β-Amyloid and Glutathione Dysregulation Cooperatively Drive Lipid Peroxidation and Ferroptosis in Neuron-Like Cells.

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by β-amyloid (Aβ) accumulation and oxidative stress, with aging being its greatest risk factor. Age-related glutathione (GSH) depletion may increase neuronal vulnerability to Aβ toxicity, but the underlying mechanisms remain unclear. Here, we investigated how impaired GSH homeostasis influences Aβ-associated neuronal injury. Human SH-SY5Y cells expressing either wild-type APP695 or the familial AD-associated APPSwe/Ind mutant were subjected to GSH depletion using complementary pharmacological (buthionine sulfoximine and dimethyl fumarate) and genetic (GCLC knockout) approaches. GSH depletion markedly sensitized APPSwe/Ind-expressing cells to ferroptotic cell death, characterized by increased lipid peroxidation, elevated malondialdehyde and 4-hydroxynonenal levels, enhanced lactate dehydrogenase release, increased transferrin receptor-1 expression, and intracellular iron accumulation. Cell death was prevented by ferrostatin-1, liproxstatin-1, and the iron chelator deferoxamine, but not by the pan-caspase inhibitor Z-VAD-FMK, confirming an iron-dependent ferroptotic mechanism. Similar findings were observed following co-treatment with Aβ oligomers and GSH depletion. Mechanistically, combined Aβ stress and GSH depletion were associated with increased chaperone-mediated autophagy (CMA) activity and reduced GPX4 protein levels. A photoactivatable CMA reporter demonstrated enhanced CMA activity and increased colocalization of GPX4 with CMA-associated puncta. Pharmacological inhibition of lysosomal function with bafilomycin A1 or treatment with the CMA inhibitor polyphyllin D rescued cell viability under these conditions. Collectively, these findings suggest a potential association among CMA activation, reduced GPX4 abundance, and increased susceptibility to ferroptotic cell death, providing new insight into how age-related redox imbalance may contribute to neuronal vulnerability in AD.