OXIDATIVE STRESS, AGING AND BRAIN CA++ TRANSPORT SYSTEMS

在这个项目中要检验的假设是，大脑中的衰老 导致质膜蛋白质的氧化变化增强 负责将Ca 2+转运入或转运出神经细胞， 这种变化的累积效应是运输功能的降低 这些蛋白质。 神经元质膜Na+/Ca ~（2+）交换体和 （Ca ~（2+）+Mg ~（2+））-ATPase是维持细胞内Ca ~（2+）含量的主要系统 跨神经元膜的Ca 2+的10/4倍梯度， 这些系统在老年大鼠膜中动力学性质变化 个脑袋 这两种转运系统都对体外反应敏感。 氧物种（ROS）。 本项目的目标是：（1）表征 氧化应激对神经元Na ~+/Ca ~（2+）交换体和血浆的影响 膜CaATP酶活性，（2）确定是否有氧化损伤 改变了交换剂的活性和结构性质， ATP酶以类似于在老化的脑膜中观察到的方式， (3)为了鉴定每个神经元膜内的肽结构域 最易受氧化诱导的Ca 2+转运蛋白 改变，并确定是否可以检测到类似的改变， 这些区域的蛋白质在老年大脑，（4）以确定 氧化应激对细胞内Ca 2 + - 在原代神经元细胞培养系统中， [Ca 2 +]/i、细胞内pH和细胞内钙离子代谢状态的调节 可以在相同的条件下监测细胞。 影响在 体外暴露于光氧化以及化学诱导的自由基 将研究突触膜Ca 2+转运系统的形成 在一定的条件下， 进行氧化改性。 高灵敏度微量分析 技术将被用来确定网站。 可能发生 将评估脑神经元中随着老化的这种改变，因为 慢性轻度氧化应激可能导致受损的神经元 这一功能在老年人口中占很大比例。 的 最后的目标是致力于获得一个更动态的图片， 氧化应激对Ca 2+调节的多种影响 在具有氧化损伤修复机制的完整细胞中， proteins. 众所周知，衰老过程，无论它是什么， 是在细胞和分子水平上，是许多人的风险因素， 病理条件，其中最糟糕的是痴呆症，如晚期- 阿尔茨海默病的发病形式。 拟议的研究将有助于 我们对氧化应激在衰老中的作用的理解 神经元功能的相关改变以及我们对 未来的潜力，更好地预防和/或管理这种压力， 可以在后期保存认知能力， 人类寿命

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.