White AR, Du T, Laughton KM, Volitakis I, Sharples RA, Xilinas ME, Hoke DE, Holsinger RM, Evin G, Cherny RA, Hill AF, Barnham KJ, Li QX, Bush AI, Masters CL. Degradation of the Alzheimer disease amyloid beta-peptide by metal-dependent up-regulation of metalloprotease activity. J Biol Chem. 2006 Jun 30;281(26):17670-80. PubMed.

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  1. The paper by White et al. demonstrates that administration of the metal ligand clioquinol with copper or zinc can induce an up-regulation of matrix metalloproteases 2 and 3 in vitro in Chinese hamster ovary and Neuro2A cells. This up-regulation leads to a reduction in secreted Aβ peptide. The work identifies a new mechanism of action for clioquinol and a potential strategy for promoting Aβ clearance, namely through metal-dependent increases in MMP activity. A beneficial effect of clioquinol in reducing plaque load has been previously demonstrated by the same group of investigators using a transgenic mouse model of AD (Cherny et al., 2001). Whether the mechanism identified in the current paper also occurs in the brain remains to be shown. The study nevertheless provides additional evidence supporting strategies designed to promote Aβ degradation and clearance in AD.

    References:

    Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA, Barnham KJ, Volitakis I, Fraser FW, Kim Y, Huang X, Goldstein LE, Moir RD, Lim JT, Beyreuther K, Zheng H, Tanzi RE, Masters CL, Bush AI. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron. 2001 Jun;30(3):665-76. PubMed.

    View all comments by Robert O. Messing

  2. Metalloproteases are increasingly recognized as important mediators of Aβ metabolism. Several of these proteases, including neprilysin (NEP), insulin-degrading enzyme (IDE), and endothelin-converting enzyme (ECE), have been convincingly shown to play a role in the catabolism of the Aβ peptide under basal conditions. In addition, some early work has also demonstrated that some of the enzymes may directly play a role in disease pathogenesis, accelerating pathology in knockout mice, while delaying plaque formation in overexpressing transgenic mice. While this makes for an attractive therapeutic target for Alzheimer disease, developing drugs that stimulate proteases is not a trivial task. In this paper, Choi et al. demonstrate that the overexpression of protein kinase C ε greatly reduces plaque pathogenesis in APPInd mice. Furthermore, they demonstrate a significant increase in ECE but not the other Aβ-degrading proteases in these transgenic mice, suggesting a potential mechanism for this effect. While the stimulation of PKCε is likely to have a multitude of other effects, given our greater understanding of this signal mediator, and pre-existing drugs that manipulate it, this kinase may make a more tangible therapeutic target for Alzheimer disease than would ECE.

    View all comments by Jin-Moo Lee

  3. This is an excellent study that not only demonstrates that PKC ε overexpression can result in fairly dramatic reductions in Aβ-associated pathology but that also provides further evidence for a critical role for endothelin converting enzyme (ECE) in modulating AD-like pathology in vivo. It will be interesting to further determine the specific ECE isoform affected by PKC ε and to determine the effect of PKC ε overexpression on behavioral phenotypes and other markers in this animal model. The development of small molecular weight activators of ECE activity may have similar effects and are actively being explored for their therapeutic potential.

    View all comments by Christopher Eckman

  4. Back in the good old days, when AD research was hyperfocused on Aβ production and aggregation, defining the mechanism of a given treatment that lowers net Aβ production was a relatively simple matter: Just check relative levels of Aβ precursor protein (APP), APP C-terminal fragments (APP-CTFs), and Aβ, and perhaps throw in a few in vitro aggregation experiments. These two papers, by showing that two established Aβ-lowering treatments act by enhancing Aβ degradation, show the errors of our past ways of thinking and offer a glimpse of the future.

    On the bright side, the sheer number of proteases implicated in Aβ degradation has dramatically increased the number of potential targets for therapeutic intervention. The sentiment expressed by an earlier commentator—that enhancing the activity of a protease is significantly more difficult than, say, blocking a secretase—was perhaps true when the list of Aβ proteases was limited to peptidases like neprilysin (NEP) and insulin-degrading enzyme (IDE). However, the latter are perhaps the exception among Aβ proteases. The activity of most proteases (plasmin and the matrix metalloproteases [MMPs] being just a couple of examples), is, in fact, exquisitely regulated by factors such as endogenous inhibitors, which can just as easily be targeted by small molecules. Scientists at Wyeth have provided the first example of the great promise of targeting protease inhibitors. At recent meetings, they have reported that inhibitors of plasminogen activator inhibitor-1, which inhibits the conversion of plasminogen to plasmin by tissue-type and urokinase-type plasminogen activator, can effectively reduce Aβ levels and reverse cognitive defects in APP transgenic mice. This early example shows that the future for AD therapies based on Aβ degradation is bright, and wide open.

    But there is some bad news with the good. Taking Aβ degradation into account raises the bar significantly in terms of defining the mechanistic basis of different treatments affecting net Aβ accumulation, as exemplified by the beautiful work of White et al. With something like 15 different proteases potentially contributing to net Aβ levels, and an unknown number that have not yet been identified, we can no longer focus simply on APP catabolites in defining mechanism. This underscores the great need for inhibitors with improved selectivity, which are particularly lacking for IDE, a point that is illustrated in the paper by White et al. To inhibit IDE, these investigators used bacitracin, a heterogeneous mixture of at least 10 cyclic dodecapeptides produced by certain strains of Bacillus subtilis. In our hands, bacitracin is not particularly potent, and it does get cleaved by IDE eventually, producing breakdown products that could inhibit other proteases non-specifically. It is also prone to batch-to-batch variability, and has biological effects other than protease inhibition. Nonetheless, bacitracin does seem to inhibit IDE reasonably specifically in short-term experiments in cells. In the experiments by White et al., bacitracin proved to be equally effective as an MMP inhibitor, yet surprisingly, they concluded that IDE was not affected based on the fact that IDE levels were unchanged. This is an odd conclusion, because changes in the levels of other proteases were not used as a criterion for the effectiveness of the corresponding inhibitors in this study. Moreover, the vast majority of IDE is present in the cytoplasm, and only a tiny minority of IDE would be expected to be in subcellular compartments relevant to Aβ degradation, so even if there were a change, it would probably be undetectable. And grinding cells or brains and looking at IDE activity would not reveal any changes occurring specifically in that tiny pool of IDE involved in Aβ degradation. Because of these issues, and because of the potential problems with bacitracin, we are left with a less-than-satisfying conclusion regarding IDE in both of the papers, at no real fault of the investigators, who simply lack the right pharmacological tools. Our lab has recently developed highly potent (low to subnanomolar IC50s) and selective IDE inhibitors, including both cell-penetrant and non-penetrant versions, which we plan to publish on soon, and we hope will aid in the molecular dissection of IDE’s function.

    In light of these two papers, and the fact that most AD research in the existing literature paid no heed to Aβ degradation, we are left to wonder whether all the conclusions are necessarily as sound as they seemed in the good old days when APP and the secretases reigned unchallenged. These new papers illustrate that it may behoove us to re-examine some of the conclusions we reached in the 1990s in the light of a more comprehensive paradigm that includes Aβ degradation.

    View all comments by Malcolm Leissring

  5. The proposal by White et al. that neuronal expression of the matrix metalloproteinases MMP2 and MMP3 is up-regulated by the metal-binding agent clioquinol (CQ) raises concerns.

    As reviewed elsewhere (1), various MMPs and notably MMP2 are up-regulated in response to injury and perturbation of neurons or other cells in various pathological settings. MMP2 and MMP3 degrade a range of extracellular matrix and other proteins. As part of an appropriately regulated “physiological” response to injury, this can be beneficial; however, MMPs are renowned for their aggressiveness and ability to cause substantial tissue damage if expression is not tightly controlled. In addition to hallmark roles in degrading the extracellular matrix, established or probable activities include altering cell adhesive properties, degrading neurotransmitter receptors, and remodeling synapses. One or more MMPs can also cause neurodegeneration and neuronal death.

    It also appears unlikely, based on the mechanisms proposed by the authors, that the reported effects of CQ would be restricted to MMP2 and MMP3 in vivo, since other cell types up-regulate other MMPs through MAPK pathways, as also reviewed elsewhere (1). The particular MMPs expressed in response to a stimulus depend on the cells being examined. The authors did not examine effects on primary astrocytes, microglia, diverse neuron types, etc.

    Indiscriminate metal chelation is also likely to exert diverse effects on other metal-dependent species in addition to the MMPs, such as the ADAMs. Except at high concentrations, metal binding agents don't usually strip ions from proteins, but they can bind ions within metalloproteins to form inactive complexes and redistribute free ions, affecting expression of metal-containing proteins (as proposed to occur by White et al.).

    However, CQ has been administered for periods up to 36 months in clinical trial patients without reported ill effects (2). Since significant long-term up-regulation of brain MMP and ADAM activity, etc., in response to CQ in these patients would result in considerable damage to brain tissue, it is improbable that CQ has the same effects in vivo over the long term that White et al. report in the short-term culture models.

    It is possible that the actual exposure of neurons to CQ in the brain is lower than in the in vitro system. Or perhaps other compensatory mechanisms involving the natural tissue inhibitors of MMPs (TIMPs) and other factors come into play in vivo to restore MMP and other metalloprotease activities to normal levels.

    Whatever the case, clearly there is considerable ignorance about the long-term effects of CQ in the brain, and it continues to be of concern that the studies of CQ have proceeded in patients before its actions have been adequately investigated in rodents or other non-human models.

    See also:

    For relevant reviews, see Matrix Metalloproteinases in the Central Nervous System. Conant K, Gottschall P (eds). Imperial College Press, London, 2005.

    References:

    Ritchie CW, Bush AI, Mackinnon A, Macfarlane S, Mastwyk M, MacGregor L, Kiers L, Cherny R, Li QX, Tammer A, Carrington D, Mavros C, Volitakis I, Xilinas M, Ames D, Davis S, Beyreuther K, Tanzi RE, Masters CL. Metal-protein attenuation with iodochlorhydroxyquin (clioquinol) targeting Abeta amyloid deposition and toxicity in Alzheimer disease: a pilot phase 2 clinical trial. Arch Neurol. 2003 Dec;60(12):1685-91. PubMed.

    View all comments by Liz Milward

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