Identifying Novel Molecular Targets for Chronic SCI

摘要 脊髓损伤(SCI)使人虚弱。星形胶质细胞是脊髓疤痕的主要成分。 明确星形胶质细胞增生症过程中的复杂分子调控对发育至关重要 有效的治疗策略。我们是第一个系统分析Long Non-One的表达谱的人 编码RNA(LncRNA)(&gt；200bp)，这是一种在纯化的星形胶质细胞中发挥重要作用的调节RNA 从成人脊髓穿过脊髓损伤阶段。在lncRNAs的作用和机制方面存在着很大的知识缺口 脊髓损伤后的星形胶质细胞增多症。2%的基因组与蛋白质编码基因有关，创新的靶向 非编码RNA可以极大地扩大可用药靶点的数量，并促进新的类别的 机械论的发现。向明确脊髓损伤和星形胶质细胞增生症机制的首要目标迈进 寻找治疗慢性脊髓损伤的新分子靶点，我们发现Zeb2os，一种高度保守的 LncRNA在星形胶质细胞增生症中起重要作用。数据表明，Zeb2os具有较高的表达水平 损伤时间与其反义蛋白编码基因ZEB2的相关性 星形胶质细胞生长因子STAT3。值得注意的是，在体中由腺相关病毒敲除Zeb2os shRNA(KD) 注射AAV可导致星形胶质细胞形态减轻，GFAP和pSTAT3表达减少 表达，并缩小病变大小。此外，原代星形胶质细胞中的Zeb2os Kd显著减少 多种基因的表达，包括GFAP、ZEB2、STAT3、Neuran、Phocacan和整合素 在疤痕的发展中。我们的RNAScope数据显示Zeb2os-FZeb2和Zeb2os-pSTAT3在 GFAP+细胞。此外，ChIP-Seq实验显示STAT3结合在ZEB2启动子区域。 值得注意的是，我们的数据和其他人的研究提供了证据，证明在损伤后，星形胶质细胞发生了变化。 从最初的有益状态(反应性星形细胞，RA)到不适应状态(疤痕形成星形细胞，SA) 表达抑制神经再生的分子。重要的是，减轻星形细胞疤痕的进展 抗整合素中和抗体促进脊髓损伤后功能改善。基于我们激动人心的 我们建议：a)检验lncRNA Zeb2os通过一种 Zeb2os/ZEB2/STAT3轴，b)破译Zeb2os通过以下方式调节星形胶质细胞的其他机制 全面确定其靶基因和途径，以及c)确定操纵Zeb2os的效果 以及AAV注射对体内解剖和功能结果的靶基因。我们的目标不是移除 疤痕；它是为了减轻后期的疤痕进展和疤痕的抑制作用 在不中断早期神经保护效果的情况下进行神经再生。这些 在脊髓损伤、星形胶质细胞生物学和基因组学方面拥有独特和强大专业知识的研究团队。总的来说， 研究将由以下人员领导 这些 一个 研究将大大促进我们对ncRNA在星形胶质细胞增生症中的功能和机制的理解，以及 从而为治疗慢性脊髓损伤提供新的分子靶点。

Abstract Spinal cord injury (SCI) is debilitating. Astrocytes are the predominant component of the spinal scar. Defining the complex molecular regulation during astrogliosis is fundamentally important for developing effective therapeutic strategies. We were the first to systematically analyze the expression profiles of long non- coding RNA (lncRNA) (>200 bp), a type of regulatory RNA that plays important roles, in purified astrocytes from adult spinal cords across SCI stages. There is a large knowledge gap in lncRNAs' roles and mechanisms in astrogliosis after SCI. As <2% of the genome is associated with protein-coding genes, innovative targeting of non-coding RNAs can greatly enlarge the number of druggable targets and facilitate new classes of mechanistic discoveries. Towards the overarching goal of defining the mechanisms of astrogliosis in SCI and identifying novel molecular targets for treating chronic SCI, we discovered that Zeb2os, a highly conserved lncRNA, plays an important functional role in astrogliosis. Data showed that Zeb2os expression has high correlation over the injury time course with its antisense protein coding gene Zeb2 and the essential astrogliosis factor Stat3. Significantly, Zeb2os shRNA knockdown (KD) in vivo by Adeno Associated Virus (AAV) injection led to attenuated astrogliosis, shown by astrocyte morphology, reduced GFAP and pSTAT3 expression, and reduced lesion size. Additionally, Zeb2os KD in primary astrocytes significantly decreased expression of many genes, including Gfap, Zeb2, Stat3, neurocan, phosphacan and integrins that are involved in scar development. Our RNAscope data demonstrated Zeb2os-FZeb2 and Zeb2os- pSTAT3 colocalization in GFAP+ cells. Furthermore, ChIP-Seq experiments revealed STAT3 bound to the Zeb2 promoter region. Notably, our data and studies by others provided evidence that, after injury, there is a change of astrocytes from an initial beneficial state (reactive astrocytes, RA) to a maladaptive state (scar-forming astrocytes, SA) expressing molecules that inhibit neuroregeneration. Importantly, attenuating astrocytic scar progression with anti-integrin neutralizing antibodies promoted functional improvement after SCI. Based on our exciting findings and others', we propose to: a) test the hypothesis that lncRNA Zeb2os regulates astrogliosis through a Zeb2os/Zeb2/Stat3 axis, b) decipher additional mechanisms by which Zeb2os regulates astrocytes by comprehensively identifying its target genes and pathways, and c) define the effects of manipulating Zeb2os and target genes on anatomical and functional outcomes in vivo by AAV injection. Our goal is not to remove the scar; it is to attenuate later stages of scar progression and the scar's inhibitory effects on neuroregeneration without interrupting neuroprotective effects at early phases. These team of investigators with unique and strong expertise in SCI, astrocyte biology and genomics. Overall, studies will be led by these a studieswill significantly advance our understanding oflncRNA function and mechanisms in astrogliosis, and thereby reveal new molecular targets for treating chronic SCI.