REGULATION OF AXON GUIDANCE BY ENA/VASP PROTEINS

描述（由申请人提供）：本申请的总体目标是阐明蛋白激酶A（PKA）引起的Ena/VASP磷酸化在发育中的中枢神经系统中轴突生长和引导中的功能。首先，将产生Ena/VASP缺失小鼠。如果由于早期产前致死性而不可能产生三重敲除小鼠，则将通过Cre/lox重组系统采用条件性敲除策略。第二，将Ena/VASP蛋白的磷酸化突变体引入Ena/VASP缺失的神经元中，并评估它们对生长锥运动性和轴突生长的影响。为了测试Ena/VASP磷酸化对轴突引导的功能，将含有磷酸化突变体的皮质神经元暴露于netrin和BDNF的梯度;已知通过PKA发出信号的两种分子。Ena/VASP蛋白还涉及结合A激酶锚定蛋白（AKAP）。为了确定哪些AKAP与皮质神经元中的Ena/VASP蛋白相关，将用PKA的II型调节亚基进行免疫共沉淀和凝胶覆盖。将克隆发现结合Ena/VASP蛋白的AKAP并用YFP标记。将用CFP-Ena/VASP蛋白进行荧光共振能量转移（FRET），以确定Ena/VASP蛋白和AKAP在生长锥中何时何地相互作用。阐明Ena/VASP蛋白在CNS发育中的功能对于理解其中定向神经元迁移和生长受损的人类CNS疾病将是重要的。

DESCRIPTION (provided by applicant): The overall goal of this application is to elucidate the function of Ena/VASP phosphorylation by protein kinase A (PKA) in axon outgrowth and guidance in the developing central nervous system. First, Ena/VASP-null mice will be generated. If it is not possible to create triple knock-out mice due to early prenatal lethality, a conditional knock-out strategy will be employed by means of the Cre/lox system of recombination. Second, phosphorylation mutants of Ena/VASP proteins will be introduced into Ena/VASP-null neurons and their effects on growth cone motility and axon outgrowth will be assessed. To test the function of Ena/VASP phosphorylation on axon guidance cortical neurons containing phosphorylation mutants will be exposed to gradients of netrin and BDNF; two molecules known to signal through PKA. Ena/VASP proteins have also been implicated in binding A kinase anchoring proteins (AKAPs). In order to determine which AKAPs associate with Ena/VASP proteins in cortical neurons co-immunoprecipitations and gel overlays will be performed with the type II regulatory subunit of PKA. AKAPs discovered to bind Ena/VASP proteins will be cloned and labeled with YFP. Fluorescent resonance energy transfer (FRET) will be performed with CFP-Ena/VASP proteins to determine where and when Ena/VASP proteins and AKAPs interact in growth cones. An elucidation of the function of Ena/VASP proteins in CNS development will be important for understanding human CNS diseases where directed neuronal migration and outgrowth are impaired.