microRNA analysis of neurons generated from patient-specific iPSCs

对患者特异性 iPSC 生成的神经元进行 microRNA 分析

基本信息

批准号：
8502556
负责人：
HERBERT M LACHMAN
金额：
$ 20.04万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8502556
关键词：
22q11 22q11.2 Address Adult Affect Alcohol abuse Animals Autopsy Behavior Behavioral Binding Biogenesis Biological Bipolar Disorder Brain Cause of Death Cell Line Cells Chromosome abnormality Chronic Cleft Palate Clinical Code Congenital Heart Defects Cytoplasm Data Detection Development Diagnosis Disease Double-Stranded RNA Down Syndrome Drug abuse Fetal Development Fibroblasts Foundations Frequencies Functional disorder Funding Gene Expression Gene Expression Profiling General Population Genes Genetic Transcription Glutamates Goals Grant Growth Health Hippocampus (Brain)Human Human Genome Knock-out Knockout Mice Learning Disabilities Leukocytes Maps Mediating Messenger RNA Methods MicroRNAs Microprocessor Mitochondria Modeling Molecular Molecular Profiling Mus National Institute of Mental Health Neurodevelopmental Disorder Neurons Nuclear Protein Nucleotides Pathway interactions Patients Pharmaceutical Preparations Physiological Play Process Proteins RNA RNA Polymerase II RNA Polymerase III RNA library RNA-Induced Silencing Complex Regulator Genes Repression Research Personnel Resort Resources Role Sampling Schizophrenia Short-Term Memory Shprintzen syndrome Small RNA Specimen Structure Study Subject Subgroup Synaptic Transmission Synaptic plasticity System Technology Transcript Transgenes Translating Translational Repression Translations Vertebral column astrogliosis autism spectrum disorder base brain cell cell type density design drug development human DICER1 protein induced pluripotent stem cell interest mRNA Expression mRNA Precursor mRNA Transcript Degradation microdeletion mouse model neurobehavioral neuron development neuropsychiatry next generation sequencing nicotine abuse novel peripheral blood prepulse inhibition research study stem synaptogenesis transcriptome sequencing treatment strategy

项目摘要

DESCRIPTION (provided by applicant): An important obstacle in understanding the molecular and physiological basis of neuropsychiatric disorders is the inaccessibility of the human brain. Investigators have had to resort to autopsy specimens and peripheral blood leukocytes to carry out such studies. Studying blood leukocytes is extremely limited in terms of extrapolating findings to abnormalities in the human brain. And, although a number of interesting findings have emerged in studying gene expression profiling in autopsy samples obtained from subjects with schizophrenia (SZ), there are numerous confounding factors that could affect data interpretation including co- morbid nicotine, alcohol and drug abuse, and chronic use of psychotropic medications. In addition, studying postmortem samples in neuropsychiatric disorders, such as SZ and autism spectrum disorders (ASD) that are both viewed as neurodevelopmental in origin, is limiting. One exciting technological advance we and others are using that could address these limitations is induced pluripotent stem cells (iPSCs). In an ongoing NIHM funded study, we have been able to cultivate human glutamatergic neurons from iPSCs using fibroblasts derived from patients with SZ and controls. That study focuses on analyzing mRNA expression profiles and carrying out electrophysiolgical studies. This current proposal is designed to capitalize on this unique biological resource for microRNA (miRNA) profiling using next generation sequencing technology (miRNA-Seq). MicroRNAs play a key role in brain development and synaptogenesis, and have been postulated to be involved in SZ pathophysiology. One of the most compelling arguments favoring a role for miRNAs in SZ comes from an analysis of velocardiofacial syndrome (VCFS), which is caused by a 22q11 microdeletion. Approximately 1/3 of patients with VCFS suffer from SZ. Although the genes responsible for the psychiatric manifestations have not been unequivocally identified, a promising candidate is DGCR8, which codes for a nuclear protein involved in miRNA biogenesis. DGCR8 combines with Drosha to form the so-called "microprocessor," which produces miRNAs from long primary miRNAs. An analysis of knockout mice by another investigator shows that hemizygosity for Dgcr8 affects behavior, alters dendritic complexity, and influences the expression of several miRNAs in the hippocampus and cortex. Since our original iPSC study includes the cultivation of iPSCs from patients with SZ with and without 22q11 deletions, as well as controls, we have a unique biological resource to expand on the mouse knockout studies. An expected finding is that RNA-Seq analysis in the neurons cultivated from iPSCs with the 22q11 deletion will show altered expression of a number of miRNAs, similar to mouse knockout model. Indeed, considering the increased complexity of gene expression in the human brain compared with lower animals, we expect that the number of miRNAs (and their targeted mRNAs) will be higher in human neurons than in the mouse model. We are especially interested in determining whether their expression is also altered in patients with SZ who do not have 22q11.2 deletions. This would point to common molecular pathways underlying disease pathophysiology, an important consideration for a genetically heterogeneous disorder like SZ. Such common pathways would be ideal targets for medication development that could be effective in a large subgroup of patients.

描述（由申请人提供）：理解神经精神疾病的分子和生理基础的重要障碍是人脑的无法获得。研究人员不得不诉诸于尸检标本和外周血白细胞进行此类研究。研究血清细胞在外推针对人脑异常方面非常有限。而且，尽管在研究从具有精神分裂症患者（SZ）受试者获得的尸检样本中的基因表达分析中出现了许多有趣的发现，但存在许多混杂因素，可能会影响数据解释，包括尼古丁尼古丁，酒精和药物滥用，以及长期使用精神药物。此外，研究神经精神疾病的死后样本，例如SZ和自闭症谱系障碍（ASD），它们均被视为原始神经发育。我们和其他人正在使用的一种激动人心的技术进步可以解决这些局限性，这是引起多能干细胞（IPSC）。在一项正在进行的NIHM资助研究中，我们能够使用源自SZ和对照患者的成纤维细胞从IPSC中培养人谷氨酸能神经元。该研究的重点是分析mRNA表达谱并进行电性研究。当前的建议旨在利用下一代测序技术（miRNA-seq）来利用这种独特的生物学资源（miRNA）分析。 microRNA在大脑发育和突触发生中起关键作用，并被认为与SZ病理生理学有关。偏爱miRNA在SZ中发挥作用的最引人注目的论点之一来自对速度综合征（VCFS）的分析，该分析是由22q11微缺失引起的。大约1/3的VCF患者患有SZ。尽管尚未明确鉴定出负责精神病表现的基因，但有前途的候选者是DGCR8，该候选者编码参与miRNA生物发生的核蛋白。 DGCR8与Drosha结合使用，形成所谓的“微处理器”，该“微处理器”产生了来自长原代miRNA的miRNA。另一位研究者对敲除小鼠的分析表明，DGCR8的半加性会影响行为，改变树突的复杂性，并影响海马和皮层中几种miRNA的表达。由于我们最初的IPSC研究包括从有或没有22Q11缺失的SZ患者的IPSC以及对照组中种植IPSC，因此我们拥有独特的生物学资源来扩展小鼠敲除研究。一个预期的发现是，从IPSC培养的具有22q11缺失的IPSC的神经元中的RNA-seq分析将显示出许多miRNA的表达，类似于小鼠敲除模型。实际上，考虑到与下动物相比，人脑中基因表达的复杂性增加，我们期望人类神经元中miRNA（及其靶向mRNA）的数量会高于小鼠模型。我们特别有兴趣确定没有22q11.2删除的SZ患者的表达是否也改变了。这将指出疾病病理生理学的基本分子途径，这是像SZ这样的遗传异质性疾病的重要考虑因素。这种常见的途径将是药物开发的理想目标，可以在大量患者亚组中有效。