RESUMO
Genomic instability and alterations in gene expression are hallmarks of eukaryotic aging. The yeast histone deacetylase Sir2 silences transcription and stabilizes repetitive DNA, but during aging or in response to a DNA break, the Sir complex relocalizes to sites of genomic instability, resulting in the desilencing of genes that cause sterility, a characteristic of yeast aging. Using embryonic stem cells, we show that mammalian Sir2, SIRT1, represses repetitive DNA and a functionally diverse set of genes across the mouse genome. In response to DNA damage, SIRT1 dissociates from these loci and relocalizes to DNA breaks to promote repair, resulting in transcriptional changes that parallel those in the aging mouse brain. Increased SIRT1 expression promotes survival in a mouse model of genomic instability and suppresses age-dependent transcriptional changes. Thus, DNA damage-induced redistribution of SIRT1 and other chromatin-modifying proteins may be a conserved mechanism of aging in eukaryotes.
Assuntos
Envelhecimento/genética , Cromatina/metabolismo , Instabilidade Genômica , Sirtuínas/genética , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Células-Tronco Embrionárias , Técnicas de Inativação de Genes , Humanos , Linfoma/metabolismo , Camundongos , Dados de Sequência Molecular , Estresse Oxidativo , Sirtuína 1 , Organismos Livres de Patógenos Específicos , Neoplasias do Timo/metabolismo , Leveduras/citologia , Leveduras/metabolismoRESUMO
We developed a procedure for the preparation of whole transcriptome cDNA libraries depleted of ribosomal RNA from only 1 microg of total RNA. The method relies on a collection of short, computationally selected oligonucleotides, called 'not-so-random' (NSR) primers, to obtain full-length, strand-specific representation of nonribosomal RNA transcripts. In this study we validated the technique by profiling human whole brain and universal human reference RNA using ultra-high-throughput sequencing.
Assuntos
Encéfalo/metabolismo , DNA Complementar/genética , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Clonagem Molecular , Humanos , RNA/genética , RNA/metabolismoRESUMO
The progressive aggregation of Amyloid-ß (Aß) in the brain is a major trait of Alzheimer's Disease (AD). Aß is produced as a result of proteolytic processing of the ß-amyloid precursor protein (APP). Processing of APP is mediated by multiple enzymes, resulting in the production of distinct peptide products: the non-amyloidogenic peptide sAPPα and the amyloidogenic peptides sAPPß, Aß40, and Aß42. Using a pathway-based approach, we analyzed a large-scale siRNA screen that measured the production of different APP proteolytic products. Our analysis identified many of the biological processes/pathways that are known to regulate APP processing and have been implicated in AD pathogenesis, as well as revealing novel regulatory mechanisms. Furthermore, we also demonstrate that some of these processes differentially regulate APP processing, with some mechanisms favouring production of certain peptide species over others. For example, synaptic transmission having a bias towards regulating Aß40 production over Aß42 as well as processes involved in insulin and pancreatic biology having a bias for sAPPß production over sAPPα. In addition, some of the pathways identified as regulators of APP processing contain genes (CLU, BIN1, CR1, PICALM, TREM2, SORL1, MEF2C, DSG2, EPH1A) recently implicated with AD through genome wide association studies (GWAS) and associated meta-analysis. In addition, we provide supporting evidence and a deeper mechanistic understanding of the role of diabetes in AD. The identification of these processes/pathways, their differential impact on APP processing, and their relationships to each other, provide a comprehensive systems biology view of the "regulatory landscape" of APP.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Técnicas Genéticas , Redes e Vias Metabólicas , RNA Interferente Pequeno/análise , Peptídeos beta-Amiloides/metabolismo , Sobrevivência Celular , Diabetes Mellitus Tipo 2/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Proteína Amiloide A Sérica/metabolismoRESUMO
Aging is accompanied by cognitive decline in a major segment of the population and is the primary risk factor for Alzheimer's disease and other prevalent neurodegenerative disorders. Despite this central role in disease pathogenesis and morbidity, the aging of the brain has not been well understood at a molecular level. This review seeks to integrate what is known about age-related cognitive and neuroanatomical changes with recent advances in understanding basic molecular mechanisms that underlie aging. An important issue is how normal brain aging transitions to pathological aging, giving rise to neurodegenerative disorders. Toxic protein aggregates have been identified as potential contributory factors, including amyloid beta-protein in Alzheimer's disease, tau in frontotemporal dementia, and alpha-synuclein in Parkinson's disease. However, current models of pathogenesis do not explain the origin of the common sporadic forms of these diseases or address the critical nexus between aging and disease. This review discusses potential approaches to unifying the systems biology of the aging brain with the pathogenesis of neurodegeneration.
Assuntos
Envelhecimento/fisiologia , Encéfalo/fisiologia , Animais , HumanosRESUMO
Alzheimer's disease and other neurodegenerative disorders of aging are characterized by clinical and pathological features that are relatively specific to humans. To obtain greater insight into how brain aging has evolved, we compared age-related gene expression changes in the cortex of humans, rhesus macaques, and mice on a genome-wide scale. A small subset of gene expression changes are conserved in all three species, including robust age-dependent upregulation of the neuroprotective gene apolipoprotein D (APOD) and downregulation of the synaptic cAMP signaling gene calcium/calmodulin-dependent protein kinase IV (CAMK4). However, analysis of gene ontology and cell type localization shows that humans and rhesus macaques have diverged from mice due to a dramatic increase in age-dependent repression of neuronal genes. Many of these age-regulated neuronal genes are associated with synaptic function. Notably, genes associated with GABA-ergic inhibitory function are robustly age-downregulated in humans but not in mice at the level of both mRNA and protein. Gene downregulation was not associated with overall neuronal or synaptic loss. Thus, repression of neuronal gene expression is a prominent and recently evolved feature of brain aging in humans and rhesus macaques that may alter neural networks and contribute to age-related cognitive changes.
Assuntos
Envelhecimento/genética , Encéfalo/metabolismo , Evolução Molecular , RNA Mensageiro/genética , Sinapses/fisiologia , Adulto , Idoso , Animais , Western Blotting , Humanos , Camundongos , Filogenia , Especificidade da Espécie , Transmissão Sináptica/genéticaRESUMO
Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.
Assuntos
Processamento Alternativo/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Anexina A7/genética , Éxons/genética , Humanos , Íntrons/genética , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Sítios de Splice de RNA/genética , Retinoblastoma/genéticaRESUMO
Alternative pre-messenger RNA (pre-mRNA) splicing plays important roles in development, physiology, and disease, and more than half of human genes are alternatively spliced. To understand the biological roles and regulation of alternative splicing across different tissues and stages of development, systematic methods are needed. Here, we demonstrate the use of microarrays to monitor splicing at every exon-exon junction in more than 10,000 multi-exon human genes in 52 tissues and cell lines. These genome-wide data provide experimental evidence and tissue distributions for thousands of known and novel alternative splicing events. Adding to previous studies, the results indicate that at least 74% of human multi-exon genes are alternatively spliced.