RESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Peptídeos beta-Amiloides/análise , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Química Encefálica , Modelos Animais de Doenças , Lipoproteínas/metabolismo , Masculino , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: Animal studies suggest that brain apolipoprotein E (apoE) levels influence amyloid-beta (Abeta) deposition and thus risk for Alzheimer's disease (AD). We have previously demonstrated that deletion of the ATP-binding cassette A1 transporter (ABCA1) in mice causes dramatic reductions in brain and cerebrospinal fluid (CSF) apoE levels and lipidation. To examine whether polymorphisms in ABCA1 affect CSF apoE levels in humans, we measured apoE in CSF taken from 168 subjects who were 43 to 91 years old and were either cognitively normal or who had mild AD. We then genotyped the subjects for ten previously identified ABCA1 single nucleotide polymorphisms (SNPs). RESULTS: In all subjects, the mean CSF apoE level was 9.09 microg/ml with a standard deviation of 2.70 microg/ml. Levels of apoE in CSF samples taken from the same individual two weeks apart were strongly correlated (r2 = 0.93, p < 0.01). In contrast, CSF apoE levels in different individuals varied widely (coefficient of variation = 46%). CSF apoE levels did not vary according to AD status, APOE genotype, gender or race. Average apoE levels increased with age by approximately 0.5 microg/ml per 10 years (r2 = 0.05, p = 0.003). We found no significant associations between CSF apoE levels and the ten ABCA1 SNPs we genotyped. Moreover, in a separate sample of 1225 AD cases and 1431 controls, we found no association between the ABCA1 SNP rs2230806 and AD as has been previously reported. CONCLUSION: We found that CSF apoE levels vary widely between individuals, but are stable within individuals over a two-week interval. AD status, APOE genotype, gender and race do not affect CSF apoE levels, but average CSF apoE levels increase with age. Given the lack of association between CSF apoE levels and genotypes for the ABCA1 SNPs we examined, either these SNPs do not affect ABCA1 function or if they do, they do not have strong effects in the CNS. Finally, we find no evidence for an association between the ABCA1 SNP rs2230806 and AD in a large sample set.
RESUMO
The low density lipoprotein receptor-related protein (LRP) is highly expressed in the brain and has been shown to alter the metabolism of amyloid precursor protein and amyloid-beta peptide (Abeta) in vitro. Previously we developed mice that overexpress a functional LRP minireceptor (mLRP2) in their brains and crossed them to the PDAPP mouse model of Alzheimer disease. Overexpression of mLRP2 in 22-month-old PDAPP mice with amyloid plaques increased a pool of carbonate-soluble Abeta in the brain and worsened memory-related behavior. In the current study, we examined the effects of mLRP2 overexpression on 3-month-old PDAPP mice that had not yet developed amyloid plaques. We found significantly higher levels of membrane-associated Abeta42 in the hippocampus of mice that overexpressed mLRP2. Using immunohistochemical methods, we observed significant intraneuronal Abeta42 in the hippocampus and frontal cortex of PDAPP mice, which frequently co-localized with the lysosomal marker LAMP-1. Interestingly, PDAPP mice lacking apolipoprotein E (apoE) had much less intraneuronal Abeta42. We also found that PC12 cells overexpressing mLRP2 cleared Abeta42 and Abeta40 more rapidly from media than PC12 cells transfected with the vector only. Preincubation of apoE3 or apoE4 with Abeta42 increased the rate of Abeta clearance, and this effect was partially blocked by receptor-associated protein. Our results support the hypothesis that LRP binds and endocytoses Abeta42 both directly and via apoE but that endocytosed Abeta42 is not completely degraded and accumulates in intraneuronal lysosomes.
Assuntos
Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/fisiologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Neurônios/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Animais , Encéfalo/metabolismo , Endocitose , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica , Lisossomos/metabolismo , Camundongos , Camundongos Transgênicos , Células PC12 , RatosRESUMO
Over 5,000 participants attended the 10th International Conference on Alzheimer's Disease (ICAD) and Related Disorders in Madrid, Spain from July 15-20, 2006. Highlights of the conference included reports on brain imaging, the discovery of mutations in the progranulin gene that cause frontotemporal dementia, the finding that neuregulin-1 is a substrate for BACE1 and new interest in the connection between Alzheimer's disease and metabolic syndromes.
RESUMO
Apolipoprotein E (apoE) genotype has a major influence on the risk for Alzheimer disease (AD). Different apoE isoforms may alter AD pathogenesis via their interactions with the amyloid beta-peptide (Abeta). Mice lacking the lipid transporter ABCA1 were found to have markedly decreased levels and lipidation of apoE in the central nervous system. We hypothesized that if Abca1-/- mice were bred to the PDAPP mouse model of AD, PDAPP Abca1-/ mice would have a phenotype similar to that of PDAPP Apoe+/- and PDAPP Apoe-/- mice, which develop less amyloid deposition than PDAPP Apoe+/+ mice. In contrast to this prediction, 12-month-old PDAPP Abca -/- mice had significantly higher levels of hippocampal Abeta, and cerebral amyloid angiopathy was significantly more common compared with PDAPP Abca1+/+ mice. Amyloid precursor protein (APP) C-terminal fragments were not different between Abca1 genotypes prior to plaque deposition in 3-month-old PDAPP mice, suggesting that deletion of Abca1 did not affect APP processing or Abeta production. As expected, 3-month-old PDAPP Abca1-/- mice had decreased apoE levels, but they also had a higher percentage of carbonate-insoluble apoE, suggesting that poorly lipidated apoE is less soluble in vivo. We also found that 12-month-old PDAPP Abca1-/- mice had a higher percentage of carbonate-insoluble apoE and that apoE deposits co-localize with amyloid plaques, demonstrating that poorly lipidated apoE co-deposits with insoluble Abeta. Together, these data suggest that despite substantially lower apoE levels, poorly lipidated apoE produced in the absence of ABCA1 is strongly amyloidogenic in vivo.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Doença de Alzheimer/patologia , Amiloide/metabolismo , Animais , Apolipoproteínas E/metabolismo , Benzotiazóis , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Ensaio de Imunoadsorção Enzimática , Deleção de Genes , Heterozigoto , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Estrutura Terciária de Proteína , Tiazóis/farmacologia , Fatores de TempoRESUMO
The apolipoprotein E (apoE) genotype is an important genetic risk factor for Alzheimer's disease (AD). In the central nervous system (CNS), most apoE is produced by astrocytes and is present in unique high-density lipoprotein (HDL)-like particles that have distinct properties from apoE derived from other sources. To develop an efficient system to produce astrocyte-derived apoE in large quantities, we produced and characterized immortalized cell lines from primary astrocyte cultures derived from human APOE knock-in mice. APOE2, APOE3, and APOE4 expressing cell lines were established that secrete apoE in HDL-like particles at similar levels, cholesterol composition, and size as those produced by primary astrocytes. In physiological buffers, astrocyte-secreted apoE3 and E4 associated equally well with amyloid-beta. Under the same conditions, only a small fraction of A beta formed sodium dodecyl sulfate (SDS)-stable complexes with apoE (E3 > E4). These immortalized astrocytes will be useful for studying mechanisms underlying the isoform-specific effects of apoE in the CNS.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/biossíntese , Apolipoproteínas E/química , Astrócitos/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/ultraestrutura , Astrócitos/ultraestrutura , Linhagem Celular Transformada , Células Cultivadas , Hipocampo/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/ultraestrutura , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/ultraestruturaRESUMO
Epidemiological studies suggest links between cholesterol metabolism and Alzheimer's disease (AD), with hypercholesterolemia associated with increased AD risk, and use of cholesterol-lowering drugs associated with decreased risk. Animal models using cholesterol-modifying dietary or pharmacological interventions demonstrate similar findings. Proposed mechanisms include effects of cholesterol on the metabolism of amyloid-beta (Abeta), the protein that deposits in AD brain. To investigate the effect of genetic alterations in plasma cholesterol on Abeta pathology, we crossed the PDAPP transgenic mouse model of AD-like cerebral amyloidosis to apolipoprotein AI-null mice that have markedly reduced plasma cholesterol levels due to a virtual absence of high density lipoproteins, the primary lipoprotein in mice. Interestingly and in contrast to models using non-physiological high fat diets or cholesterol-lowering drugs to modify plasma cholesterol, we observed no differences in Abeta pathology in PDAPP mice of the various apoAI genotypes despite robust differences in plasma cholesterol levels between the groups. Absence of apoAI also resulted in reductions in brain but not cerebrospinal fluid cholesterol, but had no effect on brain apolipoprotein E levels. These and other data suggest that it is perhaps the level of brain apolipoprotein E, not cholesterol per se, that plays a primary role in brain Abeta metabolism.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Amiloidose/metabolismo , Apolipoproteína A-I/deficiência , Colesterol/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Amiloidose/genética , Amiloidose/patologia , Animais , Apolipoproteína A-I/genética , Western Blotting , Encefalopatias/genética , Encefalopatias/metabolismo , Encefalopatias/patologia , Colesterol/análise , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Genótipo , Camundongos , Camundongos TransgênicosRESUMO
ABCA1 is an ATP-binding cassette protein that transports cellular cholesterol and phospholipids onto high density lipoproteins (HDL) in plasma. Lack of ABCA1 in humans and mice causes abnormal lipidation and increased catabolism of HDL, resulting in very low plasma apoA-I, apoA-II, and HDL. Herein, we have used Abca1-/- mice to ask whether ABCA1 is involved in lipidation of HDL in the central nervous system (CNS). ApoE is the most abundant CNS apolipoprotein and is present in HDL-like lipoproteins in CSF. We found that Abca1-/- mice have greatly decreased apoE levels in both the cortex (80% reduction) and the CSF (98% reduction). CSF from Abca1-/- mice had significantly reduced cholesterol as well as small apoE-containing lipoproteins, suggesting abnormal lipidation of apoE. Astrocytes, the primary producer of CNS apoE, were cultured from Abca1+/+, +/-, and -/- mice, and nascent lipoprotein particles were collected. Abca1-/- astrocytes secreted lipoprotein particles that had markedly decreased cholesterol and apoE and had smaller apoE-containing particles than particles from Abca1+/+ astrocytes. These findings demonstrate that ABCA1 plays a critical role in CNS apoE metabolism. Since apoE isoforms and levels strongly influence Alzheimer's disease pathology and risk, these data suggest that ABCA1 may be a novel therapeutic target.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Apolipoproteínas E/química , Astrócitos/metabolismo , Sistema Nervoso Central/metabolismo , Lipídeos/química , Transportador 1 de Cassete de Ligação de ATP , Doença de Alzheimer , Animais , Western Blotting , Córtex Cerebral/metabolismo , Líquido Cefalorraquidiano/metabolismo , Colesterol/metabolismo , Clusterina , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/metabolismo , Heterozigoto , Lipoproteínas/química , Lipoproteínas/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Chaperonas Moleculares/metabolismo , Fosfolipídeos/metabolismo , Isoformas de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/metabolismoAssuntos
Doença de Alzheimer/metabolismo , Apolipoproteínas E/metabolismo , Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Animais , Apolipoproteínas E/sangue , Apolipoproteínas E/líquido cefalorraquidiano , Modelos Animais de Doenças , Progressão da Doença , Humanos , Camundongos , Isoformas de Proteínas/sangue , Isoformas de Proteínas/líquido cefalorraquidiano , Isoformas de Proteínas/metabolismoRESUMO
Buoyant membrane fractions containing presenilin 1 (PS1), an essential component of the gamma-secretase complex, and APP CTFbeta, a gamma-secretase substrate, can be isolated from cultured cells and brain by several different fractionation procedures that are compatible with in vitro gamma-secretase assays. Analysis of these gradients for amyloid beta protein (Abeta) and CTFgamma production indicated that gamma-secretase activity is predominantly localized in these buoyant membrane microdomains. Consistent with this localization, we find that gamma-secretase activity is cholesterol dependent. Depletion of membrane cholesterol completely inhibits gamma-secretase cleavage, which can be restored by cholesterol replacement. Thus, altering cholesterol levels may influence the development of Alzheimer's disease (AD) by influencing production and deposition of Abeta within cholesterol rich membrane microdomains.