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

 描述（由申请人提供）：局灶性脑缺血（缺血性卒中）是美国最常见的残疾原因，也是第四大死亡原因。尽管人们一直在努力开发治疗该病的药物和手术方法，但组织型纤溶酶原激活剂（tPA）是目前唯一有效的治疗方法。更好地了解病理过程和发现新的靶点和疗法将大大推动该领域的发展。目前对lncRNA在脑缺血中的作用研究较少。通过在大鼠局灶性脑缺血/再灌注（IR）模型中的lncRNA阵列分析，我们鉴定了CAMK 2D相关转录本1（C2 dat 1）和2（C2 dat 2）。（C2 dat 1 -2）作为两种新的IR诱导的lncRNA，在大鼠和小鼠局灶性脑IR模型中特异性调节CaMK II δ的表达。C2 dat 1 -2 mRNA在小鼠局灶性缺血性脑损伤后皮质半影区以时间依赖性方式上调，伴随着CaMK Ⅱ δ在转录和蛋白水平的表达增加。C2 dat 1 -2和CAMK 2D的表达模式在小鼠Neuro-2a细胞中响应于体外缺血（氧-葡萄糖剥夺/再氧合，OGD/R）得到证实。敲低C2 dat 1可显著阻断CaMK Ⅱ δ的表达，并增强OGD/R诱导的细胞死亡。从机制上讲，沉默C2 dat 1后CaMK II δ表达的减少抑制了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降解的进一步抑制解释了通过消耗C2 dat 1 -2抑制NF-κB信号传导活性。因此，C2 dat 1似乎通过调节NF-κB信号通路促进神经元存活。因此，lncRNA可能是缺血性脑损伤治疗的潜在靶点。基于这些初步发现，我们推测C2 dat 1 -2是IR诱导的新型lncRNA，通过激活NF-κB信号通路调节CaMK Ⅱ δ的表达，促进神经元存活。本申请的主要目的是确定C2 dat 1 -2以及相关CaMKIIδ在IR诱导的神经元损伤中的功能和信号传导机制。长期的目标是获得更多的见解IR相关的生物过程的分子基础，并确定新的治疗靶点，在IR期间赋予神经保护。提出了两个具体的目标：具体目标1。确定C2 dat 1 -2作为新型缺血诱导的lncRNA的作用，其通过调节小鼠原代神经元培养物中CaMKIIδ的表达来促进神经元存活。具体目标2。在小鼠局灶性脑缺血模型中，确定C2 dat 1 -2的敲低是否增强IR诱导的细胞死亡，以及该效应是否通过下调CaMK II δ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.