D3 PREMRNA PROCESSING IN CHRONIC PSYCHOSIS

描述（改编自申请人的摘要）：多巴胺的D2类 受体是具有已证实的抗精神病疗效的药物的靶点， 被反复建议作为因素的病理生理学 精神分裂症 尽管研究未能证明基因上的联系， 长期住院病人死后脑组织的研究 慢性精神病患者的基因转录后水平异常， 多巴胺D3受体编码的前mRNA的加工。 这导致 D3 mRNA表达减少， 选择性剪接的截短型D3样mRNA，命名为D3 nf。 为了 对D3和D3 nf mRNA的表达进行更详细的研究 和蛋白质，为了开始检查 增强的D3 nf特异性剪接的潜在功能后果， D3前体mRNA在慢性精神病中的表达，提出：（1）进行RNase 保护试验，以确定D3和D3 nf mRNA的相对丰度 从患有以下疾病的患者死后获得的大脑的定义解剖区域 慢性精神分裂症，双相情感障碍，并从控制。 （二） 为了用D3特异性抗体进行免疫沉淀和免疫印迹实验， 单克隆抗体和D3 nf特异性多克隆抗血清，以确定 是否（在相同的解剖区域）D3和D3 nf mRNA的改变 表达也会导致相应的表达变化， 各自的蛋白质。 (3)此外，由于D3 nf在细胞中的共表达， 稳定表达D3的大鼠GH 3细胞增加D3的电泳迁移率， D3-免疫反应性（从50 kD到180 kD），并且因为这种共表达 似乎使很大一部分D3受体无法进入， 配体，稳定和诱导D3，D3 nf和D3/D3 nf的实验 表达GH 3细胞，目的是确定蛋白质 D3 nf诱导的180 kd D3免疫反应性的组成，其 翻译后修饰，其形成的动力学，及其 稳定 此外，建议进行放射性配体结合研究， 测定D3膜的（3 H）喹吡罗结合的Bmax， D3/D3 nf表达细胞。 最后，将在转染的 极化上皮细胞（MDCK）是否膜靶向的D3 蛋白质在D3 nf的存在下发生改变。 这些研究的结果 预计将提供深入了解D3表达的程度 和D3 nf mRNA和蛋白质在精神分裂症中发生改变开始 为了阐明D3受体表达的D3 nf表达的影响， 功能

DESCRIPTION (Adapted from applicant's abstract): The D2-class of dopamine receptors are targets for drugs with proven antipsychotic efficacy and have been repeatedly suggested as factors in the pathophysiology of schizophrenia. Although studies have failed to demonstrate a genetic link, studies on brain tissues obtained postmortem from long-term hospitalized patients with chronic psychosis suggest an abnormal posttranscriptional processing of dopamine D3-receptor-encoded pre-mRNA. This results in a decreased expression of D3 mRNA and an increased accumulation of an alternatively spliced, truncated D3-like mRNA, named D3nf. In order to pursue a more detailed investigation of the expression of D3 and D3nf mRNA and protein in postmortem tissues, and in order to begin to examine potential functional consequences of the enhanced D3nf-specific splicing of D3 pre-mRNA in chronic psychosis, it is proposed: (1) To perform RNase protection assays to determine the relative abundance of D3 and D3nf mRNA in defined anatomic regions of brains obtained postmortem from patients with chronic schizophrenia, bipolar affective disorders, and from controls. (2) To perform immunoprecipitation and immunoblot experiments with D3-specific monoclonal antibodies and a D3nf-specific polyclonal antiserum to determine whether (in the same anatomic regions) alterations in D3 and D3nf mRNA expression also result in corresponding changes in the expression of the respective proteins. (3) Furthermore, because co-expression of D3nf in stably D3-expressing rat GH3 cells increases the electrophoretic mobility of D3-immunoreactivity (from 50 kD to 180 kD), and because this co-expression appears to render a significant proportion of D3 receptors inaccessible to ligands, experiments on stably and inducibly D3, D3nf, and D3/D3nf expressing GH3 cells are proposed that aim at determining the protein composition of the D3nf-induced 180 kd D3-immunoreactivity, its posttranslational modifications, the kinetics of its formation, and its stability. In addition, radioligand binding studies are proposed to determine the Bmax of (3H)quinpirole binding of membranes of D3 and D3/D3nf-expressing cells. Finally, it will be tested in transfected polarized epithelial cells (MDCK) whether the membrane targeting of the D3 protein is altered in the presence of D3nf. Results of these studies are expected to provide insight into the extent to which the expression of D3 and D3nf mRNA and protein is altered in schizophrenia, and they will begin to elucidate the influence of D3nf expression of D3 receptor expression and function.