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:
1. Taha TA, El-Alwani M, Hannun YA, Obeid LM. 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. Epub 2006 Oct 12. Abstract

2. Iribarren P, Chen K, Hu J, Gong W, Cho EH, Lockett S, Uranchimeg B, Wang JM. 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. Epub 2005 Oct 11. Abstract

3. Kaneider NC, Lindner J, Feistritzer C, Sturn DH, Mosheimer BA, Djanani AM, Wiedermann CJ. 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. Epub 2004 Jun 18. Abstract

View all comments by Mary Reid

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...  Read more

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:
1. Hook VY, Kindy M, Hook G. Inhibitors of cathepsin B improve memory and reduce Abeta in transgenic Alzheimer's Disease mice expressing the wild-type, but not the Swedish mutant, beta -secretase APP site. J Biol Chem. 2008 Feb 5; Abstract

2. Mueller-Steiner S, Zhou Y, Arai H, Roberson ED, Sun B, Chen J, Wang X, Yu G, Esposito L, Mucke L, Gan L. Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease. Neuron. 2006 Sep 21;51(6):703-14. Abstract

3. Schechter I, Ziv E. Kinetic properties of cathepsin D and BACE 1 indicate the need to search for additional beta-secretase candidate(s). Biol Chem. 2008 Jan 6; Abstract

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

5. 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. Abstract

6. 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 1;4(3):231-2. Abstract

7. 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 1;4(3):233-4. Abstract

View all comments by Greg Hook

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...  Read more

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.

View all comments by Robert Vassar

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...  Read more

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:
Hook G, Hook VY, Kindy M. Cysteine protease inhibitors reduce brain beta-amyloid and beta-secretase activity in vivo and are potential Alzheimer's disease therapeutics. Biol Chem. 2007a Sep;388(9):979-83. Abstract

Hook V, Kindy M, Hook G. Cysteine protease inhibitors effectively reduce in vivo levels of brain beta-amyloid related to Alzheimer's disease. Biol Chem. 2007b Feb;388(2):247-52. Abstract

Hook V, Toneff T, Bogyo M, Greenbaum D, Medzihradszky KF, Neveu J, Lane W, Hook G, Reisine T. 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. Abstract

Hook VY, Kindy M, Hook G. 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. Epub 2008 Jan 9. Abstract

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

Knight ZA, Shokat KM. Chemical genetics: where genetics and pharmacology meet. Cell. 2007 Feb 9;128(3):425-30. Abstract

Schechter I, Ziv E. Kinetic properties of cathepsin D and BACE1 indicate the need to search for additional beta-secretase candidate(s). Biol Chem. 2008 Mar;389(3):313-20. Abstract

View all comments by Greg Hook

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...  Read more

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:
Bogyo M, Verhelst S, Bellingard-Dubouchaud V, Toba S, Greenbaum D. Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs. Chem Biol. 2000 Jan;7(1):27-38. Abstract

Mihalik R, Imre G, Petak I, Szende B, Kopper L. Cathepsin B-independent abrogation of cell death by CA-074-OMe upstream of lysosomal breakdown. Cell Death Differ. 2004 Dec;11(12):1357-60. Abstract

Montaser M, Lalmanach G, Mach L. 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. Abstract

Mueller-Steiner S, Zhou Y, Arai H, Roberson ED, Sun B, Chen J, Wang X, Yu G, Esposito L, Mucke L, Gan L. Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease. Neuron. 2006 Sep 21;51(6):703-14. Abstract

Murata M, Miyashita S, Yokoo C, Tamai M, Hanada K, Hatayama K, Towatari T, Nikawa T, Katunuma N. Novel epoxysuccinyl peptides. Selective inhibitors of cathepsin B, in vitro. FEBS Lett. 1991 Mar 25;280(2):307-10. Abstract

Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, Rowan MJ, Selkoe DJ. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature. 2002 Apr 4;416(6880):535-9. Abstract

View all comments by Li Gan

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...  Read more

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.

View all comments by Greg Hook

The primary outcome data from the reaZin study appear to be consistent with the proposed action of the intervention. The three cognitive and functional measures used for the series of secondary outcomes are appropriate, but the small size of the sample means that the study was underpowered with respect to any clinical outcome measures. The small sample size was probably responsible also for the lack of balance in baseline measures across the randomized groups. Whether one should see the preliminary clinical outcome results as encouraging is a matter of judgment. The poster presentation does not make it clear whether the composite outcome was specified a priori. If not, the meaning of the p-value of 0.15 is hard to discern. In any event, I cannot agree with the authors' conclusion that these results provide a "strong trend toward cognitive benefit favoring the treatment group."

View all comments by John Breitner