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（由适用提供）：精神分裂症（SZ）是一种终生而毁灭性的精神病，治疗方案有限，无法治愈。第3层锥体神经元的树突状脊柱密度降低是SZ验尸研究中最一致的发现之一，并且已在包括主要听觉皮层（AL）在内的多个脑区域进行了报道。据信这种细胞结构异常是SZ中的几个症状维度，包括损害社会认知和听觉幻觉的听觉处理定义。谷氨酸（GLU）信号对于树突状脊柱完整性至关重要，并且多种证据暗示SZ病理中的合成GLU信号传导。合成GLU信号网络中的蛋白质和GLU受体拮抗剂中蛋白质的许多SZ风险基因座代码可以在人类和动物模型中诱导或加剧SZ符号。这是当前的证据支持一个模型，在该模型中，遗传风险因素会在GLU信号传导蛋白网络上融合，从而导致合成结构和被认为是SZ符号的活性的损害。作为测试该模型的第一步，我利用了靶向质量光谱法（MS）方法来量化来自23 sz的Al灰质物质匀浆中的150多种合成蛋白，并量化了23 sz和匹配的对照对象。 SZ中改变的蛋白质富含基因本体学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）表明，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DE蛋白质网络均包括Na+/K+泵亚基ATP1A3。 ATP1A3突变有助于SZ的多基因伯宁，并且与快速发作肌张力障碍帕金森氏症有关，帕金森氏症是一种表现出认知障碍的综合征，其中几乎20％的患者患有以听觉幻觉为特征的合并症。同样，ATP1A3 +/-小鼠，其中ATP1A3表达仅降低15-20％，表现出认知障碍和精神病（如行为）。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改变。基于这些观察结果，我假设：在SZ受试者的Al Deep 3层中，GLU信号蛋白网络发生了变化，并且改善了ATP1A3蛋白的表达有助于这些网络变化和Al深3层脊柱丢失。我将使用新型的LASR捕获微分辨率靶向的MS（LCM-TMS）方法来检验该假设，以鉴定直接在SZ受试者的Al Deep 3层中与脊柱损失相关的GLU蛋白网络变化（AIM 1）。接下来，我将确定AT​​P1A3降低20％对AL蛋白网络和ATP1A3 +/-小鼠中脊柱密度的影响（AIM 2）。最后，我将利用定量荧光显微镜在ATP1A3中定位改变，以及SZ受试者和ATP1A3 +/-小鼠共享的选定蛋白质改变，以在SZ受试者3的Al Deep层中的锥体或抑制性细胞体内（AIM 3）。这些研究的发现绘制了疾病中的突触GLU蛋白网络障碍将支持正在进行的药物发现工作，尤其是如果以意外的方式改变了这些药物靶标的表达，这一主张由我的初步数据强烈支持。他们还将确定新的蛋白质和蛋白质共表达网络改变，以进行未来的假设检验（AIMS 1和2）。将患者的ATP1A3降低定位将支持特定细胞类型动物模型的发展（AIMS 2和3）。这些研究还将支持我在一家大型医学研究机构自己的NIH资助的实验室中研究SZ和相关神经精神疾病的突触病理学的长期职业目标。他们将在实现这一目标至关重要的三个领域提供培训 目标：1。基于MS的蛋白质组学和系统生物学方法的蛋白质网络分析方法：MS方法正在迅速发展，现在可以在一个实验中量化成千上万或蛋白质的千万辣椒。在此奖励期间，我将继续接受这些快速新兴的MS方法以及统计，生物信息和系统生物学学科的统计，对复杂蛋白质组学数据集的网络分析所需的培训。 2。遗传小鼠模型中的翻译机械研究：验尸脑组织中的描述性研究，虽然对于鉴定疾病的分子改变至关重要，但无法确定贡献 单个基因/蛋白质的病理生理学。在计划的研究中，我将学会调查 候选蛋白对合成蛋白网络和结构病理学的影响，重点是Na+/K+泵和ATP1A3的作用。 3。SZ中的皮质细胞和电路病理学：蛋白质表达对皮质功能失调的影响取决于细胞和电路定位。这是我需要在激光捕获显微解剖和定量荧光显微镜的训练，以在皮质细胞和电路的背景下研究蛋白质和蛋白质网络。为了实现这些培训目标，我已经组建了一个杰出的心态团队，以原始心态组成了罗伯特·斯威特（Robert Sweet）博士，他是SZ定量荧光显微镜和皮质细胞和电路病理学的世界领导者，以及基于MS的蛋白质组学的国际认可的专家Nathan Yates博士。包括在激光捕获，统计，生物信息学，系统生物学和NA+/K+泵生物学方面的其他专业知识的顾问中，已经增加了心态团队。在拟议的研究和培训完成时，我将是将尖端蛋白质组学，系统生物学和显微镜方法应用于人体组织和动物模型中SZ突触病理学的研究。除了作为SZ感兴趣的引人注目的蛋白质外，ATP1A3还将作为通过转化研究从MS发现中推进感兴趣的蛋白质的测试用例。