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

Abstract

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.

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

“…Similar to the DCC receptor, association with raft membranes has also been proven to be essential during axonal guidance events, as shown not only by biochemical fractionation, but also with biophysical tools. Using Fluorescence Recovery After Photobleaching (FRAP) and single-particle tracking Photoactivated Localization Microscopy (PALM), we have demonstrated that UNC5 receptors reside in raft membrane microdomains [ 100 ]. Such properties are modified after MβCD or cholesterol oxidase (ChOx) treatment [ 100 ].…”

Syntaxin-1 is necessary for UNC5/Netrin-1-dependent macropinocytosis and chemorepulsion

“…Netrin-1 exerts long-range chemorepulsion that is important for axonal guidance (Round & Stein, 2007), and plays a major role in the organization of EGL neurons during early postnatal development of the cerebellum, controlling the growth of parallel fibres (Alcantara et al, 2000;Hernaiz-Llorens et al, 2020;Przyborski et al, 1998). EGL neurons have been shown to co-express DCC, neogenin, DSCAM, UNC5B and UNC5C that sense Netrin-1 expressed in the EGL and in interneurons of the molecular layer of the cerebellum (Alcantara et al, 2000;Hernaiz-Llorens et al, 2020).…”

“…Netrin-1 exerts long-range chemorepulsion that is important for axonal guidance (Round & Stein, 2007), and plays a major role in the organization of EGL neurons during early postnatal development of the cerebellum, controlling the growth of parallel fibres (Alcantara et al, 2000;Hernaiz-Llorens et al, 2020;Przyborski et al, 1998). EGL neurons have been shown to co-express DCC, neogenin, DSCAM, UNC5B and UNC5C that sense Netrin-1 expressed in the EGL and in interneurons of the molecular layer of the cerebellum (Alcantara et al, 2000;Hernaiz-Llorens et al, 2020). To investigate whether Stx1 is necessary for Netrin-1-dependent repulsion, we co-cultured postnatal EGL explants in type I collagen 3D hydrogels, and exposed them to aggregates of either control HEK-293T cells or Netrin-1-expressing HEK-293T cells for 2 days (Alcantara et al, 2000;Hernaiz-Llorens et al, 2020).…”

“…Experiments were also approved by the ethical committee from the Generalitat of Catalonia. Postnatal cerebellums were isolated, mechanically disaggregated and trypsinized as previously described (Hernaiz-Llorens et al, 2020;Rosello-Busquets et al, 2019). Briefly, after centrifugation, neurons were resuspended in 2 mL of DMEM medium and EGL neurons were isolated by centrifugation (3000 rpm, 10 min at 4º C) in a discontinuous percoll gradient (35% and 60% of percoll).…”

“…After 48 h (2DIV) of culture, explants were fixed, and immunocytochemistry against bIIItubulin was performed. Repulsion was analysed by measuring the proximal/distal (P/D) ratio as previously described (Gil & Del Rio, 2019;Hernaiz-Llorens et al, 2020). Briefly, the P/D ratio is the fraction of axons growing in the proximal quadrant of the explant (closer to the aggregate of HEK-293T cells) versus the axons growing in the distal quadrant.…”