OXIDATIVE STRESS, AGING AND BRAIN CA++ TRANSPORT SYSTEMS

这个项目要检验的假设是大脑中的衰老 导致质膜蛋白氧化变化增强 负责将钙离子运入或运出神经细胞，以及 这种变化的累积影响是运输功能的降低 这些蛋白质中。神经元质膜Na~+/Ca~(2+)交换器和 (Ca~(2+)+Mg~(2+))-ATPase是负责维持 神经细胞膜上钙离子的10/4倍梯度，以及 这些系统在衰老大鼠膜上的动力学特性变化 大脑。这两种转运系统对体外反应都很敏感。 氧物种(ROS)。这个项目的目标是：(1)描述 氧化应激对神经细胞Na~+/Ca~(2+)交换和血浆的影响 膜CaATPase活性，(2)确定氧化损伤是否 改变交换器的活性和结构属性，以及 ATPase的方式类似于在老化的脑膜中观察到的方式， (3)确定每个神经细胞膜内的肽段。 最易受氧化诱导的钙转运蛋白 并确定类似的更改是否可以在 这些蛋白质在老年人脑中的区域，以及(4)确定 氧化应激对钙离子周转动力学的影响 在原代神经元细胞培养系统中运输蛋白质 [Ca~(2+)]/i、细胞内pH和钙代谢状态的调节 可以在相同的条件下对细胞进行监测。In的影响 体外暴露于光氧化和化学诱导的自由基 将研究突触膜钙转运系统的形成。 在这些条件下，这些蛋白质本身 进行氧化修饰。高灵敏的微量分析 将使用技术来识别这些地点。可能发生的 这种随着脑神经元老化而发生的改变将会被评估，因为 慢性轻度氧化应激可能导致神经元受损 在很大比例的老年人口中发展起来的功能。这个 最终目标致力于获得更动态的图像 氧化应激对钙调节的多种影响 在具有氧化损伤修复机制的完整细胞中 蛋白质。众所周知，衰老的过程，无论它是什么 在细胞和分子水平上，是许多人的风险因素 病理情况，其中最严重的是痴呆症，比如晚期- 阿尔茨海默病的发病形式。拟议的研究将有助于 对于我们对氧化应激在年龄中可能起到的作用的理解- 神经功能的相关改变和我们对 更好地预防和/或管理这种压力的未来潜力 可以在保持认知能力方面有很好的作用 人的寿命。

The hypothesis to be examined in this project is that aging in the brain leads to enhanced oxidative changes in the plasma membrane proteins responsible for transporting Ca2+ into or out of nerve cells and the cumulative effect of such changes is a decrease in the transport function of these proteins. The neuronal plasma membrane Na+/Ca2+ exchanger and the (Ca2+ + Mg2+)-ATPase are the major systems responsible for maintaining the 10/4-fold gradient for Ca2+ across the neuronal membrane, and the kinetic properties of these systems change in membranes from aged rat brain. Both of these transport systems are sensitive to in vitro reactive oxygen species (ROS). The goals of this project are: (1) to characterize the effects of oxidative stress on neuronal Na+/Ca2+ exchanger and plasma membrane CaATPase activity, (2) to determine whether oxidative damage alters the activity and the structural properties of the exchanger and the ATPase in a manner similar to that observed in the aging brain membranes, (3) to identify the peptide domains within each of the neuronal membrane Ca2+ transporting proteins that are most susceptible to oxidation-induced alterations and determine whether similar alterations can be detected in these regions of the proteins in aged brain, and (4) to determine the effects of oxidative stress on the kinetics of turnover of the Ca2+ - transporting proteins in primary neuronal cell culture systems in which the regulation of [Ca2+]/i, intracellular pH, and the metabolic status of the cells can be monitored under the same conditions. The effects of in vitro exposure to photo-oxidizing as well as chemically-induced radical formation on synaptic membrane Ca2+ transport systems will be studied under conditions which will indicate whether the proteins themselves undergo oxidative modification. Highly sensitive microanalytical techniques will be used to identify the sites. The possible occurrence of such modifications with aging in brain neurons will be assessed, since chronic mild oxidative stress may contribute to the compromised neuronal function which develops in a large percentage of the aged population. The final aim is devoted to obtaining a more dynamic picture of the multiplicity of effects that oxidative stress can have on Ca2+-regulation in intact cells which have repair mechanisms for oxidative damage to proteins. It is known only too well that the aging process, whatever it is at the cellular and molecular levels, is a risk factor for many pathological conditions, one of the worst being dementia such as the late- onset form of Alzheimer's Disease. The proposed studies will contribute to our understanding of the role that oxidative stress may play in age- related alterations in neuronal function and to our assessment of the future potential that better prevention and/or management of such stresses can have in preserving cognitive abilities well into the later part of the human lifespan.