s:** - Compare tau strain spreading in EXT1/EXT2 conditional knockout mice - Test whether HSPG-deficient astrocytes still form tufts vs plaques when e

SUMMARY

# s:** - Compare tau strain spreading in EXT1/EXT2 conditional knockout mice - Test whether HSPG-deficient astrocytes still form tufts vs plaques when e ## Background and Rationale This experiment investigates the role of heparan sulfate proteoglycans (HSPGs) in tau strain propagation by examining conditional knockout mice lacking EXT1/EXT2, enzymes essential for HSPG synthesis. The scientific rationale stems from growing evidence that HSPGs serve as cofactors for tau seeding and spreading betwe

METHODOLOGY NOTES

**Phase 1: Animal Preparation and Genotyping (Weeks 1-2)** • Generate EXT1/EXT2 floxed mice crossed with GFAP-Cre or Aldh1l1-Cre lines for astrocyte-specific conditional knockout (n=60 total) • Genotype all animals using PCR with primers for floxed alleles and Cre recombinase • Randomly assign to groups: Control (Cre-negative, n=15), EXT1 cKO (n=15), EXT2 cKO (n=15), EXT1/EXT2 double cKO (n=15) • Age mice to 8-10 weeks for baseline mature astrocyte populations **Phase 2: Tau Strain Preparation and Stereotactic Injection (Week 3)** • Prepare recombinant PSP-tau and CBD-tau fibrils at 2 μg/μL concentration • Perform stereotactic injection into hippocampus (AP: -2.0mm, ML: ±1.5mm, DV: -1.8mm) • Inject 2 μL of tau fibrils unilaterally, contralateral hemisphere receives vehicle control • Post-surgical monitoring for 7 days with analgesics as needed **Phase 3: Pharmacological HSPG Degradation Treatment (Weeks 4-8)** • Administer heparinase III (10 U/kg) via intracerebroventricular injectio