Macroautophagy Dysfunction in PD - Experiment Design

SUMMARY

# Macroautophagy Dysfunction in PD - Experiment Design ## Background and Rationale This clinical study investigates the central hypothesis that macroautophagy dysfunction serves as an upstream pathogenic driver of alpha-synuclein aggregation in Parkinson's disease. The research addresses the critical knowledge gap regarding the temporal relationship between autophagy impairment and protein misfolding in neurodegeneration. Using a comprehensive biomarker approach, the study will examine autophagy

METHODOLOGY NOTES

Phase 1 (Months 1-18): Generate iPSCs from 30 PD patients and 20 healthy controls, differentiate into midbrain dopaminergic neurons using established protocols. At days 35, 50, 65 post-differentiation, assess autophagy flux using bafilomycin A1 treatment (100nM, 4h) followed by LC3-II Western blot and immunofluorescence. Measure p62 accumulation, LAMP1 expression, and cathepsin B/D activity using fluorogenic substrates. Quantify alpha-synuclein aggregation via filter trap assay and Thioflavin-T staining. Assess neuronal viability using MTT assay and caspase-3 activation. Perform rescue experiments using autophagy modulators (rapamycin 100nM, trehalose 100mM). Phase 2 (Months 12-24): Recruit 100 PD patients (Hoehn-Yahr stages 1-3) and 50 age-matched controls. Collect cerebrospinal fluid and plasma samples. Measure ATG5, ATG7, Beclin-1, LC3, p62 levels via ELISA. Assess lysosomal enzymes (β-glucocerebrosidase, α-galactosidase) and alpha-synuclein species (monomeric, oligomeric, phosphory