ATP1A3 Induced Alterations to Glutamate Signaling Protein Networks in Schizophrenia

ATP1A3 诱导精神分裂症谷氨酸信号蛋白网络的改变

• 批准号: 8947117
• 负责人: Matthew L MacDonald
• 金额: $ 15.77万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-16 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8947117
• 关键词: ATP1A3 protein; Animal Model; Area; Auditory; Auditory Hallucination; Auditory area; Autopsy; Award; Behavior; Bioinformatics; Biology; Brain region; Cells; Code; Complex; Data; Data Set; Dendritic Spines; Development; Dimensions; Discipline; Disease; Drug Targeting; Dystonia 12; Excitatory Amino Acid Antagonists; Fluorescence Microscopy; Functional disorder; Funding; Future; Gene Proteins; Genes; Genetic; Glutamates; Goals; Human; Impaired cognition; Impairment; Individual; Institution; Investigation; Knockout Mice; Laboratories; Lasers; Lead; Learning; Link; Maps; Mass Spectrum Analysis; Measures; Medical Research; Mentors; Mentorship; Methods; Microdissection; Microscopy; Modeling; Molecular; Mus; Mutation; Neurobehavioral Manifestations; Neurons; Ontology; Pathologic Processes; Pathology; Pathway Analysis; Patients; Peptides; Preparation; Process; Proteins; Proteomics; Psychotic Disorders; Pump; Pyramidal Cells; Reporting; Role; Schizophrenia; Signal Pathway; Signaling Protein; Specificity; Structure; Symptoms; Synapses; Syndrome; Systems Biology; Testing; Tissue Model; Training; United States National Institutes of Health; Vertebral column; Weight; base; brain tissue; career; cell type; density; differential expression; drug discovery; genetic risk factor; glutamatergic signaling; gray matter; hippocampal pyramidal neuron; human tissue; inhibitory neuron; interest; laser capture microdissection; mouse model; neuronal cell body; neuropsychiatry; novel; novel therapeutics; protein expression; psychotic symptoms; public health relevance; research study; risk variant; signal processing; social cognition; statistics; translational study

 DESCRIPTION (provided by applicant): Schizophrenia (SZ) is a lifelong and devastating psychiatric illness with limited treatment options and no cure. Reduced dendritic spine density of layer 3 pyramidal neurons is among the most consistently reported findings in postmortem studies of SZ and has been reported in multiple brain regions including the primary auditory cortex (Al). This cytoarchitectonic abnormality is believed to underlie several symptom dimensions in SZ, including auditory processing deficits that impair social cognition and auditory hallucinations. Glutamate (Glu) signaling is essential for dendritic spine integrity and multiple lines of evidence implicate synaptic Glu signaling in SZ pathology. Many SZ risk loci code for proteins in the synaptic Glu signaling network and Glu receptor antagonists can induce or exacerbate SZ symptoms in humans and animal models. Thus, current evidence supports a model in which genetic risk factors converge on Glu signaling protein networks leading to the impairments in synaptic structure and activity believed to underlie SZ symptoms. As a first step in testing this model I utilized a targeted-mass spectrometry (MS) approach to quantify over 150 synaptic proteins in Al gray matter homogenates from 23 SZ and matched control subjects. The proteins altered in SZ were enriched for the Gene Ontology term Glutamate Signaling Pathway (p=2.5E-6, q=1.5E-3). Weighted gene co-expression network analysis (WGCNA) revealed a general decrease in the correlated expression of synaptic proteins (p=0.015). Both the dysregulated Glu Signaling Pathway and the de-correlated protein network included the Na+/K+ pump subunit ATP1A3. ATP1A3 mutations contribute to polygenic burden for SZ, and are linked to rapid onset dystonia parkinsonism, a syndrome which presents with cognitive impairments, and in which nearly 20% of patients have comorbid psychosis characterized by auditory hallucinations. Similarly, ATP1A3 +/- mice, in which ATP1A3 expression is decreased by only 15-20%, display cognitive impairments and psychosis like behaviors. My preliminary data further indicates altered expression of Glu signaling proteins in ATP1A3 +/- mouse cortex. Based on these observations I hypothesize that: The Glu signaling protein network is altered in Al deep layer 3 of SZ subjects and that decreased ATP1A3 protein expression contributes to these network alterations and Al deep layer 3 spine loss. I will test this hypothesis using a novel lasr capture microdissection-targeted MS (LCM-tMS) approach to identify Glu protein network alterations linked to spine loss directly in Al deep layer 3 of SZ subjects (Aim 1). Next, I will determine the impact of a 20% decrease in ATP1A3 on Al protein networks and spine density in ATP1A3 +/- mice (Aim 2). Finally, I will utilize quantitative fluorescence microscopy to localize alterations in ATP1A3, as well as selected protein alterations shared by SZ subjects and ATP1A3 +/- mice, to pyramidal or inhibitory cell soma within Al deep layer 3 of SZ subjects (Aim 3). Findings from these studies mapping synaptic Glu protein network impairments in disease will support ongoing drug discovery efforts, especially if homeostatic and/or pathological processes alter the expression of these drug targets in unexpected ways, an assertion strongly supported by my preliminary data. They will also identify novel protein and protein co- expression network alterations for future hypothesis testing (Aims 1 & 2). Localizing ATP1A3 reductions in patients will support the development of cell type specific animal models (Aims 2 & 3). These studies will also support my long term career goal of investigating synaptic pathology in SZ and related neuropsychiatric illnesses in my own NIH funded laboratory at a major medical research institution. They will provide training in three areas essential to achieving this goal: 1. MS based proteomic and systems biology approaches to protein network analysis: MS approaches are rapidly evolving and can now quantify tens-of-thousands of peptides from thousands or proteins in a single experiment. During this award I will receive continued training in these rapidly emerging MS methods and in the statistic, bioinformatic, and systems biology disciplines required for network analysis of complex proteomic data sets. 2. Translational mechanistic studies in genetic mouse models: Descriptive studies in postmortem brain tissue, while essential for identifying molecular alterations in disease, cannot determine the contribution of individual genes/proteins to pathophysiology. In the planned studies I will learn to investigate the effects of a candidate protein on synaptic protein networks and structural pathology, focused on the Na+/K+ pump and the role of ATP1A3. 3. Cortical cell and circuit pathology in SZ: The impact of dysregulated protein expression on cortical function depends on cell and circuit localization. Thus, I require training in laser capture microdissection and quantitative fluorescence microscopy to investigate proteins and protein networks within the context of cortical cells and circuits. To achieve these training aims I have assembled an outstanding mentorship team with primary mentor, Dr. Robert Sweet, a world leader in quantitative fluorescent microscopy and cortical cell and circuit pathology in SZ and Dr. Nathan Yates, an internationally recognized expert in MS based proteomics. The mentorship team has been augmented by the inclusion of consultants with additional expertise in laser capture, statistics, bioinformatics, systems biology, and Na+/K+ pump biology. At the completion of the proposed studies and training I will be an expert in applying cutting-edge proteomic, systems biology, and microscopy approaches to studies of SZ synaptic pathology in human tissue and animal models. Thus, in addition to serving as a compelling protein of interest in SZ, ATP1A3 will serve as a test case for advancing proteins of interest from MS discovery through translational investigations.

 描述（由申请人提供）：精神分裂症（SZ）是一种终生且具有毁灭性的精神疾病，治疗选择有限且无法治愈。第 3 层锥体神经元树突棘密度降低是 SZ 尸检研究中最一致报告的发现之一。据报道，这种细胞结构异常存在于包括初级听觉皮层 (Al) 在内的多个大脑区域，是 SZ 多种症状的基础，包括损害社会认知和认知功能的听觉处理缺陷。谷氨酸 (Glu) 信号传导对于树突棘完整性至关重要，并且多种证据表明突触 Glu 信号传导与 SZ 病理学有关。因此，目前的证据支持这样一种模型：遗传风险因素集中在 Glu 信号蛋白网络上，导致突触结构和活动受损。作为测试该模型的第一步，我利用靶向质谱 (MS) 方法对来自 23 名 SZ 和匹配对照受试者的 Al 灰质匀浆中的 150 多种突触蛋白进行了定量。基因本体术语谷氨酸信号通路（p=2.5E-6，q=1.5E-3）加权基因共表达网络分析（WGCNA）显示普遍下降。突触蛋白的相关表达（p=0.015）。失调的 Glu 信号通路和去相关蛋白网络都包括 Na+/K+ 泵亚基 ATP1A3 突变，导致 SZ 的多基因负担，并与快速发病有关。肌张力障碍帕金森症，一种表现为认知障碍的综合征，其中近 20% 的患者患有以幻听为特征的共病精神病。同样，ATP1A3 +/- 小鼠的 ATP1A3 表达仅减少 15-20%，表现出认知障碍和精神病样行为。我的初步数据表明 ATP1A3 +/- 小鼠皮质中 Glu 信号蛋白的表达进一步改变。通过这些观察，我注意到： SZ 受试者的 Al 深层 3 中的 Glu 信号蛋白网络发生了改变，并且 ATP1A3 蛋白表达的减少导致了这些网络改变和 Al 深层 3 脊柱损失。我将使用一种新型激光捕获显微切割靶向 MS (LCM-tMS) 方法来测试这一假设，以识别与 SZ 受试者的 Al 深层 3 中的脊柱损失直接相关的 Glu 蛋白网络改变（目标 1）。 ATP1A3 减少 20% 对 ATP1A3 +/- 小鼠中 Al 蛋白网络和脊柱密度的影响（目标 2）。 ATP1A3 以及 SZ 受试者和 ATP1A3 +/- 小鼠共有的选定蛋白质改变，对 SZ 受试者 Al 深层 3 内的锥体细胞或抑制细胞体进行改变（目标 3），这些研究的结果映射了疾病中的突触 Glu 蛋白网络损伤。将支持正在进行的药物发现工作，特别是如果稳态和/或病理过程以意想不到的方式改变这些药物靶标的表达，这一断言得到了我的初步数据的强烈支持。他们还将识别新的蛋白质和蛋白质。用于未来假设检验的共表达网络改变（目标 1 和 2）将支持细胞类型特异性动物模型的开发（目标 2 和 3）。在我自己的 NIH 资助的一家大型医学研究机构的实验室中，我正在研究 SZ 的突触病理学和相关的神经精神疾病。他们将提供实现这一目标所必需的三个领域的培训。 目标： 1. 基于 MS 的蛋白质组学和系统生物学方法进行蛋白质网络分析：MS 方法正在迅速发展，现在可以在一次实验中量化数千种蛋白质中的数以万计的肽。这些快速新兴的 MS 方法以及复杂蛋白质组数据集网络分析所需的统计、生物信息学和系统生物学学科。 2. 遗传小鼠模型的转化机制研究：死后大脑的描述性研究。组织虽然对于识别疾病的分子改变至关重要，但无法确定其贡献 在计划的研究中，我将学习研究个体基因/蛋白质的病理生理学。 候选蛋白对突触蛋白网络和结构病理学的影响，重点关注 Na+/K+ 泵和 ATP1A3 的作用。 3. SZ 中的皮质细胞和回路病理学：失调蛋白表达对皮质功能的影响取决于细胞和功能。因此，我需要激光捕获显微切割和定量荧光显微镜方面的培训，以研究皮质细胞和电路背景下的蛋白质和蛋白质网络。为了实现这些培训目标，我组建了一支优秀的指导团队。导师是罗伯特·斯威特博士（深圳定量荧光显微镜和皮层细胞和回路病理学领域的世界领先者）和内森·耶茨博士（国际公认的基于 MS 的蛋白质组学专家）。在激光捕获、统计学、生物信息学、系统生物学和 Na+/K+ 泵生物学方面的专业知识完成拟议的研究和培训后，我将成为应用尖端蛋白质组学、系统生物学和显微镜学的专家。因此，除了作为 SZ 中令人信服的感兴趣蛋白之外，ATP1A3 还将作为通过转化研究推进 MS 发现的感兴趣蛋白的测试用例。