A Pair of TRIM Proteins Regulate Axon Guidance in Concert

Axon guidance shapes formation of stereotyped central nervous system fiber tracts. Axons are directed by growth cones at their distal ends, which detect extracellular guidance cues through transmembrane receptors localized to the tips of bundled actin-rich filopodia. The cue netrin-1 binds to its receptor deleted in colorectal carcinoma (DCC) to induce changes in the dynamics of filopodia decorating growth cones. Alteration in filopodia is crucial to the attractive axon guidance functions of netrin-1, and requires function of the actin polymerase vasodilator stimulated phosphoprotein (VASP). Phosphorylation of VASP increases acutely in response to netrin-1, however this phosphorylation is not detected on the time scale over which filopodia respond to netrin, suggesting other regulatory mechanisms. We have identified two E3 ubiquitin ligases of the tripartite motif protein (TRIM) family, TRIM9 and TRIM67, which together regulate the nondegradative ubiquitination of VASP and are required for appropriate axonal and filopodial response to netrin-1, as well as development of netrin-1 dependent axon tracts in vivo. Both TRIMs localize to growth cone filopodia tips during development and interact directly with VASP. VASP is ubiquitinated in the presence of TRIM9, which alters the mobility and localization of VASP at filopodia tips. Deubiquitination of VASP is necessary for increase in filopodia number following netrin-1 treatment. TRIM67, conversely, reduces the ubiquitination of VASP, potentially by competing for TRIM9’s binding site for VASP. Deletion of Trim9 or Trim67 causes axons to be insensitive to guidance by netrin-1 in vitro. Both Trim deletions also alter the morphology of a netrin-1 dependent axon tract, the corpus callosum, in vivo. Deletion of Trim9 results in thickening of the tract due to excessive axon branching, while Trim67 deletion delays the approach of axons to the midline resulting in a thinner callosum. Loss of Trim67 also causes morphological changes to other brain regions, resulting in deficits in spatial memory, motor function and sensorimotor gating in adult animals. Our data suggest that this pair of TRIM proteins form an inactivating/activating, yin/yang-like regulatory mechanism for VASP in the netrin-1:DCC axon guidance signaling pathway, and are necessary for the proper formation of the brain.Doctor of Philosoph