Abstract
Neurons are the largest known cells, with complex and highly polarized morphologies and consist of a cell body (soma), several dendrites, and a single axon. The establishment of polarity necessitates initial axonal outgrowth in concomitance with the addition of new membrane to the axon's plasmalemma. Axolemmal expansion occurs by exocytosis of plasmalemmal precursor vesicles primarily at the neuronal growth cone membrane. The multiprotein exocyst complex drives spatial location and specificity of vesicle fusion at plasma membrane. However, the specific participation of its different proteins on neuronal differentiation has not been fully established. In the present work we analyzed the role of Sec3, a prominent exocyst complex protein on neuronal differentiation. Using mice hippocampal primary cultures, we determined that Sec3 is expressed in neurons at early stages prior to neuronal polarization. Furthermore, we determined that silencing of Sec3 in mice hippocampal neurons in culture precluded polarization. Moreover, using in utero electroporation experiments, we determined that Sec3 knockdown affected cortical neurons migration and morphology during neocortex formation. Our results demonstrate that the exocyst complex protein Sec3 plays an important role in axon formation in neuronal differentiation and the migration of neuronal progenitors during cortex development.
Highlights
The formation of a polarized neuron, containing a single long axon and several branching dendrites, requires the action of two interrelated processes, specification of the axon and initial axon outgrowth and the addition of new membrane to the axon’s plasmalemma
Expression and distribution of Sec3 in developing neurons In order to be involved in neuronal polarization, a protein should be expressed early before this phenomenon occurs, so we assessed wheatear Sec3 protein is expressed in cultured hippocampal pyramidal neurons at early stages of development
We determined by Western Blot analysis of cell lysates harvested at different times of culture that Sec3 is expressed previous to the polarization event, during the polarization (24 and 36h) and after the polarization; presenting a significant diminution at 72h when the cells are fully polarized (Fig. 1 b and 1c)
Summary
The formation of a polarized neuron, containing a single long axon and several branching dendrites, requires the action of two interrelated processes, specification of the axon and initial axon outgrowth and the addition of new membrane to the axon’s plasmalemma. We have previously reported that, in cultured hippocampal neurons, a universally employed method to study neuronal development and polarization, TC10 activation by IGF - 1 and the consequent assembling of Exo to the growth cone plasmalemma are critical for the control of PPVs exocytosis and, pioneer axonal outgrowth and the establishment of neuronal polarity [20] It has been shown by other colleagues that, exocyst proteins, such as Sec and Sec, are concentrated at growth cones of hipocampal neurons [14, 21, 22]. Exocyst complex protein was expressed early in developing neurons and present in the growth cone and distal axon, prominent sites for new membrane addition In these cells, loss of function of Sec repressed the establishment of neuronal polarity. The results reported in this study show that Sec is necessary for axonal outgrowth and the establishment of neuronal polarity in hippocampal neurons in culture and the early polarity switch of cortical plate neurons during cortical formation in utero
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