A lively platter of slide talks on APP processing and β-secretase (BACE) activities yielded a few interesting tidbits on the localization and regulation of β cleavage at this year’s annual meeting of the Society for Neuroscience in Atlanta, Georgia. The presentations also left the impression that cathepsin B, most recently in the news for digesting fibrillar Aβ (see ARF related news story), may yet have a role to play in the production of Aβ, as a β-secretase candidate protease.

Two talks focused on the localization and fluctuations of the BACE protein in brain. In the first, Matthew Kennedy and colleagues at Schering-Plough Research Institute, Kenilworth, New Jersey, described a radio-labeled small molecule BACE inhibitor that specifically binds BACE in mouse tissues—there was no binding to brains from BACE knockout mice. In brain sections of 6-week-old mice, they detected inhibitor binding throughout the CNS, with higher levels in the CA2/3 regions of the hippocampus and in the olfactory bulb. Using transgenic CRND8-APPswe/ind AD mice that show very early plaque deposition (10-12 weeks of age), they found that BACE levels increase after plaque deposition. In the mice, they saw a 50 percent increase in BACE inhibitor binding only after 24-30 weeks. They also saw a change in localization of the protein to high-staining foci in the cortex, which appear to colocalize with plaques.

The delay in upregulation of BACE is at odds with data presented by Jie Zhao in Robert Vassar’s lab on a new monoclonal antibody they developed to BACE. (See our ARF Madrid story for Vassar’s talk on the antibody, which the lab produced by immunizing BACE knockout mice.) Unlike all commercial antibodies tested, which either recognized multiple proteins on Western blots, or stained the brains of BACE knockout just as well as wild-type mice, the new antibody was monospecific for BACE. Zhao’s immunostaining results revealed increased BACE in the 5XFAD APP/S1 (see ARF related news story) and Tg2576 transgenic mice around amyloid plaques. The BACE elevation occurred in parallel with the rise in amyloid burden in 5XFAD mice. Increases were also seen in brain of human AD patients.

Consistent with the labeled inhibitor experiments of the Schering-Plough group, Zhao showed colocalization of BACE around amyloid plaques. Confocal views revealed BACE in an outer ring of Aβ40 that surrounded the inner, Aβ42-rich plaque. From these and other recent results, Vassar and colleagues hypothesize that energy insufficiency stress, caused, for example, by vascular hypoperfusion, induces an increase in BACE. The subsequent amyloid deposition may itself further activate BACE. Peri-plaque BACE was found in association with neuronal markers.

In a related presentation, Sarah Cole, also at Northwestern, showed some early results from crossing BACE knockouts with the 5XFAD APP/S1 strain. When Cole and colleagues looked at 4-month-old BACE-heterozygous mice, they saw a normalization of early markers of neuronal stress, namely p25 protein and phospho p38 MAP kinase. Until the mice get older, the researchers won’t know if the pathology is actually prevented, or just delayed.

Is BACE the One and Only β-secretase?
The observation that knocking out BACE1 shuts down the constitutive production of Aβ from cultured neurons and the accumulation of Aβ in FAD mouse models has been taken as proof that BACE1 is the predominant enzyme responsible for β cleavage of APP. But is BACE really the only β-secretase? Work presented by Vivian Hook, University of California, San Diego, made a strong case for cathepsin B as an additional β-secretase with a role to play in the activity-regulated secretion of Aβ.

Hook and colleagues have proposed previously that the majority of Aβ is released from cells via regulated secretion, as in the activity-dependent production and release of Aβ at synapses. According to their hypothesis, only a small amount of extracellular Aβ would derive from constitutive secretion, the BACE-dependent pathway through which Aβ makes its way into the conditioned medium of cultured neurons. The researchers, who have long studied the processing and release of neuropeptides, proposed this model based on their studies of adrenal chromaffin cells, which are widely used as a model of regulated secretion of neurotransmitters. In these cells, they have identified cathepsin B as the major β-secretase activity in secretory vesicles. BACE1 is present in the vesicles but accounts for only a small part of the total processing activity during KCl or nicotine-stimulated secretion. From these results, Hook and colleagues have proposed that cathepsin B could be the main β-secretase in neurons (Hook et al., 2002; Hook and Reisine, 2003; Hook et al., 2005).

One reason why cathepsin B has been overshadowed by BACE could be that, as Hook and colleagues demonstrated several years ago, the enzyme prefers to cleave wild-type APP, and displays much lower activity toward the altered site created by the Swedish mutation. The mutant sequence is preferred by BACE, and pops up in many of the FAD animal models which have been used to study the role of BACE in vivo. For studying β-cleavage inhibitors, Hook and coworkers favor the guinea pig model, which has a wild-type APP sequence that matches the β-secretase cleavage site in humans.

Hook used that animal to look at the role of cathepsin B in APP processing in the CNS. In her talk, she showed that infusion of selective cathepsin B inhibitor CA-074Me into the brains of guinea pigs reduced Aβ40 and Aβ42 levels by approximately one-half. In a second talk, Hook showed that another cathepsin B inhibitor, loxistatin, had the same effect after direct intracerebroventricular infusion. She saw decreased production of Aβ and the C-terminal β-secretase fragment of APP in the brain and in isolated synaptosomes. These results suggest that cathepsin B does participate in Aβ production in vivo as a β-secretase, and that inhibitors may have some potential as Aβ-lowering agents. This work is now in press in Biological Chemistry, Hook said.

A listener was quick to ask how Hook could reconcile her results with the recent paper from Li Gan showing that AD mice with the cathepsin B gene knocked out have worse plaque pathology than mice with the enzyme (see ARF related news story). In that study, the researchers found no evidence that cathepsin B acted as a secretase, but in fact found that it was able to degrade Aβ fibrils.

Hook pointed out that the AD mouse model used in that paper (the hAPP J20 line) incorporated the Swedish mutation, which could explain why the researchers saw no evidence for APP processing by cathepsin B. In animals with wild-type human APP, there could be a different outcome. Clearly, there is a lot more work to be done, but the data so far suggest that where Aβ production is concerned, BACE may be just one part of the story.—Pat McCaffrey.

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  1. Taha and colleagues (1) find that sphingosine kinase-1 is a substrate for cathepsin B. I note the studies by the Iribarren and Kaneider groups (2,3) and wonder about the effect of cathepsin B inhibitors on formyl peptide receptor-like-1/sphingosine kinase activity and whether this would alter amyloid-β1-42 uptake.

    References:

    . Sphingosine kinase-1 is cleaved by cathepsin B in vitro: identification of the initial cleavage sites for the protease. FEBS Lett. 2006 Nov 13;580(26):6047-54. PubMed.

    . CpG-containing oligodeoxynucleotide promotes microglial cell uptake of amyloid beta 1-42 peptide by up-regulating the expression of the G-protein- coupled receptor mFPR2. FASEB J. 2005 Dec;19(14):2032-4. PubMed.

    . The immune modulator FTY720 targets sphingosine-kinase-dependent migration of human monocytes in response to amyloid beta-protein and its precursor. FASEB J. 2004 Aug;18(11):1309-11. PubMed.

  2. Further to the discussion regarding cathepsin B β-secretase activity, our recent paper (1) shows that inhibitors of cathepsin B improve memory and reduce brain plaque, Aβ, and β-secretase activity in transgenic AD mice expressing human APP containing the wild-type, but not the Swedish mutant, β-secretase site. This result was expected based on the preference of cathepsin B to cleave a wild-type, but not Swedish mutant, β-secretase substrate.

    Cathepsin B's inability to efficiently cleave the Swedish mutant β-secretase substrate also predicted that knocking out cathepsin B in animals expressing APP containing that mutated site would have little or no effect on β-secretase activity of the mutated site, which is what Li Gan and colleagues found (2). Thus, our results are consistent with that finding.

    In contrast, BACE1 has such poor cleavage efficiency for the wild-type β-secretase site that Schechter and colleague postulate that there must be another β-secretase responsible for this cleavage (3). We suggest that cathepsin B has such β-secretase activity for the wild-type β-secretase site in the regulated secretory pathway.

    Others have speculated that BACE1 knockout data suggest that BACE1 is the primary if not only β-secretase (4), relying on BACE1 knockout data in support of this position (5-7). However, as we discuss in our recent paper, those data do not preclude cathepsin B β-secretase cleavage of the wild-type β-secretase site in the regulated secretory pathway (1).

    Thus, cathepsin B remains a viable alternative β-secretase candidate for cleavage of the wild-type β-secretase site, the form found in the vast majority of AD patients.

    References:

    . Inhibitors of cathepsin B improve memory and reduce beta-amyloid in transgenic Alzheimer disease mice expressing the wild-type, but not the Swedish mutant, beta-secretase site of the amyloid precursor protein. J Biol Chem. 2008 Mar 21;283(12):7745-53. PubMed.

    . Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease. Neuron. 2006 Sep 21;51(6):703-14. PubMed.

    . Kinetic properties of cathepsin D and BACE 1 indicate the need to search for additional beta-secretase candidate(s). Biol Chem. 2008 Mar;389(3):313-20. PubMed.

    . BACE1: the beta-secretase enzyme in Alzheimer's disease. J Mol Neurosci. 2004;23(1-2):105-14. PubMed.

    . 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.

    . 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.

    . BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci. 2001 Mar;4(3):233-4. PubMed.

  3. Reply to comment by Greg Hook
    I don't see solid evidence for the argument that BACE1 is not β- secretase. Dr. Hook claims that cathepsin B, not BACE1, is the β-secretase for wild-type APP. If so, then BACE1 knockouts mated to wild-type APP transgenics should still make Aβ and develop plaques. This experiment has, in fact, been done (McConlogue et al., 2007). The PDAPP mouse used in this study has the 717 London mutation, but it is wild-type at the β-secretase cleavage site, and the 717 mutation has no effect on β-secretase cleavage. McConlogue and colleagues definitively show by Aβ ELISA and immunohistochemistry that the PDAPP/BACE1 KO bigenics are devoid of Aβ and plaques. If cathepsin B were the β-secretase for wild-type APP, then there should have been no effect on Aβ and plaques in the PDAPP/BACE1 KO bigenics. I suspect that the cathepsin B inhibitors used in Hook et al., 2008 were not selective at the concentrations used and had pleiotrophic effects that altered Aβ production, clearance, or deposition by some other mechanism, as well. Knockout experiments are the cleanest way to resolve this issue.

    References:

    . 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.

    . Inhibitors of cathepsin B improve memory and reduce beta-amyloid in transgenic Alzheimer disease mice expressing the wild-type, but not the Swedish mutant, beta-secretase site of the amyloid precursor protein. J Biol Chem. 2008 Mar 21;283(12):7745-53. PubMed.

  4. I appreciate Dr. Vassar’s comment posted in response to my comment based on our new article, which supports the hypothesis that cathepsin B is a β-secretase in addition to BACE1 (Hook et al., 2008).

    Our main point is that cysteine protease inhibitors deserve further development as potential AD therapeutics because they are efficacious in two animal models relevant to AD. Significantly, they improve memory and reduce brain amyloid plaque in transgenic AD mice expressing human APP containing the wild-type β-secretase site, with reduction in brain Aβ and β-secretase activity (Hook et al., 2008). These results are supported by the effectiveness of these inhibitors to reduce brain Aβ and β-secretase activity in the normal guinea pig, which also expresses APP containing the wild-type β-secretase site (Hook et al., 2007b; Hook et al., 2007a; Hook et al., 2008). Since most AD patients have the wild-type β-secretase site, these compounds have the potential to translate into effective AD therapeutics.

    Our biochemical and pharmacological data lead us to believe that these small-molecule inhibitors act by inhibiting cathepsin B β-secretase cleavage of the wild-type β-secretase site in the regulated secretory pathway of neurons (Hook et al., 2005). Dr. Vassar disagrees based on transgenic BACE1 knockout animal experiments, which led him to conclude that BACE1 is the only β-secretase.

    Small-molecule inhibitor and gene knockout experiments are completely different, and the data obtained from each are known to often produce contradictory results (Knight and Shokat, 2007). For example, thiazolidinediones are effective type 2 diabetes drugs that enhance insulin sensitivity as PPARγ agonists, but genetic PPARγ deletion also improves insulin sensitivity. Thus, the therapeutic potential of thiazolidinediones could not be predicted by genetic analysis. So we respectfully disagree with Dr. Vassar’s assertion that knockout experiments are “the cleanest” way to resolve the mechanism by which these compounds act.

    He also asserts that the cysteine protease inhibitors are not selective in their action. To the contrary, the data show that these compounds are very selective as they have no effect in transgenic animals expressing human APP containing the Swedish mutant β-secretase site (Hook et al., 2008). A two amino acid change completely altered the drug response.

    A basic problem with the knockout mouse model is that it may not be entirely relevant to the human condition as mouse gene knockouts and the corresponding human genetic defects can have different phenotypes. For example, of nine tumor suppressor genes associated with human tumors, seven showed either different tumors or no tumors in rodents (Jacks, 1996; Schechter and Ziv, 2008).

    Nonetheless, it is our position that the BACE1 knockout mouse models have demonstrated that BACE1 has β-secretase activity for the wild-type β site. Our point is that BACE1 may not be the only β-secretase, that cathepsin B may be such an alternative β-secretase through which cysteine protease inhibitors may act, that cathepsin B may function in a different compartment than has been studied for BACE1, and therefore may have been missed in previous studies.

    The important issue to address is to find candidate therapeutic agents that can improve memory deficit for Alzheimer’s patients. Therefore, it is critical to consider all possible approaches, including the newly discovered cysteine protease inhibitors reported in our recent article (Hook et al., 2008).

    References:

    . Inhibitors of cathepsin B improve memory and reduce beta-amyloid in transgenic Alzheimer disease mice expressing the wild-type, but not the Swedish mutant, beta-secretase site of the amyloid precursor protein. J Biol Chem. 2008 Mar 21;283(12):7745-53. PubMed.

    . Cysteine protease inhibitors reduce brain beta-amyloid and beta-secretase activity in vivo and are potential Alzheimer's disease therapeutics. Biol Chem. 2007 Sep;388(9):979-83. PubMed.

    . Cysteine protease inhibitors effectively reduce in vivo levels of brain beta-amyloid related to Alzheimer's disease. Biol Chem. 2007 Feb;388(2):247-52. PubMed.

    . Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease. Biol Chem. 2005 Sep;386(9):931-40. PubMed.

    . Tumor suppressor gene mutations in mice. Annu Rev Genet. 1996;30:603-36. PubMed.

    . Chemical genetics: where genetics and pharmacology meet. Cell. 2007 Feb 9;128(3):425-30. PubMed.

    . Kinetic properties of cathepsin D and BACE 1 indicate the need to search for additional beta-secretase candidate(s). Biol Chem. 2008 Mar;389(3):313-20. PubMed.

  5. I agree that the roles of cysteine proteases in Aβ metabolism deserve in-depth investigation, and findings obtained with both genetic and pharmacological approaches should be evaluated in greater detail.

    In agreement with Dr. Vassar’s comments, the two inhibitors used in Dr. Hook’s study, CA074-Me and E-64c, are not specific for cathepsin B. E-64c is a pan-cysteine protease inhibitor, capable of inhibiting papain, cathepsins, and calpains. In contrast to CA-074 (Murata et al., 1991), which is highly specific for cathepsin B, its methyl ester derivative CA-074-Me completely lost its cathepsin B specificity (Bogyo et al., 2000), becoming capable of inhibiting cathepsin S (Bogyo et al., 2000), cathepsin L (Montaser et al., 2002), and several unidentified polypeptides (Bogyo et al., 2000). Indeed, CA-074-Me abrogated staurosporine-induced cell death in a cathepsin B-independent manner (Mihalik et al., 2004), possibly via reversing lysosomal acidification and mitochondria depolarization (Mihalik et al., 2004). This underscores the complexity of sorting out the mechanisms of any given compound.

    Carefully controlled genetic manipulations are indispensable for deciphering functions of a given protein, although caution should be taken for potential compensatory changes. Using genetic deletion and overexpression, we investigated the role of cathepsin B in Aβ metabolism. In contrast to the hypothesis that cathepsin B acts as β-secretase, our results showed that cathepsin B degrades Aβ (Mueller-Steiner et al., 2006). We assessed the effects of cathepsin B on levels of Aβ derived from either hAPP with the Swedish mutations (in J20 mice) or without (in primary neurons). In both cases, overexpression of cathepsin B reduces Aβ levels, whereas deletion of cathepsin B elevates levels of total Aβ, especially Aβ42 (Mueller-Steiner et al., 2006).

    In addition, cathepsin B overexpression in 7PA2 cells, which express hAPP lacking the Swedish mutations and secrete toxic Aβ oligomers (Walsh et al., 2002), led to profound reduction in Aβ oligomers without affecting levels of β-CTF (Sun B, Cisse M, and Gan L, unpublished observation). These studies provide direct evidence that cathepsin B does not induce β-site cleavage regardless of the presence or absence of the Swedish mutations.

    Due to the non-specific nature of the inhibitors and the lack of genetic evidence that cathepsin B has β-secretase activity, I agree with Dr. Vassar that in order to determine if the inhibitors truly act on cathepsin B, one needs to assess their efficacy (or lack thereof) in cathepsin B-null mice or neurons.

    References:

    . Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs. Chem Biol. 2000 Jan;7(1):27-38. PubMed.

    . Cathepsin B-independent abrogation of cell death by CA-074-OMe upstream of lysosomal breakdown. Cell Death Differ. 2004 Dec;11(12):1357-60. PubMed.

    . CA-074, but not its methyl ester CA-074Me, is a selective inhibitor of cathepsin B within living cells. Biol Chem. 2002 Jul-Aug;383(7-8):1305-8. PubMed.

    . Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease. Neuron. 2006 Sep 21;51(6):703-14. PubMed.

    . Novel epoxysuccinyl peptides. Selective inhibitors of cathepsin B, in vitro. FEBS Lett. 1991 Mar 25;280(2):307-10. PubMed.

    . Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature. 2002 Apr 4;416(6880):535-9. PubMed.

  6. In response to Drs. Vassar and Gan’s opinion that cathepsin B knockout experiments are the “cleanest way to resolve” if cathepsin B has a role in producing Aβ, I point out that we recently conducted such experiments and found that deleting the cathepsin B gene in transgenic mice expressing human wild-type APP results in about 70 percent and 40 percent less brain Aβ and CTFβ, respectively, than in their transgenic controls expressing cathepsin B (Hook et al., 2009). As most Alzheimer disease patients express wild-type APP, these knockout data suggest that cathepsin B has a role in producing Aβ in most Alzheimer disease patients.

    Furthermore, new data by others show that siRNA silencing cathepsin B reduces Aβ secretion by primary rat hippocampal neurons (Klein et al., 2009). Rat APP contains the human wild-type β-secretase site sequence, and thus these data are consistent with the cathepsin B gene knockout data discussed above.

    These new data, along with our previously discussed cathepsin B inhibitor data, suggest that cathepsin B has β-secretase activity for cleaving the wild-type β-secretase site and that cathepsin B inhibitors are potential Alzheimer disease therapeutics for reducing brain Aβ in most Alzheimer disease patients.

    References:

    . Genetic cathepsin B deficiency reduces beta-amyloid in transgenic mice expressing human wild-type amyloid precursor protein. Biochem Biophys Res Commun. 2009 Aug 21;386(2):284-8. PubMed.

    . Cathepsins B and L differentially regulate amyloid precursor protein processing. J Pharmacol Exp Ther. 2009 Mar;328(3):813-21. PubMed.

References

News Citations

  1. Role Reversal—AD Mouse Desperately Seeks CatB
  2. Madrid: BACE News Roundup, Part 3
  3. Paper Alert: Ramping Up FAD Mutations Puts Mouse Pathology in Overdrive

Paper Citations

  1. . Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles. J Neurochem. 2002 Apr;81(2):237-56. PubMed.
  2. . Cysteine proteases are the major beta-secretase in the regulated secretory pathway that provides most of the beta-amyloid in Alzheimer's disease: role of BACE 1 in the constitutive secretory pathway. J Neurosci Res. 2003 Nov 1;74(3):393-405. PubMed.
  3. . Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease. Biol Chem. 2005 Sep;386(9):931-40. PubMed.

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