RESUMO
The precise migration of cortical interneurons is essential for the formation and function of cortical circuits, and disruptions to this key developmental process are implicated in the etiology of complex neurodevelopmental disorders, including schizophrenia, autism and epilepsy. We have recently identified the Jun N-terminal kinase (JNK) pathway as an important mediator of cortical interneuron migration in mice, regulating the proper timing of interneuron arrival into the cortical rudiment. In the current study, we demonstrate a vital role for JNK signaling at later stages of corticogenesis, when interneurons transition from tangential to radial modes of migration. Pharmacological inhibition of JNK signaling in ex vivo slice cultures caused cortical interneurons to rapidly depart from migratory streams and prematurely enter the cortical plate. Similarly, genetic loss of JNK function led to precocious stream departure ex vivo, and stream disruption, morphological changes and abnormal allocation of cortical interneurons in vivo These data suggest that JNK signaling facilitates the tangential migration and laminar deposition of cortical interneurons, and further implicates the JNK pathway as an important regulator of cortical development.
Assuntos
Movimento Celular , Córtex Cerebral/citologia , Interneurônios/citologia , Sistema de Sinalização das MAP Quinases , Animais , Animais Recém-Nascidos , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Alternating hemiplegia of childhood (AHC) is a rare neurodevelopmental disease caused by heterozygous de novo missense mutations in the ATP1A3 gene that encodes the neuronal specific α3 subunit of the Na,K-ATPase (NKA) pump. Mechanisms underlying patient episodes including environmental triggers remain poorly understood, and there are no empirically proven treatments for AHC. In this study, we generated patient-specific induced pluripotent stem cells (iPSCs) and isogenic controls for the E815K ATP1A3 mutation that causes the most phenotypically severe form of AHC. Using an in vitro iPSC-derived cortical neuron disease model, we found elevated levels of ATP1A3 mRNA in AHC lines compared to controls, without significant perturbations in protein expression. Microelectrode array analyses demonstrated that in cortical neuronal cultures, ATP1A3+/E815K iPSC-derived neurons displayed less overall activity than neurons differentiated from isogenic mutation-corrected and unrelated control cell lines. However, induction of cellular stress by elevated temperature revealed a hyperactivity phenotype following heat stress in ATP1A3+/E815K neurons compared to control lines. Treatment with flunarizine, a drug commonly used to prevent AHC episodes, did not impact this stress-triggered phenotype. These findings support the use of iPSC-derived neuronal cultures for studying complex neurodevelopmental conditions such as AHC and provide a platform for mechanistic discovery in a human disease model.
Assuntos
Hemiplegia/metabolismo , Neurônios/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Diferenciação Celular , Células Cultivadas , Córtex Cerebral/metabolismo , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Mutação de Sentido Incorreto , Fenótipo , RNA Mensageiro/metabolismoRESUMO
Mutations in the DEPDC5 gene can cause epilepsy, including forms with and without brain malformations. The goal of this study was to investigate the contribution of DEPDC5 gene dosage to the underlying neuropathology of DEPDC5-related epilepsies. We generated induced pluripotent stem cells (iPSCs) from epilepsy patients harboring heterozygous loss of function mutations in DEPDC5. Patient iPSCs displayed increases in both phosphorylation of ribosomal protein S6 and proliferation rate, consistent with elevated mTORC1 activation. In line with these findings, we observed increased soma size in patient iPSC-derived cortical neurons that was rescued with rapamycin treatment. These data indicate that human cells heterozygous for DEPDC5 loss-of-function mutations are haploinsufficient for control of mTORC1 signaling. Our findings suggest that human pathology differs from mouse models of DEPDC5-related epilepsies, which do not show consistent phenotypic differences in heterozygous neurons, and support the need for human-based models to affirm and augment the findings from animal models of DEPDC5-related epilepsy.
Assuntos
Epilepsia Resistente a Medicamentos/genética , Proteínas Ativadoras de GTPase/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Epilepsia Resistente a Medicamentos/metabolismo , Haploinsuficiência , Humanos , Células-Tronco Pluripotentes Induzidas , Malformações do Desenvolvimento Cortical/genética , Malformações do Desenvolvimento Cortical/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Tuberous sclerosis complex (TSC) is a pediatric disorder of dysregulated growth and differentiation caused by loss of function mutations in either the TSC1 or TSC2 genes, which regulate mTOR kinase activity. To study aberrations of early development in TSC, we generated induced pluripotent stem cells using dermal fibroblasts obtained from patients with TSC. During validation, we found that stem cells generated from TSC patients had a very high rate of integration of the reprogramming plasmid containing a shRNA against TP53. We also found that loss of one allele of TSC2 in human fibroblasts is sufficient to increase p53 levels and impair stem cell reprogramming. Increased p53 was also observed in TSC2 heterozygous and homozygous mutant human stem cells, suggesting that the interactions between TSC2 and p53 are consistent across cell types and gene dosage. These results support important contributions of TSC2 heterozygous and homozygous mutant cells to the pathogenesis of TSC and the important role of p53 during reprogramming.
Assuntos
Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Perda de Heterozigosidade , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Adolescente , Adulto , Alelos , Criança , Pré-Escolar , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Genes p53 , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactente , Masculino , Mutação , RNA Interferente Pequeno/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Esclerose Tuberosa/genética , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose TuberosaRESUMO
The p75 neurotrophin receptor (p75(NTR)) mediates the death of specific populations of neurons during the development of the nervous system or after cellular injury. The receptor has also been implicated as a contributor to neurodegeneration caused by numerous pathological conditions. Because many of these conditions are associated with increases in reactive oxygen species, we investigated whether p75(NTR) has a role in neurodegeneration in response to oxidative stress. Here we demonstrate that p75(NTR) signaling is activated by 4-hydroxynonenal (HNE), a lipid peroxidation product generated naturally during oxidative stress. Exposure of sympathetic neurons to HNE resulted in neurite degeneration and apoptosis. However, these effects were reduced markedly in neurons from p75(NTR-/-) mice. The neurodegenerative effects of HNE were not associated with production of neurotrophins and were unaffected by pretreatment with a receptor-blocking antibody, suggesting that oxidative stress activates p75(NTR) via a ligand-independent mechanism. Previous studies have established that proteolysis of p75(NTR) by the metalloprotease TNFα-converting enzyme and γ-secretase is necessary for p75(NTR)-mediated apoptotic signaling. Exposure of sympathetic neurons to HNE resulted in metalloprotease- and γ-secretase-dependent cleavage of p75(NTR). Pharmacological blockade of p75(NTR) proteolysis protected sympathetic neurons from HNE-induced neurite degeneration and apoptosis, suggesting that cleavage of p75(NTR) is necessary for oxidant-induced neurodegeneration. In vivo, p75(NTR-/-) mice exhibited resistance to axonal degeneration associated with oxidative injury following administration of the neurotoxin 6-hydroxydopamine. Together, these data suggest a novel mechanism linking oxidative stress to ligand-independent cleavage of p75(NTR), resulting in axonal fragmentation and neuronal death.
Assuntos
Apoptose/fisiologia , Axônios , Estresse Oxidativo , Receptores de Fator de Crescimento Neural/fisiologia , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento , Testes de Campo VisualRESUMO
A 61-year-old man presented with 2 days of "flickering" vision. The symptom resolved with closure of the left eye. Examination demonstrated involuntary high-frequency, low-amplitude intorting movements of the left eye, consistent with superior oblique myokymia (Video 1). Ocular ductions were full, and there were no abnormal movements of the right eye.