Characterizing the function of miRNAs in neural development, synaptic plasticity and schizophrenia.

表征 miRNA 在神经发育、突触可塑性和精神分裂症中的功能。

• 批准号: 8939991
• 负责人: Zheng Li
• 金额: $ 62.97万
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939991
• 关键词: Acids; Actins; Adolescence; Adoption; Adult; Affect; Age of Onset; Alleles; Alzheimer' s Disease; Architecture; Area; Autopsy; Back; Binding; Binding Sites; Biogenesis; Brain; Brodmann' s area; Cell physiology; Cells; Clinical; Code; Cognitive deficits; Complex; Delusions; Dendrites; Dendritic Spines; Development; Diagnosis; Disease; District of Columbia; Dorsal; Environment; Etiology; Family; Functional RNA; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Predisposition to Disease; Glutamate Receptor; Goals; Hallucinations; Human; Impaired cognition; Informed Consent; Institutional Review Boards; Investigation; Ischemia; Lateral; Learning; Legal; Length; Libraries; Link; Mediating; Medical Examiners; Memory; Mental Depression; Mental disorders; Messenger RNA; Metabotropic Glutamate Receptors; MicroRNAs; Microscopic; Molecular; Morphogenesis; Mutation; N-Methylaspartate; National Institute of Mental Health; Neuraxis; Neurologic; Neurons; Nucleotides; Pathology; Phosphorylation; Play; Prevalence; Proteins; Protocols documentation; Psychopathology; RNA; RNA Binding; Regulation; Regulator Genes; Reporting; Research; Risk; Role; Sampling; Schizophrenia; Small RNA; Stress; Susceptibility Gene; Symptoms; Synapses; Synaptic Transmission; Synaptic plasticity; Technology; Testing; Translations; Twin Multiple Birth; Ubiquitination; United States National Institutes of Health; Up-Regulation; Vertebral column; Virginia; Withdrawal; base; deep sequencing; depolymerization; disability; frontal lobe; improved; insight; mRNA Transcript Degradation; neurodevelopment; neuropsychiatry; next generation sequencing; palmitoylation; protein expression; receptor; risk variant; social; tropomodulin; young adult

1. The role of miRNAs in long-lasting synaptic plasticity. microRNAs (miRNAs) are short, non-coding RNAs that bind to mRNAs to inhibit translation and/or promote mRNA degradation. Each miRNA can potentially target to hundreds of distinct mRNAs, and thousands of genes are regulated by miRNAs. miRNAs are increasingly recognized as key regulators of gene expression and have been found to play important roles in diverse cellular processes, such as the differentiation and development of cells. miRNAs are crucial for proper brain function. Hundreds of miRNAs are expressed in the brain. miRNA loss i leads to alterations in synaptic protein expression, synaptic transmission, dendritic spines, learning, and memory. Several miRNAs, such as miR-134, miR-125, miR-138, miR-132, miR-29 and miR-188, regulate the morphogenesis of dendritic spines. miRNAs are also implicated in mental disorders. For instance, mounting evidence suggests that mutations in miRNA genes and miRNA biogenesis machinery are associated with increased risk of schizophrenia (Beveridge et al., 2008). Despite the demonstrated importance of miRNAs, however, the function of the vast majority of miRNAs expressed in the brain have yet to be elucidated. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) are ionotropic glutamate receptors that mediate fast excitatory synaptic transmission in the central nervous system. AMPAR are tetramers composed of four possible subunits (GluA1-4) (Shepherd and Huganir, 2007). The number of AMPAR in synapses determines the strength of synaptic transmission, and their abnormal expression has been implicated in cognitive impairments associated with such neurological and neuropsychiatric diseases as Alzheimers disease, ischemia, schizophrenia and depression (Chang et al., 2012). AMPAR expression is regulated by synaptic activity (Grooms et al., 2006). During activity-dependent synaptic plasticity, for instance, the activation of N-methyl-D-aspartate (NMDA) or metabotropic glutamate receptors affects the abundance of synaptic AMPAR through both post-translational mechanisms (including phosphorylation, palmitoylation and ubiquitination) and local translation of dendritic mRNAs encoding AMPAR subunits (Grooms et al., 2006; Ju et al., 2004; Lu and Roche, 2012; Snyder et al., 2001; Sutton et al., 2006). Activity-dependent modulation of AMPAR is an important mechanism that tunes synaptic strength to refine synaptic connectivity during brain development and to store information in the brain during learning and memory (Shepherd and Huganir, 2007). Despite the broad recognition that AMPAR play a pivotal role in brain functions, however, molecular mechanisms underlying their regulation, especially activity-dependent local translation of AMPAR in dendrites, are only incompletely understood. In neurons, many miRNAs localize to dendrites where they can be regulated by synaptic activity (Hu et al., 2014; Schratt, 2009). For instance, NMDAR activation inhibits miR-191 expression locally in dendrites, resulting in an elevation of its target tropomodulin-2, which promotes actin depolymerization, shrinkage and elimination of dendritic spines. In view of this finding, we hypothesize that miRNAs also contribute to activity-dependent local synthesis of AMPAR in dendrites. . To test this hypothesis, we combined miRNA pull-down and computational prediction to search for miRNAs that target the AMPAR subunit GluA1. This approach leads to the identification of miR-501-3p as a GluA1 binding miRNA. Our further analysis of miR-501-3p shows that it is increased locally in dendrites following NMDAR activation, and that this upregulation of miR-501-3p is required for NMDAR-dependent inhibition of GluA1 expression, long-lasting spine shrinkage and elimination. These findings reveal that miRNAs are important regulators of activity-dependent local synthesis of dendritic AMPAR. 2. Delineating the miRNAome in schizophrenia. Schizophrenia is a debilitating mental illness with a lifetime prevalence of 4.0/1,000 worldwide, and a typical age of onset at late adolescence and young adulthood. Schizophrenia manifests as a spectrum of clinical symptoms, such as hallucinations, delusions and social withdrawal, with cognitive deficits as a core feature. The etiology of schizophrenia is unclear. Family, twin and adoption studies indicate that schizophrenia has a strong genetic component. It is thought that schizophrenia is caused by complex interactions between multiple genes and the environment. Linkage and association studies have led to the discovery of dozens of susceptibility genes. However, most risk genes have small effect sizes and are protein-coding genes. The genetic architecture of schizophrenia is still largely unclear. The recently-established next-generation sequencing technologies have significantly pushed back the limitations of prior transcriptome profiling approaches by greatly improving the throughput and depth of coverage. In addition, the superior sensitivity and quantifiability of next-generation sequencing are especially advantageous to detect low-abundant miRNAs. In this study, we applied the Illumina Solexa deep-sequencing platform, which is a next-generation sequencing technology based on massively parallel signature sequencing, to delineate the miRNA transcriptomes in schizophrenic brain. Dr. Joel Kleinman (NIMH) has provided me RNA samples isolated from postmortem brains of schizophrenic and normal control subjects. All postmortem human brains are obtained from the Offices of the Chief Medical Examiner of the District of Columbia, and of the Commonwealth of Virginia, Northern District, all with informed consent from the legal next of kin (protocol 90-M-0142 approved by the NIMH/NIH Institutional Review Board). Diagnoses, macro- and microscopic neuropathological examinations and toxicological analysis are performed on all cases. Small RNAs are extracted from Brodmann area 46 of the dorsal lateral frontal cortex (DLPFC) by using the mirVana miRNA Isolation Kit (Ambion) and used for construction of deep-sequencing libraries as described (Wu et al., 2010). DLPFC is selected because of its strong implication in the psychopathology of schizophrenia (Weinberger et al., 1986). We have completed the library construction step. We are currently conducting deep-sequencing. We will analyzed deep-sequencing results for the change of miRNAome in schizophrenia brains.

1. miRNA 在持久突触可塑性中的作用。 microRNA (miRNA) 是短的非编码 RNA，可与 mRNA 结合以抑制翻译和/或促进 mRNA 降解。每个 miRNA 都可能靶向数百个不同的 mRNA，并且数千个基因受 miRNA 调控。 miRNA 越来越被认为是基因表达的关键调节因子，并且被发现在多种细胞过程中发挥着重要作用，例如细胞的分化和发育。 miRNA 对于大脑的正常功能至关重要。数百种 miRNA 在大脑中表达。 miRNA 丢失会导致突触蛋白表达、突触传递、树突棘、学习和记忆的改变。一些 miRNA，例如 miR-134、miR-125、miR-138、miR-132、miR-29 和 miR-188，调节树突棘的形态发生。 miRNA 也与精神障碍有关。例如，越来越多的证据表明 miRNA 基因和 miRNA 生物发生机制的突变与精神分裂症风险增加相关（Beveridge 等，2008）。尽管 miRNA 的重要性已被证明，但大脑中表达的绝大多数 miRNA 的功能尚未阐明。 α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体 (AMPAR) 是离子型谷氨酸受体，介导中枢神经系统中的快速兴奋性突触传递。 AMPAR 是由四个可能的亚基 (GluA1-4) 组成的四聚体（Shepherd 和 Huganir，2007）。突触中 AMPAR 的数量决定了突触传递的强度，其异常表达与阿尔茨海默病、缺血、精神分裂症和抑郁症等神经和神经精神疾病相关的认知障碍有关（Chang 等，2012）。 AMPAR 表达受突触活动调节（Grooms 等，2006）。例如，在活动依赖性突触可塑性过程中，N-甲基-D-天冬氨酸（NMDA）或代谢型谷氨酸受体的激活通过翻译后机制（包括磷酸化、棕榈酰化和泛素化）和编码 AMPAR 亚基的树突 mRNA 的局部翻译影响突触 AMPAR 的丰度（Grooms 等， 2006年； Ju等人，2004；卢和罗氏，2012；斯奈德等人，2001；萨顿等人，2006）。 AMPAR 的活动依赖性调节是调节突触强度的重要机制，可在大脑发育过程中细化突触连接，并在学习和记忆过程中在大脑中存储信息（Shepherd 和 Huganir，2007）。尽管人们广泛认识到 AMPAR 在大脑功能中发挥着关键作用，然而，其调节的分子机制，尤其是树突中 AMPAR 的活动依赖性局部翻译，尚不完全清楚。 在神经元中，许多 miRNA 定位于树突，在那里它们可以受到突触活动的调节（Hu et al., 2014；Schratt, 2009）。例如，NMDAR 激活会抑制树突中局部 miR-191 的表达，导致其靶标原调节蛋白-2 升高，从而促进肌动蛋白解聚、收缩和树突棘消除。鉴于这一发现，我们假设 miRNA 也有助于树突中 AMPAR 的活性依赖性局部合成。 。 为了检验这一假设，我们结合 miRNA 下拉和计算预测来搜索靶向 AMPAR 亚基 GluA1 的 miRNA。这种方法导致 miR-501-3p 被鉴定为 GluA1 结合 miRNA。我们对 miR-501-3p 的进一步分析表明，NMDAR 激活后，它在树突中局部增加，并且 miR-501-3p 的这种上调是 NMDAR 依赖性 GluA1 表达抑制、持久的脊柱收缩和消除所必需的。这些发现表明 miRNA 是树突状 AMPAR 活性依赖性局部合成的重要调节因子。 2. 描绘精神分裂症中的 miRNA 组。 精神分裂症是一种使人衰弱的精神疾病，全球终生患病率为 4.0/1,000，典型发病年龄为青春期晚期和成年早期。精神分裂症表现为一系列临床症状，如幻觉、妄想和社交退缩，以认知缺陷为核心特征。精神分裂症的病因尚不清楚。家庭、双胞胎和收养研究表明，精神分裂症有很强的遗传因素。人们认为精神分裂症是由多个基因与环境之间复杂的相互作用引起的。连锁和关联研究已发现了数十种易感基因。然而，大多数风险基因的效应较小，并且是蛋白质编码基因。精神分裂症的遗传结构在很大程度上仍不清楚。 最近建立的下一代测序技术通过极大地提高通量和覆盖深度，显着克服了先前转录组分析方法的局限性。此外，新一代测序卓越的灵敏度和可定量性对于检测低丰度 miRNA 特别有利。在本研究中，我们应用Illumina Solexa深度测序平台（一种基于大规模并行特征测序的下一代测序技术）来描绘精神分裂症患者大脑中的miRNA转录组。 Joel Kleinman 博士 (NIMH) 为我提供了从精神分裂症患者和正常对照受试者死后大脑中分离出来的 RNA 样本。所有死后人类大脑均从哥伦比亚特区首席法医办公室和弗吉尼亚联邦北区首席法医办公室获得，所有这些都获得了合法近亲的知情同意（由 NIMH/NIH 机构审查委员会批准的协议 90-M-0142）。对所有病例进行诊断、宏观和微观神经病理学检查以及毒理学分析。 使用 mirVana miRNA 分离试剂盒 (Ambion) 从背外侧额皮质 (DLPFC) 的 Brodmann 区域 46 中提取小 RNA，并用于构建深度测序文库，如所述 (Wu 等人，2010)。选择 DLPFC 是因为它在精神分裂症的精神病理学中具有很强的意义（Weinberger 等，1986）。我们已经完成了图书馆建设步骤。我们目前正在进行深度测序。我们将分析精神分裂症患者大脑中 miRNAome 变化的深度测序结果。