27 Oct 2016

The 2nd Kloster Seeon Meeting on BACE Proteases in Health and Disease, held September 25-27 in Seeon near Munich, was propelled on an updraft of optimism that BACE inhibitors may work out to treat or prevent Alzheimer’s disease (see Part 1 of this series). And yet, even though no adverse events have stopped the ongoing Phase 2/3 trials, scientists are watching with bated breath because there is a long list of potential side effects that might still surface down the road. “My concern is that people may be on BACE inhibitors for decades,” said Robert Vassar, Northwestern University, Chicago. “We have to be aware of that and plan for long-term safety.” Stefan Lichtenthaler from the German Center for Neurodegenerative Diseases in Munich, who co-organized the meeting with Vassar, agreed. “We have not yet fully resolved whether BACE inhibition, per se, will lead to long-term toxicity,” he told Alzforum. The meeting tackled this issue head-on, such as with first reports on conditional knockouts of BACE1 in adult mice that enable a distinction between developmental versus adult roles of the protease. Other presentations explained how BACE processing of substrates beyond APP might affect synapses and plasticity.

Conditional KOs Debut
As industry leaders often point out, partially blocking BACE1 in adulthood and knocking it out in the embryo are wholly different things. While phenotypes have emerged in BACE1 embryonic knockouts that raise the specter of serious side effects of BACE inhibition (see table below), scientists have always wondered which of those may be rooted in developmental effects, and which truly arise in adult animals. To address this question, researchers in Vassar’s lab and in Riqiang Yan’s lab at the Lerner Research Institute, Cleveland, working independently, generated a total of four strains of conditional knockout models (cKOs). Both groups used the cre/lox system to pluck exon 2 out of the BACE gene, shutting down its expression.  

Phenotype	Embryonic Knockout Model
*unpublished. †reduced pre-pulse inhibition
	Hyde et al. (unpublished)	Cai et al., 2001	Luo et al., 2001	Roberds et al., 2001	Harrison et al., 2003	Dominguez et al., 2005
Seizures	No	Yes		Yes
Muscle Spindle Defects						Yes
Myelination Defect	Yes	Yes	Yes			Yes
Hypopigmentation	No
Spine Density Loss		Yes
Plasticity Deficits		Yes		Yes
Retinopathy		No*				Yes
Motor Control Defect	No		Yes		No	Yes
Impaired Survival	Yes	Yes	No			Yes
Weight Loss	Yes		No		Yes	Yes
Impaired Spatial and Working Memory		Yes	Yes	Yes
Altered Neurogenesis and Astrogenesis		Yes
Axonal Targeting Errors		Yes
Impaired Axonal Growth Cone						Yes
Schizophrenia-Like Phenotypes	Yes†	Yes	Yes
Hyperactive		Yes				Yes
Timid And Less Exploratory					Yes

Reported BACE1 Knockout Phenotypes. Scientists wonder which ones might be caused by chronic inhibition of BACE in older adults.

Vassar’s group engineered two different strains. In one they placed cre recombinase expression under control of the calmodulin kinase II promoter, which is active mainly in excitatory neurons in the forebrain. In the second, they ubiquitously expressed cre from the Rosa 26 promoter but made the knockout inducible with a tamoxifen-driven system. For his part, Yan’s group used the nestin promoter to drive cre expression in one strain and a tamoxifen-inducible cre driven by the ubiquitin promoter in a second. The nestin promoter turns on in neural precursors and neural stem cells, while the ubiquitin promoter is active in almost all cells. While these labs are still characterizing their mice, at Kloster Seeon they reported some notable phenotypes.

Vassar showed that unlike the embryonic BACE1 KOs, which struggle to wean and grow to be smaller than wild-type mice, the CamKII cKO mice weaned with ease and were close to normal size at three months old. They expressed no BACE1 in the cortex, a little in the hippocampus, and close to normal levels in the cerebellum. In keeping with this, the mice expressed higher levels of some BACE1 substrates in the cortex, including full-length APP, CHL1, and neuregulin 1. They had hardly any C99, the C-terminal part of APP left over after BACE cleavage.

Electrophysiologically, brain slices taken from year-old cKO mice showed normal long-term potentiation, a measure of synaptic plasticity. The mice navigated a Y maze normally, and they found the location of a hidden platform in the Morris water maze as well as control mice did in probe trials. Those are all good signs for BACE inhibitor programs, the researchers agreed. All the same, Vassar said these cKO’s may have a subtle problem with memory acquisition, as they took slightly longer to reach the platform in the first two days of learning trials.

More concerning perhaps is that, like the embryonic BACE1 knockouts, these CaMKII-driven cKOs suffered brain seizures. All six mice studied at one year of age had increased spike discharges detected by an EEG, and three of them had visible seizures as well. However, Vassar pointed out that CaMKII expression begins right after birth in these animals, making them a less faithful model of adult BACE inhibition than the inducible model.

In that vein, Vassar reported no seizure activity in the Rosa26 BACE1 knockouts when cre was induced at three, six, or nine months and the animals tested one to two weeks later. Again, histology showed that after tamoxifen, BACE1 was fully suppressed in the cortex but slightly less so in the hippocampus. These cKOs also had more APP, CHL1, and neuregulin in the brain. Myelination was normal, said Vassar. Hypomyelination is a phenotype reported for embryonic BACE knockouts. In Morris water maze probe trials, the Rosa26 BACE1 mice performed as well as controls did, but they, too, showed a slight delay in learning the position of the platform in the first day or two of training.

The males also may be weaker since they fell off a rotating rod more quickly than wild-type mice or their female siblings. Both male and female cKOs gained more weight than normal mice. This was totally unexpected since BACE1 embryonic knockouts are thinner. Vassar was perplexed by this and said it needs to be studied further. He was not sure that plumpness alone explains the males’ weaker grip on the rotarod. Hypomyelination or muscle spindle problems are also possible explanations for their apparent muscle weakness.

Yan reported that like the full embryonic BACE1 knockouts, his nestin-driven BACE1 knockouts were also smaller than normal mice. Because the nestin promoter turns on cre at around day 12 in the embryo, Yan concluded that early BACE1 expression in the neural lineage is necessary for normal growth. Yan found increased astrogenesis in the conditional knockouts as well, a phenotype that also happens in embryonic BACE1 knockouts.

In his inducible knockout, Yan and colleagues found reductions in BACE1 in the brain even before the animals were given tamoxifen, suggesting the leakage of the ubiquitin promoter that drives cre expression. This is a widely used promoter for cre-driven knockouts, but this leaky expression has never been reported before, said Yan. When they crossed these BACE1 cKO animal with 5xFAD mice, the offspring had dramatically fewer plaques at six months. This happened even without the addition of tamoxifen, in keeping with the leaky promoter hypothesis. Yan did not report exactly when this promoter begins to drive cre expression, but considered these observations highly important. “These data demonstrate that early inhibition of BACE1 will surely reduce amyloid deposition,” he told Alzforum. In addition, the BACE1 cKO had no sign of increased astrogenesis, which happens in the full embryonic knockouts. “This phenotype is more related to BACE1 function in early development rather than in the adult, indicating BACE1 inhibition will not induce this phenotype,” he concluded.

“While it’s early days in characterization, these conditional knockouts are important,” said Vassar. “Now we have tools to understand the physiological function of BACE in vivo in specific cell and tissue types, without the complication of developmental effects,” he said.

Lichtenthaler agreed. “If some phenotypes could be ruled as irrelevant for older people, we would not need to consider them potential side effects in clinical trials,” he said.—Tom Fagan

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References

News Citations

1. At 2nd Kloster Seeon Meeting, Renewed Optimism for Targeting BACE1 21 Oct 2016

Paper Citations

1. Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, Price DL, Wong PC. BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci. 2001 Mar;4(3):233-4. PubMed.
2. Luo Y, Bolon B, Kahn S, Bennett BD, Babu-Khan S, Denis P, Fan W, Kha H, Zhang J, Gong Y, Martin L, Louis JC, Yan Q, Richards WG, Citron M, Vassar R. Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci. 2001 Mar;4(3):231-2. PubMed.
3. Roberds SL, Anderson J, Basi G, Bienkowski MJ, Branstetter DG, Chen KS, Freedman SB, Frigon NL, Games D, Hu K, Johnson-Wood K, Kappenman KE, Kawabe TT, Kola I, Kuehn R, Lee M, Liu W, Motter R, Nichols NF, Power M, Robertson DW, Schenk D, Schoor M, Shopp GM, Shuck ME, Sinha S, Svensson KA, Tatsuno G, Tintrup H, Wijsman J, Wright S, McConlogue L. BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum Mol Genet. 2001 Jun 1;10(12):1317-24. PubMed.
4. Harrison SM, Harper AJ, Hawkins J, Duddy G, Grau E, Pugh PL, Winter PH, Shilliam CS, Hughes ZA, Dawson LA, Gonzalez MI, Upton N, Pangalos MN, Dingwall C. BACE1 (beta-secretase) transgenic and knockout mice: identification of neurochemical deficits and behavioral changes. Mol Cell Neurosci. 2003 Nov;24(3):646-55. PubMed.
5. Dominguez D, Tournoy J, Hartmann D, Huth T, Cryns K, Deforce S, Serneels L, Camacho IE, Marjaux E, Craessaerts K, Roebroek AJ, Schwake M, D'Hooge R, Bach P, Kalinke U, Moechars D, Alzheimer C, Reiss K, Saftig P, De Strooper B. Phenotypic and biochemical analyses of BACE1- and BACE2-deficient mice. J Biol Chem. 2005 Sep 2;280(35):30797-806. Epub 2005 Jun 29 PubMed.

Further Reading

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