Patient-Derived Stem Cells for Phosphoproteomic Profiling Neuropsychopathology

描述（由申请人提供）： ）：双相情感障碍 (BPD) 是一种神经精神疾病，定义为一生中复发和缓解的躁狂和抑郁发作。这种情绪障碍已被证明与家族倾向有很强的遗传联系。正确诊断的一个主要障碍是符合 BPD 标准的人中物质和酒精滥用/依赖的流行等混杂因素。药物滥用、中毒和戒断可能会引发情绪发作，从而重现双相情感障碍表型。通常，需要至少 6-12 个月戒除某种物质才能将情绪发作诊断为潜在的 BPD。由于潜在的极度烦躁和冲动的自杀行为，临床实践经常诉诸早期精神药物。锂盐一直是 BPD 的标准治疗方法，但由于其副作用，还开出抗惊厥药（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。人们对疾病治疗的潜在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}。对死后大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）。对于与 BPD 相关的基因表达模式尚未达成共识，因此对潜在的分子机制知之甚少。蛋白激酶途径可能会改变。锂改变大鼠海马神经元中的 MEK 和 ERK 磷酸化{Pardo，2003 #162}，降低 CREB ​​磷酸化和 CaM 激酶 IV 的活性{Tardito，2007 #163}。锂和丙戊酸盐可能会降低大鼠 GluR1 的磷酸化{Du, 2008 #164}。锂与大鼠皮质神经元中 NMDA 受体亚基 NR2B 磷酸化的降低相关{Hashimoto，2002 #159}。对大鼠大脑的研究还表明，给予锂可以减少蛋白激酶 C 从细胞质到细胞膜的易位{Hahn，1999 #158}，并抑制小鼠中的 GSK3 活性（Catapano 和 Manji，2008）。考虑到 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的限制，我们试图开发一个具有代表性的预测模型系统来探索神经细胞中蛋白质磷酸化的调节，最忠实地再现实际人类 BPD 的潜在缺陷。最近的进展使得患者特异性的人类成纤维细胞能够转化为人类诱导多能干细胞（hIPSC），这些细胞充分去分化到原始发育阶段，它们现在模仿人类胚胎干细胞（hESC），能够产生3个原始胚胎胚层，并遵循各种分化方案，成为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。我们的团队不仅能够从正常个体和患有难以建模疾病的人的成纤维细胞中产生 hIPSC，而且实际上已经针对一些神经遗传/神经精神实体（例如雷特综合症）做到了这一点，并将它们分化为神经谱系。我们建议从明确定义的 BPD 患者子集（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的患者以及患有非 BPD 的神经或精神疾病的患者的对照细胞系。开始了解 BPD 的分子基础（hIPSC 特别适合且信息丰富的一个方面）的核心是更好地了解蛋白质组和磷酸化蛋白质组水平上关键蛋白质表达的变化，特别是其磷酸化状态。我们的团队特别擅长使用 hESC 的磷酸蛋白质组学分析（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。我们相信我们可以对来自 BPD 患者的 hIPSC 应用类似的方法。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患有其他神经精神疾病的对照之间的蛋白质组和磷酸化蛋白质组差异将有助于阐明 BPD 背后的关键、诊断和潜在可药物分子机制。多维液相色谱 (MDLC) 串联质谱 (MS/MS) 是分析蛋白质组、磷酸化蛋白质组的强大工具，也是伯纳姆研究所的强项。 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。我们将使用精致的生物信息学工具（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。这种大规模分析可能会产生对照细胞和 BPD 细胞之间的许多候选分子差异，其中任何一个都可能建议改进的诊断和治疗方法。虽然我们将能够仅对少数被预测为控制多能性、神经分化和神经功能关键的蛋白质进行后续分析，但这一有价值的数据集将提供给更广泛的研究界，以便可以在这些蛋白质组和磷酸蛋白质组结果的基础上启动补充性平行研究。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，其病因尚不清楚。蛋白质组学和磷酸化蛋白质组学技术是一个强大的分析平台，可以公正地鉴定健康和患病细胞的分子谱，例如细胞。来自正常人和 BPD 患者的数据。我们建议将来自 BPD 患者的诱导多能细胞 (iPSC) 的应用与这些 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相结合，以发现 BPD 患者异常的分子基础，以及改进诊断和治疗的潜在目标。

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.