The Role of CaMKII in Cell Death and Survival Responses of Retinal Ganglion Cells

描述（由申请人提供）：视网膜疾病沉淀原因下游发生的信号转导和基因调控事件在很大程度上是未知的。然而，这些下游部位将是介入治疗的绝佳靶点。拟议研究的长期目标是确定钙调素依赖性蛋白激酶II （CaMKII）的作用，CaMKII可能是视网膜缺血等眼病启动事件的下游。CaMKII位于视网膜神经节细胞（RGCs）细胞死亡和存活途径的上游。我们知道，当使用谷氨酸受体激动剂时：1)视网膜细胞质CaMKII1发生自磷酸化；2)选择性剪接核转录物CaMKII1B增加；3) caspase-3被激活，4)细胞核和神经节细胞层的细胞死亡。我们已经证明，CaMKII的特异性抑制剂，自肽抑制肽（AIP），阻断了这些变化（1,3和4），并保护所有细胞免于细胞死亡。在细胞系辅助下的初步数据表明，AIP促进脑源性神经营养因子（BDNF）的分泌，这可能有助于挑战细胞的存活。我们也知道CaMKII1B(2)水平升高与BDNF转录增强有关。由于谷氨酸与视网膜缺血有关，CaMKII1在这种情况下也被激活，我们假设CaMKII1在视网膜缺血期间参与RGCs死亡和生存反应的通路中发挥重要作用。这将通过包括RNA干扰CaMKII1B转录本在内的方法进行测试。我们将使用大鼠视网膜缺血再灌注模型，以及原代培养的大鼠RGCs和离体视网膜外植体。目的1将确定AIP在缺血期间/之后保护rgc的时间范围。目的2将探讨细胞质CaMKII在调节RGC/视网膜中caspase-3激活、NF:B易位和BDNF释放中的作用。目的3将探讨核CaMKII1在细胞死亡/存活中的作用及其潜在机制。这些研究结果将为控制RGCs死亡和生存反应的信号转导途径和基因调控网络提供重要的见解，这将与视网膜缺血相关。公共卫生相关性：视网膜缺血是一种常见的临床实体，由于其在视网膜和脉络膜血管闭塞、前缺血性视神经病变、青光眼和糖尿病性视网膜病变等疾病中的作用而被广泛研究，但由于治疗相对无效，在发达国家仍是一种常见的致盲原因。我们建议探索与控制大鼠视网膜神经节细胞死亡和存活过程的基因调控网络相关的信号转导途径，视网膜神经节细胞的死亡是这些疾病中视力损害的主要因素。了解这些途径和网络对于开发实用的治疗干预措施非常重要。

DESCRIPTION (provided by applicant): The signal transduction and gene regulatory events which occur downstream of the precipitating causes of retinal diseases is largely unknown. Yet these downstream sites would be excellent targets for interventional therapies. The long term goal of the proposed research is to define the role of calmodulin dependent protein kinase II (CaMKII) which is likely downstream of the initiating events in eye diseases like retinal ischemia. CaMKII is upstream of the cell death and survival pathways in retinal ganglion cells (RGCs). We know that when challenged with glutamate receptor agonists: 1) retinal cytoplasmic CaMKII1 becomes autophosphorylated; 2) there is an increase in the alternatively spliced nuclear transcript, CaMKII1B; 3) caspase-3 becomes activated and 4) cells in the inner nuclear and ganglion cell layers die. We have demonstrated that the specific inhibitor of CaMKII, autocamtide inhibitory peptide (AIP), blocks these changes (1, 3 and 4) and protects all cells from cell death. Pilot data with the aid of a cell line indicates that the AIP promotes the secretion of brain derived neurotrophic factor (BDNF) which may aid the survival of challenged cells. We also know that an elevated level of CaMKII1B (2) is associated with enhanced transcription of BDNF. Since glutamate is implicated in retinal ischemia and CaMKII1 is also activated in this condition, we hypothesize that CaMKII1 plays an important role in pathways involved in the death and survival responses of RGCs during retinal ischemia. This will be tested with methods, which include RNA- interference for the CaMKII1B transcript. We will use a rat retinal ischemia-reperfusion model, as well as primary cultured rat RGCs, and ex vivo retinal explants. Aim 1 will determine the time frame in which AIP protects RGCs during/after ischemia. Aim 2 will explore the role of cytoplasmic CaMKII in regulating caspase-3 activation, NF:B translocation and BDNF release in RGC/retinas. Aim 3 will explore the role of nuclear CaMKII1 in cell death/survival and the underlying mechanisms. The results of these studies will provide important insights into the signal transduction pathways and gene regulatory networks that control death and survival responses of RGCs which will be of relevance to retinal ischemia. PUBLIC HEALTH RELEVANCE: Retinal ischemia, a common clinical entity that has been widely studied because of its proposed role in, for example, retinal and choroidal vessel occlusion, anterior ischemic optic neuropathy, glaucoma, and diabetic retinopathy, etc, remains a common cause of blindness in the developed world due to relatively ineffective treatment. We propose to explore the signal transduction pathways linked to gene regulatory networks which control cell death and survival processes in rat retinal ganglion cells, whose death is a major contributing factor for visual impairment in these diseases. Knowledge of such pathways and networks is important for the development of practical therapeutic interventions.