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.

The strongest associations of APOE genotype with disease are with conditions containing amyloid deposition including AD, Down's syndrome, cerebral amyloid angiopathy and head trauma 6181920212223. In vivo, early evidence for an involvement of apoE in AD came from immunohistochemical localization of apoE to senile plaques 24. ApoE4 increases levels of amyloid deposition in humans 618 and accelerates amyloid deposition in transgenic mice 82526. 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 2728293031. ApoE binds soluble Aβ oligomers found in the brain, plasma and CSF 3233. In vitro, apoE forms stable complexes with Aβ 34353637, alters the aggregation of various Aβ peptides 383940, modulates Aβ-induced neuroinflammation 41424344, and promotes Aβ clearance 45. In addition, Aβ42 and apoE4 act synergistically 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 464748495051. Each of these important functions is partially mediated by 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 endocytosis and cellular signaling (Figure 1) 5253. In addition to the LDLR itself, the family includes LRP/LRP1 54, megalin/LRP2 55, VLDLR 56, ApoER2/LRP8 575859, SORLA-1/LR11 6061, LRP4 62, LRP5 6364, 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 5267. 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 protein (RAP) 69. RAP is an endoplasmic reticulum (ER)-resident protein that functions in receptor folding and trafficking along the early secretory pathway and universally 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 65770717273. It is unclear which receptors are expressed on oligodendrocytes. Soluble forms of each of these receptors have been detected (see below).

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 dominant endocytosis signal for LRP is the

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Recently, several studies have demonstrated a role for some apoE receptors (specifically LRP, ApoER2, and VLDLR) as signaling molecules (see 5382 for review). VLDLR and ApoER2 transduce signals from the extracellular matrix molecule Reelin, affecting neuronal cell migration during development 83. LRP activation by ligand binding affects NMDA receptor function 848586878889. These effects are transduced through the cytoplasmic domains of receptors binding various cytoplasmic adaptor and scaffold proteins containing PID or PDZ domains, including mammalian Disabled-1 (mDab1), mDab2, FE65, JNK-interacting protein JIP-1 and JIP-2, and PSD-95 909192939495. ApoE receptor ligands also promote other intraneuronal signals via apoE receptors, including PI3 activation, ERK activation, and JNK inhibition 869697 but exactly which receptors promote which signals is unknown. ApoE receptors on glia also affect signaling pathways, affecting the state of glial activation 414298.

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 8399. Moreover, Reelin application enhanced LTP induction, which was dependent on the presence of both ApoER2 and VLDLR 97. A potential molecular mechanism for this function of ApoER2 is a 59 amino acid cytoplasmic domain that is alternatively spliced. This ApoER2 splice variant interacts with PSD-95, which is itself associated with NMDA receptor conductance 8795. 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 Reelin 87. Thus, the role of ApoER2 in LTP appears to be in the capacity of NMDA receptor modulation by increasing NMDAR conductance and thus indirectly altering intracellular calcium levels.

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 vulnerable 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 103104105106, including deficits in memory tasks 103105. In addition, apoE4 mice a exhibit greater memory impairment than apoE-knockout (apoE-KO) mice, suggesting that apoE4 confers a gain of negative function 106107.

Several studies utilizing genetically altered mice have begun to shed light on the roles of apoE in synaptic plasticity and memory formation. ApoE targeted replacement mice (apoE-TR mice) express one of the three human isoforms under the control of endogenous murine APOE promoter sequences in a conformation and at physiological 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 109110. Thus, apoE and its receptors influence NMDA receptor activity, LTP, and spatial memory.

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 species of Aβ responsible for its neural activity 5051. 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 fibrillar-induced toxicity 111112. In addition, oligomeric Aβ42-induced neurotoxicity is significantly greater in both Neuro-2A cells treated with apoE4 112113114 and primary co-cultures of wild-type (WT) neurons and glia from apoE-TR mice expressing apoE4 47112. 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.

Almost from the initial observations that apoE bound Aβ 2434, 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 endothelial 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 117118. 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, which has increased levels of several LDLR family members 121122. 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.

ApoE receptors also have been implicated in the production of Aβ. LRP interacts with APP through the intracellular adaptor protein FE65 or via direct binding to the KPI domain 90123124125126. Functionally, LRP's rapid endocytosis facilitates APP endocytic trafficking and Aβ production 127128129. Overexpression of a functional LRP minireceptor in vivo resulted in an increase of soluble Aβ in the brain 130.

The apoE receptor LRP1B, which undergoes a slow endocytosis, interacts with APP. However, unlike LRP, expression 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 compartments such that APP processing by β/γ-secretases is decreased 133134135136. Together these studies indicate that binding to APP is a common event for the LDLR family members and expression and proteolytic processing of these receptors can impact APP trafficking and processing.

LRP is synthesized as a single polypeptide chain of ~600 kDa and then cleaved in the trans-Golgi network by furin into a 515-kDa ligand-binding subunit and an 85-kDa transmembrane subunit that remain non-covalently associated with one another as they traffic to the cell surface 139. The LRP extracellular region undergoes shedding from a region close to the membrane by metalloproteinases, releasing a soluble LRP (sLRP) capable of binding ligands 140141. sLRP is detected in human plasma at nanomolar concentrations 140141 and in human CSF 142. Recent studies have also shown that the cell associated fragment of LRP can be cleaved at a third, intramembranous site by the γ-secretase, releasing its intracellular domain (ICD) 143144. These sequential cleavage events by furin, metalloproteinases, and γ-secretase closely resemble those of Notch family proteins 145146147.

Like LRP, ApoER2 and VLDLR undergo extracellular cleavages by metalloproteinases to release soluble receptors as well as C-terminal, cell-associated fragments, and these events are induced by Phorbol esters 148. Furthermore, the C-terminal fragments are cleaved by γ-secretase 149. ApoER2 and VLDLR proteolytic events are also increased by extracellular ligand binding 148. Interestingly, the different apoE alleles induced different degrees of release; both ApoER2 and VLDLR show greatest cleavage following apoE2 activation, less with apoE3 and relatively little with apoE4 148. The release of soluble forms of ApoER2 and VLDLR is affected by the presence of splice variants. ApoER2 and VLDLR both have prominent splice variants that lack the exon encoding an O-linked glycosylation site. This region is important in the regulated cleavage of transmembrane proteins 150. In addition, some ApoER2 splice variants contain an exon that encodes a furin cleavage site in the extracellular domain 59151. Furin-dependent cleavage results in extrusion of a soluble fragment of the receptor 152. Thus, cleavages of ApoER2 and VLDLR are regulated in part by alternate splicing events.

Like the other family members, LDLR exists as a soluble form 153154. LDLR shedding from the cell surface is enhanced by several stimuli, including interferon and phorbol ester. As for the other apoE receptors, this effect is dependent upon a cell surface metalloproteinase 154. Inefficient LDLR exon splicing may also contribute to soluble LDLR isoforms because LDLR ESTs have been reported which lack (i) exon 12, which causes a frameshift in the extracellular domain, resulting in a premature termination codon in exon 13, or (ii) exon 15, which encodes the LDLR O-linked glycosylation domain (BG945931 and BQ685399, respectively).

Numerous transmembrane proteins in addition to the apoE receptors have soluble forms, including growth factors and their receptors, cytokine precursors and receptors, cell adhesion molecules, enzymes, and differentiation factors 155. To gain insight into the possible mechanisms of soluble apoE receptors, we will briefly consider the functions of released extracellular domains of various other transmembrane proteins.

These potential functions of soluble receptors each apply to apoE receptors. One of the most profound implications of the production of soluble apoE receptors is the possible dominant negative effect on apoE receptor function. This action is observed with the production of soluble ApoER2. The expression of the ApoER2 variant containing the furin consensus site results in the production of soluble receptor consisting of the ligand binding domain 152. Soluble ApoER2 can effectively block Reelin binding to both ApoER2 and VLDLR and subsequent Reelin-dependent signaling in primary neuronal cells. Thus, inhibition of normal Reelin signaling through ApoER2 and VLDLR can acutely modulate other signaling mechanisms through changes in NMDA receptor activity and intracellular signaling pathways 848586878889. The effect on apoE-dependent signaling has yet to be determined, but these studies suggest that selected apoE receptor shedding would affect both specific ligand binding, as in the case of soluble ApoER2 and Reelin signaling, as well as overall apoE binding and signaling.

Other soluble apoE receptors are shown to act in the same capacity as soluble ApoER2. Soluble LRP can bind RAP in ligand blots 140 and soluble derivatives of LRP, LDLR, and VLDLR have each been shown to mediate receptor-ligand interactions 140152176177. A physiologic function has yet to be ascribed to the production of soluble apoE receptors. However, in light of the essential roles these receptors play in synaptic function, integration into numerous signal transduction pathways and their wide-range of their ligands, it is likely that this type of negative feedback would be necessary in modulating the activity of specific or multiple apoE receptor subtypes. In addition, cleavage of apoE receptors could be a necessary step in receptor turnover, affecting receptor half-lives, and in preventing uptake of apoE-containing lipoproteins. In summary, release of soluble apoE receptors from the cell surface may modulate the cell surface apoE receptor pathway through multiple mechanisms, including ligand binding away from the cell, altered cell signaling, and differences in receptor degradation.