Scientists have discovered a recessive mutation in β amyloid precursor protein that causes early onset Alzheimer disease in two siblings from a small south Italian village. Paradoxically, the mutant Aβ peptide, when mixed with wild-type, prevents aggregation. That may protect heterozygotes from developing AD, said senior author Fabrizio Tagliavini of the Carlo Besta Neurological Institute in Milan. Tagliavini, first author Giuseppe di Fede, also at Carlo Besta, and colleagues published their results in today’s Science. The work may indicate a new direction for AD pharmacotherapy using small peptides to keep Aβ from clumping.
Tagliavini and colleagues were intrigued by the case of a 36-year-old man with cognitive decline who visited their clinic. They collected DNA samples and were surprised to find that their patient was homozygous for an alanine-to-valine substitution at position 673 of APP. This corresponds to position 2 of the Aβ peptide. The man’s condition has worsened over the eight years since his symptoms began and now he cannot care for himself. His younger sister, who now suffers from mild cognitive impairment as well, also had two mutant alleles. Family members with only one mutant allele provided additional test subjects. “These heterozygotes were healthy,” Tagliavini said. “Even the oldest one, an 88-year-old lady—she is perfect.”
The A673V mutation stands in contrast to the many other known APP mutations, which are inherited in a dominant fashion. Many of those mutations flank the cleavage sites and thus still yield a wild-type Aβ peptide, Tagliavini noted, and others reside in the core of the peptide. There are two known neighboring amino-terminal mutations, at the sixth and seventh positions of Aβ (Janssen et al., 2003; Wakutani et al., 2004), which are dominant. It is not known how common the A673V mutant allele is, Tagliavini said, although he suspects it is rare. The scientists did not find the mutation in 200 healthy people and 100 people with sporadic AD.
The man had decreased Aβ1-42 and increased tau levels in his cerebrospinal fluid, similar to other people with AD. Both he and his sister showed high levels of Aβ in blood plasma, while their heterozygous family members had intermediate levels, in comparison with healthy control subjects. Fibroblasts cultured from the initial patient also released high levels of Aβ1-40 and Aβ1-42, compared to control cells. Transfected Chinese hamster ovary and COS-7 (green monkey kidney fibroblast) cells also secreted more Aβ when expressing the mutant APP, with cleavage products including Aβ1-40 and Aβ1-42 as well as Aβ11-40, Aβ11-42, and pyroglutamate AβN3pE-42. The ratio of Aβ1-40 to Aβ1-42 was unchanged. These results suggested to the scientists that the mutation alters APP processing, boosting cleavage to form Aβ.
Di Fede and colleagues then turned to biochemistry to assay the mutation’s effects on aggregation. Using synthetic peptides and laser light scattering to measure clumping, they found that the mutant peptide aggregated faster than wild-type. “The effect of the mutation was dramatic,” Tagliavini said, but left him and his colleagues puzzled. “It was impossible to understand why the heterozygous carrier did not develop disease.”
To answer their questions, the scientists mixed mutant and wild-type peptides to simulate heterozygous conditions. In these experiments, the mixtures aggregated more slowly than either individual peptide. The hybrid clumps were also unstable; they dissolved within eight minutes when diluted in buffer, which neither single peptide will do. In cultured neuroblastoma cells, the mixture of mutant and wild-type Aβ1-42 was less toxic than either alone. Approximately 85 percent of cells survived 24 hours with the peptide cocktail, compared to 80 percent for wild-type and 70 percent for mutant. In heterozygotes, then, the single mutant allele may actually protect against Alzheimer disease by preventing stable Aβ aggregates from forming, Tagliavini said.
The results underscore the importance of the amino-terminus of Aβ. Sometimes thought to be a floppy, unimportant tail (Williams et al., 2004), these and other data suggest the region is involved in aggregation. Amino-terminal mutations promote Aβ fibril elongation (Hori et al., 2007) and antibodies to the amino terminus bind and clear plaques (see ARF related news story and Bard et al., 2003). To isolate the amino-terminal effect, the authors of the current study experimented with an Aβ1-6 fragment, with and without the A673V mutation. The mutant hexapeptide bound more tightly to wild-type Aβ than the normal hexapeptide did.
The results point to potential therapies for AD, Tagliavini said. Although a hexapeptide would probably be degraded too quickly to have a protective effect, the scientists are working on ways to modify the peptide so that it lasts long enough to interfere with Aβ aggregation. The scientists have also engineered a transgenic mouse carrying the new mutation, although they have yet to discover a disease phenotype. In addition, Tagliavini hopes to discern the protein structure of the mutant Aβ in order to discover what makes it different from wild-type peptide.—Amber Dance