Female mice with apolipoprotein E4 domain interaction demonstrated impairments in spatial learning and memory performance and disruption of hippocampal cyto-architecture.

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 ß 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 APOEε4 subjects to Alzheimer's disease.

Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein.

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 ß-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.

Small molecule structure correctors abolish detrimental effects of apolipoprotein E4 in cultured neurons.

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.

Identifying polyglutamine protein species in situ that best predict neurodegeneration.

Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function to proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies, this may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that the monoclonal antibody 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular-weight conformational states assumed by expanded polyQ and disappears in higher-molecular-weight aggregated forms, such as inclusion bodies. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.

Apolipoprotein E4 domain interaction mediates detrimental effects on mitochondria and is a potential therapeutic target for Alzheimer disease.

Apolipoprotein (apo) E4 is the major genetic risk factor for late-onset Alzheimer disease (AD). ApoE4 assumes a pathological conformation through an intramolecular interaction mediated by Arg-61 in the amino-terminal domain and Glu-255 in the carboxyl-terminal domain, referred to as apoE4 domain interaction. Because AD is associated with mitochondrial dysfunction, we examined the effect of apoE4 domain interaction on mitochondrial respiratory function. Steady-state amounts of mitochondrial respiratory complexes were examined in neurons cultured from brain cortices of neuron-specific enolase promoter-driven apoE3 (NSE-apoE3) or apoE4 (NSE-apoE4) transgenic mice. All subunits of mitochondrial respiratory complexes assessed were significantly lower in NSE-apoE4 neurons compared with NSE-apoE3 neurons. However, no significant differences in levels of mitochondrial complexes were detected between astrocytes expressing different apoE isoforms driven by the glial fibrillary acidic protein promoter, leading to our conclusion that the effect of apoE4 is neuron specific. In neuroblastoma Neuro-2A (N2A) cells, apoE4 expression reduced the levels of mitochondrial respiratory complexes I, IV, and V. Complex IV enzymatic activity was also decreased, lowering mitochondrial respiratory capacity. Mutant apoE4 (apoE4-Thr-61) lacking domain interaction did not induce mitochondrial dysfunction in N2A cells, indicating that the effect is specific to apoE4-expressing cells and dependent on domain interaction. Consistent with this finding, treatment of apoE4-expressing N2A cells with a small molecule that disrupts apoE4 domain interaction restored mitochondrial respiratory complex IV levels. These results suggest that pharmacological intervention with small molecules that disrupt apoE4 domain interaction is a potential therapeutic approach for apoE4-carrying AD subjects.

Structure-dependent impairment of intracellular apolipoprotein E4 trafficking and its detrimental effects are rescued by small-molecule structure correctors.

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.

Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy.

Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.

Apolipoprotein E structure: insights into function.

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.

An optimized negative-staining protocol of electron microscopy for apoE4 POPC lipoprotein.

Apolipoprotein E (apoE), one of the major protein components of lipoproteins in the peripheral and central nervous systems, regulates cholesterol metabolism through its interaction with members of the low density lipoprotein receptor family. One key to understanding apoE function is determining the structure of lipid-bound forms of apoE. Negative-staining (NS) electron microscopy (EM) is an easy and rapid approach for studying the structure and morphology of lipid-bound forms of apoE. However, an artifact of using the conventional NS protocol is that the apoE phospholipid particles form rouleaux. In this study, we used cryo-electron microscopy (cryo-EM) to examine apoE4 palmitoyl-oleoylphosphatidylcholine (POPC) particles in a frozen-hydrated native state. By comparing the particle sizes and shapes produced by different NS protocols to those produced by cryo-EM, we propose an optimized protocol to examine apoE4 POPC particles. Statistical analysis demonstrated that the particle sizes differ by less than 5% between the optimized protocol and the cryo-EM method, with similar shapes. The high contrast and fine detail of particle images produced using this optimized protocol lend themselves to the structural study of lipid-bound forms of apoE.

VLDL lipolysis products increase VLDL fluidity and convert apolipoprotein E4 into a more expanded conformation.

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

Apolipoprotein E4 domain interaction induces endoplasmic reticulum stress and impairs astrocyte function.

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.

Putting cholesterol in its place: apoE and reverse cholesterol transport.

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.

Apolipoprotein E4 domain interaction: synaptic and cognitive deficits in mice.

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.

Amino-terminal domain stability mediates apolipoprotein E aggregation into neurotoxic fibrils.

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.

Detrimental effects of apolipoprotein E4: potential therapeutic targets in Alzheimer's disease.

As the major genetic risk factor for Alzheimer's disease, the apolipoprotein (apo) E4 isoform is a promising therapeutic target. ApoE4 likely contributes to Alzheimer's disease pathology by interacting with multiple factors through various pathways. Interactions with the amyloid beta peptide and the amyloid cascade, for example, may lead to cognitive decline and neurodegeneration. Alternatively, apoE4 might act independently of the amyloid beta peptide. Our working hypothesis is that apoE has isoform-specific effects on neuronal repair and remodeling. One or more injurious agents could result in neuronal damage, requiring neuronal repair or remodeling. The injurious agents (or "second hits") may be genetic, metabolic, or environmental. Potential therapeutic strategies include changing the structure of apoE4 to be more apoE3-like, inhibiting the protease that cleaves apoE4 into toxic fragments, and protecting mitochondria from apoE4 toxicity. Structural features that distinguish apoE4 and apoE3 determine their functional differences and hold the key to understanding how apoE4 is involved in Alzheimer's disease.

Effect of domain interaction on apolipoprotein E levels in mouse brain.

Apolipoprotein (apo) E4 is a risk factor for heart disease, Alzheimer's disease, and other forms of neurodegeneration, but the underlying mechanisms are unknown. Domain interaction, a structural property that distinguishes apoE4 from apoE2 and apoE3, results in more rapid turnover and lower plasma levels of apoE4. To determine whether domain interaction affects brain apoE levels, we analyzed brain homogenates from human apoE3 and apoE4 knock-in mice, wild-type mice, and Arg-61 apoE mice, in which domain interaction was introduced by gene targeting. As determined on Western blots, the hemibrain, cortex, hippocampus, and cerebellum of knock-in mice had 30-40% lower levels of apoE4 than apoE3, and Arg-61 mice had 25-50% lower apoE levels than wild-type mice. In the CSF, Arg-61 apoE level was 40% lower than the wild-type level. Arg-61 apoE mRNA levels were similar to or slightly higher than wild-type apoE mRNA levels. Thus, the lower Arg-61 apoE levels were not attributable to decreased mRNA levels. In culture medium from heterozygous Arg-61/wild-type and apoE4/apoE3 primary astrocytes, Arg-61 apoE and apoE4 levels were lower than wild-type apoE and apoE3, respectively, suggesting that primary astrocytes secrete lower amounts of Arg-61 apoE and apoE4. These results demonstrate that domain interaction is responsible for the lower levels of both human apoE4 and mouse Arg-61 apoE in mouse brain. Cells may recognize apoE4 and Arg-61 apoE as misfolded proteins and target them for degradation or accumulation. Thus, degradation/accumulation or lower levels of apoE4 may contribute to the association of apoE4 with Alzheimer's disease.

Diet-induced occlusive coronary atherosclerosis, myocardial infarction, cardiac dysfunction, and premature death in scavenger receptor class B type I-deficient, hypomorphic apolipoprotein ER61 mice.

BACKGROUND: Normal chow (low fat)-fed mice deficient in both the HDL receptor SR-BI and apolipoprotein E (SR-BI/apoE dKO) provide a distinctive model of coronary heart disease (CHD). They exhibit early-onset hypercholesterolemia characterized by unesterified cholesterol-rich abnormal lipoproteins (lamellar/vesicular and stacked discoidal particles), occlusive coronary atherosclerosis, spontaneous myocardial infarction, cardiac dysfunction, and premature death ( approximately 6 weeks of age). Mice in which similar features of CHD could be induced with a lipid-rich diet would represent a powerful tool to study CHD. METHODS AND RESULTS: To generate a diet-inducible model of CHD, we bred SR-BI-deficient (SR-BI KO) mice with hypomorphic apolipoprotein E mice (ApoeR61(h/h)) that express reduced levels of an apoE4-like murine apoE isoform and exhibit diet-induced hypercholesterolemia. When fed a normal chow diet, SR-BI KO/ApoeR61(h/h) mice did not exhibit early-onset atherosclerosis or CHD; the low expression level of the apoE4-like murine apoE was atheroprotective and cardioprotective. However, when fed an atherogenic diet rich in fat, cholesterol, and cholate, they rapidly developed hypercholesterolemia, atherosclerosis, and CHD, a response strikingly similar to that of SR-BI/apoE dKO mice fed a chow diet, and they died 32+/-6 days (50% mortality) after initiation of the high-fat feeding. CONCLUSIONS: The SR-BI KO/ApoeR61(h/h) mouse is a new model of diet-induced occlusive coronary atherosclerosis and CHD (myocardial infarction, cardiac dysfunction and premature death), allowing control of the age of onset, duration, severity, and possibly regression of disease. Thus, SR-BI KO/ApoeR61(h/h) mice have the potential to contribute to our understanding of CHD and its prevention and treatment.

Apolipoprotein E promotes the regression of atherosclerosis independently of lowering plasma cholesterol levels.

OBJECTIVE: The mechanisms by which apolipoprotein E (apoE) can promote the regression of atherosclerosis are not well understood. This study examined whether apoE can promote atherosclerosis regression independently of lowering plasma cholesterol levels. METHODS AND RESULTS: We studied hypomorphic apoE mice (Apoe(h/h)), which express an apoE4-like form of mouse apoE at approximately 2% to 5% of normal levels in plasma and are normolipidemic. After 18 weeks of diet-induced hypercholesterolemia, which resulted in advanced aortic atherosclerotic lesions composed of a lipid-rich layer of foam cells covering a fibrotic core, 2 groups of mice were fed a chow diet for 16 weeks. One group continued to express low levels of apoE; the other was induced to express physiological levels of plasma apoE by Cre-mediated recombination of the hypomorphic Apoe allele. In both groups, plasma cholesterol levels fell rapidly to similar levels, and histological analysis at 16 weeks revealed elimination of the foam-cell layer. However, physiological levels of plasma apoE also enhanced the removal of neutral lipids from the fibrotic cores. CONCLUSIONS: These findings demonstrate for the first time that apolipoprotein E promotes the regression of atherosclerosis independently of lowering plasma cholesterol levels. Using Apoeh/hMx1-Cre mice we have begun to address apolipoprotein E-mediated mechanisms of atherosclerosis regression. We report the existence of a cholesterol-independent role of apolipoprotein E in atherosclerosis regression. This mechanism is critical for lipid removal from the fibrotic component of the plaque but not from the foam cell-rich layer beneath the endothelium.

Comparison of the stabilities and unfolding pathways of human apolipoprotein E isoforms by differential scanning calorimetry and circular dichroism.

Differential scanning calorimetry and circular dichroism experiments were performed to study structural differences among the common isoforms of human apolipoprotein E (apoE2, apoE3, and apoE4) and their N-terminal, 22-kDa fragments. Here, we examine thermodynamic properties that characterize the structural differences among isoforms, and also differences in their unfolding behavior. The 22-kDa fragments and their full-length counterparts were found to exhibit similar differences in thermal stability (apoE4

Crystallization and preliminary X-ray diffraction analysis of apolipoprotein E-containing lipoprotein particles.

High-resolution structural information is available for several soluble plasma apolipoproteins (apos) in a lipid-free state. However, this information provides limited insight into structure-function relationships, as this class of proteins primarily performs its functions of lipid transport and modulation of lipid metabolism in a lipid-bound state on lipoprotein particles. Here, the possibility of generating homogeneous lipoprotein particles that could be crystallized was explored, opening the possibility of obtaining high-resolution structural information by X-ray crystallography. To test this possibility, apoE4 complexed with the phospholipid dipalmitoylphosphatidylcholine was chosen. Uniform particles containing 50% lipid and 50% apoE4 were obtained and crystallized using the hanging-drop method. Two crystal forms diffract to beyond 8 A resolution.