Modeling the β-secretase cleavage site and humanizing amyloid-beta precursor protein in rat and mouse to study Alzheimer's disease

Background Three amino acid differences between rodent and human APP affect medically important features, including beta-secretase cleavage of APP and A beta 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 humanAPPsequence, 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 A beta-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 rodentAPPsequence (Saito et al., Nat Neurosci 17:661-3, 2014) represent useful improvements, but need to be compared with appropriate humanized wildtype (WT) mice. Methods Computational modelling of the human beta-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(hu/hu), express APP from the endogenous promotor. We also introduced the early-onset familial Alzheimer's disease (FAD) mutation M139T into the endogenous RatPsen1 gene. Results 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 beta-secretase processing was confirmed as both humanized rodent models produce three times more (human) A beta 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 RatPsen1 gene and provide an initial characterization of A beta processing in this novel rat AD model. Conclusion The different humanized APP models (rat and mouse) expressing human A beta and PSEN1 M139T are valuable controls to study APP processing in vivo allowing the use of a human A beta ELISA which is more sensitive than the equivalent system for rodents. These animals will be made available to the research community.