Ridding the brain of toxic amyloid may require cutting off production of Aβ peptides, but another strategy that could work is to hasten the peptide’s demise. Several proteases have been shown to degrade Aβ in vivo, including neprilysin, insulin-degrading enzyme (IDE), and the metalloprotease endothelin converting enzyme (ECE). New work reveals that known plaque reducers, namely phorbol esters which activate protein kinase C and the metal chelator clioquinol (CQ), boost the activity of metalloproteases that degrade Aβ. One report, from Robert Messing and Lennart Mucke of the Gladstone Institute of Neurological Disease and University of California at San Francisco shows that overexpression of protein kinase Cε (PKCε) isoform decreases Aβ levels in mutant APP transgenic mice as a result of up-regulation of ECE in neurons. The second paper, from Ashley Bush and Colin Masters at the University of Melbourne in Australia, shows that CQ, which has anti-plaque activity in mice and has shown promising initial results in humans, can enhance extracellular Aβ degradation in cultured cells by increasing matrix metalloprotease activity. Both studies reveal novel pathways regulating Aβ proteases and open up new possibilities for the therapeutic enhancement of Aβ degradation.
In the first paper, which appears this week in the PNAS early edition, Messing and colleagues follow up on previous work showing that phorbol esters, potent activators of protein kinase C, suppress Aβ levels in cultured cells and in mouse brain. In vitro, the PKCε isoform seems to mediate this effect. To confirm if that is the case in vivo, the researchers, led by first author Doo-Sup Choi, generated transgenic mice that overexpressed human PKCε in brain by three- to fivefold, and then crossed the mice with Mucke’s H6 APP transgenic line, which carries the FAD Indiana APP mutant V707F. Double transgenics had fewer plaques as detected by thioflavin S staining and less immunoreactive Aβ. At 12-15 months of age, the PKCε-expressing animals had a 95 percent reduction in Aβ deposit area, with dramatic reductions in the extent of reactive astrocytosis and dystrophic neurites.
By several measures, APP processing was unchanged, so the researchers checked the activity of known Aβ degrading enzymes in brain. IDE and neprilysin activities were unchanged in the PKCε-APP mice, but levels of the zinc metalloprotease ECE were significantly elevated. In the hippocampus, ECE activity in PKCε/APP transgenics was roughly double that in the APP transgenic controls. To confirm that PKCε was regulating ECE, the investigators showed that phorbol ester treatment of cells transfected with PKCε boosted ECE activity.
While elevation of PKCε induced ECE and Aβ degradation, lack of PKCε did not affect levels of either protein. PKCε knockout mice maintain normal levels of ECE, and breeding those mice with the APP mice did not result in any change in plaque load. So while PKCε and ECE may not be necessary for normal regulation of Aβ, the results suggest that they might be enlisted as potential anti-amyloid agents by using strategies to increase their activity.
The second paper, which came out online April 28 as a paper in press at JBC, reports that the metal chelator clioquinol (CQ), currently in clinical testing for Alzheimer disease, can induce the degradation of Aβ in APP-expressing CHO cells. Senior investigators Bush and Masters have been investigating the mechanism of clioquinol since it was shown to reduce plaque load in APP mice (see ARF related news story). After that, a small clinical trial gave promising preliminary results in slowing cognitive decline in people with AD (see ARF related news story). Originally, the idea was that CQ, by sequestering zinc and copper ions and inhibiting them from binding to Aβ, would promote the solubilization and clearance of amyloid.
The new data challenge this idea. CQ is a lipophilic metal carrier, and lead author Anthony White shows that CQ increases metal levels in cells in vitro, and decreases their production of Aβ. Giving the cells CQ plus copper or zinc resulted in a 100-fold increase in the cellular content of the respective metals, and a 90 percent decrease in the amount of secreted Aβ peptides. The lower Aβ production was not caused by altered APP metabolism but instead was associated with a phosphoinositol-3-kinase (PI3K) and c-jun N-terminal kinase (JNK)-mediated up-regulation of the activity of two matrix metalloproteases, MMP2 and MMP3. Other MMP isoforms were not affected. Specific inhibitors of MMP2 and MMP3 blocked Aβ degradation in response to CQ-Cu. The researchers demonstrated that CQ-Cu complexes enhanced the degradation of secreted Aβ in neuronal cells by using the murine neuroblastoma cell line N2a, and human SY5Y cells with exogenously added Aβ.
The results could explain the observation by Bush in 2002 that CQ simultaneously increases CNS copper levels and lowers Aβ levels and plaque deposition in mice. If the ability of CQ-Cu to activate cell signaling pathways and boost Aβ degradation by metalloproteases can be demonstrated to occur in vivo, it will necessitate a new outlook on the role of metals and chelators like CQ in Aβ and plaque physiology.—Pat McCaffrey
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