Novel long non-coding RNAs in neuronal survival in focal cerebral ischemia

 描述(申请人提供)：局灶性脑缺血(缺血性中风)是美国最常见的致残原因和第四大死亡原因。尽管对该病的药物治疗和手术治疗都在努力，但组织纤溶酶原激活剂(TPA)仍是目前唯一有效的治疗方法。更好地了解病理过程，发现新的靶点和治疗方法，将大大推动该领域的发展。关于lncRNAs在脑缺血中的研究很少。通过对大鼠局灶性脑缺血/再灌注(IR)模型的lncRNA阵列分析，我们发现CAMK2D相关转录本1(C2dat1)和2(C2dat2)(C2dat1-2)是两个新的IR诱导的lncRNAs，它们特异性地调控大鼠局灶性脑缺血再灌注模型中CaMKIIδ的表达。在我们的初步研究中，C2dat1-2mRNAs在局灶性脑缺血后小鼠皮质半影区呈时间依赖性上调，同时伴随着CaMKIIδ在转录和蛋白水平的表达增加。C2dat1-2和CAMK2D在体外缺血(缺氧-葡萄糖剥夺/复氧，OGD/R)反应的小鼠Neuro-2a细胞中的表达模式得到证实。C2dat1基因敲除后可显著抑制CaMKIIδ的表达，增强缺氧性脑缺血再灌注诱导的细胞死亡。在机制上，沉默C2dat1后CaMKIIδ的表达减少抑制了OGD/R诱导的NF-κB信号通路的激活。进一步的分析表明，IKKα的下调和IκBα降解的进一步抑制是通过耗尽C2dat1-2而抑制NF-κB信号活性的原因。因此，C2dat1似乎通过调节NF-κB信号通路促进神经元存活。因此，lncRNAs可能成为缺血性脑损伤治疗干预的潜在靶点。基于这些初步发现，我们假设C2dat1-2是IR诱导的新的lncRNA，它调节CaMKIIδ的表达，通过激活NF-κB信号通路促进神经元存活。这一应用的主要目的是确定C2dat1-2的功能和信号机制，以及相关的CaMKIIδ在IR诱导的神经元损伤中的作用。长期目标是更深入地了解IR相关生物过程的分子基础，并确定在IR期间提供神经保护的新的治疗靶点。提出了两个特定的目的：特定的目的1.将C2dat1-2定义为新的缺血诱导的lncRNA，通过调节小鼠原代神经元培养中CaMKIIδ的表达来促进神经元的存活。特定目的2.确定C2dat1-2基因敲除是否增强了局灶性脑缺血小鼠IR诱导的细胞死亡，其作用是否通过下调CaMKIIδ介导。

 DESCRIPTION (provided by applicant): Focal cerebral ischemia (ischemic stroke) is the most common cause of disability and the fourth leading cause of death in the United State. Despite of the efforts on developing the pharmacological and surgical treatments of the disease, tissue plasminogen activator (tPA) is the only effective therapy at present. A better understanding of the pathological process and the discovery of new targets and therapies will significantly advance the field. There have been little studies on lncRNAs in cerebral ischemia. Through an lncRNA array analysis in a rat model of focal cerebral ischemia/reperfusion (IR), we have identified CAMK2D-associated transcript 1 (C2dat1) and 2 (C2dat2) (C2dat1-2) as two novel IR-induced lncRNAs that specifically regulated the expression of CaMKIIδ in rat and mouse models of focal cerebral IR. In our pilot study, C2dat1-2 mRNAs were upregulated in a time-dependent manner in mouse cortical penumbra after focal ischemic brain injury, which was accompanied by increased expression of CaMKIIδ at transcript and protein levels. The expression patterns of C2dat1-2 and CAMK2D were confirmed in mouse Neuro-2a cells in response to in vitro ischemia (oxygen-glucose deprivation/re-oxygenation, OGD/R). Knockdown of C2dat1 resulted in a significant blockade of CaMKIIδ expression, and potentiated OGD/R-induced cell death. Mechanistically, reduced CaMKIIδ expression upon silencing C2dat1 inhibited OGD/R-induced activation of the NF-κB signaling pathway. Further analysis showed that the downregulation of IKKα and further inhibition of IκBα degradation accounted for the inhibition of the NF-κB signaling activity by depleting C2dat1-2. Thus, C2dat1 appears to promote neuronal survival through regulating the NF-κB signaling pathway. Therefore, lncRNAs may be potential targets for therapeutic intervention of ischemia brain injury. Based on these preliminary findings, we hypothesize that C2dat1-2 are novel IR-induced lncRNAs that regulate the expression of CaMKIIδ to promote neuronal survival through the activation of the NF-κB signaling pathway. The primary goal of this application is to determine the function and signaling mechanisms of C2dat1-2, as well as the associated CaMKIIδ in IR- induced neuronal injury. The long-term goal is to gain more insights into the molecular bases of IR-associated biological processes and to identify novel therapeutic targets that confer neuroprotection during IR. Two specific aims are proposed: Specific Aim 1. Define the role of C2dat1-2 as novel ischemia-induced lncRNAs that promote neuronal survival by modulating CaMKIIδ expression in mouse primary neuronal cultures. Specific Aim 2. Determine if knockdown of C2dat1-2 potentiate IR-induced cell death in mouse model of focal cerebral ischemia and if the effects are mediated through down-regulation of CaMKIIδ.