Astrocytes and ischemic brain injury

描述(由申请人提供)：心脏骤停幸存者最可怕的残疾之一是神经功能障碍。即使是短暂的全脑缺血也会导致CA1海马神经元的延迟丢失，而附近的海马齿状回(DG)神经元则不会受到影响。缺乏对星形胶质细胞作用的考虑被认为是许多仅以神经元存活为目标的潜在中风治疗失败的一个因素(Nedergaard和Dirnagl，2005)。在之前的资助期，我们证明了在CA1神经元死亡之前很久的再灌流早期，海马区CA1星形胶质细胞的选择性功能障碍。我们发表了第一个证明，通过选择性地将保护蛋白靶向海马星形胶质细胞，可以实现对CA1神经元的有效保护。虽然短暂性脑缺血后迟发性CA1神经元死亡是一种细胞凋亡现象，但对脑缺血后至关重要的bcl2家族的调控还不是很清楚。BCL2家族蛋白是细胞生死决定的中心调节者(Adams等人，2007)，并通过它们对线粒体膜完整性和功能的调节来控制细胞凋亡信号。MicroRNAs(MiRNAs)是一类新的、丰富的~22核苷酸RNA，它在转录后控制基因的表达。尽管已经克隆了数百个miRNAs，但人们对它们的真正靶点和功能知之甚少。许多miRNAs以细胞特异性的方式表达，miR-29家族被认为与星形胶质细胞相关(Smirnova等人，2005年)。在这项提案中，我们将研究miR-29在脑缺血和神经保护中的作用，评估miR-29作为神经保护新靶点的操作，并评估miR-29在BCL2家族中的潜在靶点。使用计算的miRNA靶标预测算法，我们发现miR-29可能靶向几个BCL2家族成员的信使RNA，既有促凋亡的，也有抗凋亡的。目标1和目标2集中在miR-29在缺血性脑损伤中的作用。我们将首先在体内和体外研究改变miR-29表达如何改变缺血诱导的细胞死亡。目的研究脑缺血后miR-29家族成员水平的变化，包括细胞类型和前脑缺血时海马区的特异性变化。然后，我们将讨论miR-29对细胞的作用机制，重点是线粒体功能和氧化应激。将测试miR-29在选择性CA1星形胶质细胞功能障碍中的作用。除了关注miR-29在损伤转归中的作用外，我们还需要研究下游靶点。在目标3中，我们关注5个可能的miR-29靶点，它们都是BCL2家族的成员，特别是促凋亡的PUMA。这些可能是miR-29对线粒体功能和氧化应激影响的候选者。总体而言，这一建议将检验以下假设：特定的microRNAs是保护缺血性脑损伤的新的有效靶点，并促进我们对星形胶质细胞调节和损伤对心脏骤停和复苏后神经预后的贡献的理解。 与公共卫生相关：心脏骤停和复苏每年影响约45万名患者，目前几乎没有治疗选择来减少经常发生的破坏性神经并发症。这项研究的重点是开发新的方法来快速改变基因表达，以保护大脑和改善神经学结果。这种新的治疗方法可以用于靶向受影响的单个细胞类型，并可能迅速转化为临床应用。

DESCRIPTION (provided by applicant): One of the most feared disabilities in survivors of cardiac arrest is neurological impairment. Even brief global cerebral ischemia causes delayed loss of CA1 hippocampal neurons while sparing nearby hippocampal dentate gyrus (DG) neurons. Lack of consideration of the role of astrocytes is thought to be a factor in the failure o many potential stroke therapies aimed only at neuronal survival (Nedergaard and Dirnagl, 2005). In the prior funding period we demonstrated selective dysfunction of hippocampal CA1 astrocytes at early reperfusion times, long before CA1 neuron death. We published the first demonstration that potent protection of CA1 neurons can be achieved by targeting protective proteins selectively to hippocampal astrocytes. Although delayed CA1 neuronal death after transient ischemia is apoptotic, the regulation of the critically important BCL2 family following cerebral ischemia is not well understood. BCL2 family proteins are central regulators of the life/death decision in cells (Adams et al, 2007), and via their regulation of mitochondrial membrane integrity and function control apoptosis signaling. MicroRNAs (miRNAs) are a novel, abundant class of ~22-nucleotide RNAs that control gene expression post- transcriptionally. Although hundreds of miRNAs have been cloned, little is known about their real targets and functions. Numerous miRNAs are expressed in a cell-specific manner and the miR-29 family has been suggested to be astrocyte related (Smirnova et al, 2005). In this proposal we will investigate the role of miR-29 in brain ischemia and neuroprotection, evaluate manipulation of miR-29 as a novel target for neuroprotection, and assess potential targets of miR-29 in the BCL2 family. Using computational miRNA target prediction algorithms we found that miR-29 could potentially target messenger RNAs of several BCL2 family members, both pro- and anti-apoptotic. Aims 1 and 2 focus on the role of miR-29 in ischemic brain injury. We will first investigate how altering miR-29 expression changes ischemia-induced cell death in vivo and in vitro. Aim 2 will determine changes in miR-29 family member levels in response to ischemia, including cell type and hippocampal region specific changes with forebrain ischemia. We will then address the mechanisms of the miR-29 effects on cells focusing on mitochondrial function and oxidative stress. The role of miR-29 in selective CA1 astrocyte dysfunction will be tested. In addition to focusing on the role of miR-29 in outcome from injury, we need to investigate downstream targets. In Aim 3 we focus on 5 likely miR-29 targets, all members of the BCL2 family, focusing especially on pro-apoptotic PUMA. These are likely candidates for effects of miR-29 on mitochondrial function and oxidative stress. Overall this proposal will test the hypothesis that specific microRNAs are a novel and effective target for protection from ischemic brain injury, as well as advancing our understanding of the contributions of astrocyte regulation and impairment to neurological outcome following cardiac arrest and resuscitation. PUBLIC HEALTH RELEVANCE: Cardiac arrest and resuscitation affects about 450,000 patients/year and currently has few treatment options to reduce the devastating neurological complications that often occur. This research is focused on developing novel ways to rapidly change gene expression to protect the brain and improve neurological outcome. This new treatment can be used to target individual cell types that are affected, and might be able to translate rapidly to clinical applications.