Potential use of a decoy peptide for hypoxia/ischemia treatment

描述(由申请人提供)： 新生儿缺氧缺血(H/I)是新生儿脑病的主要原因，在美国，每年每1000名新生儿中就有2-4人发生脑病。虽然由此导致的死亡率下降，但儿童患严重神经疾病的比率相应增加，包括运动和学习障碍、脑瘫和癫痫。尽管H/I所致神经元损伤的机制尚不完全清楚，但已有研究表明谷氨酸受体过度兴奋所致的兴奋性毒性起着重要作用。谷氨酸受体激活的下游事件的识别一直是密集研究的对象，钙依赖的蛋白酶Calain被认为与缺血诱导的神经元损伤有关。代谢性谷氨酸受体最近成为缺血性神经元死亡的新参与者，尽管它们的作用仍然存在争议。研究集中在代谢性谷氨酸受体之一的mGluR1，因为该受体激活了两个信号级联反应，一个通过刺激磷脂酶C，合成肌醇-3-磷酸和从内部储存释放钙来导致神经退化，另一个通过PI3K-Akt途径与神经保护有关。我们最近发现，NMDA受体的激活导致Calain介导的mGluR11的C-末端结构域的截断，mGluR11是mGluR1的亚型之一。由于这种截断，mGluR11失去了它的神经保护信号，尽管它保持着正常的钙信号功能。也就是说，截短的mGluR11成为一种独有的“神经退行性受体”，提示在兴奋性毒性和神经退行性变过程中，存在一个连接NMDA受体、钙蛋白激活和mGluR1的正反馈环。此外，我们发现，在体外和体内，切割位点周围与HIV Tat多肽相连的一系列氨基酸组成的小肽对兴奋性毒性具有神经保护作用。因此，R21的应用旨在获得体内临床前数据，以开发TAT-mGluR1多肽或该多肽的修改形式，用于治疗新生儿缺氧/缺氧诱导的神经元损伤。因此，我们将在体外和体内新生儿缺氧缺血模型(特定目标1)中测试TAT-mGluR1肽的潜在神经保护作用。我们将设计并测试TAT-mGluR1类似物的神经保护作用(特定目标2)。积极的结果将鼓励这种治疗新生儿缺氧/缺血的方法的临床开发。 公共卫生相关性： 提出这项研究的理由来自于PI实验室最近的研究，这些研究已经确定了兴奋性毒性神经元损伤的新机制，以及一种有前景的神经保护肽。这一新的机制涉及一个先前未知的正反馈环的存在，该环连接了几个假设参与谷氨酸介导的神经元死亡的分子过程。这项拟议的研究将在体外和体内的新生儿缺氧/缺血性神经元损伤模型中评估该肽的神经保护作用。如果该多肽在缺氧/缺血损伤后应用时显示出神经保护作用，这些研究将提供体内概念证明，不仅用于新生儿缺氧/缺血诱导的神经元损伤，而且也用于其他形式的神经退行性疾病的神经元损伤的临床开发。

DESCRIPTION (provided by applicant): Neonatal hypoxic ischemia (H/I) is largely responsible for neonatal encephalopathy, which occurs in 2-4 out of 1000 births per year in the US. While the resulting mortality has decreased, there has been a corresponding increased rate of children developing serious neurological disorders, including motor and learning disabilities, cerebral palsy and seizures. Although the mechanisms underlying H/I-induced neuronal damage are not completely understood, it has repeatedly been proposed that excitotoxicity due to over-excitation of glutamate receptors plays a critical role. Identification of downstream events from glutamate receptor activation has been the object of intense investigation, and the calcium dependent protease calpain has been implicated in ischemia-induced neuronal damage. Metabotropic glutamate receptors have recently emerged as new players in ischemic neuronal death, although their roles remain controversial. Studies have focused on one of the metabotropic glutamate receptors, mGluR1, as this receptor activates two signaling cascades, one leading to neurodegeneration through the stimulation of phospholipase C, the synthesis of inositol-3-phosphate and the release of calcium from internal stores and another one linked to neuroprotection through the PI3K-Akt pathway. We recently found that NMDA receptor activation results in calpain-mediated truncation of the C- terminal domain of mGluR11, one of the isoforms of mGluR1. As a result of this truncation, mGluR11 loses its neuroprotective signaling, although it maintains a normal calcium signaling function. In other words, truncated mGluR11 becomes an exclusively "neurodegenerative receptors", suggesting the existence of a positive feedback loop linking NMDA receptors, calpain activation and mGluR1 in excitotoxicity and neurodegeneration. Furthermore, we found that a small peptide consisting in a sequence of amino acids surrounding the cutting site linked to the HIV tat-peptide was neuroprotective against excitotoxicity both in vitro and in vivo. This R21 application is therefore directed at obtaining in vivo preclinical data to develop the tat-mGluR1 peptide or a modified form of this peptide for therapeutic use against neonatal H/I-induced neuronal damage. We will therefore test the potential neuroprotective effects of the tat-mGluR1 peptide in an in vitro and an in vivo model of neonatal H/I (Specific Aim #1). We will design and test the neuroprotective effects of analogs of tat-mGluR1 (Specific Aim #2). Positive results will then encourage the clinical development of this approach for the treatment of neonatal hypoxia/ischemia. PUBLIC HEALTH RELEVANCE: The rationale for the proposed studies is derived from recent studies in the PI's laboratory that have identified a new mechanism for excitotoxic neuronal damage as well as a promising neuroprotective peptide. This new mechanism involves the existence of a previously unknown positive feedback loop linking several molecular processes postulated to participate in glutamate-mediated neuronal death. The proposed studies will evaluate the neuroprotective effects of the peptide in an in vitro and an in vivo model of neonatal hypoxic/ischemic neuronal damage. If the peptide shows neuroprotective effects when applied after the hypoxic/ischemic insults, these studies will provide the in vivo proof-of-concept needed to pursue the clinical development of this approach not only for neonatal hypoxia/ischemia-induced neuronal damage, but also for neuronal damage occurring in other forms of neurodegenerative diseases.