Developing a microfluidic human neurovascular unit system to investigate genetic and age-related risk factors in Alzheimer's disease

项目总结

PROJECT SUMMARY Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with severe impairment of memory, cognition and executive functions that has a tremendous health and economic burden. The lack of effective treatments to halt AD pathology necessitates development of human brain microphysiological systems to understand disease pathogenesis and develop potential therapeutics. Neurovascular unit (NVU) and blood-brain barrier (BBB) dysfunction play a key etiological role in AD progression; yet the contribution of genetic versus environmental risk factors for brain microvascular damage is unclear. In response to RFA (# PAR-20-055) this project will generate validated human pluripotent stem cells (PSC)-derived NVU cells, develop a perfused NVU 3D microphysiological system with plasma or blood from young or aged patients and compare its transcriptome profiles with the human AD brain vasculome to dissect the contribution of genetic versus age-related factors in AD. In preliminary studies, we have used CRISPR/Cas9 methodology to knock-in FAD or LOAD mutations in control PSC cell lines, developed strategies to differentiate PSC into brain microvascular endothelial cells (BMECs), pericytes or astrocytes and build brain-on-a-chip models with NVU cells and flow. In addition, our preliminary single nucleus RNA-seq of 24 human control and AD brains has identified 4 distinct BMEC populations, at the transcriptome level, one of which is positively correlated with cognitive impairment, Ab and tau accumulation. We hypothesize that synergistic interactions between genetic and environmental risk factors impair NVU function and BBB properties by altering key cellular pathways or transcription factors. We will address this hypothesis with three aims. In Aim 1, we will optimize protocols to generate BMECs via transdifferentiation from either endothelial progenitor cells or ECs, verify their molecular identity with single cell RNA-seq and validate their biological function using cellular, biochemical, imaging and functional approaches. We will incorporate BMECs into an NVU 3D microphysiological system along with hPSC-derived pericytes and astrocytes and compare cell biological, transcriptome, imaging and functional barrier properties between NVUs carrying AD-associated risk genes and isogenic controls. In Aim 2, we will leverage the data from the AMP-AD database to identify the AD brain vasculome-specific profiles associated with cognitive impairment, Ab and tau accumulation and evaluate whether AD-associated brain vasculome changes in vivo are present in the NVU 3D microphysiological system derived from hPSC lines carrying AD-associated risk genes. Finally in Aim 3, we will assess whether treatment with proteasome inhibitors that mimick loss of proteostatis, agents that produce advanced glycation end products, or dynamic flow of aged blood alters the morphology and transcriptome profiles of NVU cells and BBB transport. The proposed studies will establish a novel perfused blood/NVU 3D microphysiological system that will allow us to leverage its relevance to changes in the AD brain vasculome and examine interactions between genetic and environment factors for AD vascular pathology.