Laminin is Crucial for CNS Axon regeneration

层粘连蛋白对于中枢神经系统轴突再生至关重要

• 批准号: 6721299
• 负责人: ALFRED T MALOUF
• 金额: $ 37.75万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-15 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6721299
• 关键词: age difference; antisense nucleic acid; axon; gene expression; genetically modified animals; granule cell; growth cones; hippocampus; immunocytochemistry; in situ hybridization; innervation; integrins; laboratory mouse; laboratory rat; laminin; mossy fiber; nervous system regeneration; neurogenetics; oligonucleotides; polymerase chain reaction; protein isoforms; southern blotting; western blottings

DESCRIPTION: (Verbatim from the Applicant's Abstract) The goal of this proposal (ECM) molecules that are potent promoters of neurite outgrowth in monolayer cultures of dissociated CNS neurons. However, it has been difficult to examine whether laminins promote axon outgrowth in vivo, because the elimination of laminins expression in transgenic (knockout) animals results in an embryonic lethal phenotype. In order to examine the function of laminins in well-organized brain tissue, we have designed experiments using cultured hippocampal slices, whose anatomy and circuitry are remarkably similar to the intact hippocampus. Hippocampal slice cultures, therefore, provide a powerful model system for analyzing axon regeneration of CNS neurons under well-controlled in vitro conditions. We have chosen to study the MF pathway because it possesses profound plasticity and regenerative properties in vivo and in vitro. Indeed, MFs are capable of regenerating their normal synaptic pattern following transection in slice cultures, and they retain a plastic form of sprouting when transected in vivo. Significantly, our preliminary data suggest the laminin is expressed along the MF pathway and termination zone. The regenerative abilities of MFs make this pathway an ideal choice for examining the growth and guidance promoting effects of laminins on CNS axons. Our preliminary data demonstrate that application of antisense oligodeoxyribonuclotides (ODNs), which inhibits laminin expression, produces severe retardation and misguidance of transected MFs in slice cultures made from P4 animals. We now propose to perform a detained analysis of this abnormal regeneration using image analysis and confocal microscopy of stained MFs and terminals in fixed sections. MFs in living tissue will be analyzed using time-lapse imaging. The maximum period of MF outgrowth is between P0 and P14, and the pathway exhibits mature morphology by P21. Slices made from P21 animals will be examined to determine if MF regeneration is limited to a "critical period" that corresponds to the time of maximal MF outgrowth (P0-P14). In order to identify the specific subtypes of laminins and their integrin receptors that control axon regeneration, a detailed analysis of laminins and integrin subunit expression will be performed. We will also examine the role of laminins in directing MF outgrowth from hippocampal progenitor cells transplanted onto slice cultures. These progenitor cells have been shown to differentiate into dentate granule neurons when transplanted into the hippocampus of intact animals. Understanding the molecules that enable the mossy fiber pathway to regenerate may allow us to develop better transplantation and bridge building strategies that can be used in CNS pathways that fail to regenerate.

描述：（逐字研究申请人的摘要）该提案的目标 （ECM）是单层神经突的有效启动子的分子 解离的中枢神经系统神经元的培养物。但是，很难检查 层粘连蛋白是否在体内促进轴突的生长，因为消除了 转基因（基因敲除）动物中的层粘连蛋白表达导致胚胎 致命表型。为了检查层粘连蛋白在 组织良好的脑组织，我们已经设计了使用培养的实验 海马切片，其解剖学和电路与 完整的海马。因此，海马切片培养物提供了强大的 用于分析CNS神经元轴突再生的模型系统 良好控制的体外条件。我们选择研究MF途径 因为它在体内具有深刻的可塑性和再生特性 并在体外。实际上，MF能够再生其正常突触 切片培养物的横断后的图案，它们保留了塑料形式 在体内转移时发芽。值得注意的是，我们的初步数据 建议层粘连蛋白沿MF途径和终止区表达。这 MFS的再生能力使该途径成为检查的理想选择 层粘连蛋白对中枢神经系统轴突的生长和指导促进作用。 我们的初步数据证明了反义的应用 抑制层粘连蛋白表达的寡脱氧氧基核苷酸（ODN）会产生 切片培养物中MFS的严重迟缓和误导 来自P4动物。我们现在建议对这种异常进行拘留分析 使用染色MF的图像分析和共聚焦显微镜再生 固定部分中的终端。将使用生物组织中的MF进行分析 延时成像。 MF出生的最长时期在P0和P14之间 途径通过p21表现出成熟的形态。由P21动物制成的切片 将检查以确定MF再生是否仅限于“关键 时期“对应于最大MF出生时间（P0-P14）。按顺序 鉴定层粘连蛋白的特定亚型及其整合素受体的特定亚型 控制轴突再生，对层粘连蛋白和整联蛋白亚基的详细分析 表达式将进行。我们还将研究层粘连蛋白在 从移植到海马的祖细胞中指导MF的生长 切片文化。这些祖细胞已被证明分化为 齿状颗粒神经元移植到完整的海马 动物。了解使苔藓纤维途径到达的分子 再生可能使我们能够发展更好的移植和桥梁建设 可以在无法再生的CNS途径中使用的策略。