Therapeutic Strategies for Repairing Optic Nerve Injury

描述（由申请人提供）：这是一项使用新型的、全身可递送的小抑制肽来确定神经元内源性和细胞外抑制因子的组合靶向是否显著改善视神经损伤（ONI）后视网膜神经节细胞（RGC）轴突再生和存活的提议。切断的视神经轴突不能再生，ONI导致患者终身视力丧失。除了轴突切断后的RGC凋亡死亡之外，内在生长能力降低和抑制性分子环境都导致成熟CNS神经元再生轴突的失败。使用条件性基因敲除（KO）小鼠的研究表明，肿瘤抑制基因PTEN是一种神经元内在因子，严重限制了成年RGC的再生能力。PTEN的缺失增强了ONI后RGC轴突的生长和存活。硫酸软骨素蛋白聚糖（CSPGs）产生的胶质瘢痕是强烈抑制轴突延伸的外在因素。最近，我们和其他实验室确定LAR和PTP磷酸酶作为介导CSPG抑制的受体。它们中的任何一个的缺失部分地克服了CSPG的抑制，并刺激受损CNS轴突的生长。包括LAR在内的一些PTP也可以激活半胱天冬酶并诱导细胞凋亡。抑制PTEN和CSPG信号传导对于促进CNS轴突再生是非常有希望的，但是PTEN、LAR或PTP β的转基因缺失对于治疗患者是不可行的。我们已经设计了小的模拟肽来阻断这些抑制分子的功能，并证明了它们在体外和体内促进轴突生长的效率。由于CSPG受体似乎通过不同于PTEN信号传导的途径调节神经元功能，因此我们假设结合抑制PTEN和CSPG信号传导比单独抑制任一种更好地促进RGC轴突再生和存活。为了验证这个假设，我们将使用 小肽抑制剂单独和组合，通过与我们实验室中可用的KO小鼠的结果进行比较来验证肽的功效。我们提出通过确定以下情况来解决3个特定目的：1）在KO小鼠中，PTEN的肽阻断是否促进与转基因缺失相似的RGC轴突再生和存活; 2）两种CSPG受体的肽阻断是否比抑制一种受体促进更大的RGC轴突再生和存活;和3）用肽阻断PTEN和CSPG信号传导促进比单独靶向其中之一更大的RGC轴突再生和存活。通过同时靶向神经元内源性和环境抑制因子与小阻断肽，我们试图促进轴突再生，并减少轴突切断诱导的RGC损失，以更大程度上比单独靶向任何一个信号。我们的新策略，管理小，全身可输送的化合物损伤后，可能会促进视神经损伤的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.