Interpreting the Signaling of Notch

For this article, Eugene Russo interviewed Mark E. Fortini, an assistant professor of genetics at the University of Pennsylvania, Iva Greenwald, a Howard Hughes Medical Institute investigator and professor of biochemistry at the Columbia University College of Physicians and Surgeons, Raphael Kopan, an associate professor of molecular biology and pharmacology at Washington University in St. Louis, and Michael Wolfe, an associate professor of neurology at Harvard Medical School. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to100 times more often than the average paper of the same type and age. B. De Strooper, W. Annaert, P. Cupers, P. Saftig, K. Craessaerts, J.S. Mumm, EH Schroeter, V. Schrijvers, M.S. Wolfe, W.J. Ray, A. Goate, R. Kopan, "A presenilin-1-dependent g-secretase-like protease mediates release of Notch intracellular domain," Nature, 398:518-22, April 8, 1999. (Cited in 172 papers) G. Struhl, I. Greenwald, "Presenilin is required for activity and nuclear access of Notch in Drosophila," Nature, 398:522-25, April 8, 1999. (Cited in 133 papers) M.S. Wolfe, W.M. Xia, B.L. Ostaszewski, T.S. Diehl, W.T. Kimberly, D.J. Selkoe, "Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and g-secretase activity," Nature, 398:513-17, April 8, 1999. (Cited in 191 papers) Y.H. Ye, N. Lukinova, M.E. Fortini, "Neurogenic phenotypes and altered Notch processing in Drosophila Presenilin mutants," Nature, 398:525-9, April 8, 1999. (Cited in 120 papers) In demonstrating critical links among elements in the pathway to Alzheimer's disease (AD) onset, these four papers forged fundamental connections between the research of AD pathologists and cell biologists and made important in-roads toward potential AD therapies. As Raphael Kopan, senior author of the De Strooper et al. paper, recalls, the April 1999 findings enabled two seemingly separate fields to reach a critical point of convergence. AD pathologists had been interested in understanding the generation of ß-amyloid (Aß), the neurotoxic peptide found in the brains of AD-afflicted patients. They knew that the Aß peptides were short fragments of larger precursors called amyloid precursor proteins (APPs). AD researchers began hypothesizing as to how APPs are cleaved to generate the toxic Aß peptide sludge that accumulates in the brains of AD patients. They would discover that Aß is first cut out of APP by an enzyme dubbed ß-secretase, then by another called g-secretase, though both enzymes remained unidentified. In parallel, cell and molecular biologists were attempting to discover how cells communicate with each other during development. In 1985, Yale University researchers cloned a critical gene called Notch known to be involved in short-range communication between cells.1 Notch protein signaling is integral to a vast number of cellular processes including the generation of blood cells, the generation of T cells, and possibly even to the strengthening of synaptic connections. But the Notch-related mechanism of cell communication remained a mystery. "Here are two cells making contact with each other, reporting the contact to the nucleus," explains Kopan. "Yet there was no good handle on how that signal was being transduced, how it is that contact was being reported." Courtesy Michael Wolfe From left to right: Michael Wolfe, Thekla Diehl, Beth Ostaszewski, Taylor Kimberly, Weiming Xia, Dennis Selkoe Findings published by Iva Greenwald, author of one of the 1999 papers, started to bring together the two fields.2 Working in Caenorhabditis elegans, researchers identified a modifier of the Notch mutation and showed that the loss of a single copy of presenilin, (Sel-12), a protein notorious for its role in AD, converted these molecules back to normal. And in 1998 Bart De Strooper of the Flanders Institute of Biotechnology in Belgium showed for the first time PS1-deficient mice lose the ability to cleave APP.3 Yet researchers still understood little about PS1's role--whether it was involved in trafficking or in proteolysis (protein degradation). The April 1999 Ye et al., Struhl et al., and De Strooper et al. papers all elucidated the essential role of presenilins in Notch functioning--at the cellular or organismal level in the case of the first two papers, at a biochemical level in the case of the third. The Wolfe et al. paper revealed crucial details about the specific biochemical function of PS1. De Strooper et al. showed in vitro that mouse cells missing the ps1 gene exhibit significant reduction in the production of the APP fragments and a great reduction in proteolysis of the Notch molecule. Greenwald and colleague Gary Struhl, both from the Columbia University College of Physicians and Surgeons, as well as Ye et al., looked to Drosophila to investigate Notch signaling. While the findings of Struhl and Greenwald were essentially in line with those of De Strooper et al. (both suggested that PS1 functions in the transmembrane cleavage of Notch), those of Ye et al. had an important point of divergence: Although they did suggest that PS1 is required for the signaling of full-length Notch, they found that it was not required for signaling of a truncated form of Notch containing the transmembrane domain. However, some follow-up papers have suggested that the occurrence of presenilin-independent Notch signaling is minimal. Kopan's group showed that a mutation near the g-secretase cleavage site in mice causes a Notch-deficient phenotype.4 And earlier this year, Struhl and Greenwald showed in Drosophila--using a technique and mutant that weren't available to Ye et al. at the time--that presenilin was in fact required for signaling involving the truncated form of Notch that includes the transmembrane domain.5 Photo: Robert Klink Mark Fortini While evidence subsequent to the April 1999 papers is not in complete agreement, Mark E. Fortini, senior author of the Ye et al. paper, does admit that it now looks as though any presenilin-independent Notch signaling that's occurring is insignificant. "It's kind of like it always is in science," says Fortini, an assistant professor of genetics at the University of Pennsylvania. "You do what you can ... and then more information becomes available, better reagents, better methods, a more refined model." Kopan notes that without the benefit of biochemical methods, like sequencing peptides, fly geneticists are unable to accurately identify Notch fragments generated by mutant flies. Fortini does point to some positive impact that his group's paper had on the field: It encouraged investigators to try to correlate observed Notch-related processing defects with effects on signaling, experiments that turned out to be crucial to deciphering the pathway. The conclusions of Wolfe et al. also generated controversy. But in the two years since the papers, the group's assertions have held up against much scrutiny. In the April 1999 paper, Harvard Medical School (HMS) associate professor of neurology Michael Wolfe, along with HMS neurology professor Dennis Selkoe, went so far as to suggest that presenilins were actually cleaving APP and Notch. In fact, they made the divisive assertion that PS1 was g-secretase. The Harvard team had identified two conserved aspartates in PS1 that are critical for g-secretase activity, suggesting that PS1 is a critical cofactor for g-secretase or that it is itself g-secretase. "Gamma-secretase was this enzyme activity without a protein and presenilins were proteins without a known function," explains Wolfe. Although researchers knew that presenilins and g-secretase had some association, this paper, says Wolfe, presented the first evidence for a specific biochemical function and "offered a unifying hypothesis for the role of presenilins in g-secretase activity." Several papers have apparently proved their suspicions correct: one from Merck Research Laboratories in May 2000 showed that presenilins co-purify with g-secretase,6 and subsequent work from Merck and Wolfe's lab showed that inhibitors directed to the active site of g-secretase bind directly to presenilins.7-9 However, the definitive proof--actually purifying g-secretase as has been done with ß-secretase10--will be extremely difficult. Scientists know little about the intricacies of cleavage within the cell's lipid bilayer. And because g-secretase is a multiprotein complex made up of membrane proteins, it will be hard to crystallize. As for AD treatment implications, better knowing the identity of g-secretase should aid rational drug design by facilitating an understanding of structure-activity relationships of inhibitors. Kopan explains that reducing APP cleavage by 50 percent in AD sufferers--possibly by targeting PS1/g-secretase--will delay disease onset well beyond reasonable life expectancy. The worry is that targeting the multipurpose Notch pathway could induce major collateral damage in several other parts of the body. His group's paper showed that a near complete block of Notch proteolysis in mice does cause Notch-related symptoms. Yet these mice faired better than those completely missing the Notch gene, which suggests that toxicity levels for a related treatment could be kept at acceptable levels. "In other words," says Kopan, "you should be able to find a therapeutic window which is enough to inhibit some APP production but not enough to inhibit all of Notch production." Wolfe adds that the role of Notch in the elderly has yet to be well studied. "It could be that you can inhibit g-secretase in the aging adult and it won't have any untoward effects," he says. Eugene Russo can be contacted at erusso@the-scientist.com References 1. K.A. Wharton et al., "Nucleotide-sequence from the neurogenic locus notch implies a gene-product that shares homology with proteins containing EGF-like repeats cell," Cell, 43:567-81, 1985. 2. D. Levitan, I. Greenwald, "Facilitation of lin-12-mediated signalling by sel-12, a Caenorhabditis elegans S182 Alzheimer's disease gene," Nature, 377:351-4, 1995. 3. B. De Strooper, "Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein," Nature, 391:387-90, 1998. 4. S.S. Huppert et al., "Embryonic lethality in mice homozygous for a processing-deficient allele of Notch1," Nature, 405:966-70, 2000. 5. G. Struhl, I. Greenwald, "Presenilin-mediated transmembrane cleavage is required for Notch signal transduction in Drosophila," Proceedings of the National Academy of Science (PNAS), 98:229-34, Jan. 2, 2001. 6. Y.M. Li et al., "Presenilin 1 is linked with *-secretase activity in the detergent solubilized state," PNAS, 97:6138-43, May 23, 2000 7. Y.M. Li et al., "Photoactivated *-secretase inhibitors directed to the active site covalently label presenilin-1," Nature, 405:689-94, 2000. 8. W.P. Esler, "Transition-state analogue inhibitors of *-secretase bind directly to presenilin-1," Nature Cell Biology, 2:428-34, 2000. 9. N. Halim, "Elusive g-secretase identified," The Scientist, 14[13]:6, June 26, 2000. 10. E. Russo, "The Search for Secretases," The Scientist, 13[22]:7, Nov. 8, 1999.

Interpreting the Signaling of Notch

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