Predominance of Ferroptotic Cell Death Mechanisms in Substantia Nigra Neurodegeneration in Parkinson's Disease

Objective: The extent of neuronal loss in Parkinson's disease (PD) and the pathogenic processes underlying neuronal dysfunction and loss remain poorly understood. Here, we analyzed the expression of key molecules representing different cell death signaling pathways and their association with Lewy pathology, dopaminergic (DA) neuron loss and stage of PD progression in human postmortem brain tissue. Methods: We performed neuropathological and molecular analyses on 47 postmortem substantia nigra (SN) tissue samples from PD cases and healthy controls to investigate neuronal cell death pathways. Results: An average loss of 54% of dopaminergic neurons was found in the SN of PD cases, which correlated strongly with PD Braak staging. The apoptosis markers, cleaved subunits of caspases 3 and 8, were absent. Levels of the active necroptosis kinase, phosphorylated RIPK3 (pRIPK3), were significantly increased in advanced-stage PD. Although phosphorylated MLKL (pMLKL) levels were not significantly different, both active markers were detected in small numbers of PD neurons by immunofluorescence, suggesting focal necroptotic pathway activation. In contrast, evidence for ferroptosis was more pronounced, particularly in advanced-stage PD. This was supported by significantly increased transferrin receptor 1 (TFR1) protein levels and significantly decreased glutathione peroxidase 4 (GPX4) RNA and protein levels. Interpretation: Our findings implicate ferroptosis, and to a lesser extent necroptosis, in PD neuronal death, with ferroptosis potentially playing a larger role in advanced disease. We propose a "2-hit" model where early synucleinopathy-driven insults are amplified in advanced disease by a neuromelanin-iron-driven feedback loop, establishing ferroptosis as the predominant cell death mechanism. This stage-dependent shift provides critical insights into PD pathogenesis and suggests distinct therapeutic windows for neuroprotection. ANN NEUROL 2026;99:1415-1427.