s:** - Dose-response studies showing therapeutic window without toxicity - Cell-type specific effects across CNS populations - Demonstration that enha

SUMMARY

# s:** - Dose-response studies showing therapeutic window without toxicity - Cell-type specific effects across CNS populations - Demonstration that enha ## Background and Rationale # Expanded Experimental Description: Dose-Response and Cell-Type Specific Analysis of Dissolution Enhancement in Neurodegeneration Models The therapeutic potential of enhancing dissolution pathways in neurodegenerative disease contexts has emerged as a promising intervention strategy, yet the fundamental gap between

METHODOLOGY NOTES

**Phase 1: Cell Line Preparation and Characterization (Days 1-3)** • Establish primary neuronal cultures from hippocampal, cortical, and dopaminergic regions (n=6 wells per condition) • Culture CNS cell lines: SH-SY5Y (dopaminergic), HT22 (hippocampal), and primary astrocytes • Perform cell viability assays (MTT) and characterize baseline stress response markers (HSP70, cleaved caspase-3) • Validate cell-type specific markers via immunocytochemistry (TH, MAP2, GFAP) **Phase 2: Compound Preparation and Dose-Response Design (Day 4)** • Prepare test compound in enhanced dissolution formulation at concentrations: 0.1, 1, 10, 50, 100, 500 μM • Prepare standard formulation at same concentrations for comparison • Include vehicle controls (DMSO <0.1%) and positive controls (rotenone 100 nM for toxicity) **Phase 3: Acute Toxicity Assessment (Days 5-7)** • Treat cells with dose range for 24h, 48h, and 72h timepoints • Measure cell viability via MTT assay and LDH release at each timepoint • Ass