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）：CNS损伤后的胶质瘢痕形成改变了病变环境，从而阻碍轴突再生和可塑性，从而限制了功能恢复。反应性星形胶质细胞是神经胶质瘢痕的主要细胞组分;星形胶质细胞经历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），其在物理和化学上抑制轴突生长。抑制星形胶质细胞增生或阻止CSPG合成或降解CSPG的策略已被证明可缓解轴突生长抑制并改善功能。星形胶质细胞反应性的控制和CSPG生产的刺激所涉及的细胞内机制仍然知之甚少。最近的报道表明，磷酸二酯酶（PDE）抑制剂咯利普兰可以减少星形胶质细胞增生，我们实验室的初步数据表明，在反应性星形胶质细胞中存在PDE 4A同工酶的慢性诱导，其与胶质瘢痕的成熟平行发生。这种PDE 4亚型的位点特异性靶向可以预防星形胶质细胞增生，并为脊髓或脑损伤后的瘢痕减少提供新的治疗方向，以增强轴突可塑性和功能恢复。为了描述PDE 4A在星形胶质细胞反应性和CSPG产生中的作用，将在体外[特异性目的1]和脊髓损伤（SCI）后体内（gfap启动子驱动）[特异性目的2]通过特异性表达PDE 4A短发夹RNA（shRNA）的慢病毒载体使用干扰RNA。在星形胶质细胞培养中， PDE 4A敲低的有效性将被改进，并且PDE 4A在细胞反应性机制中的作用将被检测，所述细胞反应性机制包括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PDE 4A敲低在修复中的解剖学和功能益处。完全横断SCI模型将用于评估星形胶质细胞中的PDE 4A敲低是否防止轴突死亡和/或允许损伤部位的轴突再生，而星形胶质细胞中的分子PDE 4A抑制的功能效应将在不完全挫伤SCI范例中进行检查。 公共卫生相关性：脊髓损伤（SCI）是一种破坏性疾病，影响超过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。损伤部位周围胶质瘢痕的形成是轴突再生和功能恢复的有力障碍。尽管实验研究已经证明了靶向胶质瘢痕作为促进SCI修复的治疗方向的前景，但对负责维持胶质瘢痕的主要细胞成分星形胶质细胞的反应性以及其促细胞外基质产生的细胞内机制知之甚少。拟议的工作将通过分子操作来定义星形胶质细胞增生中环状AMP-磷酸二酯酶信号级联的作用，这是一种已知在其他细胞类型（如免疫细胞）的细胞反应性和病理功能中发挥核心作用的途径，并为神经胶质瘢痕消融提供新的治疗方向。