Researchers rely on transgenic mice to model Alzheimer's disease, but these models are far from perfect. Some can only be maintained on specific genetic backgrounds, some die young or have surprising phenotypes when crossed with other mouse lines, and in others amyloidosis drifts later in successive generations, suggesting gene expression changes from parents to offspring. Enter a new line of mice that promises to circumvent many of these problems. Researchers led by Takaomi Saido have engineered APP knock-ins. These animals recapitulate much of the pathology seen in presymptomatic AD, without overexpressing APP or interrupting other mouse genes. How do these knock-ins compare?
Join Saido at noon U.S. Eastern time on Tuesday, April 22, for a presentation on these new mice. Joining him for discussion will be Karen Hsiao Ashe, Ron DeMattos, David Holtzman, Mathias Jucker, and Mike Sasner.
Check out these knock-ins and other mouse models in our new Research Models database.
Alzforum thanks Nature Neuroscience for making Saido's paper freely available to Alzforum readers.
Over the last two decades, researchers have generated dozens of different mouse models of Alzheimer's disease. The most commonly used lines carry mutant forms of human Aβ precursor protein (APP), either alone or in combination with human presenilin, tau, or other genes. Used for both basic research and drug discovery, these animals have provided a wealth of information. However, there have been niggling doubts from the beginning that some of their phenotypes may have little to do with the human disease, and negative clinical trials later reinforced critical debate about the validity of overexpression models.
The concern arises because the human transgenes are randomly inserted into the mouse DNA, where they may disrupt essential genes or regulatory elements. For example, the Tg2576 mice only survive on a mixed B6/SJL mouse genetic background; if the transgene is maintained on a pure B6 background, the animals die prematurely, suggesting the human APP gene may be interrupting mouse DNA sequences. Transgenic mice tend to lack cell-specific alternative splicing of the APP transcript, and some strains die young of unknown causes.
Moreover, the transgenes are expressed at much higher levels than normal. Many mice with APP transgenes not only produce more Aβ than wild-type mice, but also make more full-length APP and APP fragments, including the soluble ectodomains (sAPPβ), C-terminal fragments, and the APP intracellular domain (AICD), all of which have poorly characterized functions. APP itself interferes with axonal transport because it interacts with microtubule motors. Researchers recognize that targeted replacement of the mouse genes with human versions is a cleaner approach, but such “knock-ins” have proven a tough nut to crack.
After a 12-year effort, Takaomi Saido's group at RIKEN Brain Science Institute, Wako, Japan, has made APP knock-in mice. Humanizing the Aβ sequence and introducing various mutations was time-consuming. In addition, the researchers had removed a long intron between exons 16 and 17 to make manipulations easier, but discovered that expression plummeted without this DNA. The intron probably contains important regulatory elements, said Saido.
As described in the April 13 Nature Neuroscience, first author Takashi Saito and colleagues generated three strains of knock-in mice. One, called NL, contains the Swedish mutation KM670/671NL, which enhances β-secretase cleavage of APP. A second, NL-F, carries the Swedish plus the Beyreuther/Iberian mutation I716F, which favors γ-secretase cleavage of C-terminal fragments at the 42 position and increases the Aβ42:Aβ40 ratio. The third, NL-G-F, carries the Swedish, Iberian, and the Arctic mutation E693G, which accelerates Aβ aggregation. All these mice carry humanized Aβ sequences in the mouse APP locus and are driven by normal mouse promoters. The NL-F and NL-G-F models are curated on the Alzforum Research Models database.
How do these mice compare to stalwarts such as APP23 or Tg2576? Both these lines express the Swedish mutation. Unlike APP23, which overexpresses the precursor protein sevenfold, NL and NL-F mice produce normal amounts of APP and AICD. Like APP23, they generate more β-CTFs than do wild-type mice, in keeping with the known stoking of β-secretase cleavage by the Swedish mutation. NL-F mice produced more Aβ42 than either NL or APP23 mice, leading to an accumulation of soluble and insoluble Aβ42 as the mice aged. Plaques appeared in the cortex and hippocampus at around 6 months in NL-F mice, as opposed to 12 months in the APP23 animals, and steadily grew as the knock-ins aged to 24 months. In plaques, Aβ1-42 appeared first, followed by Aβ3(pE)-42, the N-terminal truncated, pyroglutamylated form. This finding hints that full-length Aβ seeds plaques, a point of debate in the field.
Microglia and activated astrocytes accumulated around the plaques, reflecting the neuroinflammation that is also seen in the brains of people with AD. Reductions in the proteins synaptophysin and PSD95 in the cortex and hippocampus suggested the knock-in mice were losing synapses. Beginning at 18 months, NL-F mice had trouble learning the location of a treat in a Y maze. Age-matched NL mice, on the other hand, navigated the Y maze with aplomb, suggesting that the increases in Aβ42, rather than β-CTF, caused the learning and memory deficits.
In short, the NL-F mice display many of the pathologies seen in AD. Saido believes these mice can better answer questions about the pathogenesis of AD. "Conventional transgenic mice overproduce so many other APP fragments that their phenotypes could be misleading, and they make it difficult to study the underlying mechanisms driving pathology," Saido told Alzforum.
Which reported phenotypes did the knock-in mice not confirm? In other words, what parts of this extensive literature might be artifacts due to APP overexpression? The answer is far from complete. It will require further analysis of the knock-in mice and an in-depth review of the literature. That said, drawing this full comparison is important to weed the literature of reported phenotypes that could mislead scientists who use the models for mechanistic studies or drug discovery. To get the process started, Saido’s paper offers a prior phenotype from his own lab that did not hold up. He had reported previously that deficiency of the enzyme calpastatin exacerbated pathology in APP23 mice, causing the mice to die young with hyperphosphorylation of tau and somatodendritic atrophy (see Higuchi et al., 2012). While this seemed plausible at the time, crossing NL-F mice with calpastatin knockouts reproduced none of these phenotypes.
"There have been nearly 3,000 research papers using APP transgenic mice. Many describe crosses with other gene knockouts or knock-ins, such as ApoE and tau, but we are unsure if the observed phenotypes are relevant in terms of human AD pathogenesis," said Saido. He suggested much of the earlier work should be reexamined.
Because NL-F mice take 18 months to show signs of memory impairment, Saito and colleagues generated the NL-G-F mice to try to speed things up. The addition of the Arctic mutation had the desired effect, accelerating phenotypes by a factor of three. These mice initially deposit Aβ in the brain by two months, as opposed to six months of age for the NL-F mice, and falter in the Y maze at six months.
In another example of how these mice perhaps mimic human disease more faithfully than prior models, NL-G-F mice also develop subcortical amyloidosis. This recapitulates pathology seen in human carriers of the Arctic mutation, yet transgenic mice overexpressing APP with the Arctic mutation lay down no plaques in subcortical brain regions.
Saido believes the new knock--in mice simulate preclinical AD, that is, aggressive Aβ pathology with mild memory impairment but prior to overt neurofibrillary pathology. They could be used to screen for preventive medicines or biomarkers of presymptomatic disease, Saido said.
Saido offers to freely distribute these animals to academic labs and not-for profit institutions.—Tom Fagan
Research Models Citations
- Higuchi M, Iwata N, Matsuba Y, Takano J, Suemoto T, Maeda J, Ji B, Ono M, Staufenbiel M, Suhara T, Saido TC. Mechanistic involvement of the calpain-calpastatin system in Alzheimer neuropathology. FASEB J. 2012 Mar;26(3):1204-17. PubMed.
No Available Further Reading
- 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. Nature Neuroscience. Advance Online Publication, April 13, 2014