Evaluation of the lysosomal protease tripeptidyl peptidase 1 as a potential therapeutic for Alzheimer Disease

项目摘要/摘要 尽管进行了广泛的努力和投资，但仍没有治愈阿尔茨海默病(AD)的方法或有效的治疗方法 这种破坏性的疾病进展缓慢。一种潜在的治疗策略是促进 淀粉样β蛋白(Aβ)，其在大脑中的积累与疾病有关，并可能是疾病的组成部分 进程。我们最近获得了强有力的证据表明，溶酶体蛋白水解酶三肽基肽酶1(TPP1) 在Aβ纤维的降解过程中起着重要作用。我们假设，TPP1活性的增加将促进 β的降解，减缓或阻止其在大脑中的积聚，从而对 广告。我们将使用两种互补的方法在J20小鼠身上验证这一假设，这是一种转基因AD模型 这会过度产生人类Aβ，并表现出与年龄相关的斑块堆积和认知缺陷。 首先，在遗传原理验证研究中，我们将J20小鼠与我们新创造的转基因小鼠杂交 这会结构性地过度表达小鼠TPP1(活性是内源性水平的10倍左右)。第二，我们 将使用一种多肽介导的方法将重组人TPP1蛋白从血液中运送到 血脑屏障进入J20小鼠的大脑。治疗组和对照组各有30人 雌雄数量相等的动物。对于这两个目标，我们将采用相同的方法来 评估TPP1活性升高对AD表型的影响。在七个月大的时候，我们会分析 使用Morris水迷宫、新的物体识别和恐惧条件反射试验对小鼠和对照组进行治疗。 8个月后，小鼠将被安乐死，并测量脑提取液中可溶性和不溶性A-β的水平 用酶联免疫吸附试验和免疫组织化学方法检测大脑皮层和海马区的斑块。 如果TPP1的增强对J20小鼠的AD表型有积极的影响，这将提供一个强有力的 进一步探讨这一点的理论基础，长期目标是开发一种有效的AD治疗方法。其他内容 在开始人体试验之前，需要在实验室动物身上进行研究。展望未来，这是值得的 注意到靶向跨越血-脑和/或脑脊液-脑屏障的蛋白质类药物 仍然是发展阿尔茨海默病和其他神经学生物疗法的主要障碍 精神错乱。然而，在TPP1的情况下，重组蛋白通过脑室内输送到大脑 政府已经得到了联邦药品管理局和欧洲药品管理局的批准 用于治疗一种神经退行性溶酶体蓄积症。类似的交付方法应该是可行的 治疗阿尔茨海默病，并可能允许改变现有药物的用途。

PROJECT SUMMARY/ABSTRACT Despite extensive efforts and investment, there is no cure for Alzheimer disease (AD) or effective treatment to slow progression of this devastating disorder. One potential therapeutic strategy is to promote degradation of amyloid beta (Aβ), whose accumulation in the brain is associated with and may be integral to the disease process. We have recently obtained strong evidence that the lysosomal protease tripeptidyl peptidase 1 (TPP1) plays an important role in degradation of Aβ fibrils. We hypothesize that increasing activity of TPP1 will promote degradation of Aβ, slowing or preventing its accumulation in the brain with subsequent therapeutic benefits for AD. We will test this hypothesis using two complementary approaches in the J20 mouse, a transgenic AD model that overproduces human Aβ and exhibits age-dependent plaque accumulation and cognitive deficits. First, in a genetic proof-of-principle study, we will cross the J20 mouse with our newly created transgenic mouse that constitutively overexpresses mouse TPP1 (~10-fold higher activity than endogenous levels). Second, we will use a peptide-mediated method to deliver recombinant human TPP1 protein from the bloodstream across the blood-brain barrier into the brain of the J20 mouse. Treatment groups and controls will each contain 30 animals with equal numbers of males and females. For both Aims, we will employ identical approaches to evaluate the effect of elevated TPP1 activity on the AD phenotype. At the age of seven months, we will analyze treated mice and controls using the Morris water maze, novel object recognition and fear conditioning assays. At eight months, the mice will be euthanized and levels of soluble and insoluble Aβ measured in brain extracts by ELISA and plaque measured in cortex and hippocampus by immunohistochemistry. If TPP1 augmentation has a positive effect on AD phenotype in the J20 mouse, this would provide a strong rationale to explore this further with the long-term goal of developing an effective therapy for AD. Additional studies in laboratory animals would be required prior to initiation of trials in humans. Looking forward, it is worth noting that targeting protein-based drugs across the blood-brain and/or cerebrospinal fluid-brain barrier continues to be a major obstacle for the development of biologic therapeutics for AD and other neurological disorders. However, in the case of TPP1, delivery of recombinant protein to the brain by intracerebroventricular administration has been approved by both the Federal Drug Administration and the European Medicines Agency for treatment of a neurodegenerative lysosomal storage disease. A similar delivery method should be feasible for AD and may allow repurposing of an existing drug.