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Cholesterol homeostasis is of emerging therapeutic importance for Alzheimer's disease (AD). Agonists of liver-X-receptors (LXRs) stimulate several genes that regulate cholesterol homeostasis, and synthetic LXR agonists decrease neuropathological and cognitive phenotypes in AD mouse models. The cholesterol transporter ABCG1 is LXR-responsive and highly expressed in brain. In vitro, conflicting reports exist as to whether ABCG1 promotes or impedes Abeta production. To clarify the in vivo roles of ABCG1 in Abeta metabolism and brain cholesterol homeostasis, we assessed neuropathological and cognitive outcome measures in PDAPP mice expressing excess transgenic ABCG1. A 6-fold increase in ABCG1 levels did not alter Abeta, amyloid, apolipoprotein E levels, cholesterol efflux, or cognitive performance in PDAPP mice. Furthermore, endogenous murine Abeta levels were unchanged in both ABCG1-overexpressing or ABCG1-deficient mice. These data argue against a direct role for ABCG1 in AD. However, excess ABCG1 is associated with decreased levels of sterol precursors and increased levels of SREBP-2 and HMG-CoA-reductase mRNA, whereas deficiency of ABCG1 leads to the opposite effects. Although functions for ABCG1 in cholesterol efflux and Abeta metabolism have been proposed based on results with cellular model systems, the in vivo role of this enigmatic transporter may be largely one of regulating the sterol biosynthetic pathway.

Although intracellular cholesterol levels are known to influence the proteolysis of beta-amyloid precursor protein (APP), the effect of specific genes that regulate cholesterol metabolism on APP processing remains poorly understood. The cholesterol transporter ABCG1 facilitates cholesterol efflux to HDL and is expressed in brain. Notably, the human ABCG1 gene maps to chromosome 21q22.3, and individuals with Down syndrome (DS) typically manifest with Alzheimer's disease (AD) neuropathology in their 30s. Here, we demonstrate that expression of ABCG1 enhances amyloid-beta protein (Abeta) production in transfected HEK cells in a manner that requires functional cholesterol transporter activity. ABCG1-expressing cells also exhibit increased secreted APP (sAPP)alpha and sAPPbeta secretion and display increased cell surface-associated APP. These results suggest that ABCG1 increases the availability of APP as a secretase substrate for both the amyloidogenic and nonamyloidogenic pathways. In vivo, ABCG1 mRNA levels are 2-fold more abundant in DS brain compared with age- and sex-matched normal controls. Finally, both Abeta and sAPPalpha levels are increased in DS cortex relative to normal controls. These findings suggest that altered cholesterol metabolism and APP trafficking mediated by ABCG1 may contribute to the accelerated onset of AD neuropathology in DS.

Extensive data suggest that the conversion of the amyloid-beta (Abeta) peptide from soluble to insoluble forms is a key factor in the pathogenesis of Alzheimer's disease (AD). In recent years, atomic force microscopy (AFM) has provided useful insights into the physicochemical processes involving Abeta morphology, and it can now be used to explore factors that either inhibit or promote fibrillogenesis. We used ex situ AFM to explore the impact of anti-Abeta antibodies directed against different domains of Abeta on fibril formation. For the AFM studies, two monoclonal antibodies (m3D6 and m266.2) were incubated in solution with Abeta(1-42) with a molar ratio of 1:10 (antibody to Abeta) over several days. Fibril formation was analyzed quantitatively by determining the number of fibrils per microm(2) and by aggregate size analysis. m3D6, which is directed against an N-terminal domain of Abeta (amino acid residues 1-5) slowed down fibril formation. However, m266.2, which is directed against the central domain of Abeta (amino acid residues 13-28) appeared to completely prevent the formation of fibrils over the course of the experiment. Inhibition of fibril formation by both antibodies was also confirmed by thioflavin-T (ThT) fluorescence experiments carried out with Abeta(1-40) incubated for five days. However, unlike AFM results, ThT did not differentiate between the samples incubated with m3D6 versus m266.2. These results indicate that AFM can be not only reliably used to study the effect of different molecules on Abeta aggregation, but that it can provide additional information such as the role of epitope specificity of antibodies as potential inhibitors of fibril formation.

Susceptibility to the development of late-onset Alzheimer's disease is increased for individuals harboring one or more apolipoprotein E4 (apoE4) alleles. Although several isoform-specific effects of apoE have been identified, the relationship between biochemical function and risk factor assessment is unknown. Our previous studies showed that a physiologically relevant cell-derived apoE3 particle stimulates neurite outgrowth in an isoform-specific manner. In an attempt to delineate the biochemical mechanism responsible for the stimulatory effects of apoE3 on neurite outgrowth, we performed a detailed physical characterization of cell-derived apoE3 and apoE4 particles. Immunoaffinity chromatography followed by SDS-PAGE illustrated homogeneity in protein content (apoE >95%). The affinity-purified particles contained phospholipid and 1 mol of cholesterol per mole of apoE but no core lipids. Nondenaturing gradient gel electrophoresis identified two major particle populations with hydrated diameters of 8.0 and 9.2 nm. Neurite outgrowth assays performed with the affinity-purified particles resulted in similar isoform-specific differences as seen previously, apoE3 stimulatory and apoE4 neutral. Interestingly, we did not observe a reduction in apoE medium concentrations over the duration of the neurite outgrowth assays, suggesting little or no endocytic uptake. Ligand blot analysis demonstrated that the affinity-purified apoE particles bind to several Neuro-2a membrane proteins. Western blots of the Neuro-2a membrane proteins indicated that the LDL receptor, gp330, and LR8B might be involved in the apoE-binding event. These results discriminate against the lipid delivery hypothesis and suggest that the biological activity of the phospholipid apoE3 particles may be due to cell surface signaling.

For the last 30 years fibrates have been widely prescribed to treat human dyslipidemia. However, the primary mechanism by which they lower plasma lipid levels is still unknown. Studies with transgenic mice have suggested that changes in apoC-III expression levels have a dramatic influence on plasma triglyceride levels. These results suggested that fibrates could reduce lipid levels by lowering apoC-III gene expression. In the current studies, we sought to determine whether the selected fibrates, bezafibrate, clofibrate, fenofibrate, and gemfibrozil, could reduce hepatic apoC-III mRNA and plasma apoC-III levels. Chow-fed rats were orally gavaged daily with a dosing vehicle alone or with 100 mg/kg of each of the fibrates for 1 week and in addition with gemfibrozil for 2 weeks. Bezafibrate and fenofibrate lowered plasma triglyceride by approximately half and dramatically reduced hepatic apoC-III mRNA and plasma apoC-III levels. In contrast, clofibrate did not reduce plasma triglyceride levels and only partially reduced apoC-III mRNA and plasma protein levels. Gemfibrozil strongly reduced plasma triglyceride levels and had an intermediate but significant effect on apoC-III mRNA and plasma apoC-III levels. Some of the fibrates, especially gemfibrozil also reduced plasma apoC-II levels, an effect that could contribute to the observed triglyceride-lowering effect. In addition, the ratio of plasma apoE to plasma apoC-II plus apoC-III was strongly and inversely correlated with plasma triglyceride levels. As plasma apoE levels were not reduced in gemfibrozil-treated animals, this could also have contributed to the triglyceride-lowering effect of this fibrate. Fibrate-mediated triglyceride lowering was not the result of a decreased apoB or VLDL production and, therefore, suggested an enhanced VLDL remnant catabolism. Our results suggest that the mechanism by which fibrates lower plasma triglycerides is by reducing the level of hepatic apoC-III expression.

A human cDNA clone (K1) was recently isolated that encodes functional acyl-CoA:cholesterol acyltransferase (ACAT) protein (Chang et al. J. Biol. Chem. 1993. 268: 20747-20755). We used the K1 clone to screen a rabbit liver cDNA library and isolated a 919 base pair partial rabbit cDNA (ACAT14b) that was greater than 90% homologous with the human nucleotide sequence. Northern blotting using the rabbit ACAT cDNA14b revealed the existence of at least six related mRNA species (ranging from 6.2 to 1.7 kb) in various rabbit tissues. Using an RNAse protection assay, ACAT mRNA14b was detected in twelve separate rabbit organs. Adrenal gland contained the highest concentrations of ACAT mRNA14b (per microgram of total RNA) being 20-, 30-, and 50-fold higher than small intestine, aorta, and liver, respectively. Additional studies with isolated liver cell populations revealed that rabbit hepatic nonparenchymal cells contained 30-fold more ACAT mRNA14b (per microgram of total RNA) than parenchymal cells. To determine whether ACAT mRNA14b levels are regulated in vivo, rabbits were fed for 4 weeks a high fat/high cholesterol diet (HFHC; 0.5% cholesterol, 3% coconut oil, 3% peanut oil) at which point they were either kept for an additional 4 weeks on the HFHC-diet or switched to the HFHC-diet plus CI-976 (50 mg/kg), a potent and specific ACAT inhibitor; another group of rabbits was fed a chow diet for the entire 8 weeks. The HFHC-diet caused a 2- and 3-fold increase in hepatic and aortic ACAT mRNA14b levels, respectively, in comparison to chow-fed animals; there was no change in adrenal or small intestine levels. CI-976 treatment lowered ACAT mRNA14b levels by 60% and 40% in liver and aorta, respectively, in comparison to the HFHC controls; again there was no change in adrenal or small intestine levels. These data indicate that ACAT mRNA14b levels increase in a tissue specific manner in response to dietary fat and cholesterol.

Apolipoprotein A-I (apoA-I), the primary protein of high density lipoprotein, originates from intestine and liver of almost all mammalian species. In contrast to most species, intact rabbit liver is only capable of producing minute amounts of apoA-I mRNA and protein. In this report we demonstrate that purified rabbit hepatic parenchymal cells have apoA-I mRNA levels approximately 50-fold higher than intact liver after 48 h in monolayer culture. Investigations of the differential between in vivo and in vitro expression showed that conditioned media from nonparenchymal cells, a cell population essentially absent in parenchymal cell cultures, inhibited the elevation of apoA-I mRNA in a specific, concentration-dependent, and reversible fashion. Furthermore, at a concentration of nonparenchymal cell-conditioned media that inhibited apoA-I mRNA levels by > 80% compared to control, there were only slight changes in apoB, apoE, LDL receptor, LCAT, 7 alpha-hydroxylase, hepatic lipase, HMG-CoA reductase, and albumin mRNA levels. Metabolic labeling of parenchymal cell secreted proteins with [35S]methionine followed by apoA-I immunoprecipitation revealed that apoA-I synthesis and secretion corresponded to the changes observed for apoA-I mRNA. Initial biochemical characterization of the nonparenchymal cell media revealed that the inhibitory factor was > 30 kDa, heat-stable to 70 degrees C, and still active after urea denaturation and renaturation. These data suggest that, in rabbits, hepatic parenchymal-nonparenchymal communication in the form of a secreted factor may attenuate liver apoA-I expression in vivo.

The liver plays a central role in lipid metabolism and plasma lipoprotein homeostasis. This dynamic process is regulated by a variety of liver-derived proteins. However, the specific liver cells that express these proteins are largely unknown. In the current study we measured mRNA levels for 13 genes encoding proteins involved in lipid metabolism in isolated rabbit hepatic parenchymal and nonparenchymal cells. For these analyses we cloned partial rabbit cDNAs for apolipoprotein A-I (apoA-I), apolipoprotein B (apoB), apolipoprotein E (apoE), cholesteryl ester transfer protein (CETP), hepatic lipase (HL), lipoprotein lipase (LPL), HMG-CoA reductase, LDL-receptor, 7 alpha-hydroxylase, albumin, bile salt-dependent cholesteryl ester hydrolase (CEH), lecithin:cholesterol acyl transferase (LCAT), and plasminogen activator inhibitor protein-1 (PAI-1). The cDNAs provided the basis for developing quantitative RNAse protection assays for each mRNA. These assays were used to determine whether differential patterns of mRNA expression existed between liver and other tissues and between hepatic parenchymal and nonparenchymal cells. The data demonstrate a diverse range in tissue distribution and mRNA abundance. Liver expressed all mRNAs except for LPL and CEH. Messenger RNA levels in isolated liver cell populations normalized to total RNA revealed a cell segregation pattern for hepatic gene expression: parenchymal cells showed higher levels of apoA-I, apoB, apoE, albumin, LCAT, HL, and 7 alpha-hydroxylase mRNAs compared to nonparenchymal cells while nonparenchymal cells showed higher levels of CETP, LDL-receptor, HMG-CoA reductase, and PAI-1 mRNAs compared to parenchymal cells. These data demonstrate the existence of differential mRNA expression patterns in rabbit liver cell populations for genes encoding proteins affecting lipid metabolism.