Nystatin Regulates Axonal Extension and Regeneration by Modifying the Levels of Nitric Oxide

Nystatin is a pharmacological agent commonly used for the treatment of oral, mucosal and cutaneous fungal infections. Nystatin has also been extensively applied to study the cellular function of cholesterol-enriched structures because of its ability to bind and extract cholesterol from mammalian membranes. In neurons, cholesterol level is tightly regulated, being essential for synapse and dendrite formation, and for axonal guidance. However, the action of Nystatin on axon regeneration has been poorly evaluated. Here, we examine the effect of Nystatin on primary cultures of hippocampal neurons, showing how acute dose (minutes) of Nystatin increases the area of growth cones, and chronic treatment (days) enhances axon length, axon branching and axon regeneration post-axotomy. We describe two alternative signaling pathways responsible for the observed effects, and activated at different concentrations of Nystatin. At elevated concentrations, Nystatin promotes growth cone expansion through phosphorylation of Akt; whereas at low concentrations, Nystatin enhances axon length and regrowth by increasing nitric oxide levels. Together, our findings indicate new signaling pathways of Nystatin and propose this compound as a novel regulator of axon regeneration

Cholesterol Depletion Regulates Axonal Growth and Enhances Central and Peripheral Nerve Regeneration

Axonal growth during normal development and axonal regeneration rely on the action of many receptor signaling systems and complexes, most of them located in specialized raft membrane microdomains with a precise lipid composition. Cholesterol is a component of membrane rafts and the integrity of these structures depends on the concentrations present of this compound. Here we explored the effect of cholesterol depletion in both developing neurons and regenerating axons. First, we show that cholesterol depletion in vitro in developing neurons from the central and peripheral nervous systems increases the size of growth cones, the density of filopodium-like structures and the number of neurite branching points. Next, we demonstrate that cholesterol depletion enhances axonal regeneration after axotomy in vitro both in a microfluidic system using dissociated hippocampal neurons and in a slice-coculture organotypic model of axotomy and regeneration. Finally, using axotomy experiments in the sciatic nerve, we also show that cholesterol depletion favors axonal regeneration in vivo. Importantly, the enhanced regeneration observed in peripheral axons also correlated with earlier electrophysiological responses, thereby indicating functional recovery following the regeneration. Taken together, our results suggest that cholesterol depletion per se is able to promote axonal growth in developing axons and to increase axonal regeneration in vitro and in vivo both in the central and peripheral nervous systems

A conserved role for Syntaxin-1 in pre- and post-commissural midline axonal guidance in fly, chick, and mouse

Axonal growth and guidance rely on correct growth cone responses to guidance cues. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the crosstalk mechanisms between guidance and membrane dynamics and turnover. Recent studies indicate that whereas axonal attraction requires exocytosis, chemorepulsion relies on endocytosis. Indeed, our own studies have shown that Netrin-1/Deleted in Colorectal Cancer (DCC) signaling triggers exocytosis through the SNARE Syntaxin-1 (STX1). However, limited in vivo evidence is available about the role of SNARE proteins in axonal guidance. To address this issue, here we systematically deleted SNARE genes in three species. We show that loss-of-function of STX1 results in pre- and post-commissural axonal guidance defects in the midline of fly, chick, and mouse embryos. Inactivation of VAMP2, Ti-VAMP, and SNAP25 led to additional abnormalities in axonal guidance. We also confirmed that STX1 loss-of-function results in reduced sensitivity of commissural axons to Slit-2 and Netrin-1. Finally, genetic interaction studies in Drosophila show that STX1 interacts with both the Netrin-1/DCC and Robo/Slit pathways. Our data provide evidence of an evolutionarily conserved role of STX1 and SNARE proteins in midline axonal guidance in vivo, by regulating both pre- and post-commissural guidance mechanisms

Cholesterol depletion regulates axonal growth and enhances central and peripheral nerve regeneration

Axonal growth during normal development and axonal regeneration rely on the action of many receptor signaling systems and complexes, most of them located in specialized raft membrane microdomains with a precise lipid composition. Cholesterol is a component of membrane rafts and the integrity of these structures depends on the concentrations present of this compound. Here we explored the effect of cholesterol depletion in both developing neurons and regenerating axons. First, we show that cholesterol depletion in vitro in developing neurons from the central and peripheral nervous systems increases the size of growth cones, the density of filopodium-like structures and the number of neurite branching points. Next, we demonstrate that cholesterol depletion enhances axonal regeneration after axotomy in vitro both in a microfluidic system using dissociated hippocampal neurons and in a slice-coculture organotypic model of axotomy and regeneration. Finally, using axotomy experiments in the sciatic nerve, we also show that cholesterol depletion favors axonal regeneration in vivo. Importantly, the enhanced regeneration observed in peripheral axons also correlated with earlier electrophysiological responses, thereby indicating functional recovery following the regeneration. Taken together, our results suggest that cholesterol depletion per se is able to promote axonal growth in developing axons and to increase axonal regeneration in vitro and in vivo both in the central and peripheral nervous systems

Lipid raft association and subcellular localization of UNC5 Netrin-1 receptors = Associació als lípids raft i localització subcel·lular de la familia de receptors de la Netrina-1 UNC5

[eng] During brain development, UNC5 receptors play a key role in controlling axonal extension through their sensing of the guidance molecule Netrin-1. The correct positioning of receptors into specific cholesterol-enriched membrane raft microdomains is of crucial importance for the efficient transduction of the recognized signal. Whether such microdomains are required for the appropriate axonal guidance mediated by UNC5 receptors remains unknown. Here, we extend the classical biochemical characterization of raft microdomains by performing confocal microscopy, live cell FRAP analysis and sptPALM of fluorescently-tagged UNC5 receptors and we reveal for the first time differences into their membrane mobility properties. Using a combination of pharmacological and genetical approaches in primary neuronal cultures and brain cerebellar explants we further demonstrate that disrupting raft microdomains inhibit the chemorepulsive response of growth cones and axons against Netrin-1. Together, our findings indicate that the distribution of all UNC5 receptors into cholesterol-enriched raft microdomains is heterogeneous and that their specific localization have functional consequences for the axonal chemorepulsion against Netrin-1. Alternative splicing is a key mechanism in which a variety of proteins is achieved from "cut and paste" mRNA. The plethora of proteins resulting from alternative splicing processes is crucial for the proper function of certain protein isoforms. In this thesis we have detected alternative splicing in the UNC5A receptor in the nervous system. The resulting product is a UNC5A isoform that lacks the first TSPI repeat, uncovered here, to be important for cell membrane targeting. We have further analyzed the implications of losing the first TSPI repeat.[spa] Durante el desarrollo del cerebro, los receptores UNC5 juegan un papel importante en el control de la extensión axonal, guiándose a través de la molécula guía Netrina-1. El posicionamiento específico de estos receptores en microdominios de membrana llamados 'lipid raft', son de crucial importancia en la transducción de señales. Si la asociación a estos microdominios es necesaria para los procesos de guía axonal mediados por los receptores UNC5, sigue aún por resolverse. En esta tesis vamos más allá de los clásicos ensayos de fraccionamiento de membrana, utilizando técnicas como microscopia confocal, microscopia de FRAP y microscopia de super resolución. Con estas técnicas hemos descubierto diferentes propiedades de estos receptores en cuanto a la movilidad lateral y la distribución subcelular. Más allá, mediante el uso de herramientas farmacológicas y genéticas en cultivos neuronales primarios y explantes cerebelares hemos hallado que desestabilizando estos microdominios de membrana la quimiorepulsión en conos de crecimiento y axones mediada por Netrina-1 queda inhibida. El 'splicing' alternativo es un mecanismo clave en el que una variedad de proteinas se consigue gracias al mecanismo de 'corta y pega' del mRNA. El abanico de proteínas de un mismo gen que se consigue a través de este proceso, es a menudo de vital importancia para algunas isoformas proteicas. En esta tesis hemos detectado que el receptor UNC5A sufre un 'splicing' alternativo en el sistema nervioso central. El producto resultante es una isoforma corta de UNC5A que carece del dominio extracelular TSPI, el cual es importante para la llegada de este receptor a la membrana celular. Además, hemos investigado cuales son las implicaciones de perder este dominio en las funciones que lleva a cabo UNC5A en la célula

One Raft to Guide Them All, and in Axon Regeneration Inhibit Them

Central nervous system damage caused by traumatic injuries, iatrogenicity due to surgical interventions, stroke and neurodegenerative diseases is one of the most prevalent reasons for physical disability worldwide. During development, axons must elongate from the neuronal cell body to contact their precise target cell and establish functional connections. However, the capacity of the adult nervous system to restore its functionality after injury is limited. Given the inefficacy of the nervous system to heal and regenerate after damage, new therapies are under investigation to enhance axonal regeneration. Axon guidance cues and receptors, as well as the molecular machinery activated after nervous system damage, are organized into lipid raft microdomains, a term typically used to describe nanoscale membrane domains enriched in cholesterol and glycosphingolipids that act as signaling platforms for certain transmembrane proteins. Here, we systematically review the most recent findings that link the stability of lipid rafts and their composition with the capacity of axons to regenerate and rebuild functional neural circuits after damage

Growth cone repulsion to Netrin-1 depends on lipid raft microdomains enriched in UNC5 receptors

During brain development, Uncoordinated locomotion 5 (UNC5) receptors control axonal extension through their sensing of the guidance molecule Netrin-1. The correct positioning of receptors into cholesterol-enriched membrane raft microdomains is crucial for the efficient transduction of the recognized signals. However, whether such microdomains are required for the appropriate axonal guidance mediated by UNC5 receptors remains unknown. Here, we combine the use of confocal microscopy, live-cell FRAP analysis and single-particle tracking PALM to characterize the distribution of UNC5 receptors into raft microdomains, revealing differences in their membrane mobility properties. Using pharmacological and genetic approaches in primary neuronal cultures and brain cerebellar explants we further demonstrate that disrupting raft microdomains inhibits the chemorepulsive response of growth cones and axons against Netrin-1. Together, our findings indicate that the distribution of all UNC5 receptors into cholesterol-enriched raft microdomains is heterogeneous and that the specific localization has functional consequences for the axonal chemorepulsion against Netrin-1

New Partners Identified by Mass Spectrometry Assay Reveal Functions of NCAM2 in Neural Cytoskeleton Organization

Neuronal cell adhesion molecule 2 (NCAM2) is a membrane protein with an important role in the morphological development of neurons. In the cortex and the hippocampus, NCAM2 is essential for proper neuronal differentiation, dendritic and axonal outgrowth and synapse formation. However, little is known about NCAM2 functional mechanisms and its interactive partners during brain development. Here we used mass spectrometry to study the molecular interactome of NCAM2 in the second postnatal week of the mouse cerebral cortex. We found that NCAM2 interacts with >100 proteins involved in numerous processes, including neuronal morphogenesis and synaptogenesis. We validated the most relevant interactors, including Neurofilaments (NEFs), Microtubule-associated protein 2 (MAP2), Calcium/calmodulin kinase II alpha (CaMKIIα), Actin and Nogo. An in silico analysis of the cytosolic tail of the NCAM2.1 isoform revealed specific phosphorylation site motifs with a putative affinity for some of these interactors. Our results expand the knowledge of NCAM2 interactome and confirm the key role of NCAM2 in cytoskeleton organization, neuronal morphogenesis and synaptogenesis. These findings are of interest in explaining the phenotypes observed in different pathologies with alterations in the NCAM2 gene

Cholesterol Depletion Regulates Axonal Growth and Enhances Central and Peripheral Nerve Regeneration

Axonal growth during normal development and axonal regeneration rely on the action of many receptor signaling systems and complexes, most of them located in specialized raft membrane microdomains with a precise lipid composition. Cholesterol is a component of membrane rafts and the integrity of these structures depends on the concentrations present of this compound. Here we explored the effect of cholesterol depletion in both developing neurons and regenerating axons. First, we show that cholesterol depletion in vitro in developing neurons from the central and peripheral nervous systems increases the size of growth cones, the density of filopodium-like structures and the number of neurite branching points. Next, we demonstrate that cholesterol depletion enhances axonal regeneration after axotomy in vitro both in a microfluidic system using dissociated hippocampal neurons and in a slice-coculture organotypic model of axotomy and regeneration. Finally, using axotomy experiments in the sciatic nerve, we also show that cholesterol depletion favors axonal regeneration in vivo. Importantly, the enhanced regeneration observed in peripheral axons also correlated with earlier electrophysiological responses, thereby indicating functional recovery following the regeneration. Taken together, our results suggest that cholesterol depletion per se is able to promote axonal growth in developing axons and to increase axonal regeneration in vitro and in vivo both in the central and peripheral nervous systems

A conserved role for Syntaxin-1 in pre- and post-commissural midline axonal guidance in fly, chick, and mouse

Axonal growth and guidance rely on correct growth cone responses to guidance cues. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the crosstalk mechanisms between guidance and membrane dynamics and turnover. Recent studies indicate that whereas axonal attraction requires exocytosis, chemorepulsion relies on endocytosis. Indeed, our own studies have shown that Netrin-1/Deleted in Colorectal Cancer (DCC) signaling triggers exocytosis through the SNARE Syntaxin-1 (STX1). However, limited in vivo evidence is available about the role of SNARE proteins in axonal guidance. To address this issue, here we systematically deleted SNARE genes in three species. We show that loss-of-function of STX1 results in pre- and post-commissural axonal guidance defects in the midline of fly, chick, and mouse embryos. Inactivation of VAMP2, Ti-VAMP, and SNAP25 led to additional abnormalities in axonal guidance. We also confirmed that STX1 loss-of-function results in reduced sensitivity of commissural axons to Slit-2 and Netrin-1. Finally, genetic interaction studies in Drosophila show that STX1 interacts with both the Netrin-1/DCC and Robo/Slit pathways. Our data provide evidence of an evolutionarily conserved role of STX1 and SNARE proteins in midline axonal guidance in vivo, by regulating both pre- and post-commissural guidance mechanisms