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Genetics Leads the Way 2002). The pursuit of mutations that cause Notch signal- ing defects has recently yielded yet another essential to the Accomplices of Presenilins player in this pathway: the predicted seven-pass trans- membrane APH-1 (Goutte et al., 2002). Defective APH-2/nicastrin localization in aph-1 mutants hints at a possible connection to ␥-secretase activity. Presenilins mediate the ␥-secretase cleavage of Notch In a beautiful demonstration of the power of genetic transmembrane receptors as well as the transmem- screens, Francis et al. in this issue of Developmental brane ␤-amyloid precursor protein (␤APP), but they Cell report the isolation of two genes in C. elegans, aph-1 are not thought to accomplish this alone. Recent ge- and pen-2, that interact genetically with presenilins and netic screens in C. elegans, presented in this issue of play a critical role in ␥-secretase activity (Francis et al., Developmental Cell, identify two genes that are essen- 2002). Worms that are defective in both presenilin genes, tial to ␥-secretase activity and may interact with pre- hop-1 and sel-12, display all the hallmark Notch signal- senilins. ing defects: maternal-effect embryonic lethality, aber- rant somatic gonad and vulval development, and germ- line sterility. However, single sel-12 or hop-1 mutants In 1995, presenilin came in to the limelight on are fully viable and fertile. Francis et al. searched for two fronts: association with familial Alzheimer’s disease mutations that would act synergistically with a sel-12 and involvement in Notch signaling pathways in model mutation to cause germline sterility. As expected, their systems (Levitan and Greenwald, 1995; Sherrington et screen yielded new of hop-1, but they also dis- al., 1995). Since then, active research in both fields has covered mutations in two additional genes. One of these led to a unifying model in which integral membrane pre- genes was aph-1 and the other was a new gene, which senilins are responsible for the ␥-secretase activity that the authors call pen-2 (presenilin ), encoding ␤ ␤ cleaves -amyloid precursor protein ( APP) and Notch a small conserved protein with two predicted transmem- within their transmembrane domains. This proteolytic brane domains. Although the authors isolated these event is of major interest for two reasons: first, defects genes in a sensitized genetic background, mutations in in this event result in the pathogenic accumulation of either gene alone cause two bona fide Notch pheno- ␤ A peptides in Alzheimer’s patients, and second, it is types: maternal-effect embryonic lethality and defective this step during Notch signal transduction that releases vulval development, suggesting that each gene has an the Notch intracellular domain from the membrane, essential function in mediating at least some Notch sig- allowing it to move to the nucleus and regulate transcrip- naling events. It is possible that the APH-1 and PEN-2 tion. Although abundant evidence suggests that the ac- proteins are obligate components of all Notch signaling ␥ tive site of -secretase resides in the presenilin protein events in C. elegans; however, demonstration of their itself, the protein alone is not enzymatically active unless requirement in later developmental events is hampered isolated as part of a large multiprotein complex. For by the difficulty of obtaining animals lacking both mater- this reason, significant attention is currently focused on nal and zygotic gene products. deciphering the identity and function of the additional binding to the Notch stimulates two components of the presenilin complex. successive cleavages of Notch. The first, at the S2 site, Currently, the only other known component of presen- releases the extracellular domain. The remaining mem- ilin complexes is the type I transmembrane protein APH-2/ brane-tethered Notch fragment then becomes a sub- nicastrin, identified biochemically in immunoprecipi- strate for the presenilin-mediated cleavage at the S3 tates of presenilins from human cells (Yu et al., 2000). site within the transmembrane domain. This cleavage A critical role for APH-2/nicastrin in the Notch signaling releases the Notch intracellular domain, allowing its pathway had been demonstrated by its discovery in C. transit to the nucleus. In order to delineate which step elegans as a mutant that shows developmental defects in the requires aph-1 and pen-2 identical to those caused by mutations in the Notch function, Francis et al. used two constitutive versions receptor itself or in presenilins (Goutte et al., 2000). Fur- of Notch: a gain-of-function derivative of LIN-12 (one of ther experiments performed in human cells and Dro- two Notch receptors in C. elegans), which is still mem- sophila have now demonstrated clearly that the APH-2/ brane anchored and therefore dependent on S3 cleav- nicastrin protein is an essential component of the age, and a truncated LIN-12, consisting of only the intra- ␥-secretase complex that acts to cleave Notch recep- cellular portion of Notch, which therefore does not tors as well as ␤APP (Chung and Struhl, 2001; Esler et require S3 cleavage to elicit signal transduction. They al., 2002; Hu et al., 2002; Lopez-Schier and St Johnston, found that the membrane-anchored form required aph-1 Previews 7

and pen-2 activity, while the intracellular form did not. components, as suggested by the data of Francis et al., Thus, APH-1 and PEN-2 are required at or before the in which presenilin stability drops dramatically in the S3 cleavage step, as is seen for presenilins and APH-2/ absence of APH-1, PEN-2, or APH-2/nicastrin. Similarly, nicastrin in similar assays. full assembly of the complex may be essential for its Further proof that APH-1 and PEN-2 have critical roles proper trafficking to the cell surface, the site of Notch in ␥-secretase activity comes from a ␥-secretase assay intramembranous cleavage. This idea is supported by that Francis et al. developed in cultured Drosophila cells the observation that presenilins and APH-2/nicastrin fail which could be depleted of selected gene products by to localize to the cell surface if either protein is missing, RNA-mediated interference (RNAi). The assay involved and that APH-1 is also required for the cell surface local- a reporter construct whose activity was dependent on ization of APH-2/nicastrin (Chung and Struhl, 2001; the S3 cleavage of introduced Notch or ␤APP deriva- Goutte et al., 2002). tives. The endogenous ␥-secretase activity of these cells The findings of Francis et al. will certainly stimulate a was also monitored by the detection of secreted A␤40 frenzy of biochemical analysis asking whether APH-1 and A␤42 peptides resulting from ␤APP processing. In and PEN-2 can be detected in the enzymatically active these assays, ␥-secretase activity was dramatically re- ␥-secretase complex, and whether these proteins en- duced by RNAi directed at aph-1 or pen-2, as well as gage in direct interactions with the known components, by RNAi directed at presenilin or nicastrin, indicating presenilins and APH-2/nicastrin, or perhaps with the that APH-1 and PEN-2 are essential for the intramembra- substrates Notch and ␤APP. Undoubtedly, these two nous cleavage of both ␤APP and Notch. genes will receive much attention in the development These analyses demonstrate a similar requirement for of Alzheimer’s disease therapeutics, and in the pursuit of aph-1 and pen-2 in the presenilin-mediated step of the mechanism underlying intramembranous cleavage Notch signal transduction. Do they act together? A clue events. comes from a surprising source: rescue experiments performed in C. elegans with transgenic human aph-1 and human pen-2. Francis et al. found that the vulval Caroline Goutte defects of aph-1 and pen-2 mutants could be rescued Department of Biology by transgenic versions of the C. elegans genes but not Amherst College by their human counterparts. Interestingly, the human Amherst, Massachusetts 01002 genes could be coaxed into function by simultaneous introduction of both genes, and this was even further improved with the addition of a human presenilin gene. Selected Reading These data imply a functional interaction between Chung, H.M., and Struhl, G. (2001). Nat. Cell Biol. 3, 1129–1132. APH-1 and PEN-2, and between one or both of these Esler, W.P., Kimberly, W.T., Ostaszewski, B.L., Ye, W., Diehl, T.S., proteins and presenilins. Selkoe, D.J., and Wolfe, M.S. (2002). Proc. Natl. Acad. Sci. USA 99, Francis et al. have convincingly demonstrated a re- 2720–2725. quirement for two new proteins, APH-1 and PEN-2, in Francis, R., McGrath, G., Zhang, J., Ruddy, D.A., Sym, M., Apfeld, ␥-secretase activity, as evidenced in both C. elegans J., Nicoll, M., Maxwell, M., Hai, B., Ellis, M.C., et al. (2002). Dev. Cell and Drosophila cells. It is possible that APH-1 and PEN-2 3, this issue, 85–97. interact with presenilin and contribute to its stability or Goutte, C., Hepler, W., Mickey, K.M., and Priess, J.R. (2000). Devel- trafficking, but do not themselves remain associated opment 127, 2481–2492. with presenilin as it engages in ␥-secretase activity. Goutte, C., Tsunozaki, M., Hale, V.A., and Priess, J.R. (2002). Proc. However, it is tempting to speculate that these proteins Natl. Acad. Sci. USA 99, 775–779. are indeed components of the high molecular weight Hu, Y., Ye, Y., and Fortini, M.E. (2002). Dev. Cell 2, 69–78. presenilin complex. As members of this complex, they Levitan, D., and Greenwald, I. (1995). Nature 377, 351–354. may have roles as regulators, structural scaffolds, sub- Lopez-Schier, H., and St Johnston, D. (2002). Dev. Cell 2, 79–89. strate binders, or perhaps direct contributors to catalytic Sherrington, R., Rogaev, E.I., Liang, Y., Rogaeva, E.A., Levesque, activity. The predicted polytopic membrane structures G., Ikeda, M., Chi, H., Lin, C., Li, G., Holman, K., et al. (1995). Nature of APH-1 and PEN-2, like that of presenilin, could facili- 375, 754–760. tate their incorporation into an integral membrane com- Yu, G., Nishimura, M., Arawaka, S., Levitan, D., Zhang, L., Tandon, plex capable of cleaving transmembrane domains. As- A., Song, Y.Q., Rogaeva, E., Chen, F., Kawarai, T., et al. (2000). sembly of such a complex may stabilize the individual Nature 407, 48–54.