Probing Neurovascular Unit Dysfunction in Alzheimer’s Disease

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. Nearly 80% of individuals with AD present with cerebrovascular pathologies such as microinfarcts, atherosclerosis, and cerebral microbleeds. The brain vasculature, which delivers oxygen and nutrients to surrounding neurons, is regulated by the neurovascular unit (NVU). The NVU is a multicellular system composed of neurons, astrocytes, microglia, pericytes, and brain endothelial cells (ECs). While NVU dysfunction is a hallmark of AD, the interactions between brain and vascular cell types remain incompletely understood. Current in vitro NVU models often lack perfusable 3D vasculature and omit critical cell types such as microglia, highlighting the need for more physiologically relevant systems. To address this gap, the body of work presented here reports a series of NVU models of increasing cellular complexity and applies them to investigate NVU dysfunction in AD. First, we examined the effects of AD neuronal secretomes on ECs using an engineered microvessel system. We perfused microvessels with conditioned medium (CM) generated from induced pluripotent stem cell (iPSC)-derived neurons containing an AD mutation that increased amyloid-beta (Aβ) production. We observed that increased Aβ production by AD neurons strongly correlated with features of EC activation. Further, when we depleted Aβ from our CM through several methods, we saw that the EC activation we observed was attenuated. This study provided a direct link between the EC activation observed in AD and Aβ. It also established a model of indirect EC-neuron interactions in the NVU. We next investigated EC-microglia interactions by developing a suite of models, including a 2D co-culture system, a 3D microvessel model containing iPSC-derived microglia-like cells (iMGL), and a multicellular 3D NVU model comprising neurons, astrocytes, iMGL, and ECs. Using these systems, we found that iMGL supported EC structure and barrier integrity under basal conditions. We then applied an AD-mimicking neuroinflammatory stimulus, TNFα, to our microglia-vessel model and determined that iMGL could help ameliorate the EC inflammatory response and prevent EC barrier breakdown. Incorporating neurons and astrocytes into the model revealed that iMGL also enhanced neuronal morphology, highlighting their broader supportive roles within the NVU. These findings deepen our understanding of how microglia interact with the brain vasculature and provide models to uncover the mechanisms driving these interactions. Together, these studies provide novel, physiologically relevant models for probing NVU dysfunction in AD. Beyond advancing mechanistic understanding of neurovascular interactions, these platforms offer a foundation for therapeutic discovery and for examining the impact of disease genetics on NVU cell types.