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Patient-Derived Stem Cells for Phosphoproteomic Profiling Neuropsychopathology

患者来源的干细胞用于磷酸化蛋白质组学分析神经精神病理学

基本信息

批准号：
8307048
负责人：
EVAN Y SNYDER
金额：
$ 19.1万
依托单位：
SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8307048
关键词：
Abstinence Adverse effects Alcohol abuse Alcohol or Other Drugs use Animal Model Bioinformatics Biological Biological Models Biological Process Biopsy Specimen Bipolar Disorder Blood Vessels Brain CREB1 gene Carbamazepine Cell Line Cell membrane Cells Chemicals Clinic Communities Confounding Factors (Epidemiology)Consensus Convulsants Cost aspects Cytosol Data Data Set Databases Defect Dependence Development Diagnosis Diagnostic Disease Disease model Drug Delivery Systems Embryo Equilibrium Etiology Fibroblasts Future Gene Expression Generations Genes Genome Germ Layers Glean Goals Gold Grouping Health Hippocampus (Brain)Hospitals Human Individual Informed Consent Institutes Institutional Review Boards Intoxication Laboratories Lead Link Liquid Chromatography Lithium MEKs Manic Mass Spectrum Analysis Mental disorders Methods Modeling Molecular Molecular Profiling Mood Disorders Moods Mus N-Methyl-D-Aspartate Receptors Neuroglia Neurologic Neuronal Differentiation Neurons Neurophysiology - biologic function Ontology Organ Pathway Analysis Pathway interactions Patients Pattern Pharmaceutical Preparations Phenotype Phosphoproteins Phosphorylation Phosphorylation Site Phosphotransferases Population Predisposition Prevalence Protein Kinase Protein Kinase C Proteome Proteomics Protocols documentation Rattus Refractory Regulation Relapse Research Research Design Resort Resources Rett Syndrome Sampling Schizophrenia Selection Criteria Signal Pathway Signal Transduction Signal Transduction Pathway Site Skin Spinal Muscular Atrophy Staging Stem cells Substance abuse problem System Technology Therapeutic Time Tissues Undifferentiated Validation Valproic Acid Withdrawal base calmodulin-dependent protein kinase IV cell type clinical practice depressive symptoms design dopaminergic neuron follow-up genetic linkage human embryonic stem cell human embryonic stem cell line improved in vitro Model induced pluripotent stem cell insight lamotrigine meetings nerve stem cell nervous system disorder neurogenetics neuropsychiatry neuroregulation novel pluripotency predictive modeling relating to nervous system research study standard care stem cell fate suicidal tandem mass spectrometry tool topiramate valproate

项目摘要

DESCRIPTION (provided by applicant): ): Bipolar disorder (BPD) is a neuropsychiatric condition defined by a lifetime of relapsing & remitting manic & depressive episodes. This mood disorder has been shown to have strong genetic linkage with familial predisposition. A major impediment to proper diagnosis has been such confounders as the prevalence of substance & alcohol abuse/dependence among those meeting criteria for BPD. Substance abuse, intoxication & withdrawal may elicit mood episodes that recapitulate bipolar phenotypes. Typically, at least 6-12 months of abstinence from substances is required to diagnose a mood episode as underlying BPD. Clinical practice often resorts to early psychotropic medication because of potentially extreme irritability & impulsive suicidal actions. Lithium has been the standard of treatment for BPD, but, because of its side effects, anti-convulsants (including valproic acid, lamotrigine, topiramate & carbamazepine) & atypical anti-psychotics have also been prescribed. The underlying etiology & mechanisms of disease therapy are poorly understood {Rosenberg, 2007 #160}. Adequate laboratory (including animal) models have been difficult to establish {Fornito, 2009 #161}. Microarray analyses have been performed on post-mortem brain samples & compared with controls & patients with schizophrenia, but no clear distinction between neural subtypes, glia, & surrounding vascular cells has been evident (Kim & Webster, 2008). There is no consensus on gene expression patterns linked to BPD &, hence, very little insight into underlying molecular mechanisms. Protein kinase pathways may be altered. Lithium alters MEK & ERK phosphorylation {Pardo, 2003 #162}, decreases CREB phosphorylation & activity of CaM kinase IV {Tardito, 2007 #163} in rat hippocampal neurons. Lithium & valproate may reduce phosphorylation of rat GluR1 {Du, 2008 #164}. Lithium correlates with reduced phosphorylation of the NMDA receptor subunit NR2B in rat cortical neurons {Hashimoto, 2002 #159}. Studies in rat brains have also suggested that lithium administration reduces translocation of Protein Kinase C from the cytosol to the cell membrane {Hahn, 1999 #158}, & inhibits GSK3 activity in mice (Catapano & Manji, 2008). Given the diversity of implicated kinase pathways in BPD and limited by obstacles in studying human neuropsychiatric diseases, we sought to develop a representative, predictive model system to explore regulation of protein phosphorylation in neural cells that most faithfully recapitulates underlying defects of actual human BPD. Recent advances have allowed the conversion of patient-specific human fibroblasts to human induced pluripotent stem cells (hIPSCs), cells which become sufficiently dedifferentiated to a primordial developmental stage that they now emulate human embryonic stem cells (hESCs) in terms of their ability to give rise to the 3 primitive embryonic germ layers and following various differentiation protocols, to more mature, tissue- and organ-specific cell types, including those in the neural lineage. Our group not only has the ability to generate hIPSCs from fibroblasts from normal individuals & from those carrying difficult-to-model diseases, but has actually done so for some neurogenetic/neuropsychiatric entities, e.g., Rett Syndrome, & has differentiated them to neural lineages. We propose to generate hIPSCs from a well-defined subset of BPD patients (i.e., the lithium-responsive subpopulation) in order to begin more faithfully modeling BPD from a rigorous molecular mechanistic perspective using material derived from actual patients. We will include control cell lines from unaffected patients as well as patients with neurologic or psychiatric disorders that are not BPD. At the core of beginning to understand the molecular basis of BPD - and an aspect for which hIPSCs would be particularly well-suited & informative - is a better understanding of changes in the expression of key proteins, particularly their phosphorylation state, at the level of the proteome & phosphoproteome. Our team has been particularly adept at using phosphoproteomic analysis of hESCs (the 1st such analysis in the field) to identify key (including novel) drugable signal transduction pathways that influence neural differentiation. We believe we can apply a similar approach to hIPSCs from BPD patients. In other words, we hypothesize that identification of proteomic & phosphoproteomic differences between hIPSC-derived neurons from BPD vs. unaffected controls or controls with other neuropsychiatric disorders will help elucidate pivotal, diagnostic, and potentially drugable molecular mechanisms underlying BPD. Multidimensional liquid chromatography (MDLC) tandem mass spectrometry (MS/MS) is a powerful tool for analysis of proteomes, phosphoproteomes, and is a strength of the Burnham Institute. MDLC-MS/MS will be used to analyze the proteomes and phosphoproteomes of normal and patient-derived hIPSCs and their neural derivatives. We will use refined bioinformatic tools (another Burnham strength) to glean critical differences among the cell types. We will quantify total cellular proteins, with a particular focus on site- specific phophorylation. This large-scale analysis will likely yield a number of candidate molecular differences between control & BPD cells, any of which might suggest improved methods of diagnosis & treatment. While we will be able to perform follow-up analyses on only a few proteins predicted to be key to control of pluripotency, neural differentiation and neural function, this valuable dataset will be made available to the broader research community so that complementary parallel studies may be launched on the basis of these proteomic & phosphoproteomic results. PUBLIC HEALTH RELEVANCE: Bipolar disorder (BPD) is a severe and prominent societal malady with poorly understood etiology. Proteomic and phosphoproteomic technology is a powerful analytical platform allowing unbiased identification of molecular profiles of healthy and diseased cells, e.g. those from normal and BPD patients. We propose to merge the application of induced pluripotent cells (iPSCs) from BPD patients with comprehensive proteomic/phosphoproteomic analyses of these iPSCs and their neural derivatives, to discover molecular underpinnings of the abnormalities in BPD patients, as well as potential targets for improved diagnosis and treatment.

描述（由申请人提供）：双相情感障碍（BPD）是一种神经精神疾病，定义为一生中反复发作和缓解的躁狂和抑郁发作。这种情绪障碍已被证明与家族易感性有很强的遗传联系。适当诊断的一个主要障碍是在符合BPD标准的人群中普遍存在物质和酒精滥用/依赖等混杂因素。药物滥用，中毒和戒断可引起情绪发作，再现双相表现型。通常情况下，至少6-12个月的药物戒断需要诊断为潜在的BPD的情绪发作。临床实践往往诉诸于早期精神药物治疗，因为潜在的极端易怒和冲动自杀行为。锂一直是治疗BPD的标准药物，但由于其副作用，抗惊厥药（包括丙戊酸、拉莫三嗪、托吡酯和卡马西平）和非典型抗精神病药也被开了处方。潜在的病因和疾病治疗机制尚不清楚[Rosenberg， 2007 #160]。难以建立足够的实验室（包括动物）模型{Fornito， 2009 #161}。对死后脑样本进行了微阵列分析，并与对照组和精神分裂症患者进行了比较，但神经亚型、神经胶质细胞和周围血管细胞之间没有明显的区别（Kim & Webster, 2008）。关于与BPD相关的基因表达模式尚无共识，因此，对潜在分子机制的了解很少。蛋白激酶途径可能被改变。锂改变大鼠海马神经元MEK和ERK磷酸化[Pardo， 2003 #162]，降低CREB磷酸化和CaM激酶IV活性[Tardito， 2007 #163]。锂和丙戊酸盐可能降低大鼠GluR1的磷酸化[Du， 2008 #164]。锂与大鼠皮质神经元NMDA受体亚基NR2B磷酸化降低相关[Hashimoto， 2002 #159]。对大鼠大脑的研究也表明，锂可以减少蛋白激酶C从细胞质到细胞膜的转运（Hahn, 1999 #158），并抑制小鼠的GSK3活性（Catapano & Manji, 2008）。鉴于BPD中涉及的激酶途径的多样性以及研究人类神经精神疾病的障碍，我们寻求开发一个具有代表性的预测模型系统，以探索神经细胞中蛋白质磷酸化的调节，最忠实地概括了实际人类BPD的潜在缺陷。最近的进展已经允许将患者特异性人类成纤维细胞转化为人类诱导多能干细胞（hIPSCs），这些细胞已经充分去分化到原始发育阶段，现在它们在产生3个原始胚胎胚层的能力方面模仿人类胚胎干细胞（hESCs），并遵循各种分化方案，向更成熟的组织和器官特异性细胞类型，包括神经谱系中的细胞类型。我们的团队不仅有能力从正常个体和携带难以建模疾病的人的成纤维细胞中产生hipsc，而且实际上已经为一些神经遗传学/神经精神实体（例如Rett综合征）做到了这一点，并将它们分化为神经谱系。我们建议从定义明确的BPD患者亚群（即锂反应亚群）中生成hipsc，以便使用来自实际患者的材料从严格的分子机制角度开始更忠实地建模BPD。我们将包括来自未受影响患者以及非BPD的神经或精神疾病患者的对照细胞系。开始了解BPD的分子基础的核心是更好地理解关键蛋白的表达变化，特别是它们在蛋白质组和磷酸化蛋白质组水平上的磷酸化状态，这是hipsc特别适合和提供信息的一个方面。我们的团队特别擅长使用hESCs的磷蛋白质组学分析（该领域的第一个此类分析）来识别影响神经分化的关键（包括新型）可药物信号转导途径。我们相信我们可以将类似的方法应用于BPD患者的hipsc。换句话说，我们假设鉴定来自BPD的hipsc神经元与未受影响的对照组或患有其他神经精神疾病的对照组之间的蛋白质组学和磷酸化蛋白质组学差异将有助于阐明BPD的关键、诊断和潜在的可药物分子机制。多维液相色谱（MDLC）串联质谱（MS/MS）是蛋白质组学，磷蛋白质组学分析的强大工具，是Burnham研究所的优势。MDLC-MS/MS将用于分析正常和患者来源的hipsc及其神经衍生物的蛋白质组和磷酸化蛋白质组。我们将使用精细的生物信息学工具（伯纳姆的另一个优势）来收集细胞类型之间的关键差异。我们将量化总细胞蛋白，特别关注位点特异性磷酸化。这种大规模的分析可能会产生一些对照和BPD细胞之间的候选分子差异，其中任何一个都可能建议改进诊断和治疗方法。虽然我们只能对预测对多能性、神经分化和神经功能控制至关重要的少数蛋白质进行后续分析，但这个有价值的数据集将提供给更广泛的研究界，以便在这些蛋白质组学和磷蛋白质组学结果的基础上开展互补的平行研究。公共卫生相关性：双相情感障碍（BPD）是一种严重和突出的社会疾病，病因尚不清楚。蛋白质组学和磷蛋白质组学技术是一个强大的分析平台，允许对健康和病变细胞（例如正常和BPD患者）的分子谱进行无偏见的鉴定。我们建议将来自BPD患者的诱导多能细胞（iPSCs）的应用与这些iPSCs及其神经衍生物的综合蛋白质组学/磷酸化蛋白质组学分析结合起来，发现BPD患者异常的分子基础，以及改进诊断和治疗的潜在靶点。