Identifying Novel Molecular Targets for Chronic SCI

确定慢性 SCI 的新分子靶点

• 批准号: 10374478
• 负责人: JIaqian Wu-Huber
• 金额: $ 49.67万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-15 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10374478
• 关键词: Adult; Anatomy; Antisense RNA; Astrocytes; Attenuated; Behavioral; Biological Assay; Biology; Bromodeoxyuridine; CSPG3 gene; Cells; ChIP-seq; Chronic; Cicatrix; Code; Complex; Contusions; Data; Dependovirus; Development; Environment; Functional disorder; Funding; Gene Expression; Genes; Genome; Genomics; Glial Fibrillary Acidic Protein; Goals; In Vitro; Injections; Injury; Integrins; Interruption; Knowledge; Lesion; Luciferases; Measurement; Molecular; Molecular Target; Morphology; Nerve Regeneration; Pathway interactions; Phase; Phenotype; Play; Promoter Regions; Proteins; Public Health; RNA; Regulation; Regulatory Pathway; Research; Research Personnel; Role; STAT3 gene; Spinal; Spinal Cord; Spinal cord injury; Stains; Technology; Testing; Therapeutic; Time; Transgenic Mice; Untranslated RNA; astrogliosis; axon regeneration; base; druggable target; experience; experimental study; functional improvement; functional outcomes; genetic manipulation; genome-wide; improved; in vivo; innovation; interest; knock-down; mouse model; nerve injury; neutralizing antibody; novel; novel therapeutic intervention; overexpression; phosphacan; protein profiling; public health relevance; small hairpin RNA; therapeutically effective; transcriptome sequencing; viral genomics

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.

摘要 脊髓损伤（SCI）是一种严重的疾病。星形胶质细胞是脊柱瘢痕的主要成分。 确定星形胶质细胞增生过程中复杂的分子调控对于发展 有效的治疗策略。我们是第一个系统地分析长非- 编码RNA（lncRNA）（>200 bp），一种在纯化的星形胶质细胞中起重要作用的调节RNA 脊髓损伤的各个阶段。关于lncRNA的作用和机制， 星形胶质细胞增生由于<2%的基因组与蛋白质编码基因相关， 非编码RNA可以大大增加可药物靶点的数量，并促进新的药物靶点的产生。 机械的发现朝着明确SCI星形胶质细胞增生机制的总体目标， 通过鉴定治疗慢性SCI的新分子靶点，我们发现Zeb 2 os，一个高度保守的 lncRNA在星形胶质细胞增生中起重要作用。数据显示，Zeb 2 os表达量高， 其反义蛋白编码基因Zeb 2与损伤时程的相关性及必需的 星形胶质细胞增生因子Stat 3。值得注意的是，腺相关病毒体内Zeb 2 os shRNA敲低（KD） (AAV)注射导致减弱的星形胶质细胞增生，通过星形胶质细胞形态显示，GFAP和pSTAT 3减少 表达，并减少病变大小。此外，原代星形胶质细胞中的Zeb 2 os KD显著降低， 许多基因的表达，包括涉及的Gfap、Zeb 2、Stat 3、neurocan、phosphacan和整合素 在疤痕发展中的作用我们的RNAscope数据证明了Zeb 2 os-FZeb 2和Zeb 2 os-pSTAT 3共定位于 GFAP+细胞。此外，ChIP-Seq实验显示STAT 3结合到Zeb 2启动子区。 值得注意的是，我们的数据和其他人的研究提供了证据，表明损伤后，星形胶质细胞发生了变化， 从最初的有益状态（反应性星形胶质细胞，RA）到适应不良状态（瘢痕形成星形胶质细胞，SA） 表达抑制神经再生的分子重要的是，减少星形细胞瘢痕进展， 抗整联蛋白中和抗体促进SCI后的功能改善。基于我们令人兴奋的 研究结果和其他人的，我们建议：a）测试lncRNA Zeb 2 os通过调节星形胶质细胞增生的假设， Zeb 2 os/Zeb 2/Stat 3轴，B）通过以下方式破译Zeb 2 os调节星形胶质细胞的额外机制： 全面鉴定其靶基因和途径，以及c）定义操纵Zeb 2 os的效果 和靶基因对体内解剖学和功能结果的影响。我们的目标不是 疤痕;它是减弱疤痕进展的后期阶段和疤痕的抑制作用， 在早期阶段不中断神经保护作用的神经再生。这些 研究团队在SCI、星形胶质细胞生物学和基因组学方面拥有独特而强大的专业知识。总的来说， 研究将由 这些 一 这些研究将极大地促进我们对ncRNA在星形胶质细胞增生中的功能和机制的理解， 从而揭示了治疗慢性SCI的新的分子靶点。