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

描述(申请人提供)：阿尔茨海默病(AD)，一种致命的神经退行性疾病，困扰着全球近2660万人。它的特点是在脑内积聚不溶的细胞外淀粉样斑块和细胞内神经原纤维缠结，直到最近，仍被认为是脑功能障碍的主要原因。这一观点最近被重新表述，将重点放在可溶性寡聚抗体(OA？)聚集体，重点是A？42寡聚体的作用，据信会导致突触功能障碍，并最终导致AD大脑脆弱区域的神经元丢失。组织蛋白酶B是一种存在于大脑中的半胱氨酸蛋白酶，它能降解A吗？然而，在体内，用其特异性抑制剂胱抑素C(CysC)阻断其酶活性的多肽，不是增加，而是降低A？阿尔茨海默病模型中的多肽脑沉积。因为CysC蛋白的一部分(aa101-117)与其酶抑制序列不同，可以与A？并在体外抑制纤维形成，我们认为在体内产生足够高水平的这种多肽，可能会降低A？寡聚和神经毒性。我们发现：a)人工合成的CysC-A？结合肽(CysC-A？BP)与纯化的A？42特异且优先结合；b)在bAD模型中，有淀粉样斑块和OA？(TgCRND8)和一个有OA的？仅(APPE693Q)，它与A？当与体外脑切片孵育时，CysS-GFP在新皮质和海马区沉积；c)在单次鼻腔给药4天后发现类似的共同定位；以及d)在我们已经证明在大脑中几种激素高度上调的启动子的作用下，鼻腔给药CysS-GFP表达载体产生可检测到的表达水平。我们建议：1.确定在大脑中产生最佳CysC-A、BP水平的条件。这将通过比较纯化的多肽和表达多肽的构建体(未包被或包裹在微粒中)的鼻腔和鞘内传递来完成，并测试CysS启动子的哪种正激素调节剂最佳反式激活CysC-A？BP构建体。2.检测高水平的CysC-A、BP是否会减少已建立的AD中Aβ-寡聚体的沉积，以及在AD发病前对小鼠进行长期治疗是否会阻止其发展。实验条件被认为对A最有效？将利用CysC-A、BP反应的寡聚体减少来测试这种治疗对改善学习和记忆的效果。通过建议使用A？BP-纤维封闭具有有害酶抑制活性的CysC多肽，而不是完整的CysC蛋白，并通过寻找新的方法来调节和最大化CysC-A？BP在大脑中的表达，我们期望获得可能被转化为人类AD的治疗和预防方法的有益效果。 与公共卫生相关：全世界有近2700万阿尔茨海默病患者，而且这一数字还在迅速上升。这种疾病剥夺了受害者的尊严，给家庭和经济带来了难以估量的负担。我们建议在阿尔茨海默病的小鼠模型上测试一种相对简单的新方法，以防止和/或避免神经元退化和挽救功能。

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.