Apolipoprotein E in sporadic Alzheimer's disease: allelic variation and receptor interactions.

An increased prevalence of apolipoprotein E (ApoE) epsilon 4 allele exists in late onset familial Alzheimer's disease. We found, in sporadic Alzheimer's disease, that 62% of patients possessed an ApoE-epsilon 4 allele, compared with 20% of controls. ApoE-epsilon 4/4 patients had more senile plaques (SPs) than epsilon 3/3 patients. ApoE immunoreactivity of SPs was equivalent in both groups. Two receptors bind ApoE complexes, the low density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP). In normal brain, anti-LRP antibodies strongly stained neurons and lightly stained astrocytes; anti-LDL receptor antibodies stained only the neuropil and astrocytes. In Alzheimer's disease, SPs and reactive astrocytes were also strongly LRP immunoreactive. Colocalization of ApoE and LRP to SPs implies that these molecules may be involved in metabolism of components of SPs.

Demonstration by fluorescence resonance energy transfer of two sites of interaction between the low-density lipoprotein receptor-related protein and the amyloid precursor protein: role of the intracellular adapter protein Fe65.

Amyloid-beta, the major constituent of senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by proteolysis. Kunitz protease inhibitor (KPI) containing forms of APP (APP751/770) interact with a multifunctional endocytic receptor, the low-density lipoprotein receptor-related protein (LRP), which modulates its proteolytic processing affecting production of amyloid-beta. We used fluorescence resonance energy transfer (FRET) using labeled LRP and APP in H4 cell line to examine the subcellular localization and the molecular domains involved in the APP-LRP interaction. KPI-containing forms of APP (APP770) demonstrated FRET with LRP that was sensitive to the LRP inhibitor receptor-associated protein (RAP), suggesting an interaction between the extracellular domains of APP770 and LRP. APP695 also interacts with LRP to lesser degree (as measured by extracellular domain probes), and this ectodomain interaction is not altered by RAP. By using C-terminally tagged LRP and APP, we demonstrate a second site of interaction between the C termini of both APP695 and APP770 and the C terminus of LRP, and that the interactions at these regions are not sensitive to RAP. We next examined the possibility that the C-termini APP-LRP interaction was mediated by Fe65, an adaptor protein that interacts with the cytoplasmic tails of LRP and APP. FRET studies confirmed a close proximity between the amino Fe65 phosphotyrosine binding (PTB) domain and LRP cytoplasmic domain and between the carboxyl Fe65 PTB domain and the APP cytoplasmic domain. These findings demonstrate that LRP interaction with APP occurs via both extracellular and intracellular protein interaction domains.

Association of apolipoprotein J-positive beta-amyloid plaques with dystrophic neurites in Alzheimer's disease brain.

Apolipoprotein J (apoJ), also known as clusterin and SP-40,40, binds soluble beta-amyloid (Abeta and is up-regulated in the Alzheimer's disease (AD) brain. In the present study we classified apoJ-immunopositive Abeta deposits in AD temporal cortex, and found apoJ-immunoreactive plaques were often associated with dystrophic neurites. Quantitative immunohistochemical analysis of five AD brains showed that 29% of Abeta deposited in the parenchyma was associated with apoJ. Of Abeta deposits with apoJ immunopositivity, 71% were associated with phospho-tau-positive dystrophic neurites in the surrounding tissue. Conversely, 64% of phospho-tau-labeled neuritic deposits were labeled with apoJ. ApoJ was found at the core of these deposits, and co-localized with the amyloid staining agent thioflavine-S. To test the direct effects of apoJ on tau metabolism, we treated cells in culture with apoJ-containing conditioned media, and we injected apoJ-containing media into the rat hippocampus. Using both systems, we observed increases in levels of tau and phosphorylated tau. Our findings demonstrate that apoJ immunopositivity strongly correlates with the presence of amyloid and associated neuritic dystrophy in the neuropil of AD temporal cortex, and supports a model where extracellular apoJ facilitates the conversion of diffuse Abeta deposits into amyloid and enhances tau phosphorylation in neurites surrounding these of plaques.

Elevation of LDL receptor-related protein levels via ligand interactions in Alzheimer disease and in vitro.

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional receptor in the CNS that binds both apolipoprotein E (apoE) and activated alpha2-macroglobulin (alpha2M*); all 3 proteins are genetically associated with Alzheimer disease (AD). In this study we found an 85% increase in LRP levels in human AD brain frontal cortex, along with an increased level of the LRP ligands, apoE, and alpha2M. We speculated that LRP levels might be increased in response to the increased levels of its ligands, apoE, and alpha2M*. To test this hypothesis we examined the effects of alpha2M* on LRP in primary cultures. Treatment of neurons with alpha2M* significantly increased LRP levels (by 92%). This increase was prevented by coculture with receptor-associated protein (RAP), which blocks binding of LRP ligands to LRP Native alpha2M or RAP alone did not change LRP levels in vitro. We also found that alpha2M* stimulated activation of astrocytes in vitro and promoted the levels of LRP by 65%. These data indicate 1) the LRP ligand alpha2M* increases levels of LRP in primary neuronal and astrocytic cultures, 2) alpha2M*-induction of LRP levels in vitro depends on binding to LRP, and 3) LRP levels are increased in AD brain, perhaps in response to the increased levels of alpha2M.

Expression of the very low-density lipoprotein receptor (VLDL-r), an apolipoprotein-E receptor, in the central nervous system and in Alzheimer's disease.

The very low density lipoprotein receptor (VLDL-r) is a cell-surface molecule specialized for the internalization of multiple diverse ligands, including apolipoprotein E (apoE)-containing lipoprotein particles, via clathrin-coated pits. Its structure is similar to the low-density lipoprotein receptor (LDL-r), although the two have substantially different systemic distributions and regulatory pathways. The present work examines the distribution of VLDL-r in the central nervous system (CNS) and in relation to senile plaques in Alzheimer disease (AD). VLDL-r is present on resting and activated microglia, particularly those associated with senile plaques (SPs). VLDL-r immunoreactivity is also found in cortical neurons. Two exons of VLDL-r mRNA are differentially spliced in the mature receptor mRNA. One set of splice forms gives rise to receptors containing (or lacking) an extracellular O-linked glycosylation domain near the transmembrane portion of the molecule. The other set of splice forms appears to be brain-specific, and is responsible for the presence or absence of one of the cysteine-rich repeat regions in the binding region of the molecule. Ratios of the receptor variants generated from these splice forms do not differ substantially across different cortical areas or in AD. We hypothesize that VLDL-r might contribute to metabolism of apoE and apoE/A beta complexes in the brain. Further characterizations of apoE receptors in Alzheimer brain may help lay the groundwork for understanding the role of apoE in the CNS and in the pathophysiology of AD.

Progression of cerebral amyloid angiopathy: accumulation of amyloid-beta40 in affected vessels.

Cerebrovascular deposits of amyloid (cerebral amyloid angiopathy, or CAA) are generally asymptomatic, but in advanced cases, they can lead to vessel rupture and hemorrhage. The process of progression in CAA was studied by comparison of postmortem brains with asymptomatic ("mild") CAA to brains with the form of the disease associated with hemorrhage ("severe CAA"). Cortical and meningeal vessels were immunostained for beta-amyloid and examined by confocal microscopy and by systematic quantitative sampling. We focused on 2 quantitative parameters: the proportion of vessels affected by amyloid (a measure of amyloid seeding of vessels) and the amount of amyloid per affected vessel (a measure of growth of existing lesions). Surprisingly, there was no difference between the proportion of affected cortical vessels in mild and severe CAA (0.29 vs 0.32, p = 0.65), but rather an increase in the area of the 40 amino acid form of beta-amyloid per affected cortical vessel (198.5 +/- 38.7 vs 455.8 +/- 100.9 microm2/vessel, p < 0.007). Increasing doses (from 0 to 1 to 2 copies) of the apolipoprotein E epsilon4 allele were also associated with greater amyloid per vessel without change in the proportion of affected vessels within each class of CAA severity. These findings suggest that progression from asymptomatic to advanced CAA reflects progressive accumulation of amyloid in vessels previously seeded with amyloid, and that this process is selectively enhanced by apolipoprotein E epsilon4.

Quantitation of apoE domains in Alzheimer disease brain suggests a role for apoE in Abeta aggregation.

Apolipoprotein E (apoE) and apoE-derived proteolytic fragments are present in amyloid deposits in Alzheimer disease (AD) and cerebral amyloid angiopathy (CAA). In this study, we examined which apoE fragments are most strongly associated with amyloid deposits and whether apoE receptor binding domains were present. We found that both apoE2- and apoE4-specific residues were present on plaques and blood vessels in AD and CAA. We quantified Abeta plaque burden and apoE plaque burdens in 5 AD brains. ApoE N-terminal-specific and C-terminal-specific antibodies covered 50% and 74% of Abeta plaque burden, respectively (p < 0.003). Double-labeling demonstrated that the plaque cores contained the entire apoE protein, but that outer regions contained only a C-terminal fragment, suggesting a cleavage in the random coil region of apoE. Presence of N- and C-terminal apoE cleavage fragments in brain extracts was confirmed by immunoblotting. The numbers of plaques identified by the apoE N-terminal-specific antibodies and the apoE C-terminal-specific antibody were equal, but were only approximately 60% of the total Abeta plaque number (p < 0.0001). Analysis of the size distribution of Abeta and apoE deposits demonstrated that most of the Abeta-positive, apoE-negative deposits were the smallest deposits (less than 150 microm2). These data suggest that C-terminal residues of apoE bind to Abeta and that apoE may help aid in the progression of small Abeta deposits to larger deposits. Furthermore, the presence of the apoE receptor binding domain in the center of amyloid deposits could affect surrounding cells via chronic interactions with cell surface apoE receptors.

Role of the low-density lipoprotein receptor-related protein in beta-amyloid metabolism and Alzheimer disease.

Deposition of beta-amyloid (A beta), a metabolite of approximately 4 kd of the amyloid precursor protein, is a critical pathological feature in Alzheimer disease. We postulate that deposition reflects an imbalance of A beta synthesis and clearance. Several pathways that impact A beta converge on a single receptor molecule, the low-density lipoprotein receptor-related protein (LRP). This multifunctional receptor is the major neuronal receptor both for apolipoprotein E (apoE, protein; APOE, gene) and for alpha2-macroglobulin (alpha2M, protein; A2M, gene), and it mediates clearance of apoE/A beta and alpha2M/A beta complexes. The LRP also interacts with the amyloid precursor protein itself. In this review, we highlight data that support a role for LRP in A beta metabolism and hypothesize that LRP therefore plays a critical role in Alzheimer disease.

Neuropathological changes in Down's syndrome hippocampal formation. Effect of age and apolipoprotein E genotype.

BACKGROUND: The neuropathological changes of Alzheimer's disease occur universally in individuals with Down's syndrome as they reach middle age and worsen with increasing age. Thus, evaluation of patients of various ages with Down's syndrome allows one to construct a life history of the development of neuropathological changes associated with Alzheimer's disease at various points in the disease process. METHODS: We have used semiquantitative scales and quantitative computerized image analysis techniques to analyze the characteristics of neurofibrillary tangle formation and A beta amyloid deposition in the hippocampal formation and inferior temporal gyrus in 36 individuals with Down's syndrome ranging in age from 4 to 73 years. RESULTS: Neurofibrillary tangles occur in a hierarchical distribution in a circumscribed set of neuronal fields, affecting the entorhinal cortex, area CA1/subiculum, then other hippocampal subfields. Although amyloid deposition occurs more evenly in a more widespread distribution, it also accumulates over the years 30 to 50. Surprisingly, examination of the patients available older than 50 years showed no trend toward continued increased deposition of amyloid. Within this group, however, individuals who had inherited the apolipoprotein E (Apo E) epsilon 4 genotype contained more than twice the amyloid burden of individuals who did not inherit the Apo E epsilon 4 genotype. COMMENT: This large series of cases confirms earlier observations that had suggested early vulnerability of entorhinal cortex and CA1/subiculum for neurofibrillary tangles and a more widespread but specific topography of A beta deposition. Moreover, it demonstrates quantitatively that the lesions increase to a certain level and then apparently reach a plateau. The level of amyloid deposition in Down's syndrome is higher than in sporadic Alzheimer's disease. Inheritance of the Apo E epsilon 4 genotype appears to be an additional (independent) risk factor for developing higher levels of amyloid accumulation.

Lack of association of a polymorphism in the low-density lipoprotein receptor-related protein gene with Alzheimer disease.

BACKGROUND: The apolipoprotein E epsilon 4 (ApoE epsilon 4) allele is associated with an increased risk for development of Alzheimer disease (AD). We hypothesized that polymorphisms in proteins that interact with ApoE also might have an impact on the likelihood of AD developing. We examined a polymorphism in the gene for the low-density lipoprotein receptor-related protein (LRP), because LRP is a major ApoE receptor in the brain that also mediates binding and degradation of secreted Kunitz protease inhibitor forms of amyloid precursor protein. SUBJECTS AND DESIGN: The LRP genotypes in 2 groups were studied. The first group consisted of 130 patients with probable or definite AD (mean +/- SD age, 78.2 +/- 8.9 years) and 64 nondemented, control subjects (mean +/- SD age, 81.7 +/- 12.3 years) who were primarily the spouses of the patients. The second group consisted of individuals from a population-based, epidemiologic study, including 38 cognitively impaired individuals (mean +/- SD age, 79.9 +/- 4.1 years) and 93 nondemented controls (mean +/- SD age, 78.7 +/- 4.4 years). Finally, 22 brains with a neuropathological diagnosis of AD were evaluated for neuronal loss, beta-amyloid deposition, and neurofibrillary tangle number and compared with the LRP genotype. RESULTS: No genetic disequilibrium in LRP allele frequencies between controls and patients with AD or cognitive impairment was observed. No interaction between the ApoE epsilon 4 and LRP genotypes was observed in patients with AD. Moreover, the LRP genotype did not correlate with degree of neuronal loss, beta-amyloid deposition, or neurofibrillary tangle number in individuals examined using quantitative neuropathological techniques. CONCLUSION: This LRP gene polymorphism is not linked with the pathophysiological changes of AD.

Genetic association of a cystatin C gene polymorphism with late-onset Alzheimer disease.

OBJECTIVE: To determine whether the cystatin C gene (CST3) is genetically associated with late-onset Alzheimer disease (AD). DESIGN: A case-control study with 2 independent study populations of patients with AD and age-matched, cognitively normal control subjects. SETTING: The Alzheimer's Disease Research Unit at the University Hospital Hamburg-Eppendorf, Hamburg, Germany, for the initial study (n = 260). For the independent multicenter study (n = 647), an international consortium that included the Massachusetts Alzheimer's Disease Research Center at the Massachusetts General Hospital, Boston; the Scientific Institute for Research and Patient Care, Brescia, Italy; and Alzheimer's research units at the Universities of Basel and Zurich, Switzerland, and Bonn, Goettingen, and Hamburg, Germany. PARTICIPANTS: Five hundred seventeen patients with AD and 390 control subjects. MEASURES: Molecular testing of the KspI polymorphisms in the 5' flanking region and exon 1 of CST3 and the apolipoprotein E (APOE) genotype. Mini-Mental State Examination scores for both patients with AD and control subjects. RESULTS: Homozygosity for haplotype B of CST3 was significantly associated with late-onset AD in both study populations, with an odds ratio of 3.8 (95% confidence interval, 1.56-9.25) in the combined data set; heterozygosity was not associated with an increased risk. The odds ratios for CST3 B/B increased from 2.6 in those younger than 75 years to 8.8 for those aged 75 years and older. The association of CST3 B/B with AD was independent of APOE epsilon4; both genotypes independently reduced disease-free survival. CONCLUSIONS: CST3 is a susceptibility gene for late-onset AD, especially in patients aged 75 years and older. To our knowledge, CST3 B is the first autosomal recessive risk allele in late-onset AD.

Confirmation of an association between a polymorphism in exon 3 of the low-density lipoprotein receptor-related protein gene and Alzheimer's disease.

C766T, a polymorphism in exon 3 of the gene for the low-density lipoprotein receptor-related protein (LRP), was found to be associated with late-onset Alzheimer's disease (AD). We developed a PCR-restriction enzyme-based assay to analyze this allele in 234 AD patients and 103 controls. We confirmed that the LRP C766T polymorphism was in disequilibrium with AD--the C/C genotype was present in 76% of AD patients and 60% of controls (p < 0.01); however, the LRP polymorphism did not influence age at onset of AD.

Alzheimer's disease with and without coexisting Parkinson's disease changes: apolipoprotein E genotype and neuropathologic correlates.

The frequency of the apolipoprotein E (ApoE) epsilon 4 allele and its relationship with coexistent Parkinson's disease (PD) neuropathology in Alzheimer's disease (AD) have not been extensively explored. We determined ApoE genotype in 100 dementia patients with neuropathologically confirmed AD with and without concomitant Parkinson's disease (PD) changes (nigral degeneration and Lewy bodies at various sites). Fifty "AD+PD" patients were matched closely with 50 "pure AD" patients for age, sex, and duration of dementia. We found identical overrepresentation of the epsilon 4 allele in the two groups: 72% of the patients in each group had at least one ApoE epsilon 4 allele, compared with approximately 25% in the general population (p < 0.005) and in our institutional autopsy population (p < 0.001). Age at onset varied inversely with epsilon 4 allele dosage in men but not in women in both the AD and the AD+PD groups. As with amyloid deposition and plaque frequency in AD, we observed an association between epsilon 4 dosage and PD-related changes. Specifically, the severity of ubiquitin-positive neuritic change in CA2/3 of the hippocampus, but not the frequency of cortical Lewy bodies, varied significantly with epsilon 4 dosage in the AD+PD cases.

APOE genotype and gender effects on Alzheimer disease in 100 adults with Down syndrome.

BACKGROUND: Alzheimer disease (AD) neuropathology is present in Down syndrome (DS) after age 35, but dementia onset varies from ages 40 to 70 years. Because of small sample sizes and nonuniform determination of dementia, previous studies produced differing results on the influence of APOE subtypes on AD in DS. OBJECTIVE: To determine the influence of the APOE genotype and gender on development of AD in adults with DS to ascertain similarities with AD in the general population. METHODS: A total of 100 adults with DS (ages 35 to 79 years), almost all of whom were longitudinally assessed by neurologists, underwent APOE genotyping. Dementia onset was determined using criteria applied from the Tenth International Classification of Mental and Behavioral Disorders. This cohort contains the largest number of DS subjects with dementia (n = 57) in a single study, thus increasing reliability of the results. RESULTS: The epsilon2 allele frequency was 4% in those with dementia versus 13% in those without dementia (p = 0.03); epsilon4 allele frequency was 18% in those with dementia versus 13% in those without dementia (p = 0.45). Using APOE-epsilon3/3 as the reference group, the risk ratio for the development of AD at any given time was 0.34 for the APOE-epsilon2/3 group (p = 0.04) and 1.44 for the APOE-epsilon(3/4,4/4) group (p = 0.25). Women were 1.77 times as likely to dement as men at any given point in time (p = 0.04). CONCLUSIONS: The epsilon2 allele confers a protective effect, and women with DS have an increased risk for AD, as in the general population. In this sample, epsilon4 does not confer a significantly increased risk for AD in DS.

Reduced apolipoprotein epsilon 4 allele frequency in the oldest old Alzheimer's patients and cognitively normal individuals.

Recent genetic studies show that the apolipoprotein E epsilon 4 allele (ApoE-epsilon 4) is a risk factor for Alzheimer's disease (AD). If ApoE-epsilon 4 individuals develop AD as they get older, we would expect a decrease in ApoE-epsilon 4 allele frequency with increasing age. We found a marked decline in ApoE-epsilon 4 allele frequency with advancing age in both AD and cognitively normal controls (p < 0.003), although in all age groups the ApoE-epsilon 4 allele was overrepresented (p < 0.0001). Nonetheless, a few cognitively normal nonagenarians were ApoE-epsilon 4 positive. Thus, our data support two new conclusions: (1) the ApoE-epsilon 4 associated risk for AD is age-dependent, probably due to censoring by the earlier development of AD in ApoE-epsilon 4 individuals, and (2) despite the ApoE-epsilon 4 associated risk for AD, it is possible to reach extreme old age with normal cognition.

A newly identified polymorphism in the apolipoprotein E enhancer gene region is associated with Alzheimer's disease and strongly with the epsilon 4 allele.

Apolipoprotein E allele 4 (apoE epsilon 4) is a major risk factor for late-onset AD. Inheritance of this allele is associated with an earlier age of onset of dementia in individuals with AD. It is unknown whether other polymorphisms in the apoE gene may influence the effect of apoE epsilon 4 on AD. We screened portions of the promoter enhancer element and of the apoE receptor binding domain for other polymorphisms that could affect risk of AD. In particular, a C/G polymorphism at position +113 of the apoE mRNA in the apoE intron 1 enhancer element (IE1) has been recently identified. We found no other polymorphisms. We studied the relationship of the two alleles of the IE1 polymorphism with AD and found an apparent association between IE1 G and AD (n = 94; p = 0.0515). However, the IE1 G allele is also closely associated with apoE epsilon 4 (p < 0.0001). When the presence of apoE epsilon 4 is covaried, the association between the IE1 G allele and AD is no longer statistically significant (odds ratio = 1.29, 95% confidence interval: 0.44, 3.78). In contrast, epsilon 4 is still highly associated with AD when IE1 G is controlled for (odds ratio = 5.91, 95% confidence interval: 3.29, 10.63). Furthermore, there is no significant association between the age of onset of dementia and the inheritance of the G allele. We believe that the apparent association between IE1 G and AD is a consequence of the association between the epsilon 4 and IE1 G alleles.

Association of apolipoprotein E epsilon2 and vasculopathy in cerebral amyloid angiopathy.

OBJECTIVE: Hemorrhage related to cerebral amyloid angiopathy (CAA) appears to occur through a multistep pathway that includes deposition of beta-amyloid in cerebral vessels and specific vasculopathic changes in the amyloid-laden vessels, such as cracking of the vessel wall. Recent reports suggest a positive association between CAA-related hemorrhage and both the apolipoprotein E (APOE) epsilon4 allele and, unexpectedly, the APOE epsilon2 allele. Unlike APOE epsilon4, APOE epsilon2 does not appear to act through increased beta-amyloid deposition. We therefore sought to determine whether it might specifically accelerate the second step in this pathway, that is, development of the vasculopathic changes that lead to hemorrhage. METHODS: To determine the role of APOE in development of vasculopathic changes, we compared APOE genotypes in two groups of postmortem brains: 52 brains with complete amyloid replacement of vessel walls but without vasculopathic changes, and 23 brains with complete amyloid replacement of vessels with the accompanying changes of cracking of the vessel wall and paravascular leaking of blood. RESULTS: Frequency of APOE epsilon2 was significantly greater in the group with vasculopathy (0.09) than the group without (0.01, p = 0.03). The groups did not differ in mean age or extent of neuritic plaques. Analysis of a clinical series of patients with CAA-related hemorrhage confirmed an overrepresentation of APOE epsilon2 as well as an association between this allele and earlier age of first hemorrhage. CONCLUSIONS: These data suggest that APOE epsilon2 and epsilon4 might promote CAA-related hemorrhage through separate mechanisms: epsilon4 by enhancing amyloid deposition and epsilon2 by causing amyloid-laden vessels to undergo the vasculopathic changes that lead to rupture.

ApoE isoforms affect neuronal N-methyl-D-aspartate calcium responses and toxicity via receptor-mediated processes.

Apolipoprotein E (apoE) alters the pathophysiology of Alzheimer's disease, but its mechanism is not fully understood. We examined the effects of recombinant human apoE3 and apoE4 on the neuronal calcium response to N-methyl-D-aspartate (NMDA), and compared them to their toxicity. ApoE4 (100 nM) significantly increased the resting calcium (by 70%) and the calcium response to NMDA (by 185%), whereas similar changes were not obtained in apoE3-treated neurons. ApoE4, but not apoE3, also significantly increased neurotoxicity, as evidenced by enhanced lactate dehydrogenase release (by 53%) and reduced 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide levels (by 32%). ApoE4-induced changes in the calcium response to NMDA and associated neurotoxicity were blocked by coincubation with MK-801. Both the receptor-associated protein, which inhibits interaction of apoE with members of the LDL receptor family, including the low-density lipoprotein receptor-related protein (LRP), and activated alpha2-macroglobulin, another LRP ligand, prevented apoE4-induced enhancement of the calcium response to NMDA, resting calcium levels, and neurotoxicity. A tandem apoE peptide (100 nM) containing only the receptor binding region residues also eliminated the enhanced calcium signaling and neurotoxicity by apoE4. Taken together, our data demonstrate that differential effects of apoE3 and apoE4 on the calcium signaling in neurons correlate with their effect on neurotoxicity, which are secondary to receptor binding.

Expression and alternate splicing of apolipoprotein E receptor 2 in brain.

Apolipoprotein E isoforms affect the risk of developing Alzheimer's disease. Apolipoprotein E-associated risk may be related to its binding to and clearance by cell surface receptors, including members of the low-density lipoprotein receptor family. We examined the brain expression of the most recently identified member of this receptor family, apolipoprotein E receptor 2, in human brain and placenta. We analysed apolipoprotein E receptor 2 messenger RNA by reverse transcription-polymerase chain reaction and apolipoprotein E receptor 2 protein by immunohistochemistry. Four exons of the apolipoprotein E receptor 2 message were alternately spliced in both fetal and adult brain tissue. Exon 5, encoding three of the seven ligand binding repeats, was absent in the apolipoprotein E receptor 2 messenger RNA examined. Apolipoprotein E receptor 2 messages lacking exon 8, encoding an epidermal growth factor precursor repeat, exon 15, encoding the O-glycosylation region, or exon 18, encoding a cytoplasmic domain, were also present as minor splice variants in the brain and placenta. No differences were observed in the pattern of apolipoprotein E receptor 2 splicing between control and Alzheimer brains. Immunohistochemistry of mouse brain showed that apolipoprotein E receptor 2 was expressed in neurons throughout the brain, with strong expression in pyramidal neurons of the hippocampus, granule cells of the dentate gyrus, cortical neurons and Purkinje cells of the cerebellum. Thus, apolipoprotein E receptor 2 is the fourth apolipoprotein E receptor identified on neuronal cells.

Association of membrane-bound amyloid precursor protein APP with the apolipoprotein E receptor LRP.

In order to identify cell surface proteins that interact with the amyloid precursor protein (APP), we biotinylated H4 human neuroglioma cells in culture with a water soluble biotinylating agent, immunoprecipitated APP with an antibody specific to the intracellular domain, and probed the precipitated proteins with anti-biotin. In human neuroglioma cells overexpressing APP751, we found a high molecular weight protein that immunoprecipitated with APP. This band was identified as the low density lipoprotein receptor-related protein (LRP) by three criteria: first, the band immunolabeled with anti-LRP antibodies; second, the band bound the LRP receptor associated protein, RAP; and third, this band was present in LRP-expressing fibroblasts, but not LRP-deficient fibroblasts. In complementary experiments, we found that APP co-precipitated with LRP, with a preference for an isoform of APP containing the Kunitz protease inhibitor domain. Interaction of APP and LRP on the surface of living cells was demonstrated by crosslinking APP and LRP with the water-soluble cross-linking agent BS(3). APP and LRP were shown by confocal microscopy to colocalize in perinuclear structures, but to primarily remain separate in vesicles and on the cell surface. We propose that full-length APP can transiently interact with the receptor LRP on the cell surface, affecting the processing and intracellular transport of APP.