A single nuclear transcriptomic characterization of mechanisms responsible for impaired angiogenesis and blood-brain barrier function in Alzheimer’s disease

Brain perfusion and blood-brain barrier (BBB) integrity are reduced early in Alzheimer’s disease (AD). We performed single nucleus RNA sequencing of vascular cells isolated from AD and non-diseased control brains to characterize pathological transcriptional signatures responsible for this. We found that endothelial cells (EC) are enriched for expression of genes associated with susceptibility to AD. Increased β-amyloid is associated with BBB impairment and a dysfunctional angiogenic response related to a failure of increased pro-angiogenic HIF1A to increased VEGFA signalling to EC. This is associated with vascular inflammatory activation, EC senescence and apoptosis. Our genomic dissection of vascular cell risk gene enrichment provides evidence for a causal role of EC pathology in AD and suggests that reducing vascular inflammatory activation and restoring effective angiogenesis could reduce vascular dysfunction contributing to the genesis or progression of early AD. Samples from the prefrontal cortex were processed for microvessel single nuclei isolation. All steps were carried out on ice or at 4oC. Tissue was processed to first isolate microvessels and then to release single nuclei for sequencing. Tissue was dounce homogenized and then centrifuged at 1000g for 3 min. Pellets were re-suspended in 5.3% dextran and overlayed on a pre-prepared dextran gradient (4%, 12%, 16%). After centrifugation the bottom layer was subject to a second dextran gradient spin, 4200g for 20 min. The pellets were resuspended and passed through a 100um filter and then a 40um filter. The 40um filter was inverted and centrifuged at 800g for 8 minutes. The isolated microvessels were incubated with Collagenase for 20 minutes and nuclei released by grinding with a pestle and filtering through a 20um filter. *************************************************************** Submitter states that raw files are not in SRA due to patient privacy concerns. For raw files, visit the project space in https://www.synapse.org/#!Synapse:syn36812517/wiki/619350 ***************************************************************