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We have previously reported cognitive impairments in both young and old mice, particularly in female mice expressing mouse Arg-61 apoE, with a point mutation to mimic the domain interaction feature of human apoE4, as compared to the wildtype mouse (C57BL/6J) apoE. In this study, we further evaluated water maze performance in the female Arg-61 mice at an additional time point and then investigated related hippocampal cyto-architecture in these young female Arg-61 apoE mice vs. the wildtype mice. The results of behavioral performance consistently support our previous report that the young female Arg-61 apoE showed cognitive impairment versus C57BL/6J at the same age. The cyto-architectural results showed that volume of the granular cell layer (GCL) was significantly larger in both 5- and 10-month old Arg-61 apoE mice versus C57BL/6J mice. While the number of newborn calretinin-positive neurons was greater in the sub-granular zone (SGZ) in 5-month old Arg-61 mice, this number dropped significantly in 10-month old Arg-61 mice to a lower level than in age-matched C57BL/6J mice. In addition, the amyloid β species was significantly higher in 5-month old Arg-61 mice versus age-matched C57BL/6J mice. In conclusion, impaired cognitive functions in female Arg-61 apoE mice appear correlated with larger GCL volume and higher calretinin-positive cell number and suggest a compensatory cellular response that may be related to amyloid beta perturbations early in life. Therefore this study suggests a novel cyto-architectural mechanism of apoE4-dependent pathologies and increased susceptibility of APOEe4 subjects to Alzheimer’s disease.

Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.

Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC(50) of 23 and 116 nm, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.

Our previous work indicated that apolipoprotein (apo) E4 assumes a more expanded conformation in the postprandial period. The postprandial state is characterized by increased VLDL lipolysis. In this article, we tested the hypothesis that VLDL lipolysis products increase VLDL particle fluidity, which mediates expansion of apoE4 on the VLDL particle. Plasma from healthy subjects was collected before and after a moderately high-fat meal and incubated with nitroxyl-spin labeled apoE. ApoE conformation was examined by electron paramagnetic resonance spectroscopy using targeted spin probes on cysteines introduced in the N-terminal (S76C) and C-terminal (A241C) domains. Further, we synthesized a novel nitroxyl spin-labeled cholesterol analog, which gave insight into lipoprotein particle fluidity. Our data revealed that the order of lipoprotein fluidity was HDL approximately LDL

Domain interaction, a structural property of apolipoprotein E4 (apoE4), is predicted to contribute to the association of apoE4 with Alzheimer disease. Arg-61 apoE mice, a gene-targeted mouse model specific for domain interaction, have lower brain apoE levels and synaptic, functional, and cognitive deficits. We hypothesized that domain interaction elicits an endoplasmic reticulum (ER) stress in astrocytes and an unfolded protein response that targets Arg-61 apoE for degradation. Primary Arg-61 apoE astrocytes had less intracellular apoE than wild-type astrocytes, and unfolded protein response markers OASIS (old astrocyte specifically induced substance), ATF4, and XBP-1 and downstream effectors were up-regulated. ER stress appears to cause global astrocyte dysfunction as glucose uptake was decreased in Arg-61 apoE astrocytes, and astrocyte-conditioned medium promoted neurite outgrowth less efficiently than wild-type medium in Neuro-2a cell cultures. We showed age-dependent up-regulation of brain OASIS levels and processing in Arg-61 apoE mice. ER stress and astrocyte dysfunction represent a new paradigm underlying the association of apoE4 with neurodegeneration.

Apolipoprotein E4 (apoE4) is the major genetic risk factor for Alzheimer’s disease (AD) by an as yet to be defined mechanism. Since the structure or biophysical properties of a protein directly determines function, our approach to addressing mechanism is structure:function based. Domain interaction a structural property of apoE4 that distinguishes it from apoE3 is predicted to contribute to the association of apoE4 with AD. We developed a mouse model, the Arg-61 apoE model, which is specific for domain interaction. These mice display synaptic, functional, and cognitive deficits, demonstrating domain interaction is the causative factor. We present evidence that domain interaction results in stressed astrocytes that are dysfunctional and propose that dysfunctional astrocytes are an early player in apoE4-associated AD and that domain interaction is a potential therapeutic target.

Transforming growth factor-beta1 (TGF-beta1) has central functions in development, tissue maintenance, and repair and has been implicated in major diseases. We discovered that TGF-beta1 contains several amphipathic helices and hydrophobic domains similar to apolipoprotein E (apoE), a protein involved in lipoprotein metabolism. Indeed, TGF-beta1 associates with lipoproteins isolated from human plasma, cultured liver cells, or astrocytes, and its bioactivity was highest in high-density lipoprotein preparations. Importantly, lipoproteins containing the apoE3 isoform had higher TGF-beta levels and bioactivity than those containing apoE4, a major genetic risk factor for atherosclerosis and Alzheimer's disease. Because TGF-beta1 can be protective in these diseases an association with apoE3 may be beneficial. Association of TGF-beta with different types of lipoproteins may facilitate its diffusion, regulate signaling, and offer additional specificity for this important growth factor.

Despite intense interest, the molecular mechanisms underlying the association of apoE4 with Alzheimer disease are not clear. Because the function (or dysfunction) of a protein is based on its structure, this review focuses on the effects of the structural differences among the isoforms on neurodegeneration. Understanding how apoE4 structure impacts neurodegeneration is likely to provide mechanistic insight as well as potential therapeutic approaches to blunt or reduce its effects.

Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrPC) to a misfolded, pathogenic isoform (PrPSc). Prion inoculation experiments in mice expressing homologous PrPCmolecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrPC, our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.

Background Apolipoprotein E4 (apoE4), the major genetic risk factor for Alzheimer’s disease (AD) and other neurodegenerative diseases, has three structural and biophysical properties that distinguish it from the other isoforms—domain interaction, reduced stability, and lack of cysteine. Assessing their relative contributions to effects of apoE4-associated pathogenesis in AD is important from a mechanistic and therapeutic perspective, that is not possible using human apoE transgene or knock-in models. Methods We analyzed Arg-61 apoE mice, a gene-targeted model that selectively displays domain interaction. Results The mice displayed age-dependent loss of the synaptic protein synaptophysin in neocortex and hippocampus and had lower levels of the postsynaptic neuroligin-1. Activation of dentate gyrus granule neurons increased Arc expression 3.5-fold in wildtype mice but only 2.3-fold in Arg-61 mice. The losses of synaptic proteins caused a mild memory deficit in Arg-61 mice in the water-maze test. Since synaptic integrity requires efficient glutamate uptake, we measured astrocyte glutamate transporter 1 in the hippocampus. The level was reduced in Arg-61 mice, suggesting that inefficient glutamate uptake by astrocytes causes chronic excitotoxicity. Consistent with the reduced secretion of Arg-61 apoE by astrocytes in this model, cholesterol secretion was also reduced 34%. This reduction could also contribute to the synaptic deficits by limiting the availability of cholesterol for neuronal repair. Conclusions Domain interaction in the absence of other structural characteristics of apoE4 is sufficient to cause synaptic pathology and functional synaptic deficits, potentially associated with astrocyte dysfunction and impaired maintenance of neurons. Therapeutic targeting of domain interaction might blunt effects of apoE4 in neurodegenerative disease.

Apolipoprotein E (apoE) is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and infectious diseases. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, the structural study of full-length apoE is hindered by apoE's oligomerization property. Using protein-engineering techniques, we generated a monomeric, biologically active, full-length apoE. Cross-linking experiments indicate that this mutant is nearly 95-100% monomeric even at 20 mg/mL. CD spectroscopy and guanidine hydrochloride denaturation demonstrate that the structure and stability of the monomeric mutant are identical to wild-type apoE. Monomeric and wild-type apoE display similar lipid-binding activities in dimyristoylphosphatidylcholine clearance assays and formation of reconstituted high-density lipoproteins. Furthermore, the monomeric and wild-type apoE proteins display an identical LDL receptor binding activity. Availability of this monomeric, biologically active, full-length apoE allows us to collect high quality NMR data for structural determination. Our initial NMR data of lipid-free apoE demonstrates that the N-terminal domain in the full-length apoE adopts a nearly identical structure as the isolated N-terminal domain, whereas the C-terminal domain appears to become more structured than the isolated C-terminal domain fragment, suggesting a weak domain-domain interaction. This interaction is confirmed by NMR examination of a segmental labeled apoE, in which the N-terminal domain is deuterated and the C-terminal domain is double-labeled. NMR titration experiments further suggest that the hinge region (residues 192-215) that connects apoE's N- and C-terminal domains may play an important role in mediating this domain-domain interaction.

Apolipoprotein E (apoE) is a major protein component of cholesterol-transporting lipoprotein particles in the central nervous system and in plasma. Polymorphisms of apoE are associated with cardiovascular disease and with a predisposition to Alzheimer's disease and other forms of neurodegeneration. For full biological activity, apoE must be bound to a lipoprotein particle. Complexes of apoE and phospholipid mimic many of these activities. In contrast to a widely accepted discoidal model of apoA-I bound to dimyristoylphosphatidylcholine, which is based on solution studies, an X-ray diffraction study of apoE bound to dipalmitoylphosphatidylcholine (DPPC) indicated that apoE*DPPC particles are quasi-spheroidal and that the packing of the phospholipid core is similar to a micelle. Using small-angle X-ray scattering, we show that apoE*DPPC particles in solution are ellipsoidal and that the shape of the phospholipid core is compatible with a twisted-bilayer model. The proposed model is consistent with the results of mass spectrometric analysis of products of limited proteolysis. These revealed that the nonlipid-bound regions of apoE in the particle are consistent with an alpha-helical hairpin.

The three isoforms of apolipoprotein (apo) E are strongly associated with different risks for Alzheimer's disease: apoE4 > apoE3 > apoE2. Here, we show at physiological salt concentrations and pH that native tetramers of apoE form soluble aggregates in vitro that bind the amyloid dyes thioflavin T and Congo red. However, unlike classic amyloid fibrils, the aggregates adopt an irregular protofilament-like morphology and are seemingly highly alpha-helical. The aggregates formed at substantially different rates (apoE4 > apoE3 > apoE2) and were significantly more toxic to cultured neuronal cells than the tetramer. Since the three isoforms have large differences in conformational stability that can influence aggregation and amyloid pathways, we tested the effects of mutations that increased or decreased stability. Decreasing the conformational stability of the amino-terminal domain of apoE increased aggregation rates and vice versa. Our findings provide a new perspective for an isoform-specific pathogenic role for apoE aggregation in which differences in the conformational stability of the amino-terminal domain mediate neurodegeneration. (c) 2006 Elsevier Ltd. All rights reserved.

Human apolipoprotein E (apoE) is a member of the family of soluble apolipoproteins. Through its interaction with members of the low-density lipoprotein receptor family, apoE has a key role in lipid transport both in the plasma and in the central nervous system. Its three common structural isoforms differentially affect the risk of developing atherosclerosis and neurodegenerative disorders, including Alzheimer's disease. Because the function of apoE is dictated by its structure, understanding the structural properties of apoE and its isoforms is required both to determine its role in disease and for the development of therapeutic strategies.

To avoid toxic overload of cholesterol in peripheral cells, the reverse cholesterol transport pathway directs excess cholesterol through HDL acceptors to the liver for elimination. In this issue of the JCI, a study by Matsuura et al. reveals new features of this pathway, including the importance of the ATP-binding cassette transporter G1 in macrophages and apoE in cholesteryl efflux from cells to cholesterol ester-rich (CE-rich) HDL(2) acceptors (see the related article beginning on page 1435). One proposal for boosting reverse cholesterol transport has been to elevate plasma HDL levels by inhibiting CE transfer protein (CETP), which transfers CE from HDL to lower-density lipoproteins. However, there has been concern that large, CE-rich HDL(2) generated by CETP inhibition might impair reverse cholesterol transport. ApoE uniquely facilitates reverse cholesterol transport by allowing CE-rich core expansion in HDL. In lower species, these large HDLs are not atherogenic. Thus, CETP might not be essential for reverse cholesterol transport in humans, raising hope of using a CETP inhibitor to elevate HDL levels.

The premise of this review is that apolipoprotein (apo) E4 is much more than a contributing factor to neurodegeneration. ApoE has critical functions in redistributing lipids among CNS cells for normal lipid homeostasis, repairing injured neurons, maintaining synapto-dendritic connections, and scavenging toxins. In multiple pathways affecting neuropathology, including Alzheimer’s disease, apoE acts directly or in concert with age, head injury, oxidative stress, ischemia, inflammation, and excess amyloid β peptide production to cause neurological disorders, accelerating progression, altering prognosis, or lowering age of onset. We envision that unique structural features of apoE4 are responsible for apoE4-associated neuropathology. Although the structures of apoE2, apoE3, and apoE4 are in dynamic equilibrium, apoE4, which is detrimental in a variety of neurological disorders, is more likely to assume a pathological conformation. Importantly, apoE4 displays domain interaction (an interaction between the N- and C-terminal domains of the protein that results in a compact structure) and molten globule formation (the formation of stable, reactive intermediates with potentially pathological activities). In response to CNS stress or injury, neurons can synthesize apoE. ApoE4 uniquely undergoes neuron-specific proteolysis, resulting in bioactive toxic fragments that enter the cytosol, alter the cytoskeleton, disrupt mitochondrial energy balance, and cause cell death. Our findings suggest potential therapeutic strategies, including the use of “structure correctors” to convert apoE4 to an “apoE3-like” molecule, protease inhibitors to prevent the generation of toxic apoE4 fragments, and “mitochondrial protectors” to prevent cellular energy disruption.

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer's disease, and many studies have suggested that apoE has isoform-specific effects on the deposition or clearance of amyloid beta (Abeta) peptides. We examined the effects of apoE isoforms on the processing of amyloid precursor protein (APP) and on Abeta production in rat neuroblastoma B103 cells stably transfected with human wild-type APP695 (B103-APP). Lipid-poor apoE4 increased Abeta production in B103-APP cells to a greater extent than lipid-poor apoE3 (60% vs. 30%) due to more pronounced stimulation of APP recycling by apoE4 than apoE3. The difference in Abeta production was abolished by preincubating the cells with the receptor-associated protein (25 nM), which blocks the low-density lipoprotein receptor-related protein (LRP) pathway, or by reducing LRP expression by small interference RNA. The differences were also attenuated by replacing Arg-61 with threonine in apoE4 or pretreating apoE4 with small molecules, both of which abolish apoE4 intramolecular domain interaction. Thus, apoE4 appears to modulate APP processing and Abeta production through both the LRP pathway and domain interaction. These findings provide insights into why apoE4 is associated with increased risk for Alzheimer's disease and may represent a potential target for drug development.

Interaction of the amino- and carboxyl-terminal domains in apolipoprotein (apo) E, referred to as domain interaction, is predicted to be more pronounced in apoE4 than in apoE3 and to underlie the association of apoE4 with Alzheimer and cardiovascular diseases. However, direct physical proof for the domain interaction concept is lacking. To address this issue, fluorescence resonance energy transfer and electron paramagnetic resonance spectroscopy were used to probe the spatial proximity of the two domains of apoE. Both methods demonstrated that the two domains are closer in both lipid-free and phospholipid-bound apoE4 than in apoE3 as a result of domain interaction. In addition, as shown by electron paramagnetic resonance, the domains of apoE4 move apart to resemble more closely the distance in apoE3 when the isoforms are bound to triglyceride-rich emulsion particles. These results demonstrate that domain interaction is a structural property of apoE4 and that apoE adopts different conformations when complexed to different lipids.

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer and cardiovascular diseases. ApoE4 differs from the two other common isoforms (apoE2 and apoE3) by its lower resistance to denaturation and greater propensity to form partially folded intermediates. As a first step to determine the importance of stability differences in vivo, we reengineered a partially humanized variant of the amino-terminal domain of mouse apoE (T61R mouse apoE) to acquire a destabilized conformation like that of apoE4. For this process, we determined the crystal structure of wild-type mouse apoE, which, like apoE4, forms a four-helix bundle, and identified two structural differences in the turn between helices 2 and 3 and in the middle of helix 3 as potentially destabilizing sites. Introducing mutations G83T and N113G at these sites destabilized the mouse apoE conformation. The mutant mouse apoE more rapidly remodeled phospholipid than T61R mouse apoE, which supports the hypothesis that a destabilized conformation promotes apoE4 lipid binding.

The ability of apolipoprotein E (apoE) to be spared degradation in lysosomes and to recycle to the cell surface has been demonstrated by our group and others, but its physiologic relevance is unknown. In this study, we characterized apoE recycling in primary murine macrophages and probed the effects of HDL and apoA-I on this process. In cells pulsed with 125I·apoE bound to VLDL, intact apoE was found in the chase medium for up to 24 h after the pulse. Approximately 27 ± 5% of the apoE internalized during the pulse was recycled after 4 h of chase. Addition of apoA-I and HDL increased apoE recycling to 45 ± 3% and 46 ± 3%, respectively, similar to the amount of apoE recycled after pulsing the cells with 125I·apoE·HDL. In addition, apoA-I-producing macrophages from transgenic mice showed increased apoE recycling at 4 h (38 ± 3%). Increased ABCA1 expression potentiated apoE recycling, suggesting that recycling occurs via ABCA1. Finally, in the presence of apoA-I, recycled apoE exited the cells on HDL-like particles. These results suggest that apoE recycling in macrophages may be part of a larger signaling loop activated by HDL and directed at maximizing cholesterol losses from the cell.

Background: We have previously shown that chylomicron (CM)-bound lipopolysaccharide (LPS) inhibits the host innate immune response by rendering hepatocytes tolerant to pro-inflammatory cytokine stimulation. However, LPS is a complex macromolecule containing both lipid and carbohydrate domains. We hypothesized that just as lipid A confers the toxicity of LPS, it is also responsible for the immunoregulatory effect on hepatocytes. Methods: We pretreated primary rat hepatocytes for 2 h with a series of CM-LPS complexes in which the endotoxin moiety varied in its structure and/or toxicity. Subsequently, the cells were stimulated with a mixture of pro-inflammatory cytokines. Nitric oxide production was measured as an indicator of hepatocellular activation. Results: All pretreatments wherein the CM-bound complex contained the lipid A moiety readily inhibited the hepatocellular cytokine response, including CM bound to lipid A alone. In contrast, CM-LPS complexes containing detoxified LPS, which lacks the lipid A domain, had no effect on the hepatocellular response to cytokines. Conclusions: The lipid A domain of the LPS macromolecule is both sufficient and essential for the CM-mediated induction of cytokine tolerance in hepatocytes. However, this process is independent of the specific endotoxic activity of the lipid A moiety.

Our previous studies have indicated that lecithin-cholesterol acyltransferase (LCAT) contributes significantly to the apoB lipoprotein cholesteryl ester (CE) pool. Cholesterol esterification rate (CER) in apoA-I(-)(/)(-) apoE(-)(/)(-) mouse plasma was

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer disease. Although the mechanisms remain to be determined, the detrimental effects of apoE4 in neurobiology must be based on its unique structural and biophysical properties. One such property is domain interaction mediated by a salt bridge between Arg-61 in the N-terminal domain and Glu-255 in the C-terminal domain of apoE4. This interaction, which does not occur in apoE3 or apoE2, causes apoE4 to bind preferentially to certain lipoprotein particles in vitro and in vivo. Here we used fluorescence resonance energy transfer (FRET) to determine whether apoE4 domain interaction occurs in living neuronal cells. Neuro-2a cells were transfected with constructs encoding apoE3 or apoE4 in which yellow fluorescent protein (YFP) was fused to the N terminus, and cyan fluorescent protein (CFP) was fused to the C terminus. To generate a FRET signal that can be detected by spectrum confocal microscopy, the labeled N and C termini must be in close proximity (

Apolipoprotein (apo) E4 increases the risk and accelerates the onset of Alzheimer's disease (AD). However, the underlying mechanisms remain to be determined. We previously found that apoE undergoes proteolytic cleavage in AD brains and in cultured neuronal cells, resulting in the accumulation of carboxyl-terminal-truncated fragments of apoE that are neurotoxic. Here we show that this fragmentation is caused by proteolysis of apoE by a chymotrypsin-like serine protease that cleaves apoE4 more efficiently than apoE3. Transgenic mice expressing the carboxyl-terminal-cleaved product, apoE4(Δ272–299), at high levels in the brain died at 2–4 months of age. The cortex and hippocampus of these mice displayed AD-like neurodegenerative alterations, including abnormally phosphorylated tau (p-tau) and Gallyas silver-positive neurons that contained cytosolic straight filaments with diameters of 15–20 nm, resembling preneurofibrillary tangles. Transgenic mice expressing lower levels of the truncated apoE4 survived longer but showed impaired learning and memory at 6–7 months of age. Thus, carboxyl-terminal-truncated fragments of apoE4, which occur in AD brains, are sufficient to elicit AD-like neurodegeneration and behavioral deficits in vivo . Inhibiting their formation might inhibit apoE4-associated neuronal deficits.

The accumulation and aggregation of the amyloid-β peptide (Aβ) in the brain are important contributing factors to Alzheimer's disease (AD). Consequently, blocking the generation of Aβ is a potentially important treatment strategy. Recent work on the metabolism of Aβ has identified several cellular proteins and proteases that collectively promote or prevent the generation of Aβ. In addition, accumulating in vitro and in vivo evidence suggests a role for cholesterol in modulating the cellular processing of Aβ with the potential to affect AD.

The importance of hepatocyte-derived apolipoprotein (apo) E in the clearance of remnant lipoproteins in the liver is controversial. To address this controversy, we compared remnant clearance in two mouse models in which apoE is primarily derived either from hepatocytes or from an extrahepatic source. Hypomorphic apoE mice universally express reduced levels of apoE in all tissues, with the liver remaining the primary source of apoE. This mouse model of hepatocyte-derived apoE was compared with Apoe(-/-) mice transplanted with mouse bone marrow as a model of primarily non-hepatocyte-derived apoE. Immunohistochemical analysis of liver sections revealed that only the hepatocyte-derived apoE model had detectable levels of apoE on hepatic sinusoidal surfaces. The non-hepatocyte-derived apoE model with plasma apoE levels similar to those in the hepatocyte-derived model had 2-fold more total plasma cholesterol, 4-fold more total plasma triglycerides, and 8-fold higher levels of apoB48, similar to Apoe(-/-) mice. Both the hepatocyte-derived and the non-hepatocyte-derived apoE models had delayed clearance of an infused bolus of (125)I-labeled remnants compared with wild-type mice. However, after 3 h, plasma remnants reached wild-type levels only in the hepatocyte-derived apoE model, which had accumulated 70 +/- 5% of wild-type levels of remnants in the liver while the non-hepatocyte-derived apoE model had accumulated only 38 +/- 4%. These results demonstrate the existence of a role for both hepatically derived and localized apoE in remnant clearance. This role likely represents the enrichment of remnants sequestered on hepatocyte, with hepatocyte-derived apoE, facilitating their receptor-mediated internalization.

The amino-terminal domain of apolipoprotein (apo) E4 is less susceptible to chemical and thermal denaturation than the apoE3 and apoE2 domains. We compared the urea denaturation curves of the 22-kDa amino-terminal domains of the apoE isoforms at pH 7.4 and 4.0. At pH 7.4, apoE3 and apoE4 reflected an apparent two-state denaturation. The midpoints of denaturation were 5.2 and 4.3 m urea, respectively. At pH 4.0, a pH value known to stabilize folding intermediates, apoE4 and apoE3 displayed the same order of denaturation but with distinct plateaus, suggesting the presence of a stable folding intermediate. In contrast, apoE2 proved the most stable and lacked the distinct plateau observed with the other two isoforms and could be fitted to a two-state unfolding model. Analysis of the curves with a three-state unfolding model (native, intermediate, and unfolded) showed that the apoE4 folding intermediate reached its maximal concentration ( approximately 90% of the mixture) at 3.75 m, whereas the apoE3 intermediate was maximal at 4.75 m ( approximately 80%). These results are consistent with apoE4 being more susceptible to unfolding than apoE3 and apoE2 and more prone to form a stable folding intermediate. The structure of the apoE4 folding intermediate at pH 4.0 in 3.75 m urea was characterized using pepsin proteolysis, Fourier transform infrared spectroscopy, and dynamic light scattering. From these studies, we conclude that the apoE4 folding intermediate is a single molecule with the characteristics of a molten globule. We propose a model of the apoE4 molten globule in which the four-helix bundle of the amino-terminal domain is partially opened, generating a slightly elongated structure and exposing the hydrophobic core. Since molten globules have been implicated in both normal and abnormal physiological function, the differential abilities of the apoE isoforms to form a molten globule may contribute to the isoform-specific effects of apoE in disease.

We examined the effects of apolipoprotein E (apoE) domain structure and polymorphism on the kinetics of solubilization (clearance) of dimyristoyl-phosphatidylcholine multilamellar vesicles. This second order reaction consisted of two simultaneous kinetic phases; it also exhibited saturable kinetics when the apolipoprotein concentration was increased at a constant lipid concentration. Rigid connections between alpha-helices in the 4-helix bundle formed by the 22 kDa N-terminal domain of apoE reduced the reaction rate. In contrast, the more flexible interhelical connections in apoA-I and the 10 kDa C-terminal domain of apoE promoted rapid solubilization of dimyristoyl-phosphatidylcholine (DMPC) multilamellar vesicles (mLV). Full-length apoE-3 reacted at about half the rate of the C-terminal domain alone. This decrease occurred because the hinge region probably decreased the interhelical flexibility of the 10 kDa domain and because both domains are conformationally restricted when covalently linked. Furthermore, the mLV surface affinities and reaction rates of the N-terminal domain fragments of the three common apoE isoforms tended to vary inversely with the stabilities of these fragments. These results confirm the importance of apoE's structure on the kinetics of lipid interaction. They suggest that flexibility in an apolipoprotein molecule increases the time-averaged exposure of hydrophobic surface area, thereby increasing the rate of phospholipid solubilization.

In creating an allelic variant of mouse Apoe designed to resemble human apolipoprotein E4 (apoE4), we generated hypomorphic apoE (hypoE) mice that express only ∼5% of normal apoE mRNA levels in all tissues. Insertion of a neo cassette flanked by loxP sites in the third intron of Apoe reduced expression of the Arg-61 allelic variant in hypoE mice and resulted in plasma apoE levels that were ∼2–5% of normal. Unlike other mouse models with low levels of circulating apoE, hypoE mice had a nearly normal lipoprotein cholesterol profile when fed a chow diet. Further reduction of apoE expression in hypoE/Apoe−/− heterozygous mice led to an increase in remnant lipoprotein-associated cholesterol levels, demonstrating that hypoE mice express close to the threshold level of Arg-61 apoE required for a normal lipoprotein profile. Unlike wild type mice, hypoE mice were susceptible to diet-induced hypercholesterolemia, which was fully reversed within 3 weeks after resumption of a chow diet. In Mx1-Cre transgenic hypoE mice, plasma apoE levels returned to normal within 10 days after gene repair and removal of the neo cassette following induction of Cre recombinase. HypoE mice provide the opportunity for conditional gene repair by crossing with inducible or lineage/cell type-specific Cre transgenic mice, generating new models to dissect the roles of apoE in atherosclerosis regression, immunoregulation, and neurodegeneration.

Apolipoprotein (apo) E contains two structural domains, a 22-kDa (amino acids 1–191) N-terminal domain and a 10-kDa (amino acids 223–299) C-terminal domain. To better understand apoE-lipid interactions on lipoprotein surfaces, we determined the thermodynamic parameters for binding of apoE4 and its 22- and 10-kDa fragments to triolein-egg phosphatidylcholine emulsions using a centrifugation assay and titration calorimetry. In both large (120 nm) and small (35 nm) emulsion particles, the binding affinities decreased in the order 10-kDa fragment ≈ 34-kDa intact apoE4 > 22-kDa fragment, whereas the maximal binding capacity of intact apoE4 was much larger than those of the 22- and 10-kDa fragments. These results suggest that at maximal binding, the binding behavior of intact apoE4 is different from that of each fragment and that the N-terminal domain of intact apoE4 does not contact lipid. Isothermal titration calorimetry measurements showed that apoE binding to emulsions was an exothermic process. Binding to large particles is enthalpically driven, and binding to small particles is entropically driven. At a low surface concentration of protein, the binding enthalpy of intact apoE4 (−69 kcal/mol) was approximately equal to the sum of the enthalpies for the 22- and 10-kDa fragments, indicating that both the 22- and 10-kDa fragments interact with lipids. In a saturated condition, however, the binding enthalpy of intact apoE4 (−39 kcal/mol) was less exothermic and rather similar to that of each fragment, supporting the hypothesis that only the C-terminal domain of intact apoE4 binds to lipid. We conclude that the N-terminal four-helix bundle can adopt either open or closed conformations, depending upon the surface concentration of emulsion-bound apoE.

Human apolipoprotein E4 (apoE4) binds preferentially to lower density lipoproteins, including very low density lipoproteins, and is associated with increased risk of atherosclerosis and neurodegenerative disorders, including Alzheimer's disease. This binding preference is the result of the presence of Arg-112, which causes Arg-61 in the amino-terminal domain to interact with Glu-255 in the carboxyl-terminal domain. ApoE2 and apoE3, which have Cys-112, bind preferentially to high density lipoproteins (HDL) and do not display apoE4 domain interaction. Mouse apoE, like apoE4, contains the equivalent of Arg-112 and Glu-255, but lacks the critical Arg-61 equivalent (it contains Thr-61). Thus, mouse apoE does not display apoE4 domain interaction and, as a result, behaves like human apoE3, including preferential binding to HDL. To assess the potential role of apoE4 domain interaction in atherosclerosis and neurodegeneration, we sought to introduce apoE4 domain interaction into mouse apoE. Replacing Thr-61 in mouse apoE with arginine converted the binding preference from HDL to very low density lipoproteinsin vitro, suggesting that apoE4 domain interaction could be introduced into mouse apoEin vivo. Using gene targeting in embryonic stem cells, we created mice expressing Arg-61 apoE. Heterozygous Arg-61/wild-type apoE mice displayed two phenotypes found in human apoE4/E3 heterozygotes: preferential binding to lower density lipoproteins and reduced abundance of Arg-61 apoE in the plasma, reflecting its more rapid catabolism. These findings demonstrate the successful introduction of apoE4 domain interaction into mouse apoEin vivo. The Arg-61 apoE mouse model will allow the effects of apoE4 domain interaction in lipoprotein metabolism, atherosclerosis, and neurodegeneration to be determined.

Conserved lysines and arginines within amino acids 140-150 of apolipoprotein (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions. Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value. This reduction corresponds to a decrease in free energy of binding of approximately 600 cal/mol, consistent with the elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on apoE and an acidic residue on the LDLR. Binding activity was similarly reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important.Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the low density lipoprotein receptor.

Apolipoprotein (apo) E-containing high density lipoprotein particles were reported to interact in vitrowith the proteoglycan biglycan (Bg), but the direct participation of apoE in this binding was not defined. To this end, we examined thein vitro binding of apoE complexed with dimyristoylphosphatidylcholine (DMPC) to human aortic Bg before and after glycosaminoglycan (GAG) depletion. In a solid-phase assay, apoE·DMPC bound to Bg and GAG-depleted protein core in a similar manner, suggesting a protein-protein mode of interaction. The binding was decreased in the presence of 1 m NaCl and was partially inhibited by either positively (0.2 m lysine, arginine) or negatively charged (0.2 m aspartic, glutamic) amino acids. A recombinant apoE fragment representing the C-terminal 10-kDa domain, complexed with DMPC, bound as efficiently as full-length apoE, whereas the N-terminal 22-kDa domain was inactive. Similar results were obtained with a gel mobility shift assay. Competition studies using a series of recombinant truncated apoEs showed that the charged segment in the C-terminal domain between residues 223 and 230 was involved in the binding. Overall, our results demonstrate that the C-terminal domain contains elements critical for the binding of apoE to the Bg protein core and that this binding is ionic in nature and independent of GAGs.