RESUMO
Axon fasciculation is thought to be a critical step in neural circuit formation and function. Recent studies have revealed various molecular mechanisms that underlie axon fasciculation; however, the impacts of axon fasciculation, and its corollary, defasciculation, on neural circuit wiring remain unclear. Corticospinal (CS) neurons in the sensorimotor cortex project axons to the spinal cord to control skilled movements. In rodents, the axons remain tightly fasciculated in the brain and traverse the dorsal funiculus of the spinal cord. Here we show that plexinA1 (PlexA1) and plexinA3 (PlexA3) receptors are expressed by CS neurons, whereas their ligands, semaphorin-5A (Sema5A) and semaphorin-5B (Sema5B) are expressed in the medulla at the decussation site of CS axons to inhibit premature defasciculation of these axons. In the absence of Sema5A/5B-PlexA1/A3 signaling, some CS axons are prematurely defasciculated in the medulla of the brainstem, and those defasciculated CS axons aberrantly transverse in the spinal gray matter instead of the spinal dorsal funiculus. In the absence of Sema5A/Sema5B-PlexA1/A3 signaling, CS axons, which would normally innervate the lumbar spinal cord, are unbundled in the spinal gray matter, and prematurely innervate the cervical gray matter with reduced innervation of the lumbar gray matter. In both Sema5A/5B and PlexA1/A3 mutant mice (both sexes), stimulation of the hindlimb motor cortex aberrantly evokes robust forelimb muscle activation. Finally, Sema5A/5B and PlexA1/A3 mutant mice show deficits in skilled movements. These results suggest that proper fasciculation of CS axons is required for appropriate neural circuit wiring and ultimately affect the ability to perform skilled movements.SIGNIFICANCE STATEMENT Axon fasciculation is believed to be essential for neural circuit formation and function. However, whether and how defects in axon fasciculation affect the formation and function of neural circuits remain unclear. Here we examine whether the transmembrane proteins semaphorin-5A (Sema5A) and semaphorin-5B (Sema5B), and their receptors, plexinA1 (PlexA1) and plexinA3 (PlexA3) play roles in the development of corticospinal circuits. We find that Sema5A/Sema5B and PlexA1/A3 are required for proper axon fasciculation of corticospinal neurons. Furthermore, Sema5A/5B and PlexA1/A3 mutant mice show marked deficits in skilled motor behaviors. Therefore, these results strongly suggest that proper corticospinal axon fasciculation is required for the appropriate formation and functioning of corticospinal circuits in mice.
Assuntos
Semaforinas , Feminino , Masculino , Camundongos , Animais , Semaforinas/metabolismo , Fasciculação Axônica , Neurônios/metabolismo , Axônios/fisiologia , Medula Espinal/metabolismoRESUMO
Signal-induced proliferation-associated 1 (SIPA1)-like 1 (SIPA1L1; also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the PSD-95/NMDA-R-complex. However, its physiological role remains poorly understood. Here, we performed expression analyses using super-resolution microscopy (SRM) in mouse brain and demonstrated that SIPA1L1 is mainly localized to general submembranous regions in neurons, but surprisingly, not to PSD. Our screening for physiological interactors of SIPA1L1 in mouse brain identified spinophilin and neurabin-1, regulators of G-protein-coupled receptor (GPCR) signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1-/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1-/- mice showed aberrant responses to α2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation, and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Male mice were used for all experiments. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome (FXS).SIGNIFICANCE STATEMENT Signal-induced proliferation-associated 1 (SIPA1)-like 1 (SIPA1L1) is thought to regulate essential synaptic functions as a component of the PSD-95/NMDA-R-complex. In our screening for physiological SIPA1L1-interactors, we identified G-protein-coupled receptor (GPCR)-signaling regulators. Moreover, SIPA1L1 knock-out (KO) mice showed striking behavioral anomalies, which may be relevant to GPCR signaling. Our findings revealed an unexpected role of SIPA1L1, which may open new avenues for research on neuropsychiatric disorders that involve dysregulated GPCR signaling. Another important aspect of this paper is that we showed effective methods for checking PSD association and identifying native protein interactors that are difficult to solubilize. These results may serve as a caution for future claims about interacting proteins and PSD proteins, which could eventually save time and resources for researchers and avoid confusion in the field.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , N-Metilaspartato , Proteínas do Tecido Nervoso , Animais , Proteína 4 Homóloga a Disks-Large , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptor A1 de Adenosina , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Corticospinal neurons (CSNs) are located in the cortex and projecting into the spinal cord. The activation of CSNs, which is associated with skilled motor behaviors, induces the activation of interneurons in the spinal cord. Eventually, motor neuron activation is induced by corticospinal circuits to coordinate muscle activation. Therefore, elucidating how the activation of CSNs in the brain is regulated is necessary for understanding the roles of CSNs in skilled motor behaviors. However, the presynaptic partners of CSNs in the brain remain to be identified. Here, we performed transsynaptic rabies virus-mediated brain-wide mapping to identify presynaptic partners of CSNs (pre-CSNs). We found that pre-CSNs are located in all cortical layers, but major pre-CSNs are located in layer Va. A small population of pre-CSNs are also located outside the cortex, such as in the thalamus. Inactivation of layer Va neurons in Tlx3-Cre mice results in deficits in skilled reaching and grasping behaviors, suggesting that, similar to CSNs, layer Va neurons are critical for skilled movements. Finally, we examined whether the connectivity of CSNs is altered after spinal cord injury (SCI). We found that unlike connections between CNSs and postsynaptic neurons, connections between pre-CSNs and CSNs do not change after SCI.
RESUMO
The CaMKΙΙα-Cre mouse lines, possibly the most used Cre lines in neuroscience, have resulted in over 800 articles to date. Here, we demonstrate that the second most widely used CaMKΙΙα-Cre line, Tg(Camk2a-cre)2Gsc (or CamiCre), shows ectopic overexpression of synaptotagmin 2, the most efficient Ca2+ sensor for fast synchronous neurotransmitter release, in excitatory presynapses of Cre+ brains. Moreover, the upregulation of immediate-early genes and genes incorporated in bacterial artificial chromosome (BAC) transgenes, such as L-proline transporter Slc6a7, was found in Cre+ hippocampus. The copy number and integration site of the transgene are suggested to have caused the aberrant gene expression in Cre+ brains. Most importantly, CamiCre+ mice showed functional phenotypes, such as hyperactivity and enhanced associative learning, suggesting that neural activities are affected. These unexpected results suggest difficulties in interpreting results from studies using the CamiCre line and raise a warning of potential pitfalls in using Cre driver lines in general.
RESUMO
We have recently shown that beta-catenin-facilitated export of cadherins from the endoplasmic reticulum requires PX-RICS, a beta-catenin-interacting GTPase-activating protein for Cdc42. Here we show that PX-RICS interacts with isoforms of 14-3-3 and couples the N-cadherin-beta-catenin complex to the microtubule-based molecular motor dynein-dynactin. Similar to knockdown of PX-RICS, knockdown of either 14-3-3zeta or - resulted in the disappearance of N-cadherin and beta-catenin from the cell-cell boundaries. Furthermore, we found that PX-RICS and 14-3-3zeta/ are present in a large multiprotein complex that contains dynein-dynactin components as well as N-cadherin and beta-catenin. Both RNAi- and dynamitin-mediated inhibition of dynein-dynactin function also led to the absence of N-cadherin and beta-catenin at the cell-cell contact sites. Our results suggest that the PX-RICS-14-3-3zeta/ complex links the N-cadherin-beta-catenin cargo with the dynein-dynactin motor and thereby mediates its endoplasmic reticulum export.
Assuntos
Proteínas 14-3-3/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Dineínas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos/metabolismo , beta Catenina/metabolismo , Proteínas 14-3-3/genética , Animais , Antígenos CD/genética , Células COS , Caderinas/genética , Chlorocebus aethiops , Complexo Dinactina , Dineínas/genética , Retículo Endoplasmático/genética , Proteínas Ativadoras de GTPase/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Complexos Multiproteicos/genética , Transporte Proteico/fisiologia , beta Catenina/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
GABAergic dysfunction underlies many neurodevelopmental and psychiatric disorders. GABAergic synapses exhibit several forms of plasticity at both pre- and postsynaptic levels. NMDA receptor (NMDAR)-dependent inhibitory long-term potentiation (iLTP) at GABAergic postsynapses requires an increase in surface GABAARs through promoted exocytosis; however, the regulatory mechanisms and the neuropathological significance remain unclear. Here we report that the autism-related protein PX-RICS is involved in GABAAR transport driven during NMDAR-dependent GABAergic iLTP. Chemically induced iLTP elicited a rapid increase in surface GABAARs in wild-type mouse hippocampal neurons, but not in PX-RICS/RICS-deficient neurons. This increase in surface GABAARs required the PX-RICS/GABARAP/14-3-3 complex, as revealed by gene knockdown and rescue studies. iLTP induced CaMKII-dependent phosphorylation of PX-RICS to promote PX-RICS-14-3-3 assembly. Notably, PX-RICS/RICS-deficient mice showed impaired amygdala-dependent fear learning, which was ameliorated by potentiating GABAergic activity with clonazepam. Our results suggest that PX-RICS-mediated GABAAR trafficking is a key target for GABAergic plasticity and its dysfunction leads to atypical emotional processing underlying autism.
Assuntos
Proteínas Ativadoras de GTPase/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Receptores de GABA-A/fisiologia , Tonsila do Cerebelo/fisiologia , Animais , Transtorno Autístico , Células Cultivadas , Medo/fisiologia , Hipocampo/citologia , Aprendizagem/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Ácido gama-Aminobutírico/fisiologiaRESUMO
Jacobsen syndrome (JBS) is a rare congenital disorder caused by a terminal deletion of the long arm of chromosome 11. A subset of patients exhibit social behavioural problems that meet the diagnostic criteria for autism spectrum disorder (ASD); however, the underlying molecular pathogenesis remains poorly understood. PX-RICS is located in the chromosomal region commonly deleted in JBS patients with autistic-like behaviour. Here we report that PX-RICS-deficient mice exhibit ASD-like social behaviours and ASD-related comorbidities. PX-RICS-deficient neurons show reduced surface γ-aminobutyric acid type A receptor (GABAAR) levels and impaired GABAAR-mediated synaptic transmission. PX-RICS, GABARAP and 14-3-3ζ/θ form an adaptor complex that interconnects GABAAR and dynein/dynactin, thereby facilitating GABAAR surface expression. ASD-like behavioural abnormalities in PX-RICS-deficient mice are ameliorated by enhancing inhibitory synaptic transmission with a GABAAR agonist. Our findings demonstrate a critical role of PX-RICS in cognition and suggest a causal link between PX-RICS deletion and ASD-like behaviour in JBS patients.
Assuntos
Transtorno do Espectro Autista/genética , Comportamento Animal/fisiologia , Proteínas Ativadoras de GTPase/genética , Síndrome da Deleção Distal 11q de Jacobsen/genética , Transporte Proteico/genética , Receptores de GABA-A/metabolismo , Comportamento Social , Animais , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/psicologia , Comportamento Animal/efeitos dos fármacos , Clonazepam/farmacologia , Agonistas de Aminoácidos Excitatórios/toxicidade , Moduladores GABAérgicos/farmacologia , Asseio Animal , Síndrome da Deleção Distal 11q de Jacobsen/metabolismo , Síndrome da Deleção Distal 11q de Jacobsen/psicologia , Ácido Caínico/toxicidade , Camundongos , Camundongos Knockout , Percepção Olfatória/efeitos dos fármacos , Percepção Olfatória/genética , Convulsões/induzido quimicamente , Convulsões/genética , Comportamento Estereotipado/efeitos dos fármacos , Comportamento Estereotipado/fisiologia , Vocalização Animal/efeitos dos fármacos , Vocalização Animal/fisiologiaRESUMO
Cell fusion has a critical role in various developmental processes, immune response, tissue homeostasis and regeneration, and possibly, in cancer. However, the signals that regulate cell fusion remain poorly understood. In a screen for novel targets of Wnt/ß-catenin signalling, we identified glial cells missing 1 (GCM1), which encodes a transcription factor that is involved in epigenetic regulation and is critical for the fusion of syncytiotrophoblast (ST) cells. Here we show that ß-catenin/BCL9-Like (BCL9L)/T-cell factor 4 (TCF4) signalling directly targets the GCM1/syncytin pathway and thereby regulates the fusion of human choriocarcinoma cells. Furthermore, we show that the GCM1/syncytin-B pathway is significantly downregulated in the placenta of BCL9L-deficient mice and that the fusion and differentiation of ST-II cells are blocked. Our results demonstrate a signal transduction pathway that regulates cell fusion, and may provide intriguing perspectives into the various biological and pathological processes that involve cell fusion.
Assuntos
Fusão Celular , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA , Feminino , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Humanos , Masculino , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase , Gravidez , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismo , Proteínas Wnt/genética , beta Catenina/genéticaRESUMO
Cadherins mediate Ca2+-dependent cell-cell adhesion. Efficient export of cadherins from the endoplasmic reticulum (ER) is known to require complex formation with beta-catenin. However, the molecular mechanisms underlying this requirement remain elusive. Here we show that PX-RICS, a beta-catenin-interacting GTPase-activating protein (GAP) for Cdc42, mediates ER-to-Golgi transport of the N-cadherin/beta-catenin complex. Knockdown of PX-RICS expression induced the accumulation of the N-cadherin/beta-catenin complex in the ER and ER exit site, resulting in a decrease in cell-cell adhesion. PX-RICS was also required for ER-to-Golgi transport of the fibroblast growth factor-receptor 4 (FGFR4) associated with N-cadherin. PX-RICS-mediated ER-to-Golgi transport was dependent on its interaction with beta-catenin, phosphatidylinositol-4-phosphate (PI4P), Cdc42, and its novel binding partner gamma-aminobutyric acid type A receptor-associated protein (GABARAP). These results suggest that PX-RICS ensures the efficient entry of the N-cadherin/beta-catenin complex into the secretory pathway, and thereby regulates the amount of N-cadherin available for cell adhesion and FGFR4-mediated signaling.
Assuntos
Caderinas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas Ativadoras de GTPase/genética , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Transporte Proteico , Interferência de RNA , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
In our previous study, we identified RICS, a novel beta-catenin-interacting protein with the GAP activity toward Cdc42 and Rac1, and found that RICS plays an important role in the regulation of neural functions, including postsynaptic NMDA signaling and neurite outgrowth. Here we report the characterization of an N-terminal splicing variant of RICS, termed PX-RICS, which has additional phox homology (PX) and src homology 3 (SH3) domains in its N-terminal region. The PX domain of PX-RICS interacted specifically with phosphatidylinositol 3-phosphate [PtdIns(3)P], PtdIns(4)P and PtdIns(5)P. Consistent with this binding affinity, PX-RICS was found to be localized at the endoplasmic reticulum (ER), Golgi and endosomes. We also found that wild-type PX-RICS possessed much lower GAP activity than RICS, whereas a mutant form of PX-RICS whose PX domain lacks the binding ability to phosphoinositides (PIs) exhibited the GAP activity comparable to that of RICS. However, PX-RICS and RICS exhibited similar inhibitory effects on neurite elongation of Neuro-2a cells. Furthermore, we demonstrate that PX-RICS is a main isoform expressed during neural development. Our results suggest that PX-RICS is involved in early brain development including extension of axons and dendrites, and postnatal remodeling and fine-tuning of neural circuits.
Assuntos
Processamento Alternativo/genética , Proteínas Ativadoras de GTPase/metabolismo , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Caderinas/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar , Proteína 4 Homóloga a Disks-Large , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Complexo de Golgi/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Neuritos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de N-Metil-D-Aspartato/metabolismo , beta Catenina/metabolismoRESUMO
The LKB1/STK11 serine/threonine kinase is mutated in Peutz-Jeghers syndrome and various sporadic cancers such as lung adenocarcinoma. We show here that LKB1 forms a complex with LMO4, GATA-6, and Ldb1, and enhances GATA-mediated transactivation in a kinase-dependent manner. We further demonstrate that LKB1 has the potential to induce p21 expression in collaboration with LMO4, GATA-6, and Ldb1 through the p53-independent mechanism. Our findings suggest that LKB1 regulates GATA-mediated gene expression and that this activity of LKB1 may be important for its tumor suppressor function.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição GATA6/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular , Chlorocebus aethiops , Humanos , Proteínas com Domínio LIM , Ligação Proteica , Ativação Transcricional , Proteína Supressora de Tumor p53/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Regulação para CimaRESUMO
BAMBI is a transmembrane glycoprotein related to the transforming growth factor-beta (TGF-beta)-family type I receptors and functions as a negative regulator of TGF-beta signaling during development. BAMBI is induced by BMP signaling through the evolutionary conserved BMP-responsive elements in its promoter. Furthermore, we have recently shown that Wnt/beta-catenin signaling activates transcription of BAMBI and that BAMBI expression is aberrantly elevated in most colorectal carcinomas. Here, we show that BAMBI is also directly induced by TGF-beta signaling, through the three tandem repeats of 13 bp sequences containing the SMAD-binding elements, which are distinct from the BMP-responsive element. Our findings suggest that BAMBI transcription is regulated by TGF-beta signaling through direct binding of SMAD3 and SMAD4 to the BAMBI promoter.
Assuntos
Hepatoblastoma/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Ativação Transcricional , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Reguladores/genética , Hepatoblastoma/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/genética , Fatores de Crescimento Neural , Regiões Promotoras Genéticas , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Smad , Proteína Smad3 , Proteína Smad4 , Transativadores/genética , Transativadores/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND: The PSD-95 family of proteins possesses multiple protein binding domains, including three PDZ domains, an SH3 domain, a HOOK domain and a guanylate kinase-like (GK) domain. The PSD-95 proteins function as scaffolding proteins that link ion channels such as the N-methyl-d-aspartate-receptors (NMDA-Rs) with cytoskeletal networks and signalling molecules, thereby controlling synaptic plasticity and learning. RESULTS: We found that the PSD-95 family proteins interact via their GK domains with SPA-1-like protein (SPAL), a GTPase-activating protein (GAP) that is specific for Rap1. SPAL was contained within the NMDA-R-PSD-95 complex, and co-localized with PSD-95 and NMDA-R at the synapses in cultured hippocampal neurones. Furthermore, NMDA stimulation induced the dephosphorylation of SPAL in cultured hippocampal neurones. CONCLUSION: Our findings suggest that SPAL may be involved in the NMDA-mediated organization of cytoskeletal networks and signal transduction.