Signaling Mechanisms Underlying Neuroleptic Drug Actions

描述（申请人提供）：项目1将探讨典型和非典型的神经安定药物如何影响纹状体和前额皮质的蛋白磷酸化途径。预计典型和非典型神经安定药具有一些但不是全部的细胞内信号传导靶点。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差异作用。目的建立抗精神病药物对皮层和纹状体蛋白磷酸化通路的全面研究。先前的研究表明DARPP-32与神经抑制剂的作用有关。直到现在，还不能区分DARPP-32在纹状体的特定神经元细胞类型中的生化调节。这些神经元在形态上难以区分，在解剖学上相互混合。目的II的目标是获得更详细的了解特定的细胞群神经阻滞剂的作用。新的BAC转基因小鼠技术将用于在纹状体黑质、纹状体苍白球或皮质神经元中过表达表位标记的DARPP-32、RCS和mGluR5。从这些动物中进行拉下实验，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-32 KO小鼠显示出对精神抑制剂和拟精神病药的反应改变。目的III的目标是定义DARPP-32介导神经阻滞剂作用的神经元回路。Cre/loxP技术将用于产生在纹状体黑质、纹状体苍白球或皮质神经元中缺乏DARPP-32的小鼠。将在生物化学，行为和电生理测定中研究小鼠系对神经抑制剂的反应性，最后，在目标IV中，将进行与代谢型谷氨酸，5-羟色胺和毒蕈碱受体相互作用的新型蛋白质的细胞生物学研究。因此，拟议中的研究应该提供一个详细的知识，神经阻滞剂如何影响蛋白质磷酸化途径在定义的神经元电路。

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.