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Blocking BACE—Do Adult Mouse Phenotypes Predict Side Effects?

Series - BACE Proteases in Health and Disease:

Despite Eli Lilly terminating development of its BACE inhibitor LY2886721 (see Jun 2013 news story), the protease remains a popular target among pharmaceutical companies looking for the next Alzheimer's disease therapy. While BACE inhibitors may reduce production of Aß and slow or halt progression of AD, could they also wreak havoc on the aging brain? Leaders in the field debated that point at BACE Proteases in Health and Disease, a three-day meeting organized by Stefan Lichtenthaler, Technical University of Munich, October 6–8 (see part one). While many agreed that developmental phenotypes of BACE deficiency may be less relevant to therapeutic success (see part two), most researchers at the meeting expressed uneasiness that blocking normal BACE functions in adults might have unexpected consequences.

For now, those functions center around Aß precursor protein, neuregulin, and the melanin scaffold PMEL, all established BACE substrates. Jochen Herms, Ludwig Maximilians University, Munich, previously found that inhibiting γ-secretase alters dendritic spine density in wild-type mice (see Bittner et al., 2009). At Seeon, Herms reported that BACE might be involved in the formation of new dendritic spines. Using two-photon microscopy, Herms and colleagues watched spine dynamics in living mice whose neurons express enhanced green fluorescent protein (eGFP). They saw that over a period of 16 days, 3-month-old eGFP mice treated with BACE inhibitors developed by Merck (SCH-1682496) and Eli Lilly (LY2811376) formed half as many new dendritic spines as controls, and lost 10 percent of their total spine number. The effects seemed reversible, because spine numbers slowly recovered during two weeks off the drug.  Herms concluded that blocking BACE1 affects spine plasticity more profoundly than spine density.

BACE knockout animals had fewer hippocampal dendritic spines at 16 days of age, but by 32 days were undistinguishable from wild-type, suggesting that the animals may compensate for the absence of BACE1. BACE inhibitors caused no further loss of spines in BACE1 knockouts, but they did reduce spine numbers in wild-type animals. The data confirm that the compounds reduce spine numbers by binding to BACE, rather than some other target, said Herms. Do these spine losses translate into functional deficits? Herms found that BACE inhibitors reduced spontaneous and evoked neurotransmission in mice. In response to questions, Herms said he was unsure how the spine suppression might affect people with Alzheimer’s.

Another defect that raised concern was one reported on last July (see Jul 2013 news story). Carmen Birchmeier, Max Delbrück Center for Molecular Medicine, Berlin, and colleagues found that knocking out BACE1, or blocking it pharmacologically in adult mice, interferes with muscle spindles. Comprising muscle fibers and sensory neurons, spindles intersperse among the skeletal muscles and convey information about muscle tension to the brain, allowing it to coordinate movement. Birchmeier and colleagues found that BACE cleavage of the Ig-containing ß1 isoform of neuregulin supports spindle formation, and that in BACE1-negative mice spindles lose their shape and motor control is compromised. Researchers at the meeting were curious whether this effect can be reversed and what type of dose response underlies it. Birchmeier said she has yet to look at multiple doses; she used the same dose of LY2811376 that Eli Lilly used to reduce Aß production in mice. Some of the spindle deficits seem to be long-lasting, suggesting there could be some irreversible damage, Birchmeier cautioned that more work needs to be done before this is known. 

Researchers also debated the importance of pigmentation loss in adult BACE knockouts. Guillaume van Niel, Institut Curie, Paris, and colleagues reported last July that BACE2 processes the melanocyte protein PMEL, which forms a functional amyloid that binds melanin (see Rochin et al., 2013). Pigmentation failure gives BACE2-deficient animals a silvery coat, while BACE1 knockouts animals are the same color as wild-type, van Niel said. Philip Wong, Johns Hopkins University, Baltimore, reported slightly different results. In his hands the pigmentation phenotype in BACE2 knockouts was less severe. Wong said that to the naked eye the knockouts looked no different than wild-type, and only close examination revealed a subtle loss of pigmentation. "I think the key difference is the design of the animals," said Wong. Van Niel and colleagues studied a mouse engineered by researchers at Bart De Strooper's lab at KU Leuven, Belgium. That strain makes a catalytically inactive BACE2, whereas Wong's group knocked out the full gene and their mouse makes no BACE2 at all. "I suspect that because BACE2 is still there, albeit inactive, it acts as a dominant negative and prevents compensation by BACE1 in the mouse from De Strooper's lab," said Wong. The dominant negative may better mimic what happens when people are treated with non-selective BACE inhibitors, agreed Wong, because in that case the proteins are still present, though inactive.

Attendees debated what pigmentation effects might look like in people, and whether they might jeopardize BACE therapeutics. Most agreed the likely effect would be lightened skin color. "I would trade that for Alzheimer's anytime," said Wong. Van Niel suggested that potential sensitivity to melanoma, a more serious side effect, warrants further investigation.

On the other side of the coin, could there be some benefits to BACE inhibition besides reducing amyloidogenic processing of APP? Markus Stoffel, ETH Zurich, Switzerland, summarized recent findings from his lab that suggest BACE2 limits proliferation of insulin-producing ß cells in the Islet of Langerhans in the pancreas. BACE2 cleaves a transmembrane protein called TMEM27. Mice modeling maturity-onset diabetes of the young—a rare, inherited form of type 2 diabetes—downregulate this protein, while those overexpressing TMEM27 develop bigger ß cells, produce more insulin, and better control plasma glucose levels. Stoffel's group found that TMEM27 sheds an extracellular domain that likely stimulates tissue growth. In an RNAi screen to identify the sheddase that releases this domain, Stoffel found BACE2. In addition, treating obese mice with a BACE inhibitor stimulated insulin release from the pancreas and improved glucose tolerance.

Researchers agreed that the roles both BACE isoforms play in adult mice and in people are far from fully understood. Answers to some outstanding questions may come from conditionally knocking out BACE1 in adult animals. At least five different groups said that they are presently doing so. "That was fantastic to hear," said Lichtenthaler. Two years ago it seemed that nobody was having success at creating such animals. “They will be important to distinguish adult from developmental phenotypes and help us identify substrates and pathways that rely on BACE in the adult," said Lichtenthaler.—Tom Fagan

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References

News Citations

  1. Lilly Halts Phase 2 Trial of BACE Inhibitor Due to Liver Toxicity
  2. Cloistered Retreat Takes the Pulse of BACE Research
  3. BACE—Substrates, Functions, Developmental Phenotypes
  4. Paper Alert: BACE1 Required for Muscle Spindle, Motor Control

Paper Citations

  1. Bittner T, Fuhrmann M, Burgold S, Jung CK, Volbracht C, Steiner H, Mitteregger G, Kretzschmar HA, Haass C, Herms J. Gamma-secretase inhibition reduces spine density in vivo via an amyloid precursor protein-dependent pathway. J Neurosci. 2009 Aug 19;29(33):10405-9. PubMed.
  2. Rochin L, Hurbain I, Serneels L, Fort C, Watt B, Leblanc P, Marks MS, De Strooper B, Raposo G, van Niel G. BACE2 processes PMEL to form the melanosome amyloid matrix in pigment cells. Proc Natl Acad Sci U S A. 2013 Jun 25;110(26):10658-63. PubMed.

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