RESUMO
Diabetic retinopathy (DR) is a common complication of diabetes. Approximately 20% of DR patients have diabetic macular edema (DME) characterized by fluid leakage into the retina. There is a genetic component to DR and DME risk, but few replicable loci. Because not all DR cases have DME, we focused on DME to increase power, and conducted a multi-ancestry GWAS to assess DME risk in a total of 1,502 DME patients and 5,603 non-DME controls in discovery and replication datasets. Two loci reached GWAS significance (p<5x10-8). The strongest association was rs2239785, (K150E) in APOL1. The second finding was rs10402468, which co-localized to PLVAP and ANKLE1 in vascular / endothelium tissues. We conducted multiple sensitivity analyses to establish that the associations were specific to DME status and did not reflect diabetes status or other diabetic complications. Here we report two novel loci for risk of DME which replicated in multiple clinical trial and biobank derived datasets. One of these loci, containing the gene APOL1, is a risk factor in African American DME and DKD patients, indicating that this locus plays a broader role in diabetic complications for multiple ancestries. Trial Registration: NCT00473330, NCT00473382, NCT03622580, NCT03622593, NCT04108156.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Edema Macular , Humanos , Edema Macular/genética , Edema Macular/complicações , Retinopatia Diabética/genética , Retinopatia Diabética/complicações , Estudo de Associação Genômica Ampla , Apolipoproteína L1/genética , Fatores de RiscoRESUMO
The Reelin ligand regulates a Dab1-dependent signaling pathway required for brain lamination and normal dendritogenesis, but the specific mechanisms underlying these actions remain unclear. We find that Stk25, a modifier of Reelin-Dab1 signaling, regulates Golgi morphology and neuronal polarization as part of an LKB1-Stk25-Golgi matrix protein 130 (GM130) signaling pathway. Overexpression of Stk25 induces Golgi condensation and multiple axons, both of which are rescued by Reelin treatment. Reelin stimulation of cultured neurons induces the extension of the Golgi into dendrites, which is suppressed by Stk25 overexpression. In vivo, Reelin and Dab1 are required for the normal extension of the Golgi apparatus into the apical dendrites of hippocampal and neocortical pyramidal neurons. This demonstrates that the balance between Reelin-Dab1 signaling and LKB1-Stk25-GM130 regulates Golgi dispersion, axon specification, and dendrite growth and provides insights into the importance of the Golgi apparatus for cell polarization.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Complexo de Golgi/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular , Separação Celular , Células Cultivadas , Hipocampo/metabolismo , Humanos , Camundongos , Ratos , Proteína ReelinaRESUMO
Pathogenic mutations in the amyloid precursor protein (APP) gene have been described as causing early onset familial Alzheimer disease (AD). We recently identified a rare APP variant encoding an alanine-to-threonine substitution at residue 673 (A673T) that confers protection against development of AD (Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V., Bjornsson, S., Stefansson, H., Sulem, P., Gudbjartsson, D., Maloney, J., Hoyte, K., Gustafson, A., Liu, Y., Lu, Y., Bhangale, T., Graham, R. R., Huttenlocher, J., Bjornsdottir, G., Andreassen, O. A., Jönsson, E. G., Palotie, A., Behrens, T. W., Magnusson, O. T., Kong, A., Thorsteinsdottir, U., Watts, R. J., and Stefansson, K. (2012) Nature 488, 96-99). The Ala-673 residue lies within the ß-secretase recognition sequence and is part of the amyloid-ß (Aß) peptide cleavage product (position 2 of Aß). We previously demonstrated that the A673T substitution makes APP a less favorable substrate for cleavage by BACE1. In follow-up studies, we confirm that A673T APP shows reduced cleavage by BACE1 in transfected mouse primary neurons and in isogenic human induced pluripotent stem cell-derived neurons. Using a biochemical approach, we show that the A673T substitution modulates the catalytic turnover rate (V(max)) of APP by the BACE1 enzyme, without affecting the affinity (K(m)) of the APP substrate for BACE1. We also show a reduced level of Aß(1-42) aggregation with A2T Aß peptides, an observation not conserved in Aß(1-40) peptides. When combined in a ratio of 1:9 Aß(1-42)/Aß(1-40) to mimic physiologically relevant mixtures, A2T retains a trend toward slowed aggregation kinetics. Microglial uptake of the mutant Aß(1-42) peptides correlated with their aggregation level. Cytotoxicity of the mutant Aß peptides was not dramatically altered. Taken together, our findings demonstrate that A673T, a protective allele of APP, reproducibly reduces amyloidogenic processing of APP and also mildly decreases Aß aggregation. These effects could together have an additive or even synergistic impact on the risk of developing AD.
Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Alelos , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Animais , Ácido Aspártico Endopeptidases/metabolismo , Catálise , DNA Complementar/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Heterozigoto , Humanos , Concentração Inibidora 50 , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Mutação , Neurônios/metabolismo , Fragmentos de Peptídeos/genética , Ligação ProteicaRESUMO
Platelets play a crucial role in the pathogenesis of myocardial infarction (MI) by adhering to the site of a ruptured atherosclerotic plaque. The aim of this study was to screen for differences in the micro RNA (miRNA) content of platelets from patients with myocardial infarction and control patients, to investigate a possible release of miRNAs from activated platelets and to elucidate whether platelet-derived miRNAs could act as paracrine regulators of endothelial cell gene expression. Using RNA-seq, we found 9 differentially expressed miRNAs in patients compared with healthy controls, of which 8 were decreased in patients. Of these, miR-22, -185, -320b, and -423-5p increased in the supernatant of platelets after aggregation and were depleted in thrombi aspirated from MI patients, indicating the release of certain miRNAs from activated platelets. To confirm that endothelial cells could take up the released platelet miRNAs, transfer of both fluorescently labeled miRNA and exogenous cel-miR-39 from activated platelets to endothelial cells was shown. Finally, a possible paracrine role of released platelet miR-320b on endothelial cell intercellular adhesion molecule-1 expression was shown. Thus, platelets from patients with MI exhibit loss of specific miRNAs, and activated platelets shed miRNAs that can regulate endothelial cell gene expression.
Assuntos
Células Endoteliais/metabolismo , Molécula 1 de Adesão Intercelular/genética , MicroRNAs/metabolismo , Infarto do Miocárdio/genética , Ativação Plaquetária/genética , Plaquetas/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Endocitose/fisiologia , Feminino , Regulação da Expressão Gênica , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , MicroRNAs/genética , Infarto do Miocárdio/sangue , Ativação Plaquetária/fisiologia , Agregação Plaquetária/genética , TranscriptomaRESUMO
Methylation at CpG sites is a critical epigenetic modification in mammals. Altered DNA methylation has been suggested to be a central mechanism in development, some disease processes and cellular senescence. Quantifying the extent and identity of epigenetic changes in the aging process is therefore potentially important for understanding longevity and age-related diseases. In the current study, we have examined DNA methylation at >27,000 CpG sites throughout the human genome, in frontal cortex, temporal cortex, pons and cerebellum from 387 human donors between the ages of 1 and 102 years. We identify CpG loci that show a highly significant, consistent correlation between DNA methylation and chronological age. The majority of these loci are within CpG islands and there is a positive correlation between age and DNA methylation level. Lastly, we show that the CpG sites where the DNA methylation level is significantly associated with age are physically close to genes involved in DNA binding and regulation of transcription. This suggests that specific age-related DNA methylation changes may have quite a broad impact on gene expression in the human brain.
Assuntos
Envelhecimento/genética , Envelhecimento/metabolismo , Encéfalo/metabolismo , Metilação de DNA , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Ilhas de CpG , Feminino , Regulação da Expressão Gênica , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Mutations in DJ-1, PINK1 (PTEN-induced putative kinase 1) and parkin all cause recessive parkinsonism in humans, but the relationships between these genes are not clearly defined. One event associated with loss of any of these genes is altered mitochondrial function. Recent evidence suggests that turnover of damaged mitochondria by autophagy might be central to the process of recessive parkinsonism. Here, we show that loss of DJ-1 leads to loss of mitochondrial polarization, fragmentation of mitochondria and accumulation of markers of autophagy (LC3 punctae and lipidation) around mitochondria in human dopaminergic cells. These effects are due to endogenous oxidative stress, as antioxidants will reverse all of them. Similar to PINK1 and parkin, DJ-1 also limits mitochondrial fragmentation in response to the mitochondrial toxin rotenone. Furthermore, overexpressed parkin will protect against loss of DJ-1 and, although DJ-1 does not alter PINK1 mitochondrial phenotypes, DJ-1 is still active against rotenone-induced damage in the absence of PINK1. None of the three proteins complex together using size exclusion chromatography. These data suggest that DJ-1 works in parallel to the PINK1/parkin pathway to maintain mitochondrial function in the presence of an oxidative environment.
Assuntos
Autofagia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitocôndrias/genética , Mitocôndrias/fisiologia , Mutação , Proteínas Oncogênicas/genética , Estresse Oxidativo/genética , Doença de Parkinson/genética , Proteína Desglicase DJ-1 , Proteínas Quinases/genética , Rotenona/farmacologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
A fundamental challenge in the post-genome era is to understand and annotate the consequences of genetic variation, particularly within the context of human tissues. We present a set of integrated experiments that investigate the effects of common genetic variability on DNA methylation and mRNA expression in four human brain regions each from 150 individuals (600 samples total). We find an abundance of genetic cis regulation of mRNA expression and show for the first time abundant quantitative trait loci for DNA CpG methylation across the genome. We show peak enrichment for cis expression QTLs to be approximately 68,000 bp away from individual transcription start sites; however, the peak enrichment for cis CpG methylation QTLs is located much closer, only 45 bp from the CpG site in question. We observe that the largest magnitude quantitative trait loci occur across distinct brain tissues. Our analyses reveal that CpG methylation quantitative trait loci are more likely to occur for CpG sites outside of islands. Lastly, we show that while we can observe individual QTLs that appear to affect both the level of a transcript and a physically close CpG methylation site, these are quite rare. We believe these data, which we have made publicly available, will provide a critical step toward understanding the biological effects of genetic variation.
Assuntos
Encéfalo/metabolismo , Metilação de DNA , Expressão Gênica , Locos de Características Quantitativas , Ilhas de CpG , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genéticaRESUMO
It was recently reported that rs1541160 on chromosome 1q24.2 has a marked effect on survival of amyotrophic lateral sclerosis (ALS) patients by influencing KIFAP3 expression. The cohorts used in that study were collected from ALS specialty clinics. We attempted to replicate these findings in a population-based cohort of 504 Italian ALS patients. None of 140 SNPs genotyped within the KIFAP3 locus (including rs1541160) had an effect on survival (log-rank P value for rs1541160 = 0.47) or on gene expression in that region. These data illustrate the complexities associated with analyzing ALS phenotypes for association.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/genética , Proteínas do Citoesqueleto/genética , Polimorfismo de Nucleotídeo Único , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Cromossomos Humanos Par 1/genética , Estudos de Coortes , Proteínas do Citoesqueleto/metabolismo , Perfilação da Expressão Gênica/estatística & dados numéricos , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Itália , Estimativa de Kaplan-Meier , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A single amino acid change, F580Y (Legs at odd angles (Loa), Dync1h1(Loa)), in the highly conserved and overlapping homodimerization, intermediate chain, and light intermediate chain binding domain of the cytoplasmic dynein heavy chain can cause severe motor and sensory neuron loss in mice. The mechanism by which the Loa mutation impairs the neuron-specific functions of dynein is not understood. To elucidate the underlying molecular mechanisms of neurodegeneration arising from this mutation, we applied a cohort of biochemical methods combined with in vivo assays to systemically study the effects of the mutation on the assembly of dynein and its interaction with dynactin. We found that the Loa mutation in the heavy chain leads to increased affinity of this subunit of cytoplasmic dynein to light intermediate and a population of intermediate chains and a suppressed association of dynactin to dynein. These data suggest that the Loa mutation drives the assembly of cytoplasmic dynein toward a complex with lower affinity to dynactin and thus impairing transport of cargos that tether to the complex via dynactin. In addition, we detected up-regulation of kinesin light chain 1 (KLC1) and its increased association with dynein but reduced microtubule-associated KLC1 in the Loa samples. We provide a model describing how up-regulation of KLC1 and its interaction with cytoplasmic dynein in Loa could play a regulatory role in restoring the retrograde and anterograde transport in the Loa neurons.
Assuntos
Dineínas do Citoplasma/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Doenças Neurodegenerativas/metabolismo , Animais , Dineínas do Citoplasma/genética , Complexo Dinactina , Cinesinas , Camundongos , Camundongos Mutantes , Proteínas Associadas aos Microtúbulos/genética , Doenças Neurodegenerativas/genéticaRESUMO
Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Oncogênicas/metabolismo , Transtornos Parkinsonianos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Encéfalo/metabolismo , Linhagem Celular , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Genes Mitocondriais , Genes Recessivos , Glutationa/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Estresse Oxidativo , PTEN Fosfo-Hidrolase/metabolismo , Transtornos Parkinsonianos/genética , Peroxirredoxinas , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Desglicase DJ-1 , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Damage-associated microglia (DAM) profiles observed in Alzheimer's disease (AD)-related mouse models reflect an activation state that could modulate AD risk or progression. To learn whether human AD microglia (HAM) display a similar profile, we develop a method for purifying cell types from frozen cerebrocortical tissues for RNA-seq analysis, allowing better transcriptome coverage than typical single-nucleus RNA-seq approaches. The HAM profile we observe bears little resemblance to the DAM profile. Instead, HAM display an enhanced human aging profile, in addition to other disease-related changes such as APOE upregulation. Analyses of whole-tissue RNA-seq and single-cell/nucleus RNA-seq datasets corroborate our findings and suggest that the lack of DAM response in human microglia occurs specifically in AD tissues, not other neurodegenerative settings. These results, which can be browsed at http://research-pub.gene.com/BrainMyeloidLandscape, provide a genome-wide picture of microglial activation in human AD and highlight considerable differences between mouse models and human disease.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Senescência Celular/genética , Microglia/metabolismo , Microglia/patologia , Ativação Transcricional/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Bases de Dados Genéticas , Feminino , Lobo Frontal/patologia , Secções Congeladas , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Xenoenxertos , Humanos , Masculino , Camundongos , Monócitos/metabolismo , Esclerose Múltipla/patologia , Fenótipo , Reprodutibilidade dos Testes , Fatores de Risco , Lobo Temporal/patologiaRESUMO
Mutations in DJ-1 lead to a monogenic form of early onset recessive parkinsonism. DJ-1 can respond to oxidative stress, which has been proposed to be involved in the pathogenesis of sporadic Parkinson disease (PD). We have recently reported that DJ-1 interacts with mRNA in an oxidation-dependent manner. Here, we confirm interaction of DJ-1 and RNA in human brain using immunoprecipitation followed by quantitative real time PCR. We confirmed previous reports that DJ-1 is more oxidized in cortex from cases of sporadic PD compared to controls. In the same samples, protein and RNA expression was measured for four DJ-1 target genes GPx4, MAPK8IP1, ND2 and ND5. While no alterations in mRNA expression were observed, an increase in protein expression was observed in PD cases for GPx4 and MAPK8IP1. In the same patients, we saw decreased mRNA and protein levels of two mitochondrial targets, ND2 and ND5. These results suggest that these proteins undergo regulation at the post-transcriptional level that may involve translational regulation by DJ-1.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Oncogênicas/genética , Doença de Parkinson/genética , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Encéfalo/metabolismo , Eletroforese em Gel Bidimensional/métodos , Glutationa Peroxidase/genética , Humanos , Imunoprecipitação/métodos , NADH Desidrogenase/genética , Doença de Parkinson/patologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Proteína Desglicase DJ-1 , Proteína Fosfatase 2/genética , Proteínas Proto-Oncogênicas/genética , Selenoproteína W/genética , Proteína de Morte Celular Associada a bcl/genéticaRESUMO
Parkinson's disease (PD; OMIM #168600) is the second most common neurodegenerative disorder in the Western world and presents as a progressive movement disorder. The hallmark pathological features of PD are loss of dopaminergic neurons from the substantia nigra and neuronal intracellular Lewy body inclusions. Parkinsonism is typically sporadic in nature; however, several rare familial forms are linked to genetic loci, and the identification of causal mutations has provided insight into the disease process. PARK8, identified in 2002 by Funayama and colleagues, appears to be a common cause of familial PD. We describe here the cloning of a novel gene that contains missense mutations segregating with PARK8-linked PD in five families from England and Spain. Because of the tremor observed in PD and because a number of the families are of Basque descent, we have named this protein dardarin, derived from the Basque word dardara, meaning tremor.
Assuntos
Haplótipos , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Idoso , Sequência de Aminoácidos , Animais , Northern Blotting , Mapeamento Cromossômico , Clonagem Molecular , Feminino , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Dados de Sequência Molecular , Mutação , LinhagemRESUMO
Cellular cholesterol levels alter the processing of the amyloid precursor protein (APP) to produce Abeta. Activation of liver X receptors (LXRs), one cellular mechanism to regulate cholesterol homeostasis, has been found to alter Abeta levels in vitro and in vivo. To identify genes regulated by LXR, we treated human neuroblastoma cells with an LXR agonist (TO-901317) and examined gene expression by microarray. As expected, TO-901317 upregulated several cholesterol metabolism genes, but it also decreased expression of a metalloprotease inhibitor, TIMP-3. We confirmed this finding using real-time PCR and by measuring TIMP-3 protein in glia, SY5Y cells, and COS7 cells. TIMP-3 is a member of a family of metalloproteinase inhibitors and blocks A disintegrin and metalloproteinase-10 (ADAM-10) and ADAM-17, two APP alpha-secretases. We found that TIMP-3 inhibited alpha-secretase cleavage of APP and an apolipoprotein E (apoE) receptor, ApoER2. TIMP-3 decreased surface levels of ADAM-10, APP, and ApoER2. These changes were accompanied by increased APP beta-C-terminal fragment and Abeta production. These data suggest that TIMP-3 preferentially routes APP and ApoER2 away from the cell surface and alpha-secretase cleavage and encourages endocytosis and beta-secretase cleavage. In vivo, TO-901317 decreased brain TIMP-3 levels. TIMP-3 protein levels were increased in human Alzheimer's disease (AD) brain and in APP transgenic mice, suggesting that increased levels of TIMP-3 in AD may contribute to higher levels of Abeta.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Chlorocebus aethiops , Proteínas de Ligação a DNA/antagonistas & inibidores , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo , Humanos , Receptores X do Fígado , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas de Membrana/metabolismo , Camundongos , Neuroblastoma , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptores Nucleares Órfãos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , RNA Interferente Pequeno/farmacologia , Ratos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Transfecção/métodosRESUMO
The aggregation of intracellular tau protein is a major hallmark of Alzheimer's disease (AD). The extent and the stereotypical spread of tau pathology in the AD brain are correlated with cognitive decline during disease progression. Here we present an in-depth analysis of endogenous tau fragmentation in a well-characterized cohort of AD and age-matched control subjects. Using protein mass spectrometry and Edman degradation to interrogate endogenous tau fragments in the human brain, we identified two novel proteolytic sites, G323 and G326, as major tau cleavage events in both normal and AD cortex. These sites are located within the sequence recently identified as the structural core of tau protofilaments, suggesting an inhibitory mechanism of fibril formation. In contrast, a different set of novel cleavages showed a distinct increase in late stage AD. These disease-associated sites are located outside of the protofilament core sequence. We demonstrate that calpain 1 specifically cleaves at both the normal and diseased sites in vitro, and the site selection is conformation-dependent. Monomeric tau is predominantly cleaved at G323/G326 (normal sites), whereas oligomerization increases cleavages at the late-AD-associated sites. The fragmentation patterns specific to disease and healthy states suggest novel regulatory mechanisms of tau aggregation in the human brain.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Calpaína/metabolismo , Progressão da Doença , Proteínas tau/química , Proteínas tau/metabolismo , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Feminino , Humanos , Masculino , ProteóliseRESUMO
Microglia, the CNS-resident immune cells, play important roles in disease, but the spectrum of their possible activation states is not well understood. We derived co-regulated gene modules from transcriptional profiles of CNS myeloid cells of diverse mouse models, including new tauopathy model datasets. Using these modules to interpret single-cell data from an Alzheimer's disease (AD) model, we identified microglial subsets-distinct from previously reported "disease-associated microglia"-expressing interferon-related or proliferation modules. We then analyzed whole-tissue RNA profiles from human neurodegenerative diseases, including a new AD dataset. Correcting for altered cellular composition of AD tissue, we observed elevated expression of the neurodegeneration-related modules, but also modules not implicated using expression profiles from mouse models alone. We provide a searchable, interactive database for exploring gene expression in all these datasets (http://research-pub.gene.com/BrainMyeloidLandscape). Understanding the dimensions of CNS myeloid cell activation in human disease may reveal opportunities for therapeutic intervention.
Assuntos
Doença de Alzheimer/genética , Encéfalo/metabolismo , Microglia/metabolismo , Doença de Alzheimer/metabolismo , Animais , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
The recent advent of an "ecosystem" of shared biofluid sample biorepositories and data sets will focus biomarker efforts in Parkinson's disease, boosting the therapeutic development pipeline and enabling translation with real-world impact.
Assuntos
Biomarcadores/sangue , Doença de Parkinson/sangue , Estudos de Coortes , Progressão da Doença , Humanos , Doença de Parkinson/patologia , Sintomas Prodrômicos , Padrões de ReferênciaRESUMO
Common variant genome-wide association studies (GWASs) have, to date, identified >24 risk loci for Parkinson's disease (PD). To discover additional loci, we carried out a GWAS comparing 6,476 PD cases with 302,042 controls, followed by a meta-analysis with a recent study of over 13,000 PD cases and 95,000 controls at 9,830 overlapping variants. We then tested 35 loci (P < 1 × 10-6) in a replication cohort of 5,851 cases and 5,866 controls. We identified 17 novel risk loci (P < 5 × 10-8) in a joint analysis of 26,035 cases and 403,190 controls. We used a neurocentric strategy to assign candidate risk genes to the loci. We identified protein-altering or cis-expression quantitative trait locus (cis-eQTL) variants in linkage disequilibrium with the index variant in 29 of the 41 PD loci. These results indicate a key role for autophagy and lysosomal biology in PD risk, and suggest potential new drug targets for PD.
Assuntos
Estudo de Associação Genômica Ampla , Doença de Parkinson/genética , Antiparkinsonianos/farmacologia , Autofagia/genética , Estudos de Casos e Controles , Predisposição Genética para Doença , Humanos , Desequilíbrio de Ligação , Lisossomos/fisiologia , Terapia de Alvo Molecular , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/epidemiologia , Risco , Fatores de TranscriçãoRESUMO
The common p.D358A variant (rs2228145) in IL-6R is associated with risk for multiple diseases and with increased levels of soluble IL-6R in the periphery and central nervous system (CNS). Here, we show that the p.D358A allele leads to increased proteolysis of membrane bound IL-6R and demonstrate that IL-6R peptides with A358 are more susceptible to cleavage by ADAM10 and ADAM17. IL-6 responsive genes were identified in primary astrocytes and microglia and an IL-6 gene signature was increased in the CNS of late onset Alzheimer's disease subjects in an IL6R allele dependent manner. We conducted a screen to identify variants associated with the age of onset of Alzheimer's disease in APOE É4 carriers. Across five datasets, p.D358A had a meta Pâ=â3 ×10-4 and an odds ratioâ=â1.3, 95% confidence interval 1.12 -1.48. Our study suggests that a common coding region variant of the IL-6 receptor results in neuroinflammatory changes that may influence the age of onset of Alzheimer's disease in APOE É4 carriers.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Idoso , Idoso de 80 Anos ou mais , Alelos , Animais , Apolipoproteína E4/genética , Astrócitos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Técnicas de Cocultura , Estudos de Coortes , Feminino , Células HEK293 , Humanos , Interleucina-6/metabolismo , Masculino , Camundongos , Microglia/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
A common approach to understanding neurodegenerative disease is comparing gene expression in diseased versus healthy tissues. We illustrate that expression profiles derived from whole tissue RNA highly reflect the degenerating tissues' altered cellular composition, not necessarily transcriptional regulation. To accurately understand transcriptional changes that accompany neuropathology, we acutely purify neurons, astrocytes and microglia from single adult mouse brains and analyse their transcriptomes by RNA sequencing. Using peripheral endotoxemia to establish the method, we reveal highly specific transcriptional responses and altered RNA processing in each cell type, with Tnfr1 required for the astrocytic response. Extending the method to an Alzheimer's disease model, we confirm that transcriptomic changes observed in whole tissue are driven primarily by cell type composition, not transcriptional regulation, and identify hundreds of cell type-specific changes undetected in whole tissue RNA. Applying similar methods to additional models and patient tissues will transform our understanding of aberrant gene expression in neurological disease.