27 May 2006

All presenilin mutations are not created equal. While over 130 mutations in the presenilin-1 (PS1) gene cause familial, early onset Alzheimer disease (FAD), some are associated with much earlier onsets than others. Some studies suggest that the variation in age of onset (ranging from early twenties to the sixties) could be due solely to perturbations of γ-secretase activity and Aβ production (Duering et al., 2005), while others implicate additional, independent genetic and/or epigenetic factors (Larner and Doran, 2005).

To sort this out, Mark Fortini and coworkers at the National Cancer Institute in Frederick, Maryland, have turned to Drosophila. They tested the ability of 14 different presenilin (PS) mutants to fill in for missing wild-type PS during fly development. Their results, published in the May 23 issue of Current Biology, show that the capacity of a panel of presenilin mutants to rescue fly development correlates with the age of onset observed in humans. Alleles that cause disease at a younger age showed less ability to rescue the PS-null phenotype, while mutations associated with later ages of onset produced a more normal phenotype. The ability of mutants to restore normal function also correlated with their ability to support Notch cleavage and signaling. The results support the idea that among FAD patients with PS mutations, the clinical differences, particularly age of onset, are primarily determined by the mutations themselves and their effects on γ-secretase function, rather than other genetic or environmental factors. In Drosophila, PS knockout causes a prepupal lethal phenotype. Reconstitution with wild-type Drosophila PS reverses lethality and produces normal adults. Since the human PS1 gene does not rescue the fly knockout, first author Glen Seidner tested FAD-linked PS1 mutations by introducing them into the Drosophila sequence. The mutations chosen were associated with a wide range of onset ages (24-61, plus two asymptomatic mutants) and spanned the entire protein sequence. After making transgenic flies, the researchers defined eight different rescue phenotypes, ranging from no rescue, (prepupal lethality) to survival up to later developmental states, and on to full reconstitution of normal adults. Using quantitative markers for each stage, they were able to rank the PS mutants according to their functional rescue activity.

An increasing ability of the genes to complement PS loss was associated with increasing PS activity as measured by Notch signaling. By three criteria (appearance of Notch-dependent proneural clusters, immunoblot measurements of the intracellular domain cleavage product of Notch [NICD], and transcription of an NICD target gene), they showed a progressive increase in Notch activity across the phenotype spectrum, validating the use of the phenotypes as a stand in for degrees of γ-secretase activity.

The fly in the ointment, so to speak, was the random insertion of transgenes into the fly genome leading to widely varying PS expression levels, which would skew readouts of the mutants in the rescue assay. To get around this, the researchers generated from four to 16 independent insertions for each transgene, and scored each insertion separately. By this analysis, they ended up with data on 162 transgenic lines, which they used to compare onset age in humans with functional activity in flies.

Looking at the plot of the degree of morphologic rescue versus average age of onset shows an overall positive trend. Statistical analysis was tricky, because the age of onset numbers were derived from small samples (some families with just a few cases) and the phenotypes were not quantitative. Nonetheless, by two different nonparametric tests, the investigators found a significant correlation between the age of onset and biological rescue activity across all the strains.

Further statistical grouping of individual mutants put them into three distinct functional classes: strong (least functional activity), intermediate, and weak (most functional activity, and wild-type). While a few of the mutations were stronger or weaker in the fly assay than would be predicted based on their human phenotypes, overall the data support the idea that the type of PS mutation determines the severity of phenotype in both Drosophila and in people. This observation opens up the possibility of using Drosophila to explore, and perhaps even evaluate, the clinical ramifications of new or rare presenilin mutants. The flies might also be useful to search for genetic modifiers of Alzheimer disease, and perhaps other diseases as well.

The work could also help encourage a wider view of PS function and its perturbations in disease, a theme that resonates with another recent paper from the same lab (see ARF related news story). Here, the similar effect of FAD mutations on Drosophila Notch signaling and human disease onset “underscores recent proposals that in addition to APP processing, more global perturbations in pathways involving other γ-secretase substrates should be considered in early onset Alzheimer’s disease,” they write.—Pat McCaffrey

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References

News Citations

1. Protease or Not?—More to Presenilin Toxicity than Meets the (Fly's) Eye 8 May 2006

Paper Citations

1. Duering M, Grimm MO, Grimm HS, Schröder J, Hartmann T. Mean age of onset in familial Alzheimer's disease is determined by amyloid beta 42. Neurobiol Aging. 2005 Jun;26(6):785-8. PubMed.
2. Larner AJ, Doran M. Clinical phenotypic heterogeneity of Alzheimer's disease associated with mutations of the presenilin-1 gene. J Neurol. 2006 Feb;253(2):139-58. PubMed.

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