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The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis.

ABSTRACT: TRIM9 and TRIM67 are neuronally enriched E3 ubiquitin ligases essential for appropriate morphogenesis of cortical and hippocampal neurons and fidelitous responses to the axon guidance cue netrin-1. Deletion of murine Trim9 or Trim67 results in neuroanatomical defects and striking behavioral deficits, particularly in spatial learning and memory. TRIM9 and TRIM67 interact with cytoskeletal and exocytic proteins, but the full interactome is not known. Here we performed the unbiased proximity-dependent biotin identification (BioID) approach to define TRIM9 and TRIM67 protein-protein proximity network in developing cortical neurons and identified putative neuronal TRIM interaction partners. Candidates included cytoskeletal regulators, cytosolic protein transporters, exocytosis and endocytosis regulators, and proteins necessary for synaptic regulation. A subset of high-priority candidates was validated, including Myo16, Coro1A, MAP1B, ExoC1, GRIP1, PRG-1, and KIF1A. For a subset of validated candidates, we utilized total internal reflection fluorescence microscopy to demonstrate dynamic colocalization with TRIM proteins at the axonal periphery, including at the tips of filopodia. Further analysis demonstrated that the RNA interference-based knockdown of the unconventional myosin Myo16 in cortical neurons altered growth cone filopodia density and axonal branching patterns in a TRIM9- and netrin-1-dependent manner. Future analysis of other validated candidates will likely identify novel proteins and mechanisms by which TRIM9 and TRIM67 regulate neuronal form and function. [Media: see text].

SUBMITTER: Menon S

PROVIDER: S-EPMC8098814 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:During development, neurons undergo expansive growth and shape change to achieve their mature morphology and physiological function. As cellular remodeling occurs, proteins are synthesized, transported, degraded, and recycled. As such, protein levels and localization are tightly regulated. E3 ubiquitin ligases play a key role in proteostasis by altering protein localization, intracellular trafficking, and protein lifetime via the addition of ubiquitin modifiers. TRIM9 and TRIM67 are brain-enriched E3 ubiquitin ligases implicated in numerous stages of neuronal morphogenesis. We previously demonstrated both proteins are required for axon pathfinding and growth cone shape changes in developing neurons. In particular, TRIM9 and TRIM67 regulate filopodia number and stability in early stages of neuronal development. Our published in vivo studies suggest TRIM9 and TRIM67 may function at the synapse as well. We observed distinct deficits in spatial learning and memory of Trim9-/- and Trim67-/- mice in the Morris Water Maze test, compared to their littermate controls. Furthermore, adult-born neurons in the dentate gyrus in Trim9-/- mice also displayed a decreased number of dendritic spines. Here we demonstrate TRIM9 and TRIM67 localize to the post-synaptic density (PSD), a structure attached to the post-synaptic membrane in dendritic spines. We identified 148 proteins that were significantly changed (p < 0.05) in the Trim67-/- mice compared to their Trim67+/+ littermates. Gene Ontology analysis of these proteins demonstrated enrichment of several cellular pathways, including peptidyl-amino acid modification, microtubule dynamics, and Ras signal transduction. Likewise, we identified 109 proteins that were significantly changed (p < 0.05) in the Trim9-/- mice compared to their Trim9+/+ littermates. Following Gene Ontology analysis, we observed the prominent enrichment of one pathway in our significantly different proteins: the actin cytoskeleton. Changes in these actin cytoskeleton proteins were bidirectional, suggesting the presence of altered cytoskeletal architecture and organization in the Trim9-/- PSD.

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The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis.

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