RESUMEN
GNAO1 encodes G protein subunit alpha O1 (Gαo). Pathogenic variations in GNAO1 cause developmental delay, intractable seizures, and progressive involuntary movements from early infancy. Because the functional role of GNAO1 in the developing brain remains unclear, therapeutic strategies are still unestablished for patients presenting with GNAO1-associated encephalopathy. We herein report that siRNA-mediated depletion of Gnao1 perturbs the expression of transcripts associated with Rho GTPase signaling in Neuro2a cells. Consistently, siRNA treatment hampered neurite outgrowth and extension. Growth cone formation was markedly disrupted in monolayer neurons differentiated from iPSCs from a patient with a pathogenic variant of Gαo (p.G203R). This variant disabled neuro-spherical assembly, acquisition of the organized structure, and polarized signals of phospho-MLC2 in cortical organoids from the patient's iPSCs. We confirmed that the Rho kinase inhibitor Y27632 restored these morphological phenotypes. Thus, Gαo determines the self-organizing process of the developing brain by regulating the Rho-associated pathway. These data suggest that Rho GTPase pathway might be an alternative target of therapy for patients with GNAO1-associated encephalopathy.
Asunto(s)
Diferenciación Celular , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Células Madre Pluripotentes Inducidas , Neuronas , Transducción de Señal , Proteínas de Unión al GTP rho , Humanos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Neuronas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Unión al GTP rho/genética , Ratones , Animales , Quinasas Asociadas a rho/metabolismo , Organoides/metabolismo , Amidas/farmacología , PiridinasRESUMEN
An autism-associated gene Shank3 encodes multiple splicing isoforms, Shank3a-f. We have recently reported that Shank3a/b-knockout mice were more susceptible to kainic acid-induced seizures than wild-type mice at 4 weeks of age. Little is known, however, about how the N-terminal and ankyrin repeat domains (NT-Ank) of Shank3a/b regulate multiple molecular signals in the developing brain. To explore the functional roles of Shank3a/b, we performed a mass spectrometry-based proteomic search for proteins interacting with GFP-tagged NT-Ank. In this study, NT-Ank was predicted to form a variety of complexes with a total of 348 proteins, in which RNA-binding (n = 102), spliceosome (n = 22), and ribosome-associated molecules (n = 9) were significantly enriched. Among them, an X-linked intellectual disability-associated protein, Nono, was identified as a NT-Ank-binding protein. Coimmunoprecipitation assays validated the interaction of Shank3 with Nono in the mouse brain. In agreement with these data, the thalamus of Shank3a/b-knockout mice aberrantly expressed splicing isoforms of autism-associated genes, Nrxn1 and Eif4G1, before and after seizures with kainic acid treatment. These data indicate that Shank3 interacts with multiple RNA-binding proteins in the postnatal brain, thereby regulating the homeostatic expression of splicing isoforms for autism-associated genes after birth.
Asunto(s)
Ratones Noqueados , Proteínas del Tejido Nervioso , Proteínas de Unión al ARN , Animales , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Ratones , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Repetición de Anquirina , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Empalme del ARN , Encéfalo/metabolismo , Convulsiones/metabolismo , Convulsiones/genética , Convulsiones/inducido químicamente , Humanos , Unión Proteica , Ratones Endogámicos C57BLRESUMEN
Pathogenic variants in ATP1A3, the gene encoding the α3 subunit of the Na+/K+-ATPase, cause alternating hemiplegia of childhood (AHC) and related disorders. Impairments in Na+/K+-ATPase activity are associated with the clinical phenotype. However, it remains unclear whether additional mechanisms are involved in the exaggerated symptoms under stressed conditions in patients with AHC. We herein report that the intracellular loop (ICL) of ATP1A3 interacted with RNA-binding proteins, such as Eif4g (encoded by Eif4g1), Pabpc1 and Fmrp (encoded by Fmr1), in mouse Neuro2a cells. Both the siRNA-mediated depletion of Atp1a3 and ectopic expression of the p.R756C variant of human ATP1A3-ICL in Neuro2a cells resulted in excessive phosphorylation of ribosomal protein S6 (encoded by Rps6) and increased susceptibility to heat stress. In agreement with these findings, induced pluripotent stem cells (iPSCs) from a patient with the p.R756C variant were more vulnerable to heat stress than control iPSCs. Neurons established from the patient-derived iPSCs showed lower calcium influxes in responses to stimulation with ATP than those in control iPSCs. These data indicate that inefficient protein synthesis contributes to the progressive and deteriorating phenotypes in patients with the p.R756C variant among a variety of ATP1A3-related disorders.
Asunto(s)
Respuesta al Choque Térmico , Células Madre Pluripotentes Inducidas , Mitocondrias , Biosíntesis de Proteínas , ATPasa Intercambiadora de Sodio-Potasio , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Humanos , Animales , Mitocondrias/metabolismo , Ratones , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Neuronas/metabolismo , Fosforilación , Unión Proteica , Calcio/metabolismoRESUMEN
Microglia play versatile roles in progression of and protection against neuroinflammatory diseases. Little is known, however, about the mechanisms underlying the diverse reactivity of microglia to inflammatory conditions. We investigated how human induced microglia-like (iMG) cells respond to innate immune ligands. Quantitative PCR showed that poly-I:C and lipopolysaccharide (LPS) activated the expression of IL1B and TNF. Immunoreactivity of iMG did not differ between controls (n = 11) and patients with neuroinflammatory diseases (n = 24). Flow cytometry revealed that CD14high cells expressed interleukin (IL) -1ß after LPS treatment. Immunoblotting showed that poly-I:C and LPS differentially activated inflammatory pathways but commonly induced mitochondrial instability and the expression of pyruvate kinase isoform M2 (PKM2). Furthermore, a potent stimulator of PKM2 (DASA-58) alleviated IL-1ß production after LPS treatment. These data indicate that heterogeneous cell populations and mitochondrial stability underlie the divergent immunoreactivity of human iMG in environments.
Asunto(s)
Microglía , Enfermedades Neuroinflamatorias , Humanos , Microglía/metabolismo , Lipopolisacáridos/farmacología , Citometría de Flujo , Expresión GénicaRESUMEN
Epileptic seizures are distinct but frequent comorbidities in children with autism spectrum disorder (ASD). The hyperexcitability of cortical and subcortical neurons appears to be involved in both phenotypes. However, little information is available concerning which genes are involved and how they regulate the excitability of the thalamocortical network. In this study, we investigate whether an ASD-associated gene, SH3 and multiple ankyrin repeat domains 3 (Shank3), plays a unique role in the postnatal development of thalamocortical neurons. We herein report that Shank3a/b, the splicing isoforms of mouse Shank3, were uniquely expressed in the thalamic nuclei, peaking from two to four weeks after birth. Shank3a/b-knockout mice showed lower parvalbumin signals in the thalamic nuclei. Consistently, Shank3a/b-knockout mice were more susceptible to generalized seizures than wild-type mice after kainic acid treatments. Together, these data indicate that NT-Ank domain of Shank3a/b regulates molecular pathways that protect thalamocortical neurons from hyperexcitability during the early postnatal period of mice.
Asunto(s)
Trastorno del Espectro Autista , Ratones , Animales , Convulsiones , Núcleos Talámicos , Ratones Noqueados , Isoformas de Proteínas/genética , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Phelan-McDermid syndrome (PMS) is a rare genetic disorder presenting with developmental delay, epilepsy, and autism spectrum disorder (ASD). The segmental deletion of chromosome 22q13.3 affects the copy number of SHANK3, the gene encoding a scaffolding protein at the postsynaptic density. Biological studies indicate that SHANK3 plays crucial roles in the development of synaptic functions in the postnatal brain. Notably, induced pluripotent stem (iPS) cells have enabled researchers to develop brain organoids and microglia from patients and to explore the pathophysiology of neurodevelopmental disorders in human cells. Single-cell RNA sequencing of these cells revealed that human-specific genes are uniquely expressed during cortical development. Thus, patient-derived disease models are expected to identify as-yet-unidentified functions of SHANK3 in the development of human brain. These efforts may help establish a new style of translational research in pediatrics, which is expected to provide therapeutic insight for children with PMS and broader categories of disease. IMPACT: Phelan-McDermid syndrome is a prototypic model for molecular studies of autism spectrum disorder. Brain organoids are expected to provide therapeutic insight. Single-cell RNA sequencing of microglia may uncover the functional roles of human-specific genes.
Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , Pediatría , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/terapia , Trastorno Autístico/genética , Trastorno Autístico/terapia , Niño , Deleción Cromosómica , Trastornos de los Cromosomas , Cromosomas Humanos Par 22/genética , Humanos , Proteínas del Tejido Nervioso/genéticaRESUMEN
BACKGROUND: Malnutrition and vitamin deficiency are growing concerns in the clinical management of children with autism spectrum disorder (ASD). This case report presents a boy with ASD who developed vitamin A deficiency during follow-up. CASE REPORT: A 7-y-old boy had been diagnosed with ASD and developmental delay at age 18 mo. He developed convulsions associated with hypocalcemia and vitamin D deficiency at 3 y of age. Although vitamin D supplementation was continued, he was only able to eat rice, green tea, and fried potatoes from 3 y of age to age 7 y. He had started rubbing his eyes and had refused to open his eyes 9 mo before. An ophthalmologic examination showed bilateral corneal ulcers and right corneal perforation. Vitamin A was immediately supplemented with a nasogastric tube; however, his right eye was surgically enucleated against the persistent infection. LITERATURE REVIEW: A search of the relevant literature from 1993 to 2020 identified 11 cases of patients with ASD (5-17 y of age) who developed vitamin A deficiency owing to malnutrition. Only 4 cases (36%) had a full recovery in visual acuity. CONCLUSION: Vitamin A deficiency frequently causes irreversible visual impairment in children with ASD. Vigilant monitoring of vitamin levels prevents unfavorable outcomes in children with ASD and difficulty in food intake.
Asunto(s)
Trastorno del Espectro Autista , Perforación Corneal , Deficiencia de Vitamina A , Deficiencia de Vitamina D , Trastorno del Espectro Autista/complicaciones , Niño , Suplementos Dietéticos , Humanos , Masculino , Deficiencia de Vitamina A/complicacionesRESUMEN
Developmental and epileptic encephalopathy (DEE) represents a group of neurodevelopmental disorders characterized by infantile-onset intractable seizures and unfavorable prognosis of psychomotor development. To date, hundreds of genes have been linked to the onset of DEE. GNAO1 is a DEE-associated gene encoding the alpha-O1 subunit of guanine nucleotide-binding protein (GαO ). Despite the increasing number of reported children with GNAO1 encephalopathy, the molecular mechanisms underlying their neurodevelopmental phenotypes remain elusive. We herein present that co-immunoprecipitation and mass spectrometry analyses identified another DEE-associated protein, SPTAN1, as an interacting partner of GαO . Silencing of endogenous Gnao1 attenuated the neurite outgrowth and calcium-dependent signaling. Inactivation of GNAO1 in human-induced pluripotent stem cells gave rise to anomalous brain organoids that only weakly expressed SPTAN1 and Ankyrin-G. Furthermore, GNAO1-deficient organoids failed to conduct synchronized firing to adjacent neurons. These data indicate that GαO and other DEE-associated proteins organize the cytoskeletal remodeling and functional polarity of neurons in the developing brain.
Asunto(s)
Citoesqueleto/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Animales , Encéfalo/metabolismo , Encefalopatías/metabolismo , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Trastornos del Neurodesarrollo/metabolismo , Neuronas/metabolismo , FenotipoRESUMEN
BACKGROUND: Cardio-facio-cutaneous syndrome (CFCS) is a rare genetic disorder characterized by cardiovascular anomalies, dysmorphic faces, ectodermal abnormalities and developmental delays. Mutations in BRAF and other RAS-MAPK pathway-associated genes are commonly identified in patients with CFCS. While this molecular pathway is known to be associated with neuro-inflammatory conditions, only one case with CFCS has been reported thus far to develop acute encephalopathy in childhood. CASE REPORT: A 3-year-old boy with dysmorphic features and mild psychomotor delay developed acute encephalopathy. After a 45-min long, generalized seizure, the magnetic resonance imaging revealed that the restricted diffusion signals spread to the bilateral subcortical white matters on day 1 of illness. Despite the 14â¯days of intensive care, the acute symptoms of encephalopathy left him intractable epilepsy and severe neurocognitive impairments. The whole-exome sequencing analysis identified a de novo heterozygous mutation of BRAF (NM_004333:p.Thr241Met) in this case. CONCLUSION: The present case suggests that the hyperactive condition of ERK signals might augment the development of acute encephalopathy and post-encephalopathic epilepsy in childhood.
Asunto(s)
Encefalopatías/etiología , Displasia Ectodérmica/fisiopatología , Insuficiencia de Crecimiento/fisiopatología , Cardiopatías Congénitas/fisiopatología , Proteínas Proto-Oncogénicas B-raf/genética , Anomalías Múltiples/genética , Encefalopatías/complicaciones , Encefalopatías/genética , Niño , Epilepsia Refractaria/complicaciones , Displasia Ectodérmica/complicaciones , Facies , Insuficiencia de Crecimiento/complicaciones , Cardiopatías Congénitas/complicaciones , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Mutación , Proteínas Proto-Oncogénicas B-raf/fisiologíaRESUMEN
BACKGROUND: Streptococcus pyogenes is an uncommon pathogen of purpura fulminans, and the pathogenesis of S. pyogenes-purpura fulminans remains unclear because of paucity of cases. We reported a pediatric case of S. pyogenes-purpura fulminans with literature review of the disease. CASE PRESENTATION: A 3-year-old boy showed limping, lethargy and acral gangrene within 24 h. A diagnosis of S. pyogenes-purpura fulminans was made for bacterial isolation from throat and peripheral blood. Intensive therapy led to a survival with amputation of the left distal metatarsal bone, and normal development. The isolated M12 carried no mutation of csrS/R or rgg. Thrombophilia or immunodeficiency was excluded. DISCUSSION: Twelve-reported cases (9 pediatric and 3 elderly) of S. pyogenes-purpura fulminans started with shock and coagulopathy. Five patients age < 8 years had no underlying disease and survived. One youngest and two immunocompromised patients died. CONCLUSION: Streptococcus pyogenes-acute infectious purpura fulminans is a distinctive rare form of aggressive GAS infections.