Veronica Hirsch-Reinshagen

Overexpression of ABCA1 reduces amyloid deposition in the PDAPP mouse model of Alzheimer disease

APOE genotype is a major genetic risk factor for late-onset Alzheimer disease (AD). ABCA1, a member of the ATP-binding cassette family of active transporters, lipidates apoE in the CNS. Abca1(-/-) mice have decreased lipid associated with apoE and increased amyloid deposition in several AD mouse models. We hypothesized that mice overexpressing ABCA1 in the brain would have increased lipidation of apoE-containing lipoproteins and decreased amyloid deposition. To address these hypotheses, we created PrP-mAbca1 Tg mice that overexpress mouse Abca1 throughout the brain under the control of the mouse prion promoter. We bred the PrP-mAbca1 mice to the PDAPP AD mouse model, a transgenic line overexpressing a mutant human amyloid precursor protein. PDAPP/Abca1 Tg mice developed a phenotype remarkably similar to that seen in PDAPP/Apoe(-/-) mice: there was significantly less amyloid beta-peptide (Abeta) deposition, a redistribution of Abeta to the hilus of the dentate gyrus in the hippocampus, and an almost complete absence of thioflavine S-positive amyloid plaques. Analyses of CSF from PrP-mAbca1 Tg mice and media conditioned by PrP-mAbca1 Tg primary astrocytes demonstrated increased lipidation of apoE-containing particles. These data support the conclusions that increased ABCA1-mediated lipidation of apoE in the CNS can reduce amyloid burden and that increasing ABCA1 function may have a therapeutic effect on AD.

The Absence of ABCA1 Decreases Soluble ApoE Levels but Does Not Diminish Amyloid Deposition in Two Murine Models of Alzheimer Disease

ABCA1, a cholesterol transporter expressed in the brain, has been shown recently to be required to maintain normal apoE levels and lipidation in the central nervous system. In addition, ABCA1 has been reported to modulate β-amyloid (Aβ) production in vitro. These observations raise the possibility that ABCA1 may play a role in the pathogenesis of Alzheimer disease. Here we report that the deficiency of ABCA1 does not affect soluble or guanidine-extractable Aβ levels in Tg-SwDI/B or amyloid precursor protein/presenilin 1 (APP/PS1) mice, but rather is associated with a dramatic reduction in soluble apoE levels in brain. Although this reduction in apoE was expected to reduce the amyloid burden in vivo, we observed that the parenchymal and vascular amyloid load was increased in Tg-SwDI/B animals and was not diminished in APP/PS1 mice. Furthermore, we observed an increase in the proportion of apoE retained in the insoluble fraction, particularly in the APP/PS1 model. These data suggested that ABCA1-mediated effects on apoE levels and lipidation influenced amyloidogenesis in vivo.

24(S)-HYDROXYCHOLESTEROL PARTICIPATES IN A LIVER X RECEPTOR- CONTROLLED PATHWAY IN ASTROCYTES THAT REGULATES APOLIPOPROTEIN E-MEDIATED CHOLESTEROL EFFLUX

Both apolipoprotein E (apoE) and 24(S)-hydroxycholesterol are involved in the pathogenesis of Alzheimer disease (AD). It has been hypothesized that apoE affects AD development via isoform-specific effects on lipid trafficking between astrocytes and neurons. However, the regulation of the cholesterol supply of neurons via apoE-containing high density lipoproteins remains to be clarified. We show for the first time that the brain-specific metabolite of cholesterol produced by neurons, i.e. 24(S)-hydroxycholesterol, induces apoE transcription, protein synthesis, and secretion in a dose- and time-dependent manner in cells of astrocytic but not of neuronal origin. Moreover, 24(S)-hydroxycholesterol primes astrocytoma, but not neuroblastoma cells, to mediate cholesterol efflux to apoE. Similar results were obtained using the synthetic liver X receptor (LXR) agonist GW683965A, suggesting involvement of an LXR-controlled signaling pathway. A 10-20-fold higher basal LXRα and -β expression level in astrocytoma compared with neuroblastoma cells may underlie these differential effects. Furthermore, apoE-mediated cholesterol efflux from astrocytoma cells may be controlled by the ATP binding cassette transporters ABCA1 and ABCG1, since their expression was also up-regulated by both compounds. In contrast, ABCG4 seems not to be involved, because its expression was induced only in neuronal cells. The expression of sterol regulatory element-binding protein (SREBP-2), low density lipoprotein receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, and SREBP-1c was transiently up-regulated by GW683965A in astrocytes but down-regulated by 24(S)-hydroxycholesterol, suggesting that cholesterol efflux and synthesis are regulated independently. In conclusion, evidence is provided that 24(S)-hydroxycholesterol induces apoE-mediated efflux of cholesterol in astrocytes via an LXR-controlled pathway, which may be relevant for chronic and acute neurological diseases.

ATP-binding Cassette Transporter A1 Mediates the Beneficial Effects of the Liver X Receptor Agonist GW3965 on Object Recognition Memory and Amyloid Burden in Amyloid Precursor Protein/Presenilin 1 Mice

The cholesterol transpoter ATP-binding cassette transporter A1 (ABCA1) moves lipids onto apolipoproteins including apolipoprotein E (apoE), which is the major cholesterol carrier in the brain and an established genetic risk factor for late-onset Alzheimer disease (AD). In amyloid mouse models of AD, ABCA1 deficiency exacerbates amyloidogenesis, whereas ABCA1 overexpression ameliorates amyloid load, suggesting a role for ABCA1 in Aβ metabolism. Agonists of liver X receptors (LXR), including GW3965, induce transcription of several genes including ABCA1 and apoE, and reduce Aβ levels and improve cognition in AD mice. However, the specific role of ABCA1 in mediating beneficial responses to LXR agonists in AD mice is unknown. We evaluated behavioral and neuropathogical outcomes in GW3965-treated female APP/PS1 mice with and without ABCA1. Treatment of APP/PS1 mice with GW3965 increased ABCA1 and apoE protein levels. ABCA1 was required to observe significantly elevated apoE levels in brain tissue and cerebrospinal fluid upon therapeutic (33 mg/kg/day) GW3965 treatment. At 33 mg/kg/day, GW3965 was also associated with a trend toward redistribution of Aβ to the carbonate-soluble pool independent of ABCA1. APP/PS1 mice treated with either 2.5 or 33 mg/kg/day of GW3965 showed a clear trend toward reduced amyloid burden in hippocampus and whole brain, whereas APP/PS1-treated mice lacking ABCA1 failed to display reduced amyloid load in the whole brain and showed trends toward increased hippocampal amyloid. Treatment of APP/PS1 mice with either dose of GW3965 completely restored novel object recognition memory to wild-type levels, which required ABCA1. These results suggest that ABCA1 contributes to several beneficial effects of the LXR agonist GW3965 in APP/PS1 mice.

Specific Loss of Brain ABCA1 Increases Brain Cholesterol Uptake and Influences Neuronal Structure and Function

The expression of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) in the brain and its role in the lipidation of apolipoproteins indicate that ABCA1 may play a critical role in brain cholesterol metabolism. To investigate the role of ABCA1 in brain cholesterol homeostasis and trafficking, we characterized mice that specifically lacked ABCA1 in the CNS, generated using the Cre/loxP recombination system. These mice showed reduced plasma high-density lipoprotein (HDL) cholesterol levels associated with decreased brain cholesterol content and enhanced brain uptake of esterified cholesterol from plasma HDL. Increased levels of HDL receptor SR-BI in brain capillaries and apolipoprotein A-I in brain and CSF of mutant mice were evident. Cholesterol homeostasis changes were mirrored by disturbances in motor activity and sensorimotor function. Changes in synaptic ultrastructure including reduced synapse and synaptic vesicle numbers were observed. These data show that ABCA1 is a key regulator of brain cholesterol metabolism and that disturbances in cholesterol transport in the CNS are associated with structural and functional deficits in neurons. Moreover, our findings also demonstrate that specific changes in brain cholesterol metabolism can lead to alterations in cholesterol uptake from plasma to brain.

Why lipids are important for Alzheimer disease

Several lines of evidence suggest that dysregulated lipid metabolism may participate in the pathogenesis of Alzheimer’s disease (AD). Epidemiologic studies suggest that elevated mid-life plasma cholesterol levels may be associated with an increased risk of AD and that statin use may reduce the prevalence of AD. Cellular studies have shown that the levels and distribution of intracellular cholesterol markedly affect the processing of amyloid precursor protein into Aβ peptides, which are the toxic species that accumulate as amyloid plaques in the AD brain. Most importantly, genetic evidence identifies apolipoprotein E, the major cholesterol carrier in the central nervous system, as the primary genetic risk factor for sporadic AD. In humans, apoE exists as three major alleles (apoE2, apoE3, and apoE4), and inheritance of the apoE4 allele increases the risk of developing AD at an earlier age. However, exactly how apoE functions in the pathogenesis of AD remains to be fully determined. Our studies have identified that the cholesterol transporter ABCA1 is a crucial regulator of apoE levels and lipidation in the brain. Deficiency of ABCA1 leads to the loss of approximately 80% of apoE in the brain, and the residual 20% that remains is poorly lipidated. Several independent studies have shown this poorly lipidated apoE increases amyloid burden in mouse models of AD, demonstrating that apoE lipidation by ABCA1 affects key steps in amyloid deposition or clearance. Conversely, robust overexpression of ABCA1 in the brain promotes apoE lipidation and nearly eliminates the formation of mature amyloid plaques. These studies show that the lipid binding capacity of apoE is a major mechanism of its function in the pathogenesis of AD, and suggest that increasing apoE lipidation may be of therapeutic importance for this devastating disease.

Cholesterol metabolism, apolipoprotein E, adenosine triphosphate-binding cassette transporters, and Alzheimer's disease.

Purpose of review Recent evidence suggests that cholesterol metabolism participates in the pathogenesis of Alzheimers disease. Apolipoprotein E is the main lipid carrier in the brain and the best-established risk factor for late-onset Alzheimers disease. Intracellular cholesterol levels influence the generation of amyloid-β peptides, the toxic species thought to be a primary cause of Alzheimers disease. Finally, compounds that modulate cholesterol metabolism affect amyloid-β generation. This review summarizes data linking apolipoprotein E and adenosine triphosphate-binding cassette transporters to aspects of cholesterol metabolism and Alzheimers disease pathogenesis. Recent findings In vivo, the lipidation status of apolipoprotein E affects amyloid-β burden in mice with Alzheimers disease, which appears to caused by the modulation of amyloid-β deposition or clearance rather than amyloid-β production. State-of-the-art in-vivo assays reveal that amyloid-β is cleared from the brain by multiple pathways. Members of the adenosine triphosphate-binding cassette superfamily of transporters regulate lipid homeostasis and apolipoprotein metabolism in the brain, and may affect Alzheimers disease pathogenesis by modulating apolipoprotein E lipidation as well as intracellular sterol homeostasis. Summary Proteins involved in brain cholesterol metabolism may affect the pathogenesis of Alzheimers disease. Compounds that modulate the expression of these proteins may be of therapeutic benefit in Alzheimers disease.

Overexpression of Human ABCG1 Does Not Affect Atherosclerosis in Fat-Fed ApoE-Deficient Mice

Objective—The purpose of this study was to evaluate the effects of whole body overexpression of human ABCG1 on atherosclerosis in apoE−/− mice. Methods and Results—We generated BAC transgenic mice in which human ABCG1 is expressed from endogenous regulatory signals, leading to a 3- to 7-fold increase in ABCG1 protein across various tissues. Although the ABCG1 BAC transgene rescued lung lipid accumulation in ABCG1−/− mice, it did not affect plasma lipid levels, macrophage cholesterol efflux to HDL, atherosclerotic lesion area in apoE−/− mice, or levels of tissue cholesterol, cholesterol ester, phospholipids, or triglycerides. Subtle changes in sterol biosynthetic intermediate levels were observed in liver, with chow-fed ABCG1 BAC Tg mice showing a nonsignificant trend toward decreased levels of lathosterol, lanosterol, and desmosterol, and fat-fed mice exhibiting significantly elevated levels of each intermediate. These changes were insufficient to alter ABCA1 expression in liver. Conclusions—Transgenic human ABCG1 does not influence atherosclerosis in apoE−/− mice but may participate in the regulation of tissue cholesterol biosynthesis.

The cholesterol transporter ABCG1 modulates the subcellular distribution and proteolytic processing of β-amyloid precursor protein

Although intracellular cholesterol levels are known to influence the proteolysis of β-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 Alzheimers disease (AD) neuropathology in their 30s. Here, we demonstrate that expression of ABCG1 enhances amyloid-β protein (Aβ) production in transfected HEK cells in a manner that requires functional cholesterol transporter activity. ABCG1-expressing cells also exhibit increased secreted APP (sAPP)α and sAPPβ 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 Aβ and sAPPα 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.

An ABCA1-independent pathway for recycling a poorly lipidated 8.1 nm apolipoprotein E particle from glia

Lipid transport in the brain is coordinated by glial-derived lipoproteins that contain apolipoprotein E (apoE) as their primary protein. Here we show that apoE is secreted from wild-type (WT) primary murine mixed glia as nascent lipoprotein subspecies ranging from 7.5 to 17 nm in diameter. Negative-staining electron microscropy (EM) revealed rouleaux, suggesting a discoidal structure. Potassium bromide (KBr) density gradient ultracentrifugation showed that all subspecies, except an 8.1 nm particle, were lipidated. Glia lacking the cholesterol transporter ABCA1 secreted only 8.1 nm particles, which were poorly lipidated and nondiscoidal but could accept lipids to form the full repertoire of WT apoE particles. Receptor-associated-protein (RAP)-mediated inhibition of apoE receptor function blocked appearance of the 8.1 nm species, suggesting that this particle may arise through apoE recycling. Selective deletion of the LDL receptor (LDLR) reduced the level of 8.1 nm particle production by approximately 90%, suggesting that apoE is preferentially recycled through the LDLR. Finally, apoA-I stimulated secretion of 8.1 nm particles in a dose-dependent manner. These results suggest that nascent glial apoE lipoproteins are secreted through multiple pathways and that a greater understanding of these mechanisms may be relevant to several neurological disorders.