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

• 批准号: 7942983
• 负责人: EVAN Y SNYDER
• 金额: $ 69.23万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 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/7942983
• 关键词: Abstinence; Adverse effects; Alcohol abuse; Alcohol or Other Drugs use; Animal Model; B-Lymphocytes; 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; Face; Fibroblasts; Figs - dietary; Future; Gene Expression; Generations; Genes; Genome; Germ Layers; Glean; Goals; Gold; Grouping; Health; Hippocampus (Brain); Hospitals; Human; Individual; Informed Consent; Institutes; 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; Proteins; Proteome; Proteomics; Protocols documentation; Rattus; Refractory; Regulation; Relapse; Research; Research Design; Research Ethics Committees; 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; improved; in vitro Model; induced pluripotent stem cell; insight; lamotrigine; meetings; nerve stem cell; neurogenetics; neuropsychiatry; neuroregulation; novel; pluripotency; predictive modeling; public health relevance; relating to nervous system; research study; standard care; suicidal; tandem mass spectrometry; tool; topiramate; valproate

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，其生命周期是复发和恢复躁狂和抑郁发作。这种情绪障碍已被证明与家族性易感性具有很强的遗传联系。适当诊断的主要障碍是混淆者，例如满足BPD标准的物质和酗酒/依赖性的普遍性。药物滥用，醉酒和戒断可能会引起概括性双极表型的情绪发作。通常，需要至少6-12个月的戒酒，以诊断为基础BPD的情绪发作。临床实践通常是由于潜在的极端烦躁和冲动自杀行动而诉诸于早期的精神药物。锂一直是BPD治疗的标准，但由于其副作用，还开了抗抗惊厥药（包括丙戊酸，lamotrigine，tupiramake＆Carbamazepine）和非典型的抗精神药物。疾病疗法的基本病因和机制知之甚少{Rosenberg，2007＃160}。足够的实验室（包括动物）模型很难建立{Fornito，2009＃161}。与对照组和精神分裂症患者相比，已经对验尸后脑样本进行了微阵列分析，但在神经亚型，神经胶质和周围的血管细胞之间没有明显的区别（Kim＆Webster，2008年）。与BPD＆相关的基因表达模式尚无共识，因此几乎没有对基本分子机制的见解。蛋白激酶途径可能会改变。锂改变了Mek＆Erk磷酸化{Pardo，2003＃162}，减少了大鼠海马新神经元中CAM激酶IV {Tardito，2007＃163}的CREB磷酸化和活性。锂和丙戊酸可能会降低大鼠glur1 {du，2008＃164}的磷酸化。锂与大鼠皮质神经元中NMDA受体亚基NR2B的磷酸化降低相关{hashimoto，2002＃159}。大鼠大脑的研究还表明，锂给药可将蛋白激酶C的易位从细胞质到细胞膜{Hahn，1999＃158}，并抑制小鼠的GSK3活性（Catapano＆Manji，2008）。鉴于BPD中涉及激酶途径的多样性，并受到研究人类神经精神疾病的障碍的限制，我们试图开发一种代表性的预测模型系统，以探索在神经细胞中蛋白质磷酸化的调节，这些模型最忠实地倾向于重现实际人类BPD的实际缺陷。最近的进步允许将患者特异性的人成纤维细胞转化为人类诱导的多能干细胞（HIPSC），这些细胞变得足够多地被推迟到原始发育阶段，以使它们现在仿真人类胚胎干细胞（HESC）在涵盖了这些胚胎的能力以及涵盖各种类型的能力方面，以使其及其具有胚胎的分化及其类型，从在神经谱系中。我们的小组不仅有能力从正常个体的成纤维细胞中产生HIPSC，并从患有难以模仿疾病的患者中产生HIPSC，而且实际上已经为某些神经遗传学/神经精神上的实体（例如Rett综合征）而做到了这一点。我们建议从定义明确的BPD患者子集中产生HIPSC（即锂反应性亚群体），以便使用从实际患者获得的材料从严格的分子机械角度开始更忠实地对BPD进行建模。我们将包括来自未受影响的患者的控制细胞系，以及非BPD的神经或精神病患者。在开始了解BPD的分子基础的核心 - 以及HIPSC特别适合和信息性的方面是对蛋白质组和磷酸蛋白质组水平上关键蛋白质表达的变化，尤其是其磷酸化状态的变化。我们的团队特别擅长使用hESC的磷酸蛋白质组学分析（在现场中的第一个分析）来识别影响神经分化的密钥（包括新颖的）可吸毒信号转导途径。我们认为，我们可以对BPD患者的HIPSC采用类似的方法。换句话说，我们假设鉴定来自BPD的HIPSC衍生神经元与未受影响的对照或其他神经精神疾病的对照在HIPSC衍生的神经元之间存在鉴定，将有助于阐明BPD下的蛋白质组学神经元与其他神经精神疾病。多维液相色谱（MDLC）串联质谱法（MS/MS）是分析蛋白质组，磷蛋白蛋白质组的强大工具，并且是Burnham Institute的强度。 MDLC-MS/MS将用于分析正常和患者衍生的HIPSC及其神经衍生物的蛋白质组织和磷酸蛋白质组。我们将使用精致的生物信息学工具（另一种Burnham强度）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噬菌体。这种大规模的分析可能会产生对照与BPD细胞之间的许多候选分子差异，其中任何一个可能都可能提示改进的诊断和治疗方法。尽管我们将能够对仅预测的少数几个蛋白质进行后续分析，而这些蛋白质是控制多能性，神经分化和神经功能的关键，但该有价值的数据集将可用于更广泛的研究社区，以便可以根据这些蛋白质组学和磷酸蛋白质组结果的基础启动互补的平行研究。公共卫生相关性：双相情感障碍（BPD）是一种严重且突出的社会疾病，病因不足。蛋白质组学和磷蛋白质组学技术是一个强大的分析平台，允许对健康和患病细胞的分子谱公正识别，例如来自正常患者和BPD患者的患者。我们建议将这些IPSC及其神经衍生物的全面蛋白质组学/磷酸化蛋白质组学分析的BPD患者的诱导多能细胞（IPSC）合并，以发现BPD患者中异常的分子基础，以及用于改善诊断和治疗的潜在目标。