In vivo effects of apoE and clusterin on amyloid-β metabolism and neuropathology

The epsilon4 allele of apolipoprotein E APOE is a risk factor for Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), and the epsilon2 allele is associated with a decreased risk for AD. There is strong evidence to suggest that a major, if not the main, mechanism underlying the link between apoE and both AD and CAA is related to the ability of apoE to interact with the amyloid-beta (Abeta) peptide and influence its clearance, aggregation, and conformation. In addition to a number of in vitro studies supporting this concept, in vivo studies with amyloid precursor protein (APP) transgenic mice indicate that apoE and a related molecule, clusterin (also called apolipoprotein J), have profound effects on the onset of Abeta deposition, as well as the local toxicity associated with Abeta deposits both in the brain parenchyma and in cerebral blood vessels. Taken together, these studies suggest that altering the expression of apoE and clusterin in the brain or the interactions between these molecules and Abeta would alter AD pathogenesis and provide new therapeutic avenues for prevention or treatment of CAA and AD.     

Haploinsufficiency of human APOE reduces amyloid deposition in a mouse model of amyloid-β amyloidosis

The ε4 allele of the apolipoprotein E (APOE) gene is the strongest genetic risk factor for Alzheimer's disease (AD). Evidence suggests that the effect of apoE isoforms on amyloid-β (Aβ) accumulation in the brain plays a critical role in AD pathogenesis. Like in humans, apoE4 expression in animal models that develop Aβ amyloidosis results in greater Aβ and amyloid deposition than with apoE3 expression. However, whether decreasing levels of apoE3 or apoE4 would promote or attenuate Aβ-related pathology has not been directly addressed. To determine the effect of decreasing human apoE levels on Aβ accumulation in vivo, we generated human APOE isoform haploinsufficient mouse models by crossing APPPS1-21 mice with APOE isoform knock-in mice. By genetically manipulating APOE gene dosage, we demonstrate that decreasing human apoE levels, regardless of isoform status, results in significantly decreased amyloid plaque deposition and microglial activation. These differences in amyloid load between apoE3- and apoE4-expressing mice were not due to apoE4 protein being present at lower levels than apoE3. These data suggest that current therapeutic strategies to increase apoE levels without altering its lipidation state may actually worsen Aβ amyloidosis, while increasing apoE degradation or inhibiting its synthesis may be a more effective treatment approach.     

ApoE isoform-specific effects on LTP: blockade by oligomeric amyloid-beta1-42

Amyloid-beta1-42 (Abeta1-42) is crucial to Alzheimer disease (AD) pathogenesis but the conformation of the toxic Abeta species remains uncertain. AD risk is increased by apolipoprotein E4 (apoE4) and decreased by apoE2 compared with the apoE3 isoform, but whether inheritance of apoE4 represents a gain of negative or a loss of protective function is also unresolved. Using hippocampal slices from apoE knockout (apoE-KO) and human apoE2, E3, and E4 targeted replacement (apoE-TR) mice, we found that oligomeric Abeta1-42 inhibited long-term potentiation (LTP) with a hierarchy of susceptibility mirroring clinical AD risk (apoE4-TR > apoE3-TR = apoE-KO > apoE2-TR), and that comparable doses of unaggregated Abeta1-42 did not affect LTP. These data provide a novel link among apoE isoform, Abeta1-42, and a functional cellular model of memory. In this model, apoE4 confers a gain of negative function synergistic with Abeta1-42, apoE2 is protective, and the apoE-Abeta interaction is specific to oligomeric Abeta1-42.     

Amyloid beta40/42 clearance across the blood-brain barrier following intra-ventricular injections in wild-type,apoE knock-out and human apoE3 or E4 expressing transgenic mice

An important event in the pathogenesis of Alzheimer's disease (AD) is the deposition of the amyloid beta (Abeta)1-40 and 1-42 peptides in a fibrillar form, with Abeta42 typically having a greater propensity to undergo this conformational change. A major risk factor for late-onset AD is the inheritance of the apolipoprotein E (apoE) 4 allele [3,14,31]. We previously proposed that apoE may function as a "pathological chaperone" in the pathogenesis of AD (i.e. modulate the structure of Abeta, promoting or stabilizing a beta-sheet conformation), prior to the discovery of this linkage [7,40,41,42]. Data from apoE knockout / AbetaPP^(V717F) mice, has shown that the presence of apoE is necessary for cerebral amyloid formation [1,2], consistent with our hypothesis. However, in betaPP^(V717F) mice expressing human apoE3 or E4 early Abeta deposition at 9 months is suppressed, but by 15 months both human apoE expressing mice had significant fibrillar Abeta deposits with the apoE4 expressing mice having a 10 fold greater amyloid burden [8,9]. This and other data has suggested that apoE, in addition to having a facilitating role in fibril formation, may also influence clearance of Abeta peptides. In order to address if apoE affects the clearance of Abeta peptides across the blood-brain barrier (BBB) and whether there are differences in the clearance of Abeta40 versus Abeta42, we performed stereotactic, intra-ventricular micro-injections of Abeta40, Abeta42 or control peptides in wild-type, apoE knock-out (KO) or human apoE3 or apoE4 expressing transgenic mice. We found that consistent with other studies [5], Abeta40 is rapidly cleared from the brain across the BBB; however, Abeta42 is cleared much less effectively. This clearance of exogenous Abeta peptides across the BBB does not appear to be affected by apoE expression. This data suggests that Abeta42 production may favor amyloid deposition due to a reduced clearance across the BBB, compared to Abeta40. In addition, our experiments support a role of apoE as a pathological chaperone, and do not suggest an isotype specific role of apoE in exogenous Abeta peptide clearance from the CSF across the BBB.     

Transport pathways for clearance of human Alzheimer's amyloid beta-peptide and apolipoproteins E and J in the mouse central nervous system.

Amyloid beta-peptide (Abeta) clearance from the central nervous system (CNS) maintains its low levels in brain. In Alzheimer's disease, Abeta accumulates in brain possibly because of its faulty CNS clearance and a deficient efflux across the blood-brain barrier (BBB). By using human-specific enzyme-linked immunosorbent assays, we measured a rapid 30 mins efflux at the BBB and transport via the interstitial fluid (ISF) bulk flow of human-unlabeled Abeta and of Abeta transport proteins, apolipoprotein E (apoE) and apoJ in mice. We show (i) Abeta40 is cleared rapidly across the BBB via low-density lipoprotein receptor-related protein (LRP)1 at a rate of 0.21 pmol/min g ISF or 6-fold faster than via the ISF flow; (ii) Abeta42 is removed across the BBB at a rate 1.9-fold slower compared with Abeta40; (iii) apoE, lipid-poor isoform 3, is cleared slowly via the ISF flow and across the BBB (0.03-0.04 pmol/min g ISF), and after lipidation its transport at the BBB becomes barely detectable within 30 mins; (iv) apoJ is eliminated rapidly across the BBB (0.16 pmol/min g ISF) via LRP2. Clearance rates of unlabeled and corresponding 125I-labeled Abeta and apolipoproteins were almost identical, but could not be measured at low physiologic levels by mass spectrometry. Amyloid beta-peptide 40 binding to apoE3 reduced its efflux rate at the BBB by 5.7-fold, whereas Abeta42 binding to apoJ enhanced Abeta42 BBB clearance rate by 83%. Thus, Abeta, apoE, and apoJ are cleared from brain by different transport pathways, and apoE and apoJ may critically modify Abeta clearance at the BBB.     

LRP-mediated clearance of Abeta is inhibited by KPI-containing isoforms of APP

The pathogenesis of Alzheimer's disease (AD) involves the abnormal accumulation and deposition of beta-amyloid in cerebral blood vessels and in the brain parenchyma. Critical in modulating beta-amyloid deposition in brain is the flux of Abeta across the blood brain barrier. The low-density lipoprotein receptor-related protein (LRP), is a large endocytic receptor that mediates the efflux of Abeta out of brain and into the periphery. The first step in the LRP-mediated clearance of Abeta involves the formation of a complex between Abeta and the LRP ligands apolipoprotein E (apoE) or alpha(2)-macroglobulin (alpha(2)M). The Abeta/chaperone complexes then bind to LRP via binding sites on apoE or alpha(2)M. The efflux of Abeta/chaperone complexes out of the neuropil and into the periphery may be attenuated by LRP-ligands that compete with apoE or alpha(2)M for LRP binding. LRP is also the cell surface receptor for Kunitz Protease Inhibitor (KPI) containing isoforms of Abeta's parent protein, the amyloid protein precursor (APP). Protein and mRNA levels of KPI-containing APP isoforms (APP-KPI) are elevated in AD brain and are associated with increased Abeta production. In this study we show that soluble non-amyloidogenic APP-KPI can also inhibit the uptake of Abeta/alpha(2)M in a cell culture model of LRP mediated Abeta clearance. Clearance of Abeta/apoE complexes was not inhibited by APP-KPI. Our findings are consistent with studies showing that apoE and alpha(2)M have discrete binding sites on LRP. Most significantly, our data suggests that the elevated levels of APP-KPI in AD brain may attenuate the clearance of Abeta, the proteins own amyloidogenic catabolic product.     

Reducing human apolipoprotein E levels attenuates age-dependent Aβ accumulation in mutant human amyloid precursor protein transgenic mice

Apolipoprotein E4 (apoE4) plays a major role in the pathogenesis of Alzheimer's disease. Brain amyloid-β (Aβ) accumulation depends on age and apoE isoforms (apoE4 > apoE3) both in humans and in transgenic mouse models. Brain apoE levels are also isoform dependent, but in the opposite direction (apoE4 < apoE3). Thus, one prevailing hypothesis is to increase brain apoE expression to reduce Aβ levels. To test this hypothesis, we generated mutant human amyloid precursor protein transgenic mice expressing one or two copies of the human APOE3 or APOE4 gene that was knocked in and flanked by LoxP sites. We report that reducing apoE3 or apoE4 expression by 50% in 6-month-old mice results in efficient Aβ clearance and does not increase Aβ accumulation. However, 12-month-old mice with one copy of the human APOE gene had significantly reduced Aβ levels and plaque loads compared with mice with two copies, regardless of which human apoE isoform was expressed, suggesting a gene dose-dependent effect of apoE on Aβ accumulation in aged mice. Additionally, 12-month-old mice expressing one or two copies of the human APOE4 gene had significantly higher levels of Aβ accumulation and plaque loads than age-matched mice expressing one or two copies of the human APOE3 gene, suggesting an isoform-dependent effect of apoE on Aβ accumulation in aged mice. Moreover, Cre-mediated APOE4 gene excision in hippocampal astrocytes significantly reduced insoluble Aβ in adult mice. Thus, reducing, rather than increasing, apoE expression is an attractive approach to lowering brain Aβ levels.     

Cerebral β-amyloid deposition is augmented by the –491AA promoter polymorphism in non-demented elderly individuals bearing the apolipoprotein E ε4 allele

The apolipoprotein E epsilon4 allele (APOE, gene; apoE, protein) is widely accepted as a risk factor for Alzheimer's disease (AD). Our previous studies found that APOEepsilon4 promotes AD pathogenesis by fostering the early deposition of the amyloidogenic peptide Abeta in the aging brain. Recent reports suggest that polymorphisms in the upstream promoter region of APOE differentially affect the production of apoE and also may have an important influence on the probability of developing AD. In this study, we asked whether APOE promoter -491 (A/T) variants interact with APOE polymorphisms to modulate the degree of beta-amyloid- and tau-related pathology in the medial temporal lobe of the non-demented elderly. Our results confirm that APOEepsilon4 is associated with increased formation of senile plaques, cerebrovascular amyloid, and neurofibrillary tangles in the medial temporal lobe. We also found that homozygosity for A at position -491 of the APOE promoter (-491AA) correlates with increased Abeta17-24 and Abeta42 deposition in APOEepsilon4-positive cases, but not in cases lacking the epsilon4 allele. In comparison, Abeta burden is significantly less in epsilon4 carriers with the -491AT and -491TT promoter allelotypes. There was no effect of -491 polymorphisms on Abeta40 deposition (which is relatively sparse in the non-demented elderly), on the number of activated microglia, or on the amount of neurofibrillary tangles. We conclude that the amyloidogenic effects of apoE4 are exacerbated by polymorphisms in the APOE promoter that enhance apoE production.     

The nucleation growth and reversibility of Amyloid-beta deposition in vivo

The amyloid-beta (Abeta) peptide is a major constituent of the brain senile plaques that characterize Alzheimer's disease (AD). Converging observations led to the formulation of the amyloid hypothesis whereby the accumulation of soluble aggregates and insoluble Abeta deposits is the primary event in AD pathogenesis. Furthermore, the apoE4 isoform of apolipoprotein E, a major prevalent genetic risk factor of AD, is associated with increased Abeta deposition. To investigate the initial stages of the amyloid cascade in vivo and how this is affected by apoE4, we studied the effects of prolonged inhibition and subsequent reactivation of the Abeta-degrading enzyme, neprilysin, on aggregation and deposition of Abeta in apoE transgenic and control mice. The results revealed that Abeta deposition in vivo is initiated by aggregation of Abeta42, which is followed by reversible deposition of both Abeta42 and Abeta40, along with growth of the deposits, and by their subsequent irreversible fibrillization. The initiation of Abeta42 deposition is accelerated isoform-specifically by apoE4, whereas the growth and dissolution of the Abeta deposits as well as their fibrillization are similarly stimulated by the various apoE isoforms. Interestingly, Abeta deposition was associated with increased gliosis, which may reflect early pathological interactions of beta with the brain's parenchyma.     

Alzheimer's disease amyloid-beta peptide modulates apolipoprotein E isoform specific receptor binding

The major protein component of the extracellular deposits in Alzheimer's disease (AD) is a 4 kDa peptide termed amyloid-beta (Abeta). This peptide is known to bind apolipoprotein E (apoE), a key mediator of lipoprotein transport, in an isoform specific manner. Whilst these isoform specific effects on apoE are well recognized, the functional significance of this interaction is poorly understood. Here, we investigated the influence of Abeta on apoE-mediated lipoprotein binding to cells using fluorescently tagged lipoprotein-like emulsions. Using this approach, we demonstrate that Abeta enhanced the normally poor binding of apoE2 lipoprotein-like particles to fibroblasts in culture, whilst markedly reducing the binding of apoE3 and apoE4. This suggests that the action of apoE isoforms on cellular lipoprotein or cholesterol metabolism is differentially modulated by Abeta. This also suggests that Abeta may also compromise apoE function in the Alzheimer disease affected brain.     

APOE epsilon 3/ epsilon 4 heterozygotes have an elevated proportion of apolipoprotein E4 in cerebrospinal fluid relative to plasma,independent of Alzheimer's disease diagnosis

Inheritance of the apolipoprotein E (APOE) epsilon 4 allele is associated with an increased risk of Alzheimer's disease (AD). However, the risk of AD in APOE epsilon 3/ epsilon 4 heterozygotes is variable. We tested the hypothesis that the risk of AD in APOE epsilon 3/ epsilon 4 heterozygotes was linked to the relative levels of expression of apoE4 versus apoE3 protein. We measured the apoE4 isoform and total apoE using two specific enzyme-linked immunosorbent assay (ELISA) kits in three cohorts of plasma samples and two cohorts of cerebrospinal fluid samples from AD, mild cognitive impairment, and control subjects. The apoE4 ELISAs were specific as they did not detect apoE in APOE epsilon 3/epsilon 3 homozygotes and were comparable to the total apoE ELISAs in APOE epsilon 4/ epsilon 4 homozygotes. In APOE epsilon 3/ epsilon 4 individuals, the ratio of apoE4 to total apoE levels was 30-40% in plasma, suggesting a decreased production or an increased metabolism of apoE4 compared to apoE3. Surprisingly, the ratio in the CSF was reversed, with apoE4 accounting for 60-70% of the total apoE. The proportion of apoE4 in these cases did not vary by diagnosis, age of onset, or duration of AD. We conclude that the proportion of apoE4 in plasma is not predictive of AD risk in APOE epsilon 3/epsilon 4 individuals. However, the greater proportion of apoE4 in the cerebrospinal fluid suggests differential production or metabolism of the protein in the central nervous system (CNS), with the apoE4 isoform dominating.     

Role of apoE/Aβ interactions in the pathogenesis of Alzheimer’s disease and cerebral amyloid angiopathy

The ɛ4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA). The mechanism underlying this increased risk is not completely clear, yet mounting evidence supports the idea that the ability of apoE to interact with the amyloid-β (Aβ) peptide and influence its conformation and clearance plays a major role. Evidence to support this concept comes from in vitro and in vivo studies of apoE/Aβ interactions and the effects of these interactions on Aβ conformation and cellular clearance. Recent studies on the effect of murine and human apoE in APP transgenic mice provide direct evidence that apoE is critically involved in the in vivo converstion of Aβ into forms which contain high β-sheet content and associated cellular toxicity (neuritic plaques and CAA). These studies also suggest a role for human apoE in Aβ clearance in vivo.     

A Multifaceted Role for apoE in the Clearance of Beta-Amyloid across the Blood-Brain Barrier

Background: While apolipoprotein E4 (apoE4) is highly correlated with the development of Alzheimer's disease (AD), its role in AD pathology and, in particular, beta-amyloid (Aβ) removal from the brain, is not clearly defined. Objective: To elucidate the influence of apoE on the clearance of Aβ across the blood-brain barrier (BBB). Methods: Aβ(1-42) was intracerebrally administered to transgenic mice expressing human apoE isoforms and examined in the periphery. Results: apoE3 and apoE4 mice had 5 times and 2 times, respectively, more Aβ(1-42) appearing in the plasma than wild-type or apoE knockout mice, indicating an enhanced clearance of Aβ from the brain to the periphery. In vitro, unbound basolateral apoE3 (i.e., not bound to Aβ), and to a lesser extent unbound apoE4, at concentrations ≤10 nM facilitated basolateral-to-apical fluorescein-Aβ(1-42) transcytosis across a BBB model, while apoE isoforms bound to Aβ significantly disrupted Aβ transcytosis. Additionally, following apical exposure to the BBB model, we found that apoE4 bound to Aβ is able to penetrate the BBB more readily than apoE3 bound to Aβ and does so via the RAGE (receptor for advanced glycation end products) transporter. Conclusion: These studies indicate a multifaceted, isoform-dependent role for apoE in the exchange of Aβ across the BBB and may partially explain the association of apoE4 and Aβ brain accumulation in AD.     

Human and murine ApoE markedly alters A beta metabolism before and after plaque formation in a mouse model of Alzheimer's disease

The epsilon4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer's disease (AD), perhaps through effects on amyloid-beta (Abeta) metabolism. Detailed analyses of various Abeta parameters in aging APP(V717F+/-) transgenic mice expressing mouse apoE, no apoE, or human apoE2, apoE3, or apoE4 demonstrate that apoE facilitates, but is not required for, Abeta fibril formation in vivo. Human apoE isoforms markedly delayed Abeta deposition relative to mouse apoE, with apoE2 (and apoE3 to a lesser extent) having a prolonged ability to prevent Abeta from converting into fibrillar forms. Isoform-specific effects of human apoE on Abeta levels and neuritic plaque formation mimicked that observed in AD (E4 > E3 > E2). Importantly, observation of an apoE-dependent decrease in percent soluble Abeta and enrichment of Abeta in membrane microdomains prior to Abeta deposition indicates that apoE influences Abeta metabolism early in the amyloidogenic process and provides a possible novel mechanism by which apoE affects AD pathogenesis.     

Apolipoprotein E level and cholesterol are associated with reduced synaptic amyloid beta in Alzheimer’s disease and apoE TR mouse cortex

The apolipoprotein E4 allele (APOE4) contributes to Alzheimer's disease (AD) risk and APOE2 is protective, but the relevant cellular mechanisms are unknown. We have used flow cytometry analysis to measure apolipoprotein E (apoE) and amyloid beta peptide (Aβ) levels in large populations of synaptic terminals from AD and aged cognitively normal controls, and demonstrate that modest but significant increases in soluble apoE levels accompany elevated Aβ in AD cortical synapses and in an APP/PS1 rat model of AD. Dual labeling experiments document co-localization of apoE and Aβ in individual synapses with concentration of Aβ in a small population of apoE-positive synapses in both AD and controls. Consistent with a clearance role, the apoE level was higher in Aβ-positive synapses in control cases. In aged targeted replacement mice expressing human apoE, apoE2/4 synaptic terminals demonstrated the highest level of apoE and the lowest level of Aβ compared to apoE3/3 and apoE4/4 lines. In apoE2/4 terminals, the pattern of immunolabeling for apoE and Aβ closely resembled the pattern in human control cases, and elevated apoE was accompanied by elevated free cholesterol in apoE2/4 synaptic terminals. These results are consistent with a role for APOE in Aβ clearance in AD synapses, and suggest that optimal lipidation of apoE2 compared to E3 and E4 makes an important contribution to Aβ clearance and synaptic function.     

Effect of apolipoprotein AII on the interaction of apolipoprotein E with beta-amyloid: some apo(E-AII) complexes inhibit the internalization of beta-amyloid in cultures of neuroblastoma cells

Apolipoprotein (apo) E and its polymorphism are linked to the pathogenesis of late-onset and sporadic Alzheimer's disease (AD). ApoE facilitates the deposition and fibrillogenesis of beta-amyloid (Abeta), and may participate in Abeta clearance. We recently found that apo(E-AII) complex binds to Abeta much more strongly than does monomeric apoE. Here, we investigated the effect of apoAII on the interaction between apoE and Abeta. Addition of apoAII to apoE monomers increased the binding of apoE2 and apoE3 to Abeta(1-42), presumably following the formation of apo(E3-AII), apo(E2-AII), and apo(AII-E2-AII) complexes. This increased binding was not seen in the case of apoE4. When neuroblastoma cells were cultured in media containing Abeta(1-42) and a mixture of apoE3 and apoAII, intracellular Abeta was significantly reduced and cell viability was maintained at a higher level than in cells cultured without apoAII. ApoE2 itself seemed to act as an inhibitor of the endocytosis of Abeta, and we did not observe a significant effect of apoAII on the movement of Abeta in apoE2-containing medium. However, cell viability could be maintained at a higher level (as with apoE3) by adding apoAII to apoE2, despite the reduced viability of cells incubated without apoAII. In medium containing apoE4, both the amount of Abeta accumulated into cells and the cell viability were unchanged by the presence of apoAII in the medium. In addition, apoE4 itself was toxic, as previously suggested. These findings demonstrate that the type of apo(E-AII) complex present could underlie the isoform-specific role of apoE in the pathogenesis of AD.     

A novel apoE-derived therapeutic reduces vasospasm and improves outcome in a murine model of subarachnoid hemorrhage

Introduction  Recent clinical observations demonstrate that the APOE4 genotype increases the development of delayed ischemic deficit and worsens prognosis following aneurysmal subarachnoid hemorrhage (SAH). In the current study, we use targeted replacement mice expressing only human apoE3 or apoE4 to model the isoform-specific effects of apoE following SAH. We then test the hypothesis that an apoE-derived therapeutic peptide reduces vasospasm and improves functional recovery after SAH. Methods  Experimental SAH was induced in APOE3- and APOE4-targeted replacement mice. For 3 days following injury, daily functional assessments were made. Mice were then sacrificed and the cerebral vasculature visualized to quantify vasospasm. In a separate experiment, C57BI/6 mice were treated with intravenous injection of vehicle, low-dose, or high-dose apoE-mimetic peptide every 12 hours for 3 days post-SAH. Functional endopoints were assessed on a daily basis, followed by measurement of middle cerebral artery diameter. Results  Mice expressing the apoE4 isoform had greater functional deficit, mortality, cerebral edema, and vasospasm as compared with their apoE3 counterparts. Mice treated with the apoE-mimetic peptide had decreased mortality, functional deficits, and histological evidence of vasospasm as compared with vehicle-treated animals. Conclusion  Consistent with the clinical literature, the apoE4 isoform is associated with an increased incidence of vasospasm and poor functional recovery after experimental SAH. An apoE-derived peptide represents a novel therapeutic approach for the treatment of SAH. Drs. Gao and Wang contributed equally to this work     

Higher avidity binding of apolipoprotein (E-AII) complex than of apolipoprotein E monomer to beta-amyloid.

Apolipoprotein E (apoE) is believed to be closely involved in the pathogenesis of Alzheimer's disease (AD) because of its ability to bind to beta-amyloid (Abeta), the primary component of senile plaques. The presence of cystein residues in apoE2 and apoE3 allows these isoforms to form disulfide-linked complexes, such as apo(E-AII) complex and apo(AII-E-AII) complex. A 50-kDa complex [which corresponded to apo(E-AII)-Abeta, because it reacted with any of the three antibodies, anti-apoE, anti-apoAII, or anti-Abeta] was detected by immunoblot analysis in native cerebrospinal fluid (CSF) obtained from nondementia patients with the apoE phenotype E3/E3. However, a band considered to represent apoE-Abeta was not observed. The dissociation constant (Kd) values obtained for the specific binding of recombinant apoE2, apoE3, and apoE4 to Abeta(1-42) were 48.1 +/- 2.2 nM, 63.7 +/- 2.1 nM, and 75.9 +/- 1.8 nM, respectively. In contrast, the binding affinity of the partially purified apo(E3-AII) complex to Abeta(1-42) was very high, the Kd being 5.5 +/- 0.5 nM. No basic difference was observed between lipidated and nonlipidated apoE in terms of the characteristics of the binding of apoE isoforms to Abeta(1-42); however, lipidation reduced the binding capacity of each isoform in a dose-dependent manner. These findings seem consistent with the generally accepted idea that apoE4 is a risk factor for AD, insofar as only apoE4 is unable to form a complex with apoAII owing to its lack of a cystein residue. In addition, it is possible that apoE3 monomer (and possibly apoE2 monomer), like apoE4 but unlike apo(E-AII) complex, can act as a risk factor in the pathogenesis of AD.     

Cerebral small vessel disease-induced apolipoprotein E leakage is associated with Alzheimer disease and the accumulation of amyloid beta-protein in perivascular astrocytes

Apolipoprotein E (apoE) plays a role in the pathogenesis of Alzheimer disease (AD). It is involved in the receptor-mediated cellular clearance of the amyloid beta-protein (Abeta) and in the perivascular drainage of the extracellular fluid. Microvascular changes are also associated with AD and have been discussed as a possible reason for altered perivascular drainage. To further clarify the role of apoE in the perivascular and vascular pathology in AD patients, we studied its occurrence and distribution in the perivascular space, the perivascular neuropil, and in the vessel wall of AD and control cases with and without small vessel disease (SVD). Apolipoprotein E was found in the perivascular space and in the neuropil around arteries of the basal ganglia from control and AD cases disclosing no major differences. Western blot analysis of basal ganglia tissue also revealed no significant differences pertaining to the amount of full-length and C-terminal truncated apoE in AD cases compared with controls. In contrast, Abeta occurred in apoE-positive perivascular astrocytes in AD cases but not in controls. In blood vessels, apoE and immunoglobulin G were detected within the SVD-altered vessel wall. The severity of SVD was associated with the occurrence of apoE in the vessel wall and with that of Abeta in perivascular astrocytes. These results point to an important role of apoE in the perivascular clearance of Abeta in the human brain. The occurrence of apoE and immunoglobulin G in SVD lesions and in the perivascular space suggests that the presence of SVD results in plasma-protein leakage into the brain. It is therefore tempting to speculate that apoE represents a pathogenetic link between SVD and AD.