Brain microvascular dysfunction is an important feature of Alzheimer’s disease (AD). In our analysis, we identified 168 proteins whose abundance was significantly increased, and no proteins that were significantly decreased in AD. The most highly increased proteins included amyloid beta, tau, midkine, SPARC related modular calcium binding 1 (SMOC1), and fatty acid binding protein 7 (FABP7). Additionally, Gene Ontology (GO) enrichment analysis identified the enrichment of increased proteins involved in cellular detoxification and antioxidative responses. A systematic evaluation of protein functions using the UniProt database identified groupings into common functional themes including the regulation of cellular proliferation, cellular differentiation and survival, inflammation, extracellular matrix, cell stress responses, metabolism, coagulation and heme breakdown, protein degradation, cytoskeleton, subcellular trafficking, cell motility, and cell signaling. This suggests that AD brain microvessels exist in a stressed state of increased energy demand, and mount a compensatory response to ongoing oxidative and cellular damage that is associated with AD. We also used public RNAseq databases to identify cell-type enriched genes that were detected at the protein level and found no changes in abundance of these proteins between control and AD groups, indicating that changes in cellular composition of the isolated microvessels were minimal between AD and no-AD groups. Using public data, we additionally found that under half of the proteins that were significantly increased in AD microvessels had concordant changes in brain microvascular mRNA, implying substantial discordance between gene and protein levels. Together, our results offer novel insights into the molecular underpinnings of brain microvascular dysfunction in AD.

To better understand the brain microvascular molecular signatures of AD, we processed and analyzed isolated human brain microvessels by data-independent acquisition liquid chromatography with tandem mass spectrometry (DIA LC–MS/MS) to generate a quantitative dataset at the peptide and protein level. Brain microvessels were isolated from parietal cortex grey matter using protocols that preserve viability for downstream functional studies. Our cohort included 23 subjects with clinical and neuropathologic concordance for Alzheimer’s disease, and 21 age-matched controls.

All tissues were derived from brains donated for research from participants in the University of Washington (UW) Neuropathology Core. Human brain samples were collected on a continuous basis from the UW BioRepository and Neuropathology (BRaIN) laboratory and Precision Neuropathology Core, which performs rapid autopsies (postmortem interval approximately 12 h or less). For this project, upon brain removal in a rapid autopsy, a portion of the superior parietal lobule was dissected and cryostored at -80°C or processed immediately after dissection without freezing. Human microvessels (MV)s were isolated from the deidentified parietal cortex. Briefly, cerebral cortex was dissected away from subcortical white matter in cold media with 5% FBS on ice and homogenized in a Dounce type homogenizer and centrifuged at 2000g for 5 min at 4°C. Supernatant was subsequently drawn off and clean absorbent pads used to remove residual supernatant/media. The pellet was resuspended in 10 ml of 15% (w/v) dextran and centrifuged at 10,000g for 20 min at 4°C. The pellet containing enriched brain microvessels was resuspended in 1 ml Dulbecco’s Phosphate Buffered Saline, transferred to a 40 µm cell strainer and washed with 10ml of cold DPBS to remove single cells such as red blood cells. Finally, the strainer was reversed and microvessels retrieved using 2–3 ml of media or DPBS with 0.5% (w/v) BSA, then rinsed with DPBS, and centrifuged at 2000g for 3 min at 4 °C. MV pellets were washed three times in sterile DPBS to remove BSA prior to proteomic sample processing.