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: 3745500
• 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/3745500
• 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）蛋白酶连接蛋白-1（PN-1）是一种蛋白酶， 一种存在于人脑中的凝血酶抑制剂，可产生神经突延伸 在培养的神经母细胞瘤细胞中，（B）凝血酶产生神经突收缩 在培养的神经母细胞瘤细胞中，（c）PN-1和凝血酶，可逆 调节培养的星形胶质细胞的星座，（d）PN-1活性是 在AD脑中减少约6倍，和（e）PN-1-蛋白酶复合物（可能 PN-1凝血酶复合物似乎在AD脑中增加。 在一起， 这些结果表明PN-1减少（并增加）的假设 凝血酶或凝血酶样蛋白酶）可能导致病理学 通过改变神经元和星形胶质细胞来治疗AD。 我们提出的研究的一个主要目标是更多地了解 PN-1和增加PN-1-蛋白酶复合物在AD脑和学习 类似的变化是否发生在其他神经退行性疾病中。 我们将 确定这些变化是全局性的，还是局限于特定的大脑 结构. 因此，我们将测量PN-1活性、PN-1蛋白、PN-1蛋白、PN-1蛋白、PN-1蛋白和PN-1蛋白。 蛋白酶复合物和PN-1 mRNA在各种脑结构中的表达， 数字控制的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-1 mRNA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PN-1蛋白酶中存在的蛋白酶 在AD脑中发现的复合物。 上段中的研究将 确定哪些大脑部分包含复合物，如果它们发生在 其他神经退行性疾病。 我们将净化这些复合体， 酶的氨基酸组成进行鉴定， 酸性序列分析 另一个主要目标是基于我们的观察，凝血酶导致 缩短培养的大鼠海马神经元突起。 我们将 确定凝血酶是否影响神经突分支和神经突数量 节段，如果它调节神经元调节EGF，FGF和 淀粉样B蛋白。 我们还将确定凝血酶对 神经母细胞瘤细胞和神经元通过细胞表面凝血酶介导 结合位点。 我们还将纯化大鼠PN-1并分离一组单克隆抗体 Cotman博士关于PN-1对内嗅和 大鼠大脑中的海马损伤。