Mitochondrial sites of reactive oxygen species generation in cells.

描述(申请人提供)：衰老的主导理论是线粒体自由基理论，该理论认为细胞内线粒体产生的活性氧物种(ROS)是细胞损伤和衰老的主要原因。然而，这一理论充其量也是不完整的。因此，为了对其进行严格的测试，并操纵自由基的产生以促进健康寿命，充分了解线粒体自由基产生的机制和特定部位，以评估其在年龄相关疾病和衰老中的作用，最终允许合理设计有益的治疗方法是至关重要的。尽管有很多研究，但目前还缺乏这样的理解。这项研究将通过测量与衰老相关的细胞，特别是神经元、肌肉细胞和成纤维细胞中电子传递链中每个特定位置的细胞线粒体ROS的生成速度来解决这一缺陷。以前识别所涉及的位点的尝试由于一个概念性问题而导致混淆：为了定义这些位点，添加了电子传输抑制剂，但它们不可避免地扭曲了链中的电子分布和ROS产生的模式。一项关键的创新将绕过这个问题：在第一阶段使用抑制剂来定义位点，在第一阶段中，根据每个站点的ROS产生来校准内源报告信号，但在测量阶段，当几个站点可能同时运行时，当使用内源报告信号来计算单个实验条件下每个站点的ROS生成时，省略它们。需要检验的假设是，线粒体电子传输链中至少有一个位置在细胞中产生显著的ROS，是主要的氧化应激生成器。在线粒体自由基学说中，该部位ROS的产生可能是衰老及其疾病的主要原因。这一假说将通过一种严格的循序渐进的方法进行研究：首先测定模型系统(分离的线粒体)中每个位置的速率，然后在这个平台上分析细胞中产生ROS的位置。研究将有以下具体目标。(1)在没有电子传递抑制剂的情况下，建立和验证分离的线粒体电子传递链中不同位置产生ROS的测量结果。(2)用这些方法测定不同衰老相关组织、氧化不同底物的不同状态、不同年龄的线粒体中每个部位产生ROS的相对速率。(3)将这些方法移植到细胞中，并确定每个线粒体部位产生ROS的相对速率和线粒体产生ROS的数量重要性。我们将使用突触体和实验可听化细胞(包括神经元、星形胶质细胞、正常和衰老的成纤维细胞、C2C12成肌细胞和胰腺胰岛素瘤细胞)在不同的状态下氧化生理底物。公共卫生相关性：由于年龄是许多疾病的主要风险因素，了解衰老的机制可能会使我们显著减轻疾病负担，提高人类健康寿命--即使与年龄相关的细胞损害略有减少，也可能对公众健康产生广泛的好处。在正常氧化代谢过程中产生的自由基会损害细胞，并可能导致衰老。这个项目将使用一种创新的循序渐进的方法来识别、测量和比较产生这些自由基的特定细胞位置，为制定合理的策略来降低自由基的产生和减少衰老疾病开辟道路。

DESCRIPTION (provided by applicant): The dominant theory of aging is the mitochondrial free radical theory, which proposes that the production of reactive oxygen species (ROS) by intracellular mitochondria is the primary cause of cellular damage and aging. However, the theory is at best incomplete. To test it critically and to manipulate radical production to promote healthspan, it is therefore crucial to fully understand the mechanisms and specific sites of mitochondrial radical generation to assess its role in age-related diseases and aging, ultimately to allow rational design of beneficial therapies. Despite much research, such understanding is currently lacking. This study will address this deficit by measuring the rate of cellular mitochondrial ROS generation at each specific site in the electron transport chain in cells relevant to aging, particularly neurons, muscle cells and fibroblasts. Previous attempts to identify the sites involved have led to confusion because of a conceptual problem: to define the sites, electron transport inhibitors are added, but they inevitably distort electron distribution in the chain and the pattern of ROS production. This problem will be circumvented by a key innovation: using inhibitors to define sites in a first phase in which endogenous reporter signals are calibrated in terms of ROS production from each site, but omitting them in the measuring phase when the endogenous reporter signals are used to calculate ROS generation from each site under a single experimental condition when several sites may operate simultaneously. The hypothesis to be tested is that at least one site in the mitochondrial electron transport chain produces significant ROS in cells and is the main oxidative stress generator. In the mitochondrial free radical theory, ROS generation at this site would be the primary cause of aging and its diseases. The hypothesis will be investigated by a rigorous stepwise approach: first assay rates from each site in a model system (isolated mitochondria), then build on this platform to assay the sites of ROS production in cells. Studies will have the following specific aims. (1) Establish and validate measurements of ROS production from different sites in the electron transport chain of isolated mitochondria in the absence of electron transport inhibitors. (2) Using these methods, determine the relative rate of ROS production by each site in isolated mitochondria from different ageing-relevant tissues, oxidizing different substrates in different states, at different ages. (3) Transfer the methods to cells and establish the relative rate of ROS production by each mitochondrial site and the quantitative importance of mitochondrial ROS production. We will use synaptosomes and experimentally-amenable cells (including neurons, astrocytes, normal and senescent fibroblasts, C2C12 myoblasts, and pancreatic insulinoma cells oxidizing physiological substrates in different states. PUBLIC HEALTH RELEVANCE: Since age is the primary risk factor for many diseases, understanding the mechanisms of aging may allow us to significantly reduce the burden of disease and increase human healthspan - even a small decrease in age-related cellular damage may have widespread public health benefits. Free radicals generated during normal oxidative metabolism damage cells and may cause aging. This project will use an innovative stepwise approach to identify measure and compare the specific cellular sites at which these radicals are produced, opening the way for rational strategies to lower radical production and decrease the diseases of aging.