Targeting cerebrovascular endothelial cells as a therapeutic approach for amyloid pathogenesis

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 DESCRIPTION (provided by applicant): Targeting cerebrovascular endothelial cells as a therapeutic approach for amyloid pathogenesis. The accumulation of amyloid-β (Aß) in the brain blood vessels can result in the development of cerebral amyloid angiopathy (CAA). CAA is a pathological feature present concomitantly with Alzheimer's disease (AD) at a high frequency, highlighting a potentially important role for vascular Aß in dementias such as AD. While the basis by which Aß mediates deleterious effect on the blood-brain barrier (BBB) is likely multifactorial, numerous studies indicate a role for Aß mediated increases in endothelial cell permeability. The exact causes for BBB dysfunction in CAA are not well known, however impaired clearance of Aß from the brain across the BBB as well as a reduction in the efficacy of the perivascular drainage of Aβ have been proposed to enhance accumulation of cerebrovascular and parenchymal amyloid deposits in the elderly. Despite our understanding of the pathways responsible for BBB dysfunction and clearance of Aß, the availability of drugs to treat Aß pathogenesis related disorders, CAA and AD, remains lacking. The long-term goal of this project is to develop therapeutics that target the BBB to restore its function and maximize clearance of Aß from the brain, which is important to prevent or delay onset of CAA and AD. The overall goal of this project is to fully characterize an experimental endothelial BBB model as an effective high-throughput screening (HTS) format to identify therapeutics for vascular Aß pathogenesis disorders (AD, CAA and vascular dementia). The central hypothesis is that high-throughput screening (HTS) utilizing a highly novel cerebrovascular endothelial BBB model can be used to identify small molecules which beneficially regulate Aβ clearance and reduce Aβ mediated increases in BBB permeability. We will test this hypothesis by pursuing the following specific aims: 1) Utilization of a cell line-based BBB model to screen for modulators of Aβ mediated disturbances of endothelial cell function. This aim will be accomplished by investigating the following sub-aims: 1A) screen for compounds, in the presence of Aβ42 oligomers, for their effect on the gross permeability of cerebrovascular endothelial cells using Lucifer Yellow (LY) as a gross permeability marker. Compounds which reduce Aβ-mediated permeability will be advanced to Sub-Aim 1B; 1B) identify stimulators of Aβ clearance across the BBB model using the gold standard measure of iodinated Aβ as the endpoint for Aβ clearance. Hit compounds will be further examined in Aim 2. 2) Validation and mechanistic investigation of hit compounds from Aim1 for their ability to modulate expression of tight junction and Aβ clearance proteins (transport and degradation). This aim will be accomplished by testing the following sub-aims: 2A) conduct secondary confirmation and establish profiles for hits identified in Aim 1, 2B) comparison of hits kinetics for amelioration of Aβ42 oligomers induced permeability and reduced Aβ clearance in primary cerebrovascular endothelial cells with those of Sub-Aim 2A, 2C) mechanistic investigation for improved BBB tightness, integrity and Aβ clearance by hit compounds selected from 2B. 3) Test ability of the top 2 hits from Aim 2 to in vivo modulate vascular and parenchymal Aβ accumulation, and BBB integrity in a mouse model of CAA. Methods and techniques to be used to accomplish the above aims include in vitro cell culture, high-throughput screening, transport, permeability and clearance studies, Aβ kinetics, microvessels isolation from brains of wild type mice, and in vivo studies in CAA model. The data produced will provide candidate therapeutic molecules to test in future clinical studies.