TorsinA mediated regulation of Cdc42 signaling and DYT1 dystonia

项目摘要/摘要 DYT1肌张力障碍是一种毁灭性的神经运动障碍，其特征是肌肉失控。 宫缩会导致不正常的、非自愿的姿势。DYT1肌张力障碍是由Δ基因缺失(ΔGAG；DYT1E)引起的 编码与多种细胞活动(AAA+)蛋白相关的管腔ATPase的Tor1A基因 扭力A。目前尚不清楚ΔE突变是如何导致肌张力障碍的，因为 由TorsinA执行的是未知的。这项提议旨在弥合这些关键的知识差距，这将 使迫切需要的靶向治疗能够合理设计。我们最近的工作表明，扭力A是I) 通过细胞核将机械信号从细胞骨架传递到核质所必需的 核骨架和细胞骨架(LINC)复合体的包膜跨越连接物；II)信号的激活 由Rho GTPase CDC42；III)以及适当的轴突突起和生长锥形态介导。 重要的是，在DYT1患者的大脑中观察到与临床严重程度相关的轴索束中断。 肌张力障碍患者和LINC复合体和CDC42共同调节轴突延长和引导。因此，我们 假设依赖于扭蛋白A的机械力化学信号转导缺陷与DYT1有关 肌张力障碍的发病机制。我们将在培养的哺乳动物细胞和非洲爪蛙身上测试这一假设 非洲爪哇利用已建立的和新的生化、生物物理、荧光Rho GTP酶阵列 生物传感器、定量成像和蛋白质组学以及合成生物学方法。在这份提案中， 我们将定义TorsinA和它的共激活因子，内核膜蛋白LAP1，如何调节 在培养的哺乳动物细胞中组装功能性LINC复合体(目标1)。我们将确定扭力A如何 和它的另一种共激活剂，外核膜蛋白LULL1，控制着CDC42在 培养的哺乳动物细胞(目标2)。最后，我们将测试扭蛋白A依赖的机械力化学信号的作用 培养的乳突X.laevis神经元和活胚胎大脑中轴突生长过程中的转导(目标3)。 这些目标的结果将为新出现的角色提供宝贵的机械洞察力 核膜在发育和疾病中作为信号节点。此外，他们还将铺设 为未来开发治疗其他形式的癌症的新的治疗策略奠定基础 肌张力障碍、肌张力障碍加综合征，其中肌张力障碍可与另一神经科疾病同时发生 疾病，如亨廷顿病和帕金森氏症，以及其他神经学和神经精神病学 由LINC复合体蛋白突变引起的疾病包括自闭症、共济失调、双相情感障碍、痴呆、 和精神分裂症。

PROJECT SUMMARY/ABSTRACT DYT1 dystonia is a devastating neurological movement disorder characterized by uncontrolled muscle contractions that result in abnormal, involuntary postures. DYT1 dystonia is caused by a deletion (Δgag; ΔE) in the Tor1A gene encoding the luminal ATPases associated with various cellular activities (AAA+) protein torsinA. How the ΔE mutation causes DYT1 dystonia remains unclear because the basic cellular function performed by torsinA is unknown. This proposal seeks to close these critical gaps in knowledge, which will enable the rational design of urgently needed targeted therapies. Our recent work suggests that torsinA is i) required for the transduction of mechanical signals from the cytoskeleton into the nucleoplasm via the nuclear envelope spanning linker of nucleoskeleton and cytoskeleton (LINC) complex; ii) the activation of signaling mediated by the Rho GTPase Cdc42; iii) as well as proper axon outgrowth and growth cone morphology. Importantly, axonal tract disruptions that correlate with clinical severity are observed in the brains of DYT1 dystonia patients and the both the LINC complex and Cdc42 mediate axon elongation and guidance. Thus, we hypothesize that defective torsinA-dependent mechano-chemical signal transduction contributes to DYT1 dystonia pathogenesis. We will test this hypothesis in cultured mammalian cells and the African clawed frog Xenopus laevis using an array of established and novel biochemical, biophysical, fluorescent Rho GTPase biosensors, quantitative imaging and proteomics, as well as synthetic biological approaches. In this proposal, we will define how torsinA and its co-activator, the inner nuclear membrane protein LAP1, regulate the assembly of functional LINC complexes in cultured mammalian cells (Aim 1). We will determine how torsinA and its other co-activator, the outer nuclear membrane protein LULL1, control the activation of Cdc42 in cultured mammalian cells (Aim 2). Finally, we will test the role of torsinA-dependent mechano-chemical signal transduction during axon outgrowth in cultured X. laevis neurons and in the brains of living embryos (Aim 3). The results of these Aims will provide invaluable mechanistic insights into the emerging role of the nuclear envelope as a signaling node in development and disease. Furthermore, they will lay the foundation for the future development of novel therapeutic strategies for the treatment of other forms of dystonia, dystonia plus syndromes in which dystonia can occur in conjunction with another neurological disorder such as Huntington's and Parkinson's diseases, as well as other neurologic and neuropsychiatric diseases caused by mutations in LINC complex proteins including autism, ataxia, bipolar disorder, dementia, and schizophrenia.