. PKCepsilon increases endothelin converting enzyme activity and reduces amyloid plaque pathology in transgenic mice. Proc Natl Acad Sci U S A. 2006 May 23;103(21):8215-20. PubMed.

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  1. Along with neprilysin (NEP) and insulin-degrading enzyme (IDE), another metalloprotease endothelin-converting enzyme (ECE) has also been shown to degrade Aβ in both in vitro cell culture and in vivo animal models. The paper by Choi et al. crossed transgenic mice that overexpress human PKC ε isoform with APPind (V717F) mutant transgenic mice and showed a clear reduction of amyloid plaque pathology.

    The authors observed a significant decrease in Aβ levels over a 12-18-month period (with a minimal decrease in 1-3 months), but no changes in sAPPα levels. Furthermore, they found an approximately 30-40 percent increase of ECE, but not NEP or IDE, activity in cortex and a more significant twofold increase in hippocampus in the double transgenic mice, as well as a ~30 percent increase in hy926 cells overexpressing PKCε, interestingly after phorbol treatment. The authors therefore concluded that PKCε reduces Aβ by increasing ECE activity without affecting APP processing. This result, to certain extents, supports an earlier observation by Savage et al. that acute (6-12-hour) intracortical injection of phorbol esters (PMA) to APPswe Tg mouse brain reduces the levels of both Aβ and sAPPβ without significant changes in sAPPα levels.

    For more than a decade, activation of protein kinase C has been shown independently and unequivocally, by numerous laboratories including but not limited to S. Gandy, J. Buxbamn, P. Greengard, C. Haass, E Koo, D. Selkoe, V.M. Lee, M. Racchi, F. Checler, V. Bigl, I Mook-Jung, and R. Messing’s own group, to stimulate the secretion of sAPPα. The mechanisms by which PKC increases sAPPα may include direct activation of putative α-secretase TACE/ADAM and “indirect” acceleration of APP trafficking to a compartment where α-secretase is highly active. While shrouded in some controversy, the effect of PKC activation on reducing Aβ has also been widely observed.

    The observation by Choi et al. is extremely intriguing, and may provide yet another novel mechanism by which PKC, specifically the ε isoform, increases ECE activity and hence promotes Aβ degradation. How PKC, especially its various isoforms, can indeed affect the proteolytic processing of APP remains to be carefully deliberated and clarified. One of the “easiest” explanations to reconcile this issue would be that different PKC isoforms may play different roles in regulating APP/Aβ metabolism/catabolism. Lanni and colleagues showed in 2004 that PKCε’s stimulatory effect on sAPPα is coupled to cholinergic pathways, while the involvement of the α-isoform in the cholinergic receptor-mediated regulation of APP processing is negligible. It is possible that the ability to increase ECE activity is specific to the ε isoform, a Ca2+-independent isoform (whereas the conventional α-, β, and γ-, isoforms are Ca2+-dependent). In this paper, the authors only measured the steady-state level of sAPPα in the total brain homogenate but didn’t assess the processing of APP (i.e., the production/secretion of sAPPα) in primary neurons (where the PKCε was specifically overexpressed under the Thy1.2 promoter) isolated from these mice. Since the bulk of the brain is non-neuronal cells—astrocytes, fibroblasts, etc., which are known to produce high or even higher levels of sAPPα—it is possible that any neuronal increases in sAPPα production was masked. While the increased ECE activity was convincingly shown in the paper, it remains undefined how PKCε activates ECE—can PKCε directly phosphorylate ECE or alter its trafficking and hence increase its activity? In addition, since recent in vitro and in vivo studies have demonstrated that NEP and/or IDE are responsible for the vast majority, if not all, of Aβ degradation in the brain, it is surprising to see such a drastic Aβ degradation (about 80 percent, in 12-18-month-old animals) in the absence of increased NEP/IDE activity.

    To validate the reduced Aβ levels in these animals, it would be helpful to show whether brain homogenates from the PKCε transgenic can break down Aβ peptides more efficiently than those from control mice. The pathophysiological relevance of ECE-mediated Aβ degradation would be much strengthened by future discovery of decreased activity of this enzyme due to genetic mutation, age- or disease-related alterations in gene expression, or proteolytic activity in association with increased risk for AD.

    References:

    . Turnover of amyloid beta-protein in mouse brain and acute reduction of its level by phorbol ester. J Neurosci. 1998 Mar 1;18(5):1743-52. PubMed.

    . Differential involvement of protein kinase C alpha and epsilon in the regulated secretion of soluble amyloid precursor protein. Eur J Biochem. 2004 Jul;271(14):3068-75. PubMed.

  2. In their recent PNAS manuscript, Choi et al. present some very interesting findings, further suggesting a role for endothelin converting enzyme (ECE) in regulating the levels of Aβ peptides in the central nervous system. ECE is one of several potential Aβ-degrading enzymes that have been identified in brain, with other prominent candidates including neprilysin and insulin-degrading enzyme. In their present study, Choi et al. found that mice doubly transgenic for increased expression of protein kinase C ε (PKCε) and human FAD mutant AβPP exhibit decreased Aβ levels, plaque loads, and astrocytosis compared with the single transgenic human FAD mutant AβPP mice. No effects were found on AβPP levels or AβPP processing suggesting that enhanced clearance may be responsible for the reduced Aβ burden. Examination of the above-mentioned Aβ-degrading enzymes found no differences in neprilysin or insulin-degrading enzyme activities but a significant increase in ECE activity. Of note, deletion of PKCε in gene knockout mice had no effect on Aβ burden in the human FAD mutant AβPP transgenic mice. This implies that PKCε has no effect on the basal levels of ECE activity in brain.

    These findings further add to the idea that after initial production, the accumulation of Aβ in the central nervous system is largely due to inadequate clearance of the peptides from this compartment. Clearance likely involves several complementary mechanisms, including peptide degradation and transport out of the brain into the periphery. Other studies with human AβPP transgenic mouse models have similarly suggested roles for neprilysin and insulin-degrading enzyme in facilitating Aβ clearance from brain. Combined, the findings suggest that each of these mentioned, as well as other potential Aβ-degrading enzymes, contribute to maintaining Aβ levels in the central nervous system. Each may act in specific compartments within the brain or under certain circumstances, such as implicated for ECE with PKCε stimulation as shown here.

    As with any study like this, the next level of questions regards the overall physiological significance of the observations on pathological accumulation of Aβ in brain. For example, in what compartment and in what cell types does PKCε stimulate ECE expression and activity? Are other potential Aβ-degrading enzymes stimulated by PKCε? More importantly, are other Aβ clearance mechanisms enhanced by elevated PKCε expression? In this regard, it will be interesting to investigate if there is increased transport of Aβ out of the brain across the blood-brain barrier into the peripheral circulation when there is increased expression of PKCε in brain. This final point is significant since a primary role of ECE is to activate endothelin, which exerts its vasoconstrictive function at cerebral blood vessels. A first step in exploring this hypothesis would be to determine the plasma levels of human Aβ in the double transgenic mice. In any event, the work of Choi et al. further supports a role for ECE as a likely important cog on the complex wheel of Aβ clearance.

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