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