Dynamics of Tau protein, Amyloid beta oligomer, and APOE isoforms at the neurovascular unit

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This supplement will establish the utility of the µSiM-hNVU platform for mechanistic and translational studies on the mutant proteins tau, amyloid beta oligomer (Aβ), and apoE4 and their roles in blood-brain barrier (BBB) dysfunction and neuroinflammation at the neurovascular unit (NVU) in Alzheimer’s Disease (AD). The µSiM- hNVU (microphysiological system featuring a silicon membrane) is an in vitro platform we developed under the parent grant (R61/R33 HL154249) as a tool to study neurovascular injury mechanisms during sepsis (McCloskey et al., Adv Health Mater. 2022, e2200804). The µSiM is uniquely capable of high resolution and live cell microscopy and provides superior exchange of soluble factors between ‘blood’ and ‘brain’ compartments compared to other tissue chip platforms. While less is known about the role of tau proteins in the genesis of AD vascular pathology than Aβ and apoE4, there is intriguing evidence that interactions between all three factors drive neuroinflammation in the NVU microenvironment. These interactions likely relate to the common ability of these mutant proteins to bind the low-density lipoprotein receptor–related protein 1 (LRP1). The studies here will utilize the µSiM-hNVU to examine both direct effects, and competition, between Aβ, tau and apoE4, to alter BBB function, Aβ clearance, and microglial activation (as a marker of neuroinflammation). The supplement will leverage the full suite of µSiM-compatible assays and cellular resources developed in the parent award including iPSC derived quad cultures featuring endothelial cells, pericytes, astrocytes and microglia isogenic for APOE ε alleles 3/3 or 4/4. Aim 1 will examine the impact of Aβ1-42, tau and NVU APOE ε genotypes on BBB function. Studies will add Aβ1- 42 oligomers and/or tau proteins at a range of concentration to the brain side of the µSiM-hNVU while monitoring small molecule permeability, cytokine production and cellular activation. Molecular mechanisms associated with the strongest combined d microglial and BBB responses will be identified through single cell/nucelli sequencing of all 4 cell types isolated from the µSiM-hNVU. Aim 2 will assess the effect of tau and apoE3 & 4 on Aβ1-42 transport across the BBB. Tau mutants, in monomeric, pre-formed fibrils and hyper- phosphorylated forms and recombinant apoE3 & 4 proteins at a range of concentrations will be added to the brain side of the µSiM-hNVU along with a fixed concentration of Aβ1-42 oligomer to study efflux of the Aβ oligomer to the blood side and influx to the brain side. To examine the role of LRP1, we will generate an LRP1 knock out in hiPSCs with CRISPR and generate both endothelial cells and pericytes. We expect this supplement to establish the µSiM-hNVU as a new tool for mechanistic and translational studies in AD and provide preliminary data on the complex interactions of key protein risk factors driving AD-associated neuroinflammation.