RESUMO
Hyperactivation of the mechanistic target of rapamycin (mTOR) kinase, as a result of loss-of-function mutations in tuberous sclerosis complex 1 (TSC1) or TSC2 genes, causes protein synthesis dysregulation, increased cell size, and aberrant neuronal connectivity. Dysregulated synthesis of synaptic proteins has been implicated in the pathophysiology of autism spectrum disorder (ASD) associated with TSC and fragile X syndrome. However, cell type-specific translational profiles in these disease models remain to be investigated. Here, we used high-fidelity and unbiased Translating Ribosome Affinity Purification (TRAP) methodology to purify ribosome-associated mRNAs and identified translational alterations in a rat neuronal culture model of TSC. We find that expression of many stress and/or activity-dependent proteins is highly induced while some synaptic proteins are repressed. Importantly, transcripts for the activating transcription factor-3 (Atf3) and mitochondrial uncoupling protein-2 (Ucp2) are highly induced in Tsc2-deficient neurons, as well as in a neuron-specific Tsc1 conditional knock-out mouse model, and show differential responses to the mTOR inhibitor rapamycin. Gelsolin, a known target of Atf3 transcriptional activity, is also upregulated. shRNA-mediated block of Atf3 induction suppresses expression of gelsolin, an actin-severing protein, and rescues spine deficits found in Tsc2-deficient neurons. Together, our data demonstrate that a cell-autonomous program consisting of a stress-induced Atf3-gelsolin cascade affects the change in dendritic spine morphology following mTOR hyperactivation. This previously unidentified molecular cascade could be a therapeutic target for treating mTORopathies. SIGNIFICANCE STATEMENT: Tuberous sclerosis complex (TSC) is a genetic disease associated with epilepsy and autism. Dysregulated protein synthesis has been implicated as a cause of this disease. However, cell type-specific translational profiles that are aberrant in this disease are unknown. Here we show that expression of many stress and/or activity-dependent proteins is highly induced while some synaptic proteins are repressed in neurons missing the Tsc2 gene expression. Identification of genes whose translation is abnormal in TSC may provide insights to previously unidentified therapeutic targets.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Gelsolina/metabolismo , Esclerose Tuberosa/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Mutantes , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Transfecção , Esclerose Tuberosa/patologiaRESUMO
Tuberous sclerosis complex (TSC) is a disorder arising from mutation in the TSC1 or TSC2 gene, characterized by the development of hamartomas in various organs and neurological manifestations including epilepsy, intellectual disability and autism. TSC1/2 protein complex negatively regulates the mammalian target of rapamycin complex 1 (mTORC1) a master regulator of protein synthesis, cell growth and autophagy. Autophagy is a cellular quality-control process that sequesters cytosolic material in double membrane vesicles called autophagosomes and degrades it in autolysosomes. Previous studies in dividing cells have shown that mTORC1 blocks autophagy through inhibition of Unc-51-like-kinase1/2 (ULK1/2). Despite the fact that autophagy plays critical roles in neuronal homeostasis, little is known on the regulation of autophagy in neurons. Here we show that unlike in non-neuronal cells, Tsc2-deficient neurons have increased autolysosome accumulation and autophagic flux despite mTORC1-dependent inhibition of ULK1. Our data demonstrate that loss of Tsc2 results in autophagic activity via AMPK-dependent activation of ULK1. Thus, in Tsc2-knockdown neurons AMPK activation is the dominant regulator of autophagy. Notably, increased AMPK activity and autophagy activation are also found in the brains of Tsc1-conditional mouse models and in cortical tubers resected from TSC patients. Together, our findings indicate that neuronal Tsc1/2 complex activity is required for the coordinated regulation of autophagy by AMPK. By uncovering the autophagy dysfunction associated with Tsc2 loss in neurons, our work sheds light on a previously uncharacterized cellular mechanism that contributes to altered neuronal homeostasis in TSC disease.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Células Cultivadas , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Células HEK293 , Hipocampo/citologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/metabolismo , Ratos , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaRESUMO
PURPOSE: To evaluate the effects of the antiepileptic medication vigabatrin (VGB) on the retina of pigmented rats. METHODS: Scotopic and photopic electroretinograms were recorded from dark- and light-adapted Long-Evans (pigmented) and Sprague Dawley (albino) rats administered, daily, 52-55 injections of 250 mg·kg(-1)·day(-1) VGB or 25-26 injections of 500 mg·kg(-1)·day(-1) VGB, or a corresponding number of sham injections. Sensitivity and saturated amplitude of the rod photoresponse (S, Rm(P3)) and postreceptor response (1/σ, Vm) were derived, as were sensitivity and amplitude of the cone-mediated postreceptor response (1/σ(cone), Vm(cone)). The oscillatory potentials and responses to a series of flickering lights (6.25, 12.5, 25 and 50 Hz) were studied in the time and frequency domains. A subset of rats' eyes was harvested for Western blotting or histology. RESULTS: Of the parameters derived from dark-adapted ERG responses, in both pigmented and albino rats, VGB repeatedly and reliably enhanced electroretinographic parameters; no significant ERG deficits were noted. No significant alterations were observed in ER/oxidative stress or in the Akt cell death/survival pathway. There were migrations of photoreceptor nuclei toward the RPE and outgrowths of bipolar cell dendrites into the outer nuclear layer in VGB-treated rats; these were never observed in sham-treated animals. CONCLUSIONS: Although VGB is associated with retinal dysfunction in patients and VGB toxicity has been demonstrated by other laboratories in the albino rat, in our pigmented and albino rats, VGB did not induce deficits in, but rather enhanced, retinal function. Nonetheless, retinal neuronal dysplasia was observed.
Assuntos
Albinismo/fisiopatologia , Anticonvulsivantes/farmacologia , Eletrorretinografia/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/fisiologia , Epitélio Pigmentado da Retina/fisiopatologia , Vigabatrina/farmacologia , Animais , Biomarcadores/metabolismo , Western Blotting , Adaptação à Escuridão , Luz , Masculino , Ratos , Ratos Long-Evans , Ratos Sprague-DawleyRESUMO
Tuberous sclerosis complex (TSC) is a multiorgan genetic disease in which brain involvement causes epilepsy, intellectual disability, and autism. The hallmark pathological finding in TSC is the cerebral cortical tuber and its unique constituent, giant cells. However, an animal model that replicates giant cells has not yet been described. Here, we report that mosaic induction of Tsc1 loss in neural progenitor cells in Tsc1(cc) Nestin-rtTA(+) TetOp-cre(+) embryos by doxycycline leads to multiple neurological symptoms, including severe epilepsy and premature death. Strikingly, Tsc1-null neural progenitor cells develop into highly enlarged giant cells with enlarged vacuoles. We found that the vacuolated giant cells had multiple signs of organelle dysfunction, including markedly increased mitochondria, aberrant lysosomes, and elevated cellular stress. We found similar vacuolated giant cells in human tuber specimens. Postnatal rapamycin treatment completely reversed these phenotypes and rescued the mutants from epilepsy and premature death, despite prenatal onset of Tsc1 loss and mTOR complex 1 activation in the developing brain. This TSC brain model provides insights into the pathogenesis and organelle dysfunction of giant cells, as well as epilepsy control in patients with TSC.
Assuntos
Modelos Animais de Doenças , Neurônios/metabolismo , Células-Tronco/metabolismo , Esclerose Tuberosa/patologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Sirolimo/farmacologia , Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Tetralogy of Fallot (ToF) is characterized by stenosis causing partial obstruction of the right ventricular outflow tract, typically alleviated through the surgical application of a vessel patch made from a biocompatible material. In this study, we use computational simulations to compare the mechanical performance of four patch materials for various stenosis locations. Nine idealized pre-operative ToF geometries were created by imposing symmetrical stenoses on each of three anatomical sub-regions of the pulmonary arteries of three patients with previously repaired ToF. A virtual surgery methodology was implemented to replicate the steps of vessel de-pressurization, surgical patching, and subsequent vessel expansion after reperfusion. Significant differences in patch average stress (p < 0.001) were found between patch materials. Biological patch materials (porcine xenopericardium, human pericardium) exhibited higher patch stresses in comparison to synthetic patch materials (Dacron and PTFE). Observed differences were consistent across the various stenosis locations and were insensitive to patient anatomy.
Assuntos
Tetralogia de Fallot , Humanos , Animais , Suínos , Tetralogia de Fallot/cirurgia , Constrição Patológica , Ventrículos do Coração , Artéria Pulmonar , PericárdioRESUMO
CDKL5 Deficiency Disorder (CDD) is a severe encephalopathy characterized by intractable epilepsy, infantile spasms, and cognitive disabilities. The detrimental CNS manifestations and lack of therapeutic interventions represent unmet needs, necessitating identification of CDD-dependent phenotypes for in vitro disease modeling and therapeutic testing. Here, we optimized a high-content assay to quantify cilia in CDKL5-deficient neurons. Our work shows that Cdkl5-knockdown neurons have elongated cilia and uncovers cilium lengthening in hippocampi of Cdkl5 knockout mice. Collectively, our findings identify cilia length alterations under CDKL5 activity loss in vitro and in vivo and reveal elongated cilia as a robust functional phenotype for CDD.
Assuntos
Síndromes Epilépticas , Proteínas Serina-Treonina Quinases , Animais , Cílios , Síndromes Epilépticas/genética , Camundongos , Neurônios , Fenótipo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Tuberous sclerosis complex (TSC) is a neurogenetic disorder caused by loss-of-function mutations in either the TSC1 or TSC2 genes and frequently results in prominent CNS manifestations, including epilepsy, mental retardation, and autism spectrum disorder. The TSC1/TSC2 protein complex plays a major role in controlling the Ser/Thr kinase mammalian target of rapamycin (mTOR), which is a master regulator of protein synthesis and cell growth. In this study, we show that endoplasmic reticulum (ER) stress regulates TSC1/TSC2 complex to limit mTOR activity. In addition, Tsc2-deficient rat hippocampal neurons and brain lysates from a Tsc1-deficient mouse model demonstrate both elevated ER and oxidative stress. In Tsc2-deficient neurons, the expression of stress markers such as CHOP and HO-1 is increased, and this increase is completely reversed by the mTOR inhibitor rapamycin both in vitro and in vivo. Neurons lacking a functional TSC1/TSC2 complex have increased vulnerability to ER stress-induced cell death via the activation of the mitochondrial death pathway. Importantly, knockdown of CHOP reduces oxidative stress and apoptosis in Tsc2-deficient neurons. These observations indicate that ER stress modulates mTOR activity through the TSC protein complex and that ER stress is elevated in cells lacking this complex. They also suggest that some of the neuronal dysfunction and neurocognitive deficits seen in TSC patients may be attributable to ER and oxidative stress and therefore potentially responsive to agents moderating these pathways.
Assuntos
Proteínas de Transporte/metabolismo , Neurônios/fisiologia , Estresse Oxidativo/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Células Cultivadas , Pré-Escolar , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Citometria de Fluxo/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Heme Oxigenase-1/metabolismo , Hipocampo/citologia , Humanos , Lactonas/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , RNA Interferente Pequeno/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Serina/metabolismo , Sesquiterpenos/farmacologia , Serina-Treonina Quinases TOR , Treonina/metabolismo , Fatores de Tempo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Transdução Genética/métodos , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Tunicamicina/farmacologiaRESUMO
Genetic manipulations of dissociated rodent neurons provide translatable in vitro models for disease-driven phenotypes. Cilia are cellular antenna with a role in neuronal maturation and function often perturbed in neurodevelopmental disorders. Efforts for automated imaging of these microscopic protrusions are crucial given the role of cilia in the brain. We developed a cell-based assay to monitor cilia in rat hippocampal neurons using lentiviral-mediated shRNA-based gene silencing. This optimized platform can be used for high-throughput cilia imaging, disease modeling, and drug screening. For complete details on the use and execution of this protocol, please refer to Di Nardo et al. (2020).
Assuntos
Cílios/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Neurônios/fisiologia , Animais , Células Cultivadas , Cílios/metabolismo , Cílios/fisiologia , Hipocampo/diagnóstico por imagem , Hipocampo/metabolismo , Microscopia/métodos , Neurogênese , Neurônios/metabolismo , Cultura Primária de Células/métodos , RatosRESUMO
Tuberous sclerosis complex (TSC) is a neurogenetic disorder that leads to elevated mechanistic targeting of rapamycin complex 1 (mTORC1) activity. Cilia can be affected by mTORC1 signaling, and ciliary deficits are associated with neurodevelopmental disorders. Here, we examine whether neuronal cilia are affected in TSC. We show that cortical tubers from TSC patients and mutant mouse brains have fewer cilia. Using high-content image-based assays, we demonstrate that mTORC1 activity inversely correlates with ciliation in TSC1/2-deficient neurons. To investigate the mechanistic relationship between mTORC1 and cilia, we perform a phenotypic screen for mTORC1 inhibitors with TSC1/2-deficient neurons. We identify inhibitors of the heat shock protein 90 (Hsp90) that suppress mTORC1 through regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Pharmacological inhibition of Hsp90 rescues ciliation through downregulation of Hsp27. Our study uncovers the heat-shock machinery as a druggable signaling node to restore mTORC1 activity and cilia due to loss of TSC1/2, and it provides broadly applicable platforms for studying TSC-related neuronal dysfunction.
Assuntos
Cílios/metabolismo , Resposta ao Choque Térmico , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neurônios/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Envelhecimento/metabolismo , Animais , Benzoquinonas/farmacologia , Encéfalo/patologia , Regulação para Baixo/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Lactamas Macrocíclicas/farmacologia , Camundongos Knockout , Neurônios/efeitos dos fármacos , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Sirolimo/farmacologia , Fatores de Tempo , Regulação para Cima/efeitos dos fármacosRESUMO
Glucose transporter type 1 (Glut-1) facilitates glucose flux across the blood-brain-barrier. In humans, Glut-1 deficiency causes acquired microcephaly, seizures and ataxia, which are recapitulated in our Glut-1 haploinsufficient mouse model. Postnatal brain weight deceleration and development of reactive astrogliosis were significant by P21 in Glut-1(+/-) mice. The brain weight differences remained constant after P21 whereas the reactive astrocytosis continued to increase and peaked at P90. Brain immunoblots showed increased phospho-mTOR and decreased phospho-GSK3-beta by P14. After fasting, the mature Glut-1(+/-) females showed a trend towards elevated phospho-GSK3-beta, a possible neuroprotective response. Lithium chloride treatment of human skin fibroblasts from control and Glut-1 DS patients produced a 45% increase in glucose uptake. Brain imaging of mature Glut-1(+/-) mice revealed a significantly decreased hippocampal volume. These subtle immunochemical changes reflect chronic nutrient deficiency during brain development and represent the experimental correlates to the human neurological phenotype associated with Glut-1 DS.
Assuntos
Astrócitos/patologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Gliose/patologia , Transportador de Glucose Tipo 1/deficiência , Fatores Etários , Animais , Animais Recém-Nascidos , Apoptose/genética , Astrócitos/metabolismo , Peso Corporal/genética , Erros Inatos do Metabolismo dos Carboidratos/genética , Erros Inatos do Metabolismo dos Carboidratos/patologia , Proteínas de Transporte/metabolismo , Proliferação de Células , Tamanho Celular , Células Cultivadas , Dendritos/patologia , Modelos Animais de Doenças , Feminino , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/genética , Gliose/metabolismo , Transportador de Glucose Tipo 1/genética , Humanos , Hipoglicemia/genética , Hipoglicemia/patologia , Hipoglicemia/fisiopatologia , Marcação In Situ das Extremidades Cortadas/métodos , Cloreto de Lítio/farmacologia , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Knockout , Neurônios/patologia , Tamanho do Órgão/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais/genética , Pele/citologia , Serina-Treonina Quinases TORRESUMO
Neuritogenesis, the first step of neuronal differentiation, takes place as nascent neurites bud from the immediate postmitotic neuronal soma. Little is known about the mechanisms underlying the dramatic morphological changes that characterize this event. Here, we show that RhoA activity plays a decisive role during neuritogenesis of cultured hippocampal neurons by recruiting and activating its specific kinase ROCK, which, in turn, complexes with profilin IIa. We establish that this previously uncharacterized brain-specific actin-binding protein controls neurite sprouting by modifying actin stability, a function regulated by ROCK-mediated phosphorylation. Furthermore, we determine that this novel cascade is switched on or off by physiological stimuli. We propose that RhoA/ROCK/PIIa-mediated regulation of actin stability, shown to be essential for neuritogenesis, may constitute a central mechanism throughout neuronal differentiation.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Contráteis , Proteínas dos Microfilamentos/metabolismo , Neurônios/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Tamanho Celular/fisiologia , Células Cultivadas , Hipocampo/citologia , Peptídeos e Proteínas de Sinalização Intracelular , Fatores de Crescimento Neural/farmacologia , Neuritos/enzimologia , Neurônios/ultraestrutura , Profilinas , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estimulação Química , Quinases Associadas a rhoRESUMO
Somatic mutation of the MTOR gene is a genetic etiology of focal malformations of cortical development. In this issue of Neuron, Park et al. (2018) identify defective autophagy-dependent ciliogenesis/Wnt signaling as an underlying mechanism affecting neuronal migration and cortical lamination.
Assuntos
Cílios , Serina-Treonina Quinases TOR/genética , Encéfalo , Mutação , NeurôniosRESUMO
Disruption of myelination during development has been implicated in a range of neurodevelopmental disorders including tuberous sclerosis complex (TSC). TSC patients with autism display impairments in white matter integrity. Similarly, mice lacking neuronal Tsc1 have a hypomyelination phenotype. However, the mechanisms that underlie these phenotypes remain unknown. In this study, we demonstrate that neuronal TSC1/2 orchestrates a program of oligodendrocyte maturation through the regulated secretion of connective tissue growth factor (CTGF). We characterize oligodendrocyte maturation both in vitro and in vivo. We find that neuron-specific Tsc1 deletion results in an increase in CTGF secretion that non-cell autonomously stunts oligodendrocyte development and decreases the total number of oligodendrocytes. Genetic deletion of CTGF from neurons, in turn, mitigates the TSC-dependent hypomyelination phenotype. These results show that the mechanistic target of rapamycin (mTOR) pathway in neurons regulates CTGF production and secretion, revealing a paracrine mechanism by which neuronal signaling regulates oligodendrocyte maturation and myelination in TSC. This study highlights the role of mTOR-dependent signaling between neuronal and nonneuronal cells in the regulation of myelin and identifies an additional therapeutic avenue for this disease.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/fisiologia , Bainha de Mielina/fisiologia , Neurônios/fisiologia , Esclerose Tuberosa/fisiopatologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Oligodendroglia/fisiologia , Ratos , Serina-Treonina Quinases TOR/fisiologia , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Tuberous sclerosis complex (TSC) is a neurodevelopmental disease caused by TSC1 or TSC2 mutations and subsequent activation of the mTORC1 kinase. Upon mTORC1 activation, anabolic metabolism, which requires mitochondria, is induced, yet at the same time the principal pathway for mitochondrial turnover, autophagy, is compromised. How mTORC1 activation impacts mitochondrial turnover in neurons remains unknown. Here, we demonstrate impaired mitochondrial homeostasis in neuronal in vitro and in vivo models of TSC. We find that Tsc1/2-deficient neurons accumulate mitochondria in cell bodies, but are depleted of axonal mitochondria, including those supporting presynaptic sites. Axonal and global mitophagy of damaged mitochondria is impaired, suggesting that decreased turnover may act upstream of impaired mitochondrial metabolism. Importantly, blocking mTORC1 or inducing mTOR-independent autophagy restores mitochondrial homeostasis. Our study clarifies the complex relationship between the TSC-mTORC1 pathway, autophagy, and mitophagy, and defines mitochondrial homeostasis as a therapeutic target for TSC and related diseases.
Assuntos
Dinâmica Mitocondrial , Mitofagia , Modelos Biológicos , Neurônios/metabolismo , Neurônios/patologia , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Animais , Autofagia , Axônios/metabolismo , Respiração Celular , Humanos , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Mutação/genética , Células-Tronco Pluripotentes/metabolismo , Terminações Pré-Sinápticas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismoRESUMO
Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signalling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by peroxisomal biogenesis factors 19 and 5 (PEX19 and PEX5), respectively, and peroxisome-localized TSC functioned as a Rheb GTPase-activating protein (GAP) to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, and abrogated peroxisome localization, Rheb GAP activity and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS, and peroxisome-localization-deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for the TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signalling organelle involved in regulation of mTORC1.
Assuntos
Autofagia , Regulação Enzimológica da Expressão Gênica , Complexos Multiproteicos/genética , Peroxissomos/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Animais , Linhagem Celular , Células HEK293 , Humanos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas de Membrana/metabolismo , Camundongos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Ratos , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismoRESUMO
Tuberous sclerosis complex is a disease caused by mutations in the TSC1 or TSC2 genes, which encode a protein complex that inhibits mTOR kinase signaling by inactivating the Rheb GTPase. Activation of mTOR promotes the formation of benign tumors in various organs and the mechanisms underlying the neurological symptoms of the disease remain largely unknown. We found that Tsc2 haploinsufficiency in mice caused aberrant retinogeniculate projections that suggest defects in EphA receptor-dependent axon guidance. We also found that EphA receptor activation by ephrin-A ligands in neurons led to inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activity and decreased inhibition of Tsc2 by ERK1/2. Thus, ephrin stimulation inactivates the mTOR pathway by enhancing Tsc2 activity. Furthermore, Tsc2 deficiency and hyperactive Rheb constitutively activated mTOR and inhibited ephrin-induced growth cone collapse. Our results indicate that TSC2-Rheb-mTOR signaling cooperates with the ephrin-Eph receptor system to control axon guidance in the visual system.
Assuntos
Axônios/fisiologia , Movimento Celular/fisiologia , Efrina-A1/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Receptores da Família Eph/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Cones de Crescimento/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neurônios/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Ratos , Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Transdução de Sinais , Serina-Treonina Quinases TOR , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Vias Visuais/fisiologiaRESUMO
Axon formation is fundamental for brain development and function. TSC1 and TSC2 are two genes, mutations in which cause tuberous sclerosis complex (TSC), a disease characterized by tumor predisposition and neurological abnormalities including epilepsy, mental retardation, and autism. Here we show that Tsc1 and Tsc2 have critical functions in mammalian axon formation and growth. Overexpression of Tsc1/Tsc2 suppresses axon formation, whereas a lack of Tsc1 or Tsc2 function induces ectopic axons in vitro and in the mouse brain. Tsc2 is phosphorylated and inhibited in the axon but not dendrites. Inactivation of Tsc1/Tsc2 promotes axonal growth, at least in part, via up-regulation of neuronal polarity SAD kinase, which is also elevated in cortical tubers of a TSC patient. Our results reveal key roles of TSC1/TSC2 in neuronal polarity, suggest a common pathway regulating polarization/growth in neurons and cell size in other tissues, and have implications for the understanding of the pathogenesis of TSC and associated neurological disorders and for axonal regeneration.
Assuntos
Axônios , Proteínas Supressoras de Tumor/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA , Eletroporação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaRESUMO
Profilins are actin binding proteins essential for regulating cytoskeletal dynamics, however, their function in the mammalian nervous system is unknown. Here, we provide evidence that in mouse brain profilin1 and profilin2 have distinct roles in regulating synaptic actin polymerization with profilin2 preferring a WAVE-complex-mediated pathway. Mice lacking profilin2 show a block in synaptic actin polymerization in response to depolarization, which is accompanied by increased synaptic excitability of glutamatergic neurons due to higher vesicle exocytosis. These alterations in neurotransmitter release correlate with a hyperactivation of the striatum and enhanced novelty-seeking behavior in profilin2 mutant mice. Our results highlight a novel, profilin2-dependent pathway, regulating synaptic physiology, neuronal excitability, and complex behavior.
Assuntos
Comportamento Animal/fisiologia , Exocitose/fisiologia , Neurônios/fisiologia , Profilinas/fisiologia , Animais , Aprendizagem , Potenciação de Longa Duração , Memória , CamundongosRESUMO
Mammalian profilins are abundantly expressed actin monomer-binding proteins, highly conserved with respect to their affinities for G-actin, poly-L-proline, and phosphoinositides. Profilins associate with a large number of proline-rich proteins; the physiological significance and regulation of which is poorly understood. Here we show that profilin 2 associates with dynamin 1 via the C-terminal proline-rich domain of dynamin and thereby competes with the binding of SH3 ligands such as endophilin, amphiphysin, and Grb2, thus interfering with the assembly of the endocytic machinery. We also present a novel role for the brain-specific mouse profilin 2 as a regulator of membrane trafficking. Overexpression of profilin 2 inhibits endocytosis, whereas lack of profilin 2 in neurons results in an increase in endocytosis and membrane recycling. Phosphatidylinositol 4,5-bisphosphate releases profilin 2 from the profilin 2-dynamin 1 complex as well as from the profilin 2-actin complex, suggesting that profilin 2 is diverging the phosphoinositide signaling pathway to actin polymerization as well as endocytosis.