A paper in the September 21 Journal of Neuroscience suggests that two prominent phenotypes considered relevant to Alzheimer’s disease are artifacts caused by overexpression of the amyloid precursor protein (APP). Scientists led by Takaomi Saido, RIKEN Brain Science Institute, Wako, Japan, found neither elevation of the CDK5 activator p25 nor reduction in sodium channels in a knock-in mouse model expressing human APP at endogenous levels. They surmise that these phenotypes reflect too much APP, not true AD-related pathogenic processes. Other researchers were skeptical. They said the authors were overreaching in their conclusions to the current set of experiments, but were optimistic about future insights from knock-in models.
To mimic Aβ pathology in the mouse brain, many research groups have explored dozens of models that overexpress mutant human variants of APP or presenilins (see Alzforum Research Models database). However, the field has long recognized that driving up gene expression beyond physiological levels can introduce artifacts, for example by disrupting other genes near the site of transgene insertion or by overwhelming protein homeostasis, triggering apoptosis (Kuang et al., 2006).
Researchers have struggled to generate human APP knock-in (KI) models that would avoid such pitfalls. After more than a decade of trying, Saido’s lab finally succeeded, generating APPNL-F and APPNL-G-F models carrying Swedish/Iberian and Swedish/Arctic/Iberian mutations, respectively (see Apr 2014 webinar). The APPNL-F mice used in this study overproduce only Aβ, not APP, at an increased Aβ42:Aβ40 ratio, developing plaques and gliosis at six months, and cognitive impairments at 18.
About 200 scientists currently work with Saido’s KI models, and they have begun to publish their findings. Early indications suggest that mushroom spines wane in synapses as these animals age and that the orphan G protein-coupled receptor GPR3 promotes amyloidogenic processing of APP (Oct 2015 news; Huang et al., 2015). In particular, some researchers have begun checking whether the KI mice recapitulate phenotypes previously published for overexpression models. However, many researchers contacted by Alzforum said their mouse colonies are not yet old enough to examine AD-related pathology or behavior. Still other groups are waiting to receive the mice. Some bemoaned a complicated and time-consuming process to obtain the animals, available only through the RIKEN BioResource Center. In multiple comments on Alzforum, Saido has encouraged the field to use his lab’s mice to validate phenotypes published in overexpression models.
For the current study, first author Takashi Saito and colleagues examined two phenotypes previously described in some overexpression models: downregulation of the sodium channel Nav1.1 in the interneurons prompting seizures; and calcium/calpain dysfunction leading to generation of p25, which activates CDK5 to hyperphosphorylate tau, ultimately triggering synaptic and memory deficits (Oakley et al., 2006; Seo et al., 2014).
To test for the latter phenotype, Saito and colleagues compared p25 levels in homogenates of the neocortices and hippocampi from APPNL-F mice to similar preparations from APP23 mice, which overexpress APP. The knock-ins had wild-type levels of p25, while APP23 models had more than twice as much (see image). Even when Saito crossed APPNL-F mice with calpastatin-deficient animals (Cast KO), p25 levels in the offspring were normal (Takano et al., 2005). Calpastatin, the only endogenous inhibitor of calpain, normally suppresses p25 production. Saido concluded that even in Cast KO/APP knock-in mice, Aβ amyloidosis on its own fails to elevate intraneuronal calcium enough to activate calpain and trigger production of p25. Thus, Saido argues that p25 production in APP23 mice results from APP overexpression.
Other scientists were unconvinced. They noted that the conclusions hinge on one faint western blot (Figure 1) and said the experiment lacked appropriate controls, such as wild-type x Cast KO crosses. Li-Huei Tsai from Massachusetts Institute of Technology questioned if Saito and colleagues accurately measured p25. She noted that they previously reported a rise in p25 in wild-type calpastatin knockouts, and wondered why they did not see it in the APPNL-F x Calp-/- offspring (Sato et al., 2011). Saido responded that those previous results were from in vitro and ex vivo experiments that are less relevant. Tsai said data from other tissue and cell culture models indicate, as well, that Aβ generates excess p25 without APP overexpression (Zheng et al., 2005; Seo et al., 2014).
That calpain may be overactive in AD brains fits with the idea that p25 contributes to pathology, noted Tsai (see Kurbatskaya et al., 2016). The calcium-dependent protease was therapeutically targeted for Alzheimer’s when the North Chicago-based pharmaceutical company AbbVie tested the inhibitor ABT-957 in a Phase 1 trial. AbbVie has halted development of the drug. A note on clinicaltrials.gov states the compound insufficiently engaged the target. An AbbVie representative declined to elaborate.
Saito and colleagues also used western analysis to quantify Nav1.1 sodium channels. Levels in wild-type, APPNL-F, APPNL-F x Cast KO, and in APP23 were all similar and higher than in J20 mice, which overexpress APP (Verret et al., 2012). These mice begin to lose hippocampal neurons by 12 weeks of age, which is not commonly seen in APP overexpression models. Saido and colleagues took their data to mean that Nav1.1 deficiency is unique to the J20 model, and therefore an artifact of transgene expression. However, APP23 mice also overexpress APP.
Again, others were not so sure. Tsai noted that because the reduction in sodium channel expression was previously reported to be specific to interneuron subpopulations, it would be hard to detect by western blot of crude lysates (see full comment below). Saido countered that Nav1.1-positive neurons are sufficiently abundant in the hippocampus and neocortex to measure changes in the sodium channel expression with the methodology used.
Ralph Nixon at the New York University School of Medicine hypothesized that APPNL-F mice develop too mild a phenotype to see a drop in sodium channels, or a rise in p25.
Should researchers be concerned about years of data from overexpression models? Given that APPNL-F x Calp-/- mice only reproduced two of five phenotypes observed in APP23 x Calp-/- mice, i.e., amyloidosis and neuroinflammation, Saido and colleagues estimate that 60 percent of the phenotypes in APP-overexpressing mice generally could be artifacts (Saito et al., 2014). This would call into question findings from more than 3,000 papers, they wrote.
Other scientists doubt that such sweeping conclusions can be drawn just yet; after all, only two papers on the knock-ins have been published thus far on this particular topic. “It is quite a leap from reporting ‘only two of five phenotypes studied were reproduced’ to concluding that 60 percent of all phenotypes in thousands of publications are artifacts,” Mike Sasner at Jackson Laboratory in Bar Harbor, Maine, wrote to Alzforum.
Sasner said that APP knock-in models do avoid certain problems of transgenic animals, but that they have their own drawbacks. Because knock-ins express APP at near-endogenous levels, they have late-developing, mild phenotypes that make them impractical for many studies. However, he agreed that traditional APP overexpression models are not ideal for testing potential AD therapies.
Tsai pointed out that a duplication of the whole APP gene causes rare familial forms of AD, and that people with Down’s syndrome, who carry an extra copy of chromosome 21 that harbors the gene, also develop the disease. She stressed that APP overexpression is not necessarily an artificial way to model Alzheimer’s.
In addition, Nixon questioned the validity of putting multiple familial AD mutations into a single animal, as was done in both these knock-ins and some of the overexpression models. There is no indication the Swedish, Iberian, and Arctic mutations ever crop up in the same person. While Nixon agreed that overexpression causes problems, he questioned if the knock-ins are a better option. “The ultimate mouse model of Alzheimer’s disease would be the simplest and least artificial, while recapitulating the full spectrum of neuropathology,” he told Alzforum. “We still don’t have that.”
All in all, many commentators, including some who declined to be quoted, echoed concerns about protein overexpression, and agreed that the knock-in mice represent a major advance. Saido will co-chair a symposium on the animals at the Society for Neuroscience annual meeting November 12-16 in San Diego.—Gwyneth Dickey Zakaib
Research Models Citations
- Kuang E, Wan Q, Li X, Xu H, Zou T, Qi Y. ER stress triggers apoptosis induced by Nogo-B/ASY overexpression. Exp Cell Res. 2006 Jul 1;312(11):1983-8. Epub 2006 May 9 PubMed.
- Zhang H, Wu L, Pchitskaya E, Zakharova O, Saito T, Saido T, Bezprozvanny I. Neuronal Store-Operated Calcium Entry and Mushroom Spine Loss in Amyloid Precursor Protein Knock-In Mouse Model of Alzheimer's Disease. J Neurosci. 2015 Sep 30;35(39):13275-86. PubMed.
- Huang Y, Skwarek-Maruszewska A, Horré K, Vandewyer E, Wolfs L, Snellinx A, Saito T, Radaelli E, Corthout N, Colombelli J, Lo AC, Van Aerschot L, Callaerts-Vegh Z, Trabzuni D, Bossers K, Verhaagen J, Ryten M, Munck S, D'Hooge R, Swaab DF, Hardy J, Saido TC, De Strooper B, Thathiah A. Loss of GPR3 reduces the amyloid plaque burden and improves memory in Alzheimer's disease mouse models. Sci Transl Med. 2015 Oct 14;7(309):309ra164. PubMed.
- Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, Guillozet-Bongaarts A, Ohno M, Disterhoft J, Van Eldik L, Berry R, Vassar R. Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation. J Neurosci. 2006 Oct 4;26(40):10129-40. PubMed.
- Seo J, Giusti-Rodríguez P, Zhou Y, Rudenko A, Cho S, Ota KT, Park C, Patzke H, Madabhushi R, Pan L, Mungenast AE, Guan JS, Delalle I, Tsai LH. Activity-dependent p25 generation regulates synaptic plasticity and Aβ-induced cognitive impairment. Cell. 2014 Apr 10;157(2):486-98. PubMed.
- Takano J, Tomioka M, Tsubuki S, Higuchi M, Iwata N, Itohara S, Maki M, Saido TC. Calpain mediates excitotoxic DNA fragmentation via mitochondrial pathways in adult brains: evidence from calpastatin mutant mice. J Biol Chem. 2005 Apr 22;280(16):16175-84. PubMed.
- Sato K, Minegishi S, Takano J, Plattner F, Saito T, Asada A, Kawahara H, Iwata N, Saido TC, Hisanaga S. Calpastatin, an endogenous calpain-inhibitor protein, regulates the cleavage of the Cdk5 activator p35 to p25. J Neurochem. 2011 May;117(3):504-15. PubMed.
- Zheng YL, Kesavapany S, Gravell M, Hamilton RS, Schubert M, Amin N, Albers W, Grant P, Pant HC. A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons. EMBO J. 2005 Jan 12;24(1):209-20. PubMed.
- Kurbatskaya K, Phillips EC, Croft CL, Dentoni G, Hughes MM, Wade MA, Al-Sarraj S, Troakes C, O'Neill MJ, Perez-Nievas BG, Hanger DP, Noble W. Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer's disease brain. Acta Neuropathol Commun. 2016 Mar 31;4:34. PubMed.
- Verret L, Mann EO, Hang GB, Barth AM, Cobos I, Ho K, Devidze N, Masliah E, Kreitzer AC, Mody I, Mucke L, Palop JJ. Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model. Cell. 2012 Apr 27;149(3):708-21. PubMed.
- Saito T, Matsuba Y, Mihira N, Takano J, Nilsson P, Itohara S, Iwata N, Saido TC. Single App knock-in mouse models of Alzheimer's disease. Nat Neurosci. 2014 May;17(5):661-3. Epub 2014 Apr 13 PubMed.
- Saito T, Matsuba Y, Yamazaki N, Hashimoto S, Saido TC. Calpain Activation in Alzheimer's Model Mice Is an Artifact of APP and Presenilin Overexpression. J Neurosci. 2016 Sep 21;36(38):9933-6. PubMed.