Mitochondrial sites of reactive oxygen species generation in cells.

描述（由申请人提供）：衰老的主要理论是线粒体自由基理论，该理论提出细胞内线粒体产生活性氧（ROS）是细胞损伤和衰老的主要原因。然而，这个理论充其量是不完整的。为了严格地测试它并操纵自由基的产生以促进健康，因此必须充分了解线粒体自由基产生的机制和特定位点，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。尽管进行了大量研究，但目前缺乏这种理解。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点的细胞线粒体ROS生成速率来解决这一缺陷。以前的尝试，以确定所涉及的网站，导致混乱，因为一个概念上的问题：定义的网站，电子传递抑制剂的添加，但他们不可避免地扭曲电子在链中的分布和模式的活性氧生产。这个问题将通过一个关键的创新来规避：在第一阶段中使用抑制剂来定义位点，在该第一阶段中，根据来自每个位点的ROS产生来校准内源性报告信号，但是当在单个实验条件下使用内源性报告信号来计算来自每个位点的ROS产生时，当几个位点可以同时操作时，在测量阶段中省略它们。待检验的假设是，线粒体电子传递链中至少有一个位点在细胞中产生显著的ROS，并且是主要的氧化应激发生器。在线粒体自由基理论中，该位点产生的ROS是衰老及其疾病的主要原因。该假设将通过严格的逐步方法进行研究：首先从模型系统（分离的线粒体）中的每个位点测定速率，然后在此平台上构建以测定细胞中ROS产生的位点。研究将有以下具体目标。(1)建立并验证在不存在电子传递抑制剂的情况下，从分离的线粒体的电子传递链中的不同位点产生ROS的测量。(2)使用这些方法，确定在不同年龄的不同状态下氧化不同底物的不同衰老相关组织中分离的线粒体中每个位点的ROS产生的相对速率。(3)将方法转移到细胞中，并确定每个线粒体位点的ROS产生的相对速率和线粒体ROS产生的定量重要性。我们将使用突触体和实验上可接受的细胞（包括神经元、星形胶质细胞、正常和衰老的成纤维细胞、C2 C12成肌细胞和胰腺胰岛素瘤细胞）氧化不同状态的生理底物。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使我们能够显着减少疾病负担并增加人类健康寿命-即使与年龄相关的细胞损伤略有减少也可能具有广泛的公共卫生益处。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。该项目将使用创新的逐步方法来识别、测量和比较产生这些自由基的特定细胞位点，为降低自由基产生和减少衰老疾病的合理策略开辟道路。

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.