RESUMEN
The early secretory pathway and autophagy are two essential and evolutionarily conserved endomembrane processes that are finely interlinked. Although growing evidence suggests that intracellular trafficking is important for autophagosome biogenesis, the molecular regulatory network involved is still not fully defined. In this study, we demonstrate a crucial effect of the COPII vesicle-related protein TFG (Trk-fused gene) on ULK1 puncta number and localization during autophagy induction. This, in turn, affects formation of the isolation membrane, as well as the correct dynamics of association between LC3B and early ATG proteins, leading to the proper formation of both omegasomes and autophagosomes. Consistently, fibroblasts derived from a hereditary spastic paraparesis (HSP) patient carrying mutated TFG (R106C) show defects in both autophagy and ULK1 puncta accumulation. In addition, we demonstrate that TFG activity in autophagy depends on its interaction with the ATG8 protein LC3C through a canonical LIR motif, thereby favouring LC3C-ULK1 binding. Altogether, our results uncover a link between TFG and autophagy and identify TFG as a molecular scaffold linking the early secretion pathway to autophagy.
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Autofagosomas/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Western Blotting , Técnica del Anticuerpo Fluorescente , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/genética , Proteínas/genética , Interferencia de ARNRESUMEN
Upregulated signal flow through RAS and the mitogen-associated protein kinase (MAPK) cascade is the unifying mechanistic theme of the RASopathies, a family of disorders affecting development and growth. Pathogenic variants in more than 20 genes have been causally linked to RASopathies, the majority having a dominant role in promoting enhanced signaling. Here, we report that SPRED2 loss of function is causally linked to a recessive phenotype evocative of Noonan syndrome. Homozygosity for three different variants-c.187C>T (p.Arg63∗), c.299T>C (p.Leu100Pro), and c.1142_1143delTT (p.Leu381Hisfs∗95)-were identified in four subjects from three families. All variants severely affected protein stability, causing accelerated degradation, and variably perturbed SPRED2 functional behavior. When overexpressed in cells, all variants were unable to negatively modulate EGF-promoted RAF1, MEK, and ERK phosphorylation, and time-course experiments in primary fibroblasts (p.Leu100Pro and p.Leu381Hisfs∗95) documented an increased and prolonged activation of the MAPK cascade in response to EGF stimulation. Morpholino-mediated knockdown of spred2a and spred2b in zebrafish induced defects in convergence and extension cell movements indicating upregulated RAS-MAPK signaling, which were rescued by expressing wild-type SPRED2 but not the SPRED2Leu381Hisfs∗95 protein. The clinical phenotype of the four affected individuals included developmental delay, intellectual disability, cardiac defects, short stature, skeletal anomalies, and a typical facial gestalt as major features, without the occurrence of the distinctive skin signs characterizing Legius syndrome. These features, in part, characterize the phenotype of Spred2-/- mice. Our findings identify the second recessive form of Noonan syndrome and document pleiotropic consequences of SPRED2 loss of function in development.
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Mutación con Pérdida de Función , Síndrome de Noonan/genética , Fenotipo , Proteínas Represoras/genética , Alelos , Animales , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Noqueados , Pez CebraRESUMEN
Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.
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Huesos/metabolismo , Proteínas de Unión al Calcio/metabolismo , Discapacidades del Desarrollo/metabolismo , Osteogénesis/fisiología , Transducción de Señal/fisiología , Animales , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular , Línea Celular Tumoral , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Células HEK293 , Células Hep G2 , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: RASopathies are genetic syndromes affecting development and having variable cancer predisposition. These disorders are clinically related and are caused by germline mutations affecting key players and regulators of the RAS-MAPK signaling pathway generally leading to an upregulated ERK activity. Gain-of-function (GOF) mutations in PTPN11, encoding SHP2, a cytosolic protein tyrosine phosphatase positively controlling RAS function, underlie approximately 50% of Noonan syndromes (NS), the most common RASopathy. A different class of these activating mutations occurs as somatic events in childhood leukemias. METHOD: Here, we evaluated the application of a FRET-based zebrafish ERK reporter, Teen, and used quantitative FRET protocols to monitor non-physiological RASopathy-associated changes in ERK activation. In a multi-level experimental workflow, we tested the suitability of the Teen reporter to detect pan-embryo ERK activity correlates of morphometric alterations driven by the NS-causing Shp2D61G allele. RESULTS: Spectral unmixing- and acceptor photobleaching (AB)-FRET analyses captured pathological ERK activity preceding the manifestation of quantifiable body axes defects, a morphological pillar used to test the strength of SHP2 GoF mutations. Last, the work shows that by multi-modal FRET analysis, we can quantitatively trace back the modulation of ERK phosphorylation obtained by low-dose MEK inhibitor treatment to early development, before the onset of morphological defects. CONCLUSION: This work proves the usefulness of FRET imaging protocols on both live and fixed Teen ERK reporter fish to readily monitor and quantify pharmacologically- and genetically-induced ERK activity modulations in early embryos, representing a useful tool in pre-clinical applications targeting RAS-MAPK signaling.
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Síndrome de Noonan , Pez Cebra , Animales , Humanos , Adolescente , Pez Cebra/genética , Pez Cebra/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Síndrome de Noonan/genética , Mutación , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Lymphopenia, particularly when restricted to the T-cell compartment, has been described as one of the major clinical hallmarks in patients with coronavirus disease 2019 (COVID-19) and proposed as an indicator of disease severity. Although several mechanisms fostering COVID-19-related lymphopenia have been described, including cell apoptosis and tissue homing, the underlying causes of the decline in T-cell count and function are still not completely understood. OBJECTIVE: Given that viral infections can directly target thymic microenvironment and impair the process of T-cell generation, we sought to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on thymic function. METHODS: We performed molecular quantification of T-cell receptor excision circles and κ-deleting recombination excision circles to assess, respectively, T- and B-cell neogenesis in SARS-CoV-2-infected patients. We developed a system for in vitro culture of primary human thymic epithelial cells (TECs) to mechanistically investigate the impact of SARS-CoV-2 on TEC function. RESULTS: We showed that patients with COVID-19 had reduced thymic function that was inversely associated with the severity of the disease. We found that angiotensin-converting enzyme 2, through which SARS-CoV-2 enters the host cells, was expressed by thymic epithelium, and in particular by medullary TECs. We also demonstrated that SARS-CoV-2 can target TECs and downregulate critical genes and pathways associated with epithelial cell adhesion and survival. CONCLUSIONS: Our data demonstrate that the human thymus is a target of SARS-CoV-2 and thymic function is altered following infection. These findings expand our current knowledge of the effects of SARS-CoV-2 infection on T-cell homeostasis and suggest that monitoring thymic activity may be a useful marker to predict disease severity and progression.
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COVID-19 , Linfopenia , Humanos , COVID-19/metabolismo , SARS-CoV-2 , Timo , Linfopenia/genética , Gravedad del PacienteRESUMEN
Leukodystrophies are a heterogeneous group of rare inherited disorders that mostly involve the white matter of the CNS. These conditions are characterized by primary glial cell and myelin sheath pathology of variable aetiology, which causes secondary axonal degeneration, generally emerging with disease progression. Whole exome sequencing performed in five large consanguineous nuclear families allowed us to identify homozygosity for two recurrent missense variants affecting highly conserved residues of RNF220 as the causative event underlying a novel form of leukodystrophy with ataxia and sensorineural deafness. We report these two homozygous missense variants (p.R363Q and p.R365Q) in the ubiquitin E3 ligase RNF220 as the underlying cause of this novel form of leukodystrophy with ataxia and sensorineural deafness that includes fibrotic cardiomyopathy and hepatopathy as associated features in seven consanguineous families. Mass spectrometry analysis identified lamin B1 as the RNF220 binding protein and co-immunoprecipitation experiments demonstrated reduced binding of both RNF220 mutants to lamin B1. We demonstrate that RNF220 silencing in Drosophila melanogaster specifically affects proper localization of lamin Dm0, the fly lamin B1 orthologue, promotes its aggregation and causes a neurodegenerative phenotype, strongly supporting the functional link between RNF220 and lamin B1. Finally, we demonstrate that RNF220 plays a crucial role in the maintenance of nuclear morphology; mutations in primary skin fibroblasts determine nuclear abnormalities such as blebs, herniations and invaginations, which are typically observed in cells of patients affected by laminopathies. Overall, our data identify RNF220 as a gene implicated in leukodystrophy with ataxia and sensorineural deafness and document a critical role of RNF220 in the regulation of nuclear lamina. Our findings provide further evidence on the direct link between nuclear lamina dysfunction and neurodegeneration.
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Alelos , Ataxia/genética , Sordera/genética , Laminopatías/genética , Mutación/genética , Ubiquitina-Proteína Ligasas/genética , Adolescente , Secuencia de Aminoácidos , Animales , Ataxia/diagnóstico , Células COS , Niño , Chlorocebus aethiops , Sordera/diagnóstico , Drosophila , Femenino , Células HEK293 , Humanos , Laminopatías/diagnóstico , Masculino , Linaje , Adulto JovenRESUMEN
Autoimmune endocrine disorders, such as type 1 diabetes (T1D) and thyroiditis, at present are treated with only hormone replacement therapy. This emphasizes the need to identify personalized effective immunotherapeutic strategies targeting T and B lymphocytes. Among the genetic variants associated with several autoimmune disorders, the C1858T polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene, encoding for Lyp variant R620W, affects the innate and adaptive immunity. We previously exploited a novel personalized immunotherapeutic approach based on siRNA delivered by liposomes (lipoplexes) that selectively inhibit variant allele expression. In this manuscript, we improved lipoplexes carrying siRNA for variant C1858T by functionalizing them with Fab of Rituximab antibody (RituxFab-Lipoplex) to specifically target B lymphocytes in autoimmune conditions, such as T1D. RituxFab-Lipoplexes specifically bind to B lymphocytes of the human Raji cell line and of human PBMC of healthy donors. RituxFab-Lipoplexes have impact on the function of B lymphocytes of T1D patients upon CpG stimulation showing a higher inhibitory effect on total cell proliferation and IgM+ plasma cell differentiation than the not functionalized ones. These results might open new pathways of applicability of RituxFab-Lipoplexes, such as personalized immunotherapy, to other autoimmune disorders, where B lymphocytes are the prevalent pathogenic immunocytes.
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Linfocitos B/inmunología , Técnicas de Transferencia de Gen , Fragmentos Fab de Inmunoglobulinas/inmunología , Lípidos/química , Mutación/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , ARN Interferente Pequeño/administración & dosificación , Rituximab/inmunología , Secuencia de Aminoácidos , Linfocitos B/efectos de los fármacos , Línea Celular , Dicroismo Circular , Dispersión Dinámica de Luz , Humanos , Liposomas , Activación de Linfocitos/inmunología , Fenotipo , Proteolisis/efectos de los fármacos , Rituximab/química , Rituximab/farmacologíaRESUMEN
Microtubules participate in fundamental cellular processes, including chromosomal segregation and cell division, migration and intracellular trafficking. Their proper function is required for correct central nervous system development and operative preservation, and mutations in genes coding tubulins, the constituting units of microtubules, underlie a family of neurodevelopmental and neurodegenerative diseases, collectively known as 'tubulinopathies', characterized by a wide range of neuronal defects resulting from defective proliferation, migration and function. Here, we causally link a previously unreported missense mutation in TUBB2A (c.1249G>A, p.D417N), encoding one of the neuron-specific ß-tubulin isotype II, to a disorder characterized by progressive spastic paraplegia, peripheral sensory-motor polyneuropathy and ataxia. Asp417 is a highly conserved solvent-exposed residue at the site mediating binding of kinesin superfamily motors. Impaired binding to KIF1A, a neuron-specific kinesin required for transport of synaptic vesicle precursors of the disease-associated TUBB2A mutant, was predicted by structural analyses and confirmed experimentally in vitro. We show that overexpression of TUBB2AD417N disrupts the mitotic spindle bipolarity and morphology and affects the M phase entry and length. Differently from the TUBB2AN247K and TUBB2AA248V, two mutants previously identified to affect neurodevelopment, TUBB2AD417N retains the ability to assemble into microtubules. Consistent with the differential clinical and structural impact, TUBB2AA248V does not drastically affect TUBB2A binding to KIF1A, nor mitotic spindle bipolarity. Overall, our data demonstrate a pathogenic role of the p.D417N substitution that is different from previously reported TUBB2A mutations and expand the phenotypic spectrum associated with mutations in this gene.
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Discapacidad Intelectual/genética , Cinesinas/genética , Espasticidad Muscular/genética , Atrofia Óptica/genética , Paraplejía/genética , Ataxias Espinocerebelosas/genética , Degeneraciones Espinocerebelosas/genética , Tubulina (Proteína)/genética , Adolescente , Adulto , Movimiento Celular/genética , Proliferación Celular/genética , Niño , Femenino , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/fisiopatología , Masculino , Microtúbulos/genética , Microtúbulos/patología , Espasticidad Muscular/diagnóstico por imagen , Espasticidad Muscular/fisiopatología , Neuronas/metabolismo , Neuronas/patología , Atrofia Óptica/diagnóstico por imagen , Atrofia Óptica/fisiopatología , Paraplejía/fisiopatología , Polineuropatías/genética , Polineuropatías/fisiopatología , Unión Proteica , Corteza Sensoriomotora/metabolismo , Corteza Sensoriomotora/fisiopatología , Huso Acromático/genética , Ataxias Espinocerebelosas/diagnóstico por imagen , Ataxias Espinocerebelosas/fisiopatología , Degeneraciones Espinocerebelosas/fisiopatologíaRESUMEN
Mitochondrial dysfunction is a key element in the pathogenesis of neurodegenerative disorders, such as riboflavin transporter deficiency (RTD). This is a rare, childhood-onset disease characterized by motoneuron degeneration and caused by mutations in SLC52A2 and SLC52A3, encoding riboflavin (RF) transporters (RFVT2 and RFVT3, respectively), resulting in muscle weakness, ponto-bulbar paralysis and sensorineural deafness. Based on previous findings, which document the contribution of oxidative stress in RTD pathogenesis, we tested possible beneficial effects of several antioxidants (Vitamin C, Idebenone, Coenzyme Q10 and EPI-743, either alone or in combination with RF) on the morphology and function of neurons derived from induced pluripotent stem cells (iPSCs) from two RTD patients. To identify possible improvement of the neuronal morphotype, neurite length was measured by confocal microscopy after ß-III tubulin immunofluorescent staining. Neuronal function was evaluated by determining superoxide anion generation by MitoSOX assay and intracellular calcium (Ca2+) levels, using the Fluo-4 probe. Among the antioxidants tested, EPI-743 restored the redox status, improved neurite length and ameliorated intracellular calcium influx into RTD motoneurons. In conclusion, we suggest that antioxidant supplementation may have a role in RTD treatment.
Asunto(s)
Antioxidantes/farmacología , Proteínas de Transporte de Membrana/deficiencia , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Riboflavina/metabolismo , Animales , Biomarcadores , Parálisis Bulbar Progresiva , Calcio/metabolismo , Diferenciación Celular , Modelos Animales de Enfermedad , Pérdida Auditiva Sensorineural , Humanos , Células Madre Pluripotentes Inducidas/citología , Metabolismo de los Lípidos , Ratones , Ratones Noqueados , Neuronas Motoras/citología , Oxidación-Reducción , FenotipoRESUMEN
Tubulinopathies are rare neurological disorders caused by alterations in tubulin structure and function, giving rise to a wide range of brain abnormalities involving neuronal proliferation, migration, differentiation and axon guidance. TUBB is one of the ten ß-tubulin encoding genes present in the human genome and is broadly expressed in the developing central nervous system and the skin. Mutations in TUBB are responsible for two distinct pathological conditions: the first is characterized by microcephaly and complex structural brain malformations and the second, also known as "circumferential skin creases Kunze type" (CSC-KT), is associated to neurological features, excess skin folding and growth retardation. We used a combination of immunocytochemical and cellular approaches to explore, on patients' derived fibroblasts, the functional consequences of two TUBB variants: the novel mutation (p.N52S), associated with basal ganglia and cerebellar dysgenesis, and the previously reported variant (p.M73T), linked to microcephaly, corpus callosum agenesis and CSC-KT skin phenotype. Our results demonstrate that these variants impair microtubule (MT) function and dynamics. Most importantly, our studies show an altered epidermal growth factor (EGF) and transferrin (Tf) intracellular vesicle trafficking in both patients' fibroblasts, suggesting a specific role of TUBB in MT-dependent vesicular transport.
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Microtúbulos/metabolismo , Mutación/genética , Vesículas Transportadoras/metabolismo , Tubulina (Proteína)/genética , Secuencia de Aminoácidos , Movimiento Celular/efectos de los fármacos , Niño , Análisis Mutacional de ADN , Factor de Crecimiento Epidérmico/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Imagen por Resonancia Magnética , Modelos Moleculares , Nocodazol/farmacología , Fenotipo , Transporte de Proteínas , Transferrina/metabolismo , Tubulina (Proteína)/químicaRESUMEN
The intratumor heterogeneity represents one of the most difficult challenges for the development of effective therapies to treat pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG). These brain tumors are composed of heterogeneous cell subpopulations that coexist and cooperate to build a functional network responsible for their aggressive phenotype. Understanding the cellular and molecular mechanisms sustaining such network will be crucial for the identification of new therapeutic strategies. To study more in-depth these mechanisms, we sought to apply the Multifluorescent Marking Technology. We generated multifluorescent pGBM and DIPG bulk cell lines randomly expressing six different fluorescent proteins and from which we derived stable optical barcoded single cell-derived clones. In this study, we focused on the application of the Multifluorescent Marking Technology in 2D and 3D in vitro/ex vivo culture systems. We discuss how we integrated different multimodal fluorescence analysis platforms, identifying their strengths and limitations, to establish the tools that will enable further studies on the intratumor heterogeneity and interclonal interactions in pGBM and DIPG.
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Neoplasias Encefálicas/patología , Glioblastoma/patología , Glioma/patología , Neoplasias Encefálicas/metabolismo , Línea Celular , Glioblastoma/metabolismo , Glioma/metabolismo , Células HEK293 , Humanos , Proteínas Luminiscentes/metabolismo , Pediatría , Tecnología/métodosRESUMEN
SHOC2 is a scaffold protein mediating RAS-promoted activation of mitogen-activated protein kinase (MAPK) signaling in response to extracellular stimuli. A recurrent activating mutation in SHOC2 (p.Ser2Gly) causes Mazzanti syndrome, a RASopathy characterized by features resembling Noonan syndrome and distinctive ectodermal abnormalities. A second mutation (p.Met173Ile) supposed to cause loss-of-function was more recently identified in two individuals with milder phenotypes. Here, we report on the third RASopathy-causing SHOC2 mutation (c.807_808delinsTT, p.Gln269_His270delinsHisTyr), which was found associated with prenatal-onset hypertrophic cardiomyopathy. Structural analyses indicated a possible impact of the mutation on the relative orientation of the two SHOC2's leucine-rich repeat domains. Functional studies provided evidence of its activating role, revealing enhanced binding of the mutant protein to MRAS and PPP1CB, and increased signaling through the MAPK cascade. Differing from SHOC2 S2G , SHOC2 Q269_H270delinsHY is not constitutively targeted to the plasma membrane. These data document that diverse mechanisms in SHOC2 functional dysregulation converge toward MAPK signaling upregulation.
Asunto(s)
Cardiomiopatía Hipertrófica/congénito , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Adulto , Animales , Células COS , Cardiomiopatía Hipertrófica/genética , Chlorocebus aethiops , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Sistema de Señalización de MAP Quinasas , Masculino , Modelos Moleculares , Embarazo , Diagnóstico Prenatal , Conformación Proteica , Dominios Proteicos , Proteína Fosfatasa 1/metabolismo , Proteínas ras/metabolismoRESUMEN
Mitochondria are highly dynamic organelles, undergoing continuous fission and fusion. The DNM1L (dynamin-1 like) gene encodes for the DRP1 protein, an evolutionary conserved member of the dynamin family, responsible for fission of mitochondria, and having a role in the division of peroxisomes, as well. DRP1 impairment is implicated in several neurological disorders and associated with either de novo dominant or compound heterozygous mutations. In five patients presenting with severe epileptic encephalopathy, we identified five de novo dominant DNM1L variants, the pathogenicity of which was validated in a yeast model. Fluorescence microscopy revealed abnormally elongated mitochondria and aberrant peroxisomes in mutant fibroblasts, indicating impaired fission of these organelles. Moreover, a very peculiar finding in our cohort of patients was the presence, in muscle biopsy, of core like areas with oxidative enzyme alterations, suggesting an abnormal distribution of mitochondria in the muscle tissue.
Asunto(s)
Dinaminas/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Encefalomiopatías Mitocondriales/diagnóstico , Encefalomiopatías Mitocondriales/genética , Músculos/metabolismo , Músculos/patología , Biomarcadores , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Encéfalo/patología , Análisis Mutacional de ADN , Dinaminas/química , Fibroblastos/metabolismo , Estudios de Asociación Genética/métodos , Humanos , Imagen por Resonancia Magnética/métodos , Modelos Biológicos , Músculos/ultraestructura , Mutación , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Oxidative stress is involved in the pathogenesis of Duchenne muscular dystrophy (DMD), an X-linked genetic disorder caused by mutations in the dystrophin gene and characterized by progressive, lethal muscle degeneration and chronic inflammation. In this study, we explored the expression and signaling pathway of a master player of the anti-oxidant and anti-inflammatory response, namely NF-E2-related Factor 2, in muscle biopsies of DMD patients. We classified DMD patients in two age groups (Class I, 0-2 years and Class II, 2-9 years), in order to evaluate the antioxidant pathway expression during the disease progression. We observed that altered enzymatic antioxidant responses, increased levels of oxidized glutathione and oxidative damage are differently modulated in the two age classes of patients and well correlate with the severity of pathology. Interestingly, we also observed a modulation of relevant markers of the inflammatory response, such as heme oxygenase 1 and Inteleukin-6 (IL-6), suggesting a link between oxidative stress and chronic inflammatory response. Of note, using a transgenic mouse model, we demonstrated that IL-6 overexpression parallels the antioxidant expression profile and the severity of dystrophic muscle observed in DMD patients. This study advances our understanding of the pathogenic mechanisms underlying DMD and defines the critical role of oxidative stress on muscle wasting with clear implications for disease pathogenesis and therapy in human.
Asunto(s)
Distrofia Muscular de Duchenne/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/fisiología , Animales , Antioxidantes/metabolismo , Niño , Preescolar , Modelos Animales de Enfermedad , Distrofina/genética , Distrofina/metabolismo , Femenino , Glutatión/genética , Glutatión/metabolismo , Humanos , Lactante , Recién Nacido , Inflamación/genética , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Factor 2 Relacionado con NF-E2/genética , Oxidación-Reducción , Transducción de SeñalRESUMEN
In autoimmune diseases as Type 1 diabetes, the actual treatment that provides the missing hormones is not able, however, to interrupt the underlining immunological mechanism. Importantly, novel immunotherapies are exploited to protect and rescue the remaining hormone producing cells. Among probable targets of immunotherapy, the C1858T mutation in the PTPN22 gene, which encodes for the lymphoid tyrosine phosphatase (Lyp) variant R620W, reveals an autoimmunity related pathophysiological role. Our scope was to establish new C1858T PTPN22 siRNA duplexes delivered by liposomal carriers (lipoplexes) to patients' PBMC. Following lipoplexes treatment, CD3+ and CD3- immunotypes were efficiently transfected; cell integrity and viability were preserved. Specific target mRNA down-modulation was observed. After T cell receptor stimulation, in lipoplexes-treated PBMC Lyp function was restored by increased release of IL-2 in cultures. Results set-up the stage for ultimate trials in the treatment of autoimmunity based on the specific inhibitory targeting of C1858T PTPN22 by lipoplexes.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/terapia , Regulación hacia Abajo , Inmunoterapia , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , ARN Interferente Pequeño/metabolismo , Linfocitos T/metabolismo , Adolescente , Secuencia de Bases , Cationes , Niño , Preescolar , Diabetes Mellitus Tipo 1/genética , Dispersión Dinámica de Luz , Femenino , Humanos , Lípidos/química , Liposomas , Masculino , Proteína Tirosina Fosfatasa no Receptora Tipo 22/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Post-translational modulation of peptidylprolyl isomerase Pin1 might link impaired glucose metabolism and neurodegeneration, being Pin1 effectors target for the glucagon-Like-Peptide1 analog liraglutide. We tested the hypotheses in Pin1 silenced cells (SH-SY5Y) treated with 2-deoxy-d-glucose (2DG) and methylglyoxal (MG), stressors causing altered glucose trafficking, glucotoxicity and protein glycation. Rescue by liraglutide was investigated. Pin1 silencing caused increased levels of reactive oxygen species, upregulated energy metabolism as suggested by raised levels of total ATP content and mRNA of SIRT1, PGC1α, NRF1; enhanced mitochondrial fission events as supported by raised protein expression of FIS1 and DRP1. 2DG and MG reduced significantly cell viability in all the cell lines. In Pin1 KD clones, 2DG exacerbated altered mitochondrial dynamics causing higher rate of fission events. Liraglutide influenced insulin signaling pathway (GSK3b/Akt); improved cell viability also in cells treated with 2DG; but it did not revert mitochondrial dysfunction in Pin1 KD model. In cells treated with MG, liraglutide enhanced cell viability, reduced ROS levels and cell death (AnnexinV/PI); and trended to reduce anti-apoptotic signals (BAX, BCL2, CASP3). Pin1 silencing mimics neuronal metabolic impairment of patients with impaired glucose metabolism and neurodegeneration. Liraglutide rescues to some extent cellular dysfunctions induced by Pin1 silencing.
Asunto(s)
Liraglutida/farmacología , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Fármacos Neuroprotectores/farmacología , Adenosina Trifosfato/metabolismo , Apoptosis , Línea Celular Tumoral , Desoxiglucosa/toxicidad , Silenciador del Gen , Humanos , Insulina/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Factor Nuclear 1 de Respiración/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Piruvaldehído/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
Osteosarcoma is the most common primary bone cancer and the most frequent cause of bone cancer-related deaths in children and adolescents. Osteosarcoma cells are able to establish a crosstalk with resident bone cells leading to the formation of a deleterious vicious cycle. We hypothesized that osteosarcoma cells can release, in the bone microenvironment, transforming Extracellular Vesicles (EVs) involved in regulating bone cell proliferation and differentiation, thereby promoting tumor growth. We assessed EV production by three osteosarcoma cell lines with increasing aggressiveness in order to investigate their roles in the communication between osteosarcoma cells and normal recipient cells. Osteosarcoma-derived EVs were used to treat the murine fibroblast cell line NIH3T3 and to study the induction of tumor-like phenotypes. Our results showed that osteosarcoma cell lines are able to produce EVs that fuse to recipient cells, with a very high uptake efficiency. The treatment of recipient NIH3T3 with osteosarcoma-derived EVs induced substantial biological and functional effects, as an enhanced proliferation and survival capability under starved conditions, high levels of activated survival pathways, an increased migration, adhesion, and 3D sphere formation and the acquired capability to grow in an anchorage-independent manner. Moreover, in murine NIH3T3 we found human mRNAs of TNF-α, IL-6, and TGF-ß, as well as a de novo expression of murine MMP-9 and TNF-α following the treatment of human osteosarcoma-derived EVs.
Asunto(s)
Neoplasias Óseas/patología , Vesículas Extracelulares/patología , Osteosarcoma/patología , Animales , Neoplasias Óseas/metabolismo , Adhesión Celular/fisiología , Diferenciación Celular/fisiología , Línea Celular , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Vesículas Extracelulares/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Ratones , Células 3T3 NIH , Osteosarcoma/metabolismo , Fenotipo , Microambiente Tumoral/fisiologíaRESUMEN
Osteosarcoma is the most common malignant bone tumor in children and young adults. The identification of proteins which exhibit different subcellular localization in low- versus high-risk osteosarcoma can be instrumental to obtain prognostic information and to develop innovative therapeutic strategies. Beside the well-characterized membrane and cytoplasmic localization of Src protein, this study evaluated the prognostic relevance of its so-far unknown nuclear compartmentalization. We analyzed the subcellular distribution of total and activated (pY418) Src in a tissue microarray including 60 osteosarcoma samples. Immunohistochemical analyses revealed a variable pattern of Src expression and localization, ranging from negative to high-stained nuclei combined with a substantial cytoplasmic staining for total and activated forms. The analysis of Kaplan-Meier survival curves in relationship to the diverse permutations of cytoplasmic and nuclear staining suggested a correlation between Src subcellular localization and the overall survival (OS) of osteosarcoma patients. In order to explain this different subcellular localization, normal osteoblasts and three osteosarcoma cell lines were used to investigate the molecular mechanism. Once confirmed a variable Src localization also in these cell lines, we demonstrated a correlation between the N-myristoyltransferase enzymes expression and activity and the Src nuclear content. In conclusion, these results described a so-far unknown Src nuclear localization in osteosarcoma cells, suggesting that the combined detection of nuclear and cytoplasmic Src levels can be used as a prognostic marker for osteosarcoma patient survival. A correlation between the N-myristoyltransferase enzymes and the Src subcellular localization was described as well.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/enzimología , Núcleo Celular/enzimología , Osteosarcoma/enzimología , Familia-src Quinasas/metabolismo , Aciltransferasas/metabolismo , Adolescente , Adulto , Neoplasias Óseas/mortalidad , Neoplasias Óseas/patología , Neoplasias Óseas/terapia , Línea Celular Tumoral , Niño , Activación Enzimática , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Osteosarcoma/mortalidad , Osteosarcoma/patología , Osteosarcoma/terapia , Pronóstico , Procesamiento Proteico-Postraduccional , Factores de Tiempo , Análisis de Matrices Tisulares , Adulto JovenRESUMEN
To elucidate the pathogenesis of axonopathy in Friedreich's Ataxia (FRDA), a neurodegenerative disease characterized by axonal retraction, we analyzed the microtubule (MT) dynamics in an in vitro frataxin-silenced neuronal model (shFxn). A typical feature of MTs is their "dynamic instability", in which they undergo phases of growth (polymerization) and shrinkage (depolymerization). MTs play a fundamental role in the physiology of neurons and every perturbation of their dynamicity is highly detrimental for neuronal functions. The aim of this study is to determine whether MTs are S-glutathionylated in shFxn and if the glutathionylation triggers MT dysfunction. We hypothesize that oxidative stress, determined by high GSSG levels, induces axonal retraction by interfering with MT dynamics. We propose a mechanism of the axonopathy in FRDA where GSSG overload and MT de-polymerization are strictly interconnected. Indeed, using a frataxin-silenced neuronal model we show a significant reduction of neurites extension, a shift of tubulin toward the unpolymerized fraction and a consistent increase of glutathione bound to the cytoskeleton. The live cell imaging approach further reveals a significant decrease in MT growth lifetime due to frataxin silencing, which is consistent with the MT destabilization. The in vitro antioxidant treatments trigger the axonal re-growth and the increase in stable MTs in shFxn, thus contributing to identify new neuronal targets of oxidation in this disease and providing a novel approach for antioxidant therapies.