Prolyl Hydroxylation and Neuronal Cell Death

脯氨酰羟基化和神经元细胞死亡

• 批准号: 7627194
• 负责人: ROBERT S FREEMAN
• 金额: $ 33.22万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7627194
• 关键词: Address; Affect; Alzheimer' s Disease; Apoptosis; Apoptotic; BCL2L11 gene; Biochemical; Caspase; Cell Death; Cessation of life; Co-Immunoprecipitations; Data; Development; Disease; Ensure; Event; Functional disorder; Gene Expression; Goals; Hydroxylation; Hypoxia Inducible Factor; Injury; Knockout Mice; Lead; Mediating; Mediator of activation protein; Modeling; Nerve Growth Factors; Nervous system structure; Neurodegenerative Disorders; Neurons; Oxygen; PC12 Cells; Pathologic; Pathway interactions; Process; Procollagen-Proline Dioxygenase; Protein Family; Proteins; Reporting; Research; Role; Signal Transduction; Spinal cord injury; Stroke; Testing; Tumor Suppressor Genes; VHL protein; Von Hippel-Lindau Tumor Suppressor Protein; Withdrawal; base; cellular transduction; cytochrome c; deprivation; insight; nervous system development; nervous system disorder; neuron loss; neuronal survival; neurotrophic factor; novel; prevent; protein function; public health relevance; research study; transcription factor; ubiquitin-protein ligase

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.

描述（由申请人提供）：神经元死亡是一个高度调控的事件，发生在整个发育中的神经系统。在许多情况下，发育中的神经元死亡是因为它们不能获得足够的目标来源的、促进生存的因子，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和它们的目标之间建立适当数量和类型的连接是重要的。重要的是，营养因子剥夺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，并导致脊髓损伤、中风和阿尔茨海默病中出现的功能缺陷。利用一种特征明确且生理上重要的涉及ngf依赖性交感神经元的营养因子剥夺模型，我们最近发现脯氨酸羟化酶EGLN3是细胞死亡的介质。虽然EGLN3促进死亡的机制在很大程度上是未知的，但我们已经发现了EGLN3和BH3-only Bcl-2家族蛋白BIMEL之间的一种新的相互作用，BIMEL是一种由营养因子戒断诱导的细胞死亡的调节剂。初步结果显示，BIMEL在egln3缺陷神经元中诱导的死亡减少，表明这两种蛋白之间存在功能关系。共免疫沉淀实验表明，EGLN3和BIMEL分别与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，其他初步结果表明pVHL可能增强EGLN3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EGLN3协同调节营养因子剥夺诱导的细胞死亡。在Aim 1中，我们将结合基因敲除小鼠的过表达和神经元来确定NGF剥夺诱导的细胞死亡过程中EGLN3和BIMEL之间的功能关系。Aim 2的实验将确定EGLN3和BIMEL相互作用对细胞死亡的生化意义。具体的实验将确定EGLN3是否影响BIMEL的功能，BIMEL是否影响EGLN3的脯氨酸羟化酶活性，或者BIMEL是否是EGLN3的脯氨酸羟化的底物。在Aim 3中，我们将验证pVHL在营养因子剥夺过程中调节EGLN3和BIMEL蛋白水平的假设。此外，我们将测试pVHL表达在营养因子剥夺诱导的细胞死亡中是否必要。这些研究将进一步加深我们对在正常发育和神经系统疾病中导致神经元细胞死亡的机制的理解，其中营养因子剥夺导致神经元损失和功能障碍。