RESUMO
Odor perception in mammals is mediated by parallel sensory pathways that convey distinct information about the olfactory world. Multiple olfactory subsystems express characteristic seven-transmembrane G-protein-coupled receptors (GPCRs) in a one-receptor-per-neuron pattern that facilitates odor discrimination. Sensory neurons of the "necklace" subsystem are nestled within the recesses of the olfactory epithelium and detect diverse odorants; however, they do not express known GPCR odor receptors. Here, we report that members of the four-pass transmembrane MS4A protein family are chemosensors expressed within necklace sensory neurons. These receptors localize to sensory endings and confer responses to ethologically relevant ligands, including pheromones and fatty acids, in vitro and in vivo. Individual necklace neurons co-express many MS4A proteins and are activated by multiple MS4A ligands; this pooling of information suggests that the necklace is organized more like subsystems for taste than for smell. The MS4As therefore define a distinct mechanism and functional logic for mammalian olfaction.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Olfato , Animais , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Odorantes , Neurônios Receptores Olfatórios/metabolismo , FilogeniaRESUMO
The development of neural circuits relies on axon projections establishing diverse, yet well-defined, connections between areas of the nervous system. Each projection is formed by growth cones-subcellular specializations at the tips of growing axons, encompassing sets of molecules that control projection-specific growth, guidance, and target selection1. To investigate the set of molecules within native growth cones that form specific connections, here we developed growth cone sorting and subcellular RNA-proteome mapping, an approach that identifies and quantifies local transcriptomes and proteomes from labelled growth cones of single projections in vivo. Using this approach on the developing callosal projection of the mouse cerebral cortex, we mapped molecular enrichments in trans-hemispheric growth cones relative to their parent cell bodies, producing paired subcellular proteomes and transcriptomes from single neuron subtypes directly from the brain. These data provide generalizable proof-of-principle for this approach, and reveal molecular specializations of the growth cone, including accumulations of the growth-regulating kinase mTOR2, together with mRNAs that contain mTOR-dependent motifs3,4. These findings illuminate the relationships between subcellular distributions of RNA and protein in developing projection neurons, and provide a systems-level approach for the discovery of subtype- and stage-specific molecular substrates of circuit wiring, miswiring, and the potential for regeneration.
Assuntos
Axônios/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Proteoma/metabolismo , Transcriptoma/genética , Animais , Axônios/enzimologia , Processos de Crescimento Celular , Movimento Celular , Separação Celular , Feminino , Cones de Crescimento/enzimologia , Cones de Crescimento/metabolismo , Masculino , Camundongos , Proteoma/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Studies of the adult Drosophila midgut have led to many insights in our understanding of cell-type diversity, stem cell regeneration, tissue homeostasis, and cell fate decision. Advances in single-cell RNA sequencing provide opportunities to identify new cell types and molecular features. We used single-cell RNA sequencing to characterize the transcriptome of midgut epithelial cells and identified 22 distinct clusters representing intestinal stem cells, enteroblasts, enteroendocrine cells (EEs), and enterocytes. This unbiased approach recovered most of the known intestinal stem cells/enteroblast and EE markers, highlighting the high quality of the dataset, and led to insights on intestinal stem cell biology, cell type-specific organelle features, the roles of new transcription factors in progenitors and regional variation along the gut, 5 additional EE gut hormones, EE hormonal expression diversity, and paracrine function of EEs. To facilitate mining of this rich dataset, we provide a web-based resource for visualization of gene expression in single cells. Altogether, our study provides a comprehensive resource for addressing functions of genes in the midgut epithelium.
Assuntos
Sistema Digestório/metabolismo , Drosophila/metabolismo , Células-Tronco/metabolismo , Transcriptoma , Animais , Sistema Digestório/citologia , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/metabolismo , Enterócitos/metabolismo , Células Enteroendócrinas/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica , Hormônios/metabolismo , Intestinos/citologia , Células-Tronco/citologia , Fatores de Transcrição/metabolismoRESUMO
Lack of sensory hair cell (HC) regeneration in mammalian adults is a major contributor to hearing loss. In contrast, the neonatal mouse cochlea retains a transient capacity for regeneration, and forced Wnt activation in neonatal stages promotes supporting cell (SC) proliferation and induction of ectopic HCs. We currently know little about the temporal pattern and underlying mechanism of this age-dependent regenerative response. Using an in vitro model, we show that Wnt activation promotes SC proliferation following birth, but prior to postnatal day (P) 5. This age-dependent decline in proliferation occurs despite evidence that the Wnt pathway is postnatally active and can be further enhanced by Wnt stimulators. Using an in vivo mouse model and RNA sequencing, we show that proliferation in the early neonatal cochlea is correlated with a unique transcriptional response that diminishes with age. Furthermore, we find that augmenting Wnt signaling through the neonatal stages extends the window for HC induction in response to Notch signaling inhibition. Our results suggest that the downstream transcriptional response to Wnt activation, in part, underlies the regenerative capacity of the mammalian cochlea.
Assuntos
Cóclea/fisiologia , Mamíferos/fisiologia , Regeneração/genética , Transcrição Gênica , Via de Sinalização Wnt/genética , Animais , Animais Recém-Nascidos , Proliferação de Células , Transdiferenciação Celular , Embrião de Mamíferos/citologia , Epitélio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Células Labirínticas de Suporte/citologia , Células Labirínticas de Suporte/metabolismo , Masculino , Camundongos , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição TCF/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND AND AIMS: Activated hepatocytes are hypothesized to be a major source of signals that drive cirrhosis, but the biochemical pathways that convert hepatocytes into such a state are unclear. We examined the role of the Hippo pathway transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in hepatocytes to facilitate cell-cell interactions that stimulate liver inflammation and fibrosis. APPROACH AND RESULTS: Using a variety of genetic, metabolic, and liver injury models in mice, we manipulated Hippo signaling in hepatocytes and examined its effects in nonparenchymal cells to promote liver inflammation and fibrosis. YAP-expressing hepatocytes rapidly and potently activate the expression of proteins that promote fibrosis (collagen type I alpha 1 chain, tissue inhibitor of metalloproteinase 1, platelet-derived growth factor c, transforming growth factor ß2) and inflammation (tumor necrosis factor, interleukin 1ß). They stimulate expansion of myofibroblasts and immune cells, followed by aggressive liver fibrosis. In contrast, hepatocyte-specific YAP and YAP/TAZ knockouts exhibit limited myofibroblast expansion, less inflammation, and decreased fibrosis after CCl4 injury despite a similar degree of necrosis as controls. We identified cellular communication network factor 1 (CYR61) as a chemokine that is up-regulated by hepatocytes during liver injury but is expressed at significantly lower levels in mice with hepatocyte-specific deletion of YAP or TAZ. Gain-of-function and loss-of-function experiments with CYR61 in vivo point to it being a key chemokine controlling liver fibrosis and inflammation in the context of YAP/TAZ. There is a direct correlation between levels of YAP/TAZ and CYR61 in liver tissues of patients with high-grade nonalcoholic steatohepatitis. CONCLUSIONS: Liver injury in mice and humans increases levels of YAP/TAZ/CYR61 in hepatocytes, thus attracting macrophages to the liver to promote inflammation and fibrosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Hepatócitos/metabolismo , Cirrose Hepática/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Estresse Fisiológico , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Cadeia alfa 1 do Colágeno Tipo I , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Modelos Animais de Doenças , Mutação com Ganho de Função , Humanos , Cirrose Hepática/genética , Mutação com Perda de Função , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Transativadores/genética , Fatores de Transcrição/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAPRESUMO
Coordinated changes in gene expression underlie the early patterning and cell-type specification of the central nervous system. However, much less is known about how such changes contribute to later stages of circuit assembly and refinement. In this study, we employ single-cell RNA sequencing to develop a detailed, whole-transcriptome resource of gene expression across four time points in the developing dorsal lateral geniculate nucleus (LGN), a visual structure in the brain that undergoes a well-characterized program of postnatal circuit development. This approach identifies markers defining the major LGN cell types, including excitatory relay neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells. Most cell types exhibit significant transcriptional changes across development, dynamically expressing genes involved in distinct processes including retinotopic mapping, synaptogenesis, myelination, and synaptic refinement. Our data suggest that genes associated with synapse and circuit development are expressed in a larger proportion of nonneuronal cell types than previously appreciated. Furthermore, we used this single-cell expression atlas to identify the Prkcd-Cre mouse line as a tool for selective manipulation of relay neurons during a late stage of sensory-driven synaptic refinement. This transcriptomic resource provides a cellular map of gene expression across several cell types of the LGN, and offers insight into the molecular mechanisms of circuit development in the postnatal brain.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Corpos Geniculados/embriologia , Corpos Geniculados/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Transcriptoma , Animais , Axônios/fisiologia , Encéfalo/embriologia , Perfilação da Expressão Gênica , Camundongos , Microscopia Eletrônica de Varredura , Neurogênese , Retina/fisiologia , Análise de Sequência de RNA , Software , Vias Visuais/fisiologiaRESUMO
INTRODUCTION: Genome-wide association studies have led to numerous genetic loci associated with Alzheimer's disease (AD). Whole-genome sequencing (WGS) now permits genome-wide analyses to identify rare variants contributing to AD risk. METHODS: We performed single-variant and spatial clustering-based testing on rare variants (minor allele frequency [MAF] ≤1%) in a family-based WGS-based association study of 2247 subjects from 605 multiplex AD families, followed by replication in 1669 unrelated individuals. RESULTS: We identified 13 new AD candidate loci that yielded consistent rare-variant signals in discovery and replication cohorts (4 from single-variant, 9 from spatial-clustering), implicating these genes: FNBP1L, SEL1L, LINC00298, PRKCH, C15ORF41, C2CD3, KIF2A, APC, LHX9, NALCN, CTNNA2, SYTL3, and CLSTN2. DISCUSSION: Downstream analyses of these novel loci highlight synaptic function, in contrast to common AD-associated variants, which implicate innate immunity and amyloid processing. These loci have not been associated previously with AD, emphasizing the ability of WGS to identify AD-associated rare variants, particularly outside of the exome.
Assuntos
Doença de Alzheimer/genética , Frequência do Gene/genética , Predisposição Genética para Doença , Sequenciamento Completo do Genoma , Estudo de Associação Genômica Ampla , Humanos , Canais Iônicos/genética , Cinesinas/genética , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas/genéticaRESUMO
SOX5 encodes a transcription factor that is expressed in multiple tissues including heart, lung and brain. Mutations in SOX5 have been previously found in patients with amyotrophic lateral sclerosis (ALS) and developmental delay, intellectual disability and dysmorphic features. To characterize the neuronal role of SOX5, we silenced the Drosophila ortholog of SOX5, Sox102F, by RNAi in various neuronal subtypes in Drosophila. Silencing of Sox102F led to misorientated and disorganized michrochaetes, neurons with shorter dendritic arborization (DA) and reduced complexity, diminished larval peristaltic contractions, loss of neuromuscular junction bouton structures, impaired olfactory perception, and severe neurodegeneration in brain. Silencing of SOX5 in human SH-SY5Y neuroblastoma cells resulted in a significant repression of WNT signaling activity and altered expression of WNT-related genes. Genetic association and meta-analyses of the results in several large family-based and case-control late-onset familial Alzheimer's disease (LOAD) samples of SOX5 variants revealed several variants that show significant association with AD disease status. In addition, analysis for rare and highly penetrate functional variants revealed four novel variants/mutations in SOX5, which taken together with functional prediction analysis, suggests a strong role of SOX5 causing AD in the carrier families. Collectively, these findings indicate that SOX5 is a novel candidate gene for LOAD with an important role in neuronal function. The genetic findings warrant further studies to identify and characterize SOX5 variants that confer risk for AD, ALS and intellectual disability.
Assuntos
Doença de Alzheimer/genética , Esclerose Lateral Amiotrófica/genética , Deficiências do Desenvolvimento/genética , Proteínas de Drosophila/genética , Fatores de Transcrição SOXD/genética , Doença de Alzheimer/patologia , Esclerose Lateral Amiotrófica/patologia , Animais , Deficiências do Desenvolvimento/patologia , Drosophila/genética , Inativação Gênica , Estudos de Associação Genética , Humanos , Junção Neuromuscular/genética , Junção Neuromuscular/patologia , Plasticidade Neuronal/genética , Neurônios/metabolismo , Neurônios/patologia , Interferência de RNA , Via de Sinalização Wnt/genéticaRESUMO
Promoters initiate RNA synthesis, and enhancers stimulate promoter activity. Whether promoter and enhancer activities are encoded distinctly in DNA sequences is unknown. We measured the enhancer and promoter activities of thousands of DNA fragments transduced into mouse neurons. We focused on genomic loci bound by the neuronal activity-regulated coactivator CREBBP, and we measured enhancer and promoter activities both before and after neuronal activation. We find that the same sequences typically encode both enhancer and promoter activities. However, gene promoters generate more promoter activity than distal enhancers, despite generating similar enhancer activity. Surprisingly, the greater promoter activity of gene promoters is not due to conventional core promoter elements or splicing signals. Instead, we find that particular transcription factor binding motifs are intrinsically biased toward the generation of promoter activity, whereas others are not. Although the specific biases we observe may be dependent on experimental or cellular context, our results suggest that gene promoters are distinguished from distal enhancers by specific complements of transcriptional activators.
Assuntos
Proteína de Ligação a CREB/genética , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sítios de Ligação , Cromatina/genética , Proteínas de Ligação a DNA/genética , Camundongos , Neurônios/metabolismo , Ligação Proteica , Análise de Sequência de DNARESUMO
UNLABELLED: Antiretroviral therapy (ART) is successful in the suppression of HIV but cannot target and eradicate the latent proviral reservoir. The location of retroviral integration into the human genome is thought to play a role in the clonal expansion of infected cells and HIV persistence. We developed a high-throughput targeted sequence capture assay that uses a pool of HIV-specific probes to enrich Illumina libraries prior to deep sequencing. Using an expanded clonal population of ACH-2 cells, we demonstrate that this sequence capture assay has an extremely low false-positive rate. This assay assessed four cellular models commonly used to study HIV latency and latency-reversing agents: ACH-2 cells, J-Lat cells, the Bcl-2-transduced primary CD4(+)model, and the cultured TCM(central memory) CD4(+)model. HIV integration site characteristics and genes were compared between these cellular models and to previously reported patient data sets. Across these cellular models, there were significant differences in integration site characteristics, including orientation relative to that of the host gene, the proportion of clonally expanded sites, and the proportion located within genic regions and exons. Despite a greater diversity of minority integration sites than expected in ACH-2 cells, their integration site characteristics consistently differed from those of the other models and from the patient samples. Gene ontology analysis of highly represented genes from the patient samples found little overlap with HIV-containing genes from the cell lines. These findings show that integration site differences exist among the commonly used cellular models of HIV latency and in comparison to integration sites found in patient samples. IMPORTANCE: Despite the success of ART, currently there is no successful therapy to eradicate integrated proviruses. Cellular models of HIV latency are used to test the efficacy of latency-reversing agents, but it is unclear how well these models reflect HIV integration into the human genome in vivo We have developed a novel probe-based sequence enrichment assay to sequence and analyze integrated HIV. We compared HIV integration site characteristics between four cellular models and to previously described patient data sets. Significant differences were detected in the distribution of HIV integration sites between cellular models of HIV latency and compared to data sets from patient samples. The results from this study have implications for how well these cellular models of HIV infection truly reflect HIV integration in vivo and their applicability in drug discovery for novel latency-reversing agents.
Assuntos
Sondas de DNA , Infecções por HIV/genética , Infecções por HIV/virologia , HIV/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala , Integração Viral , Latência Viral , Linhagem Celular , Células Cultivadas , Mapeamento Cromossômico , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNARESUMO
Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function.
Assuntos
Quadruplex G , MicroRNAs/química , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Humanos , Mesoporfirinas/metabolismo , Camundongos , MicroRNAs/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Next-generation sequencing platforms for measuring digital expression such as RNA-Seq are displacing traditional microarray-based methods in biological experiments. The detection of differentially expressed genes between groups of biological conditions has led to the development of numerous bioinformatics tools, but so far, few exploit the expanded dynamic range afforded by the new technologies. We present edgeRun, an R package that implements an unconditional exact test that is a more powerful version of the exact test in edgeR. This increase in power is especially pronounced for experiments with as few as two replicates per condition, for genes with low total expression and with large biological coefficient of variation. In comparison with a panel of other tools, edgeRun consistently captures functionally similar differentially expressed genes.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias da Próstata/genética , RNA Neoplásico/análise , Análise de Sequência de RNA/métodos , Software , Biologia Computacional/métodos , Bases de Dados Genéticas , Humanos , Masculino , RNA Neoplásico/genéticaRESUMO
Modern DNA sequencing technologies enable geneticists to rapidly identify genetic variation among many human genomes. However, isolating the minority of variants underlying disease remains an important, yet formidable challenge for medical genetics. We have developed GEMINI (GEnome MINIng), a flexible software package for exploring all forms of human genetic variation. Unlike existing tools, GEMINI integrates genetic variation with a diverse and adaptable set of genome annotations (e.g., dbSNP, ENCODE, UCSC, ClinVar, KEGG) into a unified database to facilitate interpretation and data exploration. Whereas other methods provide an inflexible set of variant filters or prioritization methods, GEMINI allows researchers to compose complex queries based on sample genotypes, inheritance patterns, and both pre-installed and custom genome annotations. GEMINI also provides methods for ad hoc queries and data exploration, a simple programming interface for custom analyses that leverage the underlying database, and both command line and graphical tools for common analyses. We demonstrate GEMINI's utility for exploring variation in personal genomes and family based genetic studies, and illustrate its ability to scale to studies involving thousands of human samples. GEMINI is designed for reproducibility and flexibility and our goal is to provide researchers with a standard framework for medical genomics.
Assuntos
Bases de Dados Genéticas , Variação Genética , Genoma Humano , Genômica/métodos , Software , Mineração de Dados , Genótipo , HumanosRESUMO
Chronic liver injury causes fibrosis, characterized by the formation of scar tissue resulting from excessive accumulation of extracellular matrix (ECM) proteins. Hepatic stellate cell (HSC) myofibroblasts are the primary cell type responsible for liver fibrosis, yet there are currently no therapies directed at inhibiting the activity of HSC myofibroblasts. To search for potential anti-fibrotic compounds, we performed a high-throughput compound screen in primary human HSC myofibroblasts and identified 19 small molecules that induce HSC inactivation, including the polyether ionophore nanchangmycin (NCMC). NCMC induces lipid re-accumulation while reducing collagen expression, deposition of collagen in the extracellular matrix, cell proliferation, and migration. We find that NCMC increases cytosolic Ca2+ and reduces the phosphorylated protein levels of FYN, PTK2 (FAK), MAPK1/3 (ERK2/1), HSPB1 (HSP27), and STAT5B. Further, depletion of each of these kinases suppress COL1A1 expression. These studies reveal a signaling network triggered by NCMC to inactivate HSC myofibroblasts and reduce expression of proteins that compose the fibrotic scar. Identification of the antifibrotic effects of NCMC and the elucidation of pathways by which NCMC inhibits fibrosis provide new tools and therapeutic targets that could potentially be utilized to combat the development and progression of liver fibrosis.
Assuntos
Cicatriz , Células Estreladas do Fígado , Cicatriz/patologia , Colágeno/metabolismo , Éteres , Proteínas da Matriz Extracelular/metabolismo , Fibrose , Quinase 1 de Adesão Focal/metabolismo , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Compostos de EspiroRESUMO
The efficiency of cleavage of individual CRISPR/Cas9-sgRNAs remains difficult to predict based on the CRISPR target sequence alone. Different intracellular environments (dependent on cell type or cell cycle state for example) may affect sgRNA efficiency by altering accessibility of genomic DNA through DNA modifications such as epigenetic marks and DNA-binding proteins (e.g., histones) as well as alteration of the chromatin state of genomic DNA within the nucleus. We recently reported a multi-step screening method for the identification of efficient sgRNAs targeting the Herpes simplex virus (HSV-1) genome and reported a differential mechanism for viral inhibition by CRISPR-Cas9 in the latent versus lytic phase. The screening platform detailed in this protocol allows step-by-step testing of the efficiency of cleavage in a cell-free system and in the context of viral target cells such as human foreskin fibroblasts followed by functional testing of the effects of CRISPR/sgRNA on viral protein expression, replication, and reactivation. This strategy could be readily applied to other target cells such as pluripotent stem cell-derived human sensory neurons or other human DNA viruses.
RESUMO
INTRODUCTION: Genome-wide association studies have led to numerous genetic loci associated with Alzheimer's disease (AD). Whole-genome sequencing (WGS) now permit genome-wide analyses to identify rare variants contributing to AD risk. METHODS: We performed single-variant and spatial clustering-based testing on rare variants (minor allele frequency ≤1%) in a family-based WGS-based association study of 2,247 subjects from 605 multiplex AD families, followed by replication in 1,669 unrelated individuals. RESULTS: We identified 13 new AD candidate loci that yielded consistent rare-variant signals in discovery and replication cohorts (4 from single-variant, 9 from spatial-clustering), implicating these genes: FNBP1L, SEL1L, LINC00298, PRKCH, C15ORF41, C2CD3, KIF2A, APC, LHX9, NALCN, CTNNA2, SYTL3, CLSTN2. DISCUSSION: Downstream analyses of these novel loci highlight synaptic function, in contrast to common AD-associated variants, which implicate innate immunity. These loci have not been previously associated with AD, emphasizing the ability of WGS to identify AD-associated rare variants, particularly outside of coding regions.
RESUMO
With the advent of whole genome-sequencing (WGS) studies, family-based designs enable sex-specific analysis approaches that can be applied to only affected individuals; tests using family-based designs are attractive because they are completely robust against the effects of population substructure. These advantages make family-based association tests (FBATs) that use siblings as well as parents especially suited for the analysis of late-onset diseases such as Alzheimer's Disease (AD). However, the application of FBATs to assess sex-specific effects can require additional filtering steps, as sensitivity to sequencing errors is amplified in this type of analysis. Here, we illustrate the implementation of robust analysis approaches and additional filtering steps that can minimize the chances of false positive-findings due to sex-specific sequencing errors. We apply this approach to two family-based AD datasets and identify four novel loci (GRID1, RIOK3, MCPH1, ZBTB7C) showing sex-specific association with AD risk. Following stringent quality control filtering, the strongest candidate is ZBTB7C (Pinter = 1.83 × 10-7), in which the minor allele of rs1944572 confers increased risk for AD in females and protection in males. ZBTB7C encodes the Zinc Finger and BTB Domain Containing 7C, a transcriptional repressor of membrane metalloproteases (MMP). Members of this MMP family were implicated in AD neuropathology.
Assuntos
Doença de Alzheimer/genética , Análise de Dados , Bases de Dados Genéticas , Família , Estudos de Associação Genética , Loci Gênicos/genética , Estudo de Associação Genômica Ampla , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sequenciamento Completo do Genoma , Alelos , Domínio BTB-POZ/genética , Feminino , Humanos , Masculino , Metaloproteases/genética , Metaloproteases/metabolismo , Risco , Fatores Sexuais , Dedos de Zinco/genéticaRESUMO
Recent genome-wide association studies identified the angiotensin-converting enzyme gene (ACE) as an Alzheimer's disease (AD) risk locus. However, the pathogenic mechanism by which ACE causes AD is unknown. Using whole-genome sequencing, we identified rare ACE coding variants in AD families and investigated one, ACE1 R1279Q, in knockin (KI) mice. Similar to AD, ACE1 was increased in neurons, but not microglia or astrocytes, of KI brains, which became elevated further with age. Angiotensin II (angII) and angII receptor AT1R signaling were also increased in KI brains. Autosomal dominant neurodegeneration and neuroinflammation occurred with aging in KI hippocampus, which were absent in the cortex and cerebellum. Female KI mice exhibited greater hippocampal electroencephalograph disruption and memory impairment compared to males. ACE variant effects were more pronounced in female KI mice, suggesting a mechanism for higher AD risk in women. Hippocampal neurodegeneration was completely rescued by treatment with brain-penetrant drugs that inhibit ACE1 and AT1R. Although ACE variant-induced neurodegeneration did not depend on ß-amyloid (Aß) pathology, amyloidosis in 5XFAD mice crossed to KI mice accelerated neurodegeneration and neuroinflammation, whereas Aß deposition was unchanged. KI mice had normal blood pressure and cerebrovascular functions. Our findings strongly suggest that increased ACE1/angII signaling causes aging-dependent, Aß-accelerated selective hippocampal neuron vulnerability and female susceptibility, hallmarks of AD that have hitherto been enigmatic. We conclude that repurposed brain-penetrant ACE inhibitors and AT1R blockers may protect against AD.
Assuntos
Doença de Alzheimer , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Estudo de Associação Genômica Ampla , Masculino , Camundongos , Camundongos TransgênicosRESUMO
Herpes simplex virus (HSV) establishes lifelong latent infection and can cause serious human disease, but current antiviral therapies target lytic but not latent infection. We screened for sgRNAs that cleave HSV-1 DNA sequences efficiently in vitro and used these sgRNAs to observe the first editing of quiescent HSV-1 DNA. The sgRNAs targeted lytic replicating viral DNA genomes more efficiently than quiescent genomes, consistent with the open structure of lytic chromatin. Editing of latent genomes caused short indels while editing of replicating genomes produced indels, linear molecules, and large genomic sequence loss around the gRNA target site. The HSV ICP0 protein and viral DNA replication increased the loss of DNA sequences around the gRNA target site. We conclude that HSV, by promoting open chromatin needed for viral gene expression and by inhibiting the DNA damage response, makes the genome vulnerable to a novel form of editing by CRISPR-Cas9 during lytic replication.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes , Genes Virais , Herpesviridae/genética , Animais , Sequência de Bases , Linhagem Celular , Reparo do DNA/genética , DNA Viral/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/genética , Humanos , Modelos Genéticos , Mutagênese/genética , RNA Guia de Cinetoplastídeos/genética , Replicação Viral/genéticaRESUMO
The findings that amyotrophic lateral sclerosis (ALS) patients almost universally display pathological mislocalization of the RNA-binding protein TDP-43 and that mutations in its gene cause familial ALS have nominated altered RNA metabolism as a disease mechanism. However, the RNAs regulated by TDP-43 in motor neurons and their connection to neuropathy remain to be identified. Here we report transcripts whose abundances in human motor neurons are sensitive to TDP-43 depletion. Notably, expression of STMN2, which encodes a microtubule regulator, declined after TDP-43 knockdown and TDP-43 mislocalization as well as in patient-specific motor neurons and postmortem patient spinal cord. STMN2 loss upon reduced TDP-43 function was due to altered splicing, which is functionally important, as we show STMN2 is necessary for normal axonal outgrowth and regeneration. Notably, post-translational stabilization of STMN2 rescued neurite outgrowth and axon regeneration deficits induced by TDP-43 depletion. We propose that restoring STMN2 expression warrants examination as a therapeutic strategy for ALS.