Species: Rat
Genes: App
Mutations: APP K670_M671delinsNL (Swedish), APP E693G (Arctic), APP I716F (Iberian)
Modification: App: Knock-In
Disease Relevance: Alzheimer's Disease
Strain Name: N/A
Genetic Background: Sprague Dawley
Availability: Available through Bai Lu.

Summary

This rat line, created using CRISPR/Cas9 technology, carries a humanized Aβ sequence and three Alzheimer’s-linked mutations (Swedish, KM670/671NL; Arctic, E693G; and Iberian, I716F) in the endogenous rat App gene (Pang et  al., 2022). Knock-in rats express normal levels of full-length APP and APP C-terminal fragments (i.e., levels comparable to wild-type controls). Amyloid plaques appear as early as one month in homozygous knock-in brains. Although neurofibrillary tangles are not seen through 22 months of age, increased tau phosphorylation, aggregation, and conformational changes are observed, as are astrogliosis, microgliosis, and synapse loss. Notably, these animals exhibit pronounced neuron loss in the hippocampus and cortex.

The description below refers to male rats homozygous for the mutant App allele, unless stated otherwise.

Amyloid | Tau | Gliosis | Neuron Loss | Synapse Loss | Cognitive Deficits | Other | Modification Details

Amyloid

Amyloid deposition was detected in homozygous App^NL-G-F rats as young as 1 month old and in heterozygous animals as early as 4 months. Plaque burden increased with age, and progressed more rapidly in females than males, particularly after 6 months.

Tau

While App^NL-G-F rats do not develop neurofibrillary tangles, at least through 22 months of age, they do exhibit age-dependent increases in tau phosphorylation and aggregation and pathological changes in the conformation of tau.

Levels of phospho-tau recognized by monoclonal antibodies AT8 (tau doubly phosphorylated at serine 202 and threonine 205) and AT180 (tau phosphorylated at threonine 231) become elevated relative to controls between 3 and 6 months and 6 and 9 months, respectively.

Using monoclonal antibody APN-mab005, reported to recognize aggregated tau, staining was first observed in cortical neurons of 12-month-old App^NL-G-F brains and increased with age. The presence of aggregated tau was confirmed using a second reagent—the fluorescent probe PM-PBB3 (Tagai et al., 2021)—which labeled CA1 neurons in 22-month-old animals (younger ages were not examined with this tracer).

Hippocampal neurons of 22-month-old App^NL-G-F rats stained with monoclonal antibody MC1, which recognizes conformational changes in tau that may precede the appearance of neurofibrillary tangles (Jicha et al., 1999; Weaver et al., 2000). Younger ages were not examined with this antibody.

Thioflavin-S and the Bielschowsky silver stain also labeled structures in the brains of 22-month-old knock-in animals, although the staining did not resemble neurofibrillary tangles.

APN-mab005 and MC1 staining were observed in the brains of 12-month-old female App^NL-G-F rats.

Gliosis

Astrogliosis and microgliosis, particularly pronounced around amyloid plaques, were observed in homozygous knock-in rats beginning between 3 and 6 months of age. Gliosis was also seen in year-old heterozygotes (younger ages were not examined).

Neuron Loss

Compared with wild-type rats, App^NL-G-F rats had reduced brain weight, fewer neurons in the hippocampus and cortex, and enlarged lateral ventricles. At 12 months, the earliest age examined, homozygous knock-in rats had 30 percent fewer neurons in their hippocampi and 9 percent lower brain weights than wild-type rats; these differences increased to 50 percent and 16 percent, respectively, at 22 months. Markers of apoptosis and necroptosis in the brains of App^NL-G-F rats became elevated between 3 and 6 months of age, suggesting that both mechanisms contribute to neuron loss in these animals.

Synapse Loss

Biochemical, immunohistochemical, and ultrastructural evidence demonstrated the loss of synapses in App^NL-G-F rats, beginning between 3 and 6 months of age. Levels of the presynaptic marker synaptophysin and the postsynaptic marker PSD-95—quantified from western blots and compared with wild-type controls—were normal at 3 months but decreased at 9 months. Immunohistochemistry revealed the loss of synaptic markers to be most pronounced around amyloid plaques. Quantitative electron microscopy showed reductions in synaptic density, area, and perimeters in the hippocampus, entorhinal cortex, and prefrontal cortex of 6-month-old knock-in brains, compared with wild-type controls.

Cognitive Deficits

App^NL-G-F rats displayed deficits in the Morris Water Maze task and a paired associate learning task as early as 5 months and 7 months of age, respectively.

In the open-field test, the knock-in animals displayed normal locomotor activity but slightly increased anxiety.

Other

App^NL-G-F rats weighed slightly less than wild-type animals, assessed at 3, 6, and 12 months of age. The genotypes did not differ in core body temperature.

Modification Details

This rat line, created using CRISPR/Cas9 technology, carries a humanized Aβ sequence (G676R, F681Y, and R684H) and three AD-linked mutations (Swedish, KM670/671NL; Arctic, E693G; and Iberian, I716F) in the endogenous rat App gene.

Phenotype Characterization

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References

Paper Citations

1. Pang K, Jiang R, Zhang W, Yang Z, Li LL, Shimozawa M, Tambaro S, Mayer J, Zhang B, Li M, Wang J, Liu H, Yang A, Chen X, Liu J, Winblad B, Han H, Jiang T, Wang W, Nilsson P, Guo W, Lu B. An App knock-in rat model for Alzheimer's disease exhibiting Aβ and tau pathologies, neuronal death and cognitive impairments. Cell Res. 2022 Feb;32(2):157-175. Epub 2021 Nov 17 PubMed.
2. Tagai K, Ono M, Kubota M, Kitamura S, Takahata K, Seki C, Takado Y, Shinotoh H, Sano Y, Yamamoto Y, Matsuoka K, Takuwa H, Shimojo M, Takahashi M, Kawamura K, Kikuchi T, Okada M, Akiyama H, Suzuki H, Onaya M, Takeda T, Arai K, Arai N, Araki N, Saito Y, Trojanowski JQ, Lee VM, Mishra SK, Yamaguchi Y, Kimura Y, Ichise M, Tomita Y, Zhang MR, Suhara T, Shigeta M, Sahara N, Higuchi M, Shimada H. High-Contrast In Vivo Imaging of Tau Pathologies in Alzheimer's and Non-Alzheimer's Disease Tauopathies. Neuron. 2021 Jan 6;109(1):42-58.e8. Epub 2020 Oct 29 PubMed.
3. Jicha GA, Berenfeld B, Davies P. Sequence requirements for formation of conformational variants of tau similar to those found in Alzheimer's disease. J Neurosci Res. 1999 Mar 15;55(6):713-23. PubMed.
4. Weaver CL, Espinoza M, Kress Y, Davies P. Conformational change as one of the earliest alterations of tau in Alzheimer's disease. Neurobiol Aging. 2000 Sep-Oct;21(5):719-27. PubMed.

Further Reading

No Available Further Reading