Identifying Novel Molecular Targets for Chronic SCI

 描述(申请人提供)：脊髓损伤(SCI)是一种没有有效治疗的破坏性疾病。慢性脊髓损伤仍然是最难治疗的。慢性脊髓损伤的病理特征包括星形胶质细胞增多和阻碍轴突再生的抑制分子。然而，对详细的分子途径的了解仍然非常有限。例如，抑制分子在慢性期是如何调节和维持的还不清楚。目前治疗胶质细胞增生症的方法并不理想，迫切需要新的分子靶点。以前的研究通常集中在一小部分基因和通路上，因此没有提供关于SCI病理生理学基础的复杂机制的全面观点。尽管在过去的十年中，微阵列研究为脊髓损伤提供了有价值的见解，但微阵列在分辨率、动态范围和准确性方面存在局限性。RNA测序技术的最新进展使得在全球范围内定位转录区域并以前所未有的灵敏度和特异度定量分析表达成为可能。基于我们对小鼠脊髓挫伤模型急性期和亚急性期的RNA-Seq差异表达的初步研究，我们建议通过整合的RNA-Seq和网络分析来研究脊髓组织中的基因和通路与慢性脊髓损伤中胶质瘢痕的主要成分(纯化的星形胶质细胞)之间的复杂关系。我们推测，在反应性星形胶质细胞中调节或维持抑制分子的新基因和新途径(硫酸软骨素蛋白多糖、Tenascins、ePhin-B2和Sit蛋白等)。是与胶质瘢痕相关的慢性脊髓损伤抑制环境的关键。具体地说，我们建议通过在时间和空间水平上表征SCI亚慢性/慢性期的基因和剪接异构体变化来更好地理解SCI的病理生理学进展。此外，我们将使用集成网络分析的创新策略来确定新的感兴趣基因(GOI)和参与胶质细胞增殖症的途径作为新的分子靶点。我们还将把药物基因组信息纳入我们的分析中，这将成为翻译的强大工具。最后，我们将在胶质瘢痕模型中通过功能丧失和功能获得的分析来测试GOIS可能参与神经元对反应性星形胶质细胞的抑制，以了解它们的功能效应。 我们的研究首次提出利用RNA-Seq在系统水平上研究慢性脊髓损伤和胶质细胞增生症的机制，并通过创新途径和网络分析识别关键基因，并通过功能分析进行测试。全基因组分析的优势在于，作为一种从头发现的方法，它可以识别疾病过程中以前没有意识到的关键缺失环节。重要的是，我们将生成一个全面的SCI基因表达数据资源，这对研究界来说将是非常有价值的(jiaqanwulab.org/sci Browser/data)。该项目的成功完成将导致新的分子靶点的发现，并转变研究和临床范式。

 DESCRIPTION (provided by applicant): Spinal cord injury (SCI) is a devastating disease without effective treatment. The chronic SCI remains the most difficult to treat. The pathologic hallmarks in chronic SCI include increased astrogliosis and inhibitory molecules that hinder axonal regeneration. However, the understanding of the detailed molecular pathways is still very limited. For example, how inhibitory molecules are regulated and maintained in the chronic phase remains unclear. Current therapeutics for gliosis is not ideal, and new molecular targets are urgently needed. Previous studies have usually focused on a small number of genes and pathways at a time, and thus did not provide a comprehensive view of the complex mechanisms underlying SCI pathophysiology. Although, during the last decade, microarray studies have provided valuable insights into SCI, microarray suffers from limitations in resolution, dynamic range and accuracy. Recent advances in RNA-Sequencing technology make it possible to globally map transcribed regions and quantitatively analyze expression at an unprecedented level of sensitivity and specificity. Based on our preliminary studies of differential expression using RNA-Seq during acute and subacute SCI phases in mouse contusive injury models, we propose to investigate the intricate relationship of genes and pathways in the spinal cord tissue and the predominant component of the glial scar (purified astrocytes) in chronic SCI by using integrated RNA-Seq and network analyses. We hypothesize that novel genes and pathways that regulate or maintain inhibitory molecules in reactive astrocytes (chondroitin sulphate proteoglycans, tenascins, ephrin-B2 and Slit proteins etc.) are critical for the chronic SCI inhibitory environment associated with glial scar. Specifically, we propose to derive a better understanding of the progression of SCI pathophysiology by characterizing gene and splicing isoform changes in SCI subchronic/chronic phases at both temporal and spatial levels. Additionally, we will use an innovative strategy of integrated network analysis to identify novel genes of interest (GOIs) and pathways involved in gliosis as new molecular targets. We will also incorporate pharmacogenomic information into our analyses that will serve as a powerful tool for translation. Finally, we will test GOIs potentially involved in neuron inhibition of reactive astrocytes by loss- and gain-of-function assays in a glia scar model for their functional effects. Our study is the first to propose that the mechanisms of chronic SCI and gliosis be investigated at the systems level using RNA-Seq, and key genes be identified via innovative pathway and network analyses and be tested by functional assays. The advantage of the genome-wide analysis is that, as a de novo discovery approach, it can identify critical missing links in the disease processes that were not previously appreciated. Importantly, we will generate a comprehensive data resource of SCI gene expression which will be extremely valuable for the research community (jiaqianwulab.org/SCI browser/data). The successful completion of this project will lead to the discovery of novel molecular targets and shift the research and clinical paradigms.