Myotubularin PI 3-Phosphatases as Regulators of Peripheral Nerve Myelination

描述（由申请人提供）：拟议研究的总体目标是确定磷脂肌苷（PI）调节中的干扰导致髓鞘雪旺细胞异常膜运输和细胞信号传导的机制。我们在一种特殊形式的脱髓鞘性Charcot-Marie-Tooth周围神经病变（4B~ CMT4B型）的背景下研究这个问题，其特征是异常的髓鞘形成和严重的轴突变性。CMT是最常见的遗传性神经系统疾病之一，全世界每2500人中就有1人患病。这种情况导致四肢肌肉的进行性退化和感觉功能的丧失。CMT4B是由肌小管蛋白相关蛋白2 （MTMR2）或MTMR13的功能突变缺失引起的，这两种蛋白属于一个磷酸酶大家族，作为PI信号的关键调节因子。MTMR2是一种PI3 -磷酸酶，能够特异性地去磷酸化3-磷酸磷脂酰肌醇（PI3P）和3,5-二磷酸磷脂酰肌醇（PI[3,5]P2）。PI3P和PI(3,5)P2调节内体/溶酶体途径内的膜运输。因此，理论上CMT4B是由雪旺细胞的膜运输缺陷引起的。MTMR13是一种催化无活性的“假磷酸酶”，与MTMR2直接相关。MTMR13似乎是一种调节MTMR2的支架蛋白。本研究的第一个目的是确定在许旺细胞中控制PI3P和PI(3,5)P2调控的磷酸肌苷激酶-磷酸酶网络。PI激酶和磷酸酶的缺失对磷酸肌肽水平的影响将使用基于高效液相色谱的磷酸肌肽谱进行评估。同时，PI激酶和磷酸酶的缺失对髓鞘形成的影响将通过体外髓鞘培养和敲除小鼠周围神经的形态学检查来评估。本研究的第二个目的是通过确定Mtmr13激活Rab gtpase如何调节髓鞘形成，以及通过评估Mtmr2-Mtmr13复合物在调节内吞作用中的作用，来确定Mtmr13假磷酸酶在雪旺细胞膜运输中的功能。这些目标将通过生化研究来确定相互作用的Rab gtpase，以及体外髓鞘共培养来评估特定相互作用与髓鞘形成的相关性。本提案的最终目的是确定Mtmr13的哪些结构域控制其特定功能。总的来说，这些研究将使我们能够确定磷脂肌苷在髓鞘雪旺细胞中的具体关键作用。这些研究也可能为通过药理靶向PI3P-PI(3,5)P2通路来合理治疗CMT4B奠定基础。

DESCRIPTION (provided by applicant): 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.