NICOTINAMIDE MONONUCLEOTIDE ADENYLYLTRANSFERASES IN NEONATAL HYPOXIA-ISCHEMIA

描述（由申请人提供）：这个指导职业发展奖的目标是促进拉斐尔加林多博士的过渡到独立作为一个医生，科学家研究神经元死亡的神经生物学机制在受伤的发展神经元。拟议的实验将在大卫霍尔茨曼博士的指导下进行，他们将研究一个由三种NAD+代谢酶组成的家族（称为烟酰胺单核苷酸腺苷酸转移酶（NMNATs））在新生儿脑缺氧-缺血（H-I）中的潜在神经保护作用。这种形式的损伤是新生儿期死亡的最常见原因，并占围产期脑损伤所致慢性神经系统疾病的很大一部分。尽管其发病率很高，但有效的 治疗暴露于H-I的新生儿。这种缺乏有效的临床干预部分归因于我们对调节健康和受损发育大脑中神经元死亡途径的神经生物学机制和分子的不完全理解。NMNAT是已被证明具有强神经保护特性的分子，并且可能重要地参与各种中枢和外周神经系统疾病的神经退行性机制。然而，NMNATs在中枢神经系统中的神经保护特性尚未得到很好的研究，其在受伤和健康的未成熟大脑中的潜在作用甚至更不清楚。本项目的目标是验证内源性和外源性NMNAT蛋白通过增加线粒体的钙缓冲能力来保护发育中的神经元免受H-I的影响的假设。为了验证这一假设，本研究将利用各种生物化学、免疫学、分子和遗传学技术来评估这些蛋白质在受损发育神经元中的作用，其目的如下：1）利用NMNAT基因表达的免疫化学和双分子方法并采用基因敲低技术来表征内源性NMNAT在新生儿H-I后细胞死亡中的表达和作用。2)使用转基因动物模型和基因转导系统评估外源性NMNAT 1、2和3在H-I后新生儿脑中的神经保护作用。3)采用生物化学和生物发光技术研究新生儿脑中NMNAT的神经保护机制，以检查H-I和兴奋性毒性体外模型中过度表达和表达不足的NMNAT的线粒体生理学。拟议的实验可能会为我们理解调节新生儿大脑发育中神经元细胞死亡的生物过程提供新的线索。此外，这些知识可能最终导致确定新的治疗靶点，用于治疗和预防新生儿脑损伤引起的神经系统后果。

DESCRIPTION (provided by applicant): The goal of this Mentored Career Development Award is to facilitate Dr. Rafael Galindo's transition to independence as a physician-scientist studying the neurobiological mechanisms of neuronal death in injured developing neurons. The proposed experiments will be conducted under the mentorship of Dr. David Holtzman, and they will examine the potential neuroprotective role of a family of three NAD+ metabolizing enzymes, known as nicotinamide mononucleotide adenylyltransferases (NMNATs), in neonatal brain hypoxia- ischemia (H-I). This form of injury is the most common cause of death in the newborn period and accounts for a significant portion of the chronic neurological conditions attributed to brain injury in the perinatal period. Despite its high prevalence, there are only limited effective therapies available for newborns exposed to H-I. This lack of effective clinical intervention is attributed, in part, to our incomplete understanding of the neurobiological mechanisms and molecules that regulate neuronal death pathways in the healthy and injured developing brain. NMNATs are molecules that have been shown to have strong neuroprotective properties and are likely importantly involved in the neurodegenerative mechanisms of various central and peripheral neurological diseases. Nevertheless, the neuroprotective properties of NMNATs in the central nervous system have not yet been well studied, and its potential role in the injured and healthy immature brain is even less well understood. The goal of this project is to test the hypothesis that endogenous and exogenous NMNAT proteins protect developing neurons from the effects of H-I by increasing the calcium buffering capacity of mitochondria. In order to test this hypothesis, this proposal will utilized various biochemical, immunological, molecular and genetic techniques to assess the role of these proteins in injured developing neurons through the following aims: 1) To characterize the expression and role of endogenous NMNATs in cell death following neonatal H-I utilizing immunochemical and bimolecular methods of NMNAT gene expression and employing gene knock-down techniques. 2) To assess the neuroprotective role of exogenous NMNAT 1, 2 and 3 in the neonatal brain following H-I using transgenic animal models and gene transduction systems. 3) Investigate the neuroprotective mechanism of NMNAT(s) in the neonatal brain employing biochemical and bioluminescent techniques to examine mitochondrial physiology in over- and under-expressing NMNAT in vitro models of H-I and excitotoxicity. The proposed experiments will likely shed new light in our understanding of the biological processes that regulate neuronal cell death in the developing newborn brain. Furthermore, this knowledge may ultimately lead to identifying novel therapeutic targets for the treatment and prevention of the neurological consequences attributed to brain injury in the newborn.