Roles for Abl and Arg in Neuronal Development

描述(由申请人提供)：Abl和Arg非受体酪氨酸激酶调节发育中小鼠大脑中的神经元迁移和形态发生。ABL和Arg传递来自细胞表面受体的信息，促进细胞骨架重排。这项建议的目的是了解Abl和Arg调节发育中神经元细胞骨架结构和功能的分子机制。 我们的第一个目标是阐明Abl和Arg在神经元发育中的作用。我们对染色神经元的形态计量学分析表明，与野生型相比，Arg-/-海马神经元的树枝状枝减少。在大脑特异的ABL-/-Arg-/-双基因敲除小鼠的大脑皮层中，神经元排列得更紧密。这种表型可能反映了树突分枝的严重缺陷。我们还发现，在大脑特异的ABL-/-Arg-/-小鼠中，小脑颗粒神经元的子集无法正常迁移。我们将研究基因敲除小鼠和这些小鼠移植的神经元的神经元分化，以检查编码Abl和Arg信号通路组件的基因突变如何影响树突状细胞的形态发生和小脑颗粒神经元的迁移。 我们的第二个目标是了解Arg如何调节发育中的神经元的细胞骨架结构。Arg过表达导致成纤维细胞膜皱缩增加，细胞活力降低，培养的皮质神经元突起分支增多。我们发现Arg可以通过两条不同的途径调节细胞骨架结构：1)Arg C端的一半可以结合F-肌动蛋白并将F-肌动蛋白束交联到微管上；2)Arg可以磷酸化并激活p190 Rho GTP酶激活蛋白(P190RhoGAP)来抑制细胞骨架调节因子Rho。我们将在培养的成纤维细胞中表达Arg和/或p190RhoGAP突变体，以剖析这两条途径是如何对细胞骨架结构和功能起调节作用的。 我们的第三个目标是确定Abl和Arg激酶活性在发育中的神经元中是如何调节的。我们已经证明，在体外，Abl和Arg在几个位点上的磷酸化可以调节它们的激酶活性，但尚不清楚Abl和Arg如何在发育中的神经元中被磷酸化。这一领域的进展一直是有限的，因为调节发育中神经元中Abl和Arg激酶活性的细胞表面受体尚未确定。遗传和生化实验已经确定了一小部分候选受体(Robo受体，TrkB，整合素？5)，它们可能调节发育中神经元中Abl和Arg激酶的活性。我们将确定这些受体是否调节Abl和Arg激酶的活性，并检查Abl和Arg是否介导这些受体的迁移或形态发生信号。

DESCRIPTION (provided by applicant): The Abl and Arg nonreceptor tyrosine kinases regulate neuronal migration and morphogenesis in the developing mouse brain. Abl and Arg relay information from cell surface receptors to promote cytoskeletal rearrangements. The goal of this proposal is to understand the molecular mechanisms by which Abl and Arg regulate cytoskeletal structure and function in developing neurons. Our first aim is to elucidatetheroles for Abl and Arg in neuronal development. Our morphometric analysis of dye-filled neurons has shown that dendrite arbors are reduced in arg-/- hippocampal neurons relative to wild type. Neurons are more tightly packed in the cerebral cortices of brain-specific-abl-/-arg-/- double knockout mice. This phenotype may reflect a severe deficit in dendrite arborization. We also find that a subset of cerebellar granular neurons fail to migrate normally in the brain-specific-abl-/-arg-/- mice. We will study neuronal differentiation in knockout mice and in explanted neurons from these mice to examine how mutations in genes encoding components of Abl- and Arg-signaling pathways affect dendrite morphogenesis and cerebellar granular neuron migration. Our second aim is to understand how Arg regulates cytoskeletal structure in developing neurons. Arg overexpression leads to increased membrane ruffling and decreased cell motility in fibroblasts and increased neurite branching in cultured cortical neurons. We have identified two distinct pathways by which Arg can regulate cytoskeletal structure: 1) the Arg C-terminal half can bundle F-actin and crosslink F-actin bundles to microtubules; and 2) Arg can phosphorylate and activate the p190 Rho GTPase-activating protein (p190RhoGAP) to inhibit the cytoskeletal regulator Rho. We will express Arg and/or p190RhoGAP mutants in cultured fibroblasts to dissect how these two pathways contribute to the regulation of cytoskeletal structure and function. Our third aim is to determine how Abl and Arg kinase activities are regulated in developing neurons. We have shown that in vitro phosphorylation of Abl and Arg at several sites can regulate their kinase activities, but it is unclear how Abl and Arg become phosphorylated in developing neurons. Progress in this area has been limited because the cell surface receptors that regulate Abl and Arg kinase activity in developing neurons have not been identified. Genetic and biochemical experiments have identified a small number of candidate receptors (the Robo receptors, TrkB, integrin ?5) that are likely to regulate Abl and Arg kinase activity in developing neurons. We will determine whether these receptors regulate Abl and Arg kinase activity and examine whether Abl and Arg mediate migratory or morphogenetic signals from these receptors.