Palmitoylation-dependent regulation of the actin cytoskeleton in dendritic spines

树突棘中肌动蛋白细胞骨架的棕榈酰化依赖性调节

• 批准号: 8682536
• 负责人: Gareth Thomas
• 金额: $ 23.36万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8682536
• 关键词: 4-methoxy-7-nitroindolinyl-glutamate; Accounting; Actins; Acute; Acyltransferase; Address; Biological Assay; Brain; Cell Fractionation; Cell physiology; Cells; Cytoskeleton; Dendritic Spines; Development; Disease; Ensure; Enzymes; Excitatory Synapse; Fluorescence Recovery After Photobleaching; Gene Mutation; Glutamates; Grant; Health; Hippocampus (Brain); Homologous Gene; Human; Image; Impaired cognition; Impairment; Individual; Intellectual functioning disability; LIM Domain Kinase 1; Life; Light; Link; Lipids; Location; Measures; Mediating; Morphology; Nature; Nerve Degeneration; Neuronal Plasticity; Neurons; Palmitates; Palmitic Acylation Site; Phenocopy; Phenotype; Phosphotransferases; Play; Proteins; Regulation; Research; Role; Scaffolding Protein; Series; Shapes; Signal Transduction; Site; Slice; Structure; Substrate Specificity; Synapses; System; Testing; Time; Vertebral column; Viral; Work; autism spectrum disorder; cell growth regulation; cognitive function; insight; knock-down; link protein; mutant; neuron development; new therapeutic target; novel; palmitoylation; polymerization; public health relevance; research study; small hairpin RNA; transmission process; two-photon

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.

描述（由申请人提供）：本项目将研究直接棕榈酰化（脂质棕榈酸酯的共价连接）是否是LIM激酶-1（LIMK 1）调节树突棘形态和稳定性的独特能力的原因。树枝状棘是小的、富含肌动蛋白的突起，是大多数兴奋性突触的部位。树突棘肌动蛋白的空间精确调节对神经元发育和神经元可塑性的重要性是公认的。相反，受损的脊柱结构是智力残疾（ID）和自闭症谱系障碍（ASD）等疾病的标志。然而，神经元如何在空间上限制肌动蛋白调节以确保仅修饰适当的棘尚不清楚。我们发现LIMK 1是一种与高级脑功能相关的关键肌动蛋白调节因子，它直接被棕榈酰化，棕榈酰化将LIMK 1靶向于棘。棕榈酰 已知将非酶促“支架”蛋白质靶向于棘和突触，但棕榈酰化在肌动蛋白聚合的控制中以及在神经元激酶信号传导的调节中的作用完全没有描述。我们已经建立了一个shRNA介导的敲除/拯救系统，以取代内源性LIMK 1在神经元中的形式，不能棕榈酰化。我们现在提出了一系列的实验，使用这个系统，以确定棕榈酰-LIMK 1是否是正常的脊椎肌动蛋白聚合所需的功能，为脊椎特异性 形态可塑性和长期脊柱稳定性。补充实验将确定棕榈酰化是否必要且足以解释LIMK 1及其最接近的同源物LIMK 2之间的定位和功能差异，并将鉴定控制LIMK 1棕榈酰化的酶。这些实验不仅将揭示脊柱调节的新机制，而且可能揭示治疗的新靶点，以改善ID和ASD等疾病。