Therapeutic Targeting of Intracellular Mechanisms Involved in Glial Scar Formatio

描述（申请人提供）：中枢神经系统损伤后的神经胶质瘢痕改变病变环境，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胶质瘢痕的主要细胞成分；星形胶质细胞发生形态变化并产生细胞外基质，如硫酸软骨素蛋白聚糖（CSPGs），其物理和化学作用可抑制轴突生长。抑制星形胶质细胞形成或阻止CSPGs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。参与星形胶质细胞反应性控制和刺激CSPG产生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）抑制剂罗利普仑可以减少星形胶质细胞的形成，我们实验室的初步数据表明，在反应性星形胶质细胞中，PDE4A同工酶的慢性诱导与胶质瘢痕的成熟并行。PDE4亚型的位点特异性靶向可以预防星形胶质增生，并为脊髓或脑损伤后瘢痕减少提供新的治疗方向，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，将在体外[Specific Aim 1]和脊髓损伤（SCI）后的体内（gmap启动子驱动）中，通过慢病毒载体特异性表达PDE4A短发夹RNA (shRNA)，在星形胶质细胞中使用干扰RNA。在星形胶质细胞培养中，

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.