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: 9091649
• 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/9091649
• 关键词: 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; Health; Human; Impaired cognition; Impairment; Individual; Institution; Investigation; Knockout Mice; Laboratories; Lasers; Lead; Learning; Link; Maps; Mass Spectrum Analysis; Measures; Medical Research; Mental disorders; 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; new therapeutic target; novel; novel therapeutics; protein expression; psychotic symptoms; 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）是一种终生的、毁灭性的精神疾病，治疗选择有限且无法治愈。 第 3 层锥体神经元的树突棘密度降低是 SZ 死后研究中最一致报道的发现之一，并且在包括初级听觉皮层 (Al) 在内的多个大脑区域都有报道。这种细胞结构异常被认为是 SZ 多种症状的基础，包括损害社会认知和幻听的听觉处理缺陷。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 SZ 病理学中存在突触 Glu 信号传导。许多 SZ 风险位点编码突触 Glu 信号网络中的蛋白质，而 Glu 受体拮抗剂可在人类和动物模型中诱发或加剧 SZ 症状。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，据信这是 SZ 症状的基础。 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 个 SZ 和匹配对照受试者的 Al 灰质匀浆中的 150 多种突触蛋白进行定量。 SZ 中改变的蛋白质针对基因本体术语谷氨酸信号通路进行了富集（p=2.5E-6，q=1.5E-3）。 加权基因共表达网络分析（WGCNA）显示突触蛋白的相关表达普遍下降（p=0.015）。失调的 Glu 信号通路和去相关的蛋白质网络都包含 Na+/K+ 泵亚基 ATP1A3。 ATP1A3 突变会导致 SZ 的多基因负担，并与快速发作的肌张力障碍帕金森症相关，这是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。我的初步数据进一步表明 ATP1A3 +/- 小鼠皮质中 Glu 信号蛋白的表达发生了变化。基于这些观察，我假设： 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 蛋白网络变化（目标 1）。 接下来，我将确定 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）。最后，我将利用定量荧光显微镜将 ATP1A3 的变化以及 SZ 受试者和 ATP1A3 +/- 小鼠共享的选定蛋白质变化定位到 SZ 受试者的 Al 深层 3 内的锥体细胞或抑制性细胞体（目标 3）。这些绘制疾病中突触 Glu 蛋白网络损伤的研究结果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点的表达，我的初步数据强烈支持这一主张。他们还将确定新的蛋白质和蛋白质共表达网络改变，用于未来的假设检验（目标 1 和 2）。患者体内 ATP1A3 的局部减少将支持细胞类型特异性动物模型的开发（目标 2 和 3）。这些研究还将支持我的长期职业目标，即在我自己的 NIH 资助的大型医学研究机构实验室中研究 SZ 突触病理学和相关神经精神疾病。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从数千个蛋白质或蛋白质中定量数以万计的肽。在获奖期间，我将继续接受这些快速新兴的质谱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的培训。 2. 基因小鼠模型的转化机制研究：死后脑组织的描述性研究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 个体基因/蛋白质的病理生理学。在计划的学习中我将学会调查 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 的皮质细胞和回路病理学：蛋白质表达失调对皮质功能的影响取决于细胞和回路定位。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导师团队，主要导师是罗伯特·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）。导师团队的成员在激光捕获、统计学、生物信息学、系统生物学和 Na+/K+ 泵生物学方面具有额外的专业知识。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方法研究人体组织和动物模型中的 SZ 突触病理学的专家。因此，除了在 SZ 中充当引人注目的感兴趣蛋白之外，ATP1A3 还将作为通过转化研究推进 MS 发现中感兴趣蛋白的测试用例。