BACE1 knockout mice have more than their fair share of problems, but is it because they lack the enzyme during development or during adulthood? New conditional knockouts from the lab of Robert Vassar, Northwestern University, Chicago, suggest a bit of both. In the September 19 Science Translational Medicine, first author Ming-Hsuan Ou-Yang and colleagues describe how mice that lose BACE1 activity as adults avoid many troublesome characteristics of germline knockouts, such as seizures and weight loss. However, one pesky phenotype remains: The mice have shortened, somewhat disheveled mossy fiber bundles in their hippocampi. The results hint that strongly inhibiting BACE1 in adults could lead to hippocampal defects.
- Conditional BACE1 knockouts escape many of the problems of germline knockouts.
- cKOs still have short, disorganized hippocampal mossy fibers.
- Scientists caution against strong BACE1 inhibition in people.
“The work is very careful, and the data convincing,” said Riqiang Yan, University of Connecticut Health, Farmington. Yan was not involved in the work but has generated his own line of conditional BACE1 knockouts.
“The findings by the Vassar lab are extremely important for the safety of clinical trials using BACE inhibitors,” wrote Christian Haass, Ludwig-Maximilians University Munich, to Alzforum. Clinical trials that aim to almost completely inhibit BACE1 function may have a deleterious outcome, he said. Other experts suggested that lowering the dose of BACE1 inhibitors could avoid this side effect and prove safe for long-term use in normal people at risk for Alzheimer’s disease.
BACE1 works alongside γ-secretase to snip the amyloid precursor protein to release Aβ, the peptide that forms the senile plaques that characterize Alzheimer’s disease. Researchers have considered inhibiting both proteins to treat AD. In the case of γ-secretase inhibitors, this proved detrimental. Notably, in a Phase 3 trial, patients on the γ-secretase inhibitor semagacestat tended to have worse cognitive outcomes than those on placebo, along with significant psychiatric and other side effects. Scientists have attributed these changes to decreased processing of another γ-secretase substrate, Notch (Aug 2010 news; Nov 2012 news).
Cut Short. In the CA3 of the hippocampus (light green), the infrapyramidal bundle (bounded by white arrows) appears shorter in conditional knockouts (left), compared to control mice (right). [Courtesy of Ou-Yang et al., 2018; Science Translational Medicine/AAAS.]
The failure of γ-secretase inhibitors has made scientists extra cautious about inhibiting BACE1, which has many substrates beyond APP. In keeping with this, BACE1 knockouts have numerous problems. They lose myelin from their axons, develop spontaneous seizures, have trouble navigating and remembering objects, and they die younger than wild-type animals (Dominguez et al., 2005; Willem et al., 2006; Hu et al., 2010; Laird et al., 2005). However, scientists are unsure whether those traits arise from developmental deficits, or from lack of BACE1 in adulthood. In one conditional knockout created by Yan’s group, gradual reduction in BACE1 expression in the whole body starting about a month after birth avoided many of the phenotypes observed in complete knockouts, with the exception of reduced neural plasticity (Feb 2018 news). When crossed with an AD mouse model, it developed fewer plaques than transgenic controls.
Ou-Yang and colleagues took a similar approach, using the Cre/lox genetic recombination system to carve out exon 2 of BACE1. In one mouse they expressed Cre shortly after birth, limiting BACE1 loss to the forebrain on postnatal day four and beyond. The cerebellum and brain stem still had some expression. In another mouse, Ou-Yang induced Cre by injecting mice with tamoxifen. This allowed the researchers to raise the mice until full adulthood (three months of age) before they wiped out BACE1 throughout most of the body, including the cortex and hippocampus. The subcortical regions and olfactory bulb expressed some residual BACE1 in this model, too, possibly because tamoxifen did not penetrate those regions fully, the authors wrote. BACE1 expression was reduced in all mice for at least six months before behavioral testing or histopathology.
In the end, both mouse models, but especially the adult knockouts, were spared many of the problems seen in BACE1-/- knockouts. When BACE1 was knocked out just after birth, animals lived about as long as controls. They took slightly longer to learn the location of a platform in the Morris water maze, but otherwise their memories were on par with wild-type mice. These cKOs had fewer spontaneous seizures and their degree of hypomyelination was considerably less than that seen in BACE1-/- embryonic knockouts. The adult knockouts also lived as long as controls. They gained more weight than wild-type mice, but the researchers are unsure why. These mice had normal learning and memory and had no spontaneous seizures or reduced myelination in any brain region.
However, one trait of embryonic BACE1-/- mice was still apparent in both conditional knockouts. The infrapyramidal bundle (IPB)—a tract of mossy fiber axons that extends from the dentate gyrus—was uncharacteristically short. Normally, adult-born neurons in the dentate gyrus extend their axons along the ventral side of the CA3 layer of the hippocampus, forming the IPB, before they cross over the CA3 layer to its dorsal side. However, in BACE KOs, the IPB was 30 percent shorter, and crossed the CA3 layer prematurely (see image above).
Although behavioral testing picked up no learning or memory problems in the adult knockouts, the authors note that the length of the IPB in different mouse strains correlates with spatial learning (Crusio and Schwegler, 2005). Future studies should use more sensitive tests to detect any impairments, they wrote.
In a previous study, the authors attributed the IPB deficit in BACE-/- knockouts to reduced cleavage of a BACE1 substrate called close homolog of L1 (Hitt et al., 2012). CHL1 cleavage is important for guiding axons to their final destination. In the knockouts, Ou-Yang and colleagues found more full-length CHL1 in the hippocampus and less of the cleaved fragment than in control mice. The more full-length they found, the shorter the IPB. The findings indicate that BACE1 cleavage of CHL1 in adult newborn neurons is needed for axons to navigate their way through the hippocampus.
The results suggest both good news and bad news for BACE inhibitors, Vassar told Alzforum. “The good news is that most of the phenotypes we were concerned about in the germline BACE1 knockouts go away when you knock out BACE1 in adults,” he said. “The bad news is that the wiring problem remains, at least in the hippocampus.” To his mind, this does not mean BACE1 inhibitor programs are in jeopardy or that clinical trials should be stopped. “This is a note of caution; drug makers should be aware of this and consider dialing back doses,” Vassar said. The level of BACE1 knockout in these models—in the neighborhood of 90 percent—approximates that with the highest doses of BACE inhibitors given in clinical trials, he said.
Eric Siemers, formerly at Lilly and now of Siemers Integration LLC in Indianapolis, noted that the hippocampus shrank slightly in treated patients in the Phase 3 EPOCH trial of Merck’s BACE1 inhibitor, verubecestat (Dec 2017 conference news). “Whether that’s related to mossy fiber bundle shrinkage is unknown, but people need to take this paper seriously,” said Siemers. “It doesn’t mean we need to stop the [BACE1] studies, but it does mean we need to pay attention to hippocampal morphology.” He suggested that volumetric magnetic resonance measures of the hippocampus would be an important addition to trials of BACE inhibitors. In EPOCH, verubecestat provided no cognitive benefit and a subgroup analysis even suggested a slight worsening on the drug.
Philip Wong, Johns Hopkins University of Medicine, Baltimore, recommends that researchers aim to reduce BACE1 function by a maximum of 50 percent to preserve healthy BACE1 cleavage of other substrates. Companies currently exceed that in clinical trials, he said. “If you can get away with a lower dose and still be effective, you should do that.” Stefan Lichtenthaler from the German Center for Neurodegenerative Diseases, Munich, agreed. “A careful choice of the inhibitor dose may allow enough BACE1 activity to prevent such phenotypes in patients. In fact, BACE inhibitors may be more efficient in prevention than in treatment trials and a lower dose may be sufficient for prevention,” he wrote (see full comment below). Wong said it would be interesting to knock out BACE1 in 12-month-old mice, because the timing would be more comparable to shutting down BACE1 in middle-aged people with a drug.—Gwyneth Dickey Zakaib
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