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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 APOEε4 subjects to Alzheimer's disease.

Abnormal polyglutamine (polyQ) tracts are the only common feature in nine proteins that each cause a dominant neurodegenerative disorder. In Huntington's disease, tracts longer than 36 glutamines in the protein huntingtin (htt) cause degeneration. In situ, monoclonal antibody 3B5H10 binds to different htt fragments in neurons in proportion to their toxicity. Here, we determined the structure of 3B5H10 Fab to 1.9 Å resolution by X-ray crystallography. Modeling demonstrates that the paratope forms a groove suitable for binding two β-rich polyQ strands. Using small-angle X-ray scattering, we confirmed that the polyQ epitope recognized by 3B5H10 is a compact two-stranded hairpin within monomeric htt and is abundant in htt fragments unbound to antibody. Thus, disease-associated polyQ stretches preferentially adopt compact conformations. Since 3B5H10 binding predicts degeneration, this compact polyQ structure may be neurotoxic.

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.

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.

Apolipoprotein (apo) E plays a critical role in cholesterol transport, through high affinity binding to the low density lipoprotein receptor. This interaction requires apoE to be associated with a lipoprotein particle. To determine the structure of biologically active apoE on a lipoprotein particle, we crystallized dipalmitoylphosphatidylcholine particles containing two apoE molecules and determined the molecular envelope of apoE at 10 angstrom resolution. On the basis of the molecular envelope and supporting biochemical evidence, we propose a model in which each apoE molecule is folded into a helical hairpin with the binding region for the low density lipoprotein receptor at its apex.

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 (A beta) peptides. We examined the effects of apoE isoforms on the processing of amyloid precursor protein (APP) and on A beta production in rat neuroblastoma B103 cells stably transfected with human wild-type APP695 (B103-APP). Lipid-poor apoE4 increased A beta 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 A beta 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 A beta 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.

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 apoEmRNA levels were similar to or slightly higher than wild-type apoEmRNA 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.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2006.06.008 Byline: Danny M. Hatters (1)(2)(3), Clare A. Peters-Libeu (1)(2)(3), Karl H. Weisgraber (1)(2)(3) Abstract: 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. Author Affiliation: (1) Gladstone Institute of Neurological Disease and Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA (2) Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA (3) Department of Pathology, University of California, San Francisco, CA 94143, USA

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 [...]

Results demonstrate that apolipoprotein E inhibition of smooth muscle cell proliferation occurs via its binding to heparan sulfate proteoglycans. The cell migration inhibition is brought about by its binding to the low-density lipoprotein receptor related protein.

Research has been conducted on the apolipoprotein E, a lipid-transport protein in human plasma and brain. The interactions of the N-terminal domain of this apolipoprotein with enzymatically prepared heparin oligosaccharide have been explored.

The C-terminus of apolipoprotein C-I probably acts as a lipid anchor while the N-terminus contacts lecithin:cholesterol acyltransferase. Research shows that the N-terminus has a mobile hinge resulting from two lipid-binding sites.

An isoform of the apoE family of proteins, apoE3, inhibits amyloid nucleation even at low concentrations of 40 nM. The N-terminal 191 amino acids of a proteolytic fragment of apoE3 causes this inhibitory activity, which involves suppression of the formation of the amyloid fibril by the beta-amyloid protein of Alzheimer disease. The solubility, structure and growth of the amyloid fibrils remain unaffected with the action of apoE3, implying that apoE3 is not a thermodynamic inhibitor of amyloid formation.

Addition of amyloid beta protein (Abeta) to cerebrospinal fluid (CSF) of patients with Alzheimer's disease and normals reveals that transthyretin (TTR), a CSF protein, sequesters Abeta protein into stable complexes. Sequestration prevents aggregation of Abeta proteins leading to amyloid fibrils, which are associated with Alzheimer disease. In vitro studies show that apolipoprotein E does not complex with Abeta in CSF.

The binding of synthetic amyloid beta (beta/A4) peptide was compared to purified apolipoprotein E4 (apoE4) and the most common isoform, apoE3, to find if apoE4 is related to enhanced vulnerability to Alzheimer disease. The two isoforms form a complex with the beta/A4 peptide in different ways. ApoE is regionalized in the senile plaques, congophilic angiopathy and neurofibrillary tangles of Alzheimer disease.

The role of apoprotein E-rich HDL (HDL I) as preferrential substrate for hepatic lipase activated hydrolysis of phospholipids was examined. The rate of hydrolysis of apoprotein E-rich HDL and apoprotein E-poor HDL in the presence of hepatic lipase was compared as well as hepatic lipase activation in the presence of substrate and subsequent apoE adsorption. It was shown that HDL I was used more than HDL II in hepatic lipase hydrolysis and that the enzyme activation requires the intact protein. The reaction was due to protein-protein interaction and the different isoforms of HDL showed varied ability to activate the enzyme.

The proteins, apolipoprotein B and E, are located on the surface of lipoprotein molecules, which are fat and protein compounds. These apolipoproteins are involved in the binding of cholesterol-containing particles to specific lipoprotein attachment sites on cells, called receptors. This process is an important pathway for controlling the blood levels of cholesterol. An increase in blood levels of certain lipoproteins may contribute to the development of atherosclerosis, a blood vessel disorder in which lipids accumulate and cells proliferate within the arteries. Three types of lipoprotein metabolism disorders may contribute to the rise in atherogenic lipoproteins (lipoproteins that may cause atherosclerosis). Type III hyperlipoproteinemia results from certain mutations in apolipoprotein E, thereby preventing the binding of portions of very-low-density-lipoprotein (VLDL) and chylomicrons (small fat particles in the blood). Type III hyperlipoproteinemia associated with elevated levels of VLDL remnants leads to the development of atherosclerosis. The substitution of a single amino acid in apolipoprotein B prevents low-density lipoprotein (LDL) from binding to its receptor, resulting in increased blood cholesterol levels and a disorder called familial defective apolipoprotein B-100. The third disorder, familial hypercholesterolemia, is associated with increased LDL levels and the early development of atherosclerosis. It results from several mutations of the LDL receptor that interfere with the binding of LDL to its receptor. These genetic defects in lipoprotein metabolism contribute to the understanding of mechanisms responsible for normal lipoprotein metabolism and role of lipoproteins in atherosclerosis. (Consumer Summary produced by Reliance Medical Information, Inc.)