Knock-in rats with homozygous PSEN1L435F Alzheimer mutation are viable and show selective γ-secretase activity loss causing low Aβ40/42 and high Aβ43

Familial forms of Alzheimer's disease (FAD) are caused by mutations in the gene encoding amyloid precursor protein, whose processing can result in formation of β-amyloid (Aβ). FAD can also result from mutations in the presenilin 1/2 (PSEN1/2) genes, whose protein products partially compose the γ-secretase complex that cleaves Aβ from amyloid precursor protein fragments. Psen1 KO mice and knock-in (KI) mice with homozygous FAD-associated L435F mutations (Psen1LF/LF) are embryonic and perinatally lethal, precluding a more rigorous examination of the effect of Alzheimer's disease–causing Psen1 mutations on neurodegeneration. Given that the rat is a more suitable model organism with regard to surgical interventions and behavioral testing, we generated a rat KI model of the Psen1LF mutation. In this study, we focused on young Psen1LF rats to determine potential early pathogenic changes caused by this mutation. We found that, unlike Psen1LF/LF mice, Psen1LF/LF rats survive into adulthood despite loss of γ-secretase activity. Consistent with loss of γ-secretase function, Psen1LF/LF rats exhibited low levels of Aβ38, Aβ40, and Aβ42 peptides. In contrast, levels of Aβ43, a longer and potentially more amyloidogenic Aβ form, were significantly increased in Psen1LF/LF and Psen1LF/w rats. The longer survival of these KI rats affords the opportunity to examine the effect of homozygous Psen1 Alzheimer's disease–associated mutations on neurodegeneration in older animals.


Extended Experimental procedures
Generation of rats expressing the FAD Psen1 L435F mutation (Psen1 LF rats).
The rat Psen1 gene comprises 12 exons, with the ATG start codon in exon 3 and the TAG stop codon in exon 12. The CTT codon for L 435 is located in exon 12 that was selected as target site. A silent mutation (TCC to TCG) will also be introduced to prevent the binding and re-cutting of the sequence by Cas9 after homology-directed repair. gRNA targeting vector and oligo donor (with targeting sequence, flanked by 120bp homologous sequences combined on both sides) were designed as follows.

5'-CCACAGGGCCTGTGCCTTACGTTACTCCTGCTCGCCATTTTCAAGAAAGCGTTGCCGGCCTTTCCCATCTCGATCA CCTTCGGGCTCATTTTCTACTTTGCCACGGATTATCTCGTGCAGCCC-3'
Oligo donor sequence, with the mutated nucleotide in red and yellow highlight and the silent TCC to TCG mutation in red.
Cas9 mRNA, gRNA generated by in vitro transcription and oligo donor were co-injected into zygotes for production of rats carrying these knock-in (KI) mutations by homology-directed repair. To verify CRISPRinduced mutation the pups were genotyped by PCR, followed by sequence analysis. The rat Psen1 locus was amplified by PCR with the specific primers: F: TTGGTTGCGAGGCATCATGGTA and R: CCCAAGTCCAGTAGTGCAGGTGG. To identify founders carrying correctly targeted Psen1 alleles, the 456 bp long PCR products were cloned into TA vectors and 10 plasmids containing Psen1 inserts were sequenced using primer R: CCCAAGTCCAGTAGTGCAGGTGG. This analysis showed that founders Rat-ID#13 and Rat-ID#20 carried the mutant allele (WT= Wild-type): 5'-CGGCCTTTCCCATCTCGATCACCTTCGGGCTCATTTTCTACTTTG-3' Sequence of the allele carrying the FAD mutation (boxed and in red) and the silent mutation (boxed and in blue) compared to the sequence of rat Psen1. The gRNA sequence is underlined Thus, Rat-ID#13 and Rat-ID#20 were identified as a positive chimeric founder (F0-Psen1 LF rat).
Off-target analysis of targeting sequence gRNA1: GAAAATGAGCCCGAAGGTGATGG. We identified five potential off-target sites for gRNA1. Based on this analysis, Rat-ID#13 and Rat-ID#20 have been analyzed for mutations in these most likely off-target mutation sites. Mismatched bases are in red. These sites have been amplified by PCR and sequenced.