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

• 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
• 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

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，以阻止轴突再生和可塑性，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。星形胶质细胞发生形态学变化并产生细胞外基质，例如硫酸软骨素蛋白聚糖（CSPG），它们在物理和化学上抑制轴突的生长。已证明抑制星形胶质细胞增多症或防止CSPG的合成或降解的策略已被证明可以缓解轴突生长抑制并改善功能。涉及控制星形胶质反应性和CSPG产生刺激的细胞内机制仍然很少了解。最近的报道表明，磷酸二酯酶（PDE）抑制剂ROLIPRAM可以减少星形胶质细胞增多症，而我们实验室的初步数据表明，反应性星形胶质细胞中PDE4A同工酶的慢性诱导与伴随恐惧的成熟相似。然后，该PDE4同工型的位点特异性靶向可以防止星形胶质细胞增多症，并为脊髓或脑损伤后的疤痕减少提供了新的治疗方向，从而增强了轴突的可塑性和功能恢复。为了描述PDE4A在星形胶质细胞反应性和CSPG产生中的作用，将通过慢病毒载体使用干扰RNA使用PDE4A短毛RNA（SHRNA），特定在体外体内[特定的AIM 1]和VIVO（GFAP-PROMOTER驱动器驱动器）（Scinal dection sapi aim AIM）[coci）[sci）[22]在星形胶质细胞文化中， PDE4A敲低的有效性将得到完善，PDE4A在细胞反应性机理中的作用，包括a）细胞骨架重排，b）b）增强的细胞迁移，c）增加细胞增殖，d）CSPG的产生。然后在体内，这些体外效应将得到证实，以及评估的修复中PDE4A敲低的解剖和功能益处。完整的Transection 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恢复和/或允许在损伤部位进行轴突再生，而在不完整的Contime Sci Paradigm中，将检查星形胶质细胞中分子PDE4A抑制的功能效应。 公共卫生相关性：脊髓损伤（SCI）是一种毁灭性的状况，影响了超过125万美国人（克里斯托弗和达娜·里夫基金会），代表了美国的主要医疗保健成本负担。损伤部位周围的神经胶质疤痕形成代表了轴突再生和功能恢复的有效障碍。尽管实验研究表明，将神经胶质疤痕作为促进SCI修复的治疗方向的希望，但细胞内机制负责维持神经胶质疤痕，星形胶质细胞以及其抑制性细胞外基质产生的主要细胞组成的反应性。所提出的工作将通过分子操作来定义循环AMP-磷酸二酯酶信号传导在星形胶质症中的作用，这是一种已知的途径，该途径在其他细胞类型的细胞反应性和病理功能（如免疫细胞）中起着核心作用，并为闪光涂抹的新治疗方向提供了新的治疗方向。