Role Of Apolipoprotein E In Maintaining the Blood Brain Barrier Integrity - 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.
