Signaling Mechanisms Underlying Neuroleptic Drug Actions

描述(申请人提供)：项目1将探索典型和非典型抗精神病药物如何影响纹状体和前额叶皮质的蛋白质磷酸化途径。预计典型的和非典型的抗精神病药物共享一些但不是全部的细胞内信号靶点。这些相似之处可能反映了这些化合物对抗阳性症状的共同能力，而差异可能反映了它们对阴性症状和锥体外系副作用的不同作用。目的建立皮层和纹状体中受抗精神病药物调节的蛋白质磷酸化途径的综合研究。先前的研究表明，DARPP-32与抗精神病药的作用有关。到目前为止，还不可能区分DARPP-32在特定类型的纹状体神经细胞中的生化调节。这些神经元在形态上难以区分，在解剖学上是混合的。AIM II的目标是对抗精神病药物起作用的特定细胞群有更详细的了解。新的BAC转基因小鼠技术将被用于在纹状体黑质、纹状体苍白质或皮质神经元中过表达表位标记的DARPP-32、RCS和mGluR5。这些动物的下拉实验，然后进行免疫印迹或质谱分析，将允许对磷酸化事件进行细胞特异性分析。DARPP-32KO小鼠对神经阻滞剂和精神分裂药的反应发生了变化。AIM III的目标是定义DARPP-32在其中介导神经阻滞剂作用的神经元回路。Cre/loxP技术将用于产生纹状体黑质、纹状体苍白质或皮质神经元中缺乏DARPP-32的小鼠。将在生化、行为和电生理测试中研究小鼠品系对神经阻滞剂的反应性，最后，在目标IV中，将进行与代谢性谷氨酸、5-羟色胺和毒扁豆碱受体相互作用的新蛋白的细胞生物学研究。因此，这项拟议的研究应该提供关于抗精神病药物如何影响已定义的神经元回路中的蛋白质磷酸化途径的详细知识。

DESCRIPTION (provided by applicant): Project 1 will explore how typical and atypical neuroleptic drugs affect protein phosphorylation pathways in striatum and prefrontal cortex. It is anticipated that typical and atypical neuroleptics share some, but not all, intracellular signaling targets. The similarities could reflect the common ability of these compounds to counteract positive symptoms, whereas the differences could reflect their differential actions on negative symptoms and extrapyramidal side effects. Aim I will establish a comprehensive survey of protein phosphorylation pathways regulated by neuroleptics in cortex and striatum. Previous work has shown that DARPP-32 is implicated in the actions of neuroleptics. Up until the present time, it has not been possible to distinguish between the biochemical regulation of DARPP-32 in specific neuronal cell types of striatum. These neurons are morphologically indistinguishable and are intermixed anatomically. The goal of Aim II is to obtain a more detailed understanding of the specific cell population where neuroleptics act. Novel BAC transgenic mouse technology will be used to overexpress epitope-tagged DARPP-32, RCS and mGluR5 in striatonigral, striatopallidal or cortical neurons. Pull down experiments followed by immunoblotting or mass spectrometry from these animals will allow cell-specific analyses of phosphorylation events. DARPP-32 KO mice show altered responses to neuroleptics and psychotomimetics. The goal of Aim III is to define neuronal circuitries in which DARPP-32 mediates actions of neuroleptics. The Cre/loxP technology will be used to generate mice lacking DARPP-32 in striatonigral, striatopallidal or cortical neurons. The mouse lines will be studied in terms of their responsivity to neuroleptics in biochemical, behavioural and electrophysiological assays, finally, in Aim IV, studies on the cellular biology of novel proteins interacting with metabotropic glutamate, serotonin and muscarinic receptors will be conducted. Thus, the proposed studies should provide a detailed knowledge on how neuroleptics affect protein phosphorylation pathways in defined neuronal circuitries.