Identifying Novel Molecular Targets for Chronic SCI

确定慢性 SCI 的新分子靶点

• 批准号: 10532227
• 负责人: JIaqian Wu-Huber
• 金额: $ 48.04万
• 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/10532227
• 关键词: Adult; Anatomy; Antisense RNA; Astrocytes; Attenuated; Behavioral; Binding; Biological Assay; Biology; Bromodeoxyuridine; CSPG3 gene; Cells; ChIP-seq; Chronic; Cicatrix; Code; Complex; 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; Proliferating; 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; Vertebral column; Virus; astrogliosis; axon regeneration; druggable target; experience; experimental study; functional improvement; functional outcomes; genetic manipulation; genome-wide; improved; in vivo; innovation; interest; knock-down; mouse model; nerve injury; neuroprotection; neutralizing antibody; novel; novel therapeutic intervention; overexpression; phosphacan; protein profiling; public health relevance; small hairpin RNA; therapeutically effective; transcriptome sequencing

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.

抽象的 脊髓损伤 (SCI) 会使人衰弱。星形胶质细胞是脊髓疤痕的主要成分。 定义星形胶质细胞增生过程中复杂的分子调控对于开发 有效的治疗策略。我们是第一个系统分析长非表达谱的人 编码 RNA (lncRNA)（>200 bp），一种在纯化的星形胶质细胞中发挥重要作用的调节 RNA 来自 SCI 各个阶段的成人脊髓。 lncRNA的作用和机制存在很大的知识差距 SCI 后星形胶质细胞增生。由于<2% 的基因组与蛋白质编码基因相关，因此创新靶向 非编码 RNA 可以大大增加可药物靶点的数量并促进新类别的治疗 机制的发现。实现明确 SCI 中星形胶质细胞增生机制的总体目标 在确定治疗慢性 SCI 的新分子靶点时，我们发现了 Zeb2os，一种高度保守的 lncRNA在星形胶质细胞增生中发挥重要的功能作用。数据显示Zeb2os表达量高 损伤时程与其反义蛋白编码基因Zeb2的相关性及必需的 星形胶质细胞增生因子 Stat3。值得注意的是，腺相关病毒在体内敲低 Zeb2os shRNA (KD) (AAV) 注射导致星形胶质细胞增生减弱，表现为星形胶质细胞形态、GFAP 和 pSTAT3 减少 表达，并减少病变大小。此外，原代星形胶质细胞中的 Zeb2os KD 显着降低 许多基因的表达，包括相关的 Gfap、Zeb2、Stat3、neurocan、phosphacan 和整合素 在疤痕的发展中。我们的 RNAscope 数据证明了 Zeb2os-FZeb2 和 Zeb2os-pSTAT3 在 GFAP+细胞。此外，ChIP-Seq 实验显示 STAT3 与 Zeb2 启动子区域结合。 值得注意的是，我们的数据和其他人的研究提供的证据表明，受伤后，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（疤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，减轻星形胶质细胞疤痕的进展 抗整合素中和抗体促进 SCI 后功能的改善。基于我们激动人心的 的研究结果和其他人的发现，我们建议：a）检验lncRNA Zeb2os通过a调节星形胶质细胞增生的假设 Zeb2os/Zeb2/Stat3 轴，b) 破译 Zeb2os 调节星形胶质细胞的其他机制 全面识别其靶基因和途径，以及 c) 定义操纵 Zeb2os 的效果 以及 AAV 注射对体内解剖和功能结果的靶基因。我们的目标不是删除 疤痕；它是为了减弱疤痕进展的后期阶段以及疤痕对疤痕的抑制作用 神经再生而不中断早期神经保护作用。这些 研究人员团队在 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。全面的， 研究将由 这些 一个 研究将显着增进我们对星形胶质细胞增生中lncRNA功能和机制的理解，并且 从而揭示治疗慢性 SCI 的新分子靶点。