RESUMO
Zinc (Zn2+) is concentrated into pre-synaptic vesicles and co-released with neurotransmitter at some synapses. Zn2+ can accelerate assembly of the amyloid-ß peptides (Aß) and tau protein central to the neuropathological changes found in Alzheimer's disease (AD). Altered protein levels of the membrane Zn2+ transporters ZnT1, ZnT4, and ZnT6 have been reported in AD postmortem brain tissue. The present study analyzed mRNA levels of five established (LIV1, ZIP1, ZnT1, ZnT4, and ZnT6) and one potential (PRNP) Zn2+ transporter in human postmortem brain tissue from Braak-staged individuals with AD and controls using quantitative real-time PCR. Four cortical regions (middle temporal gyrus, superior occipital gyrus, superior parietal gyrus, and superior frontal gyrus) and cerebellum were examined. PRNP mRNA levels were decreased by â¼30% in all four cortical regions examined in AD patients, but unchanged in the cerebellum. In contrast, some increases in mRNA levels of the other more established Zn2+ transporters (LIV1, ZIP1, ZnT1, ZnT6) were found in AD cortex. The ratios of the mRNA levels of LIV1, ZIP1, ZnT1, ZnT4, and ZnT6/mRNA level of neuron specific enolase increased significantly as the disease progressed and Braak stage increased. Significant correlations were also identified between mRNA levels of several of the Zn2+ transporters investigated. These expression changes could either reflect or cause the altered cortical Zn2+ distribution in AD, potentially increasing the likelihood of interactions between Zn2+ and Aß or tau protein.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/metabolismo , Proteínas de Transporte/genética , RNA Mensageiro/metabolismo , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Encéfalo/patologia , Proteínas de Transporte/classificação , Proteínas de Transporte/metabolismo , Estudos de Casos e Controles , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Mudanças Depois da Morte , Estatística como Assunto , Estatísticas não ParamétricasRESUMO
Research into the cause of Alzheimer's disease (AD) has identified strong connections to cholesterol. Cholesterol and cholesterol esters can modulate amyloid precursor protein (APP) processing, thus altering production of the Abeta peptides that deposit in cortical amyloid plaques. Processing depends on the encounter between APP and cellular secretases, and is thus subject to the influence of cholesterol-dependent factors including protein trafficking, and distribution between membrane subdomains. We have directly investigated endogenous membrane beta-secretase activity in the presence of a range of membrane cholesterol levels in SH-SY5Y human neuroblastoma cells and human platelets. Membrane cholesterol significantly influenced membrane beta-secretase activity in a biphasic manner, with positive correlations at higher membrane cholesterol levels, and negative correlations at lower membrane cholesterol levels. Platelets from individuals with AD or mild cognitive impairment (n = 172) were significantly more likely to lie within the negative correlation zone than control platelets (n = 171). Pharmacological inhibition of SH-SY5Y beta-secretase activity resulted in increased membrane cholesterol levels. Our findings are consistent with the existence of a homeostatic feedback loop between membrane cholesterol level and membrane beta-secretase activity, and suggest that this regulatory mechanism is disrupted in platelets from individuals with cognitive impairment.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Plaquetas/ultraestrutura , Membrana Celular/metabolismo , Colesterol/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Plaquetas/citologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Colesterol/farmacologia , Transtornos Cognitivos/sangue , Meios de Cultura Livres de Soro/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Modelos Lineares , Masculino , Neuroblastoma/patologia , Neuroblastoma/ultraestrutura , Estatísticas não Paramétricas , Frações Subcelulares , Fatores de Tempo , beta-Ciclodextrinas/farmacologiaRESUMO
BACKGROUND: Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB. RESULTS: The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], beta-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA]) in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan(R) Gene Expression Assays). Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis. CONCLUSION: This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.
Assuntos
Encéfalo/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Humanos , RNA/metabolismo , Padrões de Referência , Reprodutibilidade dos Testes , SoftwareRESUMO
Studies suggest that activation of phosphoinositide 3-kinase-Akt may protect against neuronal cell death in Alzheimer's disease (AD). Here, however, we provide evidence of increased Akt activation, and hyperphosphorylation of critical Akt substrates in AD brain, which link to AD pathogenesis, suggesting that treatments aiming to activate the pathway in AD need to be considered carefully. A different distribution of Akt and phospho-Akt was detected in AD temporal cortex neurons compared with control neurons, with increased levels of active phosphorylated-Akt in particulate fractions, and significant decreases in Akt levels in AD cytosolic fractions, causing increased activation of Akt (phosphorylated-Akt/total Akt ratio) in AD. In concordance, significant increases in the levels of phosphorylation of total Akt substrates, including: GSK3beta(Ser9), tau(Ser214), mTOR(Ser2448), and decreased levels of the Akt target, p27(kip1), were found in AD temporal cortex compared with controls. A significant loss and altered distribution of the major negative regulator of Akt, PTEN (phosphatase and tensin homologue deleted on chromosome 10), was also detected in AD neurons. Loss of phosphorylated-Akt and PTEN-containing neurons were found in hippocampal CA1 at end stages of AD. Taken together, these results support a potential role for aberrant control of Akt and PTEN signalling in AD.
Assuntos
Doença de Alzheimer/metabolismo , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Anticorpos Monoclonais/metabolismo , Western Blotting/métodos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Citosol/metabolismo , Feminino , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Imuno-Histoquímica/métodos , Masculino , Entrevista Psiquiátrica Padronizada , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Neurônios/patologia , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Mudanças Depois da Morte , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt , Regressão Psicológica , Serina/metabolismo , Serina-Treonina Quinases TOR , Proteínas Supressoras de Tumor/metabolismo , Proteínas tau/metabolismoRESUMO
Abeta (beta-amyloid) peptides are found aggregated in the cortical amyloid plaques associated with Alzheimer's disease neuropathology. Inhibition of the proteasome alters the amount of Abeta produced from APP (amyloid precursor protein) by various cell lines in vitro. Proteasome activity is altered during aging, a major risk factor for Alzheimer's disease. In the present study, a human neuroblastoma cell line expressing the C-terminal 100 residues of APP (SH-SY5Y-SPA4CT) was used to determine the effect of proteasome inhibition, by lactacystin and Bz-LLL-COCHO (benzoyl-Leu-Leu-Leu-glyoxal), on APP processing at the gamma-secretase site. Proteasome inhibition caused a significant increase in Abeta peptide levels in medium conditioned by SH-SY5Y-SPA4CT cells, and was also associated with increased cell death. APP is a substrate of the apoptosis-associated caspase 3 protease, and we therefore investigated whether the increased Abeta levels could reflect caspase activation. We report that caspase activation was not required for proteasome-inhibitor-mediated effects on APP (SPA4CT) processing. Cleavage of Ac-DEVD-AMC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin), a caspase substrate, was reduced following exposure of SH-SY5Y-SPA4CT cells to lactacystin, and co-treatment of cells with lactacystin and a caspase inhibitor [Z-DEVD-FMK (benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone)] resulted in higher Abeta levels in medium, augmenting those seen with lactacystin alone. This study indicated that proteasome inhibition could increase APP processing specifically at the gamma-secretase site, and increase release of Abeta, in the absence of caspase activation. This indicates that the decline in proteasome function associated with aging would contribute to increased Abeta levels.
Assuntos
Acetilcisteína/análogos & derivados , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Acetilcisteína/farmacologia , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Ácido Aspártico Endopeptidases , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/química , Humanos , Complexos Multienzimáticos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Peptídeo Hidrolases/metabolismo , Inibidores de ProteassomaRESUMO
Human neuroblastoma SH-SY5Y cells stably transfected with both wild-type and exon-9 deleted (deltaE9) presenilin constructs were used to study the role of the presenilin proteins during differentiation. Cells transfected with either wild-type or deltaE9 PS1, of which the latter abolishes normal endoproteolytic cleavage of the protein, showed no obvious differences in their ability to differentiate to a neuronal-like phenotype upon treatment with retinoic acid (RA). A defined pattern of PS1 expression was observed during differentiation with both RA and the phorbol ester TPA. Full-length PS1 was shown to increase dramatically within 5-24 h of RA treatment. TPA gave an earlier and longer lasting increase in full-length PS1 levels. The intracellular distribution pattern of PS1 was markedly altered following RA treatment. Within 24h PS1 was highly up-regulated throughout the cell body around the nucleus. Between 2 and 4 weeks PS1 staining appeared punctate and also localised to the nucleus. Increases in PS1 expression upon treatment with RA and TPA were blocked by treatment with cycloheximide, indicating a role of de-novo protein synthesis in this effect. PS2 expression remained unchanged during differentiation. Levels of full-length PS1 were also seen to increase during neurogenesis and neuronal differentiation in the forebrain of first trimester human foetuses between 6.5 and 11 weeks. These combined observations support the idea that PS1 is involved in neuronal differentiation by a mechanism likely independent of endoproteolysis of the protein.