PROJECT SUMMARY. From movement to memory, the complex and wide-ranging tasks performed by the nervous system depend on the polarization of billions of neurons into two functionally distinct subcellular compartments: dendrites, which receive most of the input into the neuron, and the axon, which transmits information to other cells in the form of an action potential. Two powerful contributors to neuronal polarity are 1) the axon initial segment (AIS), a domain at the beginning of the axon that initiates action potentials, and 2) microtubules (MTs), cytoskeletal filaments involved in axon outgrowth and cargo trafficking1,2. Disrupting either of these impairs the other and causes broad downstream defects in neuronal polarity and function3–6, suggesting that the interplay between these structures is a key regulator of neuronal polarity. Intriguingly, MTs within the AIS form bundles, called “fascicles”, that are rarely observed outside the AIS7,8. This unique organization supports the idea that the AIS and MTs work together to control neuronal polarity. Therefore, investigating MT fasciculation could unlock new insights into how neurons establish and maintain polarity, as well as how that process might go awry in neurodevelopmental and neurodegenerative disorders. Though AIS MT fasciculation was first observed over 50 years ago7,8, little progress has been made in determining its function, largely because a lack of candidates for the driver of MT fasciculation made loss-of-function studies impossible. However, a recent breakthrough occurred with the discovery of the protein Tripartite Motif Containing 46 (TRIM46) at the AIS9. TRIM46 localizes to AIS MTs, and TRIM46-deficient cultured neurons lack AIS MT fasciculation9,10. They also exhibit impaired AIS formation and a range of defects in axonal outgrowth, protein distribution, and MT orientation, suggesting that TRIM46 is required for both AIS MT fasciculation and neuronal polarity more broadly9. However, these exciting claims are based on TRIM46 knockdown by shRNA, mostly in cultured neurons, so the observed defects could be attributed to shRNA toxicity or the artificial culture environment. No work has been done to investigate TRIM46 knockout in vivo. This project will leverage the Rasband lab’s extensive experience with the in vivo study of the AIS to fill this gap. The objective of this proposal is to use TRIM46 knockout mice to determine the role of TRIM46 in the nervous system and test the specific hypothesis that TRIM46 is required for AIS MT fasciculation and neuronal polarity in vivo. The proposed experiments will use immunostaining and high-resolution microscopy methods to determine the consequences of TRIM46 knockout from neuronal ultrastructure to behavior and in the contexts of development and injury. Aim 1 will determine the role of TRIM46 in AIS formation, ultrastructure, and MT fasciculation. Aim 2 will determine the role of TRIM46 in neuronal morphological, molecular, and MT polarity....