{"artifact":{"id":"experiment-exp-wiki-experiments-regulated-necrosis-parkinsons","artifact_type":"experiment","entity_ids":null,"title":"Regulated Necrosis Validation Study in Parkinson's Disease","quality_score":0.675,"created_by":"crosslink-v2","provenance_chain":null,"content_hash":null,"metadata":{"source":{"type":"manual","source_name":"wiki","extracted_by":"backfill_v1","extraction_date":"2026-04-16T01:00:16.904525Z"},"summary":"# Regulated Necrosis Validation Study in Parkinson's Disease\n## Background and Rationale\nThis 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 ","entities":{"genes":["ID"],"diseases":["Parkinson's Disease"]},"model_system":"human","_schema_version":1,"experiment_type":"clinical","primary_outcome":"Demonstration of at least 2-fold increase in phospho-MLKL and cleaved gasdermin-D immunoreactivity in PD substantia nigra compared to controls, with dose-dependent neuroprotection (>50% cell survival) using pathway inhibitors in iPSC models.","methodology_notes":"**Phase 1: In Vitro Validation (Months 1-12)**\n\n• Establish primary dopaminergic neuronal cultures from human induced pluripotent stem cells (hiPSCs) from 50 PD patients and 25 healthy controls\n• Differentiate hiPSCs into midbrain dopaminergic neurons using dual SMAD inhibition protocol over 35 days\n• Characterize neuronal populations via immunofluorescence for TH, FOXA2, and LMX1A markers\n• Induce regulated necrosis using RIPK1/RIPK3 pathway activators (TNF-α 10ng/ml + Smac mimetic 1μM + zVAD-fmk 20μM)\n• Apply necroptosis inhibitors (Necrostatin-1 30μM, GSK872 5μM) and ferroptosis modulators\n• Measure cell viability via MTT assay and LDH release at 6, 12, 24, and 48-hour timepoints\n• Quantify necrotic markers (RIPK3 phosphorylation, MLKL oligomerization) via Western blot and immunofluorescence\n• Assess mitochondrial dysfunction using TMRM staining and ATP measurement\n• Perform RNA-sequencing on treated cultures to identify regulated necrosis gene signatures\n\n**Phase 2: Drug Testing an","replication_status":"replicated","extraction_metadata":{"backfill_at":"2026-04-16T01:00:16.904533","needs_review":true,"extraction_notes":"Backfilled from wiki source (no PMID 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