The E3 Ubiquitin Ligase TRIM9 Is a Filopodia Off Switch Required for Netrin-Dependent Axon Guidance

Abstract

Summary Neuronal growth cone filopodia contain guidance receptors and contribute to axon guidance, however the mechanism by which the guidance cue netrin increases filopodia density is unknown. Here we demonstrate that TRIM9, an E3 ubiquitin ligase that localizes to filopodia tips and binds the netrin receptor DCC, interacts with and ubiquitinates the barbed-end polymerase VASP to modulate filopodial stability during netrin-dependent axon guidance. Studies with murine TRIM9+/+ and TRIM9−/− cortical neurons, along with a non-ubiquitinatable VASP mutant, demonstrate that TRIM9-mediated ubiquitination of VASP reduces VASP filopodial tip localization, VASP dynamics at tips, and filopodial stability. Upon netrin treatment, VASP is deubiquitinated, which promotes VASP tip localization and filopodial stability. TRIM9 deletion induces axon guidance defects in vitro and in vivo, whereas a gradient of deubiquitinase inhibition promotes axon turning in vitro. We conclude that a gradient of TRIM9-mediated ubiquitination of VASP creates a filopodial stability gradient during axon turning.

Trim9Deletion Alters the Morphogenesis of Developing and Adult-Born Hippocampal Neurons and Impairs Spatial Learning and Memory

“…These molecular targets of TRIM9 are likely involved in hippocampal neuron morphogenesis as well. Indeed, SNAP25, VASP, and TRIM9 are present within dendrites (Jordan et al, 2004;Lin et al, 2010;Menna et al, 2011) and may contribute to the alterations in dendritic swelling, branching density, and spine density observed here. The increase in axonal protrusions present in vivo in Trim9 Ϫ/Ϫ animals may indicate impaired cytoskeletal function, a hypothesis consistent with the involvement of TRIM9-mediated regulation of VASP (Menon et al, 2015).…”

“…TRIM9 also directly interacts with the netrin receptor DCC, and netrin stimulation inhibits the TRIM9 interaction with SNAP25, promoting exocytic and axon branching responses (Winkle et al, 2014). Second, TRIM9-mediated ubiquitination of the actin polymerase VASP decreases the stability of axonal growth cone filopodia (Menon et al, 2015). In the absence of Trim9, growth cones exhibit higher densities of stable filopodia.…”

“…Deletion of Trim9 increases hippocampal dendritic and axonal complexity in vitro TRIM9 regulates the branching and guidance of axons of developing murine cortical neurons (Winkle et al, 2014;Menon et al, 2015) and in invertebrates (Hao et al, 2010;Morikawa et al, 2011;Morf et al, 2013). TRIM9 is present in cultured hippocampal neurons, where its expression increases over time in vitro (Fig.…”

“…Deletion of Trim9 in cortical neurons is associated with elevated exocytosis, increased stability of growth cone filopodia, and loss of netrin responsiveness in vitro. These phenotypes are associated with aberrant branching and projection of cortical axons both in vitro and in vivo (Winkle et al, 2014;Menon et al, 2015), suggesting that TRIM9 regulates membrane delivery and cytoskeletal dynamics powering cortical neuron morphogenesis.…”

“…We recently identified TRIM9 as a regulator of neuronal morphogenesis in cortical neurons (Winkle et al, 2014;Menon et al, 2015). TRIM9 directly interacts with exocytic t-SNARE SNAP25 (Li et al, 2001), the actin polymerase VASP (Menon et al, 2015), and DCC, a receptor for the axon guidance cue netrin (Winkle et al, 2014).…”