s:** - Temporal analysis showing mitochondrial defects precede other pathology - Rescue experiments in isolated mitochondrial dysfunction models - Spe

SUMMARY

# s:** - Temporal analysis showing mitochondrial defects precede other pathology - Rescue experiments in isolated mitochondrial dysfunction models - Spe ## Background and Rationale # Temporal Analysis of RBP-Mediated Mitochondrial Dysfunction in Neurodegeneration The RNA-binding protein (RBP) under investigation has been implicated in neurodegenerative pathology, yet the mechanistic hierarchy of cellular dysfunction remains unclear. Current evidence suggests that mitochondrial defects may repre

METHODOLOGY NOTES

**Phase 1: Cell Line Preparation and Characterization (Days 1-7)** • Culture human neuroblastoma (SH-SY5Y) and iPSC-derived neurons in standard conditions • Transfect cells with RBP-targeting siRNA (50nM, n=6 wells per condition) • Establish control groups: scrambled siRNA, untreated cells • Validate RBP knockdown by qRT-PCR and Western blot at 24h, 48h, 72h post-transfection • Assess cell viability using MTT assay to ensure >85% viability **Phase 2: Temporal Mitochondrial Function Analysis (Days 3-14)** • Monitor mitochondrial membrane potential using TMRM staining every 24h for 10 days • Measure ATP production via luminescence assay (CellTiter-Glo) at 6h, 12h, 24h, 48h, 72h • Assess mitochondrial respiration using Seahorse XF analyzer at multiple timepoints • Evaluate mitochondrial morphology by live-cell imaging with MitoTracker Green • Quantify mitochondrial DNA copy number by qPCR every 48h **Phase 3: Downstream Pathology Assessment (Days 7-21)** • Monitor protein aggregation us