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
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
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
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.
Assuntos
Doença de Alzheimer/genética , Astrócitos/metabolismo , Endotoxemia/genética , Microglia/metabolismo , Neurônios/metabolismo , Transcrição Gênica , Transcriptoma , Adulto , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/patologia , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Endotoxemia/patologia , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Especificidade de Órgãos , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Análise de Sequência de RNARESUMO
Combining genetic insights into the pathogenesis of Parkinson's disease (PD) with findings from animal and cellular models of this disorder has advanced our understanding of the pathways that lead to the characteristic degeneration of dopaminergic neurons in the brain's nigrostriatal pathway. This has fueled an increase in candidate compounds designed to modulate these pathways and to alter the processes underlying neuronal death in this disorder. Using mitochondrial quality control and the macroautophagy/lysosomal pathways as examples, we discuss the pipeline from a comprehensive genetic architecture for PD through to clinical trials for drugs targeting pathways linked to neurodegeneration in PD. We also identify opportunities and pitfalls on the road to a clinically effective disease-modifying treatment for this disease.
Assuntos
Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Animais , Biomarcadores/metabolismo , Ensaios Clínicos como Assunto , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Desenho de Fármacos , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Substância Negra/metabolismo , Substância Negra/fisiopatologiaRESUMO
TREM and TREM-like receptors are a structurally similar protein family encoded by genes clustered on chromosome 6p21.11. Recent studies have identified a rare coding variant (p.R47H) in TREM2 that confers a high risk for Alzheimer's disease (AD). In addition, common single nucleotide polymorphisms in this genomic region are associated with cerebrospinal fluid biomarkers for AD and a common intergenic variant found near the TREML2 gene has been identified to be protective for AD. However, little is known about the functional variant underlying the latter association or its relationship with the p.R47H. Here, we report comprehensive analyses using whole-exome sequencing data, cerebrospinal fluid biomarker analyses, meta-analyses (16,254 cases and 20,052 controls) and cell-based functional studies to support the role of the TREML2 coding missense variant p.S144G (rs3747742) as a potential driver of the meta-analysis AD-associated genome-wide association studies signal. Additionally, we demonstrate that the protective role of TREML2 in AD is independent of the role of TREM2 gene as a risk factor for AD.
Assuntos
Doença de Alzheimer/genética , Variação Genética/genética , Estudo de Associação Genômica Ampla , Mutação de Sentido Incorreto/genética , Receptores Imunológicos/genética , Doença de Alzheimer/prevenção & controle , Biomarcadores/líquido cefalorraquidiano , Cromossomos Humanos Par 6 , Humanos , Metanálise como Assunto , Polimorfismo de Nucleotídeo Único/genética , Receptores Imunológicos/fisiologia , RiscoRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinson's disease (PD). Although biochemical studies have shown that certain PD mutations confer elevated kinase activity in vitro on LRRK2, there are no methods available to directly monitor LRRK2 kinase activity in vivo. We demonstrate that LRRK2 autophosphorylation on Ser(1292) occurs in vivo and is enhanced by several familial PD mutations including N1437H, R1441G/C, G2019S, and I2020T. Combining two PD mutations together further increases Ser(1292) autophosphorylation. Mutation of Ser(1292) to alanine (S1292A) ameliorates the effects of LRRK2 PD mutations on neurite outgrowth in cultured rat embryonic primary neurons. Using cell-based and pharmacodynamic assays with phosphorylated Ser(1292) as the readout, we developed a brain-penetrating LRRK2 kinase inhibitor that blocks Ser(1292) autophosphorylation in vivo and attenuates the cellular consequences of LRRK2 PD mutations in vitro. These data suggest that Ser(1292) autophosphorylation may be a useful indicator of LRRK2 kinase activity in vivo and may contribute to the cellular effects of certain PD mutations.
Assuntos
Mutação/genética , Doença de Parkinson/enzimologia , Doença de Parkinson/patologia , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Sítios de Ligação , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Proteínas Mutantes/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Doença de Parkinson/genética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transporte Proteico/efeitos dos fármacos , RatosRESUMO
The ability to specifically upregulate genes in vivo holds great therapeutic promise. Here we show that inhibition or degradation of natural antisense transcripts (NATs) by single-stranded oligonucleotides or siRNAs can transiently and reversibly upregulate locus-specific gene expression. Brain-derived neurotrophic factor (BDNF) is normally repressed by a conserved noncoding antisense RNA transcript, BDNF-AS. Inhibition of this transcript upregulates BDNF mRNA by two- to sevenfold, alters chromatin marks at the BDNF locus, leads to increased protein levels and induces neuronal outgrowth and differentiation both in vitro and in vivo. We also show that inhibition of NATs leads to increases in glial-derived neurotrophic factor (GDNF) and ephrin receptor B2 (EPHB2) mRNA. Our data suggest that pharmacological approaches targeting NATs can confer locus-specific gene upregulation effects.
Assuntos
Oligonucleotídeos Antissenso/antagonistas & inibidores , Regulação para Cima , Animais , Linhagem Celular , Cromatina/química , Cromatina/metabolismo , Éxons , Perfilação da Expressão Gênica , Genômica , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Células HEK293 , Humanos , Camundongos , Modelos Genéticos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptor EphB2/biossíntese , Análise de Sequência de DNA , Transcrição GênicaRESUMO
Hyperphosphorylation of the microtubule binding protein Tau is a feature of a number of neurodegenerative diseases, including Alzheimer's disease. Tau is hyperphosphorylated in the hippocampus of dab1-null mice in a strain-dependent manner; however, it has not been clear if the Tau phosphorylation phenotype is a secondary effect of the morbidity of these mutants. The dab1 gene encodes a docking protein that is required for normal brain lamination and dendritogenesis as part of the Reelin signaling pathway. We show that dab1 gene inactivation after brain development leads to Tau hyperphosphorylation in anatomically normal mice. Genomic regions that regulate the phospho Tau phenotype in dab1 mutants have previously been identified. Using a microarray gene expression comparison between dab1-mutants from the high-phospho Tau expressing and low-phospho Tau expressing strains, we identified Stk25 as a differentially expressed modifier of dab1-mutant phenotypes. Stk25 knockdown reduces Tau phosphorylation in embryonic neurons. Furthermore, Stk25 regulates neuronal polarization and Golgi morphology in an antagonistic manner to Dab1. This work provides insights into the complex regulation of neuronal behavior during brain development and provides insights into the molecular cascades that regulate Tau phosphorylation.
Assuntos
Genes Modificadores/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas tau/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Células HeLa , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteína Reelina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Tirosina/metabolismo , Proteínas tau/genéticaRESUMO
Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances.
Assuntos
Encéfalo/citologia , Encéfalo/patologia , Mutação , Proteínas Serina-Treonina Quinases/genética , Transcriptoma/genética , Idoso , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Doença de Parkinson/genética , Doença de Parkinson/patologia , Plasmídeos/genéticaRESUMO
Progressive supranuclear palsy (PSP) is a movement disorder with prominent tau neuropathology. Brain diseases with abnormal tau deposits are called tauopathies, the most common of which is Alzheimer's disease. Environmental causes of tauopathies include repetitive head trauma associated with some sports. To identify common genetic variation contributing to risk for tauopathies, we carried out a genome-wide association study of 1,114 individuals with PSP (cases) and 3,247 controls (stage 1) followed by a second stage in which we genotyped 1,051 cases and 3,560 controls for the stage 1 SNPs that yielded P ≤ 10(-3). We found significant previously unidentified signals (P < 5 × 10(-8)) associated with PSP risk at STX6, EIF2AK3 and MOBP. We confirmed two independent variants in MAPT affecting risk for PSP, one of which influences MAPT brain expression. The genes implicated encode proteins for vesicle-membrane fusion at the Golgi-endosomal interface, for the endoplasmic reticulum unfolded protein response and for a myelin structural component.
Assuntos
Loci Gênicos , Predisposição Genética para Doença , Variação Genética , Estudo de Associação Genômica Ampla , Paralisia Supranuclear Progressiva/genética , Tauopatias/genética , Proteínas tau/genética , Estudos de Casos e Controles , Cromossomos Humanos/genética , Estudos de Coortes , Humanos , Polimorfismo de Nucleotídeo Único/genética , Prognóstico , Fatores de Risco , Paralisia Supranuclear Progressiva/patologia , Tauopatias/patologiaRESUMO
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
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
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 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
BACKGROUND: MicroRNAs (miRNAs) have the potential to regulate diverse sets of mRNA targets. In addition, mammalian genomes contain numerous natural antisense transcripts, most of which appear to be non-protein-coding RNAs (ncRNAs). We have recently identified and characterized a highly conserved non-coding antisense transcript for beta-secretase-1 (BACE1), a critical enzyme in Alzheimer's disease pathophysiology. The BACE1-antisense transcript is markedly up-regulated in brain samples from Alzheimer's disease patients and promotes the stability of the (sense) BACE1 transcript. RESULTS: We report here that BACE1-antisense prevents miRNA-induced repression of BACE1 mRNA by masking the binding site for miR-485-5p. Indeed, miR-485-5p and BACE1-antisense compete for binding within the same region in the open reading frame of the BACE1 mRNA. We observed opposing effects of BACE1-antisense and miR-485-5p on BACE1 protein in vitro and showed that Locked Nucleic Acid-antimiR mediated knockdown of miR-485-5p as well as BACE1-antisense over-expression can prevent the miRNA-induced BACE1 suppression. We found that the expression of BACE1-antisense as well as miR-485-5p are dysregulated in RNA samples from Alzheimer's disease subjects compared to control individuals. CONCLUSIONS: Our data demonstrate an interface between two distinct groups of regulatory RNAs in the computation of BACE1 gene expression. Moreover, bioinformatics analyses revealed a theoretical basis for many other potential interactions between natural antisense transcripts and miRNAs at the binding sites of the latter.
Assuntos
MicroRNAs/metabolismo , RNA Antissenso/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Sequência de Bases , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Linhagem Celular , Ensaios de Triagem em Larga Escala , Humanos , Luciferases/metabolismo , Camundongos , MicroRNAs/genética , Dados de Sequência Molecular , Oligonucleotídeos/farmacologia , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
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) 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.