PROTEASE NEXIN 1--ALTERATIONS IN ALZHEIMER'S DISEASE

蛋白酶 Nexin 1——阿尔茨海默病的改变

• 批准号: 3767826
• 负责人: DENNIS CUNNINGHAM
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3767826
• 关键词: Alzheimer's disease; Primates; amyloid proteins; axon; brain mapping; chemical binding; dementia; enzyme complex; enzyme linked immunosorbent assay; enzyme mechanism; fibroblast growth factor; hippocampus; human tissue; immunocytochemistry; in situ hybridization; laboratory rat; messenger RNA; monoclonal antibody; neural degeneration; neuroanatomy; neuronal guidance; neurophysiology; peptidases; postmortem; protease inhibitor; protein purification; thrombin; tissue /cell culture; western blottings

These studies are based on findings that: (a) protease nexin-1 (PN-1) is a thrombin inhibitor present in human brain that produces neurite extension in cultured neuroblastoma cells, (b) thrombin produces neurite retraction in cultured neuroblastoma cells, (c) PN-1 and thrombin, reversibly regulate the stellation of cultured astrocytes, (d) PN-1 activity is reduced about 6-fold in AD brain, and (e) PN-1-protease complexes (probably PN-1 thrombin complexes appear to be increased in AD brain. Together, these results suggest the hypothesis that decreases in PN-1 (and increases in thrombin or a thrombin-like protease) could contribute to the pathology of AD by altering neurons and astrocytes. A major goal of our proposed studies is to learn more about the decrease in PN-1 and increase in PN-1-protease complexes in AD brain and to learn whether similar changes occur in other neurodegenerative diseases. WE will determine if the changes are global, or if they localized to certain brain structures. Accordingly, we will measure PN-1 activity, PN-1 protein, PN-1 protease complexes and PN-1 mRNA in various brain structures in a large number control brains and brains from individuals who died from various neurological diseases. In these studies we will also examine the microscopic distribution of PN-1 protein and PN-1 mRNA using immunohistochemistry and in situ hybridization. Another major goal is to identify the protease present in PN-1 protease complexes found in AD brain. The studies in the paragraph above will determine what brain sections contain the complexes, and if they occur in other neurodegenerative diseases. We will purify the complexes, disrupt them to release the intact protease, and identify the protease by amino acid sequence analysis. The other major goal is based on our observation that thrombin brings about shortening of neurites on cultured rat hippocampal neurons. We will determine if thrombin affects neurite branching and the number of neurite segments and if it modulates neuronal regulation by EGF, FGF and the amyloid b-protein. We will also determine if the effects of thrombin on neuroblastoma cells and neurons are mediated by cell surface thrombin binding sites. We will also purify rat PN-1 and isolate a panel of monoclonal antibodies to it for Dr. Cotman's studies on the response of PN-1 to entorhinal and hippocampal lesions in the rat brain.

这些研究基于以下发现：(A)蛋白酶Nexin-1(PN-1)是一种 人脑中存在的凝血酶抑制物可产生轴突延伸 在培养的神经母细胞瘤细胞中，(B)凝血酶产生轴突回缩 在培养的神经母细胞瘤细胞中，(C)PN-1和凝血酶，可逆地 调节培养的星形胶质细胞，(D)PN-1活性是 在AD大脑中减少了约6倍，以及(E)PN-1-蛋白酶复合体(可能 在AD脑中，PN-1凝血酶复合体似乎增加。一起， 这些结果表明，Pn-1减少(和增加 凝血酶或类凝血酶)可能与病理有关 通过改变神经元和星形胶质细胞来治疗AD。 我们建议的研究的一个主要目标是了解更多关于 阿尔茨海默病患者脑组织中PN-1和PN-1-蛋白酶复合体的增加与学习 其他神经退行性疾病是否会出现类似的变化。我们会的 确定这些变化是全局性的，还是局限于特定的大脑 结构。相应地，我们将测量PN-1活性、PN-1蛋白、PN-1 大鼠不同脑结构中的蛋白酶复合体和PN-1mRNA的表达 数字控制大脑和死于各种疾病的人的大脑 神经系统疾病。在这些研究中，我们还将研究 Pn-1蛋白和Pn-1mRNA的显微分布 免疫组织化学和原位杂交。 另一个主要目标是鉴定存在于PN-1蛋白酶中的蛋白酶 阿尔茨海默病大脑中发现的复合体。上一段中的研究将 确定包含这些复合体的大脑部分，以及它们是否出现在 其他神经退行性疾病。我们会净化这些建筑群，破坏 释放完整的蛋白水解酶，并用氨基酸鉴定其活性 酸序列分析。 另一个主要目标是基于我们观察到的凝血酶带来的 培养大鼠海马神经元上突起的缩短。我们会 确定凝血酶是否影响轴突分支和轴突数量 节段，以及它是否通过EGF、FGF3和 淀粉样蛋白B。我们还将确定凝血酶对血管内皮细胞的影响 神经母细胞瘤细胞和神经元由细胞表面凝血酶介导 结合部位。 我们还将提纯大鼠PN-1并分离一组单抗 科特曼博士关于PN-1对内脏和内脏反应的研究 大鼠脑内的海马区损伤。