Regulated Necrosis Validation Study in Parkinson's Disease

SUMMARY

# Regulated Necrosis Validation Study in Parkinson's Disease ## Background and Rationale This validation study investigates regulated necrosis pathways, including necroptosis, pyroptosis, and ferroptosis, as novel cell death mechanisms in Parkinson's disease pathogenesis. The three-phase experimental design progresses from in vitro validation to drug testing and biomarker development. Phase 1 employs patient-derived iPSC neurons and post-mortem tissue analysis to characterize regulated necrosis

METHODOLOGY NOTES

**Phase 1: In Vitro Validation (Months 1-12)** • Establish primary dopaminergic neuronal cultures from human induced pluripotent stem cells (hiPSCs) from 50 PD patients and 25 healthy controls • Differentiate hiPSCs into midbrain dopaminergic neurons using dual SMAD inhibition protocol over 35 days • Characterize neuronal populations via immunofluorescence for TH, FOXA2, and LMX1A markers • Induce regulated necrosis using RIPK1/RIPK3 pathway activators (TNF-α 10ng/ml + Smac mimetic 1μM + zVAD-fmk 20μM) • Apply necroptosis inhibitors (Necrostatin-1 30μM, GSK872 5μM) and ferroptosis modulators • Measure cell viability via MTT assay and LDH release at 6, 12, 24, and 48-hour timepoints • Quantify necrotic markers (RIPK3 phosphorylation, MLKL oligomerization) via Western blot and immunofluorescence • Assess mitochondrial dysfunction using TMRM staining and ATP measurement • Perform RNA-sequencing on treated cultures to identify regulated necrosis gene signatures **Phase 2: Drug Testing an