RESUMEN
The formation of stable adhesive contacts between pre- and post-synaptic neurons represents the initial step in synapse assembly. The cell adhesion molecule N-cadherin, the receptor tyrosine phosphatase DLAR, and the scaffolding molecule Liprin-alpha play critical, evolutionarily conserved roles in this process. However, how these proteins signal to the growth cone and are themselves regulated remains poorly understood. Using Drosophila photoreceptors (R cells) as a model, we evaluate genetic and physical interactions among these three proteins. We demonstrate that DLAR function in this context is independent of phosphatase activity but requires interactions mediated by its intracellular domain. Genetic studies reveal both positive and, surprisingly, inhibitory interactions amongst all three genes. These observations are corroborated by biochemical studies demonstrating that DLAR physically associates via its phosphatase domain with N-cadherin in Drosophila embryos. Together, these data demonstrate that N-cadherin, DLAR, and Liprin-alpha function in a complex to regulate adhesive interactions between pre- and post-synaptic cells and provide a novel mechanism for controlling the activity of Liprin-alpha in the developing growth cone.
Asunto(s)
Cadherinas/metabolismo , Proteínas de Drosophila/metabolismo , Fosfoproteínas/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Proteínas Tirosina Fosfatasas Similares a Receptores/metabolismo , Animales , Axones/metabolismo , Cadherinas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Fosfoproteínas/genética , Unión Proteica , Proteínas Tirosina Fosfatasas Similares a Receptores/genética , Sinapsis/metabolismoRESUMEN
Traumatic brain injury (TBI) is characterized by a progressive cell loss and a lack of axonal regeneration. In the central nervous system (CNS), the Rho signaling pathway regulates the neuronal response to growth inhibitory proteins and regeneration of damaged axons, and Rho activation is also correlated with an increased susceptibility to apoptosis. To evaluate whether traumatic brain injury (TBI) results in changes in Rho activation in vulnerable regions of the brain, GTP-RhoA pull down assays were performed on rat cortical and hippocampal tissue homogenates obtained from 24 h to 3 days following lateral fluid percussion brain injury (FPI). Following FPI, a significantly increased RhoA activation was observed from 24 h to 3 days post-injury in the cortex and by 3 days in the hippocampus ipsilateral to the injury. We also detected activated RhoA in the cortex and hippocampus contralateral to the injury, without concomitant changes in total RhoA levels. To determine if immediate post-traumatic events such as seizures may activate Rho, we examined RhoA activation in the brains of rats with kainic acid-induced seizures. Severe seizures resulted in bilateral RhoA activation in the cortex and hippocampus. Together, these results indicate that RhoA is activated in vulnerable brain regions following traumatic and epileptic insults to the CNS.
Asunto(s)
Lesiones Encefálicas/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Convulsiones/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Anticonvulsivantes/farmacología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Diazepam/farmacología , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Activación Enzimática/fisiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Ácido Kaínico , Masculino , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamenteRESUMEN
Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Supervivencia Celular , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Fosfatasas/metabolismo , Receptor trkA , Animales , Apoptosis/fisiología , Células Cultivadas , Activación Enzimática , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuronas/citología , Células PC12 , Fosfolipasa C gamma , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Sistema Nervioso Simpático/citología , Fosfolipasas de Tipo C/metabolismoRESUMEN
Growth inhibitory proteins in the central nervous system (CNS) block axon growth and regeneration by signaling to Rho, an intracellular GTPase. It is not known how CNS trauma affects the expression and activation of RhoA. Here we detect GTP-bound RhoA in spinal cord homogenates and report that spinal cord injury (SCI) in both rats and mice activates RhoA over 10-fold in the absence of changes in RhoA expression. In situ Rho-GTP detection revealed that both neurons and glial cells showed Rho activation at SCI lesion sites. Application of a Rho antagonist (C3-05) reversed Rho activation and reduced the number of TUNEL-labeled cells by approximately 50% in both injured mouse and rat, showing a role for activated Rho in cell death after CNS injury. Next, we examined the role of the p75 neurotrophin receptor (p75NTR) in Rho signaling. After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia. Treatment with C3-05 blocked the increase in p75NTR expression. Experiments with p75NTR-null mutant mice showed that immediate Rho activation after SCI is p75NTR dependent. Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.
Asunto(s)
Apoptosis/fisiología , Traumatismos de la Médula Espinal/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Biomarcadores , Femenino , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Modelos Biológicos , Neuroglía/metabolismo , Neuronas/metabolismo , Células PC12 , Ratas , Ratas Long-Evans , Receptor de Factor de Crecimiento Nervioso , Receptores de Factor de Crecimiento Nervioso/metabolismo , Regulación hacia ArribaRESUMEN
The activation state of Rho is an important determinant of axon growth and regeneration in neurons. Axons can extend neurites on growth inhibitory substrates when Rho is inactivated by C3-ADP-ribosyltransferase (C3). We found by Rho-GTP pull-down assay that inhibitory substrates activate Rho. To inactivate Rho, scrape-loading of C3 was necessary because it does not freely enter cells. To overcome the poor permeability of C3, we made and characterized five new recombinant C3-like chimeric proteins designed to cross the cell membrane by receptor-independent mechanisms. These proteins were constructed by the addition of short transport peptides to the carboxyl-terminal of C3 and tested using a bioassay measuring neurite outgrowth of PC-12 cells plated on growth inhibitory substrates. All five constructs stimulated neurite outgrowth but with different dose-response profiles. Biochemical properties of the chimeric proteins were examined using C3-05, the most effective construct tested. Gel shift assays showed that C3-05 retained the ability to ADP-ribosylate Rho. Western blots and immunocytochemistry were used to verify the presence of C3 inside treated cells. C3-05 was also effective at promoting neurite outgrowth in primary neuronal cultures, as well as causing the disassembly of actin stress fibers and focal adhesions complexes in fibroblasts. These studies demonstrate that the new C3-like proteins are effective in delivering biologically active C3 into different cell types, thereby, inactivating Rho.