The overall goal of the proposed research is to determine the mechanisms by which disturbances in phosphoinositide (PI) regulation lead to abnormal membrane trafficking and cellular signaling in myelinating Schwann cells. We are studying this question in the context of a specific form of demyelinating Charcot-Marie-Tooth peripheral neuropathy (type 4B~ CMT4B), which is characterized by abnormal myelination and severe axonal degeneration. CMT is one of the most common inherited neurological disorders, affecting about 1 in 2500 worldwide. This condition leads to progressive degeneration of the muscles of the extremities and loss of sensory function. CMT4B is caused by loss of function mutations in either myotubularin-related protein 2 (MTMR2) or MTMR13, which belong to a large family of phosphatases that act as key regulators of PI signaling. MTMR2 is a PI 3-phosphatase that specifically dephosphorylates phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 3,5-bisphosphate (PI[3,5]P2). PI3P and PI(3,5)P2 regulate membrane traffic within the endosomal/lysosomal pathway. Therefore, it is theorized that CMT4B arises from defects in membrane transport in Schwann cells. MTMR13 is a catalytically inactive pseudophosphatase that associates directly with MTMR2. MTMR13 appears to function as a scaffold protein that regulates MTMR2. The first aim of this proposal is to define the phosphoinositide kinase-phosphatase network that controls PI3P and PI(3,5)P2 regulation in Schwann cells. The impact of the loss of PI kinases and phosphatases on phosphoinositide levels will be evaluated using HPLC-based phosphoinositide profiling. In parallel, the impact of the loss of PI kinases and phosphatases on myelination will be assessed using in vitro myelinating cultures and morphological examination of peripheral nerves of knockout mice. The second aim of the study is to define the function of the Mtmr13 pseudophosphatase in Schwann cell membrane traffic by determining how Mtmr13's activation of Rab GTPases regulates myelination, and by assessing the role of the Mtmr2-Mtmr13 complex in the regulation of endocytosis. These goals will be accomplished using biochemical studies to identify interacting Rab GTPases, as well as in vitro myelinating co-cultures to assess the relevance of specific interactions to myelination. The final aim of this proposal is to determine which domains of Mtmr13 control its specific functions. Collectively, these studies will allow us to define the specific, critical roles of phosphoinositides in myelinating Schwann cells. These studies also may well form the basis of a rational approach to the treatment of CMT4B by pharmacological targeting of the PI3P-PI(3,5)P2 pathway.