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学基础的一个重要障碍是人脑的不可接近性。研究人员不得不依靠尸检标本和外周血白细胞来进行此类研究。研究血液白细胞在推断人脑异常方面的发现极其有限。而且，尽管在研究精神分裂症 (SZ) 受试者尸检样本的基因表达谱时出现了许多有趣的发现，但仍有许多混杂因素可能影响数据解释，包括共病尼古丁、酒精和药物滥用以及慢性病。使用精神药物。此外，对神经精神疾病（例如精神分裂症和自闭症谱系障碍（ASD））的尸检样本进行研究是有限的，这两种疾病都被视为起源于神经发育。我们和其他人正在使用的一项令人兴奋的技术进步可以解决这些限制，那就是诱导多能干细胞（iPSC）。在 NIHM 资助的一项正在进行的研究中，我们已经能够使用来自 SZ 患者和对照的成纤维细胞从 iPSC 培养人类谷氨酸能神经元。该研究的重点是分析 mRNA 表达谱并进行电生理学研究。目前的提案旨在利用这一独特的生物资源，使用下一代测序技术 (miRNA-Seq) 进行 microRNA (miRNA) 分析。 MicroRNA 在大脑发育和突触发生中发挥着关键作用，并且被认为参与了 SZ 病理生理学。支持 miRNA 在 SZ 中发挥作用的最令人信服的论据之一来自对腭心面综合征 (VCFS) 的分析，该综合征是由 22q11 微缺失引起的。大约 1/3 的 VCFS 患者患有 SZ。尽管尚未明确确定导致精神症状的基因，但一个有希望的候选基因是 DGCR8，它编码参与 miRNA 生物发生的核蛋白。 DGCR8 与 Drosha 结合形成所谓的“微处理器”，它可以从长的初级 miRNA 中产生 miRNA。另一位研究人员对基因敲除小鼠的分析表明，Dgcr8 的半合性会影响行为，改变树突复杂性，并影响海马和皮层中几种 miRNA 的表达。由于我们最初的 iPSC 研究包括培养来自有或没有 22q11 缺失的 SZ 患者以及对照的 iPSC，因此我们拥有独特的生物资源来扩展小鼠敲除研究。预期的结果是，对 22q11 缺失的 iPSC 培养的神经元进行 RNA 测序分析将显示许多 miRNA 的表达发生改变，类似于小鼠敲除模型。事实上，考虑到与低等动物相比，人类大脑中基因表达的复杂性增加，我们预计人类神经元中的 miRNA（及其目标 mRNA）数量将高于小鼠模型。我们特别感兴趣的是确定它们的表达在没有 22q11.2 缺失的 SZ 患者中是否也发生改变。这将指出疾病病理生理学的共同分子途径，这是像 SZ 这样的遗传异质性疾病的重要考虑因素。这种共同的途径将成为药物开发的理想目标，对大量患者亚组有效。