The generation and function of soluble apoE receptors in the CNS.

Abstract

More than a decade has passed since apolipoprotein E4 (APOE-epsilon4) was identified as a primary risk factor for Alzheimer 's disease (AD), yet researchers are even now struggling to understand how the apolipoprotein system integrates into the puzzle of AD etiology. The specific pathological actions of apoE4, methods of modulating apolipoprotein E4-associated risk, and possible roles of apoE in normal synaptic function are still being debated. These critical questions will never be fully answered without a complete understanding of the life cycle of the apolipoprotein receptors that mediate the uptake, signaling, and degradation of apoE. The present review will focus on apoE receptors as modulators of apoE actions and, in particular, explore the functions of soluble apoE receptors, a field almost entirely overlooked until now.

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BioMed Central
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Molecular Neurodegeneration
Open Access
Review
The generation and function of soluble apoE receptors in the CNS
G William Rebeck1, Mary Jo LaDu2, Steven Estus3,4, Guojun Bu5,6,7 and
Edwin J Weeber*8,9,10
Address: 1Department of Neuroscience, Georgetown University, Washington DC, USA, 2Department of Anatomy and Cell Biology, University of
Illinois at Chicago, Chicago, USA, 3Department of Physiology, University of Kentucky, Lexington, USA, 4Sanders-Brown Center on Aging,
University of Kentucky, Lexington, USA, 5Department of Pediatrics, Washington University, St. Louis, USA, 6Department of Cell Biology and
Physiology, Washington University, St. Louis, USA, 7Hope Center for Neurological Disorders, Washington University, St. Louis, USA, 8Department
of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, USA, 9Department of Pharmacology, Vanderbilt University, Nashville,
USA and 10Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, USA
Email: G William Rebeck - gwr2@georgetown.edu; Mary Jo LaDu - maryjo03@comcast.net; Steven Estus - sestus2@email.uky.edu;
Guojun Bu - bu@kids.wustl.edu; Edwin J Weeber* - edwin.j.weeber@vanderbilt.edu
* Corresponding author
Abstract
More than a decade has passed since apolipoprotein E4 (APOE-ε4) was identified as a primary risk
factor for Alzheimer 's disease (AD), yet researchers are even now struggling to understand how
the apolipoprotein system integrates into the puzzle of AD etiology. The specific pathological
actions of apoE4, methods of modulating apolipoprotein E4-associated risk, and possible roles of
apoE in normal synaptic function are still being debated. These critical questions will never be fully
answered without a complete understanding of the life cycle of the apolipoprotein receptors that
mediate the uptake, signaling, and degradation of apoE. The present review will focus on apoE
receptors as modulators of apoE actions and, in particular, explore the functions of soluble apoE
receptors, a field almost entirely overlooked until now.
Background
ApoE and apoE receptors
Apolipoprotein E (apoE) is a small (34-kDa) secreted
glycoprotein that associates with lipoproteins and medi-
ates uptake of these particles into target cells via receptor-
mediated endocytosis by the low density lipoprotein
(LDL) receptor family. Three commonly occurring iso-
forms have been identified in the human population due
to single nucleotide polymorphisms on the APOE gene on
chromosome 19. The apoE3 isoform (Cys112, Arg158)
occurs at the highest frequency, followed by apoE4
(Arg112, Arg158) and apoE2 (Cys112, Cys158). ApoE is a
ligand for the seven identified mammalian members of
the evolutionarily conserved low density lipoprotein
(LDL) receptor family: the low density lipoprotein recep-
tor (LDLR), apoE receptor 2 (ApoER2), the very low den-
sity lipoprotein receptor (VLDLR), multiple epidermal
growth factor (EGF) repeat-containing protein (MEGF7),
megalin, LDL-related protein-1 (LRP1) and LDL-related
protein-1b (LRP1b). APOE was initially recognized for its
importance in lipoprotein metabolism and cardiovascular
disease, however, more recently it has been studied for its
role in several biological processes not directly related to
lipoprotein transport. The following review will focus on
the role of apoE and apoE receptors in the CNS with a
focus on the processing and production of soluble apoE
receptors.
Published: 24 October 2006
Molecular Neurodegeneration 2006, 1:15 doi:10.1186/1750-1326-1-15
Received: 02 October 2006
Accepted: 24 October 2006
This article is available from: http://www.molecularneurodegeneration.com/content/1/1/15
© 2006 Rebeck et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Genetics of apoE and Alzheimer's disease
Alzheimer's disease (AD) is a complex neurodegenerative
condition characterized neuropathologically by the pres-
ence of extracellular amyloid plaques, intraneuronal neu-
rofibrillary tangles, and neuronal loss. While most AD is
sporadic in nature, two classes of genetic risk factors for
AD have been identified. The first class consists of muta-
tions responsible for the rare, familial AD (FAD). Genes
implicated in FAD include the β-amyloid protein precur-
sor (APP), presenilin-1, and presenilin-2; mutations in
these genes produce AD risk marked by autosomal domi-
nance, early disease onset, high penetrance, and relative
rarity, on the order of hundreds of families worldwide
(reviewed in [1]). Each of the mutated forms of these
genes enhances the production of amyloid-β (Aβ) peptide
[2], particularly the 42 amino acid form (Aβ42). Because
Aβ42 aggregates to form amyloid, the discovery of these
genes strongly supports the "amyloid hypothesis" [2].
A second class of AD risk factor is genetic variation that
modulates the sporadic late onset AD; at present, the sole
member of this class is the APOE gene. APOE encodes a
secreted protein of 299 amino acids important for the
transport of cholesterol. Single nucleotide polymor-
phisms (SNP) define three common alleles (ε2, ε3, and
ε4), which encode proteins that differ at two amino acids.
APOE-ε2 is associated with reduced odds and delayed
onset of AD while APOE-ε4 is associated with increased
odds and earlier onset of AD [3]. APOE-ε4 appears to
account for up to 40–50% of the genetic risk of AD [4,5].
Furthermore, APOE-ε4 is associated with increased brain
Aβ in affected individuals [6]. The effects of APOE on Aβ
burden are seen in mice as well, with studies in mice defi-
cient for apoE or transgenic for human apoE supporting a
role for apoE in Aβ fibrillogenesis and neuritic plaque for-
mation [7,8]. Importantly, the risk associated with APOE-
ε4 is modulated by other unknown genetic and environ-
mental factors.
AD has a complex etiology that encompasses environ-
mental factors as well as the genetic risk factors. The exist-
ence of environmental risk factors is demonstrated by
multiple lines of evidence, including studies of twins.
Interestingly, it was found that only about half of identical
twins have concordance for AD [9,10]. Among the envi-
ronmental factors identified, cholesterol lowering treat-
ments (reviewed in [11,12]) and anti-inflammatory drugs
(reviewed in [13]) may decrease risk of AD. Alternatively,
analysis of post-mortem AD brains showed an increased
level of traumatic brain injury compared to normal non-
AD brains, suggesting that brain trauma may significantly
increase the risk of AD [14]. ApoE is potentially involved
in each of these environmental factors.
ApoE and apoE receptors in the CNS
ApoE and cholesterol transport in the CNS
In the periphery, there are numerous lipoprotein classes
and apolipoproteins. However, in the CNS, lipoproteins
are predominantly high density and do not include the
large classes of lower density lipoproteins found in the
plasma [15]. The two major apolipoproteins present in
the cerebral spinal fluid (CSF) are apoE and apoAI; classes
of high density lipoproteins in the CSF contain either or
both of these apolipoproteins [16]. These lipoproteins
can act either to deliver cholesterol to cells, or to remove
excess cholesterol from cells [17]. The additional role of
these proteins as signaling molecules will be discussed
later in this review.
ApoE and Aβ
The strongest associations of APOE genotype with disease
are with conditions containing amyloid deposition
including AD, Down's syndrome, cerebral amyloid angio-
pathy and head trauma [6,18-23]. In vivo, early evidence
for an involvement of apoE in AD came from immunohis-
tochemical localization of apoE to senile plaques [24].
ApoE4 increases levels of amyloid deposition in humans
[6,18] and accelerates amyloid deposition in transgenic
mice [8,25,26]. Recent research has focused on soluble
oligomeric assemblies of Aβ as the proximate cause of
neuronal injury, synaptic loss and the eventual dementia
associated with AD [27-31]. ApoE binds soluble Aβ oli-
gomers found in the brain, plasma and CSF [32,33]. In
vitro, apoE forms stable complexes with Aβ [34-37], alters
the aggregation of various Aβ peptides [38-40], modulates
Aβ-induced neuroinflammation [41-44], and promotes
Aβ clearance [45]. In addition, Aβ42 and apoE4 act syner-
gistically to reduce neuronal viability in vitro and ex vivo
as measured by neurotoxicity in primary cultures and
impaired long term potentiation (LTP) in hippocampal
slice cultures [46-51]. Each of these important functions is
partially mediated by apoE receptors.
ApoE receptors
ApoE interacts with members of the LDL receptor family
on the surface of cells. The LDLR family consists of over
ten receptors that function in receptor-mediated endocy-
tosis and cellular signaling (Figure 1) [52,53]. In addition
to the LDLR itself, the family includes LRP/LRP1 [54],
megalin/LRP2 [55], VLDLR [56], ApoER2/LRP8 [57-59],
SORLA-1/LR11 [60,61], LRP4 [62], LRP5 [63,64], LRP6
[65], and LRP1B [66]. The most characteristic structural
component of the LDLR family is the cysteine-rich ligand-
binding repeats forming ligand-binding domains [52,67].
Additionally, most members of the LDLR family contain
epidermal growth factor (EGF)-like repeats and YWTD
motifs, which form β-propeller-like structures [68]. A
common feature that is shared by most members of the
LDLR family is their ability to bind the receptor-associated

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protein (RAP) [69]. RAP is an endoplasmic reticulum
(ER)-resident protein that functions in receptor folding
and trafficking along the early secretory pathway and uni-
versally antagonizes ligand-binding to all members of the
family [69].
Many of the apoE receptors have been found in the CNS.
Neurons express LDLR, LRP, ApoER2, and the VLDLR;
astrocytes express LDLR and LRP; microglia express
VLDLR and LRP [6,57,70-73]. It is unclear which receptors
are expressed on oligodendrocytes. Soluble forms of each
of these receptors have been detected (see below).
ApoE receptors and endocytosis
A major function of at least some of the apoE receptors is
clathrin-mediated endocytosis. The rapid endocytosis rate
of LRP is unique among LDLR family members. The dom-
inant endocytosis signal for LRP is the YXXL motif [74].
The two copies of the NPXY motif, which mediates LDLR
endocytosis [75], do not play significant roles in LRP
endocytosis but likely bridge interactions with adaptor
proteins for signaling and intracellular trafficking. The ini-
tial endocytosis rates of individual LDLR family members
are significantly different with half time for internaliza-
tion ranging from <0.5 min (LRP) to > 8 min (ApoER2/
VLDLR) ([76], see Table 1). These results suggest endo-
cytic functions among LDLR family members are distinct.
In addition to endocytosis, LDLR family members also
exhibit efficient recycling. In particular, sorting nexin 17,
a member of the PX-domain containing, sorting nexin
family, interacts with the proximal NPXY motif of the LRP
tail and promotes its recycling in the early endosome [77].
Other adaptor proteins, specifically Dab-1 and FE65,
affect levels of surface apoE receptors [78]. Although sig-
nificant cholesterol from the periphery does not get trans-
ported into the CNS and sufficient cholesterol is
synthesized in the CNS [79], cholesterol redistribution is
important for transport of cholesterol from glia to neu-
rons [80] and for clearance of membrane debris after CNS
damages [81].
ApoE receptors and intracellular signaling
Recently, several studies have demonstrated a role for
some apoE receptors (specifically LRP, ApoER2, and
VLDLR) as signaling molecules (see [53,82] for review).
VLDLR and ApoER2 transduce signals from the extracellu-
lar matrix molecule Reelin, affecting neuronal cell migra-
tion during development [83]. LRP activation by ligand
binding affects NMDA receptor function [84-89]. These
effects are transduced through the cytoplasmic domains
of receptors binding various cytoplasmic adaptor and
scaffold proteins containing PID or PDZ domains, includ-
ing mammalian Disabled-1 (mDab1), mDab2, FE65,
JNK-interacting protein JIP-1 and JIP-2, and PSD-95 [90-
95]. ApoE receptor ligands also promote other intraneuro-
nal signals via apoE receptors, including PI3 activation,
ERK activation, and JNK inhibition [86,96,97] but exactly
which receptors promote which signals is unknown. ApoE
receptors on glia also affect signaling pathways, affecting
the state of glial activation [41,42,98].
ApoE receptors and synaptic plasticity
These receptor-mediated processes defined in vitro are
important for brain physiological functions. The apoE
receptor antagonist RAP prevents induction of long-term
potentiation (LTP) in hippocampal slices [69]. ApoER2
and VLDLR knock-out (KO) mice have normal baseline
synaptic transmission, as measured in acute hippocampal
slices, but have subtle impairment of hippocampal LTP
[83,99]. Moreover, Reelin application enhanced LTP
induction, which was dependent on the presence of both
ApoER2 and VLDLR [97]. A potential molecular mecha-
nism for this function of ApoER2 is a 59 amino acid cyto-
plasmic domain that is alternatively spliced. This ApoER2
splice variant interacts with PSD-95, which is itself associ-
ated with NMDA receptor conductance [87,95]. Knock-in
mice exclusively expressing ApoER2 receptors that lack the
59 amino acid insert exhibit decreased LTP induction, and
no enhancement of LTP in the presence of exogenous Ree-
lin [87]. Thus, the role of ApoER2 in LTP appears to be in
the capacity of NMDA receptor modulation by increasing
LDL receptor family members
Figure 1
LDL receptor family members. LDLR, ApoER2, VLDLR
and LRP represent the four major apoE receptor family
members in the mammalian CNS. Each member contains a
single transmembrane domain, at least one ligand binding
domain, EGF repeat, YWTD β-propeller and cytoplasmic
NPxY motif.

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NMDAR conductance and thus indirectly altering intracel-
lular calcium levels.
ApoE isoforms and synaptic plasticity
Increasing evidence indicates that apoE4 itself impairs
neuronal viability. Not all cell types are susceptible to
apoE4-induced toxicity; glia are relatively resistant [100]
and only cells with a neuronal phenotype appear vulner-
able [101]. ApoE4 actually inhibits neurite outgrowth,
overrides the neurite-stimulatory effect of apoE3 and is
neurotoxic in vitro (reviewed by Teter [102]). Transgenic
expression of human apoE4 has dominant negative
behavioral effects [103-106], including deficits in mem-
ory tasks [103,105]. In addition, apoE4 mice a exhibit
greater memory impairment than apoE-knockout (apoE-
KO) mice, suggesting that apoE4 confers a gain of negative
function [106,107].
Several studies utilizing genetically altered mice have
begun to shed light on the roles of apoE in synaptic plas-
ticity and memory formation. ApoE targeted replacement
mice (apoE-TR mice) express one of the three human iso-
forms under the control of endogenous murine APOE
promoter sequences in a conformation and at physiolog-
ical levels in a temporal and spatial pattern comparable to
endogenous mouse apoE [108]. ApoE-TR mice expressing
the apoE3 isoform are identical to wild type mice in both
LTP induction and spatial learning. In contrast, mice
expressing the apoE4 isoform demonstrate compromised
LTP induction and spatial learning. Importantly, the
impaired spatial learning exhibited by apoE-deficient
mice can be rescued by infusion of human apoE3 or
apoE4 [109,110]. Thus, apoE and its receptors influence
NMDA receptor activity, LTP, and spatial memory.
ApoE, Aβ, and synaptic plasticity
In addressing the effect of apoE on Aβ-induced changes in
neuronal viability, it is unclear precisely what form of the
Aβ peptide was used in early studies because it has been
difficult to isolate and determine the conformational spe-
cies of Aβ responsible for its neural activity [50,51]. In
vitro, several recent studies have demonstrated that apoE2
and E3, but not E4, protect neurons against cell death
induced by non-fibrillar Aβ, but have no effect on fibril-
lar-induced toxicity [111,112]. In addition, oligomeric
Aβ42-induced neurotoxicity is significantly greater in
both Neuro-2A cells treated with apoE4 [112-114] and
primary co-cultures of wild-type (WT) neurons and glia
from apoE-TR mice expressing apoE4 [47,112]. Using
apoE-TR mice, oligomeric Aβ42-induced inhibition of
LTP was greatest in hippocampal slice cultures from
apoE4-TR mice, while apoE2 actually protected against
LTP impairment [46]. In vivo, crossing apoE2 transgenic
mice with APP transgenic mice prevented soluble Aβ-
induced dendritic spine loss [115].
Summary
Of the two major apolipoproteins found in the CSF, apoE
can associate to a number of extracellular molecules and
bind to four major CNS apoE receptors; VLDLR, ApoER2,
LDLR and LRP. The apoE4 isoform has garnered attention
due to the genetic association of apoE4 inheritance and
AD risk. ApoE receptors undergo rapid clathrin-mediated
endocytosis following ligand binding and have the ability
to link ligand binding to several signal transduction path-
ways. ApoE isoforms exhibit a differential affect on synap-
tic function and VLDLR and ApoER2 are shown to play a
role in synaptic plasticity and memory formation.
ApoE receptors and AD
ApoE receptors are an integral part of normal apoE metab-
olism, potentially mediating and/or modulating the
effects of apoE isoforms on AD pathological processes.
They are also important for the cellular homeostasis of
cholesterol, which may also affect Aβ production from
APP [12]. Several lines of research have implicated apoE
receptors directly in AD pathophysiology through several
mechanisms (Figure 2).
ApoE receptors endocytose Aβ
Almost from the initial observations that apoE bound Aβ
[24,34], apoE receptors have been suggested to act as
clearance mechanisms for Aβ. Since then, apoE receptors
have been found to help transport Aβ across the endothe-
lial cells forming the blood brain barrier [116] or clear Aβ
into astrocytes as a degradative process [45]. ApoE is
found on most, but not all Aβ deposits in the AD brain
[117,118]. LRP is expressed on activated astrocytes [6] and
closely associated with Aβ deposits [119]. The importance
of Aβ clearance via apoE receptors is also supported by the
significant increase in amyloid deposition observed in
transgenic APP mice deficient in the RAP gene [120],
Table 1: ApoE receptor endocytosis rates.
Receptort1/2
LRP
LDLR
ApoER2
VLDLR
0.5 min
4.8 min
8.1 min
8.2 min

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which has increased levels of several LDLR family mem-
bers [121,122]. Thus, the interactions of apoE complexes
with the apoE receptors in the CNS vitally affect not only
the metabolism of apoE, but of Aβ as well.
LRP alters APP trafficking and processing
ApoE receptors also have been implicated in the produc-
tion of Aβ. LRP interacts with APP through the intracellu-
lar adaptor protein FE65 or via direct binding to the KPI
domain [90,123-126]. Functionally, LRP's rapid endocy-
tosis facilitates APP endocytic trafficking and Aβ produc-
tion [127-129]. Overexpression of a functional LRP
minireceptor in vivo resulted in an increase of soluble Aβ
in the brain [130].
Other members of the LDLR family alter APP trafficking and
processing
The apoE receptor LRP1B, which undergoes a slow endo-
cytosis, interacts with APP. However, unlike LRP, expres-
sion of LRP1B decreases APP endocytic trafficking and
processing to Aβ [131]. ApoER2 also interacts with APP,
through an extracellular matrix molecule F-spondin [132]
and the intracellular adaptor protein FE65 [78]. These
studies suggest that conditions that stabilize APP on the
cell surface can increase α-cleavage of APP and decrease
Aβ production. Finally, several recent studies have shown
that SorLA/LR11 alters APP trafficking to discrete com-
partments such that APP processing by β/γ-secretases is
decreased [133-136]. Together these studies indicate that
ApoE receptors and AD pathophysiology
Figure 2
ApoE receptors and AD pathophysiology. ApoER2 and LRP receptors can mediate several intracellular signaling kinases
including PI3K, CDK-5, JnK and MAPK. The splice variant of ApoER2 expressing exon 19 can influence calcium influx in
response to ligand binding by modulating NMDA receptor-mediated currents via a Src-dependent mechanism. Signaling can
also be influenced by apoE receptor – APP interaction and specific apoE isoform or Reelin binding.