Molecular basis of homocysteine-glutamate receptor mediated neuronal cell death

同型半胱氨酸谷氨酸受体介导的神经元细胞死亡的分子基础

• 批准号: 8189684
• 负责人: Ranjana Poddar
• 金额: $ 18.88万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8189684
• 关键词: AMPA Receptors; Acceleration; Adverse effects; Agonist; Alzheimer' s Disease; Apoptotic; Biochemical; Blood - brain barrier anatomy; Brain; Brain Injuries; Cell Line; Cell Survival; Cessation of life; DNA Damage; Data; Development; Disease; Elderly; Electron Spin Resonance Spectroscopy; Electrophysiology (science); Event; Folic Acid; Food; Generations; Glutamate Receptor; Glutamates; Goals; Health; Homocysteine; Homocystine; Hyperhomocysteinemia; Image; Incidence; Individual; Injury; Intervention; Ischemic Stroke; Knowledge; Lead; Link; MAP Kinase Gene; MAPK1 gene; MAPK11 gene; MAPK14 gene; Mediating; Metabolic; Metabolic Diseases; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molecular; Molecular Target; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Neurons; Nitric Oxide Synthase; Nutritional; Outcomes Research; Parkinson Disease; Pathology; Pathway interactions; Peptide Signal Sequences; Phosphorylation; Plasma; Play; Population; Predisposition; Publishing; RNA Interference; Receptor Activation; Receptor Signaling; Research; Risk Factors; Role; Signal Pathway; Signal Transduction; Societies; Surface; Toxic effect; Vascular Diseases; absorption; age related; base; fluorescence imaging; fortification; nervous system disorder; neuron loss; neurotoxicity; novel; novel strategies; prevent; prospective; receptor; research study; response; therapeutic target; trafficking

DESCRIPTION (provided by applicant): Elevated plasma level of homocysteine (hyper-homocysteinemia) is a major risk factor for neurodegenerative disorders. In spite of fortification of food with folic acid as an attempt to lower homocysteine levels and reduce homocysteine related disorders, the incidence of hyper-homocysteinemia in the elderly population is still quite large. This is mainly due to lowered nutritional absorption and decreased metabolic function with advanced age. Recent studies indicate that homocysteine can penetrate the blood-brain barrier and increased levels of homocysteine in brain is associated with aggravation and acceleration of neuronal damage in ischemic stroke, Alzheimer's and Parkinson's diseases. However, the underlying signaling mechanism(s) by which homocysteine exacerbates neuronal cell death in these disorders are not well understood. One key mechanism that may be fundamental to homocysteine-induced brain damage is through stimulation of NMDAR. In this study we propose to define the intracellular targets and the specific signaling mechanism(s) that link homocysteine mediated NMDA receptor activation to neuronal cell death. Contrary to the presumption that homocysteine-mediated NMDA receptor signaling mechanisms are analogous to other NMDA receptor agonists like glutamate, our findings indicate that the effects of homocysteine on NMDA receptor activation and the downstream signaling events are quite different. Glutamate-mediated stimulation of NR2A containing NMDA receptor subtypes promotes while stimulation of NR2B containing NMDA receptor subtypes inhibits activation of ERK resulting in transient activation of ERK MAPK. In contrast, our findings show that NR2B containing NMDA receptors did not play a role in regulating homocysteine-mediated activation of ERK MAPK resulting in sustained ERK activation. Again glutamate-mediated activation of NR2A containing NMDA receptor is thought to be pro-survival while activation of NR2B containing NMDA receptor is thought to be pro-apoptotic. Our findings, on the other hand, indicate that NR2B containing NMDA receptors have no role in homocysteine- induced neurotoxicity implicating the critical involvement of NR2A containing NMDA receptor in neuronal death. Contrary to the notion that activation of ERK MAPK promotes neuronal cell survival, while p38 MAPK promotes injury, our preliminary findings also indicate a role of ERK MAPK in homocysteine-NMDA receptor mediated neuronal cell death. Experiments proposed in Specific Aim 1 will expand on these initial findings to perform functional studies to determine the role of NR2A containing NMDA receptor in homocysteine induced neuronal cell death. Our preliminary data further indicate that homocysteine-induced activation of p38 MAPK is regulated by ERK MAPK indicating that a unique crosstalk between the two MAPK pathways may play a role in homocysteine-mediated neuronal cell death. The data also indicate that both AMPAR and nNOS regulate activation of p38 and play critical roles in mediating homocysteine induced neuronal cell death. Furthermore we provide evidence for ERK MAPK dependent increased trafficking of AMPA receptor subunits to neuronal surface in response to homocysteine. Experiments in the Specific Aim 2 will evaluate the role of nNOS and AMPA receptors in mediating the crosstalk between ERK and p38 MAPK and subsequent neuronal cell death. The above studies will involve neuron culture experiments of homocysteine toxicity and utilize pharmacological, electrophysiological, Ca2+ imaging, RNAi, fluorescence imaging, electron paramagnetic resonance and biochemical approaches. The proposed studies will potentially establish an important distinction between mechanisms leading to homocysteine & glutamate toxicity. Understanding these novel molecular events triggered by homocysteine may facilitate development of targeted therapeutic approaches to reduce brain damage associated with neurodegenerative disorders in hyper-homocysteinemic individuals. PUBLIC HEALTH RELEVANCE: The goal of the proposed study is to identify the signaling pathways unique to homocysteine-mediated neuronal cell death. We hypothesize that homocysteine stimulates NR2A containing NMDA receptor to activate a crosstalk between ERK and p38 MAPK pathways that involve nNOS and AMPAR signaling. Activation of this signaling cascade eventually result in ROS generation, DNA damage and neuronal cell death. The findings will potentially demonstrate critical points of distinction between mechanisms mediated by homocysteine and other NMDAR agonists. The outcome of this research could lead to the identification of prospective molecular targets involved in the pathology of homocysteine and suggest new approaches for treatment of neurological disorders in hyper-homocysteinemic individuals.

描述（由申请方提供）：血浆同型半胱氨酸水平升高（高同型半胱氨酸血症）是神经退行性疾病的主要风险因素。尽管在食品中添加叶酸以降低同型半胱氨酸水平并减少同型半胱氨酸相关疾病，但老年人群中高同型半胱氨酸血症的发病率仍然相当高。这主要是由于随着年龄的增长，营养吸收减少和代谢功能下降。最近的研究表明，同型半胱氨酸可以穿透血脑屏障，脑内同型半胱氨酸水平的升高与缺血性中风、阿尔茨海默病和帕金森病的神经元损伤的加重和加速有关。然而，在这些疾病中，高半胱氨酸加剧神经元细胞死亡的潜在信号传导机制尚未得到很好的理解。同型半胱氨酸诱导的脑损伤的一个关键机制可能是通过刺激NMDAR。在这项研究中，我们提出了明确的细胞内目标和特定的信号转导机制，连接同型半胱氨酸介导的NMDA受体激活神经元细胞死亡。与同型半胱氨酸介导的NMDA受体信号传导机制类似于谷氨酸等其他NMDA受体激动剂的假设相反，我们的研究结果表明，同型半胱氨酸对NMDA受体激活和下游信号传导事件的影响是完全不同的。谷氨酸介导的含有NR 2A的NMDA受体亚型的刺激促进而含有NR 2B的NMDA受体亚型的刺激抑制ERK的活化，导致ERK MAPK的瞬时活化。与此相反，我们的研究结果表明，NR 2B含有NMDA受体没有发挥作用，调节同型半胱氨酸介导的ERK MAPK的激活，导致持续的ERK激活。再次，谷氨酸介导的含有NMDA受体的NR 2A的活化被认为是促存活的，而含有NMDA受体的NR 2B的活化被认为是促凋亡的。另一方面，我们的发现表明，含有NR 2B的NMDA受体在高半胱氨酸诱导的神经毒性中没有作用，这暗示了含有NR 2A的NMDA受体在神经元死亡中的关键参与。与ERK MAPK激活促进神经元细胞存活，而p38 MAPK促进损伤的观点相反，我们的初步研究结果还表明ERK MAPK在同型半胱氨酸-NMDA受体介导的神经元细胞死亡中的作用。具体目标1中提出的实验将扩展这些初步发现，以进行功能研究，以确定含NR 2A的NMDA受体在同型半胱氨酸诱导的神经元细胞死亡中的作用。我们的初步数据进一步表明，同型半胱氨酸诱导的p38 MAPK的激活是由ERK MAPK调节的，这表明两个MAPK通路之间的独特串扰可能在同型半胱氨酸介导的神经元细胞死亡中发挥作用。这些数据还表明，AMPAR和nNOS都调节p38的激活，并在介导同型半胱氨酸诱导的神经元细胞死亡中发挥关键作用。此外，我们提供的证据表明，ERK MAPK依赖性增加运输的AMPA受体亚单位的神经元表面的同型半胱氨酸的反应。具体目标2中的实验将评估nNOS和AMPA受体在介导ERK和p38 MAPK之间的串扰以及随后的神经元细胞死亡中的作用。上述研究将涉及同型半胱氨酸毒性的神经元培养实验，并利用药理学、电生理学、Ca 2+成像、RNAi、荧光成像、电子顺磁共振和生物化学方法。拟议的研究将可能建立导致同型半胱氨酸和谷氨酸毒性的机制之间的重要区别。了解这些由同型半胱氨酸引发的新分子事件可能有助于开发靶向治疗方法，以减少高同型半胱氨酸血症患者神经退行性疾病相关的脑损伤。 公共卫生相关性：拟议研究的目标是确定同型半胱氨酸介导的神经元细胞死亡的独特信号通路。我们假设同型半胱氨酸刺激含有NR 2A的NMDA受体激活ERK和p38 MAPK通路之间的串扰，涉及nNOS和AMPAR信号。这种信号级联的激活最终导致ROS产生、DNA损伤和神经元细胞死亡。这些发现可能会证明同型半胱氨酸和其他NMDAR激动剂介导的机制之间的关键区别点。这项研究的结果可能会导致识别参与同型半胱氨酸的病理学的前瞻性分子靶点，并提出治疗高同型半胱氨酸血症患者神经系统疾病的新方法。