RESUMO
Genomic DNA that resides in the nuclei of mammalian neurons can be as old as the organism itself. The life span of nuclear RNAs, which are critical for proper chromatin architecture and transcription regulation, has not been determined in adult tissues. In this work, we identified and characterized nuclear RNAs that do not turn over for at least 2 years in a subset of postnatally born cells in the mouse brain. These long-lived RNAs were stably retained in nuclei in a neural cell type-specific manner and were required for the maintenance of heterochromatin. Thus, the life span of neural cells may depend on both the molecular longevity of DNA for the storage of genetic information and also the extreme stability of RNA for the functional organization of chromatin.
Assuntos
Encéfalo , Cromatina , RNA Nuclear , Animais , Camundongos , Encéfalo/metabolismo , Regulação da Expressão Gênica , Heterocromatina/genética , RNA Nuclear/genéticaRESUMO
Caloric restriction (CR) extends organismal lifespan and health span by improving glucose homeostasis mechanisms. How CR affects organellar structure and function of pancreatic beta cells over the lifetime of the animal remains unknown. Here, we used single nucleus transcriptomics to show that CR increases the expression of genes for beta cell identity, protein processing, and organelle homeostasis. Gene regulatory network analysis link this transcriptional phenotype to transcription factors involved in beta cell identity (Mafa) and homeostasis (Atf6). Imaging metabolomics further demonstrates that CR beta cells are more energetically competent. In fact, high-resolution light and electron microscopy indicates that CR reduces beta cell mitophagy and increases mitochondria mass, increasing mitochondrial ATP generation. Finally, we show that long-term CR delays the onset of beta cell aging and senescence to promote longevity by reducing beta cell turnover. Therefore, CR could be a feasible approach to preserve compromised beta cells during aging and diabetes.
RESUMO
Caloric restriction (CR) extends organismal lifespan and health span by improving glucose homeostasis mechanisms. How CR affects organellar structure and function of pancreatic beta cells over the lifetime of the animal remains unknown. Here, we used single nucleus transcriptomics to show that CR increases the expression of genes for beta cell identity, protein processing, and organelle homeostasis. Gene regulatory network analysis link this transcriptional phenotype to transcription factors involved in beta cell identity (Mafa) and homeostasis (Atf6). Imaging metabolomics further demonstrates that CR beta cells are more energetically competent. In fact, high-resolution light and electron microscopy indicates that CR reduces beta cell mitophagy and increases mitochondria mass, increasing mitochondrial ATP generation. Finally, we show that long-term CR delays the onset of beta cell aging and senescence to promote longevity by reducing beta cell turnover. Therefore, CR could be a feasible approach to preserve compromised beta cells during aging and diabetes.
RESUMO
During apoptosis, caspases degrade 8 out of ~30 nucleoporins to irreversibly demolish the nuclear pore complex. However, for poorly understood reasons, caspases are also activated during cell differentiation. Here, we show that sublethal activation of caspases during myogenesis results in the transient proteolysis of four peripheral Nups and one transmembrane Nup. 'Trimmed' NPCs become nuclear export-defective, and we identified in an unbiased manner several classes of cytoplasmic, plasma membrane, and mitochondrial proteins that rapidly accumulate in the nucleus. NPC trimming by non-apoptotic caspases was also observed in neurogenesis and endoplasmic reticulum stress. Our results suggest that caspases can reversibly modulate nuclear transport activity, which allows them to function as agents of cell differentiation and adaptation at sublethal levels.
Assuntos
Caspases , Poro Nuclear , Diferenciação Celular , Apoptose , Estresse do Retículo EndoplasmáticoRESUMO
The LINC complex tethers the cell nucleus to the cytoskeleton to regulate mechanical forces during cell migration, differentiation, and various diseases. The function of LINC complexes relies on the interaction between highly conserved SUN and KASH proteins that form higher-order assemblies capable of load bearing. These structural details have emerged from in vitro assembled LINC complexes; however, the principles of in vivo assembly remain obscure. Here, we report a conformation-specific SUN2 antibody as a tool to visualize LINC complex dynamics in situ. Using imaging, biochemical, and cellular methods, we find that conserved cysteines in SUN2 undergo KASH-dependent inter- and intra-molecular disulfide bond rearrangements. Disruption of the SUN2 terminal disulfide bond compromises SUN2 localization, turnover, LINC complex assembly in addition to cytoskeletal organization and cell migration. Moreover, using pharmacological and genetic perturbations, we identify components of the ER lumen as SUN2 cysteines redox state regulators. Overall, we provide evidence for SUN2 disulfide bond rearrangement as a physiologically relevant structural modification that regulates LINC complex functions.
Assuntos
Membrana Nuclear , Proteínas Nucleares , Citoesqueleto/metabolismo , Dissulfetos/metabolismo , Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Camundongos , AnimaisRESUMO
Peripheral heterochromatin positioning depends on nuclear envelope associated proteins and repressive histone modifications. Here we show that overexpression (OE) of Lamin B1 (LmnB1) leads to the redistribution of peripheral heterochromatin into heterochromatic foci within the nucleoplasm. These changes represent a perturbation of heterochromatin binding at the nuclear periphery (NP) through a mechanism independent from altering other heterochromatin anchors or histone post-translational modifications. We further show that LmnB1 OE alters gene expression. These changes do not correlate with different levels of H3K9me3, but a significant number of the misregulated genes were likely mislocalized away from the NP upon LmnB1 OE. We also observed an enrichment of developmental processes amongst the upregulated genes. ~74% of these genes were normally repressed in our cell type, suggesting that LmnB1 OE promotes gene de-repression. This demonstrates a broader consequence of LmnB1 OE on cell fate, and highlights the importance of maintaining proper levels of LmnB1.
Assuntos
Heterocromatina , Lamina Tipo B , Heterocromatina/genética , Heterocromatina/metabolismo , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Núcleo Celular/metabolismo , Expressão GênicaRESUMO
Pancreatic islet beta cells are essential for maintaining glucose homeostasis. To understand the impact of aging on beta cells, we performed meta-analysis of single-cell RNA sequencing datasets, transcription factor (TF) regulon analysis, high-resolution confocal microscopy, and measured insulin secretion from nondiabetic donors spanning most of the human life span. This revealed the range of molecular and functional changes that occur during beta cell aging, including the transcriptional deregulation that associates with cellular immaturity and reorganization of beta cell TF networks, increased gene transcription rates, and reduced glucose-stimulated insulin release. These alterations associate with activation of endoplasmic reticulum (ER) stress and autophagy pathways. We propose that a chronic state of ER stress undermines old beta cell structure function to increase the risk of beta cell failure and type 2 diabetes onset as humans age.
RESUMO
The vascular system is a key player for the maintenance of healthy tissues, suggesting how the physiological decline of blood vessel functionality during aging could be a major contributor of organ degeneration. While basic research studies have begun to pinpoint potential mechanisms of vascular aging, it is now critical to translate them into therapeutically relevant options. Microphysiological systems represent a powerful tool to precisely control which combinations of stimuli are provided to in vitro reconstructed blood vessels and to analyze their functional consequences. After highlighting key aspects of vascular aging, this review discusses in vitro models that are able to recapitulate relevant features of blood vessel damage during aging. Strategies to improve current in vitro systems so that they will more faithfully recapitulate vascular aging are proposed, emphasizing the importance of combining in vivo models with microphysiological systems for an effective translation of vascular aging biomarkers and therapies to the clinical level.
Assuntos
Envelhecimento , Dispositivos Lab-On-A-Chip , HumanosRESUMO
Nuclear pore complexes (NPCs) bridge the nucleus and the cytoplasm and are indispensable for crucial cellular activities, such as bidirectional molecular trafficking and gene transcription regulation. The discovery of long-lived proteins (LLPs) in NPCs from postmitotic cells raises the exciting possibility that the maintenance of NPC integrity might play an inherent role in lifelong cell function. Age-dependent deterioration of NPCs and loss of nuclear integrity have been linked to age-related decline in postmitotic cell function and degenerative diseases. In this review, we discuss our current understanding of NPC maintenance in proliferating and postmitotic cells, and how malfunction of nucleoporins (Nups) might contribute to the pathogenesis of various neurodegenerative and cardiovascular diseases.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Envelhecimento , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
In order to combat molecular damage, most cellular proteins undergo rapid turnover. We have previously identified large nuclear protein assemblies that can persist for years in post-mitotic tissues and are subject to age-related decline. Here, we report that mitochondria can be long lived in the mouse brain and reveal that specific mitochondrial proteins have half-lives longer than the average proteome. These mitochondrial long-lived proteins (mitoLLPs) are core components of the electron transport chain (ETC) and display increased longevity in respiratory supercomplexes. We find that COX7C, a mitoLLP that forms a stable contact site between complexes I and IV, is required for complex IV and supercomplex assembly. Remarkably, even upon depletion of COX7C transcripts, ETC function is maintained for days, effectively uncoupling mitochondrial function from ongoing transcription of its mitoLLPs. Our results suggest that modulating protein longevity within the ETC is critical for mitochondrial proteome maintenance and the robustness of mitochondrial function.
Assuntos
Transporte de Elétrons/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Camundongos , Fosforilação OxidativaRESUMO
Understanding basic mechanisms of aging holds great promise for developing interventions that prevent or delay many age-related declines and diseases simultaneously to increase human healthspan. However, a major confounding factor in aging research is the heterogeneity of the aging process itself. At the organismal level, it is clear that chronological age does not always predict biological age or susceptibility to frailty or pathology. While genetics and environment are major factors driving variable rates of aging, additional complexity arises because different organs, tissues, and cell types are intrinsically heterogeneous and exhibit different aging trajectories normally or in response to the stresses of the aging process (e.g., damage accumulation). Tackling the heterogeneity of aging requires new and specialized tools (e.g., single-cell analyses, mass spectrometry-based approaches, and advanced imaging) to identify novel signatures of aging across scales. Cutting-edge computational approaches are then needed to integrate these disparate datasets and elucidate network interactions between known aging hallmarks. There is also a need for improved, human cell-based models of aging to ensure that basic research findings are relevant to human aging and healthspan interventions. The San Diego Nathan Shock Center (SD-NSC) provides access to cutting-edge scientific resources to facilitate the study of the heterogeneity of aging in general and to promote the use of novel human cell models of aging. The center also has a robust Research Development Core that funds pilot projects on the heterogeneity of aging and organizes innovative training activities, including workshops and a personalized mentoring program, to help investigators new to the aging field succeed. Finally, the SD-NSC participates in outreach activities to educate the general community about the importance of aging research and promote the need for basic biology of aging research in particular.
Assuntos
Fragilidade , Gerociência , Envelhecimento , HumanosRESUMO
Vascular dysfunctions are a common feature of multiple age-related diseases. However, modeling healthy and pathological aging of the human vasculature represents an unresolved experimental challenge. Here, we generated induced vascular endothelial cells (iVECs) and smooth muscle cells (iSMCs) by direct reprogramming of healthy human fibroblasts from donors of different ages and Hutchinson-Gilford Progeria Syndrome (HGPS) patients. iVECs induced from old donors revealed upregulation of GSTM1 and PALD1, genes linked to oxidative stress, inflammation and endothelial junction stability, as vascular aging markers. A functional assay performed on PALD1 KD VECs demonstrated a recovery in vascular permeability. We found that iSMCs from HGPS donors overexpressed bone morphogenetic protein (BMP)-4, which plays a key role in both vascular calcification and endothelial barrier damage observed in HGPS. Strikingly, BMP4 concentrations are higher in serum from HGPS vs. age-matched mice. Furthermore, targeting BMP4 with blocking antibody recovered the functionality of the vascular barrier in vitro, hence representing a potential future therapeutic strategy to limit cardiovascular dysfunction in HGPS. These results show that iVECs and iSMCs retain disease-related signatures, allowing modeling of vascular aging and HGPS in vitro.
Assuntos
Células Endoteliais/fisiologia , Glutationa Transferase/genética , Miócitos de Músculo Liso/fisiologia , Fosfoproteínas Fosfatases/genética , Progéria/genética , Envelhecimento/fisiologia , Animais , Glutationa Transferase/metabolismo , Humanos , Camundongos , Fosfoproteínas Fosfatases/metabolismoRESUMO
In recent years, the nuclear pore complex (NPC) has emerged as a key player in genome regulation and cellular homeostasis. New discoveries have revealed that the NPC has multiple cellular functions besides mediating the molecular exchange between the nucleus and the cytoplasm. In this review, we discuss non-transport aspects of the NPC focusing on the NPC-genome interaction, the extreme longevity of the NPC proteins, and NPC dysfunction in age-related diseases. The examples summarized herein demonstrate that the NPC, which first evolved to enable the biochemical communication between the nucleus and the cytoplasm, now doubles as the gatekeeper of cellular identity and aging.
Assuntos
Envelhecimento/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Envelhecimento/genética , Senilidade Prematura/genética , Senilidade Prematura/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Proteína de Ligação a CREB/metabolismo , Senescência Celular , Genoma , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genéticaRESUMO
During mitosis, transcription of genomic DNA is dramatically reduced, before it is reactivated during nuclear reformation in anaphase/telophase. Many aspects of the underlying principles that mediate transcriptional memory and reactivation in the daughter cells remain unclear. Here, we used ChIP-seq on synchronized cells at different stages after mitosis to generate genome-wide maps of histone modifications. Combined with EU-RNA-seq and Hi-C analyses, we found that during prometaphase, promoters, enhancers, and insulators retain H3K4me3 and H3K4me1, while losing H3K27ac. Enhancers globally retaining mitotic H3K4me1 or locally retaining mitotic H3K27ac are associated with cell type-specific genes and their transcription factors for rapid transcriptional activation. As cells exit mitosis, promoters regain H3K27ac, which correlates with transcriptional reactivation. Insulators also gain H3K27ac and CCCTC-binding factor (CTCF) in anaphase/telophase. This increase of H3K27ac in anaphase/telophase is required for posttranscriptional activation and may play a role in the establishment of topologically associating domains (TADs). Together, our results suggest that the genome is reorganized in a sequential order, in which histone methylations occur first in prometaphase, histone acetylation, and CTCF in anaphase/telophase, transcription in cytokinesis, and long-range chromatin interactions in early G1. We thus provide insights into the histone modification landscape that allows faithful reestablishment of the transcriptional program and TADs during cell division.
Assuntos
Cromatina/metabolismo , Código das Histonas/genética , Histonas/metabolismo , Mitose/genética , Processamento de Proteína Pós-Traducional/genética , Ativação Transcricional/genética , Animais , Pontos de Checagem do Ciclo Celular/genética , Cromossomos/genética , Cromossomos/metabolismo , Elementos Facilitadores Genéticos , Genoma/genética , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de TempoRESUMO
Aging of the circulatory system correlates with the pathogenesis of a large spectrum of diseases. However, it is largely unknown which factors drive the age-dependent or pathological decline of the vasculature and how vascular defects relate to tissue aging. The goal of the study is to design a multianalytical approach to identify how the cellular microenvironment (i.e., fibroblasts) and serum from healthy donors of different ages or Alzheimer disease (AD) patients can modulate the functionality of organ-specific vascular endothelial cells (VECs). Long-living human microvascular networks embedding VECs and fibroblasts from skin biopsies are generated. RNA-seq, secretome analyses, and microfluidic assays demonstrate that fibroblasts from young donors restore the functionality of aged endothelial cells, an effect also achieved by serum from young donors. New biomarkers of vascular aging are validated in human biopsies and it is shown that young serum induces angiopoietin-like-4, which can restore compromised vascular barriers. This strategy is then employed to characterize transcriptional/functional changes induced on the blood-brain barrier by AD serum, demonstrating the importance of PTP4A3 in the regulation of permeability. Features of vascular degeneration during aging and AD are recapitulated, and a tool to identify novel biomarkers that can be exploited to develop future therapeutics modulating vascular function is established.
Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Microvasos/metabolismo , Idoso , Feminino , Humanos , Masculino , Técnicas Analíticas MicrofluídicasRESUMO
Nucleoporin 93 (Nup93) expression inversely correlates with the survival of triple-negative breast cancer patients. However, our knowledge of Nup93 function in breast cancer besides its role as structural component of the nuclear pore complex is not understood. Combination of functional assays and genetic analyses suggested that chromatin interaction of Nup93 partially modulates the expression of genes associated with actin cytoskeleton remodeling and epithelial to mesenchymal transition, resulting in impaired invasion of triple-negative, claudin-low breast cancer cells. Nup93 depletion induced stress fiber formation associated with reduced cell migration/proliferation and impaired expression of mesenchymal-like genes. Silencing LIMCH1, a gene responsible for actin cytoskeleton remodeling and up-regulated upon Nup93 depletion, partially restored the invasive phenotype of cancer cells. Loss of Nup93 led to significant defects in tumor establishment/propagation in vivo, whereas patient samples revealed that high Nup93 and low LIMCH1 expression correlate with late tumor stage. Our approach identified Nup93 as contributor of triple-negative, claudin-low breast cancer cell invasion and paves the way to study the role of nuclear envelope proteins during breast cancer tumorigenesis.
Assuntos
Citoesqueleto de Actina/genética , Proliferação de Células/genética , Proteínas com Domínio LIM , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Neoplasias de Mama Triplo Negativas/genética , Citoesqueleto de Actina/metabolismo , Carcinogênese/genética , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Interferência de RNA , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The inner nuclear membrane (INM) is a subdomain of the endoplasmic reticulum (ER) that is gated by the nuclear pore complex. It is unknown whether proteins of the INM and ER are degraded through shared or distinct pathways in mammalian cells. We applied dynamic proteomics to profile protein half-lives and report that INM and ER residents turn over at similar rates, indicating that the INM's unique topology is not a barrier to turnover. Using a microscopy approach, we observed that the proteasome can degrade INM proteins in situ. However, we also uncovered evidence for selective, vesicular transport-mediated turnover of a single INM protein, emerin, that is potentiated by ER stress. Emerin is rapidly cleared from the INM by a mechanism that requires emerin's LEM domain to mediate vesicular trafficking to lysosomes. This work demonstrates that the INM can be dynamically remodeled in response to environmental inputs.
Assuntos
Estresse do Retículo Endoplasmático , Proteínas de Membrana/análise , Mioblastos/química , Mioblastos/fisiologia , Membrana Nuclear/química , Proteínas Nucleares/análise , Proteoma/análise , Animais , Linhagem Celular , Vesículas Citoplasmáticas/metabolismo , Lisossomos/metabolismo , Camundongos , Transporte Proteico , ProteômicaRESUMO
Most neurons are not replaced during an animal's lifetime. This nondividing state is characterized by extreme longevity and age-dependent decline of key regulatory proteins. To study the lifespans of cells and proteins in adult tissues, we combined isotope labeling of mice with a hybrid imaging method (MIMS-EM). Using 15N mapping, we show that liver and pancreas are composed of cells with vastly different ages, many as old as the animal. Strikingly, we also found that a subset of fibroblasts and endothelial cells, both known for their replicative potential, are characterized by the absence of cell division during adulthood. In addition, we show that the primary cilia of beta cells and neurons contains different structural regions with vastly different lifespans. Based on these results, we propose that age mosaicism across multiple scales is a fundamental principle of adult tissue, cell, and protein complex organization.
Assuntos
Envelhecimento/genética , Senescência Celular/genética , Mosaicismo , Especificidade de Órgãos/genética , Animais , Cílios/metabolismo , Células Endoteliais/metabolismo , Feminino , Fibroblastos/metabolismo , Células Secretoras de Insulina/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos , Neurônios/metabolismo , Pâncreas/metabolismoRESUMO
Many adult tissues contain postmitotic cells as old as the host organism. The only organelle that does not turn over in these cells is the nucleus, and its maintenance represents a formidable challenge, as it harbors regulatory proteins that persist throughout adulthood. Here we developed strategies to visualize two classes of such long-lived proteins, histones and nucleoporins, to understand the function of protein longevity in nuclear maintenance. Genome-wide mapping of histones revealed specific enrichment of long-lived variants at silent gene loci. Interestingly, nuclear pores are maintained by piecemeal replacement of subunits, resulting in mosaic complexes composed of polypeptides with vastly different ages. In contrast, nondividing quiescent cells remove old nuclear pores in an ESCRT-dependent manner. Our findings reveal distinct molecular strategies of nuclear maintenance, linking lifelong protein persistence to gene regulation and nuclear integrity.
Assuntos
Regulação da Expressão Gênica/fisiologia , Histonas/metabolismo , Mitose/fisiologia , Poro Nuclear/metabolismo , Animais , Linhagem Celular , Estudo de Associação Genômica Ampla , Camundongos , Fatores de TempoRESUMO
The genome is packaged and organized nonrandomly within the 3D space of the nucleus to promote efficient gene expression and to faithfully maintain silencing of heterochromatin. The genome is enclosed within the nucleus by the nuclear envelope membrane, which contains a set of proteins that actively participate in chromatin organization and gene regulation. Technological advances are providing views of genome organization at unprecedented resolution and are beginning to reveal the ways that cells co-opt the structures of the nuclear periphery for nuclear organization and gene regulation. These genome regulatory roles of proteins of the nuclear periphery have important influences on development, disease and ageing.