Regulation of dendritic spine shape and synapse and dendrite stability by Arg

描述(由申请人提供)：神经回路在神经发育障碍中不能正常发育，在神经退行性疾病中过早退化。我们已经证明，树突棘的形态发生以及后来的突触和树突的稳定性需要Abl相关基因(Arg)非受体酪氨酸激酶，它作用于整合素黏附受体的下游，介导细胞骨架结构的变化。我们试图了解整合素是如何通过Arg及其效应器来控制神经回路的形成和维持的。我们的第一个目标是阐明Arg在树突棘形态、突触稳定性和树突维持中的作用。在出生后第21天，Arg-/-小鼠大脑皮质和海马区的树突和突触发育正常，但树突棘不能正常成熟，导致突触和树突丢失，出生后42天行为障碍。我们将利用电生理学和分析Arg-/-小鼠突触和树突的三维重建来研究Arg信号通路的丧失如何影响突触和树突的形成、稳定性和功能。我们还将使用条件失活的Arg等位基因和可诱导的Arg转基因来确定Arg信号何时是正常突触发育所必需的，并防止突触丢失和树突变性。我们的第二个目标是了解整合素如何激活Arg来调节突触和树突的稳定性。我们的工作表明，整合素通过Arg介导细胞骨架结构的变化，但我们不知道Arg是如何在体内招募到整合素异源二聚体来实现激酶激活的。我们将验证这一假说，并得到初步数据的支持，即整合素21或23细胞质尾巴直接与Arg结合以介导激酶激活。含有21或23个亚基的整合素在体内调节突触的形成和树突的稳定性，但目前尚不清楚哪些特定的整合素异源二聚体通过Arg调节突触和树突的维持。我们将监测Arg信号通路，并分析整合素21和23突变小鼠的突触和树突结构，以确定哪些整合素通过Arg调控树突棘的形态发生和突触/树突的稳定性。我们的第三个目标是确定Arg如何通过其效应器发出信号来调节突触和树突的稳定性。Arg是体内正常树突棘形态发生所必需的，但我们不知道Arg是如何协调这些过程所需的细胞骨架变化的。我们的生化研究已经确定了几种底物(p190RhoGAP、Cortactin、Myosin IIB)，Arg通过这些底物促进细胞骨架结构的变化。我们将研究通过Arg的整合素信号如何影响这些Arg底物在培养的皮质神经元中的分布。我们还将测试在已建立的海马神经元培养中，底物的RNAi敲除是否影响树突棘结构以及突触和树突的稳定性。公共卫生相关性：在神经发育障碍(如智力低下)中，神经回路不能正常发育，在神经退行性疾病(如阿尔茨海默病)中，神经回路过早退化。突触功能缺陷和/或突触数量减少会导致树突节段的丢失和神经回路的退化。我们将研究一种生物化学途径，调节突触的形态发生和功能，并防止大脑中神经回路的退化。

DESCRIPTION (provided by applicant): Neural circuits do not develop properly in neurodevelopmental disorders and degrade prematurely in neurodegenerative disorders. We have shown that dendritic spine morphogenesis and later synapse and dendrite stability requires the Abl-related gene (Arg) nonreceptor tyrosine kinase, which acts downstream of integrin adhesion receptors to mediate changes in cytoskeletal structure. We seek to understand how integrins signal through Arg and its effectors to control the formation and maintenance of neural circuitry. Our first aim is to elucidate the roles for Arg in dendritic spine morphology, synapse stability, and dendrite maintenance. Dendrites and synapses develop normally through postnatal day 21 in the arg-/- mouse cortex and hippocampus, but dendritic spines do not mature properly, leading to synapse and dendrite loss and behavioral deficits by postnatal day 42. We will use electrophysiology and analyze three-dimensional reconstructions of synapses and dendrites in arg-/- mice to examine how the loss of Arg-signaling pathways compromises the formation, stability, and function of synapses and dendrites. We will also use a conditionally inactivatable arg allele and an inducible arg transgene to determine when Arg signaling is required for proper synapse development and to protect against synapse loss and dendritic degeneration. Our second aim is to understand how integrins activate Arg to regulate synapse and dendrite stability. Our work has shown that integrins act through Arg to mediate changes in cytoskeletal structure, but we do not understand how Arg is recruited to integrin heterodimers to achieve kinase activation in vivo. We will test the hypothesis, supported by preliminary data, that integrin 21 or 23 cytoplasmic tails bind directly to Arg to mediate kinase activation. Integrins containing 21 or 23 subunits regulate synapse formation and dendrite stability in vivo, but it is unclear which specific integrin heterodimers act through Arg to regulate synapse and dendrite maintenance. We will monitor Arg signaling pathways and analyze synapse and dendrite structure in integrin 21 and 23 mutant mice to determine which integrins act through Arg to control dendritic spine morphogenesis and synapse/dendrite stability. Our third aim is to determine how Arg signals through its effectors to regulate synapse and dendrite stability. Arg is required for proper dendritic spine morphogenesis in vivo, but we do not understand how Arg coordinates the cytoskeletal changes required for these processes. Our biochemical studies have identified several substrates (p190RhoGAP, cortactin, myosin IIB) through which Arg acts to promote changes in cytoskeletal structure. We will examine how integrin signaling through Arg affects the distribution of these Arg substrates in cultured cortical neurons. We will also test whether RNAi knockdown of the substrates affects dendritic spine structure and synapse and dendrite stability in established hippocampal neuronal cultures. PUBLIC HEALTH RELEVANCE: Neural circuits do not develop properly in neurodevelopmental disorders, such as mental retardation, and degrade prematurely in neurodegenerative disorders, such as Alzheimer's Disease. Defects in synapse function and/or reductions in synapse number lead to the loss of dendrite segments and degeneration of neural circuits. We will study a biochemical pathway that regulates synapse morphogenesis and function and protects against degeneration of neural circuits in the brain.