Controlled expression of a cystatin C-peptide as therapy for Alzheimer's disease

描述（由申请人提供）：阿尔茨海默病（AD）是一种致命的神经退行性疾病，困扰着全球近2660万人。其特征在于不溶性的细胞外淀粉样蛋白斑块和细胞内神经纤维缠结在脑中的积累，直到最近，这被认为是脑功能障碍的主要原因。这一观点最近已被重新表述，重点是可溶性寡聚抗体（oA？）聚合物，强调的作用，A？42寡聚体，其被认为在AD脑的脆弱区域中引起突触功能障碍和最终的神经元损失。组织蛋白酶B，一种存在于大脑中的半胱氨酸蛋白酶，可以降解A？肽，但是，阻止其酶活性在体内与其特异性抑制剂，胱抑素C（CysC），而不是增加，降低A？AD模型中的肽脑沉积。因为CysC蛋白的一部分（aa 101 - 117），不同于其酶抑制序列，可以结合到A？并抑制原纤维形成在体外，我们建议，产生足够高的水平，这种肽在体内，可能会减少A？寡聚化和神经毒性。我们展示了a）合成的CysC-A？-结合肽（CysC-A？BP）结合特异性和优先纯化的A？42，B）在B AD小鼠模型中，一个具有淀粉样蛋白斑块和oA？（TgCRND 8）和一个oA？只有（APPE 693 Q），它与A？当与脑切片体外孵育时，在新皮层和海马中沉积，c）在肽的单次鼻内递送后4天发现类似的共定位，和d）在我们已经显示在脑中被几种激素高度上调的启动子下鼻内递送CysS-GFP表达构建体产生可检测的表达水平。我们建议：1.确定产生最佳水平的CysC-A的条件？脑内的BP这将通过比较鼻内鞘内递送纯化的肽和肽表达构建体，未涂覆或封装在微粒中，并测试哪种CysS启动子的阳性激素调节剂最佳地反式激活CysC-A？BP结构。2.为了测试是否高水平的CysC-A？BP将降低A？-低聚物在已建立的AD中的沉积以及在AD发作前对小鼠进行慢性治疗是否会阻止其发展。实验条件认为最有效的A？寡聚体减少响应CysC-A？BP将用于测试该治疗对学习和记忆改善的影响。建议使用A？BP-原纤维阻断CysC肽，而不是完整的CysC蛋白，其有害的酶抑制活性，并通过寻找新的方法来提供调节和最大化CysC-A？BP在大脑中的表达，我们希望实现有益的效果，可能会转化为治疗和预防方法，以AD在人类。 公共卫生相关性：全世界有近2700万人患有阿尔茨海默病，而且这个数字还在迅速上升。这种疾病剥夺了受害者的尊严，给家庭和经济带来了难以形容的负担。我们建议在阿尔茨海默病的小鼠模型中测试一种相对简单的预防和/或避免神经元变性和挽救功能的新方法。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD), a fatal neurodegenerative disorder, afflicts close to 26.6 million people worldwide. It is characterized by accumulation in the brain of insoluble, extracellular amyloid plaques and intracellular neurofibrillary tangles considered to be, until recently, the main cause of brain dysfunction. This view has been recently reformulated to focus on soluble oligomeric Ab (oA?) aggregates, with emphasis on the role of A?42 oligomers, which are believed to cause synaptic dysfunction and eventual neuronal loss in the vulnerable regions of AD brains. Cathepsin B, a cysteine protease present in the brain, can degrade A? peptides, however, blocking its enzymatic activity in vivo with its specific inhibitor, cystatin C (CysC), rather than increasing, lowered A? peptide brain deposition in AD models. Because a portion of the CysC protein (aa101- 117), distinct from its enzyme inhibitory sequence, can bind to A? and inhibit fibril formation in vitro, we propose that generating sufficiently high levels of this peptide in vivo, might reduce A? oligomerization and neurotoxicity. We showed a) that synthetic CysC-A?-Binding Peptide (CysC-A?BP) binds specifically and preferentially to purified A?42, b) that in b AD mouse models, one with amyloid plaques and oA? (TgCRND8) and one with oA? only (APPE693Q), it co-localizes with the A? deposits in neocortex and hippocampus when incubated with brain sections in vitro, c) that similar co-localization is found 4 days after a single intranasal delivery of the peptide and d) that intranasal delivery of a CysS-GFP-expressing construct under a promoter that we have shown to be highly upregulated in the brain by several hormones produces detectable expression levels. We propose: 1. To identify conditions that generate optimal levels of CysC-A?BP in the brain. This will be done by comparing intranasal to intrathecal delivery of purified peptide and peptide-expressing construct, uncoated or encapsulated in microparticles, and to test which positive hormonal regulators of the CysS promoter optimally transactivate the CysC-A?BP construct. 2. To test whether high levels of CysC-A?BP will reduce A?-oligomer deposits in established AD and whether chronic treatment of mice before AD onset will prevent its development. Experimental conditions deemed most effective in A? oligomer reduction in response to CysC-A?BP will be utilized to test the effect this treatment has on improvement of learning and memory. By proposing to use an A?BP-fibril blocking CysC peptide, rather than the complete CysC protein, with its detrimental enzyme inhibitory activity, and by finding novel ways to deliver regulate and maximize CysC-A?BP expression in the brain, we expect to achieve beneficial effects that might be translated into therapeutic and preventive approaches to AD in humans. PUBLIC HEALTH RELEVANCE: Close to 27 million people live with Alzheimer's disease worldwide and the number is rapidly rising. This disease robs its victims of dignity and places untold burden on families and on the economy. We propose to test in a mouse model of Alzheimer's disease a relatively simple novel method of preventing and/or averting the neuronal degeneration and of rescuing function.