Isolation and Characterization of Cholinesterases From Plaques and Tangles

描述(申请人提供)：自从用组织化学方法证明阿尔茨海默病(AD)的病理特征斑块和缠结含有胆碱能酶乙酰胆碱酯酶(AChE)和丁基胆碱酯酶(BUCHE)的活性以来，已经有20多年了。同样的方法学表明，这些AD相关胆碱酯酶(AD-CHE)与正常神经元胞体和轴突中的胆碱酯酶相比具有不同的性质，可能起源于神经胶质细胞，并可能具有异常活性。生化研究表明CHES可能参与了AD的病理过程，特别是与斑块的形成及其主要成分--淀粉样蛋白(A)肽的产生有关。有人建议，除了目前旨在改善AD皮质胆碱能缺陷的ChE抑制剂治疗外，抑制斑块和缠结中的ChE也是可取的，或许更有益，因为它们可能参与了疾病的过程。然而，关于斑块和缠结中的Ches的研究目前处于停滞状态，因为迄今为止唯一能够区分AD-Ches和正常Ches的方法是基于组织切片中活性的组织化学演示。目前还没有专门从斑块和缠结中分离斑块的方法。因此，没有关于AD-Ches的生化特性以及它们是否在疾病病理中做出特定贡献的直接信息。此外，能够特异性靶向AD-CHES的CHE抑制剂的开发和临床前测试需要一种从组织中分离这些酶的方法。利用我们实验室最近的观察，一些CHS在细胞外基质蛋白硫酸乙酰肝素蛋白多糖(HSPG)的帮助下锚定在组织(如肌肉)上，并且HSPG也存在于斑块和缠结中，本申请中的实验将测试两种从斑块、缠结和胶质细胞中分离CHE的新方法。这项拟议研究的具体目标将检验以下假设：1.固定和新鲜冰冻组织切片中来自斑块、缠结和神经胶质细胞的胆碱酯酶，而不是神经元和轴突的胆碱酯酶，将随着时间的推移被被动释放到存储缓冲区中。2.固定和新鲜冰冻组织中的斑块、缠结和胶质细胞的胆碱酯酶，而不是固定和新鲜冰冻组织中的神经元和轴突的胆碱酯酶，在各种糖胺多聚糖、肝素酶或蛋白酶处理后将被释放。3.所释放的胆碱酯酶将表现出与AD脑内斑块、缠结和神经胶质的组织化学和生化相同的分子、酶特性和抑制模式。AD-Ches的成功分离和鉴定将有助于对这些ChE物种可能的有害影响进行机制研究；将有助于设计和测试专门针对AD-Ches的新抑制剂；并可能导致将AD-Ches作为血液和脑脊液中AD的生物标志物的研究。阿尔茨海默病的斑块和缠结含有胆碱能酶乙酰胆碱酯酶(AChE)和丁基胆碱酯酶(BUCHE)，这些酶的性质发生了改变，已被认为参与了疾病的过程。然而，目前还没有从斑块和缠结中专门分离AChE和BuChE的方法，本申请旨在填补这一空白。从斑块和缠结中成功分离和鉴定AChE和BuChE，将有助于对这些酶种类可能有害影响的机制研究，将有助于设计和测试针对斑块和缠结中胆碱酯酶的新抑制剂，并可能导致将斑块和缠结胆碱酯酶作为血液和脑脊液中AD的生物标志物的研究。

DESCRIPTION (provided by applicant): It has been over two decades since it was demonstrated using histochemical methods that plaques and tangles, the pathological hallmarks of Alzheimer's disease (AD), contain activity specific to cholinergic enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The same methodology demonstrated that these AD-related cholinesterases (AD-ChE) possess different properties when compared with cholinesterases found in normal neuronal cell bodies and axons, are likely to originate in glial cells and may possess unusual activities. Biochemical studies have indicated that ChEs may be involved in the pathology of AD, particularly as it relates to the genesis of plaques and production of their main constituent, the amyloid- (A) peptide. It has been suggested that in addition to current ChE inhibitor therapies aimed at ameliorating the cortical cholinergic deficit in AD, it would also be desirable and perhaps more beneficial to inhibit ChEs in plaques and tangles, due to their possible participation in the disease process. However, research regarding the ChEs in plaques and tangles is currently at a stand-still because the only methods to date that have allowed distinction between AD-ChEs and normal ChEs are based on histochemical demonstration of activity in tissue sections. There is no method available to specifically isolate ChEs from plaques and tangles. Therefore, there is no direct information on the biochemical properties of AD-ChEs and whether they make a specific contribution to disease pathology. Furthermore, the development and pre-clinical testing of ChE inhibitors that can specifically target AD-ChEs requires a method which can isolate these enzymes from tissue. Taking advantage of recent observations at our laboratory, the fact that some ChEs are anchored to tissues (such as muscle) with the aide of extracellular matrix proteins heparan sulphate proteoglycans (HSPG) and that HSPG are also present in plaques and tangles, the experiments in the present application will test two novel methods for isolation of ChEs from plaques, tangles and glia. The specific aims of the proposed research will test the following hypotheses: 1. Cholinesterases from plaques, tangles and glial cells but not neurons and axons in fixed and fresh frozen tissue sections will be passively released into storage buffer over time. 2. Cholinesterases from plaques, tangles and glial cells but not neurons and axons in fixed and fresh frozen tissue will be released upon treatment with various glycosaminoglycans, heparinases or proteases. 3. Released cholinesterases will display molecular and enzymatic characteristics and inhibition patterns identical to those demonstrated histochemically in plaques, tangles and glia and biochemically in AD brain. The successful isolation and characterization of AD-ChEs will allow mechanistic studies of the possible detrimental effects of these ChE species; will aide in design and testing of new inhibitors specifically targeting AD-ChEs; and, is likely to lead to investigation of AD-ChEs as biomarkers of AD in blood and CSF. Plaques and tangles of Alzheimer's disease contain the cholinergic enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with altered properties that have been suggested to participate in the disease process. However, currently there are no methods available to isolate AChE and BuChE specifically from plaques and tangles, and the present application aims to fill in this gap. The successful isolation and characterization of AChE and BuChE from plaques and tangles will allow mechanistic studies of the possible detrimental effects of these enzyme species, will aide in design and testing of new inhibitors specifically targeting cholinesterases in plaques and tangles, and is likely to lead to investigation of plaque and tangle cholinesterases as biomarkers of AD in blood and cerebrospinal fluid.