LITHIUM REGULATION OF BRAIN PROTEIN KINASE C SUBSTRATES

我们最近报道，慢性锂可以深刻地改变 大鼠的细胞内浓度。这是一次 肉豆蔻酰化丙氨酸富含C激酶底物(MARCKS)蛋白出现 锂在临床中的作用时程平行 双相情感障碍的治疗。从我们的实验室收集证据 还有一些人认为PKC与锂在大脑中的长期作用有关 以及对它的一些主要磷蛋白底物的调节， 包括马克在内。为了将这项研究推进到下一阶段 检验我们关于锂的作用机制的假设 调节这种PKC底物，我们必须启动一种 生理相关的细胞系统，服从于复杂 实验方法和没有活体模型的限制。 在这个小的拨款提案中，我们将建立一个新的细胞模型系统 在培养中使用永生化的海马细胞，并利用 当前的细胞异质性启动亚克隆策略以 分离和稳定表型特性作为一种宝贵的资源 我们未来的研究。例如，虽然我们已经确认了Marcks 在这些细胞的早期传代中表达，我们预计 该基因表达水平不同的亚克隆的建立 磷蛋白。因为锂在大脑中的作用似乎取决于 在限制获得肌醇的情况下，我们将制定类似的条件 在我们的细胞系统中接触到锂。我们将在体内复制我们的 在这些海马细胞中的发现，并检查转录， 锂离子改变的翻译和翻译后机制 MARCKS在海马区的调控和表达。这些 研究将为攻读更多专业提供必要的基础 在未来提供资金，以审查行动的可能性 Marcks上的锂被其他已知的主要PKC底物共享 敬布莱恩。此外，我们将处于测试的绝佳位置 我们的假设是Marcks和/或其他 磷蛋白可能在慢性锂的作用中起关键作用 M受体与磷脂酶C偶联的调节作用 大脑的关键区域。

We have recently reported that chronic lithium profoundly alters the intracellular concentration of the rat. This reduction in a Myristoylated Alanine Rich C Kinase Substrate (MARCKS) protein appears to parallel the time course of action of lithium in the clinical treatment of bipolar disorder. Accumulating evidence from our laboratory and others implicates PKC in the long term action of lithium in the brain as well as the regulation of some of its major phosphoprotein substrates, including MARCKS. In order to move this research to the next phase of examining our hypotheses regarding the mechanism of action of lithium in regulating this PKC substrate, we must initiate the development of a physiologically relevant cell system, amenable to sophisticated experimental approaches and devoid of the limitations of in vivo models. In this Small Grant proposal we will establish a new cell model system using immortalized hippocampal cells in culture, and take advantage of current cellular heterogeneity to initiate a subcloning strategy to isolate and stabilize phenotypic properties as a valuable resource for our future studies. For example, while we have identified MARCKS expression in an early passage of these cells, we anticipate the development of subclones with differential levels of expression of this phosphoprotein. Inasmuch as lithium's action in brain appears to depend upon limited access to inositol, we will develop analogous conditions for lithium exposure in our cell system. We will replicate our in vivo findings in these hippocampal cells and examine transcriptional, translational and posttranslational mechanisms by which lithium alters the regulation and expression of MARCKS in the hippocampus. These studies will provide the necessary foundation to pursue more major funding in the future to examine the possibility that the action of lithium on MARCKS is shared by other known major PKC substrates unique to brain. In addition, we will then be in an excellent position to test our hypothesis that alterations in MARCKS and/or the other phosphoproteins may play a critical role in the action of chronic lithium on the regulation of muscarinic receptors coupled to phospholipase C in key regions of the brain.