Myotubularin PI 3-Phosphatases as Regulators of Peripheral Nerve Myelination

描述(申请人提供)：拟议研究的总体目标是确定磷脂酰肌醇(PI)调节紊乱导致髓鞘雪旺细胞异常膜转运和细胞信号转导的机制。我们是在一种特殊形式的脱髓鞘Charcot-Marie-Tooth周围神经病(4B~CMT4B型)的背景下研究这一问题的，该疾病的特征是髓鞘异常和严重的轴突变性。多发性肌萎缩侧索硬化症是最常见的遗传性神经疾病之一，全世界每2500人中就有1人患病。这种情况会导致四肢肌肉的进行性退化和感觉功能的丧失。CMT4B是由肌管蛋白相关蛋白2(MTMR2)或MTMR13功能突变引起的，这两个蛋白属于作为PI信号关键调节因子的磷酸酶家族。MTMR2是磷脂酰肌醇3-磷酸(PI3P)和磷脂酰肌醇3，5-二磷酸(PI[3，5]P2)的特异性脱磷酸酶。PI3P和PI(3，5)P2调节内体/溶酶体途径的膜运输。因此，推测CMT4B是由雪旺细胞膜转运缺陷引起的。MTMR13是一种催化失活的“假磷酸酶”，直接与MTMR2结合。MTMR13似乎是一种调控MTMR2的支架蛋白。这个建议的第一个目的是确定控制雪旺细胞中PI3P和PI(3，5)P2调控的磷脂酰肌醇激酶-磷酸酶网络。磷脂酰肌醇激酶和磷酸酶的缺失对肌醇磷脂水平的影响将使用基于高效液相色谱的肌醇磷脂图谱进行评估。同时，将通过体外髓鞘培养和对基因敲除小鼠周围神经的形态检查来评估PI激酶和磷酸酶丢失对髓鞘形成的影响。本研究的第二个目的是通过确定Rab GTP酶的Mtmr13‘S激活如何调节髓鞘形成，以及通过评估Mtmr2-Mtmr13复合体在调节细胞内吞作用中的作用，来确定Mtmr13假性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鉴定相互作用的Rab GTP酶，以及在体外髓鞘共培养中评估特定相互作用与髓鞘形成的相关性。这项提案的最终目的是确定Mtmr13的哪些结构域控制其特定功能。总而言之，这些研究将使我们能够确定磷脂酰肌醇在髓鞘雪旺细胞中的特定关键作用。这些研究也可能为通过药物靶向PI3P-PI(3，5)P2通路治疗CMT4B奠定了基础。

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.