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Introduction: Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters, RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide), which are involved in different redox reactions, including the energetic metabolism processes occurring in mitochondria. To date, human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons, which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far, no attention has been dedicated to astrocytes. Results and discussion: Here, we demonstrate that in vitro differentiation of astrocytes, which guarantee trophic and metabolic support to neurons, from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons, RTD2 does not compromise the morpho-functional features of astrocytes.
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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
Riboflavin Transporter Deficiency (RTD) is a rare genetic, childhood-onset disease. This pathology has a relevant neurological involvement, being characterized by motor symptoms, ponto-bulbar paralysis and sensorineural deafness. Such clinical presentation is associated with muscle weakness and motor neuron (MN) degeneration, so that RTD is considered part of the MN disease spectrum. Based on previous findings demonstrating energy dysmetabolism and mitochondrial impairment in RTD induced Pluripotent Stem cells (iPSCs) and iPSC-derived MNs, here we address the involvement of intrinsic apoptotic pathways in disease pathogenesis using these patient-specific in vitro models by combined ultrastructural and confocal analyses. We show impaired neuronal survival of RTD iPSCs and MNs. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) documents severe alterations in patients' cells, including deranged mitochondrial ultrastructure, and altered plasma membrane and nuclear organization. Occurrence of aberrantly activated apoptosis is confirmed by immunofluorescence and TUNEL assays. Overall, our work provides evidence of a role played by mitochondrial dysfunction in RTD, and identifies neuronal apoptosis as a contributing event in disease pathogenesis, indicating intrinsic apoptosis pathways as possible relevant targets for more effective therapeutical approaches.
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Microtubules are dynamic cytoskeletal structures involved in several cellular functions, such as intracellular trafficking, cell division and motility. More than other cell types, neurons rely on the proper functioning of microtubules to conduct their activities and achieve complex morphologies. Pathogenic variants in genes encoding for α and ß-tubulins, the structural subunits of microtubules, give rise to a wide class of neurological disorders collectively known as "tubulinopathies" and mainly involving a wide and overlapping range of brain malformations resulting from defective neuronal proliferation, migration, differentiation and axon guidance. Although tubulin mutations have been classically linked to neurodevelopmental defects, growing evidence demonstrates that perturbations of tubulin functions and activities may also drive neurodegeneration. In this study, we causally link the previously unreported missense mutation p.I384N in TUBA1A, one of the neuron-specific α-tubulin isotype I, to a neurodegenerative disorder characterized by progressive spastic paraplegia and ataxia. We demonstrate that, in contrast to the p.R402H substitution, which is one of the most recurrent TUBA1A pathogenic variants associated to lissencephaly, the present mutation impairs TUBA1A stability, reducing the abundance of TUBA1A available in the cell and preventing its incorporation into microtubules. We also show that the isoleucine at position 384 is an amino acid residue, which is critical for α-tubulin stability, since the introduction of the p.I384N substitution in three different tubulin paralogs reduces their protein level and assembly into microtubules, increasing their propensity to aggregation. Moreover, we demonstrate that the inhibition of the proteasome degradative systems increases the protein levels of TUBA1A mutant, promoting the formation of tubulin aggregates that, as their size increases, coalesce into inclusions that precipitate within the insoluble cellular fraction. Overall, our data describe a novel pathogenic effect of p.I384N mutation that differs from the previously described substitutions in TUBA1A, and expand both phenotypic and mutational spectrum related to this gene.
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De novo CLTC mutations underlie a spectrum of early-onset neurodevelopmental phenotypes having developmental delay/intellectual disability (ID), epilepsy, and movement disorders (MD) as major clinical features. CLTC encodes the widely expressed heavy polypeptide of clathrin, a major component of the coated vesicles mediating endocytosis, intracellular trafficking, and synaptic vesicle recycling. The underlying pathogenic mechanism is largely unknown. Here, we assessed the functional impact of the recurrent c.2669C > T (p.P890L) substitution, which is associated with a relatively mild ID/MD phenotype. Primary fibroblasts endogenously expressing the mutated protein show reduced transferrin uptake compared to fibroblast lines obtained from three unrelated healthy donors, suggesting defective clathrin-mediated endocytosis. In vitro studies also reveal a block in cell cycle transition from G0/G1 to the S phase in patient's cells compared to control cells. To demonstrate the causative role of the p.P890L substitution, the pathogenic missense change was introduced at the orthologous position of the Caenorhabditis elegans gene, chc-1 (p.P892L), via CRISPR/Cas9. The resulting homozygous gene-edited strain displays resistance to aldicarb and hypersensitivity to PTZ, indicating defective release of acetylcholine and GABA by ventral cord motor neurons. Consistently, mutant animals show synaptic vesicle depletion at the sublateral nerve cords, and slightly defective dopamine signaling, highlighting a generalized deficit in synaptic transmission. This defective release of neurotransmitters is associated with their secondary accumulation at the presynaptic membrane. Automated analysis of C. elegans locomotion indicates that chc-1 mutants move slower than their isogenic controls and display defective synaptic plasticity. Phenotypic profiling of chc-1 (+/P892L) heterozygous animals and transgenic overexpression experiments document a mild dominant-negative behavior for the mutant allele. Finally, a more severe phenotype resembling that of chc-1 null mutants is observed in animals harboring the c.3146 T > C substitution (p.L1049P), homologs of the pathogenic c.3140 T > C (p.L1047P) change associated with a severe epileptic phenotype. Overall, our findings provide novel insights into disease mechanisms and genotype-phenotype correlations of CLTC-related disorders.
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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
Vesicle biogenesis, trafficking and signaling via Endoplasmic reticulum-Golgi network support essential developmental processes and their disruption lead to neurodevelopmental disorders and neurodegeneration. We report that de novo missense variants in ARF3, encoding a small GTPase regulating Golgi dynamics, cause a developmental disease in humans impairing nervous system and skeletal formation. Microcephaly-associated ARF3 variants affect residues within the guanine nucleotide binding pocket and variably perturb protein stability and GTP/GDP binding. Functional analysis demonstrates variably disruptive consequences of ARF3 variants on Golgi morphology, vesicles assembly and trafficking. Disease modeling in zebrafish validates further the dominant behavior of the mutants and their differential impact on brain and body plan formation, recapitulating the variable disease expression. In-depth in vivo analyses traces back impaired neural precursors' proliferation and planar cell polarity-dependent cell movements as the earliest detectable effects. Our findings document a key role of ARF3 in Golgi function and demonstrate its pleiotropic impact on development.
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Trastornos del Neurodesarrollo , Pez Cebra , Humanos , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Aparato de Golgi/metabolismo , Retículo Endoplásmico/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismoRESUMEN
Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma. The Fusion-Positive (FP) subtype expresses the chimeric protein PAX3-FOXO1 (P3F) while the Fusion-Negative (FN) is devoid of any gene translocation. FP-RMS and metastatic FN-RMS are often unresponsive to conventional therapy. Therefore, novel therapeutic approaches are needed to halt tumor progression. NOTCH signaling has oncogenic functions in RMS and its pharmacologic inhibition through γ-secretase inhibitors blocks tumor growth in vitro and in vivo. Here, we show that NOTCH signaling blockade resulted in the up-regulation and phosphorylation of the MET oncogene in both RH30 (FP-RMS) and RD (FN-RMS) cell lines. Pharmacologic inhibition of either NOTCH or MET signaling slowed proliferation and restrained cell survival compared to control cells partly by increasing Annexin V and CASP3/7 activation. Co-treatment with NOTCH and MET inhibitors significantly amplified these effects and enhanced PARP1 cleavage in both cell lines. Moreover, it severely hampered cell migration, colony formation, and anchorage-independent growth compared to single-agent treatments in both cell lines and significantly prevented the growth of FN-RMS cells grown as spheroids. Collectively, our results unveil the overexpression of the MET oncogene by NOTCH signaling targeting in RMS cells and show that MET pathway blockade sensitizes them to NOTCH inhibition.
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Lamin A, a main constituent of the nuclear lamina, is involved in mechanosignaling and cell migration through dynamic interactions with the LINC complex, formed by the nuclear envelope proteins SUN1, SUN2 and the nesprins. Here, we investigated lamin A role in Ewing Sarcoma (EWS), an aggressive bone tumor affecting children and young adults. In patients affected by EWS, we found a significant inverse correlation between LMNA gene expression and tumor aggressiveness. Accordingly, in experimental in vitro models, low lamin A expression correlated with enhanced cell migration and invasiveness and, in vivo, with an increased metastatic load. At the molecular level, this condition was linked to altered expression and anchorage of nuclear envelope proteins and increased nuclear retention of YAP/TAZ, a mechanosignaling effector. Conversely, overexpression of lamin A rescued LINC complex organization, thus reducing YAP/TAZ nuclear recruitment and preventing cell invasiveness. These effects were also obtained through modulation of lamin A maturation by a statin-based pharmacological treatment that further elicited a more differentiated phenotype in EWS cells. These results demonstrate that drugs inducing nuclear envelope remodeling could be exploited to improve therapeutic strategies for EWS.
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Membrana Nuclear , Sarcoma de Ewing , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismoRESUMEN
The C1858T variant of the protein tyrosine phosphatase N22 (PTPN22) gene is associated with pathophysiological phenotypes in several autoimmune conditions, namely, Type 1 diabetes and autoimmune thyroiditis. The R620W variant protein, encoded by C1858T, leads to a gain of function mutation with paradoxical reduced T cell activation. We previously exploited a novel personalized immunotherapeutic approach based on siRNA delivered by liposomes (lipoplexes, LiposiRNA) that selectively inhibit variant allele expression. In this manuscript, we functionalize lipoplexes carrying siRNA for variant C1858T with a high affinity ligand of Siglec-10 (Sig10L) coupled to lipids resulting in lipoplexes (LiposiRNA-Sig10L) that enhance delivery to Siglec-10 expressing immunocytes. LiposiRNA-Sig10L lipoplexes more efficiently downregulated variant C1858T PTPN22 mRNA in PBMC of heterozygous patients than LiposiRNA without Sig10L. Following TCR engagement, LiposiRNA-Sig10L more significantly restored IL-2 secretion, known to be paradoxically reduced than in wild type patients, than unfunctionalized LiposiRNA in PBMC of heterozygous T1D patients.
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Diabetes Mellitus Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 22 , Autoinmunidad , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/terapia , Humanos , Factores Inmunológicos , Inmunoterapia , Leucocitos Mononucleares/metabolismo , Ácido N-Acetilneuramínico , Monoéster Fosfórico Hidrolasas , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 22/metabolismo , ARN Interferente Pequeño/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido SiálicoRESUMEN
The cells possess several mechanisms to counteract the over-production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), including enzymes such as superoxide dismutase, catalase and glutathione peroxidase. Moreover, an important sensor involved in the anti-oxidant response is KEAP1-NRF2-ARE signaling complex. Under oxidative stress (OS), the transcription factor NRF2 can dissociate from the KEAP1-complex in the cytosol and translocate into the nucleus to promote the transcriptional activation of anti-oxidant genes, such as heme oxygenase 1 and NADPH quinone oxidoreductase. Within this context, the activation of NRF2 response is further regulated by BACH1, a transcription repressor, that compete with the KEAP1-NRF2-ARE complex. In this work, we focused on the role of BACH1/NRF2 ratio in the regulation of the anti-oxidant response, proposing their antithetical relation as a valuable target for a therapeutic strategy to test drugs able to exert neuroprotective effects, notably in aging and neurodegenerative diseases. Among these, Down syndrome (DS) is a complex genetic disorder characterized by BACH1 gene triplication that likely results in the impairment of NRF2 causing increased OS. Our results revealed that BACH1 overexpression alters the BACH1/NRF2 ratio in the nucleus and disturbs the induction of antioxidant response genes ultimately resulting in the accumulation of oxidative damage both in Ts2Cje mice (a mouse model of DS) and human DS lymphoblastoid cell lines (LCLs). Based on this evidence, we tested Caffeic Acid Phenethyl Ester (CAPE) and the synthetic analogue VP961, which have been proven to modulate NRF2 activity. We showed that CAPE and VP961 administration to DS LCLs was able to promote NRF2 nuclear translocation, which resulted in the amelioration of antioxidant response. Overall, our study supports the hypothesis that BACH1 triplication in DS subjects is implicated in the alteration of redox homeostasis and therapeutic strategies to overcome this effect are under investigation in our laboratory.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Síndrome de Down , Factor 2 Relacionado con NF-E2 , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Ácidos Cafeicos , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Alcohol Feniletílico/análogos & derivadosRESUMEN
Besides its structural properties in the nucleoskeleton, Lamin A/C is a mechanosensor protein involved in perceiving the elasticity of the extracellular matrix. In this study we provide evidence about Lamin A/C-mediated regulation of osteosarcoma cell adhesion and spreading on substrates with tissue-specific elasticities. Our working hypothesis is based on the observation that low-aggressive and bone-resident SaOS-2 osteosarcoma cells express high level of Lamin A/C in comparison to highly metastatic, preferentially to the lung, osteosarcoma 143B cells, thereby suggesting a role for Lamin A/C in tumor cell tropism. Specifically, LMNA gene over-expression in 143B cells induced a reduction in tumor cell aggressiveness in comparison to parental cells, with decreased proliferation rate and reduced migration capability. Furthermore, LMNA reintegration into 143B cells changed the adhesion properties of tumor cells, from a preferential tropism toward the 1.5 kPa PDMS substrate (resembling normal lung parenchyma) to the 28 kPa (resembling pre-mineralized bone osteoid matrix). Our study suggests that Lamin A/C expression could be involved in the organ tropism of tumor cells, thereby providing a rationale for further studies focused on the definition of cancer mechanism of metastatization.
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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
PCDH19-related epilepsy is a rare genetic disease caused by defective function of PCDH19, a calcium-dependent cell-cell adhesion protein of the cadherin superfamily. This disorder is characterized by a heterogeneous phenotypic spectrum, with partial and generalized febrile convulsions that are gradually increasing in frequency. Developmental regression may occur during disease progression. Patients may present with intellectual disability (ID), behavioral problems, motor and language delay, and a low motor tone. In most cases, seizures are resistant to treatment, but their frequency decreases with age, and some patients may even become seizure-free. ID generally persists after seizure remission, making neurological abnormalities the main clinical issue in affected individuals. An effective treatment is lacking. In vitro studies using patient-derived induced pluripotent stem cells (iPSCs) reported accelerated neural differentiation as a major endophenotype associated with PCDH19 mutations. By using this in vitro model system, we show that accelerated in vitro neurogenesis is associated with a defect in the cell division plane at the neural progenitors stage. We also provide evidence that altered PCDH19 function affects proper mitotic spindle orientation. Our findings identify an altered equilibrium between symmetric versus asymmetric cell division as a previously unrecognized mechanism contributing to the pathogenesis of this rare epileptic encephalopathy.
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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
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
The cytoskeletal network plays a crucial role in differentiation, morphogenesis, function and homeostasis of the nervous tissue, so that alterations in any of its components may lead to neurodegenerative diseases. Riboflavin transporter deficiency (RTD), a childhood-onset disorder characterized by degeneration of motor neurons (MNs), is caused by biallelic mutations in genes encoding the human riboflavin (RF) transporters. In a patient- specific induced Pluripotent Stem Cells (iPSCs) model of RTD, we recently demonstrated altered cell-cell contacts, energy dysmetabolism and redox imbalance.The present study focusses on cytoskeletal composition and dynamics associated to RTD, utilizing patients' iPSCs and derived MNs. Abnormal expression and distribution of α- and ß-tubulin (α- and ß-TUB), as well as imbalanced tyrosination of α-TUB, accompanied by impaired ability to repolymerize after nocodazole treatment, were found in RTD patient-derived iPSCs. Following differentiation, MNs showed consistent changes in TUB content, which was associated with abnormal morphofunctional features, such as neurite length and Ca++ homeostasis, suggesting impaired differentiation.Beneficial effects of RF supplementation, alone or in combination with the antioxidant molecule N-acetyl-cystine (NAC), were assessed. RF administration resulted in partially improved cytoskeletal features in patients' iPSCs and MNs, suggesting that redundancy of transporters may rescue cell functionality in the presence of adequate concentrations of the vitamin. Moreover, supplementation with NAC was demonstrated to be effective in restoring all the considered parameters, when used in combination with RF, thus supporting the therapeutic use of both compounds.
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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
In this study, we explored whether Nutlin-3a, a well-known, nontoxic small-molecule compound antagonizing the inhibitory interaction of MDM2 with the tumor suppressor p53, may restore ligands for natural killer (NK) cell-activating receptors (NK-AR) on neuroblastoma cells to enhance the NK cell-mediated killing. Neuroblastoma cell lines were treated with Nutlin-3a, and the expression of ligands for NKG2D and DNAM-1 NK-ARs and the neuroblastoma susceptibility to NK cells were evaluated. Adoptive transfer of human NK cells in a xenograft neuroblastoma-bearing NSG murine model was assessed. Two data sets of neuroblastoma patients were explored to correlate p53 expression with ligand expression. Luciferase assays and chromatin immunoprecipitation analysis of p53 functional binding on PVR promoter were performed. Primary neuroblastoma cells were also treated with Nutlin-3a, and neuroblastoma spheroids obtained from one high-risk patient were assayed for NK-cell cytotoxicity. We provide evidence showing that the Nutlin-3a-dependent rescue of p53 function in neuroblastoma cells resulted in (i) increased surface expression of ligands for NK-ARs, thus rendering neuroblastoma cell lines significantly more susceptible to NK cell-mediated killing; (ii) shrinkage of human neuroblastoma tumor masses that correlated with overall survival upon adoptive transfer of NK cells in neuroblastoma-bearing mice; (iii) and increased expression of ligands in primary neuroblastoma cells and boosting of NK cell-mediated disaggregation of neuroblastoma spheroids. We also found that p53 was a direct transcription factor regulating the expression of PVR ligand recognized by DNAM-1. Our findings demonstrated an immunomodulatory role of Nutlin-3a, which might be prospectively used for a novel NK cell-based immunotherapy for neuroblastoma.