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

Abstract

BackgroundThree amino acid differences between rodent and human APP affect medically important features, including β-secretase cleavage of APP and Aβ peptide aggregation(1–3). 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(4,5). Knock-in strategies which introduce clinical mutants in a humanized endogenous rodent APP sequence(6) represent useful improvements, but need to be compared with appropriate humanized wildtype (WT) mice. MethodsComputational 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 Apphu/hu, 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.ResultsWe 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(6). 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.Conclusion 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.

Iron Responsive Element (IRE)-mediated responses to iron dyshomeostasis in Alzheimer’s disease

“…We make an important distinction between these fAD-like models that resemble the genetic state of human fAD as closely as possible and other knock-in models that invoke genetic states which have never been observed in human fAD (e.g. models possessing homozygous mutations that have only been observed in a heterozygous state in humans and/or several fAD-causative mutations simultaneously as described by Saito et al [51] or Serneels et al [52]). Despite the comparatively more subtle pathology seen in fAD-like knock-in models, because they more closely mimic the genetic state of fAD, it would be expected that gene expression analysis of knock-in models may reveal informative early molecular changes that are relevant to early fAD states in human brains.…”

An App knock-in rat model for Alzheimer’s disease exhibiting Aβ and tau pathologies, neuronal death and cognitive impairments

“…Unfortunately, these rat models, which are with single App mutation leading to little Aβ deposits, do not exhibit significant AD pathologies. [39][40][41][42] Accordingly, it is highly desirable to develop alternative models that can faithfully recapitulate majority of the pathogenic mechanisms in AD using knock-in technology. In this study, we have generated an App knock-in rat line harboring Swedish-Beyreuther/ Iberian-Arctic mutations using the CRISPR/Cas9 technology.…”

Genetic mapping of APP and amyloid-β biology modulation by trisomy 21

“…Mouse APP differs in sequence from the human protein. In the amyloid-β region these differences both reduce the protein’s cleavage by β-secretase and the tendency of the amyloid-β generated to aggregate [14] thus limiting our ability to determine how changes to biology affect accumulation of amyloid-β – a key early aspect of AD. Whereas the over- and mis-expression of APP in transgenic mouse models may cause artefactual phenotypes, masking the modulatory effect of the extra copy of Hsa21 genes and causing elevated mortality which may confound data interpretation [13, 15].…”