Neuroplasticity, Caloric Restriction & Dopamine Function

16.卡路里限制和衰老：这项研究反映了PI的一个新的研究领域，并将重点放在神经化学通路的变化如何可能作为卡路里限制对大脑老化影响的中介这一基本问题上。具体地说，我们想要确定神经递质神经元细胞氧化还原状态的变化是否可能介导热量限制对老年大脑神经可塑性的影响。神经递质神经元已知在脑损伤后调节轴突生长和突触替换。多巴胺神经元是我们研究的重点，因为1)纹状体皮质损伤后的突触替换是由纹状体中的多巴胺细胞调制的，2)多巴胺神经元是特别的 随着年龄的增长易受氧化损伤，这是由于在多巴胺代谢过程中产生有毒的自由基，并形成可修饰细胞蛋白质和损害多巴胺神经元的多巴胺醌。具体地说，我们将测试这一假设，即Iowerinq细胞水平的抗氧化剂qlutathione(GSH)作为一种改变中纹状体多巴胺神经元细胞氧化还原状态的策略，会导致多巴胺氧化增加和蛋白质半胱氨酰儿茶酚的形成，从而破坏脑损伤后多巴胺神经元和扰乱神经可塑性的多巴胺调制。我们将通过一个组合来检验我们的假设 体外和体内实验模型，选择性地表达DOX诱导的多巴胺神经元中GSH水平的降低。目的1将使用GSH水平降低的PC12细胞来评估降低多巴胺神经元中细胞GSH对多巴胺自氧化的调节以及暴露于一氧化氮稳态浓度升高后蛋白质半胱氨酰儿茶酚的形成的影响，这是脑损伤后常见的情况。目的2将表征已知的参与调节纹状体突起生长和突触形成的候选分子(即pCREB、BDNF、GAP-43、SCG-10)在单侧皮质纹状体损伤后上调的时间顺序的变化，该跨基因小鼠的多巴胺能中纹状体神经元中GSH水平降低。来自这些先导研究的数据将为进一步的机制研究提供基础，这些机制研究将使用质谱仪确定哪些特定蛋白质在氧化应激条件下遭受损害或失去功能能力，以及未来的体内研究，以确定卡路里限制是否可以防止多巴胺神经元中与年龄相关的蛋白质修饰。

16. Caloric Restriction and Aging: This study reflects a new area of research for the PI and will focus on the fundamental question of how changes in neurochemical pathways may act as possible mediators of the caloric restriction effect on brain aging. Specifically, we want to determine whether alteration of the cellular redox state of neurotransmitter neurons, that are known to modulate neurite outgrowth and synapse replacement after brain injury, may mediate the caloric restriction effect on neural plasticity in the aged brain. Dopamine neurons are the focus of our investigation because 1) synapse replacement in the striatum after a corticostriatal lesion is modulated by mesostriatal dopamine cells, and 2) dopamine neurons are particularly vulnerable to oxidative insult with age due to the generation of toxic free radicals during dopamine metabolism and the formation of dopamine quinones that can modify cellular proteins and damage dopamine neurons. Specifically, we will test the hypothesis that Iowerinq cellular levels of the antioxidant .qlutathione (GSH), as a strategy to alter the cellular redox state of mesostriatal dopamine neurons, leads to an increase in dopamine oxidation and the formation of protein cysteinyl-catechols that can damaqe dopamine neurons and disrupt dopamine modulation of neural plasticity after brain iniury. We will test our hypothesis through a combination of in vitro and in vivo experimental models that selectively express dox-inducible reductions in the level of GSH in dopamine neurons. Aim 1 will use PC12 cells with reduced levels of GSH to assess the effect of lowering cellular GSH in dopamine neurons on the regulation of dopamine autoxidation and the formation of protein cysteinyl-catechols after exposure to an elevation in the steady-state concentration of nitric oxide, a condition typically seen after brain injury. Aim 2 will characterize changes in the temporal schedule of the upregulation of candidate molecules known to participate in the regulation of neurite outgrowth and synaptogenesis (i.e., pCREB, BDNF, GAP-43, SCG-10) in the striatum after a unilateral corticostriatal lesion in transqenic mice with reduced levels of GSH in dopaminergic mesostriatal neurons. Data from these pilot studies will provide the basis for additional mechanistic studies that will identify which particular proteins undergo damage or lose their functional capacity under conditions of oxidative stress using mass spectrometry, as well as, future in vivo studies to determine if age-related protein modifications in dopamine neurons are prevented by caloric restriction.