Identification of Novel Drug Targets For Use in Preventing Deafness Caused by NF2

描述(申请人提供)：神经纤维瘤病2型(NF2)是一种以双侧前庭神经鞘瘤发展为特征的肿瘤疾病，也称为听神经瘤。所有NF2患者中有98%经历部分至完全听力丧失。NF2的治疗在监测肿瘤生长和缓慢但渐进性听力丧失、手术切除影响脑干功能的较大肿瘤和完全永久性耳聋之间取得平衡。人工耳蜗和听性脑干植入物已经被用于部分恢复患者的听力，取得了不同的成功。目前尚缺乏在保持听力的同时减缓或逆转肿瘤生长的治疗方法。这一建议验证了一种假设，即纠正缺乏NF2基因产物Schwannomin/Merlin功能的多余神经鞘瘤细胞的细胞骨架缺陷，将允许这些细胞与轴突相互作用，并接收促进其分化和/或凋亡的信号。识别直接结合肌动蛋白并调节雪旺细胞形态的蛋白质至关重要。这些蛋白质可以作为药物的靶点，修复神经鞘瘤细胞中的肌动蛋白动力学，恢复轴突接触。或者，修改肌动蛋白调节蛋白的药物可能会促进细胞死亡，这是细胞质分裂和有丝分裂纺锤体组织失败的结果。Schwanomin/Merlin的一个功能是抑制p21激活的蛋白激酶(PAK)的CDC42/RAC激活。我们将研究PAK信号通路中的LIM激酶(LIMK)和Cofilin，这两个末端靶标。LIMK是PAK的底物，因此其活性被预测在神经鞘瘤中很高。Cofilin是一种普遍表达的肌动蛋白结合因子，它能解聚f-肌动蛋白，并为新的肌动蛋白聚合创造成核位点。LIMK对丝氨酸-3的磷酸化抑制了Cofilin的功能。我们的初步研究表明，LIMK和Cofilin调节雪旺细胞中肌动蛋白的动力学和功能。此外，我们的结果表明LIMK和Cofilin在Schwannomin/Merlin的下游起作用。我们建议进行以下研究：1)确定这些蛋白在控制正常大鼠雪旺细胞肌动蛋白聚合和细胞功能中的作用；2)利用nf2ex2缺失的小鼠干细胞建立NF2的体外模型，以确定Schwannomin/Merlin失活是否导致LIMK和Cofilin活性的去调节以及SC功能的丧失；3)确定LIMK和Cofilin的调节剂是否恢复了nf2ex2缺失的SCs的形态和功能。这些研究是验证LIMK和/或Cofilin作为药物靶标用于开发旨在保护听力的NF2有效治疗方法的初步步骤。这项建议中的工作与2型神经纤维瘤病引起的听力损失有关。这种疾病的特征是双侧听神经鞘瘤的发展和98%的患者听力丧失。作为这一建议的结果，我们希望建立一个具有良好特性的NF2体外模型，并提出一个新的治疗方向，即肌动蛋白修饰蛋白LIMK和Cofilin。

DESCRIPTION (provided by applicant): Neurofibromatosis type 2 (NF2) is a tumor disorder characterized by development of bilateral vestibular schwannomas also called acoustic neuromas. 98% of all NF2 patients experience partial to complete loss of hearing. Treatment for NF2 is balanced between monitoring tumor growth and the slow but progressive loss of hearing with surgical removal of larger tumors impinging on brainstem function and complete and permanent deafness. Cochlear and auditory brainstem implants have been used to partially restore hearing in a subset of patients with varying success. Therapeutics that slow or reverse tumor growth whilst maintaining hearing are currently lacking. This proposal tests the hypothesis that correcting the cytoskeletal defects in supernumerary schwannoma cells lacking function of the nf2 gene product, schwannomin/merlin, will allow these cells to interact with axons and receive cues promoting their differentiation and/or apoptosis. Identifying proteins that directly bind actin and regulate Schwann cell morphology is of the utmost importance. These proteins can serve as targets for drugs that will repair actin dynamics in schwannoma cells and restore axonal contact. Alternatively, drugs that modify actin regulatory proteins could promote cell death as a result of failed cytokinesis and mitotic spindle organization. One function of schwanomin/merlin is to inhibit Cdc42/Rac activation of p21 activated kinase (PAK). We will investigate LIM kinase (LIMK) and cofilin, terminal targets in a PAK signaling pathway. LIMK is a substrate for PAK, thus its activity is predicted to be high in schwannomas. Cofilin is a ubiquitously expressed actin-binding factor that depolymerizes f-actin and creates nucleation sites for new actin polymerization. Cofilin's function is inhibited by phosphorylation on serine-3 by LIMK. Our preliminary studies demonstrate that LIMK and cofilin modulate actin dynamics and function in Schwann cells. Moreover, our results suggest that LIMK and cofilin act down-stream of Schwannomin/merlin. We propose studies to: 1) identify the role of these proteins in controlling actin polymerization and cellular function in normal rat Schwann cells, 2) establish an in vitro model for NF2 using nf2ex2deleted mouse SCs to determine if inactivation of schwannomin/merlin leads to de-regulation of LIMK and cofilin activity and loss of SC function, and 3) determine if modulators of LIMK and cofilin restore the morphology and function of nf2ex2deleted SCs. These studies are initial steps in validating LIMK and/or cofilin as drug targets for development of an effective treatment for NF2 aimed at preserving hearing. The work in this proposal is relevant to the loss of hearing caused by Neurofibromatosis type 2. This disorder is characterized by development of bilateral acoustic schwannomas and loss of hearing in 98% of patients. As an outcome of this proposal, we hope to create a well characterized in vitro model for NF2 and advance a novel therapeutic direction, the actin modifying proteins LIMK and cofilin.