PROTEIN CARBONYLATION AND AXONAL DAMAGE IN EAE

描述（由申请人提供）：我们的长期目标是定义和描述多发性硬化症组织损伤的机制，多发性硬化症是人类中枢神经系统最常见的脱髓鞘疾病。近年来，氧化应激与多发性硬化症及其动物模型实验性变应性脑脊髓炎（EAE）的病理生理有关。活性氧（ROS）由活化的巨噬细胞/小胶质细胞释放或由神经细胞内功能失调的线粒体内源性产生。虽然有无可置疑的实验证据表明氧化应激在这些疾病中起因果作用，但ROS产生组织损伤的确切机制尚不清楚。活性氧积累的一个主要后果是非酶将醛或酮引入蛋白质的特定氨基酸残基（即羰基化）。基于最近关于其他系统中蛋白质羰基化的分子和细胞后果的报道以及我们实验室的一些重要发现，我们假设EAE中氧化应激的一个主要结果是神经元蛋白质的羰基化，这有助于组织损伤和轴突损伤。我们还提出了抑制蛋白质羰基化将是治疗这种疾病的想法。为了验证我们的假设，我们将测量缓解/复发EAE小鼠脊髓和大脑中蛋白质羰基的水平与疾病的病理标志（炎症、神经元死亡、轴突损伤和脱髓鞘）的关系。这些研究将采用最先进的生化和免疫细胞化学技术。然后，我们将通过氧化还原蛋白质组学确定疾病炎症和退行性阶段的氧化蛋白，并将从产生的羰基化氨基酸残基的类型确定氧化蛋白的化学性质和起源。最后，我们将研究各种羰基清除剂和代谢抑制剂在EAE动物中预防组织损伤和轴突损伤的能力。如果成功，这些研究将揭示氧化应激导致脱髓鞘疾病慢性残疾的新的分子机制。公共卫生相关性：多发性硬化症（MS）是一种神经系统疾病，在美国大约每700名年轻人中就有1人受到影响。我们最近观察到，一种叫做羰基化的特殊氧化过程改变了多发性硬化症患者的几种脑蛋白。这些修饰影响蛋白质功能，并可能导致这种毁灭性疾病的组织损伤。通过小鼠多发性硬化症模型，我们将确定降低蛋白质羰基化的药物是否能有效预防组织损伤和神经功能障碍。我们设想在未来，这些药物可以与抗氧化剂、抗炎药或神经活性物质联合使用，以改善慢性MS的临床管理。

DESCRIPTION (provided by applicant): Our long-term goal is to define and characterize the mechanisms underlying tissue injury in multiple sclerosis, the most common demyelinating disease of the central nervous system in humans. In recent years, oxidative stress has been implicated in the pathophysiology of both multiple sclerosis and its animal model, experimental allergic encephalomyelitis (EAE). Reactive oxygen species (ROS) are released by activated macrophages/microglia or are endogenously generated by dysfunctional mitochondria in the nerve cells. Although there is unquestionable experimental evidence demonstrating that oxidative stress plays a causal role in these disorders, the precise mechanism(s) by which ROS produces tissue damage is far from clear. A major consequence of ROS accumulation is the non-enzymatic introduction of aldehydes or ketones into specific amino acid residues of proteins (i.e. carbonylation). Based on recent reports regarding the molecular and cellular consequences of protein carbonylation in other systems and a number of important findings from our laboratory, we hypothesize that a major outcome of oxidative stress in EAE is the carbonylation of neuronal proteins, which contributes to tissue damage and axonal injury. We also put forth the idea that inhibition of protein carbonylation will be therapeutic in this disease. To test our hypothesis, we will measure the levels of protein carbonyls in the spinal cord and brain of remitting/relapsing EAE mice in relationship to well-established pathological hallmarks of the disease (inflammation, neuronal death, axonal damage and demyelination). These studies will employ state-of the-art biochemical and immunocytochemical techniques. We will then identify the oxidized proteins in the inflammatory and degenerative stages of the disease by redox proteomics, and will ascertain both the chemical nature and origin of the oxidizing species from the type of carbonylated amino acid residues produced. Finally, we will examine the ability of various carbonyl scavengers and metabolic inhibitors to prevent tissue injury and axonal damage in EAE animals. If successful, these studies will uncover a novel molecular mechanism by which oxidative stress causes chronic disability in demyelinating disorders. PUBLIC HEALTH RELEVANCE: Multiple sclerosis (MS) is a neurological disorder that affects approximately 1 in 700 young adults in the US. We have recently observed that a special type of oxidative process called carbonylation modifies several brain proteins from MS patients. These modifications affect protein function and likely contribute to tissue injury in this devastating disease. Using a mouse model of MS, we will determine whether drugs that reduce protein carbonylation can effectively prevent tissue damage and neurological deficits. We envision that in the future these agents could be administered in combination with antioxidants, anti-inflammatory or neuroactive substances for an improved clinical management of chronic MS.