Zinc‐Mediated Lysosomal Destabilization Links Mitochondrial Damage to Neuronal Death in a Cellular <scp>MPP</scp> ⁺ Model of Parkinson's Disease

Dysregulation of autophagy and lysosomal function is central to Parkinson's disease (PD), yet the upstream mechanisms leading to lysosomal failure remain unclear. Across primary mouse cortical neurons, MT-3 deficient primary mouse astrocytes, human iPSC-derived midbrain dopaminergic neurons, and Rho⁰ CHO cells lacking mitochondrial respiration, we investigated how mitochondrial stress perturbs zinc (Zn²⁺) homeostasis and lysosomal integrity. We identify intracellular zinc as a critical mediator linking mitochondrial dysfunction to lysosomal membrane permeabilization (LMP) and neuronal death. Inhibition of mitochondrial complex I by 1-methyl-4-phenylpyridinium (MPP⁺) elevated reactive oxygen species (ROS) and intracellular zinc, jointly driving LMP. Blocking either ROS or zinc markedly attenuated lysosomal damage and cell death, demonstrating that both act upstream of LMP. To define zinc regulation, we examined metallothionein-3 (MT-3), a brain-enriched zinc-binding protein. MT-3-deficient astrocytes were more vulnerable to MPP⁺ and zinc overload (ZnCl₂) but paradoxically resistant to hydrogen peroxide (H₂O₂), suggesting that MT-3 buffers cytosolic zinc during mitochondrial injury or extracellular zinc influx yet can release bound zinc under oxidative conditions. Using Rho⁰ cells, we show that MPP⁺ toxicity depends on mitochondrial ROS, as loss of mitochondrial function nearly abolished cell death. However, Rho⁰ cells were highly sensitive to ZnCl₂ and H₂O₂ and exhibited markedly reduced lysosomal abundance, indicating limited capacity to sequester zinc and increased susceptibility to zinc-mediated injury. These findings support a coordinated system in which lysosomes and zinc-binding proteins maintain zinc homeostasis. When cytosolic zinc rises, its accumulation within lysosomes induces LMP and accelerates cell death. Collectively, our results identify intracellular zinc as an upstream trigger of lysosomal dysfunction and neurodegeneration. Zinc-mediated LMP provides a mechanistic link between mitochondrial injury, impaired autophagic flux, and α-synuclein pathology in PD. Enhancing zinc homeostasis and lysosomal resilience may offer promising therapeutic strategies.