Species: Rat
Genes: APP, PSEN1
Mutations: APP K670_M671delinsNL (Swedish), APP V717I (London), PSEN1 M146L (A>C)
Modification: APP: Virus; PSEN1: Virus
Disease Relevance: Alzheimer's Disease
Strain Name: N/A
Genetic Background: Wistar
Availability: Viral vectors are available through AgenT SAS under collaboration or partnership agreements.

Summary

Adeno-associated viral (AAV) vectors separately encoding human APP with the Swedish and London mutations and human PSEN1 with the M146L mutation were injected bilaterally into the hippocampi of young adult (8-week-old) rats. AAV-AD rats develop amyloid plaques and cerebral amyloid angiopathy, accrue hyperphosphorylated tau, and exhibit progressive behavioral impairments.

Only male rats were used in the study reported here. Controls rats were injected with only with AAV carrying M146L PSEN1.

Full-length human PSEN1 and full-length human APP did not accumulate in the hippocampi of AAV-AD rats. However, levels of a 30-kDa N-terminal fragment of PSEN1—a component of the active γ–secretase complex—were elevated, as were levels of APP cleavage products. Measured one month after virus injection, the levels of CTFβ—the APP C-terminal fragment generated by BACE cleavage—were approximately double those measured in the hippocampi of controls, and levels of soluble Aβ40 and Aβ42 were elevated approximately 10- and 27- fold, respectively. While steady-state levels of CTFβ and Aβ40 in the hippocampus remained stable over time, levels of Aβ42 progressively increased from one to 30 months post-injection, reaching concentrations similar to those measured in hippocampal samples from AD patients.

Levels of Aβ were also measured in cerebrospinal fluid (CSF) collected from the cisterna magna, and both Aβ40 and Aβ42 were found to be elevated in AAV-AD animals. At one month post-injection, levels of Aβ40 were approximately sixfold higher in CSF from AAV-AD rats (6.00 ± 1.03 pg/mL) compared with controls (0.94 ± 0.34 pg/mL), while levels of Aβ42 were approximately 15-fold higher (control: 0.35 ± 0.09 pg/mL; AAV-AD: 5.46 ± 0.97 pg/mL). Levels of CSF Aβ40 and Aβ42 increased from one to eight months post-injection, but there was a trend toward a decline in the levels of both peptides when measured at 30 months post-injection.

There was a progressive increase in the amount of hyperphosphorylated endogenous rat tau. (In the descriptions that follow, the amino acid numbers refer to the 752-amino-acid isoform of rat tau (Uniprot P19332-1); for reference, the corresponding amino acids in the 441-amino-acid isoform of human tau (Uniprot P10636-8) are given in brackets.) Phosphorylation at Ser733 [Ser422] and Thr492 [Thr181] was apparent by eight months post-injection. At 30 months, there were further increases in the levels of tau phosphorylated at these two sites, and hyperphosphorylation was observed at additional sites (Ser513 [Ser202], Thr523 [Thr212], Thr528 [Thr217], Thr542 [Thr231], and Thr542/Ser546 [Thr231/Ser235]).

Neuropathology

At 30 months post-injection, amyloid plaques were observed in the hippocampi of AAV-AD rats, primarily in the subiculum. Cerebral amyloid angiopathy was also present at this time. Scattered neurons in the hippocampus displayed immunoreactivity for human APP. The appearance of hippocampal neurons immunostained with antibodies AT8 (recognizes tau phosphorylated at Ser513/Thr516 [Ser202/Thr205]) and AT100 (recognizes tau phosphorylated at Thr523/Ser525 [Thr212/Ser214]) at 30 months post-injection suggests the presence of (pre)tangle-like structures, but silver staining is required to confirm the presence of actual neurofibrillary tangles. No astrogliosis was seen up to 30 months post-injection.

Behavior

At three months post-injection, AAV-AD and control rats performed similarly in a battery of behavioral tests. At eight months post-injection, AAV-AD rats spent less time in the center of the open field than did controls, although both groups traveled similar total distances. The groups did not differ in their performances in the Y-maze or novel-object-recognition tests. In the Morris water maze, AAV-AD rats learned the location of the hidden platform as well as did controls. In a probe trial (memory test) conducted four hours after the last training session, both groups also performed similarly, but the AAV-AD rats did not perform as well as controls in probe tests administered three and five days after training.

Electrophysiology

Synaptic plasticity at Schaffer collateral-CA1 synapses was assessed in hippocampal slices obtained from rats three or eight months after virus injection. At three months post-injection, long-term potentiation (LTP) and long-term depression (LTD) were similar in AAV-AD and control rats. However, there appeared to be an increase in tonic glutamate currents in CA1 pyramidal neurons, believed to reflect the activation of extrasynaptic NMDA receptors, in AAV-AD animals. At eight months post-injection, LTP was impaired in hippocampal slices obtained from AAV-AD rats, but LTD and the amplitude of the tonic glutamate current did not differ from controls.

Modification Details

Human APP751 cDNA containing the Swedish and London mutations and human PSEN1 cDNA containing the M146L mutation were cloned separately into AAV2 plasmids with CAG promoters to generate AAV2-CAG-APPSL or AAV2-CAG-PS1M146L. The AAV packaging plasmid was replaced with a plasmid construct containing the rep gene of AAV2 and the cap gene of AAV9. Viral vectors were injected bilaterally into the hippocampi of eight-week male Wistar rats.

Availability

Viral vectors are available through AgenT SAS under collaboration or partnership agreements. Contact Jérôme Braudeau: jerome.braudeau@agent-biotech.com.

Phenotype Characterization

COMMENTS / QUESTIONS

1. • Jérôme Braudeau
     
   • Posted: 27 Feb 2019

   In the AAV-AD rat model, the majority of the hippocampal cells have no genetic modification, making it a relevant model for the non-genetic forms of the disease that represent more than 92 percent of cases (Prince et al., 2015). Indeed, the technology used is not based on a transgenic approach. Because disease induction is conducted only on adult animals, AAV-AD rats do not suffer from developmental compensation or genetic drift. 

   Moreover, the pattern of APP expression in AAV-AD model may mimic both genomic mosaicism and APP gene recombination recently described in the sporadic form of human AD, in which an increase in copy number was observed for the APP gene in a limited subset of neurons (Bushman et al., 2015) and an appearance of somatic mutations known to be associated with familial form of Alzheimer’s disease was described (Lee et al., 2018). The AAV-AD rat model could thus be considered as a closer model of the sporadic form of AD than transgenic animals.

   Its pathophysiological relevance has been validated by comparing it to postmortem samples of AD patients. The concentration of Aβ42 peptide gradually increases to reach, at the late stage, concentrations comparable to those measured in the hippocampi of AD patients. As hyperphosphorylation of the endogenous tau protein gradually takes place, the memory capacity simultaneously declines, reproducing the chronology of events progression seen in clinics. Amyloid plaques and cerebral amyloid angiopathy develop only in aged AAV-AD rats. Intraneuronal aggregates of hyperphosphorylated tau protein confirm a full commitment of the tau pathology (Audrain et al., 2017).

   All these features make the AAV-AD rat model a powerful tool to better predict the preventive drug efficacy during clinical trials. It could thus accelerate the development of therapies, specifically acting during silent AD phases, for secondary prevention. This model also constitutes a study system to characterize new biomarkers or panels of biomarkers of early diagnosis, disease progression, target engagement, and drug efficacy.

   References:

   Bushman DM, Kaeser GE, Siddoway B, Westra JW, Rivera RR, Rehen SK, Yung YC, Chun J. Genomic mosaicism with increased amyloid precursor protein (APP) gene copy number in single neurons from sporadic Alzheimer's disease brains. Elife. 2015 Feb 4;4 PubMed.

   Audrain M, Souchet B, Alves S, Fol R, Viode A, Haddjeri A, Tada S, Orefice NS, Joséphine C, Bemelmans AP, Delzescaux T, Déglon N, Hantraye P, Akwa Y, Becher F, Billard JM, Potier B, Dutar P, Cartier N, Braudeau J. βAPP Processing Drives Gradual Tau Pathology in an Age-Dependent Amyloid Rat Model of Alzheimer's Disease. Cereb Cortex. 2017 Oct 18;:1-18. PubMed.

   Lee MH, Siddoway B, Kaeser GE, Segota I, Rivera R, Romanow WJ, Liu CS, Park C, Kennedy G, Long T, Chun J. Somatic APP gene recombination in Alzheimer's disease and normal neurons. Nature. 2018 Nov;563(7733):639-645. Epub 2018 Nov 21 PubMed.

   Prince M, Wimo A, Guerchet M, Ali GC, Wu YT, Prina M. The Global Impact of Dementia: An analysis of prevalence, incidence, cost and trends. World Alzheimer Report 2015, Alzheimer’s Disease International, London.

   View all comments by Jérôme Braudeau

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References

External Citations

1. AgenT SAS

Further Reading