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

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. 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. This project will generate validated human pluripotent stem cells (PSC)-derived NVU cells, develop a perfused microphysiological NVU 3D 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. We have used CRISPR/Cas9 methodology to knock-in FAD or LOAD mutations in control PSC cell lines, and developed strategies to differentiate PSC-derived cells into brain microvascular endothelial cells (BMECs), pericytes or

astrocytes and build brain-on-a-chip models with NVU cells and flow. Moreover, our preliminary single nucleus RNA-seq of human AD brains has identified 4 distinct BMEC populations, at the transcriptome level, one of which is positively correlated with cognitive impairment, A and tau accumulation. We hypothesize that synergistic

interactions between genetic and environmental risk factors impair NVU function and BBB properties by altering key neurovascular signaling 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 primed ECs, verify their

molecular identity with single cell RNA-seq and validate their biological function using relevant cellular, imaging and functional approaches. We will incorporate BMECs into an NVU microfluidic 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 isolate the vasculome compartment from human post-mortem brains and assess differences in vasculome profiles from control and AD brains. We will evaluate whether AD-associated transcriptional changes in BMECs, pericytes

and astrocytes in vivo are present in the microphysiological NVU 3D system derived from hPSC lines carrying AD-associated risk genes. Finally in Aim 3, we will assess whether treatment with proteasome inhibitors (to mimic loss of proteostatis), agents that produce advanced glycation end products, or dynamic flow of aged versus

young blood from control or AD patients alters the morphology and transcriptome profiles of NVU cells and transport across the BBB. This Diversity Supplement will allow us to extend these studies to characterize LOAD mutant astrocytes more deeply as well as see how induction of senescence modulates these phenotypes both

in monolayer and in 3D NVUs. 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 the interactions between genetic and environment factors for AD vascular pathology.