p as an upstream regulator of ATP6AP2-dependent lysosomal dysfunction in prion infection. Inhibition of miR-148a-3p may restore lysosomal clearance capacity and reduce PrPSc accumulation, suggesting a potential regulatory strategy for PrDs.