RESUMO
Complex diseases often involve the interplay between genetic and environmental factors. Charcot-Marie-Tooth type 2 neuropathies (CMT2) are a group of genetically heterogeneous disorders, in which similar peripheral neuropathology is inexplicably caused by various mutated genes. Their possible molecular links remain elusive. Here, we found that upon environmental stress, many CMT2-causing mutant proteins adopt similar properties by entering stress granules (SGs), where they aberrantly interact with G3BP and integrate into SG pathways. For example, glycyl-tRNA synthetase (GlyRS) is translocated from the cytoplasm into SGs upon stress, where the mutant GlyRS perturbs the G3BP-centric SG network by aberrantly binding to G3BP. This disrupts SG-mediated stress responses, leading to increased stress vulnerability in motoneurons. Disrupting this aberrant interaction rescues SG abnormalities and alleviates motor deficits in CMT2D mice. These findings reveal a stress-dependent molecular link across diverse CMT2 mutants and provide a conceptual framework for understanding genetic heterogeneity in light of environmental stress.
Assuntos
Doença de Charcot-Marie-Tooth , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , Animais , Camundongos , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Citoplasma , Neurônios Motores , Proteínas com Motivo de Reconhecimento de RNA/metabolismoRESUMO
The neuromuscular junction (NMJ) is a synapse between motoneurons and skeletal muscles to control motor behavior. Acetylcholine receptors (AChRs) are restricted at the synaptic region for proper neurotransmission. Mutations in the mitochondrial CHCHD10 protein have been identified in multiple neuromuscular disorders; however, the physiological roles of CHCHD10 at NMJs remain elusive. Here, we report that CHCHD10 is highly expressed at the postsynapse of NMJs in skeletal muscles. Muscle conditional knockout CHCHD10 mice showed motor defects, abnormal neuromuscular transmission and NMJ structure. Mechanistically, we found that mitochondrial CHCHD10 is required for ATP production, which facilitates AChR expression and promotes agrin-induced AChR clustering. Importantly, ATP could effectively rescue the reduction of AChR clusters in the CHCHD10-ablated muscles. Our study elucidates a novel physiological role of CHCHD10 at the peripheral synapse. It suggests that mitochondria dysfunction contributes to neuromuscular pathogenesis.
Assuntos
Proteínas Mitocondriais/genética , Músculo Esquelético/metabolismo , Doenças da Junção Neuromuscular/genética , Receptores Colinérgicos/genética , Agrina/farmacologia , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Neurônios Motores/metabolismo , Músculo Esquelético/patologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/genética , Sinapses/genética , Transmissão Sináptica/genéticaRESUMO
Four-and-a-half LIM domain protein 1 (FHL1) is a member of the FHL protein family that serves as a scaffold protein to maintain normal cellular structure and function. Its mutations have been implicated in multiple muscular diseases. These FHL1 related myopathies are characterized by symptoms such as progressive muscle loss, rigid or bent spine, even cardiac or respiratory failure in some patients, which implies pathological problems not only in muscles, but also in the nervous system. Moreover, decreased FHL1 protein level has been found in patients with FHL1 mutations, indicating the protein loss-of-function as a pathological cause of such diseases. These findings suggest the significance of understanding the systemic role of FHL1 in the homeostasis of nervous system and muscle. Here we reported that Fhl1 loss in C2C12 myotubes obscured acetylcholine receptor (AChR) clustering in addition to myotube fusion, which was associated with impaired MuSK phosphorylation. Mechanistically, myostatin-SMAD2/3 signaling was enhanced, whereas IGF-PI3K-AKT signaling was suppressed in Fhl1-/- C2C12 myotubes. Reversion of these molecular alterations rescued AChR clustering and differentiation deficits. These data outline a systemic regulation of AChR clustering and myotube fusion by FHL1, which may offer clues for mechanism study and development of therapeutic strategies to treat FHL1 related myopathies.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Miostatina/metabolismo , Junção Neuromuscular/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Folistatina/farmacologia , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/metabolismoRESUMO
Neuromuscular junctions (NMJs) are peripheral synapses between motoneurons and skeletal muscle fibers that are critical for the control of muscle contraction. Dysfunction of these synapses has been implicated in congenital myasthenic syndrome (CMS). In vertebrates, agrin-LRP4-MuSK signaling plays a critical role in acetylcholine receptor (AChR) clustering and NMJ formation. The adaptor protein DOK7 is the downstream substrate of MuSK and also a cytoplasmic activator of MuSK. The role of DOK7 in the promotion of AChR clustering and the mechanisms involved have been well studied; however, the negative regulation of DOK7 after MuSK activation remains unknown. Anaphase-promoting complex 2 (APC2), the core subunit of APC/C E3 ligase complex, was originally believed to regulate cell-cycle transitions. Here, we show that APC2 is enriched at post-synapse of NMJs in postmitotic myotubes. In response to agrin stimulation, APC2 negatively regulates AChR clustering by promoting the ubiquitination of DOK7 at lysine 243 for its proteolytic degradation, which relies on MuSK kinase activity and the phosphorylation of tyrosine 106 in DOK7. Thus, this study provides a mechanism whereby agrin signaling is negatively regulated as part of vertebrate NMJ homeostasis.
Assuntos
Agrina/metabolismo , Subunidade Apc2 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Proteólise , Transdução de Sinais , Ubiquitinação , Agrina/genética , Animais , Subunidade Apc2 do Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclo Celular , Linhagem Celular , Camundongos , Fibras Musculares Esqueléticas/citologia , Proteínas Musculares/genéticaRESUMO
During aging, acetylcholine receptor (AChR) clusters become fragmented and denervated at the neuromuscular junction (NMJ). Underpinning molecular mechanisms are not well understood. We showed that LRP4, a receptor for agrin and critical for NMJ formation and maintenance, was reduced at protein level in aged mice, which was associated with decreased MuSK tyrosine phosphorylation, suggesting compromised agrin-LRP4-MuSK signaling in aged muscles. Transgenic expression of LRP4 in muscles alleviated AChR fragmentation and denervation and improved neuromuscular transmission in aged mice. LRP4 ubiquitination was augmented in aged muscles, suggesting increased LRP4 degradation as a mechanism for reduced LRP4. We found that sarcoglycan α (SGα) interacted with LRP4 and delayed LRP4 degradation in cotransfected cells. AAV9-mediated expression of SGα in muscles mitigated AChR fragmentation and denervation and improved neuromuscular transmission in aged mice. These observations support a model where compromised agrin-LRP4-MuSK signaling serves as a pathological mechanism of age-related NMJ decline and identify a novel function of SGα in stabilizing LRP4 for NMJ stability in aged mice.SIGNIFICANCE STATEMENT This study provides evidence that LRP4, a receptor of agrin that is critical for NMJ formation and maintenance, is reduced at protein level in aged muscles. Transgenic expression of LRP4 in muscles ameliorates AChR fragmentation and denervation and improves neuromuscular transmission in aged mice, demonstrating a critical role of the agrin-LRP4-MuSK signaling. Our study also reveals a novel function of SGα to prevent LRP4 degradation in aged muscles. Finally, we show that NMJ decline in aged mice can be mitigated by AAV9-mediated expression of SGα in muscles. These observations provide insight into pathological mechanisms of age-related NMJ decline and suggest that improved agrin-LRP4-MuSK signaling may be a target for potential therapeutic intervention.
Assuntos
Envelhecimento , Músculo Esquelético/metabolismo , Junção Neuromuscular/metabolismo , Receptores Colinérgicos/metabolismo , Receptores de LDL/metabolismo , Sarcoglicanas/metabolismo , Animais , Feminino , Proteínas Relacionadas a Receptor de LDL , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/inervação , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
Yes-associated protein (Yap) is a major effector of the Hippo pathway that regulates cell proliferation and differentiation during development and restricts tissue growth in adult animals. However, its role in synapse formation remains poorly understood. In this study, we characterized Yap's role in the formation of the neuromuscular junction (NMJ). In HSA-Yap-/- mice where Yap was mutated specifically in muscle cells, AChR clusters were smaller and were distributed in a broader region in the middle of muscle fibers, suggesting that muscle Yap is necessary for the size and location of AChR clusters. In addition, HSA-Yap-/- mice also exhibited remarkable presynaptic deficits. Many AChR clusters were not or less covered by nerve terminals; miniature endplate potential frequency was reduced, which was associated with an increase in paired-pulse facilitation, indicating structural and functional defects. In addition, muscle Yap mutation prevented reinnervation of denervated muscle fibers. Together, these observations indicate a role of muscle Yap in NMJ formation and regeneration. We found that ß-catenin was reduced in the cytoplasm and nucleus of mutant muscles, suggesting compromised ß-catenin signaling. Both NMJ formation and regeneration deficits of HSA-Yap-/- mice were ameliorated by inhibiting ß-catenin degradation, further corroborating a role of ß-catenin or Wnt-dependent signaling downstream of Yap to regulate NMJ formation and regeneration.SIGNIFICANCE STATEMENT This paper explored the role of Yes-associated protein (Yap) in neuromuscular junction (NMJ) formation and regeneration. Yap is a major effector of the Hippo pathway that regulates cell proliferation and differentiation during development and restricts tissue growth in adult animals. However, its role in synapse formation remains poorly understood. We provide evidence that muscle Yap mutation impairs both postsynaptic and presynaptic differentiation and function and inhibits NMJ regeneration after nerve injury, indicating a role of muscle Yap in these events. Further studies suggest compromised ß-catenin signaling as a potential mechanism. Both NMJ formation and regeneration deficits of HSA-Yap-/- mice were ameliorated by inhibiting ß-catenin degradation, corroborating a role of ß-catenin or Wnt-dependent signaling downstream of Yap to regulate NMJ formation and regeneration.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Força Muscular/fisiologia , Músculo Esquelético/fisiologia , Regeneração Nervosa/fisiologia , Junção Neuromuscular/fisiologia , Fosfoproteínas/metabolismo , Transmissão Sináptica/fisiologia , Animais , Proteínas de Ciclo Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/inervação , Receptores Colinérgicos/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Sinalização YAP , beta Catenina/metabolismoRESUMO
Cortical lamination is crucial for the assembly of cerebellar circuitry. In this process, granule neurons (GNs) migrate along Bergmann glia (BG), which are specialized astroglial cells, from the external granule layer to the internal granule layer. However, the molecular mechanisms underlying BG development are not well understood. Here, we show that GFAP::Cre;Erbb3(F/F) mice, which lack Erbb3 in both radial glia and neurons, exhibit impairments in balance and motor coordination. Cerebellar lamination is aberrant, with misplaced Purkinje neurons and GN clusters. These phenotypes were not observed in Math1::CreER(T2);Erbb3(F/F) mice, where the Erbb3 gene was deleted in GNs, suggesting involvement of non-neuronal Erbb3 in cerebellar lamination. Mechanistic studies indicate that ERBB3 is crucial for the proliferation of BG, which are required for GN migration. These observations identify a crucial role for ERBB3 in cerebellar lamination and reveal a novel mechanism that regulates BG development.
Assuntos
Proliferação de Células/fisiologia , Cerebelo/embriologia , Neuroglia/fisiologia , Neurônios/fisiologia , Receptor ErbB-3/metabolismo , Análise de Variância , Animais , Western Blotting , Cerebelo/citologia , Primers do DNA/genética , Camundongos , Camundongos Knockout , Neuroglia/citologia , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Bone mass is maintained by balanced activity of osteoblasts and osteoclasts. Lrp4 (low-density lipoprotein receptor related protein 4) is a member of the LDL receptor family, whose mutations have been identified in patients with high-bone-mass disorders, such as sclerosteosis and van Buchem diseases. However, it remains unknown whether and how Lrp4 regulates bone-mass homeostasis in vivo. Here we provide evidence that Lrp4-null mutation or specific mutation in osteoblast-lineage cells increased cortical and trabecular bone mass, which was associated with elevated bone formation and impaired bone resorption. This phenotype was not observed in osteoclast-selective Lrp4 knockout mice. Mechanistic studies indicate that loss of Lrp4 function in osteoblast-lineage cells increased serum levels of sclerostin, a key factor for bone-mass homeostasis that interacts with Lrp4, but abolished the inhibition of Wnt/ß-catenin signaling and osteoblastic differentiation by sclerostin. Concomitantly, sclerostin induction of RANKL (receptor activator of nuclear kappa B ligand) was impaired, leading to a lower ratio of RANKL over OPG (osteoprotegerin) (a key factor for osteoclastogenesis). Taken together, these results support the view for Lrp4 as a receptor of sclerostin to inhibit Wnt/ß-catenin signaling and bone formation and identify Lrp4 as a critical player in bone-mass homeostasis.
Assuntos
Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Receptores de LDL/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Aminoácidos/sangue , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Reabsorção Óssea/sangue , Diferenciação Celular , Linhagem da Célula , Fêmur/diagnóstico por imagem , Fêmur/patologia , Glicoproteínas/sangue , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Relacionadas a Receptor de LDL , Camundongos Knockout , Músculos/metabolismo , Especificidade de Órgãos , Osteoblastos/patologia , Osteocalcina/metabolismo , Osteoclastos/patologia , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Receptores de LDL/deficiência , Células Estromais/metabolismo , Células Estromais/patologia , Via de Sinalização Wnt , Microtomografia por Raio-X , beta Catenina/metabolismoRESUMO
INTRODUCTION: The prevalence and characteristics of agrin and low-density lipoprotein-related receptor protein 4 (LRP4) antibody-positive amyotrophic lateral sclerosis (ALS) patients were studied. METHODS: We tested 82 ALS patients and 59 controls for agrin and LRP4 antibodies using enzyme-linked immunoassay (ELISA). RESULTS: We found that 13.8% of ALS patients had agrin antibodies, and 9.8% had LRP4 antibodies. Women with ALS are twice as likely as men to have antibodies. Agrin-positive ALS patients are younger than agrin-negative ALS patients. CONCLUSIONS: Antibodies to agrin and LRP4 are found in ALS patients. It must be determined whether these antibodies are pathogenic. Because antibody-positive patients have upper as well as lower motor neuron findings, the antibodies' effects cannot be explained solely by their actions at the neuromuscular junction. A breakdown in interneuronal signaling may be the cause of ALS. Further research is needed to resolve this question. Muscle Nerve, 2016 Muscle Nerve 55: 430-432, 2017.
Assuntos
Agrina/imunologia , Esclerose Lateral Amiotrófica/sangue , Autoanticorpos/sangue , Lipoproteínas LDL/imunologia , Fatores Etários , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Fatores SexuaisRESUMO
ErbB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2), a receptor tyrosine kinase of the ErbB family, is overexpressed in around 25% of breast cancers. In addition to forming a heterodimer with other ErbB receptors in response to ligand stimulation, ErbB2 can be activated in a ligand-independent manner. We report here that Erbin, an ErbB2-interacting protein that was thought to act as an antitumor factor, is specifically expressed in mammary luminal epithelial cells and facilitates ErbB2-dependent proliferation of breast cancer cells and tumorigenesis in MMTV-neu transgenic mice. Disruption of their interaction decreases ErbB2-dependent proliferation, and deletion of the PDZ domain in Erbin hinders ErbB2-dependent tumor development in MMTV-neu mice. Mechanistically, Erbin forms a complex with ErbB2, promotes its interaction with the chaperon protein HSP90, and thus prevents its degradation. Finally, ErbB2 and Erbin expression correlates in human breast tumor tissues. Together, these observations establish Erbin as an ErbB2 regulator for breast tumor formation and progression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Receptor ErbB-2/metabolismo , Adulto , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Progressão da Doença , Feminino , Deleção de Genes , Técnicas de Silenciamento de Genes , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , Ligação ProteicaRESUMO
The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires neuronal agrin that acts by binding to LRP4 to stimulate MuSK. Mutations have been identified in agrin, MuSK, and LRP4 in patients with congenital myasthenic syndrome, and patients with myasthenia gravis develop antibodies against agrin, LRP4, and MuSK. However, it remains unclear whether the agrin signaling pathway is critical for NMJ maintenance because null mutation of any of the three genes is perinatal lethal. In this study, we generated imKO mice, a mutant strain whose LRP4 gene can be deleted in muscles by doxycycline (Dox) treatment. Ablation of the LRP4 gene in adult muscle enabled studies of its role in NMJ maintenance. We demonstrate that Dox treatment of P30 mice reduced muscle strength and compound muscle action potentials. AChR clusters became fragmented with diminished junctional folds and synaptic vesicles. The amplitude and frequency of miniature endplate potentials were reduced, indicating impaired neuromuscular transmission and providing cellular mechanisms of adult LRP4 deficiency. We showed that LRP4 ablation led to the loss of synaptic agrin and the 90 kDa fragments, which occurred ahead of other prejunctional and postjunctional components, suggesting that LRP4 may regulate the stability of synaptic agrin. These observations demonstrate that LRP4 is essential for maintaining the structural and functional integrity of the NMJ and that loss of muscle LRP4 in adulthood alone is sufficient to cause myasthenic symptoms.
Assuntos
Junção Neuromuscular/fisiologia , Junção Neuromuscular/ultraestrutura , Receptores de LDL/deficiência , Animais , Humanos , Proteínas Relacionadas a Receptor de LDL , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de LDL/fisiologiaRESUMO
Lysosomal storage disorders (LSDs), which are characterized by genetic and metabolic lysosomal dysfunctions, constitute over 60 degenerative diseases with considerable health and economic burdens. However, the mechanisms driving the progressive death of functional cells due to lysosomal defects remain incompletely understood, and broad-spectrum therapeutics against LSDs are lacking. Here, we found that various gene abnormalities that cause LSDs, including Hexb, Gla, Npc1, Ctsd and Gba, all shared mutual properties to robustly autoactivate neuron-intrinsic cGAS-STING signalling, driving neuronal death and disease progression. This signalling was triggered by excessive cytoplasmic congregation of the dsDNA and DNA sensor cGAS in neurons. Genetic ablation of cGAS or STING, digestion of neuronal cytosolic dsDNA by DNase, and repair of neuronal lysosomal dysfunction alleviated symptoms of Sandhoff disease, Fabry disease and Niemann-Pick disease, with substantially reduced neuronal loss. We therefore identify a ubiquitous mechanism mediating the pathogenesis of a variety of LSDs, unveil an inherent connection between lysosomal defects and innate immunity, and suggest a uniform strategy for curing LSDs.
Assuntos
Doenças por Armazenamento dos Lisossomos , Doença de Niemann-Pick Tipo C , Humanos , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Lisossomos/metabolismo , Imunidade Inata , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismoRESUMO
Neuregulin 1 (NRG1) is an axon-derived factor that is critical for Schwann cell (SC) development and myelinogenesis in a manner dependent on transmembrane tyrosine kinases ErbB2 and ErbB3. Recent studies suggest that NRG1 signaling plays a role in remyelination of regenerated nerves after injury. In this study, we investigated the role of Erbin, a protein that interacts with ErbB2 in remyelination of injured nerves. We show that Erbin expression increased dramatically in injured nerves. Myelinated axons were fewer, and g-ratios of those that were myelinated were increased in erbin(-/-) mice, which were impaired in functional recovery from nerve injury. These results indicate a necessary role of Erbin in remyelination of regenerating axons. Erbin ablation had little effect on numbers of BrdU-labeled and TUNEL-labeled SCs, suggesting mechanisms independent of altered proliferation or apoptosis. We demonstrated that Erbin mutant mice were impaired in raising or maintaining the levels of ErbB2 and in producing NRG1 in axons. Together, these observations demonstrate that Erbin is required for remyelination of regenerated axons after injury, probably by regulating ErbB2 and NRG1 levels, identifying a novel player in regulating remyelination.
Assuntos
Axônios/fisiologia , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica/fisiologia , Bainha de Mielina/metabolismo , Regeneração Nervosa/fisiologia , Neuropatia Ciática/patologia , Animais , Axônios/ultraestrutura , Bromodesoxiuridina , Proteínas de Transporte/genética , Morte Celular/genética , Morte Celular/fisiologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/genética , Marcação In Situ das Extremidades Cortadas , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteína Básica da Mielina/metabolismo , Regeneração Nervosa/genética , Neuregulina-1/metabolismo , RNA Mensageiro/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Recuperação de Função Fisiológica/genética , Recuperação de Função Fisiológica/fisiologia , Neuropatia Ciática/complicações , Fatores de Tempo , Degeneração Walleriana/etiologia , Degeneração Walleriana/metabolismo , Degeneração Walleriana/patologiaRESUMO
Bone morphogenetic protein (BMP) signaling plays a crucial role in maintaining the pluripotency of mouse embryonic stem cells (ESCs) and has negative effects on ESC neural differentiation. However, it remains unclear when and how BMP signaling executes those different functions during neural commitment. Here, we show that a BMP4-sensitive window exists during ESC neural differentiation. Cells at this specific period correspond to the egg cylinder stage epiblast and can be maintained as ESC-derived epiblast stem cells (ESD-EpiSCs), which have the same characteristics as EpiSCs derived from mouse embryos. We propose that ESC neural differentiation occurs in two stages: first from ESCs to ESD-EpiSCs and then from ESD-EpiSCs to neural precursor cells (NPCs). We further show that BMP4 inhibits the conversion of ESCs into ESD-EpiSCs during the first stage, and suppresses ESD-EpiSC neural commitment and promotes non-neural lineage differentiation during the second stage. Mechanistic studies show that BMP4 inhibits FGF/ERK activity at the first stage but not at the second stage; and IDs, as important downstream genes of BMP signaling, partially substitute for BMP4 functions at both stages. We conclude that BMP signaling has distinct functions during different stages of ESC neural commitment.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Neurogênese , Animais , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camundongos , FosforilaçãoRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating motoneuron disease, in which lower motoneurons lose control of skeletal muscles. Degeneration of neuromuscular junctions (NMJs) occurs at the initial stage of ALS. Dipeptide repeat proteins (DPRs) from G4C2 repeat-associated non-ATG (RAN) translation are known to cause C9orf72-associated ALS (C9-ALS). However, DPR inclusion burdens are weakly correlated with neurodegenerative areas in C9-ALS patients, indicating that DPRs may exert cell non-autonomous effects, in addition to the known intracellular pathological mechanisms. Here, we report that poly-GA, the most abundant form of DPR in C9-ALS, is released from cells. Local administration of poly-GA proteins in peripheral synaptic regions causes muscle weakness and impaired neuromuscular transmission in vivo. The NMJ structure cannot be maintained, as evidenced by the fragmentation of postsynaptic acetylcholine receptor (AChR) clusters and distortion of presynaptic nerve terminals. Mechanistic study demonstrated that extracellular poly-GA sequesters soluble Agrin ligands and inhibits Agrin-MuSK signaling. Our findings provide a novel cell non-autonomous mechanism by which poly-GA impairs NMJs in C9-ALS. Thus, targeting NMJs could be an early therapeutic intervention for C9-ALS.
Assuntos
Esclerose Lateral Amiotrófica , Animais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/veterinária , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Agrina , Dipeptídeos/metabolismoRESUMO
Expansion of the hexanucleotide repeat GGGGCC in the C9orf72 gene is the most common genetic factor in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Poly-Gly-Ala (poly-GA), one form of dipeptide repeat proteins (DPRs) produced from GGGGCC repeats, tends to form neurotoxic protein aggregates. The C9orf72 GGGGCC repeats and microglial receptor TREM2 are both associated with risk for ALS/FTD. The role and regulation of TREM2 in C9orf72-ALS/FTD remain unclear. Here, we found that poly-GA proteins activate the microglial NLRP3 inflammasome to produce interleukin-1ß (IL-1ß), which promotes ADAM10-mediated TREM2 cleavage and inhibits phagocytosis of poly-GA. The inhibitor of the NLRP3 inflammasome, MCC950, reduces the TREM2 cleavage and poly-GA aggregates, resulting in the alleviation of motor deficits in poly-GA mice. Our study identifies a crosstalk between NLRP3 and TREM2 signaling, suggesting that targeting the NLRP3 inflammasome to sustain TREM2 is an approach to treat C9orf72-ALS/FTD.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Animais , Camundongos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Dipeptídeos/metabolismo , Expansão das Repetições de DNA , Demência Frontotemporal/genética , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas/genéticaRESUMO
Neuregulin 1 (NRG1) and its receptor ErbB4 are both susceptibility genes of schizophrenia. However, little is known about the underlying mechanisms of their malfunction. Although ErbB4 is enriched in GABAergic interneurons, the role of NRG1 in excitatory synapse formation in these neurons remains poorly understood. We showed that NRG1 increased both the number and size of PSD-95 puncta and the frequency and amplitude of miniature EPSCs (mEPSCs) in GABAergic interneurons, indicating that NRG1 stimulates the formation of new synapses and strengthens existing synapses. In contrast, NRG1 treatment had no effect on either the number or size of excitatory synapses in glutamatergic neurons, suggesting its synaptogenic effect is specific to GABAergic interneurons. Ecto-ErbB4 treatment diminished both the number and size of excitatory synapses, suggesting that endogenous NRG1 may be critical for basal synapse formation. NRG1 could stimulate the stability of PSD-95 in the manner that requires tyrosine kinase activity of ErbB4. Finally, deletion of ErbB4 in parvalbumin-positive interneurons led to reduced frequency and amplitude of mEPSCs, providing in vivo evidence that ErbB4 is important in excitatory synaptogenesis in interneurons. Together, our findings suggested a novel synaptogenic role of NRG1 in excitatory synapse development, possibly via stabilizing PSD-95, and this effect is specific to GABAergic interneurons. In light of the association of the genes of both NRG1 and ErbB4 with schizophrenia and dysfunction of GABAergic system in this disorder, these results provide insight into its potential pathological mechanism.
Assuntos
Potenciais Pós-Sinápticos Excitadores/fisiologia , Interneurônios/efeitos dos fármacos , Neuregulina-1/farmacologia , Sinapses/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Biofísica , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Contagem de Células/métodos , Células Cultivadas , Córtex Cerebral/citologia , Cicloeximida/farmacologia , Proteína 4 Homóloga a Disks-Large , Estimulação Elétrica , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Receptores ErbB/genética , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Glutamato Descarboxilase/genética , Proteínas de Fluorescência Verde/genética , Humanos , Técnicas In Vitro , Interneurônios/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Neuregulina-1/metabolismo , Técnicas de Patch-Clamp/métodos , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor ErbB-4 , Sinapses/fisiologia , Fatores de Tempo , Transfecção/métodos , Tirfostinas/farmacologiaRESUMO
Neuregulin 1 (NRG1) is a trophic factor that has been implicated in neural development, neurotransmission, and synaptic plasticity. NRG1 has multiple isoforms that are generated by usage of different promoters and alternative splicing of a single gene. However, little is known about NRG1 isoform composition profile, whether it changes during development, or the underlying mechanisms. We found that each of the six types of NRG1 has a distinct expression pattern in the brain at different ages, resulting in a change in NRG1 isoform composition. In both human and rat, the most dominant are types III and II, followed by either type I or type V, while types IV and VI are the least abundant. The expression of NRG1 isoforms is higher in rat brains at ages of E13 and P5 (in particular type V), suggesting roles in early neural development and in the neonatal critical period. At the cellular level, the majority of NRG1 isoforms (types I, II, and III) are expressed in excitatory neurons, although they are also present in GABAergic neurons and astrocytes. Finally, the expression of each NRG1 isoform is distinctly regulated by neuronal activity, which causes significant increase in type I and IV NRG1 levels. Neuronal activity regulation of type IV expression requires a CRE cis-element in the 5' untranslated region (UTR) that binds to CREB. These results indicate that expression of NRG1 isoforms is regulated by distinct mechanisms, which may contribute to versatile functions of NRG1 and pathologic mechanisms of brain disorders such as schizophrenia.
Assuntos
Córtex Cerebral/fisiologia , Neuregulina-1/genética , Neurônios/fisiologia , Isoformas de Proteínas/genética , Fatores Etários , Análise de Variância , Animais , Astrócitos/citologia , Astrócitos/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Córtex Cerebral/citologia , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Neuregulina-1/metabolismo , Neurônios/citologia , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The neuromuscular junction (NMJ), a peripheral synaptic connection between motoneurons and skeletal muscle fibers, controls movement. Dysregulation of NMJs has been implicated in various motor disorders. Because of their large size and easy accessibility, NMJs have been extensively investigated in the neuroscience field and have greatly contributed to our understanding of the fundamental principles of synapses in the central nervous system. Researchers have tried multiple ways to develop models to recreate NMJs. Rapid progress in the research and development of tissue-like organoids has made it possible to produce human NMJ three-dimensional (3D) models in vitro, providing an additional powerful strategy to study NMJs. Here, we introduce the most recent advances of human embryonic stem cell- or induced pluripotent stem cell-derived organoids to model 3D NMJs.
Assuntos
Transtornos Motores , Organoides , Humanos , Neurônios Motores , Fibras Musculares Esqueléticas , Músculo Esquelético , Junção NeuromuscularRESUMO
BACKGROUND: Neuromuscular junctions (NMJs) are peripheral synapses connecting motoneurons and skeletal myofibers. At the postsynaptic side in myofibers, acetylcholine receptor (AChR) proteins are clustered by the neuronal agrin signal. Meanwhile, several nuclei in each myofiber are specially enriched around the NMJ for postsynaptic gene transcription. It remains mysterious that how gene expressions in these synaptic nuclei are systematically regulated, especially by motoneurons. RESULTS: We found that synaptic nuclei have a distinctive chromatin structure and gene expression profiling. Synaptic nuclei are formed during NMJ development and maintained by motoneuron innervation. Transcriptome analysis revealed that motoneuron innervation determines the distinct expression patterns in the synaptic region and non-synaptic region in each multinucleated myofiber, probably through epigenetic regulation. Myonuclei in synaptic and non-synaptic regions have different responses to denervation. Weighted gene co-expression network analysis revealed that the histone lysine demethylases Kdm1a is a negative regulator of synaptic gene expression. Inhibition of Kdm1a promotes AChR expression but impairs motor functions. CONCLUSION: These results demonstrate that motoneurons innervation determines the distinct gene expressions in multinucleated myofibers. Thus, dysregulation of nerve-controlled chromatin structure and muscle gene expression might cause muscle weakness and atrophy in motoneuron degenerative disorders.