Experiment Proposal (Crux): What is the evidence that blood-brain barrier (BBB) permeability changes serve as early biomarkers f [60426-210049]

AIMS

HYPOTHESES

PROTOCOL SUMMARY

Step 1: Deconvolve CAV1+ endothelial cell subpopulations from SEA-AD snRNA-seq using reanalysis with caveolin-1 as a discriminating feature, comparing NC vs early-AD vs late-AD subgroups; compute co-expression modules of CAV1 with claudin-5, PDGFR-β, GLUT1, and inflammatory genes (IL1B, TNF-α) per cell. Step 2: Stratify donors by CAV1 co-expression signature (CAV1-high-inflammatory vs CAV1-high-barrier-protective) and test for associations with ante-mortem DCE-MRI BBB leakage data and CSF/plasma GFAP, NFL, and S100B levels where available. Step 3: Validate CAV1 spatial resolution in post-mortem BA9/MTG tissue from matched donors using multiplexed RNAscope + immunofluorescence for claudin-5, PDGFR-β, and amyloid/tau pathology burden. Step 4: In an in-vitro BBB-on-chip model (iPSC-derived brain endothelial/pericyte co-culture), modulate CAV1 expression via CRISPRi (loss-of-function) and CAV1-OE (gain-of-function) to test (a) baseline trans-endothelial electrical resistance and fluorescein flux, (b) response to inflammatory challenge (TNF-α/IL-1β), and (c) recovery kinetics after insult removal. Step 5: Quantify CAV1 phosphorylation status (Y14 vs S80) in AD vs control endothelial nuclei as a biochemical read-out distinguishing caveolae-mediated transcytosis (pathogenic) from signalingadaptation (compensatory).

PREDICTED OBSERVATIONS

If CAV1 is compensatory (H1 true): CAV1-high endothelial cells will co-express barrier-preserving genes (GLUT1, SLC2A1; PDGFR-β), cluster in early-AD Braak stage donors, precede detectable GFAP/NFL elevation in CSF, and show increased TEER after CAV1-OE in-vitro with reduced post-inflammatory leakage. If CAV1 is pathogenic (H2 true): CAV1+ cells will co-express pro-inflammatory modules and vesicular trafficking genes, correlate with DCE-MRI leakage within the same donor, increase monotonically with late-AD stages, and CAV1-OE will accelerate barrier breakdown and increase fluorescein flux after cytokine challenge.

FALSIFICATION CRITERIA

CAV1 upregulation would be confirmed as non-compensatory (H2) if: (a) CAV1+ endothelial cells in early-AD show no barrier-preserving gene co-expression but instead show CD31 downregulation and IL1B/TNF co-induction; (b) CAV1 expression correlates positively with concurrent DCE-MRI leakage, not preceded by it; (c) CAV1-OE in-vitro fails to improve TEER under any condition and accelerates fluorescein flux. Conversely, CAV1 is confirmed compensatory if: (a) CAV1+ cells cluster by GLUT1+/PDGFR-β+ signature in early-AD before detectable GFAP/NFL elevation; (b) DCE-MRI leakage is greater in CAV1-low endothelial clusters within the same donor; (c) CAV1-OE reduces transcytosis flux and improves recovery post-inflammatory challenge.

DATASET DEPENDENCIES