Gradual shifts in gene transcription steer neurodevelopmental programs, however this occurs too slowly to account for rapid developmental changes in neuronal form and function triggered by extracellular cues. Alternatively, post-translational ubiquitination alters protein function, localization and half-lif on timescales that permit the rapid responses critical to many stages of neuronal development. For example, axon guidance and synaptogenesis stimulated by the secreted axon guidance cue netrin involve protein ubiquitination. However the mechanism mediating ubiquitination, the substrates of ubiquitination, and the consequences of ubiquitination have yet to be defined. As defective axon guidance and synaptic function underlie several neurological conditions, defining these cellular and molecular mechanisms is a critical next step in order to therapeutically modulate neuronal responses on relevant timescales. Our recent work identified the brain-enriched mammalian E3 ubiquitin ligases, TRIM9 and TRIM67, as competing determinants downstream of the axon guidance and synaptogenesis promoting cue, netrin. TRIM9 and TRIM67 have >75% sequence similarity, and we found that both interact with the netrin receptor DCC. Through interactions we have identified with cytoskeletal and exocytic regulatory proteins, TRIM9 and TRIM67 spatio-temporally modulate cytoskeletal dynamics and membrane trafficking to rapidly tune neuronal form and function. Using murine models that lack TRIM9 or TRIM67, we have identified striking neural phenotypes in vivo. These include aberrant generation of neurons, anomalous neuronal morphology, circuit disruptions, and overt hippocampal-dependent learning deficits. To determine how these ubiquitin ligases control neuronal development and function, identifying their substrates of ubiquitination and the outcome of ubiquitination is necessary. Here we propose an unbiased dual proteomic approach to identify substrates ubiquitinated by TRIM9 and TRIM67, and subsequently validate these candidates using in vitro ubiquitination assays. In this novel approach we first compare changes in the ubiquitome of wildtype, TRIM9-/-, TRIM67-/-, and TRIM67-/-/TRIM9-/- cortical neurons. Next, we fuse TRIM9 and TRIM67 to a promiscuous biotin ligase to biotinylate and identify proximal proteins. We validate top hits using in vitro ubiquitination assays. Finally we identify changes in the expression and localization of validated substrates in cortical neurons isolated from single and double knockout embryos. Since E3 ubiquitin ligases are amenable to pharmacological manipulation, we expect this study to identify druggable targets for tuning neuronal development and function.
Nerves must reach and innervate appropriate targets to establish neural connectivity underlying function and behavior. Damage to the nerves within the adult brain and spinal column by injury or disease is often irreparable. We seek a comprehensive understanding of how nerve fibers reach and connect to multiple targets during development expecting this will illuminate the fundamental defects in neurological syndromes as well as develop rehabilitative therapies for spinal cord injury.
|Plooster, Melissa; Menon, Shalini; Winkle, Cortney C et al. (2017) TRIM9-dependent ubiquitination of DCC constrains kinase signaling, exocytosis, and axon branching. Mol Biol Cell 28:2374-2385|