Mitochondrial Redox Systems in Neurogenic Hypertension

描述（由申请人提供）：脑血管紧张素II（AngII）信号转导失调与心血管疾病（包括心力衰竭和高血压）的发病机制有关。血管紧张素II循环水平的增加可通过作用于血脑屏障缺陷的脑区域（称为脑室周围器官（CVO））而导致神经源性高血压的发展。CVO的激活可以改变中枢心血管输出，包括交感神经兴奋。为了更好地理解AngII在神经源性高血压发展中的中心作用并鉴定该疾病的新治疗靶点，研究AngII在脑中的神经元内信号传导机制是必要的。以前，我们和其他人确定了活性氧（ROS），特别是超氧化物自由基（O2.-），由NADPH氧化酶产生，作为AngII刺激的中枢神经元中的重要信号中间体。然而，额外的O2来源。包括线粒体，这是氧气的主要场所。在大多数细胞中，还有待研究。此外，NADPH氧化酶和O2之间的潜在联系。在神经元内的信号传导的血管紧张素II仍有待阐明。在此，我们提出了一系列分子、生物化学和综合心血管生理学实验来验证我们的假设，即AngII敏感神经元中的NADPH氧化酶有助于线粒体O2-的增加。其又通过作用于已知控制神经元放电和交感神经兴奋的氧化还原敏感蛋白而介导AngII依赖性高血压。我们将在三个具体目标中检验这一假设。在具体目标1中，我们将研究NADPH氧化酶在产生O2中的作用。在AngII刺激的神经元中。这一目标建立在我们的初步数据基础上，这些数据表明AngII增加了线粒体O2。NADPH氧化酶催化亚单位（Nox 4）存在于神经元的线粒体中。在具体目标2中，我们将研究生物产生O2的机制。调节血管紧张素II诱导的神经元激活。这一目标得到了我们新的初步数据的支持，表明线粒体O2清除的增加。通过腺病毒介导的锰超氧化物歧化酶（MnSOD）的过度表达，O2.-特异性靶向线粒体的清除酶，减弱AngII诱导的神经元钾电流抑制。最后，在具体目标3中，我们将通过研究脑内产生的O2的作用来扩展我们先前的观察，即脑中MnSOD过表达抑制急性中枢AngII诱导的升压反应。在AngII依赖性神经源性高血压的小鼠模型中，在脑中介导慢性交感神经兴奋和高血压的发展。这些研究将为AngII的神经元内信号传导机制提供新的信息，并可能识别神经元定位的O2。作为血管紧张素II依赖性神经心血管疾病（如高血压）的重要治疗靶点。 公共卫生相关性：高血压和心力衰竭是两种与大脑异常刺激相关的心血管疾病。自由基和氧化剂产生的脑细胞称为神经元已被证明是参与这些心血管疾病。通过研究和确定产生这些氧化剂的特定神经元细胞位置，该项目将促进开发新疗法的机会，这些新疗法可能需要针对特定的细胞位置，以改善高血压的治疗。

DESCRIPTION (provided by applicant): Dysregulation of brain angiotensin II (AngII) signaling is implicated in the pathogenesis of cardiovascular diseases, including heart failure and hypertension. Increased circulating levels of AngII can lead to the development of neurogenic hypertension by acting on blood-brain-barrier deficient brain regions, known as circumventricular organs (CVOs). Activation of the CVOs can alter central cardiovascular outputs including sympatho-excitation. To better understand the central actions of AngII in the development of neurogenic hypertension and to identify novel therapeutic targets of the disease, it is essential to investigate the intra- neuronal signaling mechanisms of AngII in the brain. Previously, we and others identified reactive oxygen species (ROS), particularly superoxide radicals (O2.-), generated by NADPH oxidase as important signaling intermediates in central neurons stimulated with AngII. However, additional sources of O2.-- including mitochondria, which are the primary sites for O2.- generation in most cells, have yet to be investigated. In addition, a potential link between NADPH oxidase and mitochondria-derived O2.- in the intra-neuronal signaling of AngII remains to be elucidated. Herein, we propose a series of molecular, biochemical, and integrative cardiovascular physiological experiments to test our hypothesis that a mitochondria-localized NADPH oxidase in AngII-sensitive neurons contributes to an increase in mitochondrial O2.-, which in turn mediates AngII-dependent hypertension by acting on redox-sensitive proteins known to control neuronal firing and sympatho-excitation. We will test this hypothesis in three Specific Aims. In Specific Aim 1, we will investigate the role of NADPH oxidase in producing mitochondria-localized O2.- in AngII-stimulated neurons. This aim builds upon our preliminary data showing that AngII increases mitochondrial O2.- in cultured neurons and that a NADPH oxidase catalytic subunit (Nox4) is present in mitochondria of neurons. In Specific Aim 2, we will examine the mechanisms by which mitochondrial-produced O2.- regulates AngII- induced neuronal activation. This aim is supported by our new preliminary data indicating that increased scavenging of mitochondrial O2.- via adenoviral-mediated overexpression of manganese superoxide dismutase (MnSOD), the O2.- scavenging enzyme specifically targeted to mitochondria, attenuates AngII- induced inhibition of neuronal potassium current. Finally, in Specific Aim 3, we will expand our previous observation that MnSOD overexpression in the brain inhibits the acute central AngII-induced pressor response by investigating the role of mitochondrial-produced O2.- in the brain in mediating the chronic sympatho-excitation and the development of hypertension in a mouse model of AngII-dependent neurogenic hypertension. These studies will provide new information on the intra-neuronal signaling mechanisms of AngII, and may identify mitochondrial-localized O2.- in neurons as important therapeutic targets in AngII-dependent neuro-cardiovascular diseases, such as hypertension. PUBLIC HEALTH RELEVANCE: Hypertension and heart failure and are two cardiovascular diseases associated with abnormal stimulation in the brain. Free radicals and oxidants produced in brain cells called neurons have been shown to be involved in these cardiovascular diseases. By investigating and identifying the specific neuronal cell location where these oxidants are generated this project will advance opportunities to develop new therapeutics that may need to be targeted to specific cellular locations for the improved treatment of hypertension.