Caracterización del papel de WIP en morfogénesis neuronal

Abstract

ABSTRACT The great complexity of the nervous system is sustained by the highly polarized morphology of neurons. For this reason is important that neuritogenesis occurs at the right place and at the right moment in order to establish correct connections with proper targets. Several environmental cues converge on intracellular events, as signaling transduction, exocytic and endocytic mechanisms and cytoskeletal rearrangements, to modulate neuritogenesis. Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP), is an actin-binding protein involved in the regulation of actin polymerization in cells such as fibroblasts and lymphocytes. In spite of its recognized function in non-neuronal cells, the role of WIP in the central nervous system has never been examined before. We used WIP-deficient mice to examine the function of WIP in neurons using imaging techniques and gain-of-function and loss-of-function in vitro studies and in utero electroporation as in vivo approach. Herein we describe the expression of WIP in embryonic and adult murine neural cells. In this thesis work we report that WIP-/- differentiating primary hippocampal neurons exhibit enlargement of somas, and overgrowth of neuritic and dendritic branches that are more evident in early developmental stages. WIP deficiency also increases the amplitude and the frequency of miniature excitatory postsynaptic currents, suggesting that WIP-/- neurons contain more mature synapses than WT neurons. Our data describe that loss of WIP in primary neurons alters the subcellular distribution of actin nucleation promoting factors with WIP-binding capacity, such as N-WASP and cortactin. Inhibition of N-WASP activity by wiskostatin treatment approaches the phenotype of WIP-/- immature neurons to that of control cells, supporting the contribution of WIP to the regulation of N-WASP activity in neuritogenesis. Our results demonstrate an essential role for the cooperative action of WIP-cortactin-N-WASP to control neuronal morphogenesis and the adequate formation of neuronal networks. Moreover, in this work we show that the increased neuritic branching and soma area described in WIP-/- immature primary neurons correlate with a significative reduction in the mTORC1/p70S6K pathway activity. Blocking mTORC1 kinase activity with rapamycin at early stages of neuronal differentiation induces neuritic branching overgrowth and soma enlargement in WT neurons. The absence of WIP also associates with a enhancement of the PI3K/Akt/mTORC1 pathway in E17 murine cortex. In summary, these findings reveal WIP as a new negative regulator of neuronal and synaptic maturation.