LITHIUM REGULATION OF BRAIN PROTEIN KINASE C SUBSTRATES

描述(改编自申请人的摘要)：本建议书代表 我们所从事的研究的延续和自然延伸 为我们最初的发现提供了额外的支持，该发现确定了一个 脑中蛋白激酶C(PKC)的重要磷酸蛋白底物 (Marcks；肉豆蔻酰化富含丙氨酸C激酶底物)作为分子 慢性锂在海马体中的作用靶点。我们建议 继续我们对慢性锂对马克作用的研究 亚克隆海马区永生化细胞的调控作用 在前一个授权期内，在肌醇限制下进行研究 专门设计的条件，以提供与生理相关的 模特。使用特定的单抗和重组DNA策略 在我们实验室开发的，我们将在这个细胞模型中确定 转录和转录后细胞内事件的序列 介导锂诱导的MARCKS表达下调。 我们实验室的初步研究最近提供了证据 支持PKC介导的MARCKS基因在这些细胞中的失稳。 我们的建议旨在研究锂的这种作用在 大脑通过：(1)同时检测PKC磷蛋白底物 (MRP/GAP-43)在大脑中，这与Marcks的一些特性相同 蛋白质，以及(2)追求我们的初步发现丙戊酸盐(哪种 似乎具有与锂相似的临床特性可能有一个共同之处 MARCKS基因表达调控的作用机制。此外 我们将进一步确认我们的发现在生理上的相关性 原代海马细胞以及我们已经有的大鼠模型 Marcks在选择性区域高表达的初步数据 用原位杂交法检测正常海马区。与我们的 参与斯坦利基金会神经病理联合会和NIMH 脑库，我们将把我们的研究扩展到死后脑组织 有充分证据和特征的躁郁症患者 (MDI)。我们将能够第一次检查这一关系 锂和Marcks在人脑中的调节之间 确立Marcks在MDI病理生物学中的作用。我们的发现 锂诱导的对这种磷蛋白表达的调节， 源于其在受体偶联PI信号和PKC中的作用 激活，为我们提供了一个窗口，通过它我们可以开始了解 长期服用锂对神经可塑性事件的下游分子影响 涉及细胞骨架重塑和信号转导的调制， 这可能与其对这种疾病的治疗效果有关。 此外，我们预计这些研究最终也将使我们能够 启动合理的药物发现努力，为下一代的发展 一代情绪稳定剂。

DESCRIPTION (Adapted from applicant's abstract): This proposal represents the continuation and natural extension of our studies in which we have provided additional support for our original discovery that identified a prominent phosphoprotein substrate for protein kinase C (PKC) in brain (MARCKS; Myristoylated Alanine Rich C Kinase Substrate) as a molecular target for the action of chronic lithium in hippocampus. We propose to continue our investigations of the action of chronic lithium on MARCKS regulation using immortalized hippocampal cells which have been subcloned during the previous grant period and are studied under inositol-limiting conditions specifically designed to provide a physiologically relevant model. Using specific monoclonal antibodies and recombinant DNA strategies developed in our laboratory we will determine in this cell model the sequence of transcriptional and posttranscriptional intracellular events mediating the lithium-induced down-regulation of MARCKS expression. Preliminary studies in our laboratory have recently provided evidence in support of a PKC-mediated destabilization of MARCKS mRNA in these cells. Our proposal seeks to examine the uniqueness of this action of lithium in the brain by: (1) concomitantly examining PKC phosphoprotein substrates (MRP/GAP-43) in brain which share some of the properties of the MARCKS protein, and (2) pursuing our preliminary findings that valproate (which appears to possess similar clinical properties to lithium may share a common mechanism of action vis a vis regulation of MARCKS expression. In addition we will further confirm the physiological relevance of our findings in both primary hippocampal cells as well as a rat model where we already have preliminary data of a heightened expression of MARCKS in selective regions of normal hippocampus using in situ hybridization. In concert with our participation in the Stanley Foundation Neuropathology Consortium, and NIMH Brain Bank, we will be extending our studies to postmortem brain tissue from patients with well documented and characterized manic-depressive illness (MDI). We will be able to examine for the first time the relationship between lithium and MARCKS regulation in human brain and potentially establish a role for MARCKS in the pathobiology of MDI. Our discovery of the lithium-induced regulation of the expression of this phosphoprotein, stemming from its action in receptor-coupled PI signaling and PKC activation, offers us a window through which we can begin to understand the downstream molecular impact of long-term lithium on neuroplastic events involving cytoskeletal remodeling and modulation of signal transduction, which may be associated with its therapeutic efficacy in this disorder. Moreover, we anticipate these studies will also ultimately enable us to initiate rational drug discovery efforts for the development of the next generation of mood stabilizers.