Not the Usual Suspects: Tracking BACE Inhibition, Axon Role

BACE1 has been a tough nut to crack. This β-secretase enzyme starts the process of cleaving APP into neurotoxic Aβ, making it an attractive target for Alzheimer’s disease therapeutics, and yet companies have struggled for 10 years to develop effective inhibitors (see, e.g., ARF related news story). One of the challenges is figuring out if drugs are having the desired effect in the brain. In the February 6 PLoS One, researchers led by Niklas Mattsson and Erik Portelius at the University of Gothenburg, Mölndal, Sweden, report that inhibiting the enzyme creates a distinctive signature of Aβ peptides in the cerebrospinal fluid (CSF). This CSF fingerprint could be a useful tool for tracking the efficacy of inhibitors in clinical trials, the authors suggest. In less positive news, scientists led by Robert Vassar at Northwestern University, Chicago, Illinois, recently uncovered a novel role for BACE in axon guidance, raising a possible caution for trials of BACE1 inhibitors. The data appeared in the December 28 Molecular Neurodegeneration.

Mattsson and colleagues looked for signs of BACE1 inhibition in several different cell lines, some of which expressed wild-type human APP, some human APP with the Swedish mutation, and some another form of mutant human APP (APP751 with the V717F familial mutation). The authors treated the cultures with two different BACE1 inhibitors: the commercially available BACE1 inhibitor IV and, in collaboration with pharmaceutical company AstraZeneca, Södertälje, Sweden, the inhibitor AZ-20. Normally, BACE1 cuts APP at position 1 of the Aβ peptide, and γ-secretase then cleaves the protein somewhere around position 40-49 to create the common Aβ fragments. As expected, with BACE1 inhibition, levels of Aβ40 and Aβ42, as well as those of a host of other Aβ peptides, dropped in the cells. Notably, in most assays the relative decline in the minor peptide Aβ1-34 was much greater than that of the other peptides. Aβ1-34 is cut at both ends by BACE1, Mattsson said, which may make it particularly vulnerable to the lack of the enzyme. The results were consistent across cell lines and treatments.

Conversely, BACE1 inhibition strikingly and consistently increased levels of the rare Aβ5-40 in all cell lines. This suggests the existence of another enzyme, unmasked by BACE1 inhibition, that cuts at position 5, although its identity remains a mystery. Some previous studies have found Aβ5-40 in amyloid plaques in human AD brains (see Takeda et al., 2004; Portelius et al., 2010). Further research on this rare peptide may yield more insights into APP processing, Mattsson suggested, adding that it is not yet known if it plays a role in AD.

To study BACE1 inhibition in vivo, the authors used dogs. Canine Aβ is identical to human, and dogs release similar Aβ cleavage products into their CSF (see Portelius et al., 2010). The researchers collaborated with pharmaceutical company Novartis in Basel, Switzerland, to treat the animals with two experimental BACE1 inhibitors. A separate group of dogs received BACE1-inhibitor S from collaborators at Janssen Research and Development, Beerse, Belgium. In agreement with the cell culture results, levels of Aβ5-40 soared in the CSF, while most other Aβ peptides dropped, with the decline in Aβ1-34 particularly steep. Overall, the authors found that an increased ratio of CSF Aβ5-40/Aβ1-34 is a more sensitive marker of BACE1 inhibition than are declines in Aβ40 or Aβ42, and can completely separate placebo and treatment groups.

“The reported data are amazing, and demonstrate that simplistic views of proteolytic processing of APP are all but untenable,” Fred Van Leuven at KULeuven, Belgium, wrote to ARF. For his part, Mattsson said the data further reinforce that it is worth looking at peptides other than Aβ40 and Aβ42.

The paper by Vassar and colleagues focused on potential side effects of BACE1 inhibition. The enzyme has other substrates besides APP, and recent studies have turned up several axon guidance molecules as possible targets (see ARF related news story on Hemming et al., 2009). To investigate further, first author Tharinda Rajapaksha examined the axons of olfactory sensory neurons in BACE1 knockout mice. Each axon expresses a particular odorant receptor (out of a suite of around 1,000), and needs to hook up to a specific, corresponding glomerulus in the olfactory bulb for odors to be relayed correctly to the cortex. Rajapaksha and colleagues found that olfactory bulb glomeruli in the knockouts were misshapen and smaller than those in wild-type mice, hinting that many olfactory axons failed to reach their targets. Using fluorescent tags, the authors looked at the behavior of axons containing specific odorant receptors and discovered that at least some of these hooked up to the wrong glomeruli. This kind of miswiring might affect the mouse’s ability to smell that particular odor, Vassar noted, adding that one of the next steps will be to test this.

Vassar plans to look at whether BACE1 aids axon guidance in other neuronal cell types as well. He also wants to find the BACE1 substrates involved and dissect the molecular mechanisms. It is not yet clear how the defects in knockout mice might apply to BACE1 inhibitor therapy in adult humans. However, Vassar pointed out that some neuronal populations, such as olfactory and hippocampal neurons, regenerate throughout life, implying new wiring could be affected by BACE1 inhibition. Vassar told ARF he remains enthusiastic about BACE1 inhibition as a therapeutic strategy, but he also believes scientists should proceed with caution. “The drug companies need to take this [the data] into consideration in their clinical trials and be on the lookout for these types of problems,” he suggested.

Johan Lundkvist at AstraZeneca, who is a coauthor on the PLoS One paper, notes that Vassar’s paper provides “novel and important insights,” but more data are needed on how it might translate to trials of BACE1 inhibitors. “We have learned that only a limited decrease in BACE activity (BACE+/- mice) is sufficient to affect the development of amyloid pathology,” Lundkvist wrote to ARF. “It will therefore be interesting to learn…whether small molecule-mediated inhibition of BACE, at doses efficacious in lowering Aβ production, has any impact on the sensory neuronal network of the olfactory system.” (See full comment below.)—Madolyn Bowman Rogers

Comments

  1. This study by Rajapaksha et al. provides novel, very interesting, and important insights to the role of BACE in Neurobiology. Rajapaksha and coauthors applied an elegant genetic approach to explore the role of BACE in the olfactory system. Analysis of BACE-/- mice revealed smaller olfactory bulbs and mistargeted olfactory sensory neurons (OSNs), suggesting that BACE is involved in the process of axonal guidance of OSNs. The data are intriguing and, as the authors state in the paper, it will be interesting to learn whether the neuronal phenotype also impacts olfactory function, whether BACE plays a similar role in other neuronal subtypes, and what are the substrates/signaling pathways involved that cause the phenotype. From other BACE gene targeting studies (1), we have learned that only a limited decrease in BACE activity (BACE+/- mice) is sufficient to affect the development of amyloid pathology. It will therefore be interesting to learn whether the OSN phenotype is also present in BACE+/- mice and, from a drug discovery perspective, whether small molecule-mediated inhibition of BACE, at doses efficacious in lowering Aβ production, has any impact on the sensory neuronal network of the olfactory system.

    References:

    McConlogue L, Buttini M, Anderson JP, Brigham EF, Chen KS, Freedman SB, Games D, Johnson-Wood K, Lee M, Zeller M, Liu W, Motter R, Sinha S. Partial reduction of BACE1 has dramatic effects on Alzheimer plaque and synaptic pathology in APP Transgenic Mice. J Biol Chem. 2007 Sep 7;282(36):26326-34. PubMed.

    View all comments by Johan Lundkvist
    • User Profile Image Vivian Hook
      University of California, San Diego
    • Posted:

    This article by Mattsson et al. is authored by an outstanding group, which investigated the effects of BACE1 inhibitors on Aβ peptide forms in media secreted from cultured neurons and in CSF after in-vivo treatment of dogs. Interestingly, the data suggest that the BACE1 inhibitors result in an altered Aβ peptide pattern, with an increase in the ratio of Aβ5-40:Aβ1-34. However, several aspects of the study should be carefully considered. Firstly, the wide range of dog ages, and the few number of dogs per group, make conclusions from the data difficult, since it is known that Aβ levels in CSF differ depending on the age of the dog (Head et al., 2010). Secondly, because a cocktail of protease inhibitors was not included in sample preparations, it is not known if the profiles of Aβ peptides observed are from the endogenous condition or if they became degraded during the in-vitro methods. Thirdly, use of standards for quantitative mass spectrometry is typical in the field, but standards were not evident in this study. Fourthly, the cell culture data reflect only Aβ produced in the constitutive, and not regulated, secretory pathway; since a major portion of Aβ is released from the regulated secretory pathway, which utilizes cathepsin B for Aβ production (Klein et al., 2009), nothing can be inferred regarding the role of cathepsin B in producing Aβ from the study’s finding that an inhibitor of cathepsin B had no effect on constitutive Aβ secretion. Finally, the authors should indicate whether they, or other groups, have data indicating that BACE1 inhibitors improve memory deficits, the critical goal of Alzheimer’s therapeutics. It will be of interest for the authors to continue these studies to examine these important features of Aβ research.

    References:

    Head E, Pop V, Sarsoza F, Kayed R, Beckett TL, Studzinski CM, Tomic JL, Glabe CG, Murphy MP. Amyloid-beta peptide and oligomers in the brain and cerebrospinal fluid of aged canines. J Alzheimers Dis. 2010;20(2):637-46. PubMed.

    Klein DM, Felsenstein KM, Brenneman DE. Cathepsins B and L differentially regulate amyloid precursor protein processing. J Pharmacol Exp Ther. 2009 Mar;328(3):813-21. PubMed.

    View all comments by Vivian Hook

  3. The study by Mattsson et al. provides clear and important evidence that specific generation of Aβ5-x is a general phenomenon under the condition of BACE1 inhibition. The authors emphasize that the Aβ pattern may be useful in monitoring the in-vivo effects of BACE1 inhibitors. However, it remains an open question by what mechanism Aβ5-x is produced.

    In our previous studies (Takeda et al., 2004; Murayama et al., 2007), we demonstrated that neuroblastoma SH-SY5Y cells expressing the caspase-cleaved form of amyloid precursor protein (APP) lacking the C-terminal 31 amino acids preferentially produce Aβ5-40/42. We showed that treatment of the cells with TAPI-I, which can inhibit α-secretase, decreases Aβ5-40 and increases Aβ1-40, suggesting that α-secretase-like proteases are involved in the generation of Aβ5-40/42. We additionally observed that treatment of SH-SY5Y cells expressing wild-type APP with a BACE1 inhibitor, OM99-2, decreased Aβ1-40 with an increase of Aβ5-40, consistent with the results by Mattsson et al. Therefore, inhibition of BACE1 may lead to the distinct processing of APP between Phe4 and Arg5, possibly mediated by α-secretase-like proteases.

    Although it is not clear whether Aβ5-x has a pathological role, our previous data indicate that Aβ5-x is particularly deposited in intermediate vessels with amyloid angiopathy in Alzheimer’s disease (AD) brains. The findings by Mattsson et al. imply that clinical application of BACE1 inhibitors to AD patients could induce the generation of Aβ5-x in the brain, which might influence the pathological processes. Thus, the pathophysiological property of this N-terminally truncated Aβ species needs further clarification.

    References:

    Takeda K, Araki W, Akiyama H, Tabira T. Amino-truncated amyloid beta-peptide (Abeta5-40/42) produced from caspase-cleaved amyloid precursor protein is deposited in Alzheimer's disease brain. FASEB J. 2004 Nov;18(14):1755-7. PubMed.

    Murayama KS, Kametani F, Tabira T, Araki W. A novel monoclonal antibody specific for the amino-truncated beta-amyloid Abeta5-40/42 produced from caspase-cleaved amyloid precursor protein. J Neurosci Methods. 2007 Apr 15;161(2):244-9. PubMed.

    View all comments by Wataru Araki

  4. BACE1 inhibitor developers should take note of this paper by Rajapaksha et al. It is a novel finding that raises valid questions about potential BACE1 inhibitor drugs. Rajapaksha and colleagues looked only into the olfactory system. It's a good start that provided very interesting results; however, investigators should look into effects of BACE1 in more critical areas of the brain.

    View all comments by Cindy Zhu

Make a Comment

To make a comment you must login or register.

References

News Citations

  1. Barcelona: Out of Left Field—Hit to The Eye Kills BACE Inhibitor
  2. Dual Secretase Approach Shows Promise in AD Mice

Paper Citations

  1. Takeda K, Araki W, Akiyama H, Tabira T. Amino-truncated amyloid beta-peptide (Abeta5-40/42) produced from caspase-cleaved amyloid precursor protein is deposited in Alzheimer's disease brain. FASEB J. 2004 Nov;18(14):1755-7. PubMed.
  2. Portelius E, Bogdanovic N, Gustavsson MK, Volkmann I, Brinkmalm G, Zetterberg H, Winblad B, Blennow K. Mass spectrometric characterization of brain amyloid beta isoform signatures in familial and sporadic Alzheimer's disease. Acta Neuropathol. 2010 Aug;120(2):185-93. PubMed.
  3. Portelius E, Van Broeck B, Andreasson U, Gustavsson MK, Mercken M, Zetterberg H, Borghys H, Blennow K. Acute effect on the Aβ isoform pattern in CSF in response to γ-secretase modulator and inhibitor treatment in dogs. J Alzheimers Dis. 2010;21(3):1005-12. PubMed.
  4. Hemming ML, Elias JE, Gygi SP, Selkoe DJ. Identification of beta-secretase (BACE1) substrates using quantitative proteomics. PLoS One. 2009;4(12):e8477. PubMed.

Further Reading

Primary Papers

  1. Rajapaksha TW, Eimer WA, Bozza TC, Vassar R. The Alzheimer's β-secretase enzyme BACE1 is required for accurate axon guidance of olfactory sensory neurons and normal glomerulus formation in the olfactory bulb. Mol Neurodegener. 2011;6:88. PubMed.
  2. Mattsson N, Rajendran L, Zetterberg H, Gustavsson M, Andreasson U, Olsson M, Brinkmalm G, Lundkvist J, Jacobson LH, Perrot L, Neumann U, Borghys H, Mercken M, Dhuyvetter D, Jeppsson F, Blennow K, Portelius E. BACE1 inhibition induces a specific cerebrospinal fluid β-amyloid pattern that identifies drug effects in the central nervous system. PLoS One. 2012;7(2):e31084. PubMed.

Annotate

To make an annotation you must Login or Register.