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疤痕改变病变环境，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胶质瘢痕的主要细胞成分；星形胶质细胞经历形态变化并产生细胞外基质，如硫酸软骨素蛋白多糖(CSPGs)，它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合成或降解CSPG的策略已被证明可以解除对轴突生长的抑制，改善功能。参与控制星形胶质细胞反应性和刺激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)抑制剂罗力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PDE4A同工酶的慢性诱导，这种诱导与胶质瘢痕的成熟平行发生。这种PDE4亚型的定位靶向可以防止星形胶质细胞增生，并为脊髓或脑损伤后瘢痕的减少提供新的治疗方向，从而增强轴突的可塑性和功能恢复。为了阐明PDE4A在星形胶质细胞反应性和CSPG产生中的作用，我们将通过慢病毒载体表达的PDE4A短发夹状RNA(ShRNA)在体外[特异性目标1]和体内(GFAP启动子驱动)[特异性目标2]来研究PDE4A在星形胶质细胞反应和CSPG产生中的作用。在星形胶质细胞培养中， PDE4A基因敲除的有效性将得到改进，并将研究PDE4A在细胞反应机制中的作用，包括A)细胞骨架重排，B)促进细胞迁移，C)促进细胞增殖，D)产生CSPG。然后在体内，这些体外效应将得到证实，以及PDE4A基因敲除在修复中的解剖学和功能益处的评估。完整的脊髓横断损伤模型将被用来评估星形胶质细胞中PDE4A基因敲除是否阻止轴突死亡和/或允许轴突跨损伤部位再生，而PDE4A分子抑制在星形胶质细胞中的功能效应将在不完全挫伤SCI范式中进行检验。

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.