Palmitoylation-dependent regulation of the actin cytoskeleton in dendritic spines

描述(申请人提供)：这个项目将解决是否直接棕榈酰化，脂棕榈酸酯的共价连接，解释了LIM Kinase-1(LIMK1)调节树突棘的形态和稳定性的独特能力。树突棘是一种小的富含肌动蛋白的突起，是大多数兴奋性突触的位置。树突棘肌动蛋白的空间精确调控对神经元发育和神经元可塑性的重要性已得到充分证实。相反，脊柱结构受损是智力残疾(ID)和自闭症谱系障碍(ASDS)等疾病的标志。然而，神经元如何在空间上限制肌动蛋白的调控，以确保只修改适当的脊椎，目前尚不清楚。我们发现，LIMK1是一种与高级大脑功能相关的关键肌动蛋白调节因子，它直接棕榈酰化，并将LIMK1靶向脊椎。棕榈酰化 已知的是将非酶“支架”蛋白靶向脊柱和突触，但棕榈酰化在控制肌动蛋白聚合和调节神经元激酶信号转导中的作用完全未知。我们已经建立了一个shRNA介导的击倒/挽救系统，以一种不能棕榈酰化的形式取代神经元中的内源性LIMK1。我们现在建议使用这个系统进行一系列实验，以确定Palmitoyl-LIMK1是否在功能上是正常的脊柱肌动蛋白聚合所必需的，对于脊柱特异的 形态可塑性，并有助于脊柱的长期稳定。补充实验将确定棕榈酰化是否必要且足以解释LIMK1与其最接近的同源物LIMK2在定位和功能上的差异，并将确定控制LIMK1棕榈酰化的酶。这些实验不仅将阐明脊柱调节的新机制，还可能揭示改善ID和ASDS等疾病的治疗新靶点。

DESCRIPTION (provided by applicant): This project will address whether direct palmitoylation, the covalent attachment of the lipid palmitate, accounts for the unique ability of LIM Kinase-1 (LIMK1) to regulate the morphology and stability of dendritic spines. Dendritic spines are small, actin-rich protrusions that are the sites of most excitatory synapses. The importance of spatially precise regulation of dendritic spine actin for neuronal development and for neuronal plasticity is well established. Conversely, impaired spine structure is a hallmark of conditions such as Intellectual Disability (ID) and Autism Spectrum Disorders (ASDs). However, how neurons spatially restrict actin regulation to ensure that only appropriate spines are modified is unclear. We have found that LIMK1, a key actin regulator linked to higher brain function, is directly palmitoylated and that palmitoylation targets LIMK1 to spines. Palmitoylation is known to target non-enzymatic 'scaffold' proteins to spines and synapses, but roles for palmitoylation in the control of actin polymerization, and in the regulation of neuronal kinase signaling are completely undescribed. We have established an shRNA-mediated knockdown/rescue system to replace endogenous LIMK1 in neurons with a form that cannot be palmitoylated. We now propose a series of experiments using this system to determine whether palmitoyl-LIMK1 is functionally required for normal spine actin polymerization, for spine- specific morphological plasticity, and for long-term spine stability. Complementary experiments will determine whether palmitoylation is necessary and sufficient to explain differences in localization and function between LIMK1 and its closest homolog LIMK2 and will identify the enzyme that controls LIMK1 palmitoylation. These experiments will not only shed light on new mechanisms of spine regulation but may reveal new targets for therapy to ameliorate conditions such as ID and ASDs.