Species: Rat
Genes: App
Mutations: APP K670_M671delinsNL (Swedish)
Modification: App: Knock-In
Disease Relevance: Alzheimer's Disease
Strain Name: N/A
Genetic Background: Long-Evans
Availability: Available through Luciano D'Adamio.

Summary

The KM670/671NL (“Swedish”) double mutation in APP was linked to AD in two large Swedish pedigrees. The mutation is located immediately adjacent to the β-secretase site and results in increased β-secretase processing of APP (Cai et al., 1993; Rabe et al., 2011). This knock-in rat model carries the Swedish mutation and a humanized Aβ sequence within the endogenous rat App gene. The initial characterization of these animals showed a gene-dose-dependent increase in steady-state levels of β-secretase cleavage products (sAPPβ and CTFβ), Aβ40, and Aβ42, as well as alterations in glutamatergic synaptic transmission (Tambini et al., 2019).

Rats homozygous for the humanized Aβ sequence, without the Swedish mutation, were also generated to serve as controls; these animals are referred to as App^h/h. Animals that carry the Swedish mutation on an App^h/h background are referred to as App^s/s and App^s/h, when homozygous or heterozygous for the Swedish mutation, respectively.

For the initial characterization of these rats, mRNA and protein levels were measured in the brains of 21-day-old animals.

Expression of mutant App in these knock-in rats is under the control of natural regulatory elements. Levels of App mRNA in brain were similar in App^s/s, App^h/h, and wild-type animals (App^w/w) (Tambini et al., 2019).

Steady-state levels of immature (unglycosylated) APP were similar in the brains of App^s/s, App^s/h, and App^h/h rats. However, levels of the mature protein were lower in the brains of animals homozygous for the Swedish mutation compared with control App^h/h brains, perhaps due to increased cleavage of the mutant protein by β-secretase (Tambini et al., 2020).

β-secretase (BACE) cleavage generates a soluble N-terminal fragment, sAPPβ, and an intramembrane C-terminal fragment, CTFβ. Levels of CTFβ were elevated in the brains of App^s/s rats, compared with App^h/h animals (Tambini et al., 2019; Tambini et al., 2020). The brains of males heterozygous for the Swedish mutation also contained more CTFβ than the brains of control App^h/h males, but a small group of heterozygous females (five rats per genotype) did not show this increase (Tambini et al., 2020). A gene-dose-dependent increase in sAPPβ was observed in both sexes (i.e., App^s/s > App^s/h > App^h/h) (Tambini et al., 2020), consistent with the initial finding in an earlier study that examined only homozygotes and pooled the sexes (Tambini et al., 2019).

Cleavage of APP by α-secretase similarly generates a soluble N-terminal fragment, sAPPα, and an intramembrane C-terminal fragment, CTFα. Levels of CTFα did not differ between the three genotypes (App^s/s, App^s/h, App^h/h), but levels of sAPPα were decreased in the brains of rats carrying the Swedish mutation (Tambini et al., 2020). Less sAPPα was found in males homozygous or heterozygous  for the mutation, compared with control App^h/h males, although only homozygous App^s/s females showed this decrease relative to control females.

Aβ40 and Aβ42 levels were increased in a gene-dose-dependent manner (App^s/s > App^s/h > App^h/h) in both sexes (Tambini et al., 2020), consistent with the earlier study that examined only homozygotes and pooled the sexes (Tambini et al., 2019). The Aβ42/Aβ40 ratio did not differ between the genotypes (Tambini et al., 2019; Tambini et al., 2020).

Neuropathology

No plaques or neurofibrillary tangles were observed in any of the three genotypes (App^s/s, App^s/h, or App^h/h) at 15 days or three months of age (Tambini et al., 2019).

Neuron loss was not apparent in young rats carrying the Swedish mutation: Neuron densities in several cortical regions and in the hippocampus, assessed qualitatively, did not differ between the three genotypes (Tambini et al., 2019).

Electrophysiology

Recordings from CA1 pyramidal neurons in hippocampal slices from 5- to 8-week-old rats revealed that glutamatergic synaptic transmission is augmented in rats carrying the Swedish mutation (Tambini et al., 2019).

Two indicators of presynaptic function at excitatory synapses were altered in rats homozygous for the Swedish mutation: The frequency of miniature excitatory postsynaptic currents (mEPSCs) was increased and paired-pulse facilitation (PPF) was decreased in App^s/s rats, compared with App^h/h controls. App^s/h heterozygotes also showed deficits in PPF, compared with App^h/h controls, although the frequency of mEPSCs did not differ between these two genotypes.

The amplitude of mEPSCs was elevated in brain slices from rats homozygous for the Swedish mutation, compared with App^h/h controls, suggesting an additional postsynaptic effect.

Electrophysiological characteristics of inhibitory synapses (amplitude, frequency, and decay time of miniature inhibitory postsynaptic currents and PPF) did not differ between genotypes.

Modification Details

CRISPR/Cas9 was used to edit the endogenous rat App gene, generating a humanized Aβ sequence through the following mutations (amino acid positions numbered as in Aβ1–40): G5R (GGA > CGA), F10Y (TTC > TAC), R13H (CGC > CAT). The Swedish double mutation, KM670/671NL (numbered as in the 770-amino-acid isoform of human APP), located two amino acids N-terminally to the BACE1 cleavage site, was also introduced: AAG ATG > AAT CTG.

APP-mutation carriers were crossed five times to wild-type Long-Evans rats to reduce the probability of off-target mutations, then male and female F5-App^s/w rats (animals heterozygous for the humanized Aβ sequence and the Swedish mutation) were bred to generate rats homozygous for humanized Aβ and the Swedish mutation (App^s/s).

Related Strains

App^h knock‐in. This knock-in rat model carries a humanized Aβ sequence within the endogenous rat App gene.

App^p knock‐in. This knock-in rat model carries the Icelandic mutation and a humanized Aβ sequence within the endogenous rat App gene.

Phenotype Characterization

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References

Mutations Citations

1. APP K670_M671delinsNL (Swedish)
2. APP A673T (Icelandic)

Research Models Citations

1. App knock-in (humanized Aβ)
2. App knock‐in (Icelandic mutation and humanized Aβ)

Paper Citations

1. Cai XD, Golde TE, Younkin SG. Release of excess amyloid beta protein from a mutant amyloid beta protein precursor. Science. 1993 Jan 22;259(5094):514-6. PubMed.
2. Rabe S, Reichwald J, Ammaturo D, De Strooper B, Saftig P, Neumann U, Staufenbiel M. The Swedish APP mutation alters the effect of genetically reduced BACE1 expression on the APP processing. J Neurochem. 2011 Oct;119(1):231-9. PubMed.
3. Tambini MD, Yao W, D'Adamio L. Facilitation of glutamate, but not GABA, release in Familial Alzheimer's APP mutant Knock-in rats with increased β-cleavage of APP. Aging Cell. 2019 Dec;18(6):e13033. Epub 2019 Sep 9 PubMed.
4. Tambini MD, Norris KA, D'Adamio L. Opposite changes in APP processing and human Aβ levels in rats carrying either a protective or a pathogenic APP mutation. Elife. 2020 Feb 5;9 PubMed.

Other Citations

1. Luciano D'Adamio

Further Reading

No Available Further Reading