Alzheimer’s disease (AD) is a progressive neurodegenerative disease likely caused by a combination
of both genetic and environmental factors. Of the genetic risk factors identified, the 34 kDa protein,
apolipoprotein (apo) E4, is of significance importance as APOE4 carriers account for 65-80% of all
AD cases. Although apoE4 plays a normal role in lipoprotein transport, how it contributes to AD
pathogenesis is currently unknown. Emerging data suggests that apoE4 is sensitive to proteolytic
cleavage and thus contributes to the underlying molecular pathology associated with AD possibly
through a loss of function. Using a site-directed antibody to cleaved apoE4 we have recently
determined an amino-terminal cleavage fragment of apoE4 of 17 KDa can be formed following
incubation of full-length apoE4 with matrix metalloproteinase-9 (MMP-9) that localizes to the nucleus
of microglia of the human AD brain. The goal of this proposal is to expand those findings by
determining the mechanisms by which a recombinantly-produced, His-tagged fragment of apoE4
(nApoE41-151) is taken up by microglia, traffics to the nucleus, and alters gene expression. We
hypothesize that the trafficking of this fragment to the nucleus serves a pathophysiological function
that regulates the expression of genes related to microglia activation and cell death. Experiments
described in Aim 1a will rigorously test the hypothesis that nApoE41-151 is taken up by cells through a
specific receptor-mediated pathway involving the low-density lipoprotein (LDL) receptor or the LDL
receptor-related protein (LRP-1). In Aim 1b, parallel experiments will also assess whether trafficking
of nApoE41-151 to the nucleus occurs aided by the use of our site-directed cleavage antibody that
specifically detects this apoE4 fragment as well as an anti-His antibody. We propose experiments to
elucidate the pathway by which nApoE41-151 traffics from the cytoplasm to the nucleus, presumably
following receptor-mediated endocytosis.
Experiments outlined in Aim 2 will determine what transcriptional effects if any this apoE4 fragment
has once localized within the nucleus. In Aim 2a, we will first characterize the potential binding of
nApoE41-151 to a possible enhancer region of nuclear DNA 5’ to a novel, uncharacterized gene that
was isolated following chromatin immunoprecipitation in preliminary studies. These experiments will
analyze the potential binding kinetics, in vitro, utilizing several techniques that allow for the
characterization of interactions of the DNA sequence and nApoE41-151. In addition, in Aim 2b, we will
determine if this sequence serves a potential regulator of transcription using a luciferase reporter
gene construct. Finally, in Aim 2c we will examine overall gene expression in BV2 microglial cells
following treatment with nApoE41-151 following purification of RNA samples and transcriptome
analysis. Because microglia produce apoE4, our hypothesis is this fragment is part of a feedback
loop that regulates the expression of APOE4 gene or may lead to transcriptional regulation of other
genes that contribute to cell death or microglia activation. Data from this proposal could uncover a
novel pathophysiological role for apoE4 and lead to a better understanding as to why inheritance of
this gene enhances AD risk.