Therapeutic Strategies for Repairing Optic Nerve Injury

描述(申请人提供)：这是一项使用新颖的、系统可释放的小抑制肽来确定联合靶向神经元内部和细胞外抑制因子是否显著改善视神经损伤(ONI)后视网膜神经节细胞(RGC)轴突再生和存活的建议。切断的视神经轴突不能再生，ONI会导致患者终身视力丧失。除了轴突切断后RGC的凋亡性死亡外，固有生长能力的降低和分子环境的抑制都是导致成熟的中枢神经系统神经元无法再生轴突的原因。使用条件性基因敲除(KO)小鼠的研究表明，肿瘤抑制基因PTEN是一种神经内源性因子，严重限制成年视网膜节细胞的再生能力。PTEN基因缺失可促进ONI后RGC轴突的生长和存活。胶质瘢痕中产生的硫酸软骨素蛋白多糖(CSPGs)是强烈抑制轴突延伸的外在因素。最近，我们和其他实验室发现LAR和PTP�磷酸酶是介导CSPG抑制的受体。它们中的任何一个的缺失部分克服了CSPG的抑制作用，并刺激了受损中枢神经系统轴突的生长。一些PTPs，包括LAR，也可以激活caspase并诱导细胞凋亡。抑制PTEN和CSPG信号转导对促进中枢神经系统轴突再生很有希望，但转基因缺失PTEN、LAR或PTP�用于治疗是不可行的。我们设计了小分子模拟肽来阻断这些抑制分子的功能，并在体外和体内证明了它们促进轴突生长的有效性。由于CSPG受体似乎通过与PTEN信号不同的途径来调节神经元功能，我们假设联合抑制PTEN和CSPG信号可以更好地促进RGC轴突的再生和存活，而不是单独抑制其中之一。为了检验这一假设，我们将使用 小肽抑制剂单独和组合，通过与我们实验室提供的KO小鼠的结果比较来验证多肽的有效性。我们建议通过确定以下三个特定目标来解决：1)多肽阻断PTEN是否促进与转基因缺失类似的KO小鼠RGC轴突的再生和存活；2)多肽阻断两个CSPG受体比抑制一个受体更能促进RGC轴突的再生和存活；3)多肽同时阻断PTEN和CSPG信号可促进更大的RGC轴突再生和存活，而不是单独针对其中之一。通过同时靶向神经元内源性和环境抑制因子和小的阻断多肽，我们试图促进轴突再生，并在更大程度上减少轴突切断引起的RGC损失，而不是单独靶向任何一个信号。我们的新策略是在损伤后给予小的、系统可释放的化合物，这可能会促进视神经损伤实用组合疗法的发展。

DESCRIPTION (provided by applicant): This is a proposal that uses novel, systemically deliverable, small inhibitory peptides to determine whether combined targeting of neuron-intrinsic and extracellular inhibitory factors markedly improves retinal ganglion cell (RGC) axon regeneration and survival after optic nerve injury (ONI). Severed optic axons fail to regenerate and ONI leads to life-long visual loss in patients. In addition to apoptotic RGC death following axotomy, both a reduced intrinsic growth capacity and an inhibitory molecular environment contribute to failure of mature CNS neurons to regenerate their axons. Studies using conditional knockout (KO) mice suggest that the tumor suppressor gene PTEN is one neuron-intrinsic factor that critically restricts the regenerative capacity of adult RGCs. Deletion of PTEN enhanced RGC axon growth and survival after ONI. Chondroitin sulfate proteoglycans (CSPGs) generated in glial scars are extrinsic factors that strongly suppress axon extension. Recently, we and other labs identified LAR and PTP� phosphatases as receptors that mediate CSPG inhibition. Deletion of either of them partially overcomes suppression by CSPGs and stimulates growth of injured CNS axons. Some PTPs, including LAR, can also activate caspases and induce cell apoptosis. Suppressing PTEN and CSPG signaling is very promising for promoting CNS axon regeneration, but transgenic deletion of PTEN, LAR or PTP� is not feasible for treating patients. We have designed small mimetic peptides to block functions of these inhibitory molecules and demonstrated their efficiency in promoting axon growth in vitro and in vivo. Because CSPG receptors appear to regulate neuronal functions via pathways different from PTEN signaling, we hypothesize that combining inhibition of PTEN and CSPG signaling promotes RGC axon regeneration and survival better than inhibiting either alone. To test this hypothesis, we will use small peptide inhibitors alone and in combinations, validating the peptide efficacy by comparison to results with KO mice available in our lab. We propose to address 3 Specific Aims by determining whether: 1) peptide blockade of PTEN promotes similar RGC axon regeneration and survival as transgenic deletion in KO mice; 2) peptide blockade of two CSPG receptors promotes greater RGC axon regeneration and survival than inhibiting one receptor; and 3) blocking both PTEN and CSPG signaling with peptides promotes greater RGC axon regeneration and survival than targeting either one alone. By simultaneously targeting neuron-intrinsic and environmental inhibitory factors with small blocking peptides, we attempt to promote axon regeneration and to reduce axotomy-induced RGC loss to greater degrees than by targeting either signal individually. Our novel strategy of administering small, systemically deliverable compounds post-injury may facilitate development of practical combinatorial therapy for optic nerve injury.