MOLECULAR CHANGES IN NEURODEGENERATION

神经变性中的分子变化

基本信息

批准号：
6243673
负责人：
TSUNAO SAITOH
金额：
$ 14.17万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-06-01 至 1998-05-31
项目状态：
已结题

项目摘要

Research for the last few years in our laboratory has revealed that one of the physiological functions of APP is to regulate cellular functions and survival. In particular, APP has been found to be a trophic molecule that regulates neurite extension in vitro and synaptic formation/structure in vivo. Considering that in many instances trophic factors protect neurons from damages caused by various injuries, APP is a prominent candidate for the intervention of neuronal injuries. Therefore, we propose to study the involvement of APP in several models of neuronal and synaptic degeneration and regeneration. In Specific Aim 1, we will determine the degree of APP involvement in the rabbit spinal cord ischemia model (RSCIM). Our hypothesis is that APP synthesis and degradation are affected by ischemia as a part of the protective mechanisms to counteract the damage induced by ischemia. APP isoforms before and after ischemia will be quantified and localized, where possible, at both protein and mRNA levels using various techniques including Western blotting, immunohistochemistry, Northern blotting, slot blotting, RT-PCR technique, and in situ hybridization. This work serves as a foundation for Specific Aim 2 where we will optimize the procedure for the APP-peptide therapy using RSCIM. This study is based on our recent finding that a short peptide fragment representing the active trophic domain of APP antagonizes the ischemia-induced damage in rabbit spinal cords. We will evaluate ischemic injury using clinical, biochemical, molecular biological, and morphological criteria. Morphological criteria include neuronal and synaptic counts. In Specific Aim 3, we will examine a potential APP involvement in the sprouting reactions in the molecular layer of the dentate gyrus after the perforant path (PP) lesion. We will ask which neurons produce APP in response to PP lesion, and also if the infusion of a peptide which interferes with the function of APP inhibits the sprouting. Specific Aim 4 will test the hypothesis that the effect of NGF and beta-FGF to rescue medioseptal neurons from degeneration after the transection of fimbria-fornix (FF) is partially through the activation of putative APP pathways. We will investigate the effect of APP peptide infusion on the medioseptal neurons after FF lesion. Then, we will attempt to antagonize the effects of NGF and bFGF by co-infusing a peptide that completes with APP and blocks its function. Finally, in Specific Aim 5, we will test the potential effect of APP agonists and antagonists in the protection by growth factors from glutamate-induced neuronal toxicity employing the in vitro culture model.

我们实验室过去几年的研究表明， APP的生理功能之一是调节细胞功能和生存。特别是，APP被发现是一种营养分子调节体外神经突延伸和突触体内的形成/结构。考虑到在许多情况下营养因素保护神经元免受各种损伤造成的损害，APP是干预神经元损伤的杰出候选者。因此，我们建议研究APP在几个模型中的参与神经元和突触变性和再生。特定目标 1、我们将确定APP对兔脊髓的参与程度脊髓缺血模型（RSCIM）。我们的假设是 APP 合成和作为保护性机制的一部分，降解会受到缺血的影响抵消缺血引起的损伤的机制。应用亚型缺血前后的情况将被量化和定位，其中使用各种技术在蛋白质和 mRNA 水平上实现可能包括Western blotting、免疫组织化学、Northern blotting、slot 印迹、RT-PCR 技术和原位杂交。这项工作服务于作为具体目标 2 的基础，我们将在其中优化程序使用 RSCIM 进行 APP 肽治疗。这项研究是基于我们最近发现代表活性的短肽片段 APP营养结构域对抗兔缺血损伤脊髓。我们将使用临床评估缺血性损伤，生化、分子生物学和形态学标准。形态学标准包括神经元和突触计数。具体来说目标 3，我们将检查潜在的 APP 参与萌芽过程穿通后齿状回分子层的反应路径（PP）病变。我们将询问哪些神经元产生 APP 来响应 PP 损伤，以及如果输注干扰肽 APP的作用是抑制发芽。具体目标 4 将测试假设 NGF 和 β-FGF 对挽救中间隔的作用穹窿伞 (FF) 横断后变性的神经元部分是通过激活假定的 APP 途径。我们将研究APP肽输注对中间隔神经元的影响 FF 病变后。然后，我们将尝试拮抗NGF的作用和 bFGF 通过共注入肽来完成 APP 并阻断其功能。最后，在具体目标5中，我们将测试潜在的效果 APP激动剂和拮抗剂在生长因子保护中的作用采用体外培养模型研究谷氨酸诱导的神经元毒性。