GDP/GTP Exchange Factors: Nucleus Accumbens Plasticity

描述（由申请人提供）： 长期滥用可卡因等物质会导致腹侧被盖区/背核神经元的结构变化。树突分支、棘密度和棘形态的变化发生在神经元中，这些神经元包括这种共同的奖励途径，这些变化可能是成瘾特征性行为变化的基础。虽然DARPP-32（多巴胺和cAMP调节的磷酸化蛋白，32 kDa）和PP-1（蛋白磷酸酶-1）在整合撞击中型棘神经元的信号中起关键作用，但尚不清楚结构变化是如何调节的。Rho家族小GTP结合蛋白和GDP/GTP交换因子家族以及控制其活化的GTP酶活化蛋白是细胞骨架动力学的关键调节因子。我们的初步数据显示，慢性可卡因治疗成年雄性大鼠后，Kalirin，Rho家族GDP/GTP交换因子的表达大幅增加。器官型切片培养物中Kalirin的外源表达增加了棘密度，而Kalirin水平的反义介导的降低导致棘密度的降低。我们将首先完成对慢性可卡因暴露对卡林的影响的探索。将通过对对照大鼠和可卡因处理大鼠制备的提取物进行免疫学和生物化学分析，鉴定Kalirin-7相互作用物。将通过蛋白水解酶的串联质谱分析鉴定Kalirin-7在对照和可卡因处理条件下磷酸化的主要位点。将使用器官型切片培养物和原代神经元培养物的生物射弹转染来评价Kalirin-7表达变化后在中等多刺神经元中观察到的棘样结构的改变。将使用时间推移成像和GFP标记的Kalirin检查脊柱动力学。我们的初步数据表明，几个良好描述的可卡因反应信号蛋白与Kalirin相互作用。Cdk 5使Kalirin-7与DARPP-32和Pak一起沿着磷酸化。Kalirin激活肌动蛋白聚合的关键调节因子Pak并与其形成复合物。磷酸化和灭活Pak的蛋白激酶A也磷酸化Kalirin。PP-1是一种定位于棘的DARPP-32靶标，也与Kalirin结合。我们的假设是Kalirin-7通过与这些调节剂的相互作用，整合了不同信号通路对棘形成的影响。

DESCRIPTION (provided by applicant): Chronic abuse of substances such as cocaine leads to structural changes in the neurons of the ventral tegmental area/nucleus accumbens. The changes in dendritic branching, spine density and spine morphology that occur in the neurons comprising this common reward pathway presumably underlie some of the behavioral changes that characterize addiction. Although it is clear that DARPP-32 (Dopamine and cAMP-Regulated PhosphoProtein, 32 kDa) and PP-1 (Protein phosphatase-1) play key roles in integrating the signals impinging on medium spiny neurons, it is not yet clear how structural changes are regulated. Rho family small GTP binding proteins and the families of GDP/GTP exchange factors and GTPase activating proteins that control their activation are key regulators of cytoskeletal dynamics. Our preliminary data reveal a substantial increase in expression of Kalirin, a Rho family GDP/GTP exchange factor, following chronic cocaine treatment of adult male rats. Exogenous expression of Kalirin in organotypic slice cultures increases spine density, while antisense-mediated reductions in Kalirin levels result in a decrease in spine density. We will first complete our exploration of the effects of chronic cocaine exposure on Kalirin. Kalirin-7 interactors will be identified through immunological and biochemical analysis of extracts prepared from control and cocaine-treated rats. The major sites at which Kalirin-7 is phosphorylated under control and cocaine-treated conditions will be identified by tandem mass spectroscopic analysis of proteolytic digests. The alterations in spine-like structures observed in medium spiny neurons following changes in Kalirin-7 expression will be evaluated using biolistic transfection of organotypic slice cultures and primary neuronal cultures. Spine dynamics will be examined using time lapse imaging and GFP-tagged Kalirin. Our preliminary data demonstrate that several well-described cocaine-responsive signaling proteins interact with Kalirin. Cdk5 phosphorylates Kalirin-7 along with DARPP-32 and Pak. Kalirin activates and forms a complex with Pak, a key regulator of actin polymerization. Protein kinase A, which phosphorylates and inactivates Pak, also phosphorylates Kalirin. PP-1, a DARPP-32 target localized to spines, also binds to Kalirin. It is our hypothesis that Kalirin-7, through its interactions with these regulators, integrates the effects of diverse signaling pathways on spine formation.