🧫 Experiment Protocol
Validationproposed
SUMMARY
# Alpha-Synuclein Staging and Spreading in DLB — Spatial Propagation Mapping
## Background and Rationale
Spatial propagation mapping study to define alpha-synuclein spreading patterns in dementia with Lewy bodies (DLB), testing whether cortical-first spreading distinguishes DLB from Parkinson's disease dementia (PDD) where spreading is brainstem-first.
**Protocol**: Post-mortem brain mapping in 40 DLB cases and 40 PDD cases across 20 brain regions (Braak staging + cortical and limbic sampling).
METHODOLOGY NOTES
**Phase 1: Cell Line Preparation and Alpha-Synuclein Introduction (Days 1-3)**
• Maintain H4 human neuroglioma cells and differentiated SH-SY5Y neuroblastoma cells at 37°C, 5% CO2
• Seed cells in 96-well imaging plates at 5×10^4 cells/well for time-course analysis
• Prepare recombinant alpha-synuclein preformed fibrils (PFFs) at 5 μg/mL concentration
• Introduce fluorescently-labeled alpha-synuclein (Alexa Fluor 488) to donor cells via lipofection
• Establish co-culture system with 1:1 ratio of donor (alpha-synuclein+) to recipient cells
**Phase 2: Spatial Propagation Monitoring (Days 4-21)**
• Perform live-cell confocal microscopy every 48 hours using 63x oil immersion objective
• Track alpha-synuclein aggregation using ThioflavinS staining (1 μg/mL, 30 min incubation)
• Measure cell-to-cell transfer using proximity ligation assay (PLA) for alpha-synuclein interactions
• Quantify spreading distance using automated image analysis (minimum 200 cells per timepoint)
• Apply sequential st