A new conditional knockout mouse offers clues on the effects of dialing down β-secretase, a.k.a. BACE1, a target for potential Alzheimer’s therapies. Researchers led by Riqiang Yan, Lerner Research Institute, Cleveland, report in the February 14 Journal of Experimental Medicine that the mice, which gradually lose expression of BACE1 soon after birth, appear healthy but for weakened neural plasticity. When crossed to a mouse model of AD, their postnatal reduction in BACE1 not only halted Aβ plaque formation, but also appeared to eliminate existing plaques. This led to claims in the press that AD can be completely reversed by targeting BACE (e.g., Feb 15 Newsweek), which came two days after Merck announced the termination of its BACE inhibitor verubecestat (Feb 2018 news). Alzforum covered preliminary data from Yan’s study in 2016, when he presented them at the second Kloster Seeon meeting on BACE proteases in health and disease in Germany (Oct 2016 news).
- In a conditional BACE knockout, expression begins falling in early adulthood.
- Apart from weak LTP, knockouts appear healthy.
- When crossed with 5xFAD mice, the knockout appears to ablate amyloid plaques.
“I am delighted to see that someone finally made the conditional BACE1 knockout mouse,” said Joanna Jankowsky, Baylor College of Medicine in Houston. Stefan Lichtenthaler, German Center for Neurodegenerative Diseases in Munich, was also enthusiastic. “It is particularly intriguing to see that BACE1 inhibition not only reduces plaque growth, but may even shrink existing plaques,” he wrote (see full comment below). However, researchers noted important limitations of this study, as well.
BACE1 embryonic knockouts develop brain and muscle defects, display abnormal behaviors, and often die young (Vassar et al., 2014; Barão et al., 2016; Hou et al., 2017). Researchers developing therapies that target BACE1 have long wondered to what extent these problems stem from developmental errors versus harmful effects later in life.
Vanishing Act? From postnatal day 75 (top) to 300 (bottom), brain amyloid plaques (red arrows) grow in number and size in 5xFAD mice (left)), but dwindle in 5xFAD mice crossed to BACE1 conditional knockouts (right). [© Hu et al., J Exp Med, 2018.]
To address this question, first author Xiangyou Hu engineered an inducible BACE1 knockout. He used a tamoxifen-inducible cre driven by the ubiquitin promoter, which is active in nearly all cells, to control the removal of exon 2 from the BACE gene. To the researchers’ surprise, even before giving these animals tamoxifen, brain BACE1 levels started dropping around 30 days after birth, falling to 50 percent at two months, and to 20 percent at four months of age. “The cre expression turned out to be very leaky,” explained Yan, now at the University of Connecticut, Farmington. Though eliminating the model’s manipulability, this technical fluke had a silver lining in that it avoided potential side effects from tamoxifen.
Joining a collection of BACE transgenic mouse models, these new conditional knockouts (cKOs) have a normal lifespan and, so far, appear free of the deficits that afflict embryonic KOs. Five-month-old cKOs sport well-myelinated axons in their corpora callosa, sciatic nerves, and optic nerves, and the astrocytes in their dentate gyri are similar in number and type to those of healthy controls; all these features are known to be disrupted in embryonic knockouts. Also, unlike embryonic KOs, the cKOs do not experience spontaneous seizures. However, the cKOs have elevated levels of full-length neuregulin, a BACE1 substrate involved in brain development and synaptic plasticity. Perhaps because of this, long-term potentiation evoked by stimulation in the hippocampus was lower than LTP by about half in wild-type controls.
To model the consequences of losing BACE1 in AD, the researchers crossed the cKOs with 5xFAD mice, which start accumulating amyloid plaques at 2.5 months of age. They compared plaque pathology using the 6E10 antibody, which recognizes plaques, APP, and soluble Aβ peptides. At 2.5 months, plaques in the cortices of the 5xFAD/BACE cKO mice numbered, on average, 24 per sagittal slice, compared with 5xFAD controls. Plaques steadily grew in size and number in the controls. By 10 months of age, plaques numbered almost 800 in the controls, but were essentially undetectable if BACE was absent (see image above).
“The brain has some system to eliminate amyloid plaques if you turn off the spigot,” said Yan. This idea has been tested before, with mixed results. Suppressing γ-secretase reduced pre-existing amyloid plaques in APP transgenics, whereas suppressing APP itself did not (Jul 2005 news; Jankowsky et al., 2005; Mar 2013 news).
Jochen Herms, German Center for Neurodegenerative Diseases, Munich, recently reported that the BACE1 inhibitor NB-360 blocks new plaque formation but can’t eliminate existing plaques (Jan 2018 news). Herms was not convinced plaques had vanished in Yan’s new crosses. He believes the fading of the 6E10 signal might reflect reduced intraneuronal accumulation of APP fragments, since 6E10 binds not only Aβ, but also BACE cleavage products such as C99. “The [loss of 6E10 binding] does not necessarily mean that existing plaques have been cleared,” Herms said, adding that it is difficult to understand exactly what is happening since, for example, one would expect intracellular, full-length APP to increase with BACE loss. Both he and Jankowsky would have liked to see a more specific marker for fibrillary Aβ aggregates used. Yan told Alzforum he obtained essentially identical findings using thioflavin S, which binds to fibrillary plaques. However, he analyzed only a few such samples, and these data were not included in the paper.
As plaques dwindled in the cKO/5xFAD mice, gliosis as per anti-GFAP antibodies and dystrophic neurites detected by anti-RTN3 antibodies both faded. Moreover, the weak hippocampal LTP seen in 5xFAD mice was partially rescued. After stimulation, excitatory postsynaptic potentials climbed by 21 percent in the crosses, compared with 5 percent in the 5xFAD mice. That’s despite weakened LTP seen in the BACE cKO alone. By comparison, LTP increases 60 percent in wild-type mice, and 28 percent in BACE cKOs.
What do the findings mean for the clinical future of BACE1 inhibitors? Ulf Neumann, Novartis, Basel, Switzerland, noted that the cKO/5xFAD mice were still young when BACE1 dipped more than 50 percent. “They may produce Aβ deposits that are ‘easy’ to clear, with comparatively few densely compact plaques,” he said. In contrast, AD patients who have harbored plaques for many decades may have irreversible damage, he said. While he thinks there are caveats in interpreting the data, Neumann said the study can be taken as another hint that early, and very early, anti-Aβ treatments have chances of success.
Yan acknowledged that the mice’s young age is a limitation. “We are now making additional cre lines to control BACE1 expression more tightly in late adult stages,” he said.—Marina Chicurel
- Merck Axes Verubecestat for Prodromal AD, Researchers Say ‘Go Earlier’
- What Exactly Does BACE Do in Adults?
- Amyloid Hypothesis—Closing the Spigot Helps, Temporarily
- Inducible APP Mice—Cognition Restored in Advanced Amyloidosis?
- BACE Block Nips New Plaques in the Bud, Old Ones Keep Growing
Research Models Citations
- Vassar R, Kuhn PH, Haass C, Kennedy ME, Rajendran L, Wong PC, Lichtenthaler SF. Function, therapeutic potential and cell biology of BACE proteases: current status and future prospects. J Neurochem. 2014 Jul;130(1):4-28. Epub 2014 Apr 19 PubMed.
- Barão S, Moechars D, Lichtenthaler SF, De Strooper B. BACE1 Physiological Functions May Limit Its Use as Therapeutic Target for Alzheimer's Disease. Trends Neurosci. 2016 Mar;39(3):158-69. Epub 2016 Jan 30 PubMed.
- Hou H, Fan Q, He W, Suh H, Hu X, Yan R. BACE1 Deficiency Causes Abnormal Neuronal Clustering in the Dentate Gyrus. Stem Cell Reports. 2017 Jul 11;9(1):217-230. Epub 2017 Jun 29 PubMed.
- Jankowsky JL, Slunt HH, Gonzales V, Savonenko AV, Wen JC, Jenkins NA, Copeland NG, Younkin LH, Lester HA, Younkin SG, Borchelt DR. Persistent amyloidosis following suppression of Abeta production in a transgenic model of Alzheimer disease. PLoS Med. 2005 Dec;2(12):e355. Epub 2005 Nov 15 PubMed.
No Available Further Reading
- Hu X, Das B, Hou H, He W, Yan R. BACE1 deletion in the adult mouse reverses preformed amyloid deposition and improves cognitive functions. J Exp Med. 2018 Mar 5;215(3):927-940. Epub 2018 Feb 14 PubMed.