Anyone with an ATM card knows how easy it is to drain a bank account when withdrawals outstrip deposits. The same applies to brain amyloid: even if production holds steady, boosting clearance can deplete toxic amyloid-β (Aβ). The discovery over the past several years of a host of proteases that degrade Aβ—the list includes neprilysin, insulin-degrading enzyme, endothelin-converting enzymes 1 and 2, plasmin, cathepsin B, and angiotensin-converting enzyme—led to the speculation that decreased activity of one or more Aβ proteases might cause some cases of Alzheimer disease. By extension, enhancing their activity would constitute a treatment for the disease.
Both ideas find support in a pair of papers looking at the effects of changing the activity of neprilysin, a major Aβ-degrading protease. In the first paper, out this week in PLoS Medicine, Dennis Selkoe at Boston’s Brigham and Women’s Hospital and Ole Isacson of McLean Hospital in Belmont, both in Massachusetts, take a gene therapy approach to delivering neprilysin to the brain in a mouse model of AD. The result is a dramatic clearance of amyloid plaques.
The second paper, in the July issue of the American Journal of Pathology, from Selkoe and Wesley Farris at the University of Pittsburgh School of Medicine in Pennsylvania, demonstrates the converse: when neprilysin activity is even partially decreased by gene knockout in human APP-expressing mice, the investigators find enhanced Aβ deposition. Decreases in neprilysin activity have been reported during aging and in some people with late-onset AD (Hellstrom-Lindahl et al., 2006), and the new results add evidence to the idea that diminished proteolytic clearance could cause or contribute to amyloid buildup in AD.
Farris and colleagues crossed J9 transgenic mice, which express human APP with two AD mutations, with neprilysin knockout mice. Mice missing one or both neprilysin genes showed elevated levels of whole brain and plasma Aβ peptides, with an increase in dimers and hippocampal plaque size and number. In the heterozygous knockouts, a 50 percent reduction in activity was sufficient to increase amyloid load fivefold. In these mice, the half-life of soluble brain Aβ was prolonged, indicating that its degradation was slowed. Homozygous animals lacking all neprilysin developed amyloid angiopathy in addition to plaques. The findings demonstrate an important role for proteolysis in setting the levels of Aβ, and suggest that the lower levels seen in some people with AD could contribute to amyloid load.
If decreased neprilysin causes AD, then increasing the activity of the enzyme should set things right. That is the idea behind the PLoS paper. First author Matthew Hemming engineered mouse fibroblasts to produce a soluble, secreted form of neprilysin and then implanted them into aged J20 mice that had already developed advanced amyloid pathology. When the animals were examined 28 days later, their plaques proved to have been cleared at the site of injection, as well as in surrounding tissue. Apparently, neprilysin diffused out from the cells in the graft and dissolved surrounding amyloid deposits.
“The results support the use of Aβ-degrading proteases as a means to therapeutically lower Aβ levels and encourage further exploration of ex vivo gene delivery for the treatment of AD,” the authors conclude. The idea of using fibroblasts to deliver a therapeutic protein to the brain is not new: a similar approach is in early testing in humans to deliver nerve growth factor to support cholinergic neurons (see ARF related news story).
Neprilysin is but one of several proteases implicated in Aβ accumulation, and any one could be a reasonable target for therapeutics, according to Christopher Eckman of the Mayo Clinic in Jacksonville, Florida. “Enhancing protease activity to lower Aβ is a viable approach,” Eckman says, “But it is not as much on people’s radar screens, because of the general belief that there will be side effects.” The reason for that belief is that none of the proteases are solely dedicated to Aβ. “Neprilysin, IDE, and ECE all degrade lots of proteins,” Eckman says, “so it is surprising that upregulation doesn’t have much effect on the animals. This gives us hope that this is a reasonable approach.”
The identification of multiple Aβ-degrading enzymes is good news, Eckman says. “This gives us a number of enzymes to play with. The proteases so far identified all have slightly different properties. The key to which one will succeed will not be its ability to degrade Aβ —they all do that. The question will be, do they have a minimal side effect profile?” he explained.
Historically, degradation has been the neglected side of the Aβ equation. Hundreds of labs have worked on the pathways that lead to Aβ production, including the β- and γ-secretases. Fewer have studied the proteases that degrade Aβ. That may be changing, as genetic evidence accumulates for the role of proteases in AD. A recent report showed decreased IDE activity in one AD family with linkage to the chromosomal region containing the IDE gene (Kim et al., 2007). Also, as the prospects for increasing protease activity with either gene therapy or allosteric small molecules improve, more companies are starting to get interested. The bottom line is that the proteases offer an alternative pathway to limit Aβ, and, as Eckman says, “The more things we have in our arsenal to decrease Aβ, the better.”—Pat McCaffrey
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