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1000例新生儿中有2-4例发生新生儿脑病。虽然由此造成的死亡率有所下降，但患严重神经系统疾病的儿童比例相应增加，包括运动和学习障碍、大脑性麻痹和癫痫。虽然H/I诱导的神经元损伤的机制尚未完全了解，但已多次提出，由于谷氨酸受体的过度兴奋引起的兴奋性毒性起着关键作用。谷氨酸受体激活的下游事件的鉴定一直是深入研究的对象，并且钙依赖性蛋白酶钙蛋白酶与缺血诱导的神经元损伤有关。代谢型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。研究集中在代谢型谷氨酸受体之一mGluR 1上，因为该受体激活两个信号级联，一个通过磷脂酶C的刺激导致神经变性，肌醇-3-磷酸的合成和钙从内部储存的释放，另一个通过PI 3 K-Akt途径与神经保护有关。我们最近发现，NMDA受体激活导致钙蛋白酶介导的mGluR 11（mGluR 1的同种型之一）C末端结构域的截短。由于这种截短，mGluR 11失去了其神经保护信号，尽管它保持了正常的钙信号功能。换句话说，截短的mGluR 11成为唯一的“神经退行性受体”，表明在兴奋性毒性和神经退行性变中存在连接NMDA受体、钙蛋白酶激活和mGluR 1的正反馈环。此外，我们发现，一个小的肽组成的切割位点周围的氨基酸序列连接到HIV的tat肽是神经保护兴奋性毒性在体外和体内。因此，该R21申请旨在获得体内临床前数据，以开发tat-mGluR 1肽或该肽的修饰形式，用于治疗新生儿H/I诱导的神经元损伤。因此，我们将在新生儿H/I的体外和体内模型中测试tat-mGluR 1肽的潜在神经保护作用（具体目标#1）。我们将设计和测试tat-mGluR 1类似物的神经保护作用（具体目标#2）。积极的结果将鼓励这种方法用于治疗新生儿缺氧/缺血的临床开发。 公共卫生相关性： 拟议研究的基本原理来自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有前途的神经保护肽。这种新的机制涉及到一个以前未知的正反馈环的存在，连接几个分子过程假设参与谷氨酸介导的神经元死亡。所提出的研究将在新生儿缺氧/缺血性神经元损伤的体外和体内模型中评估肽的神经保护作用。如果该肽在缺氧/缺血损伤后应用时显示出神经保护作用，则这些研究将提供追求该方法的临床开发所需的体内概念验证，所述临床开发不仅用于新生儿缺氧/缺血诱导的神经元损伤，而且用于在其他形式的神经变性疾病中发生的神经元损伤。

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.