Selective mitochondrial damage and dysfunction in cholesterol-exposed neuronal cells: Role of mitochondrial lipid...

Studies have revealed an association between elevated neuronal cholesterol and neuronal dysfunction, in particular, mitochondrial impairment. However, the mechanism by which cholesterol disrupts neuronal mitochondrial function remains unclear, which prompts our current investigation. Using cultured HT22 mouse hippocampal neuronal cells as an in-vitro model, we found that the unmetabolized cholesterol, rather than its ester derivatives, can alter the MTT activity in cultured neuronal cells in a concentration-dependent manner, with an apparent IC₅₀ ≤ 1 μM. At low micromolar concentrations (≤10 μM), cholesterol selectively disrupts mitochondrial function without causing overt cell death or reducing cell density. Functional and structural analyses revealed increased mitochondrial lipid peroxidation, loss of mitochondrial membrane potential, opening of the mitochondrial permeability transition pore, disruption of mitochondrial membrane integrity and ultrastructure, reduced mitochondrial density, and decreased cellular ATP levels. Seahorse-based bioenergetic profiling further demonstrated marked reductions in basal respiration, maximal respiratory capacity, and ATP-linked respiration, indicating a broad impairment of mitochondrial oxidative metabolism. In contrast, higher cholesterol concentrations (100 μM) induced overt cytotoxicity. Furthermore, genes involved in cholesterol biosynthesis (e.g., HMGCR, HMGCS1) and transport (e.g., STARD4, ABCA1), as well as mitochondrial energy metabolism pathways, are altered in cholesterol-treated neuronal cells. These results suggest that free cholesterol at very low concentrations can induce selective mitochondrial toxicity in cultured neurons and impairs mitochondrial ATP production. These findings shed light on the crucial role of dysregulated cholesterol homeostasis in the pathogenesis of neurodegenerative diseases and also form the basis for therapeutic interventions.