RESUMO
TRPM4 is a non-selective cation channel activated by intracellular Ca2+ but only permeable to monovalent cations, its activation regulates membrane potential and intracellular calcium. This channel participates in the migration and adhesion of non-excitable cells and forms an integral part of the focal adhesion complex. In neurons, TRPM4 expression starts before birth and its function at this stage is not clear, but it may function in processes such as neurite development. Here we investigate the role of TRPM4 in neuritogenesis. We found that neurons at DIV 0 express TRPM4, the inhibition of TRPM4 using 9-Ph reduces neurite number and slows the progression of neurite development, keeping neurons in stage 1. The genetic suppression of TRPM4 using an shRNA at later stages (DIV2) reduces neurite length. Conversely, at DIV 0, TRPM4 inhibition augments the Cch-induced Ca2 + i increase, altering the calcium homeostasis. Together, these results show that TRPM4 participates in progression of neurite development and suggest a critical role of the calcium modulation during this stage of neuronal development.
Assuntos
Cálcio , Córtex Cerebral , Neuritos , Neurogênese , Canais de Cátion TRPM , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Animais , Neuritos/metabolismo , Neuritos/efeitos dos fármacos , Cálcio/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Neurônios/metabolismoRESUMO
Regulation of directed axon guidance and branching during development is essential for the generation of neuronal networks. However, the molecular mechanisms that underlie interstitial (or collateral) axon branching in the mammalian brain remain unresolved. Here, we investigate interstitial axon branching in vivo using an approach for precise labeling of layer 2/3 callosal projection neurons (CPNs). This method allows for quantitative analysis of axonal morphology at high acuity and also manipulation of gene expression in well-defined temporal windows. We find that the GSK3ß serine/threonine kinase promotes interstitial axon branching in layer 2/3 CPNs by releasing MAP1B-mediated inhibition of axon branching. Further, we find that the tubulin tyrosination cycle is a key downstream component of GSK3ß/MAP1B signaling. These data suggest a cell-autonomous molecular regulation of cortical neuron axon morphology, in which GSK3ß can release a MAP1B-mediated brake on interstitial axon branching upstream of the posttranslational tubulin code.
Assuntos
Proteínas de Transporte , Tubulina (Proteína) , Animais , Tubulina (Proteína)/metabolismo , Proteínas de Transporte/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Microtúbulos/metabolismo , Axônios/metabolismo , Células Cultivadas , MamíferosRESUMO
Regulation of directed axon guidance and branching during development is essential for the generation of neuronal networks. However, the molecular mechanisms that underlie interstitial axon branching in the mammalian brain remain unresolved. Here, we investigate interstitial axon branching in vivo using an approach for precise labeling of layer 2/3 callosal projection neurons (CPNs), allowing for quantitative analysis of axonal morphology at high acuity and also manipulation of gene expression in well-defined temporal windows. We find that the GSK3ß serine/threonine kinase promotes interstitial axon branching in layer 2/3 CPNs by releasing MAP1B-mediated inhibition of axon branching. Further, we find that the tubulin tyrosination cycle is a key downstream component of GSK3ß/MAP1B signaling. We propose that MAP1B functions as a brake on axon branching that can be released by GSK3ß activation, regulating the tubulin code and thereby playing an integral role in sculpting cortical neuron axon morphology.