MOLECULAR CHANGES IN NEURODEGENERATION

我们实验室过去几年的研究表明，有一种 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 调节轴突在体外和突触中的延伸 体内形成/结构。考虑到在许多情况下营养丰富 APP是一款保护神经元免受各种损伤的因子 神经损伤干预的杰出候选人。 因此，我们建议研究APP在几个模型中的参与 神经元和突触的退化和再生。以特定的目标 1，我们将确定APP在兔脊髓的受累程度 脊髓缺血模型(RSCIM)。我们的假设是，应用程序合成和 降解受缺血的影响是保护性的一部分 抗缺血损伤的机制。APP异构体 缺血前后将被量化和定位，其中 可能的，在蛋白质和信使核糖核酸水平使用各种技术 包括蛋白质印迹、免疫组织化学、Northern印迹、狭缝 斑点杂交、RT-PCR技术和原位杂交。这部作品服务于 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-多肽治疗。这项研究是基于我们的 最近发现，一段代表活性的短肽片段 APP营养结构域拮抗兔脑缺血损伤 脊髓。我们将用临床评估缺血性损伤， 生化、分子生物学和形态标准。 形态标准包括神经元和突触计数。具体而言 目标3，我们将检查潜在的应用程序参与萌芽 穿孔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，如果输注一种干扰 APP的作用是抑制萌发。《特定目标4》将测试 神经生长因子和β-成纤维细胞生长因子挽救中隔的假说 穹隆海马伞切断后神经元的变性 部分是通过激活可能的APP通路。我们会 观察APP多肽输注对大鼠中隔神经元的影响 FF损毁后。然后，我们将尝试对抗NGF的影响 通过与APP共同输注一种多肽来阻断其 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作用中的作用 采用体外培养模型研究谷氨酸诱导的神经元毒性。

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.