Friday, November 22, 2024
11/22/2024
Project Summary Of the several metabolic and immunological privileges that the brain enjoys, autonomy in cholesterol regulation is one of the important features. This privilege is afforded by the presence of the blood brain barrier (BBB) that is composed of a monolayer of brain microvasculature endothelial cells (BMVEC) which have cholesterol-rich tight junctions (TJ). It is not known if the central nervous system or the vasculature provides cholesterol to the BMVEC. Recent studies show that apolipoprotein E (apoE)-null mice display severely increased BBB permeability, which is aggravated with aging. ApoE is polymorphic with apoE3 considered anti- atherogenic and apoE4 associated with risk for Alzheimer's disease (AD). Both serve as ligands for lipoprotein receptor mediated endocytosis thereby lowering plasma cholesterol. We hypothesize that apoE plays a critical role in maintaining the integrity of the BBB in an isoform-specific manner by providing cholesterol to the TJ in BMVEC. In parallel, we propose that age-related oxidative stressors impair the ability of apoE to mediate the function of supplying cholesterol thereby compromising the BBB integrity. To test this hypothesis, in Aim 1 we will evaluate the isoform-specific role of apoE in maintaining the BBB integrity using polarized BMVEC as mono- or co-cultures with astrocytes secreting apoE3- or apoE4- high density lipoprotein (HDL). They will be treated with apoE3 or apoE4-HDL with fluorescent cholesterol supplied from the basolateral or apical sides and cell associated fluorescence visualized by fluorescence microscopy using TJ markers and quantified by flow cytometry. TJ complexes will be captured by immunoprecipitation to quantify levels of TJ markers and cholesterol. Lastly, cellular internalization of cholesterol will be evaluated to determine if there are preferred uptake routes via the LDLr family. In Aim 2, the effect of oxidized apoE3 and apoE4 on uptake and TJ integrity will be followed, with additional focus on scavenger receptors that play a role in internalizing oxidized lipoproteins. This is relevant since heightened oxidative stress is considered one of the hallmarks of aging. In Aim 3, the apoE isoform- and oxidative stress-related alterations to the proteomic and lipidomic profiles of TJ complexes will be determined by mass spectrometry. It allows us to develop an interactome map and identify candidate proteins that may be associated with the aberrant behavior of apoE4. A diverse group of undergraduate and graduate students will be involved from experimental design to data collection and analysis, manuscript preparation and conference presentations. An HSI, CSULB has seen a transformative change in research culture after ~10 years of the NIH Building Infrastructure Leading to Diversity (BUILD) initiative. It is well suited to engage a large number of students from diverse backgrounds in meaningful research leading to careers in biomedical sciences. Employing innovative and inter-disciplinary approaches, findings from this proposal will provide timely opportunities to identify potential mechanism-based intervention strategies to address AD that affects millions of aging populations in the US and elsewhere.
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