INHIBITION AND DE-INHIBITION OF NEURITE OUTGROWTH

神经突生长的抑制和去抑制

• 批准号: 2269199
• 负责人: DAVID F MUIR
• 金额: $ 9.7万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2269199
• 关键词: Schwann cells; antibody; axon; biological models; cell growth regulation; chemical binding; chick embryo; dendrites; embryo /fetus tissue /cell culture; gel electrophoresis; glycosidases; growth inhibitors; immunochemistry; immunoelectron microscopy; laboratory rabbit; laboratory rat; laminin; molecular weight; mucopolysaccharides; nervous system regeneration; neurogenesis; neurotrophic factors; protein sequence; proteoglycan; receptor binding; stromelysin; synthetic peptide

Outgrowth by neuronal axons is a major feature of development and regeneration in the nervous system. In peripheral nerve, Schwann cells can promote axonal outgrowth by depositing stimulatory cues on their cell surface and into the surrounding basal lamina. Laminin (LN) is known to be one of the most potent of these promoters. Recent evidence suggests that inhibitory cues play an important role in restricting and directing axonal growth. The PI has found that rat Schwann cells secrete a proteoglycan (Neurite-Inhibiting Factor or NIF) which complexes with laminin and blocks its neurite-promoting activity in vitro. In addition, when the NIF-LN complex is exposed to proteases produced by neurons or Schwann cells, the inhibitory proteoglycan component is abolished resulting in the unmasking of LN's stimulatory activity. Based on these findings we propose the following thesis for the regulation of axonal outgrowth: i) Schwann cells present to neurons a matrix of stimulatory and inhibitory components; ii) the stimulatory component LN can exist in an inactive complex with NIF; iii) proteolysis is required to unmask the stimulatory potential of the NIF-LN complex and; iv) proteases are released by neurons and Schwann cells which degrade NIF and in effect activate the neurite-promoting ability of the LN. The aims of this project are: (1) to determine the homogeneity of our NIF preparation by electrophoretic analysis, characterizations of the glycosaminoglycan side-chains and amino acid sequencing of the core protein. Synthetic peptides corresponding to unique amino acid sequences will be used to produce monospecific antibodies against the core protein of NIF; (2) to determine if NIF binds to LN through side-chain or core protein interactions. Defined fragments of LN which contain neurite- promoting or cell-binding sites will be used to determine which regions of LN NIF interacts with and whether both the cell attachment and neurite-promoting activities of LN are inhibited by NIF. In addition, we will attempt to describe how NIF inhibits LN's activity by determining the affect that NIF binding to LN has on LN binding to neuronal surface receptors; (3) to determine if stromelysin/transin and other neural proteases can degrade NIF and de-inhibit the neurite-promoting potential of the NIF-LN complex and if the release of proteases by neurons can de- inhibit NIF-LN complexes during neurite outgrowth. Similarly, we will determine if neurons secrete heparan sulfate degrading endoglycosidases (heparanases) that can de-inhibit NIF-LN substrates; and (4) to determine by immunolocalizations and direct interventions if the inhibitory proteoglycan and proteoglycanases actively regulate axonal outgrowth in normal nerve and in models of neural regeneration.

神经元轴突的生长是发育的主要特征， 神经系统的再生。 在周围神经中，雪旺细胞 可以通过在细胞上沉积刺激信号来促进轴突生长 表面并进入周围的基底层。 层粘连蛋白（LN）是已知的 是这些促进剂中最有效的一种。 最近的证据表明 抑制性线索在限制和引导 轴突生长 PI发现大鼠雪旺细胞分泌一种 蛋白聚糖（神经突抑制因子或NIF），其与 层粘连蛋白并阻断其体外促进神经突的活性。 此外，本发明还提供了一种方法， 当NIF-LN复合物暴露于由神经元产生的蛋白酶时， 雪旺氏细胞，抑制性蛋白多糖成分被废除 导致LN的刺激活性的暴露。 基于这些 研究结果我们提出了以下论文的调节轴突 生长：i）雪旺细胞向神经元呈现刺激性基质， 和抑制成分; ii）刺激成分LN可以存在于 与NIF的非活性复合物; iii）需要蛋白水解来揭示NIF的活性。 NIF-LN复合物的刺激潜力，和; iv）蛋白酶， 神经元和雪旺氏细胞释放NIF， 激活LN的促神经突起能力。 本项目的目的是：（1）确定我们的同质性 通过电泳分析制备NIF， 糖胺聚糖侧链和核心的氨基酸测序 蛋白 对应于独特氨基酸序列的合成肽 将用于生产针对核心蛋白的单特异性抗体 （2）确定NIF是否通过侧链或核心与LN结合 蛋白质相互作用 含有神经突的LN的确定片段- 促进或细胞结合位点将用于确定哪些区域 LN NIF的相互作用，以及细胞附着和 LN的促神经突起活性被NIF抑制。 此外，本发明还提供了一种方法， 我们将试图描述NIF如何抑制LN的活性， NIF与LN结合对LN与神经元表面结合影响 受体;（3）确定是否溶基质素/转蛋白和其他神经 蛋白酶可以降解NIF并解除对神经突促进潜力的抑制 NIF-LN复合物，如果神经元蛋白酶的释放可以减少- 在神经突生长期间抑制NIF-LN复合物。 同样，我们将 确定神经元是否分泌硫酸乙酰肝素降解内切糖苷酶 （4）确定可以去抑制NIF-LN底物的乙酰肝素酶（heparanase）;和 通过免疫定位和直接干预， 蛋白聚糖和蛋白聚糖酶积极调节轴突生长， 正常神经和神经再生模型。