Regulation And Processing Of Amyloid Precursor Protein G

淀粉样前体蛋白 G 的调控和加工

基本信息

批准号：
6667922
负责人：
JOHN W KUSIAK
金额：
--
依托单位：
NATIONAL INSTITUTE ON AGING
依托单位国家：
美国
项目类别：
财政年份：
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/6667922
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

项目摘要

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-beta 17-42 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. Recent evidence has suggested that A-beta peptides may kill cells by increased oxidative stress. Mice lacking the p66 isoform of ShcA adaptor protein (p66Shc) are less susceptible to oxidative stress and have an extended life span. We showed that A-beta peptides induced p66shc phosphorylation and caused cell death in two cell lines. However, cells over-expressing a dominant-negative p66Shc protein were more resistant to A-beta-induced cell death. A-beta induced the phosphorylation (inactivation) of forkhead FKHRL1 and FKHR transcription factors in cells over-expressing wild-type p66Shc, but not in cells over-expressing the dominant-negative p66Shc, suggesting that A-beta peptide inactivated forkhead in a p66Shc-dependent manner. These results show that phosphorylation of p66Shc plays an important role in A-beta toxicity and redox regulation of forkhead proteins. Overall these results provide a rationale for targeting particular elements of apoptotic pathways, APP processing, and A-beta-induced signaling cascades for therapeutic intervention in AD.

工作总结：该项目的主要重点是发现淀粉样蛋白前体蛋白（APP）在阿尔茨海默氏病（AD）的病因和病理学中的作用。该蛋白质的正常生理作用也正在研究中。 APP对于研究很重要，因为APP的处理以及APP突变和Presenilin突变对APP处理的影响直接对AD中A-Beta肽的生产和细胞外沉积的增加。 APP的α-和β-分泌酶处理还产生了两种大型N末端分泌形式的蛋白质，它们可能分别具有神经营养或神经毒性特性。 AD患者的大脑表现出降低的突触连通性以及选择性和大量神经元细胞损失。我们有兴趣检查该细胞死亡所涉及的机制。 APP中的罕见突变导致AD的早期发作，常染色体显性形式（FAD）。以前，我们表明，通过稳定转染PC12细胞或腺病毒介导的原代皮质神经元的基因转移，对APP的FAD突变形式的过表达导致了几天内凋亡细胞死亡的增加。最近，我们一直在研究几种A-beta肽对人类神经母细胞瘤细胞的毒性作用以及在A-Beta肽处理后被激活的信号通路。低浓度的A-Beta 1-42通过ELISA测量的凋亡杀死了SH-SY5Y和IMR-32细胞。 A-Beta 1-40的效力要少得多。 A-Beta 17-42，由APP的顺序α-和γ-分泌酶切割源自APP，剂量依赖性地杀死这些细胞。 A-beta 17-42以前被认为是非淀粉样蛋白生成应用程序加工而不是有毒肽的产物。最近的证据表明，这种肽在AD大脑的斑块中积聚，尽管目前尚不清楚其在AD病理学上的重要性。 A-beta 17-42激活的caspase-8和caspase-3，以及诱导的PARP裂解，在导致凋亡细胞死亡的一系列事件中很重要。选择性caspase-8和caspase-3抑制剂完全阻断了A-beta 17-42诱导的神经元死亡。 A-Beta 17-42激活的C-Jun N末端激酶（JNK）约2倍。对主导裂化SEK1构建体的过表达（一种磷酸化和激活JNK的蛋白激酶）防止A-beta 17-42诱导的神经元死亡50-70％。结果表明，A-BETA 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肽可能是从APP蛋白水解衍生而来的另外有毒肽，并且可能在AD的神经元细胞损耗特征中起作用。我们还使用人神经元SH-SY5Y细胞分析了A-BetA毒性的早期信号传导机制。我们专注于有丝分裂原激活的蛋白激酶和PI-3激酶/AKT级联反应。 A-BETA 1-42治疗导致剂量依赖性细胞死亡，弱激活了Akt和ERK，但对P38激酶没有影响。但是，A-Beta 1-42对ERK和AKT的激活显然没有在A-β毒性中起作用，因为分别对这些激酶途径的特异性抑制剂U0126和Wortmannin对A-Beta诱导的神经元死亡没有影响。但是，A-Beta 1-42诱导的JNK激活大约两倍，并且似乎在A-beta诱导的神经元死亡中起着关键作用，因为显性阴性构建体SEK-1 SEK-1阻止了JNK激活并保护了细胞死亡。胰岛素样生长因子-1（IGF-1）剂量依赖于A-beta毒性的剂量依赖性细胞通过强烈激活ERK和AKT并阻止A-beta诱导的JNK激活。特定的GO/GI抑制剂百日咳毒素（PT）也通过阻止A-beta诱导的JNK激活来保护A-beta毒性。这些结果表明，A-BETA肽部分可以激活PT敏感的G蛋白，从而导致JNK激活。这可能是A-beta毒性的重要且早期的事件，也是AD病理学神经元丧失的早期信号通路。最后，我们一直在研究缩短的，分泌的应用程序的效果，这些应用程序是从APP的最初β-分泌酶处理中得出的。该蛋白质称为分泌的应用Beta（SAPPB）。我们生成了过表达正宗的萨普和萨普布的细胞系，并将这些蛋白质分泌到周围的介质中。我们发现，将含有SAPPB的条件培养基添加到NGF分化的PC12细胞或原发性皮质神经元时，导致凋亡细胞死亡，而含有萨帕的条件培养基则没有这种作用。特定于萨普布的抗体防止了细胞死亡，并略微截断，高度纯化的萨普布（Sappb）也会导致细胞死亡。这些后者的结果强烈表明，SAPPB本身是一种源自β-分泌酶应用程序加工的其他有毒蛋白。最近的证据表明，A-Beta肽可能通过增加的氧化应激杀死细胞。缺乏SHCA衔接蛋白（P66SHC）p66同工型的小鼠不易受到氧化应激的影响，并且具有延长的寿命。我们表明，A-Beta肽诱导P66SHC磷酸化并在两种细胞系中导致细胞死亡。然而，过表达显性阴性P66SHC蛋白的细胞对A-Beta诱导的细胞死亡的抗性更大。 A-beta induced the phosphorylation (inactivation) of forkhead FKHRL1 and FKHR transcription factors in cells over-expressing wild-type p66Shc, but not in cells over-expressing the dominant-negative p66Shc, suggesting that A-beta peptide inactivated forkhead in a p66Shc-dependent manner.这些结果表明，p66SHC的磷酸化在A-beta毒性和分叉蛋白的氧化还原调节中起重要作用。总的来说，这些结果为靶向凋亡途径，应用程序处理和A-Beta诱导的信号级联的特定要素提供了理由，以在AD中进行治疗干预。