Neural-derived extracellular vesicles microRNAs in bipolar disorder: a peripheral window into the brain

项目总结/摘要 双相情感障碍是一种慢性且通常严重的精神疾病，具有很强的遗传基础。但即使 随着分子遗传学研究的重大进展，与该疾病相关的少数遗传变异 解释只有一小部分的遗传性，这导致了假设的参与后生 BD的病理生理学改变虽然最近的研究试图确定表观遗传生物标志物， BD，由于表观遗传改变的组织特异性和对表观遗传改变的偏倚评估， 候选基因或通过微阵列进行全基因组研究。由于生物标志物的研究 反复关注不一定反映大脑变化的外围测量， 生物学和临床相关的发现受到了严重阻碍。这项研究将填补这一重要空白 通过测量大脑释放的血浆来源的细胞外囊泡（EV）中的表观遗传标记。 具体来说，我们将重点关注最近提出的microRNA和其他非编码RNA， 介导BD的重要机制，并可能整合基因和环境刺激。值得注意的是，微RNA- 填充的EV是由神经组织释放的，作为细胞间通信的一种方法，可能是关键的参与者。 在将表观遗传标记转移到生殖细胞中，最终有助于代际和跨代遗传 BD的传播。我们的工作假设是BD患者将显示特定miRNA的改变， 与对照相比，血浆神经源性EV中的其他非编码RNA转录物。为了验证这一点，我们将首先 通过分析来自60名健康对照者和60名BD患者血液样本，鉴定BD患者神经特异性EV microRNA 第一类病人（已收集及储存于本署）（目标1）。在神经源性EV 免疫沉淀和表征，RNA将被分离，下一代测序文库将被 在Illumina NextSeq仪器上制备并测序，其中在大约100 μ L处具有1x 75 bp单端读段。 每个样本1000 - 1500万次读取。显著改变的miRNA将通过实时PCR进行验证， 将通过功能途径评估差异表达转录本的推定生物学相关性 分析。此外，将使用miRNA表达数据构建机器学习模型，以预测 个体样本是否属于BD或对照组，允许建立临床 有用的预测性生物特征，可以在现场立即产生影响。第二步，我们将调查 神经元EVs转录组标记物与临床、神经解剖学和神经认知的相关性 每个受试者可用的参数，最终目标是确定新的临床相关性- 在疾病的内表型中鉴定EV标志物（目的2）。这种大脑特异性标记的鉴定 将可能为BD的科学研究开辟新的途径。

Project Summary/Abstract Bipolar disorder is a chronic and often severe psychiatric disorder with a strong genetic basis. However, even with significant progress made by molecular genetic studies, the few genetic variants associated with the disorder explain only a small portion of its heritability, which led to the hypothesis of the involvement of epigenetic alterations in BD’s pathophysiology. While recent studies have attempted to identify epigenetic biomarkers in BD, most results are limited due to the tissue specificity of epigenetic alterations and the biased assessments of candidate genes or genome-wide investigations through microarrays. Since the search for biomarkers have repetitively focused on peripheral measures that do not necessarily reflect brain alterations, significant biologically- and clinically-relevant findings have been significantly hindered. This study will fill this important gap by measuring epigenetic markers in plasma-derived extracellular vesicles (EVs) released by the brain. Specifically, we will focus on microRNAs and other non-coding RNAs that have been recently proposed to mediate important mechanisms in BD and may integrate gene and environment stimuli. Of note, microRNAs- filled EVs are released by the neural tissue as a method of cell-to-cell communication and may be the key players in transferring epigenetic markers to germ cells, ultimately contributing to the inter- and transgenerational transmission of BD. Our working hypothesis is that patients with BD will show alterations in specific miRNAs and other non-coding RNA transcripts in plasma neural-derived EVs compared to controls. To test this, we will initially identify neural-specific EVs microRNAs in BD by analyzing blood samples from 60 healthy controls and 60 BD type I patients (already collected and stored in our Department) (Aim 1). After neural-derived EVs immunoprecipitation and characterization, RNA will be isolated and next generation sequencing libraries will be prepared and sequenced on an Illumina NextSeq instrument with 1x75 bp single-end reads at an approximate depth of 10-15 million reads per sample. Significantly altered miRNAs will be validated by real-time PCR and the putative biological relevance of the differentially expressed transcripts will be assessed by functional pathway analyses. In addition, a machine learning model will be built using the miRNA expression data in order to predict whether an individual sample belongs to the BD or control group, allowing for the establishment of a clinically useful predictive biosignature that can have immediate impact in the field. On a second step we will investigate the correlation between neuronal EVs transcriptome markers with clinical, neuroanatomical, and neurocognitive parameters available for each subject, with the ultimate goal of identifying the clinical relevance of the newly- identified EVs markers in endophenotypes of illness (Aim 2). The identification of such brain-specific markers will likely open new avenues for scientific investigation of BD.