Centre for Brain and Disease Research, Flanders Institute for Biotechnology (VIB), Leuven, Belgium.
Department of Neurosciences and Leuven Brain Institute, KU Leuven, Leuven, Belgium.
Mol Neurodegener. 2020 Oct 19;15(1):60. doi: 10.1186/s13024-020-00399-z.
Three amino acid differences between rodent and human APP affect medically important features, including β-secretase cleavage of APP and Aβ peptide aggregation (De Strooper et al., EMBO J 14:4932-38, 1995; Ueno et al., Biochemistry 53:7523-30, 2014; Bush, 2003, Trends Neurosci 26:207-14). Most rodent models for Alzheimer's disease (AD) are, therefore, based on the human APP sequence, expressed from artificial mini-genes randomly inserted in the rodent genome. While these models mimic rather well various biochemical aspects of the disease, such as Aβ-aggregation, they are also prone to overexpression artifacts and to complex phenotypical alterations, due to genes affected in or close to the insertion site(s) of the mini-genes (Sasaguri et al., EMBO J 36:2473-87, 2017; Goodwin et al., Genome Res 29:494-505, 2019). Knock-in strategies which introduce clinical mutants in a humanized endogenous rodent APP sequence (Saito et al., Nat Neurosci 17:661-3, 2014) represent useful improvements, but need to be compared with appropriate humanized wildtype (WT) mice.
Computational modelling of the human β-CTF bound to BACE1 was used to study the differential processing of rodent and human APP. We humanized the three pivotal residues we identified G676R, F681Y and R684H (labeled according to the human APP770 isoform) in the mouse and rat genomes using a CRISPR-Cas9 approach. These new models, termed mouse and rat App, express APP from the endogenous promotor. We also introduced the early-onset familial Alzheimer's disease (FAD) mutation M139T into the endogenous Rat Psen1 gene.
We show that introducing these three amino acid substitutions into the rodent sequence lowers the affinity of the APP substrate for BACE1 cleavage. The effect on β-secretase processing was confirmed as both humanized rodent models produce three times more (human) Aβ compared to the original WT strain. These models represent suitable controls, or starting points, for studying the effect of transgenes or knock-in mutations on APP processing (Saito et al., Nat Neurosci 17:661-3, 2014). We introduced the early-onset familial Alzheimer's disease (FAD) mutation M139T into the endogenous Rat Psen1 gene and provide an initial characterization of Aβ processing in this novel rat AD model.
The different humanized APP models (rat and mouse) expressing human Aβ and PSEN1 M139T are valuable controls to study APP processing in vivo allowing the use of a human Aβ ELISA which is more sensitive than the equivalent system for rodents. These animals will be made available to the research community.
在 APP 中，氨基酸残基的三个差异影响到一些医学上重要的特征，包括β-分泌酶对 APP 的切割和 Aβ 肽的聚集（De Strooper 等人，EMBO J 14:4932-38, 1995；Ueno 等人，Biochemistry 53:7523-30, 2014；Bush，2003，Trends Neurosci 26:207-14）。因此，大多数阿尔茨海默病（AD）的啮齿动物模型都是基于人类 APP 序列，由人工合成的小型基因随机插入啮齿动物基因组中表达。虽然这些模型很好地模拟了疾病的各种生化方面，如 Aβ 聚集，但它们也容易受到过表达的影响，并因插入的小型基因的位置或附近的基因受到影响而发生复杂的表型改变（Sasaguri 等人，EMBO J 36:2473-87, 2017；Goodwin 等人，Genome Res 29:494-505, 2019）。将临床突变引入人源化内源性啮齿动物 APP 序列的基因敲入策略（Saito 等人，Nat Neurosci 17:661-3, 2014）代表了一种有用的改进，但需要与适当的人源化野生型（WT）小鼠进行比较。
我们使用计算建模的方法研究了人类β-CTF 与 BACE1 的结合，以研究不同物种 APP 的不同加工方式。我们使用 CRISPR-Cas9 方法在鼠和大鼠基因组中对我们鉴定出的三个关键残基 G676R、F681Y 和 R684H（根据人类 APP770 同工型进行标记）进行了人源化。这些新的模型被称为鼠和大鼠 App，它们从内源性启动子表达 APP。我们还将早发性家族性阿尔茨海默病（FAD）突变 M139T 引入内源性 Rat Psen1 基因。
我们发现，将这三个氨基酸替换引入到啮齿动物序列中会降低 APP 底物与 BACE1 切割的亲和力。β-分泌酶加工的效果得到了证实，因为这两种人源化的啮齿动物模型产生的（人类）Aβ 比原始 WT 株多三倍。这些模型是研究转基因或基因敲入突变对 APP 加工影响的合适对照或起点（Saito 等人，Nat Neurosci 17:661-3, 2014）。我们将早发性家族性阿尔茨海默病（FAD）突变 M139T 引入内源性 Rat Psen1 基因，并对这种新型大鼠 AD 模型中的 Aβ 加工进行了初步表征。
表达人类 Aβ 和 PSEN1 M139T 的不同人源化 APP 模型（鼠和大鼠）是研究体内 APP 加工的有价值的对照，可用于使用比啮齿动物更敏感的人类 Aβ ELISA。这些动物将提供给研究社区。
