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

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 astrocytic endfeet being essential for efficient cerebrospinal fluid flow and protein clearance. The hypothesis is that loss of AQP4 function impairs tau clearance and exacerbates strain-specific spreading patterns characteristic of Progressive Supranuclear Palsy (PSP) and Corticobasal Degeneration (CBD). The experimental design involves injecting human-derived PSP and CBD tau strains into the substantia nigra and motor cortex of AQP4 knockout mice, followed by pharmacological rescue experiments using compounds that restore AQP4 polarization. Advanced imaging techniques will track tau propagation over time, while biochemical assays will quantify tau burden and clearance rates.

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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 astrocytic endfeet being essential for efficient cerebrospinal fluid flow and protein clearance. The hypothesis is that loss of AQP4 function impairs tau clearance and exacerbates strain-specific spreading patterns characteristic of Progressive Supranuclear Palsy (PSP) and Corticobasal Degeneration (CBD). The experimental design involves injecting human-derived PSP and CBD tau strains into the substantia nigra and motor cortex of AQP4 knockout mice, followed by pharmacological rescue experiments using compounds that restore AQP4 polarization. Advanced imaging techniques will track tau propagation over time, while biochemical assays will quantify tau burden and clearance rates. The rescue component will test whether re-establishing proper AQP4 function can ameliorate tau pathology, providing mechanistic insights into the relationship between glymphatic dysfunction and tauopathy progression. This study addresses fundamental questions about how astrocytic water homeostasis influences neurodegenerative disease progression and whether targeting AQP4 function represents a viable therapeutic approach.

This experiment directly tests predictions arising from the following hypotheses:

• SASP-Driven Aquaporin-4 Dysregulation
• Aquaporin-4 Polarization Rescue
• Glymphatic System-Enhanced Antibody Clearance Reversal
• Circadian Glymphatic Entrainment via Targeted Orexin Receptor Modulation
• Aquaporin-4 Polarization Enhancement via TREK-1 Channel Modulation

Experimental Protocol

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 FRET pairs

Phase 3: Stereotaxic Tau Injection and AQP4 Modulation (Week 4)
• Anesthetize mice with isoflurane and perform stereotaxic injection of tau fibrils (2μL, 1μg total) into left hippocampus (coordinates: -2.0mm AP, -1.5mm ML, -2.0mm DV)
• Administer vehicle or TGN-020 (AQP4 modulator, 30mg/kg i.p. daily) starting 1 day post-injection
• Monitor injection sites via bioluminescence imaging in subset of mice receiving fluorescently-labeled tau
• Perform post-surgical monitoring and analgesic administration for 3 days

Phase 4: Longitudinal Assessment (Weeks 5-16)
• Conduct weekly behavioral testing: rotarod performance, Y-maze spontaneous alternation, and Morris water maze (weeks 8, 12, 16)
• Collect CSF samples via cisterna magna puncture at 4, 8, and 12 weeks post-injection (n=6 per group per timepoint)
• Perform MRI imaging at 8 and 16 weeks to assess brain volume and ventricular size
• Monitor body weight and general health status biweekly

Phase 5: Terminal Analysis and Tissue Processing (Week 17)
• Sacrifice mice via transcardial perfusion with 4% paraformaldehyde
• Collect brain tissue and process for: immunohistochemistry (AT8, MC1, AQP4), Western blotting, and biochemical fractionation
• Perform stereological quantification of tau pathology in hippocampus, entorhinal cortex, and connected regions
• Analyze CSF samples for total tau, phospho-tau, and inflammatory markers via ELISA and Simoa assays

Expected Outcomes

Increased tau pathology spread in AQP4 knockout mice: AQP4-/- mice will show 40-60% greater tau burden (AT8+ area) compared to wild-type mice at 12 weeks post-injection with both PSP and CBD strains (p<0.01, effect size d>0.8)

Impaired CSF tau clearance in knockout mice: AQP4-/- mice will demonstrate 2-3 fold higher CSF tau concentrations at all timepoints compared to wild-type controls, with area under the curve analysis showing significantly delayed clearance kinetics (p<0.001)

Differential strain-specific spreading patterns: PSP tau will preferentially spread to motor cortex and brainstem regions while CBD tau will show greater cortical involvement, with this pattern being exaggerated 2-fold in AQP4-/- mice compared to controls

Pharmacological rescue with TGN-020: Treatment with AQP4 modulator will restore perivascular AQP4 polarization and reduce tau pathology by 30-50% in both genotypes, with greater rescue efficacy in wild-type mice (interaction p<0.05)

Behavioral deficits correlating with pathology: AQP4-/- mice will show significantly worse performance on rotarod (20-30% reduction) and spatial memory tasks (15-25% impairment) that correlates with regional tau burden (r>0.6, p<0.01)

Ventricular enlargement in knockout mice: MRI analysis will reveal 15-25% larger ventricular volume in AQP4-/- mice by 16 weeks, indicating compromised CSF dynamics and brain atrophy (p<0.001, Cohen's d>1.0)

Success Criteria

• Statistical power requirement: Achieve >80% power to detect 30% difference in tau pathology between genotypes with α=0.05, confirmed by post-hoc power analysis

• Genotype validation: >95% accuracy in AQP4 genotyping confirmed by both PCR and Western blot, with complete absence of AQP4 protein in knockout mice

• Tau injection success: >90% of injected mice show detectable tau pathology at injection site by week 4, with spreading to at least 2 connected brain regions by week 12

• AQP4 modulator efficacy: TGN-020 treatment must demonstrate >50% restoration of perivascular AQP4 polarization index compared to vehicle controls (p<0.01)

• CSF collection success: Successful CSF collection from >80% of planned samples with <5% hemolysis contamination and detectable tau levels above assay sensitivity (0.1 pg/mL)

• Reproducible strain differences: PSP and CBD tau strains must show statistically distinct spreading patterns (p<0.05) and regional preference indices differing by >2-fold between strain types