Regulation And Processing Of Amyloid Precursor Protein G

工作概述：本项目的一个主要重点是发现淀粉样蛋白前体蛋白（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的加工以及APP突变和早老素突变对APP加工的影响直接关系到AD中a - β肽的产生和胞外沉积的增加，因此研究APP非常重要。APP的α -和β -分泌酶加工也产生两种大的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。阿尔茨海默病患者的大脑表现出突触连通性下降和选择性和大量的神经元细胞损失。我们对研究这种细胞死亡的机制很感兴趣。罕见的APP突变导致早发，常染色体显性形式的AD （FAD）。在此之前，我们发现，通过稳定转染PC12细胞或通过腺病毒介导的原代皮质神经元基因转移，APP的FAD突变形式的过表达会导致凋亡细胞数天内死亡增加。最近，我们一直在研究几种a - β肽对人类神经母细胞瘤细胞的毒性作用以及在a - β肽治疗后激活的信号通路。ELISA检测低浓度a - β 1-42通过凋亡杀死SH-SY5Y和IMR-32细胞。a - 1-40的效力要小得多。a- β 17-42通过α -和γ -分泌酶连续切割APP而产生，剂量依赖性地杀死这些细胞凋亡。a - β 17-42以前被认为是非淀粉样变性APP加工的产物，而不是一种有毒的肽。最近的证据表明，这种肽在阿尔茨海默病脑斑块中积累，尽管它在阿尔茨海默病病理中的重要性目前尚不清楚。a -beta17-42激活caspase-8和caspase-3，并诱导PARP裂解，这是导致细胞凋亡的一系列事件的重要蛋白。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。过表达显性干扰型SEK1构建体（一种磷酸化并激活JNK的蛋白激酶）对a - β 17-42诱导的神经元死亡有50- 70%的保护作用。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APP蛋白水解产生的另一种毒性肽，可能在AD的神经元细胞损失特征中起作用。我们还利用人类神经元SH-SY5Y细胞分析了a - β毒性的早期信号机制。我们专注于丝裂原活化蛋白激酶和PI-3激酶/Akt级联反应。a - β 1-42治疗导致剂量依赖性细胞死亡，可弱激活Akt和ERK，但对p38激酶无影响。然而，a- β 1-42对ERK和Akt的激活显然没有在a- β毒性中发挥作用，因为这些激酶途径的特异性抑制剂U0126和wortmannin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激活约两倍，并且似乎在a- β诱导的神经元死亡中起关键作用，因为显性阴性构建体SEK-1阻断JNK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，剂量依赖性地保护细胞免受a- β毒性。一种特异性的Go/Gi抑制剂百日咳毒素（pertussis toxin， PT）也通过阻断A- β诱导的JNK激活来保护A- β的毒性。这些结果表明，a - β肽可以部分激活pt敏感的g蛋白，从而激活JNK。这可能是a - β毒性的重要早期事件，也是阿尔茨海默病病理中神经元丢失的早期信号通路。最后，我们一直在研究源自APP初始β -分泌酶加工的一种缩短的、分泌形式的APP的影响。这种蛋白质被称为分泌型APP β (sAPPb)。我们产生了过表达真实sAPPa和sAPPb的细胞系，并将这些蛋白质分泌到周围介质中。我们发现含有sAPPb的条件培养基，当添加到NGF分化的PC12细胞或原代皮质神经元中时，导致细胞凋亡死亡，而含有sAPPa的条件培养基则没有这种影响。一种针对sAPPb的抗体阻止了细胞死亡，一种稍微截断的、高度纯化的sAPPb也会导致细胞死亡。这些结果强烈表明sAPPb本身是β -分泌酶APP加工过程中产生的另一种有毒蛋白。总的来说，这些结果为针对凋亡通路的特定元件以及APP加工进行AD治疗干预提供了理论依据。

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.