Proposed experiment from debate on Mitochondrial transfer between astrocytes and neurons

SUMMARY

# Proposed experiment from debate on Mitochondrial transfer between astrocytes and neurons ## Background and Rationale Tunneling nanotubes (TNTs) represent a critical mechanism for intercellular communication and organelle transfer between astrocytes and neurons during neurodegeneration. GAP43, a growth-associated protein, plays a pivotal role in membrane dynamics and cytoskeletal organization that could influence TNT formation and stability. This falsification experiment aims to test the hypoth

METHODOLOGY NOTES

**Phase 1: Cell Culture Preparation (Days 1-3)** • Culture primary rat cortical astrocytes and neurons in separate dishes using DMEM supplemented with 10% FBS and 1% penicillin/streptomycin • Maintain cultures at 37°C, 5% CO2 until 70-80% confluence • Prepare GAP43-overexpressing astrocytes via lentiviral transduction (MOI=5) with pLenti-GAP43-GFP vector • Generate GAP43 knockdown astrocytes using shRNA targeting GAP43 (3 different sequences) with scrambled control • Confirm transfection efficiency >80% via fluorescence microscopy at 48h post-transduction **Phase 2: TNT Visualization and Stability Analysis (Days 4-6)** • Seed astrocytes (2×10⁴ cells/well) and neurons (1×10⁴ cells/well) in co-culture on glass-bottom dishes • Stain with CellTracker Red (astrocytes) and CellTracker Green (neurons) for 30 minutes • Perform live-cell imaging using confocal microscopy with environmental chamber (37°C, 5% CO2) • Acquire time-lapse images every 2 minutes for 4 hours to assess TNT dynamics • Q