Oxidative Stress and Glutamate Excitotoxicity on Aged Dopaminergic Neruons

描述(由申请人提供)：本提案请求支持一项全面的培训计划，该计划将使Heather Boger博士能够拓宽、增强和完善她的技术技能，这是她从事富有成效的独立研究生涯所必需的。在该奖项的指导阶段(1年至2年)，博格博士将接受一个合作导师团队的多方面培训，其中包括分子生物学、电化学、纳米颗粒(微球)的生产和统计学方面的培训。在独立阶段(3-5年)提出的研究计划建立在这种培训的基础上，并更具体地侧重于涉及神经退化的机制。衰老和帕金森病(PD)是一种已知的神经退行性疾病，可导致运动障碍，与黑质胶质细胞源性神经营养因子(GDNF)减少、氧化应激增加以及基底节回路间接途径增加(包括多巴胺D2受体表达增加，黑质谷氨酸从过度活跃的丘脑核神经元释放增加，黑质纹状体多巴胺表达和功能降低)有关。我们已经证明，GDNF基因减少的小鼠模型有早期起病的运动功能障碍，并有证据表明间接途径功能增加，例如纹状体D2受体表达增加和黑质纹状体表达加速下降。然而，GDNF基因的部分缺失是否会影响黑质谷氨酸释放和氧化应激随年龄增长而变化尚不清楚。因此，本研究建议的总体假设是，GDNF的内源性缺失通过氧化应激增加STN-黑质谷氨酸的兴奋性毒性，从而加强黑质纹状体DAR能系统功能障碍。针对这一假说，已制定了三个特定目标：目的1)急性给予GDNF可改变GDNF遗传减少的小鼠的神经元反应，目的2)长期给予GDNF将减轻部分GDNF对DA功能的增龄效应，以及目的3)GDNF的可获得性减少导致的DA能丢失的进展情况扰乱黑质从丘脑底核输入的谷氨酸，从而导致氧化应激增加和持续的DAR能功能障碍。这些研究的结果将有助于深入了解与GDNF部分丢失相关的早发性多巴胺能损失的机制，并可能确定减少氧化应激、黑质纹状体多巴胺损失以及因衰老和帕金森病而发生的运动功能障碍的治疗靶点。此外，这些研究的结果将有助于指导博格博士未来在系统神经科学领域的独立研究。 公共卫生相关性：胶质细胞系衍生神经营养因子(GDNF)在帕金森氏病患者中减少，我们已经证明，GDNF部分丢失的小鼠会加速与年龄相关的运动功能和黑质纹状体多巴胺的丧失，但GDNF减少和多巴胺丢失之间的关系尚不清楚。我们认为，由于长期减少GDNF导致的多巴胺丢失导致谷氨酸从丘脑底核释放到黑质，从而导致氧化应激和持续的多巴胺能损伤。此外，外源性GDNF的应用将减轻由于终生减少GDNF而导致的谷氨酸毒性增加所引起的多巴胺能损伤。

DESCRIPTION (provided by applicant): This proposal requests support for a comprehensive training plan that will enable Heather Boger, PhD, to broaden, enhance, and refine her technical skills that are necessary for a productive independent research career. Dr. Boger will receive multifaceted training during the mentored phase (Year 1-2) of the award from a team of collaborating mentors that include training in molecular biology, electrochemistry, production of nanoparticles (microspheres), and statistics. The research plan that is proposed during the independent phase (Year 3-5) builds on this training and focuses more specifically on mechanisms involved with neurodegeneration. Aging and Parkinson's disease (PD), a known neurodegenerative disease resulting in motor impairments, has been associated with a reduction of glial cell line-derived neurotrophic factor (GDNF) in the substantia nigra, increased oxidative stress, and an increase in the indirect pathway of the basal ganglia circuitry (including increase dopamine D2 receptor expression, increased nigral glutamate release from overactive subthalamic nucleus neurons, and decreased nigrostriatal dopamine expression and function). We have demonstrated that a mouse model with a genetic reduction of GDNF have early-onset motor dysfunction and evidence of increased indirect pathway function, such as increased striatal D2 receptor expression and accelerated decline in nigrostriatal expression. However, it is not known whether a partial gene deletion of GDNF impacts subthalamic nucleus glutamate release into the substantia nigra and oxidative stress with age. Therefore, the overall hypothesis of this research proposal is that the intrinsic GDNF loss enhances nigrostriatal DAergic system dysfunction by increasing STN-nigral glutamate excitotoxicity via oxidative stress. To address this hypothesis, three specific aims have been formulated: Aim 1) The neuronal response to acute administration of GDNF is altered in mice with a genetic reduction of GDNF, Aim 2) Chronic administration of GDNF will alleviate the age-related effects of a partial loss of GDNF on DA function, and Aim 3) The progress DAergic loss due to less availability of GDNF dysregulates the glutamatergic input from the subthalamic nucleus in the substantia nigra, resulting in elevated oxidative stress and continued DAergic dysfunction. Findings from these studies will provide insight into the mechanisms underlying early-onset dopaminergic loss associated with a partial loss of GDNF and may identify therapeutic targets to reduce oxidative stress, nigrostriatal dopamine loss, and motor dysfunction occurring with aging and parkinsonism. In addition, results from these studies will serve to guide Dr. Boger's future independent research in the area of systems neuroscience. PUBLIC HEALTH RELEVANCE: Glial cell line-derived neurotrophic factor (GDNF) is reduced in Parkinson's disease patients and we have shown that mice with a partial loss of GDNF have accelerated age-related loss of motor function and nigrostriatal dopamine, but the relationship between GDNF reductions and dopamine loss is unknown. We propose that the dopamine loss as a result of long-term reduction of GDNF results in increased glutamate release from the subthalamic nucleus into the substantia nigra resulting in oxidative stress and continued dopaminergic damage. Furthermore, exogenous GDNF administration will alleviate the dopaminergic damage associated with increased glutamate toxicity as a result of a life-long reduction of GDNF.