Cholesterol overload promotes mitochondria-mediated apoptosis with impaired mitochondrial unfolded protein response and mitophagy in SH-SY5Y cells

Cholesterol accumulation in the brain has been implicated in mitochondrial dysfunction and neurodegeneration; however, its specific effects on mitochondrial quality control pathways, including the mitochondrial unfolded protein response (UPR^mt) and mitophagy, remain poorly defined. In this study, SH-SY5Y human neuroblastoma cells were treated with 25 or 50 μg/mL water-soluble cholesterol for 24 h. UPR^mt, mitophagy, and inflammasome activation were assessed using molecular and cellular approaches, including immunoblotting, quantitative RT-PCR, and fluorescence-based imaging. Cholesterol treatment increased intracellular cholesterol levels up to 1.7-fold and induced dose-dependent cytotoxicity and apoptosis. UPRmt was suppressed, as evidenced by reduced expression of mitochondrial chaperones and proteases. In parallel, cholesterol impaired mitophagy by disrupting autophagic flux, leading to the accumulation of damaged mitochondria. This was accompanied by increased cytosolic mitochondrial DNA (mtDNA), caspase-1 activation, and interleukin-1β secretion. These findings indicate that impaired mitochondrial clearance promotes mtDNA release, thereby linking mitochondrial dysfunction to inflammasome activation. Collectively, cholesterol overload disrupts UPRmt and mitophagy, thereby promoting mitochondrial dysfunction, inflammasome activation, and neuronal apoptosis.