RESUMO
Ribonucleoprotein (RNP) assemblies are ubiquitous in eukaryotic cells and have functions throughout RNA transcription, splicing, and stability. Of the RNA-binding proteins that form RNPs, TAR DNA-binding protein of 43 kD (TDP43) is of particular interest due to its essential nature and its association with disease. TDP43 plays critical roles in RNA metabolism, many of which require its recruitment to RNP granules such as stress granules, myo-granules, and neuronal transport granules. Moreover, the presence of cytoplasmic TDP43-positive inclusions is a pathological hallmark of several neurodegenerative diseases. Despite the pervasiveness of TDP43 aggregates, TDP43 mutations are exceedingly rare, suggesting that aggregation may be linked to dysregulation of TDP43 function. Oligomerization is a part of normal TDP43 function; thus, it is of interest to understand what triggers the irreversible aggregation that is seen in disease. Herein, we examine TDP43 functions, particularly in RNP granules, and the mechanisms which may explain pathological TDP43 aggregation.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Doenças Neurodegenerativas/patologia , Agregados Proteicos/fisiologia , Humanos , Doenças Neurodegenerativas/metabolismoRESUMO
UNLABELLED: The vaccinia virus B1R gene encodes a highly conserved protein kinase that is essential for the poxviral life cycle. As demonstrated in many cell types, B1 plays a critical role during viral DNA replication when it inactivates the cellular host defense effector barrier to autointegration factor (BAF or BANF1). To better understand the role of B1 during infection, we have characterized the growth of a B1-deficient temperature-sensitive mutant virus (Cts2 virus) in U2OS osteosarcoma cells. In contrast to all other cell lines tested to date, we found that in U2OS cells, Cts2 viral DNA replication is unimpaired at the nonpermissive temperature. However, the Cts2 viral yield in these cells was reduced more than 10-fold, thus indicating that B1 is required at another stage of the vaccinia virus life cycle. Our results further suggest that the host defense function of endogenous BAF may be absent in U2OS cells but can be recovered through either overexpression of BAF or fusion of U2OS cells with mouse cells in which the antiviral function of BAF is active. Interestingly, examination of late viral proteins during Cts2 virus infection demonstrated that B1 is required for optimal processing of the L4 protein. Finally, execution point analyses as well as electron microscopy studies uncovered a role for B1 during maturation of poxviral virions. Overall, this work demonstrates that U2OS cells are a novel model system for studying the cell type-specific regulation of BAF and reveals a role for B1 beyond DNA replication during the late stages of the viral life cycle. IMPORTANCE: The most well characterized role for the vaccinia virus B1 kinase is to facilitate viral DNA replication by phosphorylating and inactivating BAF, a cellular host defense responsive to foreign DNA. Additional roles for B1 later in the viral life cycle have been postulated for decades but are difficult to examine directly due to the importance of B1 during DNA replication. Here, we demonstrate that in U2OS cells, a B1 mutant virus escapes the block in DNA replication observed in other cell types and, instead, this mutant virus exhibits impaired late protein accumulation and incomplete maturation of new virions. These data provide the clearest evidence to date that B1 is needed for multiple critical junctures in the poxviral life cycle in a manner that is both dependent on and independent of BAF.
Assuntos
Replicação do DNA , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Osteoblastos/virologia , Proteínas Quinases/genética , Vaccinia virus/genética , Proteínas Virais/genética , Animais , Fusão Celular , Linhagem Celular Tumoral , DNA Viral/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/imunologia , Osteoblastos/imunologia , Proteínas Quinases/imunologia , Transdução de Sinais , Vaccinia virus/imunologia , Proteínas Virais/imunologia , Vírion/genética , Vírion/crescimento & desenvolvimento , Vírion/imunologia , Replicação Viral/genéticaRESUMO
The nuclear RNA-binding protein TDP43 is integrally involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Previous studies uncovered N-terminal TDP43 isoforms that are predominantly cytosolic in localization, highly prone to aggregation, and enriched in susceptible spinal motor neurons. In healthy cells, however, these shortened (s)TDP43 isoforms are difficult to detect in comparison to full-length (fl)TDP43, raising questions regarding their origin and selective regulation. Here, we show that sTDP43 is created as a byproduct of TDP43 autoregulation and cleared by nonsense mediated RNA decay (NMD). The sTDP43-encoding transcripts that escape NMD can lead to toxicity but are rapidly degraded post-translationally. Circumventing these regulatory mechanisms by overexpressing sTDP43 results in neurodegeneration in vitro and in vivo via N-terminal oligomerization and impairment of flTDP43 splicing activity, in addition to RNA binding-dependent gain-of-function toxicity. Collectively, these studies highlight endogenous mechanisms that tightly regulate sTDP43 expression and provide insight into the consequences of aberrant sTDP43 accumulation in disease.
RESUMO
Skeletal muscle formation is a complex process that requires tight spatiotemporal control of key myogenic factors. Emerging evidence suggests that RNA processing is crucial for the regulation of these factors, and that multiple post-transcriptional regulatory pathways work dependently and independently of one another to enable precise control of transcripts throughout muscle development and repair. Moreover, disruption of these pathways is implicated in neuromuscular disease, and the recent development of RNA-mediated therapies shows enormous promise in the treatment of these disorders. We discuss the overlapping post-transcriptional regulatory pathways that mediate muscle development, how these pathways are disrupted in neuromuscular disorders, and advances in RNA-mediated therapies that present a novel approach to the treatment of these diseases.
Assuntos
Desenvolvimento Muscular/fisiologia , Doenças Musculares , Doenças Neuromusculares , Processamento Pós-Transcricional do RNA , Processamento Alternativo , Animais , Humanos , MicroRNAs , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/etiologia , Doenças Musculares/metabolismo , Doenças Musculares/prevenção & controle , Doenças Neuromusculares/etiologia , Doenças Neuromusculares/metabolismo , Doenças Neuromusculares/prevenção & controle , Poliadenilação , RNA/metabolismoRESUMO
Cortical hyperexcitability and mislocalization of the RNA-binding protein TDP43 are highly conserved features in amyotrophic lateral sclerosis (ALS). Nevertheless, the relationship between these phenomena remains poorly defined. Here, we showed that hyperexcitability recapitulates TDP43 pathology by upregulating shortened TDP43 (sTDP43) splice isoforms. These truncated isoforms accumulated in the cytoplasm and formed insoluble inclusions that sequestered full-length TDP43 via preserved N-terminal interactions. Consistent with these findings, sTDP43 overexpression was toxic to mammalian neurons, suggesting neurodegeneration arising from complementary gain- and loss-of-function mechanisms. In humans and mice, sTDP43 transcripts were enriched in vulnerable motor neurons, and we observed a striking accumulation of sTDP43 within neurons and glia of ALS patients. Collectively, these studies uncover a pathogenic role for alternative TDP43 isoforms in ALS, and implicate sTDP43 as a key contributor to the susceptibility of motor neurons in this disorder.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/biossíntese , Neurônios Motores/metabolismo , Neuroglia/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Neurônios Motores/patologia , Neuroglia/patologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genéticaRESUMO
Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to assess factors that influence neuronal fate. These assays require the observation of separate populations of cells at discrete time points. As a result, individual cells cannot be followed prospectively over time, severely limiting the ability to discriminate whether subcellular events, such as puncta formation or protein mislocalization, are pathogenic drivers of disease, homeostatic responses, or merely coincidental phenomena. Single-cell longitudinal microscopy overcomes these limitations, allowing the researcher to determine differences in survival between populations and draw causal relationships with enhanced sensitivity. This video guide will outline a representative workflow for experiments measuring single-cell survival of rat primary cortical neurons expressing a fluorescent protein marker. The viewer will learn how to achieve high-efficiency transfections, collect and process images enabling the prospective tracking of individual cells, and compare the relative survival of neuronal populations using Cox proportional hazards analysis.
Assuntos
Microscopia de Fluorescência/métodos , Neurônios/citologia , Animais , Morte Celular , Sobrevivência Celular , Córtex Cerebral/citologia , Neurônios/metabolismo , Modelos de Riscos Proporcionais , Ratos Long-EvansRESUMO
Ribonucleic acid (RNA) homeostasis is dynamically modulated in response to changing physiological conditions. Tight regulation of RNA abundance through both transcription and degradation determines the amount, timing, and location of protein translation. This balance is of particular importance in neurons, which are among the most metabolically active and morphologically complex cells in the body. As a result, any disruptions in RNA degradation can have dramatic consequences for neuronal health. In this chapter, we will first discuss mechanisms of RNA stabilization and decay. We will then explore how the disruption of these pathways can lead to neurodegenerative disease.
Assuntos
Doenças Neurodegenerativas/metabolismo , Estabilidade de RNA/fisiologia , RNA Mensageiro , HumanosRESUMO
The nuclear RNA-binding protein TDP43 is integrally involved in RNA processing. In accord with this central function, TDP43 levels are tightly regulated through a negative feedback loop, in which TDP43 recognizes its own RNA transcript, destabilizes it, and reduces new TDP43 protein production. In the neurodegenerative disorder amyotrophic lateral sclerosis (ALS), cytoplasmic mislocalization and accumulation of TDP43 disrupt autoregulation; conversely, inefficient TDP43 autoregulation can lead to cytoplasmic TDP43 deposition and subsequent neurodegeneration. Because TDP43 plays a multifaceted role in maintaining RNA metabolism, its mislocalization and accumulation interrupt several RNA processing pathways that in turn affect RNA stability and gene expression. TDP43-mediated disruption of these pathways-including alternative mRNA splicing, non-coding RNA processing, and RNA granule dynamics-may directly or indirectly contribute to ALS pathogenesis. Therefore, strategies that restore effective TDP43 autoregulation may ultimately prevent neurodegeneration in ALS and related disorders.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/genética , Estabilidade de RNA , Processamento Alternativo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Humanos , Neurônios Motores/metabolismo , RNA/genética , RNA/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative disorders marked in most cases by the nuclear exclusion and cytoplasmic deposition of the RNA binding protein TDP43. We previously demonstrated that ALS-associated mutant TDP43 accumulates within the cytoplasm, and that TDP43 mislocalization predicts neurodegeneration. Here, we sought to prevent neurodegeneration in ALS/FTD models using selective inhibitor of nuclear export (SINE) compounds that target exportin-1 (XPO1). SINE compounds modestly extend cellular survival in neuronal ALS/FTD models and mitigate motor symptoms in an in vivo rat ALS model. At high doses, SINE compounds block nuclear egress of an XPO1 cargo reporter, but not at lower concentrations that were associated with neuroprotection. Neither SINE compounds nor leptomycin B, a separate XPO1 inhibitor, enhanced nuclear TDP43 levels, while depletion of XPO1 or other exportins had little effect on TDP43 localization, suggesting that no single exporter is necessary for TDP43 export. Supporting this hypothesis, we find overexpression of XPO1, XPO7 and NXF1 are each sufficient to promote nuclear TDP43 egress. Taken together, our results indicate that redundant pathways regulate TDP43 nuclear export, and that therapeutic prevention of cytoplasmic TDP43 accumulation in ALS/FTD may be enhanced by targeting several overlapping mechanisms.