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OBJECTIVES: We aimed to explore the prevalence of peroneal neuropathy in children during coronavirus disease-19 (COVID-19) pandemic. BACKGROUND: Since the COVID-19 outbreak, many children worldwide have experienced a dramatic lifestyle changes, including conducting most daily activities indoors. Peroneal nerve palsy is one of the most common entrapment neuropathies and circumstances as prolonged immobilization or leg crossing predisposes an individual to peroneal neuropathy. METHODS: This is a case-control retrospective study that included patients referred to our neurophysiology clinic with foot drop. We compared the prevalence of spontaneous peroneal neuropathy 1 year before (April 2019/March 2020) and 1 year during the COVID-19 pandemic (April 2020/March 2021); and we also continued collecting data prospectively between April and September 2021 analysis the whole pandemic period. RESULTS: Totally, 399 patient clinical notes and NCS/EMG reports were reviewed, 220 were evaluated 1 year before and 179 1 year during COVID-19 pandemic. During the COVID-19 pandemic, there was a higher prevalence of peroneal neuropathy (odds ratio 4.74, 95%CI 1.30-17.25, p = 0.0183). In the COVID group (n = 11), mean age was 14 years and 63.4% were males. Mean age was 15 years and 66.7% were males in the Control group (n = 3). There was a significant difference in the time from symptoms onset to the neurophysiology assessment, with a mean time of 14 days in the Control group and 87.5 days in the COVID group. CONCLUSIONS: This study provides evidence that during the COVID-19 pandemic period, there was a higher prevalence of peroneal neuropathy among children. Strategies to prevent peroneal neuropathy should be recommened to this age group.
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COVID-19 , Neuropatias Fibulares , Masculino , Humanos , Criança , Adolescente , Feminino , Neuropatias Fibulares/epidemiologia , Neuropatias Fibulares/diagnóstico , Pandemias , Estudos Retrospectivos , Prevalência , COVID-19/epidemiologiaRESUMO
PURPOSE: Pediatric Ambulatory Continuous Peripheral Nerve Block (ACPNB) programs are a safe and effective pain management modality that can reduce patient length of stay (LOS) while ensuring optimal, multimodal pain management at home after surgery. Our institution previously solely used electronic infusion pumps to deliver local anesthetic via peripheral nerve catheters, requiring postoperative inpatient admissions for pain management. We aimed to enhance postoperative pain management and decrease hospital LOS after orthopedic foot and ankle surgery through implementation of an ACPNB program. DESIGN: An ACPNB program was developed and implemented for pediatric patients undergoing foot and ankle reconstruction surgery. METHODS: We provide a detailed description of the multidepartment collaboration led by the acute pain service (APS) and orthopedics that resulted in the development and implementation of a pediatric ACPNB program using portable, elastomeric devices for patients undergoing reconstructive foot and ankle surgery. Implementation tools, including caregiver and nursing education resources, a data collection log, a process map, and staff surveys are shared. FINDINGS: Twenty-eight patients received elastomeric devices during the 12 months of data collection. All 28 patients who required a continuous peripheral nerve block (CPNB) for pain management following foot and ankle reconstruction surgery received their block via an elastomeric device rather than an electronic hospital infusion pump. All patients and caregivers expressed positive satisfaction with pain management after hospital discharge. No patient with an elastomeric device required scheduled opioids for pain management by the end of their hospital admission. LOS on the orthopedic inpatient unit for foot and ankle surgery decreased by 58%, representing an estimated 29 days and $27,557.88 saved. A majority (96.4%) of staff survey respondents reported feeling satisfied with their overall experience working with an elastomeric device. CONCLUSIONS: The successful implementation of a pediatric ACPNB program has led to positive patient outcomes, including a significant decrease in hospital LOS and health system cost savings for this patient population.
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Anestésicos Locais , Bloqueio Nervoso , Dor Pós-Operatória , Criança , Humanos , Anestésicos Locais/administração & dosagem , Hospitais , Tempo de Internação , Bloqueio Nervoso/métodos , Dor Pós-Operatória/tratamento farmacológico , Nervos PeriféricosRESUMO
Polycystin-1 (PC-1) is a transmembrane protein, encoded by the PKD1 gene, mutated in autosomal dominant polycystic kidney disease (ADPKD). This common genetic disorder, characterized by cyst formation in both kidneys, ultimately leading to renal failure, is still waiting for a definitive treatment. The overall function of PC-1 and the molecular mechanism responsible for cyst formation are slowly coming to light, but they are both still intensively studied. In particular, PC-1 has been proposed to act as a mechanosensor, although the precise signal that activates the mechanical properties of this protein has been long debated and questioned. In this review, we report studies and evidence of PC-1 function as a mechanosensor, starting from the peculiarity of its structure, through the long journey that progressively shed new light on the potential initiating events of cystogenesis, concluding with the description of PC-1 recently shown ability to sense the mechanical stimuli provided by the stiffness of the extracellular environment. These new findings have potentially important implications for the understanding of ADPKD pathophysiology and potentially for designing new therapies.NEW & NOTEWORTHY Polycystin-1 has recently emerged as a possible receptor able to sense extracellular stiffness and to negatively control the cellular actomyosin contraction machinery. Here, we revisit a large body of literature on autosomal dominant polycystic kidney disease providing a new possible mechanistic view on the topic.
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Matriz Extracelular/metabolismo , Rim/metabolismo , Mecanotransdução Celular , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Microambiente Celular , Matriz Extracelular/patologia , Predisposição Genética para Doença , Humanos , Rim/patologia , Mutação , Fenótipo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Conformação Proteica , Relação Estrutura-Atividade , Canais de Cátion TRPP/química , Canais de Cátion TRPP/genéticaRESUMO
Exogenous IgE acts as an adjuvant in tumor vaccination in mice, and therefore a direct role of endogenous IgE in tumor immunosurveillance was investigated. By using genetically engineered mice, we found that IgE ablation rendered mice more susceptible to the growth of transplantable tumors. Conversely, a strengthened IgE response provided mice with partial or complete resistance to tumor growth, depending on the tumor type. By genetic crosses, we showed that IgE-mediated tumor protection was mostly lost in mice lacking FcεRI. Tumor protection was also lost after depletion of CD8(+) T cells, highlighting a cross-talk between IgE and T cell-mediated tumor immunosurveillance. Our findings provide the rationale for clinical observations that relate atopy with a lower risk for developing cancer and open new avenues for the design of immunotherapeutics relevant for clinical oncology.
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Imunoglobulina E/imunologia , Vigilância Imunológica/imunologia , Neoplasias/imunologia , Receptores de IgE/imunologia , Adjuvantes Imunológicos , Animais , Engenharia Genética , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Receptores de IgE/deficiênciaRESUMO
The IgE-mediated immune system activation can be redirected to combat tumors. Mouse and human IgE have been shown to provide a potent adjuvant effect in antitumor vaccination, with a crucial role played by FcεRI. This effect results from T cell-mediated adaptive immune response. Modified vaccinia virus Ankara (MVA) has been used to infect IgE-loaded tumor cells. These results led to a shift toward a highly safe protocol employing membrane IgE (mIgE), thus eliminating any possible anaphylactogenicity caused by circulating IgE. Evidence that human mIgE and a truncated version lacking IgE Fabs (tmIgE) bind and activate FcεRI has been fundamental and forms the core of this report. Human tmIgE has been engineered into a recombinant MVA (rMVA-tmIgE), and the expression of tmIgE and its transport to the surface of rMVA-tmIgE-infected cells has been detected by Western blot and cytofluorimetry, respectively. FcεRI activation by tmIgE has been confirmed by the release of ß-hexosaminidase in a cell-to-cell contact assay using human FcεRI-transfected RBL-SX38 cells. The rMVA-tmIgE antitumor vaccination strategy has been investigated in FcεRIα(-/-) human FcεRIα(+) mice, with results indicating a level of protection comparable to that obtained using soluble human IgE tumor cell loading. The rMVA-tmIgE vector represents a device that suits safe IgE-based antitumor vaccines, harboring the possibility to couple tmIgE with other gene insertions that might enhance the antitumor effect, thus bringing the field closer to the clinics.
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Vacinas Anticâncer/imunologia , Membrana Celular/imunologia , Imunoglobulina E/imunologia , Neoplasias/imunologia , Vaccinia virus , Animais , Vacinas Anticâncer/biossíntese , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Feminino , Humanos , Imunoglobulina E/biossíntese , Imunoglobulina E/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , VacinaçãoRESUMO
LAMB2 gene disorders present with different phenotypes. Pierson syndrome (PS) is a common phenotype associated with LAMB2 variants. Neuromuscular phenotype has been reported including hypotonia and developmental delay. However, neuromuscular junction abnormalities represented as congenital myasthenic syndrome (CMS) was reported in one adult patient only. Here, in this paper, we present two pediatric cases with a severe presentation of PS and have CMS so expanding the knowledge of LAMB2 related phenotypes. The first patient had hypotonia and global developmental delay. Targeted genetic testing panel demonstrated homozygous pathogenic variant in the LAMB2 gene (c.5182C>T, pGln1728*) which was reported by Maselli et al. 2009. Repetitive nerve stimulation (RNS) showed a decremental response at low frequency of 3 Hz. On the other hand, the second patient had profound weakness since birth. Tri-Whole exome sequencing showed homozygous pathogenic variant in the LAMB2 gene c.2890C>T, pArg964*. A trial of salbutamol did not improve the symptoms. Both patients passed away from sequala of PS. The spectrum of phenotypic changes associated with LAMB2 mutations is still expanding, and further investigation into the various clinical and morphologic presentations associated with these mutations is important to better identify and manage affected individuals.
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Síndromes Miastênicas Congênitas , Humanos , Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/fisiopatologia , Síndromes Miastênicas Congênitas/diagnóstico , Masculino , Feminino , Anormalidades do Olho/genética , Anormalidades do Olho/complicações , Laminina/genética , Fenótipo , Mutação , Anormalidades Múltiplas/genética , Lactente , Doenças da Junção Neuromuscular/genética , Pré-Escolar , Síndrome Nefrótica , Distúrbios PupilaresRESUMO
Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare autoimmune neurological disorder seen in both pediatric and adult populations. CIDP typically presents with progressive and persistent weakness over at least 4 weeks in addition to sensory symptoms in the extremities. Although CIDP shares common clinical features between children and adults, it sometimes presents as a distinct clinical entity in children that requires close attention and recognition. A major caveat when diagnosing a child with CIDP is the clinical and diagnostic overlap with inherited neuropathies, most commonly Charcot-Marie-Tooth disease (CMT). Demyelinating CMT (dCMT) and CIDP might share similar clinical presentations, and sometimes it might be difficult to differentiate them on the basis of the electrodiagnostic findings or cerebrospinal fluid (CSF) albumino-cytological dissociation. This indeed merits early consideration for genetic testing in patients who do not respond to conventional CIDP therapies. Current treatment options for CIDP include intravenous immunoglobulins (IVIG), corticosteroids (CS), and plasmapheresis (PLEX). The need for novel therapies is essential in instances where patients continue to have symptoms despite the standard therapies or due to adverse effects of long-term use of standard therapies such as CS. This paper reviews the challenges in the diagnosis of CIDP in children and the current as well as novel therapies for CIDP.
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There are more than 10,000 individual rare diseases and most are without therapy. Personalized genetic therapy represents one promising approach for their treatment. We present a road map for individualized treatment of an ultra-rare disease by establishing a gene replacement therapy developed for a single patient with hereditary spastic paraplegia type 50 (SPG50). Through a multicenter collaboration, an adeno-associated virus-based gene therapy product carrying the AP4M1 gene was created and successfully administered intrathecally to a 4-year-old patient within 3 years of diagnosis as part of a single-patient phase 1 trial. Primary endpoints were safety and tolerability, and secondary endpoints evaluated efficacy. At 12 months after dosing, the therapy was well tolerated. No serious adverse events were observed, with minor events, including transient neutropenia and Clostridioides difficile gastroenteritis, experienced but resolved. Preliminary efficacy measures suggest a stabilization of the disease course. Longer follow-up is needed to confirm the safety and provide additional insights on the efficacy of the therapy. Overall, this report supports the safety of gene therapy for SPG50 and provides insights into precision therapy development for rare diseases. Clinical trial registration: NCT06069687 .
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Dependovirus , Terapia Genética , Paraplegia Espástica Hereditária , Humanos , Dependovirus/genética , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/terapia , Terapia Genética/métodos , Pré-Escolar , Masculino , Vetores Genéticos/genética , Resultado do TratamentoRESUMO
Spinal muscular atrophy (SMA) is a neuromuscular genetic disorder caused by the loss of lower motor neurons leading to progressive muscle weakness and atrophy. With the rise of novel therapies and early diagnosis on newborn screening (NBS), the natural history of SMA has been evolving. Earlier therapeutic interventions can modify disease outcomes and improve survival. The role of treatment in infants born preterm is an important question given the importance of early intervention. In this study, we discuss the case of an infant born at 32 weeks who was diagnosed with SMA on NBS and was treated with Spinraza® (Nusinersen) and Zolgensma® (Onasemnogene abeparvovec-xioi) within the first 2 months of life. With the scarce evidence that currently exists, clinicians should be aware of the efficacy and safety impact of early therapy particularly in the preterm infant.
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OBJECTIVE: Spinal muscular atrophy (SMA) is a neuromuscular disorder that manifests with motor deterioration and respiratory complications. The paradigm of care is shifting as disease-modifying therapies including nusinersen, onasemnogene abeparvovec and risdiplam alter the disease trajectory of SMA. The objective of this study was to explore caregivers' experiences with disease-modifying therapies for SMA. DESIGN: Qualitative study including semistructured interviews with caregivers of children with SMA who received disease-modifying therapies. Interviews were audio recorded, transcribed verbatim, coded and analysed using content analysis. SETTING: The Hospital for Sick Children (Toronto, Canada). RESULTS: Fifteen family caregivers of children with SMA type 1 (n=5), type 2 (n=5) and type 3 (n=5) participated. There were two emerging themes and several subthemes (in parentheses): (1) inequities in access to disease-modifying therapies (variable regulatory approvals, prohibitively expensive therapies and insufficient infrastructure) and (2) patient and family experience with disease-modifying therapies (decision making, hope, fear and uncertainty). CONCLUSION: The caregiver experience with SMA has been transformed by the advent of disease-modifying therapies. Consistent and predictable access to disease-modifying therapies is a major concern for caregivers of children with SMA but is influenced by regulatory approvals, funding and eligibility criteria that are heterogenous across jurisdictions. Many caregivers described going to great lengths to access therapies, highlighting issues related to justice, such as equity and access. This diverse population reflects contemporary patients and families with SMA; their broad experiences may inform the healthcare delivery of other emerging orphan drugs.
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Atrofia Muscular Espinal , Atrofias Musculares Espinais da Infância , Criança , Humanos , Cuidadores , Atrofia Muscular Espinal/tratamento farmacológico , Atrofias Musculares Espinais da Infância/tratamento farmacológico , Pesquisa Qualitativa , IncertezaRESUMO
Depriving cells of nutrients triggers an energetic crisis, which is resolved by metabolic rewiring and organelle reorganization. Primary cilia are microtubule-based organelles at the cell surface, capable of integrating multiple metabolic and signalling cues, but their precise sensory function is not fully understood. Here we show that primary cilia respond to nutrient availability and adjust their length via glutamine-mediated anaplerosis facilitated by asparagine synthetase (ASNS). Nutrient deprivation causes cilia elongation, mediated by reduced mitochondrial function, ATP availability and AMPK activation independently of mTORC1. Of note, glutamine removal and replenishment is necessary and sufficient to induce ciliary elongation or retraction, respectively, under nutrient stress conditions both in vivo and in vitro by restoring mitochondrial anaplerosis via ASNS-dependent glutamate generation. Ift88-mutant cells lacking cilia show reduced glutamine-dependent mitochondrial anaplerosis during metabolic stress, due to reduced expression and activity of ASNS at the base of cilia. Our data indicate a role for cilia in responding to, and possibly sensing, cellular glutamine levels via ASNS during metabolic stress.
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Aspartato-Amônia Ligase , Glutamina , Glutamina/metabolismo , Aspartato-Amônia Ligase/metabolismo , Cílios/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Spinal muscular atrophy (SMA) is a genetic disorder that may result in neuromuscular weakness and respiratory insufficiency. Gene replacement therapy has changed the trajectory of this condition, but long-term outcomes related to sleep disordered breathing are not known. METHODS: This was a retrospective review of infants with SMA identified via newborn screening who subsequently received onasemnogene abeparvovec at the Hospital for Sick Children (Ontario, Canada). Polysomnograms were conducted at the time of confirmed diagnosis as well as regularly thereafter. RESULTS: Eleven children (4 female) were identified via newborn screen (7 with 2 copies of the SMN2 gene and 4 with 3 copies of the SMN2 gene) and received onasemnogene abeparvovec at a median age of 3.6 weeks. All eleven infants met criteria for sleep disordered breathing based on their first completed polysomnograms but improved over time. Three infants required respiratory technology, including a premature infant who was prescribed nocturnal supplemental oxygen therapy for central sleep apnea and two symptomatic infants with neuromuscular weakness who required nocturnal noninvasive ventilation. We did not find a correlation between motor scores and polysomnogram parameters. CONCLUSION: Children treated with onasemnogene abeparvovec have reduced sleep disordered breathing over time. Polysomnograms revealed abnormal parameters in all children, but the clinical significance of these findings was unclear for children who were asymptomatic for sleep disordered breathing or neuromuscular weakness. These results highlight the need to evaluate both motor scores and respiratory symptoms to ensure a holistic evaluation of clinical status.
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Atrofia Muscular Espinal , Síndromes da Apneia do Sono , Apneia do Sono Tipo Central , Atrofias Musculares Espinais da Infância , Criança , Recém-Nascido , Humanos , Lactente , Feminino , Triagem Neonatal , Síndromes da Apneia do Sono/diagnóstico , Síndromes da Apneia do Sono/terapia , Apneia do Sono Tipo Central/diagnóstico , Apneia do Sono Tipo Central/terapia , Ontário , Atrofias Musculares Espinais da Infância/diagnóstico , Atrofias Musculares Espinais da Infância/genética , Atrofias Musculares Espinais da Infância/terapiaRESUMO
Background and Objectives: The introduction of spinal muscular dystrophy (SMA)-modifying therapies, such as antisense oligonucleotide therapy, has changed the natural history of SMA. Most reports on treatment outcomes have focused on motor scores and respiratory function. The objective of this study is to document the development and progression of scoliosis in patients with SMA1 treated with nusinersen. Methods: A descriptive single-center study was conducted in patients with SMA1 who were treated with nusinersen before 6 months of age. Data were collected on patients who met criteria, including age at the first nusinersen dose, number of nusinersen doses, degree of scoliosis, respiratory parameters, feeding route, and motor scores at baseline and follow-up. The Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP-INTEND) was subanalyzed using axial (AxS) and appendicular motor (ApS) scores to evaluate a possible correlation between scoliosis and axial muscle strength. Results: From our cohort, 31 percent (11/35) of patients had a diagnosis of SMA1. Sixty-three percent (7/11) met the inclusion criteria. All patients (7/7) showed initial improvement in their CHOP-INTEND scores in correlation with improvement on the ApS. Despite this, most patients did not show improvement in the AxS. Subsequently, all patients developed scoliosis in the first year of life with Cobb angles that ranged between 18° and 60°. Furthermore, total CHOP-INTEND scores had dropped in 2 patients alongside the development of a Cobb angle of >40°. Discussion: Despite the significant improvement in functional motor assessment in patients with SMA1, there is a progression of significant scoliosis despite treatment. Subsequently, lack or minimal improvement on the axial CHOP-INTEND scores may predict worsening on the total motor scores.
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Working with C57BL/6 mouse tumor models, we had previously demonstrated that vaccination with IgE-coated tumor cells can protect against tumor challenge, an observation that supports the involvement of IgE in antitumor immunity. The adjuvant effect of IgE was shown to result from eosinophil-dependent priming of the T cell-mediated adaptive immune response. The protective effect is likely to be mediated by the interaction of tumor cell-bound IgE with receptors, which then trigger the release of mediators, recruitment of effector cells, cell killing and tumor Ag cross-priming. It was therefore of utmost importance to demonstrate the strict dependence of the protective effect on IgE receptor activation. First, the protective effect of IgE was confirmed in a BALB/c tumor model, in which IgE-loaded modified VV Ankara-infected tumor cells proved to be an effective cellular vaccine. However, the protective effect was lost in Fc(epsilon)RIalpha(-/-) (but not in CD23(-/-)) knockout mice, showing the IgE-Fc(epsilo)nRI interaction to be essential. Moreover, human IgE (not effective in BALB/c mice) had a protective effect in the humanized knockin mouse (Fc(epsilon)RIalpha(-/-) hFc(epsilon)RIalpha(+)). This finding suggests that the adjuvant effect of IgE could be exploited for human therapeutics.
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Adjuvantes Imunológicos/fisiologia , Adjuvantes Imunológicos/uso terapêutico , Antineoplásicos/uso terapêutico , Imunoglobulina E/fisiologia , Imunoglobulina E/uso terapêutico , Receptores de IgE/fisiologia , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Adjuvantes Imunológicos/metabolismo , Animais , Antígenos de Neoplasias/imunologia , Antineoplásicos/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/administração & dosagem , Reagentes de Ligações Cruzadas/metabolismo , Reagentes de Ligações Cruzadas/uso terapêutico , Feminino , Técnicas de Introdução de Genes , Humanos , Imunoglobulina E/metabolismo , Leucemia Basofílica Aguda/imunologia , Leucemia Basofílica Aguda/patologia , Leucemia Basofílica Aguda/terapia , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Linfoma de Células T/terapia , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ratos , Receptores de IgE/deficiência , Receptores de IgE/genéticaRESUMO
Mutations in the NPHP1 gene, coding for human nephrocystin-1 (NPHP1), cause the autosomal recessive disease nephronophthisis, the most common cause of end-stage renal disease in children and adolescents. The function and structure of NPHP1 are still poorly characterized. NPHP1 presents a modular structure well in keeping with its role as an adaptor protein: it harbors an SH3 domain flanked by two glutamic acid-rich regions and a conserved C-terminal nephrocystin homology domain (NHD). Similar to other NPHP protein family members, its N-terminus contains a putative coiled-coil domain (NPHP1CC) that is supposed to play an important role in NPHP1 self-association and/or protein-protein interactions. Structural studies proving its structure and its oligomerization state are still lacking. Here we demonstrate that NPHP1CC is monomeric in solution and unexpectedly folds into an autonomous domain forming a three-stranded antiparallel coiled coil suitable for protein-protein interactions. Notably, we found that the NPHP1CC shares remarkable structural similarities with the three-stranded coiled coil of the BAG domain protein family, which is known to mediate the anti-apoptotic function of these proteins, suggesting a possible similar role for NPHP1CC. In agreement with this hypothesis, we show that in the context of the full-length protein the NPHP1CC is fundamental to regulate resistance to apoptotic stimuli.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas do Citoesqueleto/metabolismo , Sequência de Aminoácidos , Animais , Cães , Humanos , Células Madin Darby de Rim Canino , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Dobramento de Proteína , Alinhamento de SequênciaRESUMO
Polycystin-1 (PC-1) and 2 (PC-2) are the products of the PKD1 and PKD2 genes, which are mutated in Autosomal Dominant Polycystic Kidney Disease (ADPKD). They form a receptor/channel complex that has been suggested to function as a mechanosensor, possibly activated by ciliary bending in the renal tubule, and resulting in calcium influx. This model has recently been challenged, leaving the question as to which mechanical stimuli activate the polycystins still open. Here, we used a SILAC/Mass-Spec approach to identify intracellular binding partners of tagged-endogenous PC-1 whereby we detected a class of interactors mediating regulation of cellular actomyosin contraction. Accordingly, using gain and loss-of-function cellular systems we found that PC-1 negatively regulates cellular contraction and YAP activation in response to extracellular stiffness. Thus, PC-1 enables cells to sense the rigidity of the extracellular milieu and to respond appropriately. Of note, in an orthologous murine model of PKD we found evidence of increased actomyosin contraction, leading to enhanced YAP nuclear translocation and transcriptional activity. Finally, we show that inhibition of ROCK-dependent actomyosin contraction by Fasudil reversed YAP activation and significantly improved disease progression, in line with recent studies. Our data suggest a possible direct role of PC-1 as a mechanosensor of extracellular stiffness.
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Actomiosina/fisiologia , Canais de Cátion TRPP/fisiologia , Animais , Modelos Animais de Doenças , Cães , Espaço Extracelular/metabolismo , Imunofluorescência , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Imunoprecipitação , Células Madin Darby de Rim Canino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Doenças Renais Policísticas/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Renal cell carcinomas (RCCs) are common cancers diagnosed in more than 350,000 people each year worldwide. Several pathways are de-regulated in RCCs, including mTORC1. However, how mTOR drives tumorigenesis in this context is unknown. The lack of faithful animal models has limited progress in understanding and targeting RCCs. Here, we generated a mouse model harboring the kidney-specific inactivation of Tsc1. These animals develop cysts that evolve into papillae, cystadenomas, and papillary carcinomas. Global profiling confirmed several metabolic derangements previously attributed to mTORC1. Notably, Tsc1 inactivation results in the accumulation of fumarate and in mTOR-dependent downregulation of the TCA cycle enzyme fumarate hydratase (FH). The re-expression of FH in cellular systems lacking Tsc1 partially rescued renal epithelial transformation. Importantly, the mTORC1-FH axis is likely conserved in human RCC specimens. We reveal a role of mTORC1 in renal tumorigenesis, which depends on the oncometabolite fumarate.
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Carcinoma de Células Renais/metabolismo , Fumaratos/metabolismo , Neoplasias Renais/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Células Cultivadas , Feminino , Fumarato Hidratase/genética , Fumarato Hidratase/metabolismo , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Endogâmicos C57BL , Regulação para CimaRESUMO
Cystic kidney diseases (CKD) is a class of disorders characterized by ciliary dysfunction and, therefore, belonging to the ciliopathies. The prototype CKD is autosomal dominant polycystic kidney disease (ADPKD), whose mutated genes encode for two membrane-bound proteins, polycystin-1 (PC-1) and polycystin-2 (PC-2), of unknown function. Recent studies on CKD-associated genes identified new mechanisms of morphogenesis that are central for establishment and maintenance of proper renal tubular diameter. During embryonic development in the mouse and lower vertebrates a convergent-extension (CE)-like mechanism based on planar cell polarity (PCP) and cellular intercalation is involved in "sculpting" the tubules into a narrow and elongated shape. Once the appropriate diameter is established, further elongation occurs through oriented cell division (OCD). The polycystins (PCs) regulate some of these essential processes. In this review we summarize recent work on the role of PCs in regulating cell migration, the cytoskeleton, and front-rear polarity. These important properties are essential for proper morphogenesis of the renal tubules and the lymphatic vessels. We highlight here several open questions and controversies. Finally, we try to outline some of the next steps required to study these processes and their relevance in physiological and pathological conditions.
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Nuclear retinoic acid (RA) receptors (RARs) activate gene expression through dynamic interactions with coregulators in coordination with the ligand and phosphorylation processes. Here we show that during RA-dependent activation of the RARalpha isotype, the p160 coactivator pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3 is phosphorylated by p38MAPK. SRC-3 phosphorylation has been correlated to an initial facilitation of RARalpha-target genes activation, via the control of the dynamics of the interactions of the coactivator with RARalpha. Then, phosphorylation inhibits transcription via promoting the degradation of SRC-3. In line with this, inhibition of p38MAPK markedly enhances RARalpha-mediated transcription and RA-dependent induction of cell differentiation. SRC-3 phosphorylation and degradation occur only within the context of RARalpha complexes, suggesting that the RAR isotype defines a phosphorylation code through dictating the accessibility of the coactivator to p38MAPK. We propose a model in which RARalpha transcriptional activity is regulated by SRC-3 through coordinated events that are fine-tuned by RA and p38MAPK.
Assuntos
Acetiltransferases/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Oncogênicas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Receptores do Ácido Retinoico/metabolismo , Transativadores/metabolismo , Transcrição Gênica/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Células COS , Chlorocebus aethiops , Regulação da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Histona Acetiltransferases , Humanos , Camundongos , Complexos Multiproteicos/metabolismo , Coativador 3 de Receptor Nuclear , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptor alfa de Ácido Retinoico , Transcrição Gênica/efeitos dos fármacos , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
In eukaryotic cells, liganded RAR gamma 2/RXR alpha heterodimers activate the transcription of retinoic acid (RA) target genes and then are degraded through the ubiquitin-proteasome pathway. In this study, we dissected the role of the RAR gamma 2 and RXR alpha partners as well as of their respective AF-1 and AF-2 domains in the processes of transactivation and degradation. RAR gamma 2 is the "engine" initiating transcription and its own degradation subsequent to ligand binding. Integrity of its AF-2 domain and phosphorylation of its AF-1 domain are required for both the degradation and the transactivation of the receptor. Deletion of the whole AF-1 domain does not impair these processes but shifts the receptor toward other proteolytic pathways through RXR alpha. In contrast, RXR alpha plays only a modulatory role, cooperating with RAR gamma 2 through its AF-2 domain and its phosphorylated AF-1 domain in both the transcription activity and the degradation of the RAR gamma 2/RXR alpha heterodimers. Our results underline that the AF-1 and AF-2 domains of each heterodimer partner cooperate with one other and that this cooperation is relevant for both the transcription and degradation processes.