Enhancing axon regeneration by multi-inhibitor blocking

描述(由申请人提供)： 成年哺乳动物的中枢神经系统(CNS)是轴突再生的深层抑制环境。这在很大程度上是由于中枢神经系统损伤环境中的多个轴突再生抑制物(ARI)所致。其中包括Nogo、髓鞘相关糖蛋白(MAG)、少突胶质细胞髓磷脂糖蛋白(OMGp)和硫酸软骨素蛋白多糖(CSPG)。每个ARI与神经或轴突表面的互补配体结合，阻止轴突再生。对ARI及其配体的快速了解提供了以前未曾预料到的机会来阻止它们的行动，并潜在地增强轴突再生。 不同已知的ARL在体内阻断轴突再生的相对作用尚不清楚。这项R21探索性/发展性赠款申请描述了一种高度定向的短期计划，在一个具有良好特征的活体脊髓损伤模型中，单独和联合阻断已知的ARI。建议的研究利用了有关ARI分子性质的最新发现。这些结果可能会指导未来的研究，因为它们将提供在同一损伤模型中并排阻断不同抑制系统的效果的比较数据。根据结果，他们可能会鼓励未来的努力，将重点放在多个抑制剂的阻断上，而不是单一的系统阻断上。建议方法的“探索性/发展性”方面包括(I)在单一病变模型中阻断多个抑制剂；以及(Ii)使用糖生物学的工具作为实验疗法。每个ARI和/或其神经细胞表面配体(S)都是糖基化的。此外，糖基化在抑制轴突再生中起着关键的结构和功能作用。Nogo的神经细胞配体NgR是一种糖基磷脂酰肌醇(GPI)连接的糖蛋白，OMGp也是如此。MAG是一种唾液酸结合蛋白，可与神经细胞表面神经节苷脂GD1a和GT1b结合，也可与NGR结合。最后，CSPG的糖链介导了其对轴突再生的抑制。糖生物学工具与新的生化工具相结合，为阻断ARI和增强体内轴突再生提供了可行的手段。为此，我们将使用以下糖基酶：磷脂酰肌醇特异性磷脂酶C(PI-PLC)，切割NGR和OMgp的GPI锚定；神经氨酸酶，切割GDLA和GT1b；软骨素酶ABC，切割CSPG。每种酶的生化效果将通过免疫组织化学进行监测，并将在大鼠脊髓损伤(背侧半横断)后确定其对再生的影响。阻断ARI的糖、肽和抗体也将被用来进一步提高这些研究的价值。由此得到的数据可能为体内模型中不同ARI的相对贡献提供洞察，并可能支持对促进中枢神经系统损伤后轴突再生的新方法的评估。

DESCRIPTION (provided by applicant): The adult mammalian central nervous system (CNS) is a profoundly inhibitory environment for axon regeneration. This is due, in significant measure, to multiple axon regeneration inhibitors (ARI's) in the milieu of a CNS injury. These include Nogo, myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp) and chondroitin sulfate proteoglycans (CSPG). Each ARI binds to complementary ligands on the nerve or axon surface, halting axon regeneration. Rapidly emerging knowledge of ARI's and their ligands provides previously unanticipated opportunities to block their actions and potentially enhance axon regeneration. The relative contributions of the different known ARl's in blocking axon regeneration in vivo are unknown. This R21 Exploratory/Developmental Grant application describes a highly directed, short-term program to block the known ARI's, individually and in combination, in a well characterized in vivo spinal cord injury model. The proposed studies take advantage of the latest findings regarding the molecular nature of ARI's. The results may direct future studies, in that they will provide comparative data on the effects of blocking the different inhibitory systems side by side in the same lesion model. Depending on the outcome, they may encourage future efforts to focus on multi inhibitor blocking rather than on blocking single systems. The "exploratory/developmental" aspects of the proposed approach include (i) multi-inhibitor blocking in a single lesion model; and (ii) the use of the tools of glycobiology as experimental therapeutics. Each ARI and/or its nerve cell surface ligand(s) are glycosylated. Moreover, glycosylation plays key structural and functional roles in inhibiting axon regeneration. The nerve cell ligand for Nogo, NgR, is a glycosylphosphatidylinositol (GPI)-Iinked glycoprotein, as is OMgp. MAG is a sialic acid binding protein that binds to nerve cell surface gangliosides GDla and GTlb, as well as to NgR. Finally, the sugar chains of CSPG mediate its inhibition of axon regeneration. The tools of glycobiology, in concert with novel biochemical tools, provide feasible means to block ARI's and enhance axon regeneration in vivo. We will use the following glyco-enzymes for this purpose: phosphatidylinositol-specific phospholipase C (PI-PLC), to cleave the GPI anchors of NgR and OMgp, neuraminidase to cleave GDla and GTlb, and chondroitinaseABC to cleave CSPG. The biochemical efficacy of each enzyme will be monitored immunohistochemically, and their effects on regeneration will be determined after spinal cord lesion (dorsal hemisection) in rats. ARI-blocking saccharides, peptides, and antibodies will also be used to further enhance the value of these studies. The resulting data may provide insight on the relative contributions of the different ARI's in an in vivo model, and may support evaluation of new approaches to enhance axon regeneration after CNS injury.