By David N. Leff

A new player has pulled up a chair in the cutthroat card game of Alzheimer¿s disease (AD). It¿s a natural enzyme that goes by the name of neprilysin ¿ alias neutral zinc metalloendopeptidase.

¿What neprilysin does in the mammalian body,¿ observed cell and molecular biologist Norma Gerard, at Harvard Medical School in Boston, ¿is degrade relatively small, often insoluble peptides into smaller, soluble dipeptides. I was just looking through a literature search of Alzheimer¿s disease some years ago,¿ Gerard recalled, ¿and came up with a number of reports not of neprilysin specifically but of antagonists to that enzyme as inhibiting the degradation of toxic beta amyloid.¿

This stubby, 42-amino-acid, denatured protein, Ab for short, is the active rogue molecule of AD¿s two hallmarks ¿ senile neuritic plaques wrapped around the disease¿s dying neurons, and tangles poisoning those brain cells from within.

Norma Gerard and her husband, cell and molecular biologist Craig Gerard, are co-authors of a paper in today¿s Science, dated May 25, 2001, which bears the title: ¿Metabolic regulation of brain Ab by neprilysin.¿ Seven of its eight other authors are scientists at the RIKEN Brain Science Institute in Wako-shi, Japan.

¿What we found in this study,¿ Norma Gerard told BioWorld Today, ¿is that knockout mice with a targeted deletion of the neprilysin gene accumulate more of the toxic beta amyloid that forms those insoluble plaques and tangles. Wild-type mice, equipped with normal amounts of the enzyme, show no such amyloid-beta buildups. We also showed ¿ which is novel ¿ that neprilysin is responsible for degrading the amyloid to smaller peptides that are nontoxic, soluble, and can be easily cleared from the brain.¿

Therapeutic Outlook Relies On Enzyme Levels

¿This suggests possibilities down the line for therapy of Alzheimer¿s,¿ she continued, ¿perhaps in our ability to regulate expression of neprilysin. This is because it appears that the brain tissues in some people with AD show a decrease in neprilysin levels. So, if we understood a little bit better how expression of the gene is regulated, we might have some opportunity of keeping their levels therapeutically up. Our findings also suggest that it might someday be possible to identify individuals at risk for Alzheimer¿s, based on variations in their neprilysin gene.

¿There¿s been data over the years,¿ Gerard went on, ¿showing that inhibitors of neprilysin can increase levels of the toxic beta amyloid peptide. So the obvious thing for us to do was knock out the gene for the enzyme in mice, and see what differences that made.¿

Whereupon, the Gerards deleted the functional neprilysin gene from mice, transfected the replacement construct into embryonic stem cells, and implanted those cells into embryonic blastocysts ¿ the usual KO procedure.

¿Our latest finding,¿ she observed, ¿suggests that these knockout mice have worse AD symptoms.¿

When the team examined the brains of their neprilysin-deficient animals, they found that levels of amyloid beta ¿ unchecked by the missing enzyme ¿ concentrated in the descending order of hippocampus, cortex, hypothalamus/striatum and cerebellum. ¿These brain regions,¿ their paper reported, ¿correlated with the vulnerability to Ab depositions in brains of humans with AD.¿ The hippocampus, cerebral center of memory, learning and cognition, is the bull¿s eye targeted by the pathogenic amyloid.

¿Our observations,¿ the Science article concluded, ¿suggest that even partial down-regulation of neprilysin activity, which could be caused by aging, can contribute to AD development by promoting Ab accumulation.¿

Nuts And Bolts Of AD Turn Two Ways

The mother molecule of AD¿s brain etiology is a lengthy amino-acid chain called APP ¿ amyloid precursor protein. Several enzymes discovered in the 1990s ¿ notably, two presenilins and two secretases ¿ have guilty hands in carving amyloid beta peptide out of APP to perpetrate those plaques and tangles. This has led to the ongoing search for therapeutic drugs capable of inhibiting those culpable enzymes. So far, they have run into some unwanted side effects.

Neprilysin ¿ judging by its KO mice ¿ seems to work the other way around, chopping up and spitting out that Ab by peptide degradation, instead of trying to prevent its synthesis in the first place. Neurogeneticist Rudolph Tanzi, at Harvard-affiliated Massachusetts General Hospital, observed anent the neprilysin buzz, ¿It¿s just as important to find the degradation pathway as the synthetic pathway.¿ He is quoted in a Science ¿News of the Week¿ commentary accompanying the Gerard/RIKEN paper. It¿s title: ¿New clue to the cause of Alzheimer¿s.¿

¿Tanzi¿s group at Harvard,¿ the editorial reported, ¿has found hints of . . . a weak association between human Alzheimer¿s and the region on chromosome 3 where the neprilysin gene is located. It was just short of statistical significance, however, and he hasn¿t published the work.¿

The Japanese team, the commentary recorded, ¿identified neprilysin as a possible Ab-degrading enzyme about a year ago. They injected mouse brains with Ab and also with a series of specific protease inhibitors to see which would reduce the breakdown of the peptide. Because neprilysin had the greatest activity against Ab in test tube studies, the scientists collaborated with Craig and Norma Gerard¿s group at Harvard Medical School. They had knocked out the neprilysin gene in mice to see how that affected the animals¿ ability to degrade the peptide.

¿When injected into the brains of normal animals, Ab is broken down within about 30 minutes. But in the KO mice, the researchers found, almost all of the pathogenic peptide persisted. By crossing those mice with normal animals, the team produced mice that have just one active copy of the neprilysin gene. These heterozygotic animals yielded intermediate results. More of the injected Ab persisted than in normal animals, but less than in the complete knockouts.

¿That¿s important,¿ the Japanese senior author, Takaomi Saido, pointed out, ¿because it indicates that even partial reduction of neprilysin activity, which could be caused by aging, will elevate Ab and thus cause Alzheimer¿s.¿