RESUMO
Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:
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Caenorhabditis elegans/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Neoplasias/fisiopatologia , Elongação Traducional da Cadeia Peptídica , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Neoplasias Encefálicas/fisiopatologia , Caenorhabditis elegans/genética , Sobrevivência Celular , Transformação Celular Neoplásica , Quinase do Fator 2 de Elongação/genética , Privação de Alimentos , Glioblastoma/fisiopatologia , Células HeLa , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Transplante de Neoplasias , Fator 2 de Elongação de Peptídeos/metabolismo , Transplante HeterólogoRESUMO
Chronic inflammasome activation in mononuclear phagocytes (MNPs) promotes fibrosis in various tissues, including the kidney. The cellular and molecular links between the inflammasome and fibrosis are unclear. To address this question, we fed mice lacking various immunological mediators an adenine-enriched diet, which causes crystal precipitation in renal tubules, crystal-induced inflammasome activation, and renal fibrosis. We found that kidney fibrosis depended on an intrarenal inflammasome-dependent type 3 immune response driven by its signature transcription factor Rorc (retinoic acid receptor-related orphan receptor C gene), which was partially carried out by type 3 innate lymphoid cells (ILC3s). The role of ILCs in the kidney is less well known than in other organs, especially that of ILC3. In this article, we describe that depletion of ILCs or genetic deficiency for Rorc attenuated kidney inflammation and fibrosis. Among the inflammasome-derived cytokines, only IL-1ß expanded ILC3 and promoted fibrosis, whereas IL-18 caused differentiation of NKp46+ ILC3. Deficiency of the type 3 maintenance cytokine, IL-23, was more protective than IL-1ß inhibition, which may be explained by the downregulation of the IL-1R, but not of the IL-23R, by ILC3 early in the disease, allowing persistent sensing of IL-23. Mechanistically, ILC3s colocalized with renal MNPs in vivo as shown by multiepitope-ligand cartography. Cell culture experiments indicated that renal ILC3s caused renal MNPs to increase TGF-ß production that stimulated fibroblasts to produce collagen. We conclude that ILC3s link inflammasome activation with kidney inflammation and fibrosis and are regulated by IL-1ß and IL-23.
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Coxsackievirus B3 (CVB3) encodes proteinases that are essential for processing of the translated viral polyprotein. Viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. While some host protein substrates of the CVB3 3C and 2A cysteine proteinases have been identified, the full repertoire of targets is not known. Here, we utilize an unbiased quantitative proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) to conduct a global analysis of CVB3 protease-generated N-terminal peptides in both human HeLa and mouse cardiomyocyte (HL-1) cell lines infected with CVB3. We identified >800 proteins that are cleaved in CVB3-infected HeLa and HL-1 cells including the viral polyprotein, known substrates of viral 3C proteinase such as PABP, DDX58, and HNRNPs M, K, and D and novel cellular proteins. Network and GO-term analysis showed an enrichment in biological processes including immune response and activation, RNA processing, and lipid metabolism. We validated a subset of candidate substrates that are cleaved under CVB3 infection and some are direct targets of 3C proteinase in vitro. Moreover, depletion of a subset of TAILS-identified target proteins decreased viral yield. Characterization of two target proteins showed that expression of 3Cpro-targeted cleaved fragments of emerin and aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 modulated autophagy and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, respectively. The comprehensive identification of host proteins targeted during virus infection provides insights into the cellular pathways manipulated to facilitate infection. IMPORTANCE: RNA viruses encode proteases that are responsible for processing viral proteins into their mature form. Viral proteases also target and cleave host cellular proteins; however, the full catalog of these target proteins is incomplete. We use a technique called terminal amine isotopic labeling of substrates (TAILS), an N-terminomics to identify host proteins that are cleaved under virus infection. We identify hundreds of cellular proteins that are cleaved under infection, some of which are targeted directly by viral protease. Revealing these target proteins provides insights into the host cellular pathways and antiviral signaling factors that are modulated to promote virus infection and potentially leading to virus-induced pathogenesis.
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Infecções por Coxsackievirus , Enterovirus Humano B , Proteólise , Enterovirus Humano B/metabolismo , Humanos , Camundongos , Animais , Células HeLa , Infecções por Coxsackievirus/virologia , Infecções por Coxsackievirus/metabolismo , Proteínas Virais/metabolismo , Proteômica/métodos , Interações Hospedeiro-Patógeno , Proteases Virais 3C/metabolismo , Linhagem Celular , Proteases Virais/metabolismo , Poliproteínas/metabolismoRESUMO
Intestinal Peyer's patches are essential lymphoid organs for the generation of T cell-dependent immunoglobulin A (IgA) for gut homeostasis. Through the use of interleukin 17 (IL-17) fate-reporter mice, we found here that endogenous cells of the TH17 subset of helper T cells in lymphoid organs of naive mice 'preferentially' homed to the intestines and were maintained independently of IL-23. In Peyer's patches, such TH17 cells acquired a follicular helper T cell (TFH cell) phenotype and induced the development of IgA-producing germinal center B cells. Mice deficient in TH17 cells failed to generate antigen-specific IgA responses, which provides evidence that TH17 cells are the crucial subset required for the production of high-affinity T cell-dependent IgA.
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Imunoglobulina A/imunologia , Nódulos Linfáticos Agregados/imunologia , Células Th17/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Feminino , Centro Germinativo/citologia , Centro Germinativo/imunologia , Imunoglobulina A/biossíntese , Imunoglobulina A Secretora/imunologia , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Interleucina-23/genética , Interleucina-23/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Knockout , Nódulos Linfáticos Agregados/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Th17 cells are most abundant in the gut, where their presence depends on the intestinal microbiota. Here, we examined whether intestinal Th17 cells contribute to extra-intestinal Th17 responses in autoimmune kidney disease. We found high frequencies of Th17 cells in the kidneys of patients with antineutrophil cytoplasmatic antibody (ANCA)-associated glomerulonephritis. We utilized photoconversion of intestinal cells in Kaede mice to track intestinal T cell mobilization upon glomerulonephritis induction, and we found that Th17 cells egress from the gut in a S1P-receptor-1-dependent fashion and subsequently migrate to the kidney via the CCL20/CCR6 axis. Depletion of intestinal Th17 cells in germ-free and antibiotic-treated mice ameliorated renal disease, whereas expansion of these cells upon Citrobacter rodentium infection exacerbated pathology. Thus, in some autoimmune settings, intestinal Th17 cells migrate into target organs, where they contribute to pathology. Targeting the intestinal Th17 cell "reservoir" may present a therapeutic strategy for these autoimmune disorders.
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Doenças Autoimunes/imunologia , Quimiotaxia de Leucócito/imunologia , Glomerulonefrite/imunologia , Receptores de Lisoesfingolipídeo/imunologia , Células Th17/imunologia , Animais , Citrobacter rodentium , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/imunologia , Citometria de Fluxo , Humanos , Intestinos/imunologia , Rim/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Esfingosina-1-FosfatoRESUMO
Internal ribosomal entry sites (IRESs) engage with the eukaryotic translation apparatus to promote end-independent initiation. We identified a conserved class of â¼150 nt long intergenic region (IGR) IRESs in dicistrovirus genomes derived from members of the phyla Arthropoda, Bryozoa, Cnidaria, Echinodermata, Entoprocta, Mollusca and Porifera. These IRESs, exemplified by Wenling picorna-like virus 2, resemble the canonical cricket paralysis virus (CrPV) IGR IRES in comprising two nested pseudoknots (PKII/PKIII) and a 3'-terminal pseudoknot (PKI) that mimics a tRNA anticodon stem-loop base-paired to mRNA. However, they are â¼50 nt shorter than CrPV-like IRESs, and PKIII is an H-type pseudoknot that lacks the SLIV and SLV stem-loops that are primarily responsible for the affinity of CrPV-like IRESs for the 40S ribosomal subunit and that restrict initial binding of PKI to its aminoacyl (A) site. Wenling-class IRESs bound strongly to 80S ribosomes but only weakly to 40S subunits. Whereas CrPV-like IRESs must be translocated from the A site to the peptidyl (P) site by elongation factor 2 for elongation to commence, Wenling-class IRESs bound directly to the P site of 80S ribosomes, and decoding begins without a prior translocation step. A chimeric CrPV clone containing a Wenling-class IRES was infectious, confirming that the IRES functioned in cells.
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Sítios Internos de Entrada Ribossomal , Vírus de RNA , Sequência de Bases , DNA Intergênico/genética , DNA Intergênico/metabolismo , Ribossomos/metabolismo , Vírus de RNA/genética , RNA Viral/metabolismo , Biossíntese de ProteínasRESUMO
BACKGROUND: Pulsed field ablation is a novel nonthermal cardiac ablation modality using ultra-rapid electrical pulses to cause cell death by a mechanism of irreversible electroporation. Unlike the traditional ablation energy sources, pulsed field ablation has demonstrated significant preferentiality to myocardial tissue ablation, and thus avoids certain thermally mediated complications. However, its safety and effectiveness remain unknown in usual clinical care. METHODS: MANIFEST-PF (Multi-National Survey on the Methods, Efficacy, and Safety on the Post-Approval Clinical Use of Pulsed Field Ablation) is a retrospective, multinational, patient-level registry wherein patients at each center were prospectively included in their respective center registries. The registry included all patients undergoing postapproval treatment with a multielectrode 5-spline pulsed field ablation catheter to treat atrial fibrillation (AF) between March 1, 2021, and May 30, 2022. The primary effectiveness outcome was freedom from clinical documented atrial arrhythmia (AF/atrial flutter/atrial tachycardia) of ≥30 seconds on the basis of electrocardiographic data after a 3-month blanking period (on or off antiarrhythmic drugs). Safety outcomes included the composite of acute (<7 days postprocedure) and latent (>7 days) major adverse events. RESULTS: At 24 European centers (77 operators) pulsed field ablation was performed in 1568 patients with AF: age 64.5±11.5 years, female 35%, paroxysmal/persistent AF 65%/32%, CHA2DS2-VASc 2.2±1.6, median left ventricular ejection fraction 60%, and left atrial diameter 42 mm. Pulmonary vein isolation was achieved in 99.2% of patients. After a median (interquartile range) follow-up of 367 (289-421) days, the 1-year Kaplan-Meier estimate for freedom from atrial arrhythmia was 78.1% (95% CI, 76.0%-80.0%); clinical effectiveness was more common in patients with paroxysmal AF versus persistent AF (81.6% versus 71.5%; P=0.001). Acute major adverse events occurred in 1.9% of patients. CONCLUSIONS: In this large observational registry of the postapproval clinical use of pulsed field technology to treat AF, catheter ablation using pulsed field energy was clinically effective in 78% of patients with AF.
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Fibrilação Atrial , Flutter Atrial , Ablação por Cateter , Veias Pulmonares , Humanos , Feminino , Pessoa de Meia-Idade , Idoso , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/cirurgia , Fibrilação Atrial/tratamento farmacológico , Estudos Retrospectivos , Volume Sistólico , Função Ventricular Esquerda , Resultado do Tratamento , Flutter Atrial/etiologia , Sistema de Registros , Ablação por Cateter/efeitos adversos , Ablação por Cateter/métodos , RecidivaRESUMO
Mucosal-associated invariant T (MAIT) cells are T cells that express a semi-invariant αß T-cell receptor (TCR), recognizing non-peptide antigens, such as microbial-derived vitamin B2 metabolites, presented by the nonpolymorphic MHC class I related-1 molecule. Like NKT cells and γδT cells, MAIT cells belong to the group of innate-like T cells that combine properties of the innate and adaptive immune systems. They account for up to 10% of the blood T-cell population in humans and are particularly abundant at mucosal sites. Beyond the emerging role of MAIT cells in antibacterial and antiviral defenses, increasing evidence suggests additional functions in noninfectious settings, including immune-mediated inflammatory diseases and tissue repair. Here, we discuss recent advances in the understanding of MAIT cell functions in sterile tissue inflammation, with a particular focus on autoimmunity, chronic inflammatory diseases, and tissue repair.
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Células T Invariantes Associadas à Mucosa , Humanos , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Inflamação , AutoimunidadeRESUMO
Viruses have evolved diverse strategies to evade the host innate immune response and promote infection. The retinoic acid-inducible gene I (RIG-I)-like receptors RIG-I and MDA5 are antiviral factors that sense viral RNA and trigger downstream signal via mitochondrial antiviral-signaling protein (MAVS) to activate type I interferon expression. 14-3-3ε is a key component of the RIG-I translocon complex that interacts with MAVS at the mitochondrial membrane; however, the exact role of 14-3-3ε in this pathway is not well understood. In this study, we demonstrate that 14-3-3ε is a direct substrate of both the poliovirus and coxsackievirus B3 (CVB3) 3C proteases (3Cpro) and that it is cleaved at Q236↓G237, resulting in the generation of N- and C-terminal fragments of 27.0 and 2.1 kDa, respectively. While the exogenous expression of wild-type 14-3-3ε enhances IFNB mRNA production during poly(I:C) stimulation, expression of the truncated N-terminal fragment does not. The N-terminal 14-3-3ε fragment does not interact with RIG-I in co-immunoprecipitation assays, nor can it facilitate RIG-I translocation to the mitochondria. Probing the intrinsically disordered C-terminal region identifies key residues responsible for the interaction between 14-3-3ε and RIG-I. Finally, overexpression of the N-terminal fragment promotes CVB3 infection in mammalian cells. The strategic enterovirus 3Cpro-mediated cleavage of 14-3-3ε antagonizes RIG-I signaling by disrupting critical interactions within the RIG-I translocon complex, thus contributing to evasion of the host antiviral response. IMPORTANCE Host antiviral factors work to sense virus infection through various mechanisms, including a complex signaling pathway known as the retinoic acid-inducible gene I (RIG-I)-like receptor pathway. This pathway drives the production of antiviral molecules known as interferons, which are necessary to establish an antiviral state in the cellular environment. Key to this antiviral signaling pathway is the small chaperone protein 14-3-3ε, which facilitates the delivery of a viral sensor protein, RIG-I, to the mitochondria. In this study, we show that the enteroviral 3C protease cleaves 14-3-3ε during infection, rendering it incapable of facilitating this antiviral response. We also find that the resulting N-terminal cleavage fragment dampens RIG-I signaling and promotes virus infection. Our findings reveal a novel viral strategy that restricts the antiviral host response and provides insights into the mechanisms underlying 14-3-3ε function in RIG-I antiviral signaling.
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Infecções por Picornaviridae , Picornaviridae , Animais , Cisteína Endopeptidases/metabolismo , Proteína DEAD-box 58/metabolismo , Imunidade Inata , Mamíferos , Peptídeo Hidrolases/metabolismo , Picornaviridae/metabolismo , Transdução de Sinais , Tretinoína , Proteínas Virais/metabolismo , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/virologia , Proteases Virais 3CRESUMO
Viruses have evolved mechanisms to modulate cellular pathways to facilitate infection. One such pathway is the formation of stress granules (SG), which are ribonucleoprotein complexes that assemble during translation inhibition following cellular stress. Inhibition of SG assembly has been observed under numerous virus infections across species, suggesting a conserved fundamental viral strategy. However, the significance of SG modulation during virus infection is not fully understood. The 1A protein encoded by the model dicistrovirus, Cricket paralysis virus (CrPV), is a multifunctional protein that can bind to and degrade Ago-2 in an E3 ubiquitin ligase-dependent manner to block the antiviral RNA interference pathway and inhibit SG formation. Moreover, the R146 residue of 1A is necessary for SG inhibition and CrPV infection in both Drosophila S2 cells and adult flies. Here, we uncoupled CrPV-1A's functions and provide insight into its underlying mechanism for SG inhibition. CrPV-1A mediated inhibition of SGs requires the E3 ubiquitin-ligase binding domain and the R146 residue, but not the Ago-2 binding domain. Wild-type but not mutant CrPV-1A R146A localizes to the nuclear membrane which correlates with nuclear enrichment of poly(A)+ RNA. Transcriptome changes in CrPV-infected cells are dependent on the R146 residue. Finally, Nup358/RanBP2 is targeted and degraded in CrPV-infected cells in an R146-dependent manner and the depletion of Nup358 blocks SG formation. We propose that CrPV utilizes a multiprong strategy whereby the CrPV-1A protein interferes with a nuclear event that contributes to SG inhibition in order to promote infection.
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Proteínas Virais , Replicação Viral , Animais , Proteínas Virais/metabolismo , Grânulos de Estresse , Linhagem Celular , Drosophila , Grânulos Citoplasmáticos/metabolismoRESUMO
Staphylococcus aureus is frequently detected in patients with sepsis and thus represents a major health burden worldwide. CD4+ T helper cells are involved in the immune response to S. aureus by supporting antibody production and phagocytosis. In particular, Th1 and Th17 cells secreting IFN-γ and IL-17A, are involved in the control of systemic S. aureus infections in humans and mice. To investigate the role of T cells in severe S. aureus infections, we established a mouse sepsis model in which the kidney was identified to be the organ with the highest bacterial load and abundance of Th17 cells. In this model, IL-17A but not IFN-γ was required for bacterial control. Using Il17aCre × R26YFP mice we could show that Th17 fate cells produce Th17 and Th1 cytokines, indicating a high degree of Th17 cell plasticity. Single cell RNA-sequencing of renal Th17 fate cells uncovered their heterogeneity and identified a cluster with a Th1 expression profile within the Th17 cell population, which was absent in mice with T-bet/Tbx21-deficiency in Th17 cells (Il17aCre x R26eYFP x Tbx21-flox). Blocking Th17 to Th1 transdifferentiation in Th17 fate cells in these mice resulted in increased S. aureus tissue loads. In summary, we highlight the impact of Th17 cells in controlling systemic S. aureus infections and show that T-bet expression by Th17 cells is required for bacterial clearance. While targeting the Th17 cell immune response is an important therapeutic option in autoimmunity, silencing Th17 cells might have detrimental effects in bacterial infections.
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Sepse , Infecções Estafilocócicas , Proteínas com Domínio T/metabolismo , Animais , Plasticidade Celular , Humanos , Interleucina-17 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Staphylococcus aureus , Células Th1 , Células Th17RESUMO
Although the cytokine interleukin 9 (IL-9) was discovered decades ago, it remains one of the most enigmatic cytokines identified so far, in particular because its functional activities remain far from clear. Breakthroughs made through the use of IL-9 reporter mice have allowed the identification of cell types that produce IL-9 in vivo and, contrary to expectations based on previous results obtained in vitro, it is not T cells but instead a previously unknown type of innate lymphoid cell, called the 'ILC2 cell', that is the main cell type that expresses IL-9 in vivo. In this perspective, we put forward a hypothesis about the potential biological functions of IL-9 in the immune system and beyond.
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Interleucina-9/imunologia , Células Th2/imunologia , Animais , Sobrevivência Celular/imunologia , Humanos , CamundongosRESUMO
Innate lymphoid cells (ILCs) that express NK cell receptors (NCRs) and the transcription factor T-bet populate nonlymphoid tissues and are crucial in immune responses against viral infections and malignancies. Recent studies highlighted the heterogeneity of this ILC population and extended their functional spectrum to include important roles in tissue homeostasis and autoimmunity. In this article, we provide detailed profiling of NCR+T-bet+ ILC populations in the murine kidney, identifying conventional NK (cNK) cells and type 1 ILCs (ILC1s) as the two major subsets. Induction of renal inflammation in a mouse model of glomerulonephritis did not substantially influence abundance or phenotype of cNK cells or ILC1s in the kidney. For functional analyses in this model, widely used depletion strategies for total NCR+ ILCs (anti-NK1.1 Ab application) and cNK cells (anti-asialoGM1 serum application) were unreliable tools, because they were accompanied by significant off-target depletion of kidney NKT cells and CD8+ T cells, respectively. However, neither depletion of cNK cells and ILC1s in NKT cell-deficient mice nor specific genetic deletion of cNK cells in Ncr1 Cre/wt × Eomes fl/fl mice altered the clinical course of experimental glomerulonephritis. In summary, we show in this article that cNK cells and ILC1s are dispensable for initiation and progression of immune-mediated glomerular disease and advise caution in the use of standard Ab depletion methods to study NCR+ ILC function in mouse models.
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Glomerulonefrite , Imunidade Inata , Animais , Linfócitos T CD8-Positivos , Rim , Células Matadoras Naturais , CamundongosRESUMO
Stroke results in neuronal cell death, which causes long-term disabilities in adults. Treatment options are limited and rely on a narrow window of opportunity. Apoptosis inhibitors demonstrate efficacy in improving neuronal cell survival in animal models of stroke. However, many inhibitors non-specifically target apoptosis pathways and high doses are needed for treatment. We explored the use of a novel caspase-3/7 inhibitor, New World Laboratories (NWL) 283, with a lower IC50 than current caspase-3/7 inhibitors. We performed in vitro and in vivo assays to determine the efficacy of NWL283 in modulating cell death in a preclinical model of stroke. In vitro and in vivo assays show that NWL283 enhances cell survival of neural precursor cells. Delivery of NWL283 following stroke enhances endogenous NPC migration and leads to increased neurogenesis in the stroke-injured cortex. Furthermore, acute NWL283 administration is neuroprotective at the stroke injury site, decreasing neuronal cell death and reducing microglia activation. Coincident with NWL283 delivery for 8 days, stroke-injured mice exhibited improved functional outcomes that persisted following cessation of the drug. Therefore, we propose that NWL283 is a promising therapeutic warranting further investigation to enhance stroke recovery.
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Isquemia Encefálica , AVC Isquêmico , Células-Tronco Neurais , Acidente Vascular Cerebral , Animais , Camundongos , Sobrevivência Celular , Caspase 3 , Acidente Vascular Cerebral/tratamento farmacológico , Apoptose , Neurogênese/fisiologia , Camundongos Endogâmicos C57BL , Isquemia Encefálica/tratamento farmacológicoRESUMO
Traumatic spinal cord injury (SCI) is a leading cause of permanent neurologic disabilities in young adults. Functional impairments after SCI are substantially attributed to the progressive neurodegeneration. However, regeneration of spinal-specific neurons and circuit re-assembly remain challenging in the dysregulated milieu of SCI because of impaired neurogenesis and neuronal maturation by neural precursor cells (NPCs) spontaneously or in cell-based strategies. The extrinsic mechanisms that regulate neuronal differentiation and synaptogenesis in SCI are poorly understood. Here, we perform extensive in vitro and in vivo studies to unravel that SCI-induced upregulation of matrix chondroitin sulfate proteoglycans (CSPGs) impedes neurogenesis of NPCs through co-activation of two receptor protein tyrosine phosphatases, LAR and PTPσ. In adult female rats with SCI, systemic co-inhibition of LAR and PTPσ promotes regeneration of motoneurons and spinal interneurons by engrafted human directly reprogramed caudalized NPCs (drNPC-O2) and fosters their morphologic maturity and synaptic connectivity within the host neural network that culminate in improved recovery of locomotion and sensorimotor integration. Our transcriptomic analysis of engrafted human NPCs in the injured spinal cord confirmed that inhibition of CSPG receptors activates a comprehensive program of gene expression in NPCs that can support neuronal differentiation, maturation, morphologic complexity, signal transmission, synaptic plasticity, and behavioral improvement after SCI. We uncovered that CSPG/LAR/PTPσ axis suppresses neuronal differentiation in part by blocking Wnt/ß-Catenin pathway. Taken together, we provide the first evidence that CSPGs/LAR/PTPσ axis restricts neurogenesis and synaptic integration of new neurons in NPC cellular therapies for SCI. We propose targeting LAR and PTPσ receptors offers a promising clinically-feasible adjunct treatment to optimize the efficacy and neurologic benefits of ongoing NPC-based clinical trials for SCI.SIGNIFICANCE STATEMENT Transplantation of neural precursor cells (NPCs) is a promising approach for replacing damaged neurons after spinal cord injury (SCI). However, survival, neuronal differentiation, and synaptic connectivity of transplanted NPCs within remain challenging in SCI. Here, we unravel that activation of chondroitin sulfate proteoglycan (CSPG)/LAR/PTPσ axis after SCI impedes the capacity of transplanted human NPCs for replacing functionally integrated neurons. Co-blockade of LAR and PTPσ is sufficient to promote re-generation of motoneurons and spinal V1 and V3 interneurons by engrafted human caudalized directly reprogramed NPCs (drNPC-O2) and facilitate their synaptic integration within the injured spinal cord. CSPG/LAR/PTPσ axis appears to suppress neuronal differentiation of NPCs by inhibiting Wnt/ß-Catenin pathway. These findings identify targeting CSPG/LAR/PTPσ axis as a promising strategy for optimizing neuronal replacement, synaptic re-connectivity, and neurologic recovery in NPC-based strategies.
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Células-Tronco Neurais , Traumatismos da Medula Espinal , Animais , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Feminino , Humanos , Neurônios Motores/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Ratos , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , beta CateninaRESUMO
Viral protein genome-linked (VPg) protein plays an essential role in protein-primed replication of plus-stranded RNA viruses. VPg is covalently linked to the 5' end of the viral RNA genome via a phosphodiester bond typically at a conserved amino acid. Whereas most viruses have a single VPg, some viruses have multiple VPgs that are proposed to have redundant yet undefined roles in viral replication. Here, we use cricket paralysis virus (CrPV), a dicistrovirus that has four nonidentical copies of VPg, as a model to characterize the role of VPg copies in infection. Dicistroviruses contain two main open reading frames (ORFs) that are driven by distinct internal ribosome entry sites (IRESs). We systematically generated single and combinatorial deletions and mutations of VPg1 to VPg4 within the CrPV infectious clone and monitored viral yield in Drosophila S2 cells. Deletion of one to three VPg copies progressively decreased viral yield and delayed viral replication, suggesting a threshold number of VPgs for productive infection. Mass spectrometry analysis of CrPV VPg-linked RNAs revealed viral RNA linkage to either a serine or threonine in VPg, mutations of which in all VPgs attenuated infection. Mutating serine 4 in a single VPg abolished viral infection, indicating a dominant negative effect. Using viral minigenome reporters that monitor dicistrovirus 5' untranslated (UTR) and IRES translation revealed a relationship between VPg copy number and the ratio of distinct IRES translation activities. We uncovered a novel viral strategy whereby VPg copies in dicistrovirus genomes compensate for the relative IRES translation efficiencies to promote infection. IMPORTANCE Genetic duplication is exceedingly rare in small RNA viral genomes, as there is selective pressure to prevent RNA genomes from expanding. However, some small RNA viruses encode multiple copies of a viral protein, most notably an unusual viral protein that is linked to the viral RNA genome. Here, we investigate a family of viruses that contains multiple viral protein genome-linked proteins and reveal a novel viral strategy whereby viral protein copy number counterbalances differences in viral protein synthesis mechanisms.
Assuntos
Dicistroviridae , Genoma Viral , Biossíntese de Proteínas , Infecções por Vírus de RNA , RNA Viral , Proteínas Virais , Regiões 5' não Traduzidas/genética , Animais , Linhagem Celular , Dicistroviridae/genética , Dicistroviridae/metabolismo , Drosophila/citologia , Drosophila/virologia , Genoma Viral/genética , Sítios Internos de Entrada Ribossomal/genética , Mutação , Infecções por Vírus de RNA/virologia , RNA Viral/genética , Serina/metabolismo , Treonina/metabolismo , Carga Viral , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
All viruses must usurp host ribosomes for viral protein synthesis. Dicistroviruses utilize an intergenic region internal ribosome entry site (IGR IRES) to directly recruit ribosomes and mediate translation initiation from a non-AUG start codon. The IGR IRES adopts a three-pseudoknot structure that comprises a ribosome binding domain of pseudoknot II and III (PKII and PKIII), and a tRNA-like anticodon domain (PKI) connected via a short, one to three nucleotide hinge region. Recent cryo-EM structural analysis of the dicistrovirus Taura syndrome virus (TSV) IGR IRES bound to the ribosome suggests that the hinge region may facilitate translocation of the IRES from the ribosomal A to P site. In this study, we provide mechanistic and functional insights into the role of the hinge region in IGR IRES translation. Using the honeybee dicistrovirus, Israeli acute paralysis virus (IAPV), as a model, we demonstrate that mutations of the hinge region resulted in decreased IRES-dependent translation in vitro. Toeprinting primer extension analysis of mutant IRESs bound to purified ribosomes and in rabbit reticulocyte lysates showed defects in the initial ribosome positioning on the IRES. Finally, using a hybrid dicistrovirus clone, mutations in the hinge region of the IAPV IRES resulted in decreased viral yield. Our work reveals an unexpected role of the hinge region of the dicistrovirus IGR IRES coordinating the two independently folded domains of the IRES to properly position the ribosome to start translation. IMPORTANCE Viruses must use the host cell machinery to direct viral protein expression for productive infection. One such mechanism is an internal ribosome entry site that can directly recruit host cell machinery. In this study, we have identified a novel sequence in an IRES that provides insight into the mechanism of viral gene expression. Specifically, this novel sequence promotes viral IRES activity by directly guiding the host cell machinery to start gene expression at a specific site.
Assuntos
Dicistroviridae , Sítios Internos de Entrada Ribossomal , Viroses , Vírus , Animais , Dicistroviridae/genética , Dicistroviridae/metabolismo , Sítios Internos de Entrada Ribossomal/genética , Mutação , Biossíntese de Proteínas , Coelhos , Ribossomos/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Viroses/metabolismo , Viroses/virologia , Vírus/genéticaRESUMO
AIM: To study the effect of sulfatide on gene expression and proliferation of human primary fibroblasts induced by insulin, insulin-like growth factor-1 and human growth hormone. MATERIALS AND METHODS: Human primary fibroblasts were exposed to 1, 3 and 30 µM of sulfatide or its precursor galactosylceramide (GalCer). Proliferation was determined by 3 H-thymidine incorporation and gene expression via microarray analysis. RESULTS: Sulfatide and GalCer reduced the growth rate of fibroblasts by 32%-82% when exposed to 0.5 nM insulin. After challenge with 120 µM of H2 O2 , sulfatide reduced membrane leakage. Fibroblast gene expression was altered by sulfatide in gene pathways associated with cell cycle/growth, transforming growth factor-ß function, and encoding of proteins involved in intracellular signalling. NFKBIA, a key control element in NF-кB regulation, was decreased 2-fold by sulfatide. CONCLUSIONS: Sulfatide strongly inhibits fibroblast growth. We therefore suggest the addition of sulfatide to injectable commercial insulin formulations, which would reduce adverse fibroblast growth and improve well-being in patients with diabetes.
Assuntos
Insulina , Sulfoglicoesfingolipídeos , Humanos , Insulina/farmacologia , Insulina/metabolismo , Sulfoglicoesfingolipídeos/metabolismo , Sulfoglicoesfingolipídeos/farmacologia , Insulina Regular Humana , Fibroblastos/metabolismo , Estresse OxidativoRESUMO
BACKGROUND: Pulsed-field ablation (PFA) is a novel ablation modality for atrial fibrillation (AF) ablating myocardium by electroporation without tissue-heating. With its different mechanism of tissue ablation, it is assumed that lesion creation is divergent to thermal energy sources. 68Ga-fibroblast-activation protein inhibitor (FAPI) PET/CT targets FAP-alpha expressed by activated fibroblasts. We aimed to assess 68Ga-FAPI uptake in pulmonary veins as surrogate for ablation damage after PFA and cryoballoon ablation (CBA). METHODS: 26 patients (15 PFA, 11 CBA) underwent 68Ga-FAPI-PET/CT after ablation. Standardized uptake values (SUV) and fibroblast-activation volumes of localized tracer uptake were assessed. RESULTS: Patient characteristics were comparable between groups. In PFA, focal FAPI uptake was only observed in 3/15 (20%) patients, whereas in the CBA cohort, 10/11 (90.9%) patients showed atrial visual uptake. We observed lower values of SUVmax (2.85 ± 0.56 vs 4.71 ± 2.06, P = 0.025) and FAV (1.13 ± 0.84 cm3 vs 3.91 ± 2.74 cm3, P = 0.014) along with a trend towards lower SUVpeak and SUVmean in PFA vs CBA patients, respectively. CONCLUSION: Tissue response with respect to fibroblast activation seems to be less pronounced in PFA compared to established thermal ablation systems. This functional assessment might contribute to a better understanding of lesion formation in thermal and PFA ablation potentially contributing to better safety outcomes.
Assuntos
Veias Pulmonares , Humanos , Veias Pulmonares/diagnóstico por imagem , Veias Pulmonares/cirurgia , Radioisótopos de Gálio , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Terapia com Eletroporação , FibroblastosRESUMO
Apoptosis is a critical process for the maintenance of cell populations, and involves mitochondrial depolarization, the sequential cleavage of caspase-9 and -3, followed by the externalization of phosphatidylserine (PS) on the plasma membrane. The actin cytoskeleton and its accessory proteins are known regulators of apoptotic signaling in nucleated cells but their roles in platelet apoptosis are undefined. Filamin A (FLNA) is a ubiquitously expressed actin-crosslinking protein that also serves as an intracellular signaling scaffold. Here we used platelets from mice with a platelet-specific FLNA deficiency (Flnafl/Y, Pf4-cre/+, termed platelet-specific knockout) to test the role of FLNA in platelet apoptosis. Treatment with the BH3-mimetic drug ABT-737 induced caspase-3 cleavage and PS exposure in platelets from floxed mice (Flnafl/Y, termed control) but these effects were essentially abrogated in FLNA-null platelets (platelet-specific knockout). Protein kinase C (PKC), a known FLNA ligand, was also activated by ABT-737, and PKC's phosphorylation of its downstream substrates was attenuated in FLNA-null platelets. The PKC inhibitor bisindolylmaleimide (BIM) also reduced caspase-3 cleavage, thus essentially phenocopying the FLNA-null platelets. Notably, the caspase-3 cleavage defect in FLNA-null platelets was rescued by the PKC-activating phorbol ester PMA, suggesting that FLNA and PKC share a common pathway in regulating platelet apoptosis. Mitochondrial depolarization and caspase-9 cleavage were unaffected by BIM treatment, suggesting that PKC specifically controls the downstream caspase-3 point of the pro-apoptotic signaling pathway. These data point to a novel role for FLNA in the regulation of platelet apoptosis, thus providing an improved understanding of how circulating platelet counts are maintained.