microRNA analysis of neurons generated from patient-specific iPSCs

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一个重要障碍是人类大脑的不可及性。研究人员不得不利用尸检标本和外周血白细胞来进行此类研究。研究血液白细胞在将发现外推到人类大脑异常方面极其有限。而且，尽管在研究从精神分裂症（SZ）受试者获得的尸检样本中的基因表达谱中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长期使用精神药物。此外，研究神经精神疾病的尸检样本，如SZ和自闭症谱系障碍（ASD），这两者都被视为神经发育的起源，是有限的。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）。在一项正在进行的NIHM资助的研究中，我们已经能够使用来自SZ患者和对照组的成纤维细胞从iPSC中培养人多巴胺能神经元。这项研究的重点是分析mRNA表达谱和进行电生理学研究。目前的提案旨在利用这种独特的生物资源，使用下一代测序技术（miRNA-Seq）进行microRNA（miRNA）分析。microRNA在脑发育和突触发生中起关键作用，并已被假定参与SZ病理生理学。支持miRNAs在SZ中发挥作用的最令人信服的论点之一来自对velocardiofacial综合征（VCFS）的分析，这是由22 q11微缺失引起的。大约1/3的VCFS患者患有SZ。虽然负责精神病表现的基因尚未明确确定，但一个有希望的候选者是DGCR 8，它编码参与miRNA生物合成的核蛋白。DGCR 8与Drosha结合形成所谓的“微处理器”，它从长的初级miRNA产生miRNA。另一位研究人员对基因敲除小鼠的分析表明，Dgcr 8的半合子影响行为，改变树突的复杂性，并影响海马和皮质中几种miRNA的表达。由于我们最初的iPSC研究包括培养来自SZ患者（有和没有22 q11缺失）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。一个预期的发现是，从具有22 q11缺失的iPSC培养的神经元中的RNA-Seq分析将显示许多miRNA的表达改变，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（及其靶向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是否也改变了SZ患者谁没有22q11.2缺失。这将指向疾病病理生理学基础的共同分子途径，这是像SZ这样的遗传异质性疾病的重要考虑因素。这些共同的途径将是药物开发的理想目标，可以在一个大的患者亚组中有效。

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.