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1.
Nature ; 631(8021): 645-653, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38987596

RESUMEN

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.


Asunto(s)
Células Dendríticas , Homeostasis , Megacariocitos , Trombopoyesis , Animales , Femenino , Humanos , Masculino , Ratones , Apoptosis , Plaquetas/citología , Médula Ósea , Linaje de la Célula , Proliferación Celular , Células Dendríticas/inmunología , Células Dendríticas/citología , Retroalimentación Fisiológica , Inmunidad Innata , Microscopía Intravital , Megacariocitos/citología , Megacariocitos/inmunología , Ratones Endogámicos C57BL , SARS-CoV-2/inmunología , COVID-19/inmunología , COVID-19/fisiopatología , COVID-19/virología
2.
J Biol Chem ; 300(2): 105648, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38219816

RESUMEN

Cellular plasticity is crucial for adapting to ever-changing stimuli. As a result, cells consistently reshape their translatome, and, consequently, their proteome. The control of translational activity has been thoroughly examined at the stage of translation initiation. However, the regulation of ribosome speed in cells is widely unknown. In this study, we utilized a timed ribosome runoff approach, along with proteomics and transmission electron microscopy, to investigate global translation kinetics in cells. We found that ribosome speeds vary among various cell types, such as astrocytes, induced pluripotent human stem cells, human neural stem cells, and human and rat neurons. Of all cell types studied, mature cortical neurons exhibit the highest rate of translation. This finding is particularly remarkable because mature cortical neurons express the eukaryotic elongation factor 2 (eEF2) at lower levels than other cell types. Neurons solve this conundrum by inactivating a fraction of their ribosomes. As a result, the increase in eEF2 levels leads to a reduction of inactive ribosomes and an enhancement of active ones. Processes that alter the demand for active ribosomes, like neuronal excitation, cause increased inactivation of redundant ribosomes in an eEF2-dependent manner. Our data suggest a novel regulatory mechanism in which neurons dynamically inactivate ribosomes to facilitate translational remodeling. These findings have important implications for developmental brain disorders characterized by, among other things, aberrant translation.


Asunto(s)
Biosíntesis de Proteínas , Ribosomas , Animales , Humanos , Ratas , Neuronas/metabolismo , Procesamiento Proteico-Postraduccional , Ribosomas/metabolismo , Ratones , Ratones Endogámicos C57BL
3.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-35105800

RESUMEN

Apoptosis is widely believed to be crucial for epithelial cell death and shedding in the intestine, thereby shaping the overall architecture of the gastrointestinal tract, but also regulating tolerance induction, pinpointing a role of apoptosis intestinal epithelial cell (IEC) turnover and maintenance of barrier function, and in maintaining immune homeostasis. To experimentally address this concept, we generated IEC-specific knockout mice that lack both executioner caspase-3 and caspase-7 (Casp3/7ΔIEC), which are the converging point of the extrinsic and intrinsic apoptotic pathway. Surprisingly, the overall architecture, cellular landscape, and proliferation rate remained unchanged in these mice. However, nonapoptotic cell extrusion was increased in Casp3/7ΔIEC mice, compensating apoptosis deficiency, maintaining the same physiological level of IEC shedding. Microbiome richness and composition stayed unaffected, bearing no sign of dysbiosis. Transcriptome and single-cell RNA sequencing analyses of IECs and immune cells revealed no differences in signaling pathways of differentiation and inflammation. These findings demonstrate that during homeostasis, apoptosis per se is dispensable for IEC turnover at the top of intestinal villi intestinal tissue dynamics, microbiome, and immune cell composition.


Asunto(s)
Apoptosis , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Células Epiteliales/enzimología , Homeostasis , Mucosa Intestinal/enzimología , Transducción de Señal , Animales , Caspasa 3/genética , Caspasa 7/genética , Ratones , Ratones Transgénicos
4.
Proteomics ; 24(10): e2300384, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38185761

RESUMEN

The alpha7 nicotinic acetylcholine receptor (α7 nAChR; CHRNA7) is expressed in the nervous system and in non-neuronal tissues. Within the central nervous system, it is involved in various cognitive and sensory processes such as learning, attention, and memory. It is also expressed in the cerebellum, where its roles are; however, not as well understood as in the other brain regions. To investigate the consequences of absence of CHRNA7 on the cerebellum proteome, we performed a quantitative nano-LC-MS/MS analysis of samples from CHRNA7 knockout (KO) mice and corresponding wild type (WT) controls. Liver, an organ which does not express this receptor, was analyzed, in comparison. While the liver proteome remained relatively unaltered (three proteins more abundant in KOs), 90 more and 20 less abundant proteins were detected in the cerebellum proteome of the KO mice. The gene ontology analysis of the differentially abundant proteins indicates that the absence of CHRNA7 leads to alterations in the glutamatergic system and myelin sheath in the cerebellum. In conclusion, our dataset provides new insights in the role of CHRNA7 in the cerebellum, which may serve as a basis for future in depth-investigations.


Asunto(s)
Cerebelo , Proteoma , Receptor Nicotínico de Acetilcolina alfa 7 , Animales , Ratones , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/genética , Cerebelo/metabolismo , Cromatografía Liquida/métodos , Hígado/metabolismo , Ratones Noqueados , Proteoma/metabolismo , Proteoma/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem
5.
J Am Soc Nephrol ; 34(3): 394-411, 2023 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-36857499

RESUMEN

SIGNIFICANCE STATEMENT: Cells undergoing necrosis release extracellular high mobility group box (HMGB)-1, which triggers sterile inflammation upon AKI in mice. Neither deletion of HMGB1 from tubular epithelial cells, nor HMGB1 antagonism with small molecules, affects initial ischemic tubular necrosis and immediate GFR loss upon unilateral ischemia/reperfusion injury (IRI). On the contrary, tubular cell-specific HMGB1 deficiency, and even late-onset pharmacological HMGB1 inhibition, increased functional and structural recovery from AKI, indicating that intracellular HMGB1 partially counters the effects of extracellular HMGB1. In vitro studies indicate that intracellular HMGB1 decreases resilience of tubular cells from prolonged ischemic stress, as in unilateral IRI. Intracellular HMGB1 is a potential target to enhance kidney regeneration and to improve long-term prognosis in AKI. BACKGROUND: Late diagnosis is a hurdle for treatment of AKI, but targeting AKI-CKD transition may improve outcomes. High mobility group box-1 (HMGB1) is a nuclear regulator of transcription and a driver of necroinflammation in AKI. We hypothesized that HMGB1 would also modulate AKI-CKD transition in other ways. METHODS: We conducted single-cell transcriptome analysis of human and mouse AKI and mouse in vivo and in vitro studies with tubular cell-specific depletion of Hmgb1 and HMGB1 antagonists. RESULTS: HMGB1 was ubiquitously expressed in kidney cells. Preemptive HMGB1 antagonism with glycyrrhizic acid (Gly) and ethyl pyruvate (EP) did not affect postischemic AKI but attenuated AKI-CKD transition in a model of persistent kidney hypoxia. Consistently, tubular Hmgb1 depletion in Pax8 rtTA, TetO Cre, Hmgb1fl/fl mice did not protect from AKI, but from AKI-CKD transition. In vitro studies confirmed that absence of HMGB1 or HMGB1 inhibition with Gly and EP does not affect ischemic necrosis of growth-arrested differentiated tubular cells but increased the resilience of cycling tubular cells that survived the acute injury to oxidative stress. This effect persisted when neutralizing extracellular HMGB1 with 2G7. Consistently, late-onset HMGB1 blockade with EP started after the peak of ischemic AKI in mice prevented AKI-CKD transition, even when 2G7 blocked extracellular HMGB1. CONCLUSION: Treatment of AKI could become feasible when ( 1 ) focusing on long-term outcomes of AKI; ( 2 ) targeting AKI-CKD transition with drugs initiated after the AKI peak; and ( 3 ) targeting with drugs that block HMGB1 in intracellular and extracellular compartments.


Asunto(s)
Lesión Renal Aguda , Proteína HMGB1 , Insuficiencia Renal Crónica , Humanos , Animales , Ratones , Riñón , Regeneración , Células Epiteliales , Estrés Oxidativo , Ácido Glicirrínico
6.
Reproduction ; 166(3): 221-234, 2023 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-37432973

RESUMEN

In brief: Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is expressed by various murine ovarian cells. Morphological and molecular investigations, including a proteomic study of adult Chrna7 knockout (KO) mouse ovaries, reveal the roles of these receptors in the local regulation of the ovary. Abstract: Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is involved in cellular functions ranging from synaptic transmission in neurons to regulation of inflammation, cell growth and metabolism to cell death in other cells. Our qPCR results and other studies indicated that nAChRa7 is expressed in the adult mouse ovary, while in situ hybridization and single-cell sequencing data suggested this expression may be shared by several ovarian cells, including fibroblast-like and steroidogenic stroma cells, macrophages and oocytes of small follicles. To explore a possible involvement of nAChRa7 in ovarian functions, we evaluated ovarian morphology of Chrna7-null mutant adult mice (KO) and wildtype mice (WT; 3 months, metestrus) by performing immunohistochemistry, qPCR studies, measurements of serum progesterone and proteomic analyses. The evaluation of serial sections indicated fewer primordial follicles but similar numbers of primary, secondary and tertiary follicles, as well as corpora lutea in KO and WT mice. Atresia was unchanged. Serum progesterone and mRNA levels of proliferation and most apoptosis markers were not changed, yet two typical macrophage markers were elevated. Furthermore, the proteomes of KO ovaries were significantly altered with 96 proteins increased and 32 decreased in abundance in KOs compared to WTs. Among the elevated proteins were markers for stroma cells. Hence, the lack of nAChRa7 causes changes in small follicle counts and alterations of the ovarian stroma cells. The ovarian phenotype of Chrna7 mutant mice links this channel protein to the local regulation of ovarian cells, including stroma cells.


Asunto(s)
Ovario , Receptores Nicotínicos , Animales , Femenino , Ratones , Ratones Noqueados , Ovario/metabolismo , Fenotipo , Progesterona/metabolismo , Proteómica , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo
7.
Mol Cancer ; 21(1): 199, 2022 10 13.
Artículo en Inglés | MEDLINE | ID: mdl-36229873

RESUMEN

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer entities including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity.Here, we show in the EwS model that - capitalizing on neomorphic DNA-binding preferences - the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes.We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor entities typically considered to be immunologically 'cold'.Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.


Asunto(s)
Sarcoma de Ewing , Sarcoma , Antígenos de Superficie/uso terapéutico , Línea Celular Tumoral , Niño , ADN , Ganciclovir/uso terapéutico , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-15/genética , Interleucina-15/metabolismo , Interleucina-15/uso terapéutico , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Sarcoma/genética , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/terapia , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Timidina Quinasa/uso terapéutico
8.
Glia ; 69(1): 165-181, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32744730

RESUMEN

Unlike microglia and NG2 glia, astrocytes are incapable of migrating to sites of injury in the posttraumatic cerebral cortex, instead relying on proliferation to replenish their numbers and distribution in the affected region. However, neither the spectrum of their proliferative repertoire nor their postinjury distribution has been examined in vivo. Using a combination of different thymidine analogs and clonal analysis in a model of repetitive traumatic brain injury, we show for the first time that astrocytes that are quiescent following an initial injury can be coerced to proliferate after a repeated insult in the cerebral cortex grey matter. Interestingly, this process is promoted by invasion of monocytes to the injury site, as their genetic ablation (using CCR2-/- mice) increased the number of repetitively dividing astrocytes at the expense of newly proliferating astrocytes in repeatedly injured parenchyma. These differences profoundly affected both the distribution of astrocytes and recovery period for posttraumatic behavior deficits suggesting key roles of astrocyte self-renewal in brain repair after injury.


Asunto(s)
Astrocitos , Animales , Lesiones Traumáticas del Encéfalo , Ratones , Ratones Endogámicos C57BL , Monocitos , Neuroglía
9.
Mol Hum Reprod ; 27(7)2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-33993290

RESUMEN

Extracellular ATP has been described to be involved in inflammatory cytokine production by human testicular peritubular cells (HTPCs). The ectonucleotidases ENTPD1 and NT5E degrade ATP and have been reported in rodent testicular peritubular cells. We hypothesized that if a similar situation exists in human testis, ATP metabolites may contribute to cytokine production. Indeed, ENTPD1 and NT5E were found in situ and in vitro in HTPCs. Malachite green assays confirmed enzyme activities in HTPCs. Pharmacological inhibition of ENTPD1 (by POM-1) significantly reduced pro-inflammatory cytokines evoked by ATP treatment, suggesting that metabolites of ATP, including adenosine, are likely involved. We focused on adenosine and detected three of the four known adenosine receptors in HTPCs. One, A2B, was also found in situ in peritubular cells of human testicular sections. The A2B agonist BAY60-6583 significantly elevated levels of IL6 and CXCL8, a result also obtained with adenosine and its analogue NECA. Results of siRNA-mediated A2B down-regulation support a role of this receptor. In mouse peritubular cells, in contrast to HTPCs, all four of the known adenosine receptors were detected; when challenged with adenosine, cytokine expression levels significantly increased. Organotypic short-term testis cultures yielded comparable results and indicate an overall pro-inflammatory action of adenosine in the mouse testis. If transferable to the in vivo situation, our results may implicate that interference with the generation of ATP metabolites or interference with adenosine receptors could reduce inflammatory events in the testis. These novel insights may provide new avenues for treatment of sterile inflammation in male subfertility and infertility.


Asunto(s)
Adenosina/fisiología , Testículo/metabolismo , 5'-Nucleotidasa/metabolismo , Adenosina/farmacología , Adenosina Trifosfato/metabolismo , Adenosina-5'-(N-etilcarboxamida)/farmacología , Adulto , Aminopiridinas/farmacología , Animales , Apirasa/antagonistas & inhibidores , Apirasa/fisiología , Células Cultivadas , Citocinas/metabolismo , Proteínas Ligadas a GPI/metabolismo , Humanos , Infertilidad Masculina/metabolismo , Infertilidad Masculina/terapia , Inflamación , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Receptor de Adenosina A2B/fisiología , Receptores Purinérgicos P1/análisis , Receptores Purinérgicos P1/metabolismo , Testículo/citología
10.
Brain Behav Immun ; 95: 429-443, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33895286

RESUMEN

Loss of appetite (anorexia) is a typical behavioral response to infectious diseases that often reduces body weight. Also, anorexia can be observed in cancer and trauma patients, causing poor quality of life and reduced prospects of positive therapeutic outcomes. Although anorexia is an acute symptom, its initiation and endocrine regulation during antiviral immune responses are poorly understood. During viral infections, plasmacytoid dendritic cells (pDCs) produce abundant type I interferon (IFN-I) to initiate first-line defense mechanisms. Here, by targeted ablation of pDCs and various in vitro and in vivo mouse models of viral infection and inflammation, we identified that IFN-I is a significant driver of somatostatin (SST). Consequently, SST suppressed the hunger hormone ghrelin that led to severe metabolic changes, anorexia, and rapid body weight loss. Furthermore, during vaccination with Modified Vaccinia Ankara virus (MVA), the SST-mediated suppression of ghrelin was critical to viral immune response, as ghrelin restrained the production of early cytokines by natural killer (NK) cells and pDCs, and impaired the clonal expansion of CD8+ T cells. Thus, the hormonal modulation of ghrelin through SST and the cytokine IFN-I is fundamental for optimal antiviral immunity, which comes at the expense of calorie intake.


Asunto(s)
Apetito , Ghrelina , Interferón Tipo I/inmunología , Somatostatina/inmunología , Virosis/inmunología , Animales , Linfocitos T CD8-positivos , Células Dendríticas , Inmunidad Innata , Ratones , Calidad de Vida
11.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-34445704

RESUMEN

RNA-binding proteins (RBPs) are essential regulators controlling both the cellular transcriptome and translatome. These processes enable cellular plasticity, an important prerequisite for growth. Cellular growth is a complex, tightly controlled process. Using cancer cells as model, we looked for RBPs displaying strong expression in published transcriptome datasets. Interestingly, we found the Pumilio (Pum) protein family to be highly expressed in all these cells. Moreover, we observed that Pum2 is regulated by basic fibroblast growth factor (bFGF). bFGF selectively enhances protein levels of Pum2 and the eukaryotic initiation factor 4E (eIF4E). Exploiting atomic force microscopy and in vitro pulldown assays, we show that Pum2 selects for eIF4E mRNA binding. Loss of Pum2 reduces eIF4E translation. Accordingly, depletion of Pum2 led to decreased soma size and dendritic branching of mature neurons, which was accompanied by a reduction in essential growth factors. In conclusion, we identify Pum2 as an important growth factor for mature neurons. Consequently, it is tempting to speculate that Pum2 may promote cancer growth.


Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Factor 4E Eucariótico de Iniciación/genética , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Expresión Génica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía de Fuerza Atómica/métodos , Neurogénesis/fisiología , Unión Proteica/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Transcriptoma/genética
12.
Cell Tissue Res ; 381(1): 83-98, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32140928

RESUMEN

It has been established that inflammation plays an important role in bone formation and bone loss. Although a lot is known about the role of TNF-α in bone health, very little is understood about TNF-ß, also called lymphotoxin. In this report, we examine the effect of TNF-ß on osteogenic differentiation of mesenchymal stem cells (MSCs) and its modulation by resveratrol. Monolayer and high-density cultures of MSCs were treated with osteogenic induction medium with/without TNF-ß, Sirt1 inhibitor nicotinamide (NAM), antisense oligonucleotides against Sirt1 (ASO) and/or Sirt1 stimulator resveratrol. We found that TNF-ß inhibits, in a similar way to NAM or Sirt1-ASO, the early stage of osteogenic differentiation of MSCs and this was accompanied with downregulation of bone-specific matrix, ß1-integrin, Runx2 and with upregulation of NF-κB phosphorylation and NF-κB-regulated gene products involved in the inflammatory, degradative processes and apoptosis. However, resveratrol reversed TNF-ß- and NAM-suppressed MSCs osteogenesis by activation of Sirt1 and Runx2 that led to osteoblast differentiation. Furthermore, downregulation of Sirt1 by mRNA inhibited the effect of resveratrol, highlighting the important impact of this enzyme in the TNF-ß signaling pathway. Finally, resveratrol was able to manifest its effect both by suppression of TNF-ß-induced NF-κB and through direct activation of the Sirt1 and Runx2 pathway. Thus, through these studies, we present a mechanism by which a T cell-derived cytokine, TNF-ß can affect bone formation through modulation of MSCs differentiation that involves NF-κB, Sirt1, Runx2 and resveratrol reversed TNF-ß-promoted impairments in MSCs osteogenesis.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Linfotoxina beta/farmacología , Células Madre Mesenquimatosas/citología , Osteoblastos , Osteogénesis , Resveratrol/farmacología , Animales , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Perros , FN-kappa B/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Sirtuina 1/metabolismo
13.
J Am Soc Nephrol ; 30(10): 1857-1869, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31296606

RESUMEN

BACKGROUND: Serum oxalate levels suddenly increase with certain dietary exposures or ethylene glycol poisoning and are a well known cause of AKI. Established contributors to oxalate crystal-induced renal necroinflammation include the NACHT, LRR and PYD domains-containing protein-3 (NLRP3) inflammasome and mixed lineage kinase domain-like (MLKL) protein-dependent tubule necroptosis. These studies examined the role of a novel form of necrosis triggered by altered mitochondrial function. METHODS: To better understand the molecular pathophysiology of oxalate-induced AIK, we conducted in vitro studies in mouse and human kidney cells and in vivo studies in mice, including wild-type mice and knockout mice deficient in peptidylprolyl isomerase F (Ppif) or deficient in both Ppif and Mlkl. RESULTS: Crystals of calcium oxalate, monosodium urate, or calcium pyrophosphate dihydrate, as well as silica microparticles, triggered cell necrosis involving PPIF-dependent mitochondrial permeability transition. This process involves crystal phagocytosis, lysosomal cathepsin leakage, and increased release of reactive oxygen species. Mice with acute oxalosis displayed calcium oxalate crystals inside distal tubular epithelial cells associated with mitochondrial changes characteristic of mitochondrial permeability transition. Mice lacking Ppif or Mlkl or given an inhibitor of mitochondrial permeability transition displayed attenuated oxalate-induced AKI. Dual genetic deletion of Ppif and Mlkl or pharmaceutical inhibition of necroptosis was partially redundant, implying interlinked roles of these two pathways of regulated necrosis in acute oxalosis. Similarly, inhibition of mitochondrial permeability transition suppressed crystal-induced cell death in primary human tubular epithelial cells. PPIF and phosphorylated MLKL localized to injured tubules in diagnostic human kidney biopsies of oxalosis-related AKI. CONCLUSIONS: Mitochondrial permeability transition-related regulated necrosis and necroptosis both contribute to oxalate-induced AKI, identifying PPIF as a potential molecular target for renoprotective intervention.


Asunto(s)
Lesión Renal Aguda/patología , Necrosis por Permeabilidad de la Transmembrana Mitocondrial , Necroptosis , Lesión Renal Aguda/inducido químicamente , Animales , Células Cultivadas , Humanos , Masculino , Ratones , Oxalatos/administración & dosificación
14.
Int J Mol Sci ; 21(10)2020 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-32429128

RESUMEN

The hippocampus is central for higher cognition and emotions. In patients suffering from neuropsychiatric or neurodegenerative diseases, hippocampal signaling is altered causing cognitive defects. Thus, therapeutic approaches aim at improving cognition by targeting the hippocampus. Enhanced physical activity (EPA) improves cognition in rodents and humans. A systematic screen, however, for expression changes in the hippocampus along the dorso-ventral axis is missing, which is a prerequisite for understanding molecular mechanisms. Here, we exploited label free mass spectrometry to detect proteomic changes in the hippocampus of male mice upon voluntary wheel running. To identify regional differences, we examined dorsal and ventral CA1, CA3 and dentate gyrus hippocampal subregions. We found metabolic enzymes and actin binding proteins, such as RhoA, being upregulated in the hippocampus upon EPA suggesting a coordination between metabolism and cytoskeleton remodeling; two pathways essential for synaptic plasticity. Strikingly, dorsal and ventral hippocampal subregions respond differentially to EPA. Together, our results provide new insight into proteomic adaptations driven by physical activity in mice. In addition, our results suggest that dorsal and ventral hippocampus, as well as hippocampal subregions themselves, contribute differently to this process. Our study therefore provides an important resource for studying hippocampal subregion diversity in response to EPA.


Asunto(s)
Hipocampo/metabolismo , Actividad Motora , Proteoma/metabolismo , Envejecimiento/fisiología , Animales , Masculino , Espectrometría de Masas , Ratones Endogámicos C57BL , Neurogénesis , Condicionamiento Físico Animal , Proteómica
15.
Int J Mol Sci ; 21(7)2020 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-32244288

RESUMEN

OBJECTIVE: The majority of chemotherapeutic agents stimulate NF-κB signaling that mediates cell survival, proliferation and metastasis. The natural turmeric non-curcuminoid derivate Calebin A has been shown to suppress cell growth, invasion and colony formation in colorectal cancer cells (CRC) by suppression of NF-κB signaling. Therefore, we hypothesized here that Calebin A might chemosensitize the TNF-ß-treated tumor cells and potentiates the effect of 5-Fluorouracil (5-FU) in advanced CRC. MATERIALS AND METHODS: CRC cells (HCT116) and their clonogenic 5-FU chemoresistant counterparts (HCT116R) were cultured in monolayer or alginate-based 3D tumor environment culture and were treated with/without Calebin A, TNF-ß, 5-FU, BMS-345541 and DTT (dithiothreitol). RESULTS: The results showed that TNF-ß increased proliferation, invasion and resistance to apoptosis in chemoresistant CRC cells. Pretreatment with Calebin A significantly chemosensitized HCT116R to 5-FU and inhibited the TNF-ß-induced enhanced efforts for survival, invasion and anti-apoptotic effects. We found further that Calebin A significantly suppressed TNF-ß-induced phosphorylation and nuclear translocation of p65-NF-κB, similar to BMS-345541 (specific IKK inhibitor) and NF-κB-induced tumor-promoting biomarkers (NF-κB, ß1-Integrin, MMP-9, CXCR4, Ki67). This was associated with increased apoptosis in HCT116 and HCT116R cells. Furthermore, blocking of p65-NF-κB stimulation by Calebin A was imparted through the downmodulation of p65-NF-κB binding to the DNA and this suppression was turned by DTT. CONCLUSION: Our findings indicate, for the first time, that Calebin A chemosensitizes human CRC cells to chemotherapy by targeting of the p65-NF-κB signaling pathway.


Asunto(s)
Cinamatos/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Fluorouracilo/metabolismo , Linfotoxina-alfa/metabolismo , Monoterpenos/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Cinamatos/farmacología , Neoplasias del Colon/patología , Neoplasias Colorrectales/patología , Fluorouracilo/farmacología , Humanos , Linfotoxina-alfa/farmacología , FN-kappa B/metabolismo , Fosforilación , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo
16.
Trends Genet ; 32(12): 788-800, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27817871

RESUMEN

Local protein expression at synapses is a prerequisite for learning in mammalian neurons. It has been shown that a subset of RNAs is localized in dendrites. These transcripts are first assembled into ribonucleoprotein particles in the cell body and are then transported along the cytoskeleton to or near synapses in a translationally repressed state. However, we know very little about the underlying mechanisms of local translation as well as potential protein degradation. Research in the last years showed many features of general translation. One very interesting aspect with raising attention is co-translational folding, a process that guides protein folding during ribosome elongation. In this review, we propose that translation speed is influenced by the codon usage of localized transcripts, which in turn affects protein folding and ultimately degradation efficiency. Together, these processes significantly contribute to synaptic proteome changes and synaptic plasticity. Furthermore, we envision that co-translational misfolding could contribute to neurodegenerative diseases.


Asunto(s)
Neuronas , Biosíntesis de Proteínas/genética , Procesamiento Proteico-Postraduccional/genética , ARN/genética , Animales , Dendritas/genética , Regulación del Desarrollo de la Expresión Génica , Mamíferos/genética , Plasticidad Neuronal/genética , Proteoma/genética
17.
Cell Commun Signal ; 17(1): 87, 2019 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-31358011

RESUMEN

BACKGROUND: The understanding of lysosomes has been expanded in recent research way beyond their view as cellular trash can. Lysosomes are pivotal in regulating metabolism, endocytosis and autophagy and are implicated in cancer. Recently it was discovered that the lysosomal V-ATPase, which is known to induce apoptosis, interferes with lipid metabolism in cancer, yet the interplay between these organelles is poorly understood. METHODS: LC-MS/MS analysis was performed to investigate lipid distribution in cells. Cell survival and signaling pathways were analyzed by means of cell biological methods (qPCR, Western Blot, flow cytometry, CellTiter-Blue). Mitochondrial structure was analyzed by confocal imaging and electron microscopy, their function was determined by flow cytometry and seahorse measurements. RESULTS: Our data reveal that interfering with lysosomal function changes composition and subcellular localization of triacylglycerids accompanied by an upregulation of PGC1α and PPARα expression, master regulators of energy and lipid metabolism. Furthermore, cardiolipin content is reduced driving mitochondria into fission, accompanied by a loss of membrane potential and reduction in oxidative capacity, which leads to a deregulation in cellular ROS and induction of mitochondria-driven apoptosis. Additionally, cells undergo a metabolic shift to glutamine dependency, correlated with the fission phenotype and sensitivity to lysosomal inhibition, most prominent in Ras mutated cells. CONCLUSION: This study sheds mechanistic light on a largely uninvestigated triangle between lysosomes, lipid metabolism and mitochondrial function. Insight into this organelle crosstalk increases our understanding of mitochondria-driven cell death. Our findings furthermore provide a first hint on a connection of Ras pathway mutations and sensitivity towards lysosomal inhibitors.


Asunto(s)
Muerte Celular , Metabolismo de los Lípidos , Mitocondrias/metabolismo , Muramidasa/metabolismo , Línea Celular Tumoral , Humanos , Estrés Oxidativo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores
18.
Int J Mol Sci ; 20(7)2019 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-30979012

RESUMEN

Staufen2 (Stau2) is an RNA-binding protein that is involved in dendritic spine morphogenesis and function. Several studies have recently investigated the role of Stau2 in the regulation of its neuronal target mRNAs, with particular focus on the hippocampus. Here, we provide evidence for Stau2 expression and function in cerebellar Purkinje cells. We show that Stau2 downregulation (Stau2GT) led to an increase of glutamate receptor ionotropic delta subunit 2 (GluD2) in Purkinje cells when animals performed physical activity by voluntary wheel running compared with the age-matched wildtype (WT) mice (C57Bl/6J). Furthermore, Stau2GT mice showed lower performance in motor coordination assays but enhanced motor learning abilities than did WT mice, concomitantly with an increase in dendritic GluD2 expression. Together, our results suggest the novel role of Stau2 in Purkinje cell synaptogenesis in the mouse cerebellum.


Asunto(s)
Envejecimiento , Encéfalo/fisiología , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Células de Purkinje/metabolismo , Proteínas de Unión al ARN/genética , Receptores de Glutamato/genética , Animales , Cerebelo/citología , Cerebelo/fisiología , Femenino , Eliminación de Gen , Masculino , Ratones Endogámicos C57BL , Actividad Motora , Células de Purkinje/citología , ARN Mensajero/genética , Receptores de Glutamato/análisis
19.
J Proteome Res ; 17(4): 1677-1689, 2018 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-29560722

RESUMEN

High-dose ionizing radiation is known to induce adverse effects such as inflammation and fibrosis in the heart. Transcriptional regulators PPARα and TGFß are known to be involved in this radiation response. PPARα, an anti-inflammatory transcription factor controlling cardiac energy metabolism, is inactivated by irradiation. The pro-inflammatory and pro-fibrotic TGFß is activated by irradiation via SMAD-dependent and SMAD-independent pathways. The goal of this study was to investigate how altering the level of PPARα influences the radiation response of these signaling pathways. For this purpose, we used genetically modified C57Bl/6 mice with wild type (+/+), heterozygous (+/-) or homozygous (-/-) PPARα genotype. Mice were locally irradiated to the heart using doses of 8 or 16 Gy; the controls were sham-irradiated. The heart tissue was investigated using label-free proteomics 20 weeks after the irradiation and the predicted pathways were validated using immunoblotting, ELISA, and immunohistochemistry. The heterozygous PPARα mice showed most radiation-induced changes in the cardiac proteome, whereas the homozygous PPARα mice showed the least changes. Irradiation induced SMAD-dependent TGFß signaling independently of the PPARα status, but the presence of PPARα was necessary for the activation of the SMAD-independent pathway. These data indicate a central role of PPARα in cardiac response to ionizing radiation.


Asunto(s)
Corazón/efectos de la radiación , Miocardio/metabolismo , PPAR alfa/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Genotipo , Heterocigoto , Ratones , Ratones Endogámicos C57BL , Miocardio/química , PPAR alfa/genética , Proteómica , Transducción de Señal , Proteínas Smad/metabolismo
20.
Neurobiol Learn Mem ; 150: 107-115, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29496644

RESUMEN

Staufen2 (Stau2) is a double-stranded RNA-binding protein (RBP) involved in posttranscriptional gene expression control in neurons. In flies, staufen contributes to learning and long-term memory formation. To study the impact of mammalian Stau2 on behavior, we generated a novel gene-trap mouse model that yields significant constitutive downregulation of Stau2 (Stau2GT). In order to investigate the effect of Stau2 downregulation on hippocampus-dependent behavior, we performed a battery of behavioral assays, i.e. open field, novel object recognition/location (NOR/L) and Barnes maze. Stau2GT mice displayed reduced locomotor activity in the open field and altered novelty preference in the NOR and NOL paradigms. Adult Stau2GT male mice failed to discriminate between familiar and newly introduced objects but showed enhanced spatial novelty detection. Additionally, we observed deficits in discriminating different spatial contexts in a Barnes maze assay. Together, our data suggest that Stau2 contributes to novelty preference and explorative behavior that is a driver for proper spatial learning in mice.


Asunto(s)
Conducta Exploratoria/fisiología , Hipocampo/metabolismo , Aprendizaje/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo , Reconocimiento en Psicología/fisiología , Animales , Conducta de Elección/fisiología , Ratones , Ratones Noqueados , Actividad Motora/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN/genética
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