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/伏隔核神经元的结构变化。组成这种共同奖赏通路的神经元中出现的树突分支、棘突密度和棘突形态的变化可能是成瘾的一些行为变化的基础。虽然DARPP-32(多巴胺和cAMP调节的磷酸蛋白，32 kDa)和PP-1(蛋白磷酸酶-1)在整合冲击中等刺突神经元的信号方面发挥着关键作用，但结构变化是如何调控的尚不清楚。Rho家族、小GTP结合蛋白以及控制其激活的GDP/GTP交换因子和GTP酶激活蛋白家族是细胞骨架动力学的关键调节因子。我们的初步数据显示，成年雄性大鼠在慢性可卡因治疗后，Kalirin的表达显著增加，Kalirin是一种Rho家族GDP/GTP交换因子。在器官型切片培养中外源表达Kalirin会增加脊柱密度，而反义介导的Kalirin水平降低会导致脊柱密度下降。我们将首先完成对长期接触可卡因对卡利林影响的探索。通过对对照组和可卡因处理组大鼠提取液的免疫学和生化分析，将鉴定Kalirin-7相互作用因子。在对照和可卡因处理的条件下，Kalirin-7被磷酸化的主要部位将通过蛋白水解物的串联质谱分析来确定。随着Kalirin-7表达的变化，在中等刺突神经元中观察到的棘样结构的变化将通过器官型切片培养和原代神经元培养的生物转染来评估。脊柱动力学将使用时间推移成像和GFP标记的Kalirin进行检查。我们的初步数据表明，几个描述良好的可卡因反应信号蛋白与Kalirin相互作用。CDK5与DARPP-32和Pak一起磷酸化Kalirin-7。Kalirin激活并与肌动蛋白聚合的关键调节因子Pak形成复合体。蛋白激酶A使Pak磷酸化并使其失活，也使Kalirin磷酸化。PP-1是一种定位于脊柱的DARPP-32靶标，也与Kalirin结合。我们的假设是，Kalirin-7通过与这些调控因子的相互作用，整合了不同信号通路对脊柱形成的影响。

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.