Regulation And Processing Of Amyloid Precursor Protein G

淀粉样前体蛋白 G 的调控和加工

• 批准号: 6508408
• 负责人: JOHN W KUSIAK
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 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/6508408
• 关键词: Alzheimer's disease; aging; amyloid proteins; apoptosis; developmental genetics; disease /disorder etiology; gene induction /repression; gene mutation; human tissue; neurogenetics; neuropathology; pathologic process; protein metabolism

Summary of Work: A major focus of this project is to discover the role of the Amyloid Precursor Protein (APP) in the etiology and pathology of Alzheimer's Disease (AD). The normal physiological role of this protein is also under investigation. APP is important to study since the processing of APP and the effect of APP mutations and Presenilin mutations on APP processing bear directly on the increased production and extracellular deposition of A-beta peptides in AD. The alpha- and beta-secretase processing of APP also generates two large N-terminal secreted forms of the protein, which may have neurotrophic or neurotoxic properties, respectively. Brains of AD patients exhibit decreased synaptic connectivity and selective and massive neuronal cell loss. We are interested in examining the mechanisms involved in this cell death. Rare mutations in APP lead to an early onset, autosomal dominant form of AD (FAD). Previously, we showed that over-expression of FAD mutant forms of APP by either stably transfecting PC12 cells or by adenovirus-mediated gene transfer of primary cortical neurons led to increased apoptotic cell death over several days. More recently, we have been studying the toxic effects of several A-beta peptides on human neuroblastoma cells and the signaling pathways that are activated upon A-beta peptide treatment. Low concentrations of A-beta 1-42 killed SH-SY5Y and IMR-32 cells by apoptosis as measured by an ELISA. A-beta 1-40 was much less potent. A-beta 17-42, derived from APP by sequential alpha- and gamma-secretase cutting of APP, dose-dependently killed these cells apoptotically. A-beta 17-42 previously was thought to be a product of non-amyloidogenic APP processing and not a toxic peptide. Recent evidence shows that this peptide accumulates in the plaques of AD brains although its importance in AD pathology is unclear at this time. A-beta17-42 activated caspase-8 and caspase-3, and induced PARP cleavage, proteins important in a cascade of events leading to apoptotic cell death. Selective caspase-8 and caspase-3 inhibitors completely blocked A-beta 17-42 induced neuronal death. A-beta 17-42 activated c-jun N-terminal kinase (JNK) approximately two fold. Over-expression of a dominant-interfering SEK1 construct (a protein kinase that phosphorylates and activates JNK) protected against A-beta 17-42 -induced neuronal death by 50-70 %. The results demonstrate that A-beta 17-42 peptide induced neuronal apoptosis via a Fas-like/caspase-8 activation pathway. The results also suggest that p3 peptide may be an additional toxic peptide derived from APP proteolysis and may have a role in the neuronal cell loss characteristic of AD. We also analyzed the early signaling mechanisms of A-beta toxicity using human neuronal SH-SY5Y cells. We have focused on mitogen-activated protein kinases and the PI-3 kinase/Akt cascades. A-beta 1-42 treatment, which resulted in a dose-dependent cell death, weakly activated Akt and ERK, but had no effect on p38 kinase. However, this activation of ERK and Akt by A-beta 1-42 apparently did not play a role in A-beta toxicity since specific inhibitors of these kinase pathways, U0126 and wortmannin respectively, had no influence on A-beta-induced neuronal death. However A-beta 1-42-induced JNK activation by about two fold and seemed to play a critical role in A-beta-induced neuronal death since the dominant-negative construct SEK-1 blocked JNK activation and protected against cell death. Insulin-like growth factor-1 (IGF-1) dose-dependently protected cells from A-beta toxicity by strongly activating ERK and Akt and blocking A-beta-induced JNK activation. A specific Go/Gi inhibitor, pertussis toxin, (PT) also protected against A-beta toxicity by blocking A-beta-induced JNK activation. These results suggested that A-beta peptides in part could activate PT-sensitive-G-proteins leading to JNK activation. This may be an important and early event of A-beta toxicity and an early signaling pathway underlying neuronal loss in AD pathology. Finally, we have been examining the effects of a shortened, secreted form of APP derived from the initial beta-secretase processing of APP. This protein is called secreted APP beta (sAPPb). We generated cell lines over-expressing authentic sAPPa and sAPPb and which secrete these proteins into the surrounding media. We found that conditioned media containing sAPPb, when added to either NGF differentiated PC12 cells or primary cortical neurons, led to an apoptotic cell death, while conditioned media containing sAPPa had no such effect. An antibody specific to sAPPb prevented the cell death and a slightly truncated, highly purified form of sAPPb also caused cell death. These latter results strongly suggested that sAPPb itself is an additional toxic protein derived from beta-secretase APP processing. Overall these results provide a rationale for targeting particular elements of apoptotic pathways as well as APP processing for therapeutic intervention in AD.

工作总结：本项目的一个主要重点是发现淀粉样前体蛋白（APP）在阿尔茨海默病（AD）的病因和病理中的作用。这种蛋白质的正常生理作用也在研究中。APP是重要的研究，因为APP的加工和APP突变和早老素突变对APP加工的影响直接影响AD中A-β肽的增加的产生和细胞外沉积。APP的α-和β-分泌酶加工还产生两种大的N-末端分泌形式的蛋白质，其可能分别具有神经营养或神经毒性特性。AD患者的脑表现出突触连接性降低和选择性和大量神经元细胞损失。我们有兴趣研究这种细胞死亡的机制。APP中的罕见突变导致早发性常染色体显性形式的AD（FAD）。以前，我们表明，无论是稳定转染的PC12细胞或腺病毒介导的基因转移的原代皮层神经元的APP的FAD突变形式的过度表达，导致细胞凋亡数天内增加。最近，我们一直在研究几种A-β肽对人神经母细胞瘤细胞的毒性作用以及A-β肽治疗后激活的信号通路。如通过ELISA测量的，低浓度的A-β 1 - 42通过凋亡杀死SH-SY5Y和IMR-32细胞。A-β 1 - 40的效力要小得多。通过APP的连续α-和γ-分泌酶切割从APP衍生的A-β 17 - 42，剂量依赖性地杀死这些细胞。以前认为A-β 17 - 42是非淀粉样蛋白生成APP加工的产物，而不是毒性肽。最近的证据表明，这种肽积累在AD大脑斑块，虽然它的重要性在AD病理学是不清楚的。A-β 17 - 42激活半胱天冬酶-8和半胱天冬酶-3，并诱导PARP裂解，这些蛋白质在导致凋亡性细胞死亡的级联反应中很重要。选择性caspase-8和caspase-3抑制剂完全阻断A-β 17 - 42诱导的神经元死亡。A-β 17 - 42激活c-jun N-末端激酶（JNK）约两倍。显性干扰SEK1构建体（一种磷酸化和激活JNK的蛋白激酶）的过表达可使A-β 17 - 42诱导的神经元死亡保护50 - 70%。结果表明，A-β 17 - 42肽通过Fas样/caspase-8激活途径诱导神经元凋亡。结果还表明，p3肽可能是一个额外的毒性肽来源于APP蛋白水解，并可能在AD的神经元细胞损失的特点的作用。 我们还使用人类神经元SH-SY5Y细胞分析了A-β毒性的早期信号传导机制。我们主要关注丝裂原活化蛋白激酶和PI-3激酶/Akt级联反应。A-β 1 - 42处理导致剂量依赖性细胞死亡，弱激活Akt和ERK，但对p38激酶没有影响。然而，A-β 1 - 42对ERK和Akt的激活显然在A-β毒性中不起作用，因为这些激酶途径的特异性抑制剂U0126和渥曼青霉素分别对A-β诱导的神经元死亡没有影响。然而，A-β 1 - 42诱导JNK活化约两倍，并且似乎在A-β诱导的神经元死亡中起关键作用，因为显性负性构建体SEK-1阻断JNK活化并保护免于细胞死亡。胰岛素样生长因子-1（IGF-1）通过强烈激活ERK和Akt并阻断A-β诱导的JNK激活，剂量依赖性地保护细胞免受A-β毒性。特异性Go/Gi抑制剂百日咳毒素（PT）也通过阻断A-β诱导的JNK活化来保护免受A-β毒性。这些结果表明，A-β肽可以部分激活PT敏感性G蛋白，导致JNK激活。这可能是A-β毒性的重要早期事件，也是AD病理学中神经元丢失的早期信号通路。 最后，我们一直在研究一种缩短的分泌型APP的作用，这种APP来源于APP最初的β-分泌酶加工。这种蛋白质被称为分泌型APP β（sAPPb）。我们产生了过表达真正的sAPPa和sAPPb的细胞系，并将这些蛋白质分泌到周围培养基中。我们发现，含有sAPPb的条件培养基，当添加到任何NGF分化的PC12细胞或原代皮层神经元，导致细胞凋亡死亡，而含有sAPPa的条件培养基没有这样的效果。特异性sAPPb的抗体防止细胞死亡和轻微截短，高度纯化的形式的sAPPb也引起细胞死亡。这些后一结果强烈表明，sAPPb本身是一种额外的毒性蛋白质来源于β-分泌酶APP加工。总体而言，这些结果提供了一个基本原理，针对特定元素的凋亡途径，以及APP加工的治疗干预AD。