Therapeutic Targeting of Intracellular Mechanisms Involved in Glial Scar Formatio

参与神经胶质疤痕形成的细胞内机制的治疗靶向

基本信息

批准号：
8386059
负责人：
Damien D. Pearse
金额：
$ 19.13万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-06-01 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8386059
关键词：
Abate Ablation Affect American Area Astrocytes Axon Brain Cell Death Cell Proliferation Cell physiology Cells Chondroitin Sulfate Proteoglycan Chronic Cicatrix Contusions Cyclic AMP DNA Sequence Rearrangement Data Effectiveness Environment Enzymes Extracellular Matrix Family Foundations Growth Health Care Costs Immune Immune Cell Activation In Vitro Inflammatory Injury Isoenzymes Laboratories Lentivirus Vector Lesion Lipopolysaccharides Mediating Microglia Modeling Molecular Neuroglia Pathologic Processes Pathway interactions Phosphodiesterase Inhibitors Phosphorylation Play Prevention strategy Production Protein Isoforms Proteins RNA Interference Recovery of Function Regulation Reporting Role Rolipram Signal Transduction Silver Site Spinal Cord Spinal Cord transection injury Spinal cord injury Therapeutic Tissues Tumor Necrosis Factor-alpha Viral Vector Work astrogliosis attenuation axon growth axon regeneration cell motility cell type central nervous system injury cytokine functional outcomes functional restoration improved improved functioning in vivo inhibitor/antagonist injured injury and repair macrophage migration novel novel therapeutics phosphodiesterase IV phosphoric diester hydrolase prevent promoter repaired research study small hairpin RNA therapeutic development therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Glial scarring following CNS injury alters the lesion environment so as to impede axonal regeneration and plasticity, thereby limiting functional restitution. Reactive astrocytes are the main cellular component of the glial scar; astrocytes undergo morphological changes and produce extracellular matrix, such as chondroitin sulfate proteoglycans (CSPGs), which physically and chemically inhibit axon growth. Strategies that inhibit astrogliosis or prevent the synthesis of, or degrade, CSPGs have been demonstrated to relieve axon growth inhibition and improve function. Intracellular mechanisms involved in the control of astrocyte reactivity and the stimulation of CSPG production remain poorly understood. Recent reports have shown that the phosphodiesterase (PDE) inhibitor Rolipram can reduce astrogliosis and Preliminary Data from our laboratory indicates that there is a chronic induction of the PDE4A isozyme in reactive astrocytes that occurs in parallel with the maturation of the glial scar. Site-specific targeting of this PDE4 isoform may then prevent astrogliosis and offer a novel therapeutic direction for scar reduction after injury to the spinal cord or brain so as to enhance axon plasticity and functional recovery. To delineate the role of PDE4A in astrocyte reactivity and CSPG production, interference RNA will be used via lentiviral vector expressed PDE4A short-hairpin RNA (shRNA) specifically within astrocytes in vitro [Specific Aim 1] and in vivo (gfap-promoter driven) after spinal cord injury (SCI) [Specific Aim 2]. In astrocyte cultures, the effectiveness of PDE4A knockdown will be refined and the role of PDE4A in mechanisms of cellular reactivity, including A) cytoskeletal rearrangements, B) enhanced cell migration, C) increased cell proliferation and, D) the production of CSPGs, will be examined. Then in vivo, these in vitro effects will be corroborated as well as the anatomical and functional benefits of PDE4A knockdown in repair assessed. A complete transection SCI model will be used to assess if PDE4A knockdown in astrocytes prevents axon dieback and/or allows axonal regeneration across the injury site, while the functional effects of molecular PDE4A inhibition in astrocytes will be examined in an incomplete contusive SCI paradigm. PUBLIC HEALTH RELEVANCE: Spinal cord injury (SCI) is a devastating condition that affects more than 1.25 million Americans (Christopher and Dana Reeve Foundation), representing a major health care cost burden to the US. The formation of a glial scar around the injury site represents a potent obstacle to axonal regeneration and functional restitution. Although experimental studies have demonstrated the promise of targeting the glial scar as a therapeutic direction for promoting SCI repair, the intracellular mechanisms responsible for maintaining the reactivity of the major cellular constituent of the glial scar, the astrocyte, as well as its inhibtory extracellular matrix production are poorly understood. The proposed work will define, through molecular manipulations, a role for the cyclic AMP- phosphodiesterase signaling cascade in astrogliosis, a pathway known to play a central role in the cellular reactivity and pathological functioning of other cell types such as immune cells, as well as provide a novel therapeutic direction for glial scar ablation.

描述（由申请人提供）：中枢神经系统损伤后的神经胶质疤痕改变了病变环境，从而阻碍轴突再生和可塑性，从而限制功能恢复。反应性星形胶质细胞是神经胶质疤痕的主要细胞成分；星形胶质细胞经历形态变化并产生细胞外基质，例如硫酸软骨素蛋白聚糖（CSPG），它可以通过物理和化学方式抑制轴突生长。抑制星形胶质细胞增生或阻止 CSPG 合成或降解的策略已被证明可以减轻轴突生长抑制并改善功能。涉及控制星形胶质细胞反应性和刺激 CSPG 产生的细胞内机制仍知之甚少。最近的报告表明，磷酸二酯酶 (PDE) 抑制剂咯利普兰 (Rolipram) 可以减少星形胶质细胞增生，我们实验室的初步数据表明，反应性星形胶质细胞中存在 PDE4A 同工酶的慢性诱导，这与神经胶质疤痕的成熟同时发生。这种 PDE4 同种型的位点特异性靶向可能会预防星形胶质细胞增生，并为脊髓或大脑损伤后减少疤痕提供新的治疗方向，从而增强轴突可塑性和功能恢复。为了描述 PDE4A 在星形胶质细胞反应性和 CSPG 产生中的作用，将通过慢病毒载体表达 PDE4A 短发夹 RNA (shRNA)，在体外 [具体目标 1] 和体内（gfap 启动子驱动）中使用干扰 RNA，特别是在星形胶质细胞内脊髓损伤 (SCI) [具体目标 2]。在星形胶质细胞培养物中，将完善 PDE4A 敲低的有效性，并检查 PDE4A 在细胞反应机制中的作用，包括 A) 细胞骨架重排、B) 增强的细胞迁移、C) 增加的细胞增殖和 D) CSPG 的产生。然后在体内，这些体外效应将得到证实，并评估 PDE4A 敲低在修复中的解剖学和功能益处。完整的横切 SCI 模型将用于评估星形胶质细胞中的 PDE4A 敲低是否可以防止轴突枯死和/或允许轴突在损伤部位再生，而星形胶质细胞中分子 PDE4A 抑制的功能作用将在不完全挫伤性 SCI 范例中进行检查。公共健康相关性：脊髓损伤 (SCI) 是一种毁灭性的疾病，影响着超过 125 万美国人（克里斯托弗和达纳·里夫基金会），是美国主要的医疗保健费用负担。损伤部位周围神经胶质疤痕的形成是轴突再生和功能恢复的潜在障碍。尽管实验研究已证明以神经胶质疤痕为靶点作为促进 SCI 修复的治疗方向有希望，但负责维持神经胶质疤痕主要细胞成分星形胶质细胞的反应性及其抑制性细胞外基质产生的细胞内机制人们了解甚少。拟议的工作将通过分子操作来定义环AMP-磷酸二酯酶信号级联在星形胶质细胞增生中的作用，该途径已知在其他细胞类型（例如免疫细胞）的细胞反应性和病理功能中发挥核心作用。为胶质疤痕消融提供新的治疗方向。