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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：该项目的一个主要焦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的加工过程值得研究，APP突变和早老素突变对APP加工的影响直接关系到AD患者A-β多肽的产生和细胞外沉积的增加。APP的α-和β-分泌酶处理还会产生两种大的N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。阿尔茨海默病患者的大脑表现出突触连接性降低和选择性大量神经细胞丢失。我们对研究这种细胞死亡的机制很感兴趣。APP的罕见突变导致早发性常染色体显性AD(FAD)。在此之前，我们发现，通过稳定地转染PC12细胞或通过腺病毒介导的原代皮质神经元基因转移，FAD突变形式的APP的过度表达会在几天内导致细胞凋亡增加。最近，我们一直在研究几种A-β多肽对人神经母细胞瘤细胞的毒性作用，以及在A-β多肽处理后激活的信号通路。低浓度A-β1-42可诱导SH-SY5Y和IMR-32细胞发生凋亡。A-β1-40的效力要小得多。APP通过顺次切割APP的α和伽马分泌酶而产生的A-β17-42，以剂量依赖的方式杀死这些细胞。A-β17-42此前被认为是非淀粉样变性APP加工的产物，而不是一种有毒的多肽。最近的证据表明，这种多肽积聚在AD大脑的斑块中，尽管它在AD病理中的重要性目前尚不清楚。A-β17-42激活caspase-8和caspase-3，并诱导PARP裂解，这两种蛋白质在一系列导致细胞凋亡的事件中起重要作用。选择性caspase-8和caspase-3抑制剂可完全阻断A-β17-42诱导的神经元死亡。A-β17-42激活的c-jun氨基末端激酶(JNK)约为JNK的两倍。显性干扰SEK1结构(一种磷酸化并激活JNK的蛋白激酶)的过度表达对A-β17-42诱导的神经元死亡有50-70%的保护作用。结果表明，A-β17-42肽通过Fas样蛋白/caspase-8激活途径诱导神经细胞凋亡。提示P3多肽可能是APP蛋白分解所产生的一种额外的毒性多肽，可能在AD的神经细胞丢失特征中起一定作用。我们还利用人神经元SH-SY5Y细胞分析了A-β毒性的早期信号机制。我们的重点是丝裂原激活的蛋白激酶和PI-3激酶/Akt级联。A-β1-42诱导的细胞死亡呈剂量依赖性，对Akt和ERK有微弱的激活作用，但对p38激酶无明显影响。然而，A-β1-42对ERK和Akt的这种激活显然没有在A-β毒性中起作用，因为这两个激酶通路的特异性抑制剂U0126和Wortmannin对A-β诱导的神经元死亡没有影响。然而，A-β1-42诱导的JNK活性约为A-β1-42的两倍，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-负性结构SEK-1阻断了JNK的激活并保护细胞免于死亡。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，以剂量依赖的方式保护细胞免受A-β毒性。一种特异性的GO/GI抑制剂百日咳毒素(PT)也通过阻断A-β诱导的JNK激活来保护A-β毒性。这些结果提示，A-β多肽可以部分激活PT敏感的G-蛋白，从而导致JNK的激活。这可能是A-β毒性的一个重要的早期事件，也可能是AD病理中神经元丢失的一个早期信号通路。最后，我们一直在研究APP的一种缩短的、分泌形式的APP的影响，这种APP是从APP的初始β-分泌酶处理中衍生出来的。这种蛋白质被称为分泌型APPβ(SAPPb)。我们建立了过表达正品Sappa和sAPPB的细胞系，并将这些蛋白分泌到周围的培养液中。我们发现，无论是NGF分化的PC12细胞还是原代皮质神经元，当加入含有sAPPB的条件培养液时，都会导致细胞凋亡，而含有Sappa的条件培养液没有这种作用。一种针对sAPPB的抗体阻止了细胞死亡，一种略微截短的、高度纯化的sAPPB形式也导致了细胞死亡。后者的结果有力地表明，sAPPB本身是一种额外的有毒蛋白，来自于β-分泌酶APP的加工。最近的证据表明，A-β多肽可能通过增加氧化应激来杀死细胞。缺乏p66亚型SHCA适配器蛋白(P66Shc)的小鼠不太容易受到氧化应激的影响，并有更长的寿命。我们发现A-β多肽诱导了p66shc的磷酸化，并导致了两种细胞系的细胞死亡。然而，过度表达显性负p66Shc蛋白的细胞对A-β诱导的细胞死亡更具抵抗力。在高表达野生型p66Shc的细胞中，A-β诱导叉头FKHRL1和FKHR转录因子的磷酸化(失活)，而在过表达显性-阴性p66Shc的细胞中则不诱导，提示A-β肽以p66Shc依赖的方式使叉头失活。这些结果表明，p66Shc的磷酸化在叉头蛋白的A-β毒性和氧化还原调节中起重要作用。总体而言，这些结果为AD的治疗干预提供了靶向凋亡途径、APP处理和A-β诱导的信号级联的特定元件的理论基础。