s:** - Test tau spreading in AQP4 knockout vs wild-type mice with PSP/CBD strains - Rescue AQP4 polarization pharmacologically and measure tau patholo

SUMMARY

# s:** - Test tau spreading in AQP4 knockout vs wild-type mice with PSP/CBD strains - Rescue AQP4 polarization pharmacologically and measure tau patholo ## Background and Rationale This experiment examines the role of aquaporin-4 (AQP4) polarization in tau pathology spreading by comparing AQP4 knockout mice with wild-type controls using PSP and CBD tau strains. AQP4 is the predominant water channel in astrocytes and plays a crucial role in glymphatic clearance, with its polarization to astrocyti

METHODOLOGY NOTES

**Phase 1: Animal Preparation and Genotyping (Weeks 1-2)** • Breed AQP4 knockout (AQP4-/-) and wild-type (AQP4+/+) C57BL/6J mice to generate 60 mice per genotype (120 total) • Perform genotyping via PCR using primers specific for AQP4 deletion cassette • Age mice to 8-10 weeks and perform baseline behavioral assessment using rotarod and open field tests • Randomize mice into treatment groups: Vehicle control, PSP tau, CBD tau, PSP tau + AQP4 modulator, CBD tau + AQP4 modulator (n=12 per group per genotype) **Phase 2: Tau Strain Preparation and Characterization (Week 3)** • Prepare recombinant PSP-derived 4R tau fibrils and CBD-derived mixed 3R/4R tau fibrils using established seeding protocols • Characterize tau strains via thioflavin T binding, electron microscopy, and Western blot for conformational specificity • Sonicate fibrils to 100-200nm fragments and quantify by Bradford assay (target: 2μg/μL) • Validate seeding capacity using HEK293T biosensor cells expressing tau-CFP/YFP FRE