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Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.
Assuntos
Miocárdio/metabolismo , Biossíntese de Proteínas , Adolescente , Adulto , Idoso , Animais , Códon/genética , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fases de Leitura Aberta/genética , RNA Circular/genética , RNA Circular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ribossomos/genética , Ribossomos/metabolismo , Adulto JovemRESUMO
In systemic lupus erythematosus (SLE) loss of immune tolerance, autoantibody production and immune complex deposition are required but not sufficient for organ damage1. How inflammatory signals are initiated and amplified in the setting of autoimmunity remains elusive. Here, we set out to dissect layers and hierarchies of autoimmune kidney inflammation in order to identify tissue-specific cellular hubs that amplify auto-inflammatory responses. Using high-resolution single-cell profiling of kidney immune and parenchymal cells, in combination with antibody blocking and genetic deficiency, we show that tissue-resident NKp46+ innate lymphoid cells (ILC) are crucial signal amplifiers of disease-associated macrophage expansion and epithelial cell injury in lupus nephritis, downstream of autoantibody production. NKp46 signaling in a distinct subset of ILC1 instructed an unconventional immune-regulatory transcriptional program, which included the expression of the myeloid cell growth factor CSF2. CSF2 production by NKp46+ ILC promoted the population expansion of monocyte-derived macrophages. Blockade of the NKp46 receptor (using the antibody mNCR1.152) or genetic deficiency of NKp46 abrogated epithelial cell injury. The same cellular and molecular patterns were operative in human lupus nephritis. Our data support that NKp46+ ILC1 promote parenchymal cell injury by granting monocyte-derived macrophages access to epithelial cell niches. NKp46 activation in ILC1 thus constitutes a previously unrecognized, critical tissue rheostat that amplifies organ damage in autoimmune hosts, with broad implications for inflammatory pathologies and therapies.
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Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.
Assuntos
Miocárdio/citologia , Análise de Célula Única , Transcriptoma , Adipócitos/classificação , Adipócitos/metabolismo , Adulto , Enzima de Conversão de Angiotensina 2/análise , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Células Epiteliais/classificação , Células Epiteliais/metabolismo , Epitélio , Feminino , Fibroblastos/classificação , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Átrios do Coração/anatomia & histologia , Átrios do Coração/citologia , Átrios do Coração/inervação , Ventrículos do Coração/anatomia & histologia , Ventrículos do Coração/citologia , Ventrículos do Coração/inervação , Homeostase/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Miócitos Cardíacos/classificação , Miócitos Cardíacos/metabolismo , Neurônios/classificação , Neurônios/metabolismo , Pericitos/classificação , Pericitos/metabolismo , Receptores de Coronavírus/análise , Receptores de Coronavírus/genética , Receptores de Coronavírus/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Células Estromais/classificação , Células Estromais/metabolismoRESUMO
The existence of naturally occurring ribosome heterogeneity is now a well-acknowledged phenomenon. However, whether this heterogeneity leads to functionally diverse 'specialized ribosomes' is still a controversial topic. Here, we explore the biological function of RPL3L (uL3L), a ribosomal protein (RP) paralogue of RPL3 (uL3) that is exclusively expressed in skeletal muscle and heart tissues, by generating a viable homozygous Rpl3l knockout mouse strain. We identify a rescue mechanism in which, upon RPL3L depletion, RPL3 becomes up-regulated, yielding RPL3-containing ribosomes instead of RPL3L-containing ribosomes that are typically found in cardiomyocytes. Using both ribosome profiling (Ribo-seq) and a novel orthogonal approach consisting of ribosome pulldown coupled to nanopore sequencing (Nano-TRAP), we find that RPL3L modulates neither translational efficiency nor ribosome affinity towards a specific subset of transcripts. In contrast, we show that depletion of RPL3L leads to increased ribosome-mitochondria interactions in cardiomyocytes, which is accompanied by a significant increase in ATP levels, potentially as a result of fine-tuning of mitochondrial activity. Our results demonstrate that the existence of tissue-specific RP paralogues does not necessarily lead to enhanced translation of specific transcripts or modulation of translational output. Instead, we reveal a complex cellular scenario in which RPL3L modulates the expression of RPL3, which in turn affects ribosomal subcellular localization and, ultimately, mitochondrial activity.
Ribosomes are macromolecular machines responsible for protein synthesis in all living beings. Recent studies have shown that ribosomes can be heterogeneous in their structure, possibly leading to a specialized function. Here, we focus on RPL3L, a ribosomal protein expressed exclusively in striated muscles. We find that the deletion of the Rpl3l gene in a mouse model triggers a compensation mechanism, in which the missing RPL3L protein is replaced by its paralogue, RPL3. Furthermore, we find that RPL3-containing ribosomes establish closer interactions with mitochondria, cellular organelles responsible for energy production, leading to higher energy production when compared with RPL3L-containing ribosomes. Finally, we show that the RPL3RPL3L compensation mechanism is also triggered in heart disease conditions, such as hypertrophy and myocardial infarction.
Assuntos
Coração , Mitocôndrias , Proteínas Ribossômicas , Ribossomos , Animais , Camundongos , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Myocardial fibrosis is a key pathologic feature of hypertrophic cardiomyopathy (HCM). However, the fibrotic pathways activated by HCM-causing sarcomere protein gene mutations are poorly defined. Because lysophosphatidic acid is a mediator of fibrosis in multiple organs and diseases, we tested the role of the lysophosphatidic acid pathway in HCM. Lysphosphatidic acid receptor 1 (LPAR1), a cell surface receptor, is required for lysophosphatidic acid mediation of fibrosis. We bred HCM mice carrying a pathogenic myosin heavy-chain variant (403+/-) with Lpar1-ablated mice to create mice carrying both genetic changes (403+/- LPAR1 -/-) and assessed development of cardiac hypertrophy and fibrosis. Compared with 403+/- LPAR1WT, 403+/- LPAR1 -/- mice developed significantly less hypertrophy and fibrosis. Single-nucleus RNA sequencing of left ventricular tissue demonstrated that Lpar1 was predominantly expressed by lymphatic endothelial cells (LECs) and cardiac fibroblasts. Lpar1 ablation reduced the population of LECs, confirmed by immunofluorescence staining of the LEC markers Lyve1 and Ccl21a and, by in situ hybridization, for Reln and Ccl21a. Lpar1 ablation also altered the distribution of fibroblast cell states. FB1 and FB2 fibroblasts decreased while FB0 and FB3 fibroblasts increased. Our findings indicate that Lpar1 is expressed predominantly by LECs and fibroblasts in the heart and is required for development of hypertrophy and fibrosis in an HCM mouse model. LPAR1 antagonism, including agents in clinical trials for other fibrotic diseases, may be beneficial for HCM.
Assuntos
Cardiomiopatia Hipertrófica , Receptores de Ácidos Lisofosfatídicos/genética , Animais , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Proteínas de Transporte , Modelos Animais de Doenças , Células Endoteliais/patologia , Fibrose , Hipertrofia/patologia , CamundongosRESUMO
The relative contribution of the effector molecules produced by T cells to tumour rejection is unclear, but interferon-γ (IFNγ) is critical in most of the analysed models. Although IFNγ can impede tumour growth by acting directly on cancer cells, it must also act on the tumour stroma for effective rejection of large, established tumours. However, which stroma cells respond to IFNγ and by which mechanism IFNγ contributes to tumour rejection through stromal targeting have remained unknown. Here we use a model of IFNγ induction and an IFNγ-GFP fusion protein in large, vascularized tumours growing in mice that express the IFNγ receptor exclusively in defined cell types. Responsiveness to IFNγ by myeloid cells and other haematopoietic cells, including T cells or fibroblasts, was not sufficient for IFNγ-induced tumour regression, whereas responsiveness of endothelial cells to IFNγ was necessary and sufficient. Intravital microscopy revealed IFNγ-induced regression of the tumour vasculature, resulting in arrest of blood flow and subsequent collapse of tumours, similar to non-haemorrhagic necrosis in ischaemia and unlike haemorrhagic necrosis induced by tumour necrosis factor. The early events of IFNγ-induced tumour ischaemia resemble non-apoptotic blood vessel regression during development, wound healing or IFNγ-mediated, pregnancy-induced remodelling of uterine arteries. A better mechanistic understanding of how solid tumours are rejected may aid the design of more effective protocols for adoptive T-cell therapy.
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Vasos Sanguíneos/crescimento & desenvolvimento , Hipóxia Celular/imunologia , Interferon gama/imunologia , Isquemia/imunologia , Neoplasias/irrigação sanguínea , Neoplasias/imunologia , Remodelação Vascular , Animais , Vasos Sanguíneos/imunologia , Vasos Sanguíneos/metabolismo , Linhagem Celular Tumoral , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Interferon gama/biossíntese , Microscopia Intravital , Isquemia/metabolismo , Isquemia/patologia , Masculino , Camundongos , Necrose , Neoplasias/metabolismo , Neoplasias/patologia , Receptores de Interferon/metabolismo , Células Estromais/imunologia , Células Estromais/metabolismo , Especificidade por Substrato , Cicatrização , Receptor de Interferon gamaRESUMO
Fibrosis is a common pathology in cardiovascular disease. In the heart, fibrosis causes mechanical and electrical dysfunction and in the kidney, it predicts the onset of renal failure. Transforming growth factor ß1 (TGFß1) is the principal pro-fibrotic factor, but its inhibition is associated with side effects due to its pleiotropic roles. We hypothesized that downstream effectors of TGFß1 in fibroblasts could be attractive therapeutic targets and lack upstream toxicity. Here we show, using integrated imaging-genomics analyses of primary human fibroblasts, that upregulation of interleukin-11 (IL-11) is the dominant transcriptional response to TGFß1 exposure and required for its pro-fibrotic effect. IL-11 and its receptor (IL11RA) are expressed specifically in fibroblasts, in which they drive non-canonical, ERK-dependent autocrine signalling that is required for fibrogenic protein synthesis. In mice, fibroblast-specific Il11 transgene expression or Il-11 injection causes heart and kidney fibrosis and organ failure, whereas genetic deletion of Il11ra1 protects against disease. Therefore, inhibition of IL-11 prevents fibroblast activation across organs and species in response to a range of important pro-fibrotic stimuli. These results reveal a central role of IL-11 in fibrosis and we propose that inhibition of IL-11 is a potential therapeutic strategy to treat fibrotic diseases.
Assuntos
Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patologia , Fibrose/metabolismo , Fibrose/patologia , Interleucina-11/metabolismo , Animais , Comunicação Autócrina , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/induzido quimicamente , Coração , Humanos , Interleucina-11/antagonistas & inibidores , Interleucina-11/genética , Subunidade alfa de Receptor de Interleucina-11/deficiência , Subunidade alfa de Receptor de Interleucina-11/genética , Rim/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Miocárdio/metabolismo , Miocárdio/patologia , Escores de Disfunção Orgânica , Biossíntese de Proteínas , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Transgenes/genéticaRESUMO
Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model.
Assuntos
Cardiomiopatias/genética , Edição de Genes , Mutação de Sentido Incorreto , Proteínas Quinases/genética , Fatores Etários , Animais , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Conectina/metabolismo , Predisposição Genética para Doença , Heterozigoto , Homozigoto , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/metabolismo , Proteólise , Proteoma , Transcriptoma , Função Ventricular EsquerdaRESUMO
Persistent NF-κB activation is a hallmark of the malignant Hodgkin/Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL). Genomic lesions, Epstein-Barr virus infection, soluble factors, and tumor-microenvironment interactions contribute to this activation. Here, in an unbiased approach to identify the cHL cell-secreted key factors for NF-κB activation, we have dissected the secretome of cultured cHL cells by chromatography and subsequent mass spectrometry. We identified lymphotoxin-α (LTA) as the causative factor for autocrine and paracrine activation of canonical and noncanonical NF-κB in cHL cell lines. In addition to inducing NF-κB, LTA promotes JAK2/STAT6 signaling. LTA and its receptor TNFRSF14 are transcriptionally activated by noncanonical NF-κB, creating a continuous feedback loop. Furthermore, LTA shapes the expression of cytokines, receptors, immune checkpoint ligands and adhesion molecules, including CSF2, CD40, PD-L1/PD-L2, and VCAM1. Comparison with single-cell gene-activity profiles of human hematopoietic cells showed that LTA induces genes restricted to the lymphoid lineage, as well as those largely restricted to the myeloid lineage. Thus, LTA sustains autocrine NF-κB activation, impacts activation of several signaling pathways, and drives expression of genes essential for microenvironmental interactions and lineage ambiguity. These data provide a robust rationale for targeting LTA as a treatment strategy for cHL patients.
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Doença de Hodgkin/imunologia , Janus Quinase 2/imunologia , Linfotoxina-alfa/imunologia , NF-kappa B/imunologia , Fator de Transcrição STAT6/imunologia , Linhagem Celular , Regulação Neoplásica da Expressão Gênica , Doença de Hodgkin/genética , Humanos , Linfotoxina-alfa/genética , Células de Reed-Sternberg/imunologia , Células de Reed-Sternberg/metabolismo , Transdução de Sinais , Ativação TranscricionalRESUMO
BACKGROUND: Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global posttranscriptional mechanisms underlying fibroblast-to-myofibroblast conversion in the heart have not been explored. METHODS: Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. The integration with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients demonstrates that these posttranscriptional mechanisms are also active in the diseased fibrotic human heart. RESULTS: We generated nucleotide-resolution translatome data during the transforming growth factor ß1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation, and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in posttranscriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested the same posttranscriptional regulatory network was underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as Pumilio RNA binding family member 2 (PUM2) and Quaking (QKI) that work in concert to regulate the translation of target transcripts in human diseased hearts. Furthermore, silencing of both PUM2 and QKI inhibits the transition of fibroblasts toward profibrotic myofibroblasts in response to transforming growth factor ß1. CONCLUSIONS: We reveal widespread translational effects of transforming growth factor ß1 and define novel posttranscriptional regulatory networks that control the fibroblast-to-myofibroblast transition. These networks are active in human heart disease, and silencing of hub genes limits fibroblast activation. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure.
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Cardiopatias/genética , Cardiopatias/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Biossíntese de Proteínas/genética , Proteínas de Ligação a RNA/genética , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Perfilação da Expressão Gênica/métodos , Cardiopatias/patologia , Humanos , Análise de Sequência de RNA/métodos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Histone modifications are epigenetic marks that play fundamental roles in many biological processes including the control of chromatin-mediated regulation of gene expression. Little is known about interindividual variability of histone modification levels across the genome and to what extent they are influenced by genetic variation. We annotated the rat genome with histone modification maps, identified differences in histone trimethyl-lysine levels among strains, and described their underlying genetic basis at the genome-wide scale using ChIP-seq in heart and liver tissues in a panel of rat recombinant inbred and their progenitor strains. We identified extensive variation of histone methylation levels among individuals and mapped hundreds of underlying cis- and trans-acting loci throughout the genome that regulate histone methylation levels in an allele-specific manner. Interestingly, most histone methylation level variation was trans-linked and the most prominent QTL identified influenced H3K4me3 levels at 899 putative promoters throughout the genome in the heart. Cis- acting variation was enriched in binding sites of distinct transcription factors in heart and liver. The integrated analysis of DNA variation together with histone methylation and gene expression levels showed that histoneQTLs are an important predictor of gene expression and that a joint analysis significantly enhanced the prediction of gene expression traits (eQTLs). Our data suggest that genetic variation has a widespread impact on histone trimethylation marks that may help to uncover novel genotype-phenotype relationships.
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Epigênese Genética , Variação Genética , Genoma , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Histonas/genética , Fígado/metabolismo , Masculino , Metilação , Miocárdio/metabolismo , Regiões Promotoras Genéticas , Locos de Características Quantitativas , Ratos , Ratos Endogâmicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In much animal research, genetic variation is rather avoided than used as a powerful tool to identify key regulatory genes in complex phenotypes. Adult hippocampal neurogenesis is one such highly complex polygenic trait, for which the understanding of the molecular basis is fragmented and incomplete, and for which novel genetic approaches are needed. In this study, we aimed at marrying the power of the BXD panel, a mouse genetic reference population, with the flexibility of a cell culture model of adult neural precursor proliferation and differentiation. We established adult-derived hippocampal precursor cell cultures from 20 strains of the BXD panel, including the parental strains C57BL/6J and DBA/2J. The rates of cell proliferation and neuronal differentiation were measured, and transcriptional profiles were obtained from proliferating cultures. Together with the published genotypes of all lines, these data allowed a novel systems genetics analysis combining quantitative trait locus analysis with transcript expression correlation at a cellular level to identify genes linked with the differences in proliferation. In a proof-of-principle analysis, we identified Lrp6, the gene encoding the coreceptor to Frizzled in the Wnt pathway, as a potential negative regulator of precursor proliferation. Overexpression and siRNA silencing confirmed the regulatory role of Lrp6. As well as adding to our knowledge of the pathway surrounding Wnt in adult hippocampal neurogenesis, this finding allows the new appreciation of a negative regulator within this system. In addition, the resource and associated methodology will allow the integration of regulatory mechanisms at a systems level.
Assuntos
Diferenciação Celular/genética , Hipocampo/citologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/biossíntese , Neurogênese/genética , Neurônios/citologia , Animais , Técnicas de Cultura de Células , Proliferação de Células/genética , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Neurônios/metabolismo , RNA Interferente Pequeno/genética , Via de Sinalização Wnt/genéticaRESUMO
Infection of newborn rats with Borne disease virus (BDV) results in selective degeneration of granule cell neurons of the dentate gyrus (DG). To study cellular countermechanisms that might prevent this pathology, we screened for rat strains resistant to this BDV-induced neuronal degeneration. To this end, we infected hippocampal slice cultures of different rat strains with BDV and analyzed for the preservation of the DG. Whereas infected cultures of five rat strains, including Lewis (LEW) rats, exhibited a disrupted DG cytoarchitecture, slices of three other rat strains, including Sprague-Dawley (SD), were unaffected. However, efficiency of viral replication was comparable in susceptible and resistant cultures. Moreover, these rat strain-dependent differences in vulnerability were replicated in vivo in neonatally infected LEW and SD rats. Intriguingly, conditioned media from uninfected cultures of both LEW and SD rats could prevent BDV-induced DG damage in infected LEW hippocampal cultures, whereas infection with BDV suppressed the availability of these factors from LEW but not in SD hippocampal cultures. To gain further insights into the genetic basis for this rat strain-dependent susceptibility, we analyzed DG granule cell survival in BDV-infected cultures of hippocampal neurons derived from the F1 and F2 offspring of the crossing of SD and LEW rats. Genome-wide association analysis revealed one resistance locus on chromosome (chr) 6q16 in SD rats and, surprisingly, a locus on chr3q21-23 that was associated with susceptibility. Thus, BDV-induced neuronal degeneration is dependent on the host genetic background and is prevented by soluble protective factors in the disease-resistant SD rat strain.
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Vírus da Doença de Borna/fisiologia , Giro Denteado/virologia , Degeneração Neural/virologia , Neurônios/virologia , Animais , Animais Recém-Nascidos , Fatores Biológicos/química , Fatores Biológicos/farmacologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Mapeamento Cromossômico , Cromossomos de Mamíferos/genética , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Giro Denteado/metabolismo , Giro Denteado/patologia , Resistência à Doença/genética , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/virologia , Interações Hospedeiro-Patógeno , Masculino , Degeneração Neural/genética , Degeneração Neural/prevenção & controle , Neurônios/metabolismo , Neurônios/patologia , Polimorfismo de Nucleotídeo Único , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Solubilidade , Especificidade da Espécie , Técnicas de Cultura de TecidosRESUMO
The spontaneously hypertensive rat (SHR) is the most widely studied animal model of hypertension. Scores of SHR quantitative loci (QTLs) have been mapped for hypertension and other phenotypes. We have sequenced the SHR/OlaIpcv genome at 10.7-fold coverage by paired-end sequencing on the Illumina platform. We identified 3.6 million high-quality single nucleotide polymorphisms (SNPs) between the SHR/OlaIpcv and Brown Norway (BN) reference genome, with a high rate of validation (sensitivity 96.3%-98.0% and specificity 99%-100%). We also identified 343,243 short indels between the SHR/OlaIpcv and reference genomes. These SNPs and indels resulted in 161 gain or loss of stop codons and 629 frameshifts compared with the BN reference sequence. We also identified 13,438 larger deletions that result in complete or partial absence of 107 genes in the SHR/OlaIpcv genome compared with the BN reference and 588 copy number variants (CNVs) that overlap with the gene regions of 688 genes. Genomic regions containing genes whose expression had been previously mapped as cis-regulated expression quantitative trait loci (eQTLs) were significantly enriched with SNPs, short indels, and larger deletions, suggesting that some of these variants have functional effects on gene expression. Genes that were affected by major alterations in their coding sequence were highly enriched for genes related to ion transport, transport, and plasma membrane localization, providing insights into the likely molecular and cellular basis of hypertension and other phenotypes specific to the SHR strain. This near complete catalog of genomic differences between two extensively studied rat strains provides the starting point for complete elucidation, at the molecular level, of the physiological and pathophysiological phenotypic differences between individuals from these strains.
Assuntos
Hipertensão/genética , Animais , Códon de Terminação , Dosagem de Genes , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Ratos , Ratos Endogâmicos SHR , Transcrição GênicaRESUMO
Many studies have shown that abdominal adiposity is more strongly related to health risks than peripheral adiposity. However, the underlying pathways are still poorly understood. In this cross-sectional study using data from RNA-sequencing experiments and whole-body MRI scans of 200 participants in the EPIC-Potsdam cohort, our aim was to identify novel genes whose gene expression in subcutaneous adipose tissue has an effect on body fat mass (BFM) and body fat distribution (BFD). The analysis identified 625 genes associated with adiposity, of which 531 encode a known protein and 487 are novel candidate genes for obesity. Enrichment analyses indicated that BFM-associated genes were characterized by their higher than expected involvement in cellular, regulatory and immune system processes, and BFD-associated genes by their involvement in cellular, metabolic, and regulatory processes. Mendelian Randomization analyses suggested that the gene expression of 69 genes was causally related to BFM and BFD. Six genes were replicated in UK Biobank. In this study, we identified novel genes for BFM and BFD that are BFM- and BFD-specific, involved in different molecular processes, and whose up-/downregulated gene expression may causally contribute to obesity.
RESUMO
In humans and mice, L-arginine:glycine amidinotransferase (AGAT) and its metabolites homoarginine (hArg) and creatine have been linked to cardiovascular disease (CVD), specifically myocardial infarction (MI) and heart failure (HF). The underlying molecular and regulatory mechanisms, however, remain unclear. To identify potential pathways of cardiac AGAT metabolism, we sequenced microRNA (miRNA) in left ventricles of wild-type (wt) compared to AGAT-deficient (AGAT-/-) mice. Using literature search and validation by qPCR, we identified eight significantly regulated miRNAs in AGAT-/- mice linked to atherosclerosis, MI and HF: miR-30b, miR-31, miR-130a, miR-135a, miR-148a, miR-204, miR-298, and let-7i. Analysis of Gene Expression Omnibus (GEO) data confirmed deregulation of these miRNAs in mouse models of MI and HF. Quantification of miRNA expression by qPCR in AGAT-/- mice supplemented with creatine or hArg revealed that miR-30b, miR-31, miR-130a, miR-148a, and miR-204 were regulated by creatine, while miR-135a and miR-298 showed a trend of regulation by hArg. Finally, bioinformatics-based target prediction showed that numerous AGAT-dependent genes previously linked to CVD are likely to be regulated by the identified miRNAs. Taken together, AGAT deficiency and hArg/creatine supplementation are associated with cardiac miRNA expression which may influence cardiac (dys)function and CVD.
Assuntos
Insuficiência Cardíaca , MicroRNAs , Infarto do Miocárdio , Amidinotransferases , Animais , Arginina/metabolismo , Creatina/metabolismo , Homoarginina/metabolismo , Camundongos , MicroRNAs/genética , Infarto do Miocárdio/genéticaRESUMO
Pathogenic variants in genes that cause dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM) convey high risks for the development of heart failure through unknown mechanisms. Using single-nucleus RNA sequencing, we characterized the transcriptome of 880,000 nuclei from 18 control and 61 failing, nonischemic human hearts with pathogenic variants in DCM and ACM genes or idiopathic disease. We performed genotype-stratified analyses of the ventricular cell lineages and transcriptional states. The resultant DCM and ACM ventricular cell atlas demonstrated distinct right and left ventricular responses, highlighting genotype-associated pathways, intercellular interactions, and differential gene expression at single-cell resolution. Together, these data illuminate both shared and distinct cellular and molecular architectures of human heart failure and suggest candidate therapeutic targets.
Assuntos
Displasia Arritmogênica Ventricular Direita , Cardiomiopatia Dilatada , Insuficiência Cardíaca , Análise de Célula Única , Transcriptoma , Displasia Arritmogênica Ventricular Direita/genética , Atlas como Assunto , Cardiomiopatia Dilatada/genética , Núcleo Celular/genética , Insuficiência Cardíaca/genética , Ventrículos do Coração , Humanos , RNA-SeqRESUMO
Patient-derived xenograft (PDX) tumor models represent a valuable platform for identifying new biomarkers and novel targets, to evaluate therapy response and resistance mechanisms. This study aimed at establishment, characterization and therapy testing of colorectal carcinoma-derived PDX. We generated 49 PDX and validated identity between patient tumor and corresponding PDX. Sensitivity of PDX toward conventional and targeted drugs revealed that 92% of PDX responded toward irinotecan, 45% toward 5-FU, 65% toward bevacizumab, and 61% toward cetuximab. Expression of epidermal growth factor receptor (EGFR) ligands correlated to the sensitivity toward cetuximab. Proto-oncogene B-RAF, EGFR, Kirsten rat sarcoma virus oncogene homolog gene copy number correlated positively with cetuximab and erlotinib sensitivity. The mutational analyses revealed an individual mutational profile of PDX and mainly identical profiles of PDX from primary tumor vs corresponding metastasis. Mutation in PIK3CA was a determinant of accelerated tumor doubling time. PDX with wildtype Kirsten rat sarcoma virus oncogene homolog, proto-oncogene B-RAF, and phosphatidylinositol-4,5-bisphosphate 3-kinaseM catalytic subunit alfa showed higher sensitivity toward cetuximab and erlotinib. To study the molecular mechanism of cetuximab resistance, cetuximab resistant PDX models were generated, and changes in HER2, HER3, betacellulin, transforming growth factor alfa were observed. Global proteome and phosphoproteome profiling showed a reduction in canonical EGFR-mediated signaling via PTPN11 (SHP2) and AKT1S1 (PRAS40) and an increase in anti-apoptotic signaling as a consequence of acquired cetuximab resistance. This demonstrates that PDX models provide a multitude of possibilities to identify and validate biomarkers, signaling pathways and resistance mechanisms for clinically relevant improvement in cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Xenoenxertos , Animais , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Variações do Número de Cópias de DNA , Resistencia a Medicamentos Antineoplásicos , Imunofluorescência , Perfilação da Expressão Gênica , Instabilidade Genômica , Humanos , Imuno-Histoquímica , Camundongos , Terapia de Alvo Molecular , Mutação , Medicina de Precisão/métodos , Proteoma , Proteômica , Proto-Oncogene Mas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Little is known about the impact of trans-acting genetic variation on the rates with which proteins are synthesized by ribosomes. Here, we investigate the influence of such distant genetic loci on the efficiency of mRNA translation and define their contribution to the development of complex disease phenotypes within a panel of rat recombinant inbred lines. RESULTS: We identify several tissue-specific master regulatory hotspots that each control the translation rates of multiple proteins. One of these loci is restricted to hypertrophic hearts, where it drives a translatome-wide and protein length-dependent change in translational efficiency, altering the stoichiometric translation rates of sarcomere proteins. Mechanistic dissection of this locus across multiple congenic lines points to a translation machinery defect, characterized by marked differences in polysome profiles and misregulation of the small nucleolar RNA SNORA48. Strikingly, from yeast to humans, we observe reproducible protein length-dependent shifts in translational efficiency as a conserved hallmark of translation machinery mutants, including those that cause ribosomopathies. Depending on the factor mutated, a pre-existing negative correlation between protein length and translation rates could either be enhanced or reduced, which we propose to result from mRNA-specific imbalances in canonical translation initiation and reinitiation rates. CONCLUSIONS: We show that distant genetic control of mRNA translation is abundant in mammalian tissues, exemplified by a single genomic locus that triggers a translation-driven molecular mechanism. Our work illustrates the complexity through which genetic variation can drive phenotypic variability between individuals and thereby contribute to complex disease.
Assuntos
Cardiomegalia/genética , Iniciação Traducional da Cadeia Peptídica , Locos de Características Quantitativas , RNA Mensageiro/genética , RNA Nucleolar Pequeno/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Animais , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Variação Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Biogênese de Organelas , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Transgênicos , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Ribossomos/patologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sarcômeros/metabolismo , Sarcômeros/patologiaRESUMO
The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+ -load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow-up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non-failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca2+ -binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+ -scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.