Project Summary/Abstract The fact that both duplication and deletion of the Peripheral Myelin Protein 22 (PMP22) gene cause dysmyelinating peripheral neuropathy illustrates the importance of PMP22 for peripheral myelin integrity. PMP22 duplication causes Charcot-Marie-Tooth Disease Type 1A (CMT1A) and PMP22 deletion causes Hereditary Neuropathy with Liability to Pressure Palsies (HNPP). Although CMT1A and HNPP are the most common inherited peripheral neuropathies, research on them is underfunded and there are currently no disease-modifying treatments. This is largely due to the fact that PMP22 function and the consequence of altered PMP22 expression remain unclear. Thus, there is a critical need to expand knowledge of what PMP22 does in myelin, how it is regulated and when precise expression is required as a means to improve therapeutic potential of CMT1A and HNPP. This proposal aims to utilize cutting-edge techniques, including conditional mouse models and super resolution microscopy, and knowledge of cell adhesion and membrane biophysics to advance understanding of CMT1A and HNPP pathomechanisms. My central hypothesis is that PMP22 gene dosage and lipid raft association govern localization and organization of myelin adherens and tight junctions; a function that is most critical during development. In Aim 1, I will determine how PMP22 regulates adhesion junction organization in peripheral nerve myelin during development and aging with super resolution and electron microscopy. I will aid the interpretation of these studies by evaluating the effects of altered PMP22 expression on prototypical adherens and tight junctions in Madin-Darby Canine Kidney (MDCK) epithelial cells. The temporal requirement for precise PMP22 expression in myelin will also be defined by generating powerful conditional mouse models of CMT1A and HNPP. In Aim 2, I will determine how palmitoylation impacts PMP22 lipid raft association and regulation of adhesion junctions and define the biophysical properties of PMP22 within the plasma membrane using MDCK and Schwann cell models of CMT1A and HNPP and advanced biophysical methods. This K22 Career Transition Award will provide me with additional training, mentorship and expertise in cell adhesion, membrane biophysics and microscopy, thereby enabling my proposed research and facilitating my transition to independence. This training will complement my previous training in cell and molecular neurobiology and my current peripheral nerve training, and the expertise acquired during Phase I will be applied to more complex models in Phase II to expand mechanistic details. Completion of these aims will accelerate progress towards my long-term goal of developing an independent academic research career studying pathomechanisms of CMT1A and HNPP as a means to improve their therapeutic potential. The training and mentorship provided by this award will expand my technical skills and expertise, positioning me for success as an independent inves...