Excitatory signaling and oxidative phosphorylation alterations in schizophrenia

 描述(由申请人提供)：精神分裂症是一种复杂的疾病，缺乏有效的治疗方案来治疗患者所经历的普遍和衰弱的认知障碍。自从我的博士训练开始以来，我一直对识别导致这些损伤的潜在回路改变非常感兴趣，以便它们可以得到治疗。尽管我的关注点很单一，但在我的科学生涯中，用于研究这个问题的方法和概念框架已经演变，而这个K01应用程序代表着这一演变的下一个重要步骤。这个K01应用包括研究、临床、 和职业发展，以及教学/培训机会，以最大限度地提高我成功过渡到独立的能力。我的长期职业目标是：1)确定和描述导致精神分裂症患者皮质功能障碍的分子和细胞变化；2)使用动物模型确定这些观察到的病理变化的可能原因；3)利用这些发现开发针对精神分裂症患者认知障碍的病理生理学药物疗法；4)在一流大学建立一个独立的研究实验室，以实现这些目标；以及5)指导学生和博士后研究员，为下一代科学家和创新者做出贡献。我的短期职业目标包括1)掌握拟议的研究技术，包括激光捕获人类和啮齿动物皮层单细胞群体的显微解剖，人类前额叶皮质组织的电子显微镜线粒体分析，以及用于啮齿动物研究的由设计药物(DREADDS)范式独家激活的shRNA和设计受体(DREADDS)范例；2)掌握实验设计，以确定疾病的发现是原因、补偿、后果还是困惑；以及3)成功地从匹兹堡大学的教员过渡到助理教授。重要的是，通过K01奖项完成这些短期目标开始解决长期目标1-3，并将我带到实现剩余长期目标的轨道上。目前的培训计划还增加了[[针对每个主要培训目标的正式课程]]、侧重于科学职业发展的互动式讲习班和教学经验，以进一步拓宽我的职业技能集。匹兹堡大学医学院的精神病学系是实现这些短期和长期目标的理想环境。该科室在临床研究、治疗和培训方面处于全国领先地位。在精神病学主席大卫·刘易斯博士的主要指导下，我将完全访问他的实验室和当前应用程序所需的所有基础设施支持。工作记忆是精神分裂症患者的一种核心认知功能受损，依赖于前额叶皮质(PFC)电路的激活。因此，被诊断为精神分裂症的人在执行工作记忆任务时，PFC激活减少。这种较低的PFC激活似乎是疾病病理生理学的组成部分，而不仅仅是表现不佳的反映。因此，必须确定导致精神分裂症患者PFC神经元活性降低的细胞和电路变化，以便确定适当的治疗靶点。这项研究计划的重点是确定两种离散的可能的分子/生理障碍中的哪一种可能是导致精神分裂症患者PFC损害的上游事件。支持神经元兴奋是大脑中最耗能的活动，由线粒体通过氧化磷酸化(OXPHOS)合成ATP提供。越来越多的证据表明，在精神分裂症患者的PFC中，末端和限速的氧磷酶--细胞色素C氧化酶(COX)的表达水平较低。因此，这一K01应用的研究目标是确定导致精神分裂症受试者PFC中COX水平较低的潜在机制。COX降低可能是神经元兴奋性的慢性降低导致受影响神经元的ATP需求降低(假说1)，或者是由于COX表达不足而削弱了所有神经元的代谢能力(假说2)。区分这些替代方案对于确定精神分裂症皮质功能障碍的合适治疗靶点具有重要意义，因为H1表示靶向兴奋增强，而H2表示增强COX表达以恢复线粒体呼吸。为了检验哪一种假说最受影响个体的研究结果的支持，我们使用激光显微解剖技术对三个不同神经元群体的样本进行了解剖，并使用定量聚合酶链式反应来测量COX相关转录本的表达(Aim 1.1)，并使用体视学电子显微镜对精神分裂症和健康对照受试者的PFC中线粒体的丰度和形态进行了量化(Aim 1.2)。然而，因为不能用人类死后组织来确定因果关系，所以在目标2和3中使用了实验动物模型来直接测试H1和H2的机制。在目标2中，DREADD药物遗传学技术被用来诱导PFC锥体细胞兴奋的长期减少，并对目标1的每一项措施进行了评估。在目标3中，病毒传递shRNA的方法被用来损害COX在PFC中的可用性，并评估了目标1中的每一项指标。总之，这些目的提供了疾病现象的明确特征，并扩展到动物模型的概念验证研究，以提供令人信服的、收敛的和决定性的数据，说明在疾病中起作用的机制是什么。

 DESCRIPTION (provided by applicant): Schizophrenia is a complex disorder lacking an effective treatment option for the pervasive and debilitating cognitive impairments experienced by patients. I have been acutely interested in identifying the underlying circuitry alterations tha contribute to these impairments, so that they may be treated, since the start of my doctoral training. Though my focus has been singular, the approaches and conceptual framework used to study this problem have evolved over my scientific career, and this K01 application represents the next major step in that evolution. This K01 application includes research, clinical, and career development, along with teaching/training opportunities to maximize my ability to successfully transition to independence. My long term career goals are to 1) identify and describe the molecular and cellular alterations that contribute to cortical dysfunction in schizophrenia, 2) determine the possible causes of these observed pathologies using animal models, 3) use these findings to develop pathophysiology-based pharmacological treatments for cognitive impairment in schizophrenia patients, 4) establish an independent research laboratory at a top-tier university to accomplish these goals, and 5) mentor students and postdoctoral fellows to contribute to the next generation of scientists and innovators. My short term career goals include 1) master the research techniques proposed, including laser capture microdissection of single cell populations from human and rodent cortex, electron microscopic mitochondrial analysis of human prefrontal cortical tissue, and shRNA and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) paradigms for rodent studies; 2) master experimental design to determine whether disease findings are a cause, compensation, consequence or confound; and 3) successfully transition as a faculty member from Instructor to Assistant Professor at the University of Pittsburgh. Importantly, completing these short terms goals via this K01 award begins to address long term goals 1 - 3, and puts me on a trajectory to accomplish the remaining long term objectives. The current training plan is also augmented with [[formal courses that address each of the major training goals]], interactive workshops focused on scientific career development, and teaching experiences to further broaden my career skill set. The Department of Psychiatry at the University of Pittsburgh's School of Medicine is an ideal environment in which to accomplish these short and long term goals. This department is a national leader in clinical research, treatment and training. Under the primary mentorship of Dr. David Lewis, Chair of Psychiatry, I will have full access to his laboratory and all of the infrastructure support required for the current application. Working memory is a core cognitive function impaired in schizophrenia that depends upon activation of prefrontal cortex (PFC) circuitry. Accordingly, individuals diagnosed with schizophrenia show reduced PFC activation while performing working memory tasks. This lower PFC activation appears to be an integral part of the disease pathophysiology, and not simply a reflection of poor performance. Thus, the cellular and circuitry alterations that underlie lower PFC neuronal activity in schizophrenia must be determined in order to identify appropriate therapeutic targets. This research proposal focuses on determining which of two discrete possible molecular/physiological disturbances is a likely upstream event leading to PFC impairments in schizophrenia. Supporting neuronal excitation represents the largest energy-consuming activity in the brain, supplied by ATP synthesis in mitochondria via oxidative phosphorylation (OXPHOS). Accumulating evidence indicates that expression of the terminal and rate-limiting OXPHOS enzyme, cytochrome c oxidase (COX), is lower in the PFC of schizophrenia subjects. Thus, the research goal of this K01 application is to determine the underlying mechanism contributing to lower levels of COX in the PFC of schizophrenia subjects. Lower COX could be a consequence of chronic reductions in neuronal excitation that lower ATP demand in the affected neurons (Hypothesis 1), or due to deficient COX expression that impairs metabolic capacity in all neurons (Hypothesis 2). Distinguishing between these alternatives has important implications for identifying appropriate therapeutic targets for cortical dysfunction in schizophrenia, as H1 indicates targeted enhancement of excitation, whereas H2 indicates enhancing COX expression to recover mitochondrial respiration. In order to test which hypothesis is most supported by findings in affected individuals, laser microdissection is used to dissect samples of three distinct neuronal populations and quantitative PCR is used to measure the expression of COX-related transcripts (Aim 1.1), and stereological electron microscopy is used to quantify mitochondrial abundance and morphology (Aim 1.2) in the PFC of schizophrenia and healthy comparison subjects. However, because cause- and-effect relationships cannot be determined using human postmortem tissue, experimental animal models are used in Aims 2 and 3 to directly test the mechanisms of H1 and H2. In Aim 2, DREADD pharmacogenetic technique is used to induce long-term reductions in PFC pyramidal cell excitation, and each measure from Aim 1 is assessed. In Aim 3, viral delivery of shRNA approach is used to impair COX availability in the PFC, and each measure from Aim 1 is assessed. Together, these Aims provide a definitive characterization of the disease phenomenon, and extend to proof-of-concept studies in animal models to provide compelling, convergent and conclusive data regarding which mechanism is operative in the illness.