Prolyl Hydroxylation and Neuronal Cell Death

描述（由申请人提供）：神经元死亡是一种高度调节的事件，发生在整个发育中的神经系统中。在许多情况下，发育中的神经元死亡是因为它们未能获得足够的靶源性生存促进因子，如神经生长因子（NGF）。这种类型的发育程序性细胞死亡对于确保在神经元及其靶点之间建立适当数量和类型的连接非常重要。重要的是，营养因子缺乏也是成熟神经系统损伤和疾病的常见后果。在这种情况下，神经元死亡是病理性的，并有助于脊髓损伤，中风和阿尔茨海默病中看到的功能缺陷。使用一个良好的特点和生理上重要的模型，营养因子剥夺涉及神经生长因子依赖的交感神经元，我们最近确定了脯氨酰羟化酶EGLN 3作为介质的细胞死亡。虽然EGLN 3促进死亡的机制在很大程度上是未知的，我们已经发现了EGLN 3和BH 3-only Bcl-2家族蛋白BIMEL之间的一种新的相互作用，BIMEL是一种由营养因子撤退诱导的细胞死亡的既定调节因子。初步结果显示，在EGLN 3缺陷的神经元中，BIMEL诱导的死亡减少，表明这两种蛋白质之间存在功能关系。免疫共沉淀实验证明EGLN 3和BIMEL各自与von Hippel-Lindau肿瘤抑制蛋白pVHL相互作用。pVHL在交感神经元中的表达促进细胞死亡，而其他初步结果表明，pVHL可以增强EGLN 3和BIMEL蛋白的稳定性。基于这些新的观察结果，我们假设pVHL，BIMEL和EGLN 3协调调节营养因子剥夺诱导的细胞死亡。在目标1中，我们将使用过表达和来自敲除小鼠的神经元的组合来确定在NGF剥夺诱导的细胞死亡期间EGLN 3和BIMEL之间的功能关系。目的2中的实验将确定EGLN 3和BIMEL之间相互作用对细胞死亡的生化意义。具体实验将确定EGLN 3是否影响BIMEL的功能，BIMEL是否影响EGLN 3的脯氨酰羟化酶活性，或者BIMEL是否是EGLN 3的脯氨酰羟化的底物。在目标3中，我们将检验pVHL在营养因子剥夺期间调节EGLN 3和BIMEL蛋白水平的假设。此外，我们将测试pVHL表达是否是营养因子剥夺诱导的细胞死亡所必需的。这些研究将进一步加深我们对正常发育过程中导致神经元细胞死亡的机制的理解，以及在营养因子缺乏导致神经元损失和功能障碍的神经系统疾病中。 公共卫生相关性：营养因子剥夺诱导的细胞死亡不仅对神经系统的正常发育至关重要，而且还导致伴随中风、脊髓损伤和神经退行性疾病的神经元的损失和功能障碍。这个项目将描述新的机制，调节神经营养因子剥夺引起的细胞死亡。从该项目中获得的信息将进一步加深我们对正常发育的理解，并有助于确定旨在预防病理性神经元细胞死亡的治疗新靶点。

DESCRIPTION (provided by applicant): Neuronal death is a highly regulated event that occurs throughout the developing nervous system. In many cases, developing neurons die because they fail to receive adequate supplies of target-derived, survival promoting factors such as nerve growth factor (NGF). This type of developmentally programmed cell death is important for ensuring that the proper numbers and types of connections are established between neurons and their targets. Importantly, trophic factor deprivation is also a frequent consequence of injury and disease in the mature nervous system. In such cases, the neuronal death is pathologic and contributes to the functional deficits seen in spinal cord injury, stroke, and Alzheimer's disease. Using a well-characterized and physiologically important model for trophic factor deprivation involving NGF-dependent sympathetic neurons, we recently identified the prolyl hydroxylase EGLN3 as a mediator of cell death. While the mechanism by which EGLN3 promotes death is largely unknown, we have discovered a novel interaction between EGLN3 and the BH3-only Bcl-2 family protein BIMEL, an established regulator of cell death induced by trophic factor withdrawal. Preliminary results reveal that death induced by BIMEL is reduced in EGLN3-deficient neurons, suggesting a functional relationship exists between these two proteins. Co-immunoprecipitation experiments demonstrate that EGLN3 and BIMEL each interact with the von Hippel-Lindau tumor suppressor protein pVHL. Expression of pVHL in sympathetic neurons promotes cell death while other preliminary results suggest that pVHL may enhance EGLN3 and BIMEL protein stability. Based on these novel observations, we hypothesize that pVHL, BIMEL and EGLN3 function coordinately to regulate trophic factor deprivation-induced cell death. In Aim 1 we will use a combination of over-expression and neurons from knockout mice to determine the functional relationship between EGLN3 and BIMEL during NGF deprivation-induced cell death. Experiments in Aim 2 will determine the biochemical significance of the interaction between EGLN3 and BIMEL for cell death. Specific experiments will determine if EGLN3 influences the function of BIMEL, if BIMEL affects the prolyl hydroxylase activity of EGLN3, or if BIMEL is a substrate for prolyl hydroxylation by EGLN3. In Aim 3, we will test the hypothesis that pVHL regulates EGLN3 and BIMEL protein levels during trophic factor deprivation. In addition, we will test if pVHL expression is necessary for trophic factor deprivation-induced cell death. These studies will further our understanding of the mechanisms that lead to neuronal cell death during normal development and in nervous systems disorders where trophic factor deprivation contributes to neuronal loss and dysfunction. PUBLIC HEALTH RELEVANCE: Trophic factor deprivation-induced cell death is not only critical for proper development of the nervous system but it also contributes to the loss and dysfunction of neurons that accompanies stroke, spinal cord injury, and neurodegenerative disease. This project will characterize new mechanisms that regulate cell death caused by neurotrophic factor deprivation. Information gained from this project will further our understanding of normal development and could help identify new targets for therapies aimed at preventing pathological neuronal cell death.