Stockholm. One of the unresolved issues in the debate about γ-secretase revolves around the other members of the complex besides presenilin and nicastrin. If they were all isolated and better understood, it might be possible to reconstitute γ-secretase activity in vitro, a definitive experiment to prove the identity of γ-secretase.

A poster and a talk at the 8th International Conference on Alzheimer’s Disease and Related Disorders today presented new information on other protein members of the presenilin complex. The poster, by Jinhe Li at Pharmacia Corporation in Kalamzoo, Michigan, and colleagues there and at Exelixis Inc. in South San Francisco, described how C. elegans-based genetic screens for presenilin enhancers yielded three genes (aph-1, aph-2/nicastrin, and pen-2) that are required for presenilin function in Notch cleavage. The authors cloned the human homologs (aph-1a, aph-1b) and report that they are broadly expressed in different tissues. They also mapped aph-1a, pen-2 and nicastrin to chromosome regions reported to probably harbor AD risk genes. The work appeared in the July Developmental Cell.

The researchers used yeast two-hybrid assays and co-immunoprecipitation to detect interactions between aph-1b, pen-2, nicastrin , and presenilin 1. Aph-1, aph-1b and nicastrin appeared to reside in the ER in a pattern that resembles previous PS-1 localization studies. The poster proposes a complex consisting of presenilin, aph-1a, aph-1b, aph-2/nicastrin, and pen2. To test whether these new proteins can modulate Aβ generation, the scientists transfected HEK293 cells with aph-1a, aph-1b, and pen-2 versions bearing small deletions or substitutions and showed that these alterations affected Aβ levels.

Finally, the poster showed on nicastrin/pen-2 double knockout mice, showing they are embryonic lethal in much the same way as are presenilin1/2-double knockout mice, presumably due to impaired notch signaling.

As likely members of the γ-secretase complex, aph-1a, aph-1b, and pen-2 could be targets for drug development, the authors write, even as additional biological targets for γ-secretase keep emerging.

Working with Dennis Selkoe, Michael Wolfe, and others, Taylor Kimberly of Brigham and Women’s Hospital expanded these findings to his work with Chinese hamster ovary cells. Kimberly reported today that when overexpressing all three, aph-1, pen-2, and presenilin-1, γ-secretase activity shoots up in extracts of these cells when he added the substrate. In parallel, the authors saw increased generation of both Aβ and AICD, the intracellular cytoplasmic fragment of APP. (Nicastrin was already present in excess quantities in these cells.) “With these two additional components in hand, I now believe the γ-secretase complex is complete. We can now try to reconstitute activity in vitro,” said Selkoe.—Gabrielle Strobel


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