There are times in the course of scientific enterprise when researchers must re-evaluate their hypotheses in the face of new, unexpected data. Two papers published this week offer the Alzheimer’s field just those kinds of contrary results. In the February 3 Journal of Neuroscience, researchers from Washington University in St. Louis, Missouri, report that the γ-secretase complex, long assumed to require nicastrin to recognize and cleave amyloid precursor protein, gets by without it. And in a paper posted online by PNAS, scientists from the University of California in Santa Barbara present evidence that p25, long thought to be a hyperactive, tau-phosphorylating cleavage product of p35, has identical kinetics to its parent.

“Science is about observation and about not really accepting your dogma to the degree that it forces you to ignore what is right in front of your own eyes,” said Raphael Kopan, senior author on the Journal of Neuroscience paper. “That is how progress is made.”

No Nicastrin? No Problem
For Guojun Zhao, first author on the Kopan group paper, progress started with an unexpected, unexplainable band on a Western blot. It was the right size for the product of γ-secretase cleavage of Notch, but the cell line he was using lacked nicastrin, thought to be required for the enzyme’s activity.

In addition to Notch receptors, γ-secretase cleaves the amyloid precursor protein (APP), producing toxic Aβ fragments. The enzyme complex contains four proteins: presenilin, nicastrin, Pen2, and Aph1. Presenilin contains the catalytic site. Pen2 and Aph1 are of uncertain, but essential function. And everybody knows that nicastrin provides the substrate recognition site (see ARF related news story on AbstractShah et al., 2005).

But lately, there has been some controversy over nicastrin’s precise contribution. In 2008, researchers from Bart De Strooper’s laboratory at KU Leuven in Belgium reported that nicastrin contributes to maturation of the complex, but not its activity (Chávez-Gutiérrez et al., 2008). Zhao chose to pursue that pesky band.

He added γ-secretase inhibitors to his cells. “And, lo and behold, the band went away,” Kopan said. To further test the enzyme’s activity, Zhao transfected embryonic fibroblasts from a nicastrin-knockout mouse with a γ-secretase substrate. When he treated the cells with protease inhibitors, the cleavage product band intensified. The results suggest that γ-secretase minus nicastrin is active, but the enzyme or its cleavage product are normally degraded by the proteasome. Using protease inhibitors to reveal the cleavage product was the “key trick,” said Michael Wolfe of Harvard Medical School, who was not involved in the research. Without those inhibitors, the cleavage product would be easy to miss. The researchers confirmed their results in a second nicastrin-negative cell line, and with APP instead of Notch as a substrate. Without nicastrin, the cells were able to cleave an APP substrate and release Aβ40 into the media.

“We thought the complex required all four components, and that was that, so I was dubious,” Wolfe said. He was surprised to find the paper convincing, he told ARF. It is difficult to reconcile the current work with past papers suggesting nicastrin is crucial for substrate recognition, but Wolfe suggested nicastrin might assist in substrate recognition without being absolutely necessary. “We need to revise our thinking about how the enzyme is assembled,” Kopan said. The authors suggest that nicastrin acts to stabilize the enzyme. “It is still essential, but not for the function we thought,” Kopan said. He suggested that working with a three-part complex might simplify biochemical and structural studies. Nicastrin is the largest component of the complex, and he suggested crystallizing three proteins might be easier than four, for example.

P25 Not More Potent
Another cleavage product relevant to neurodegenerative disease is p25. Its parent is the longer p35, a membrane-bound regulatory subunit of Cdk5. Cdk5/p35 is an essential neural kinase. When Cdk5 instead hooks up with p25, a cytoplasmic proteolysis product, the two wreak havoc, phosphorylating tau, causing DNA damage, and promoting neurodegeneration. The p25 form accumulates in the brains of people with AD and, because it is not readily degraded, promotes constitutive activity of Cdk5 (Patrick et al., 1999). Cdk5/p25 has also been linked to Parkinson disease (Smith et al., 2003) and amyotrophic lateral sclerosis (Nguyen et al., 2001).

“There is a common belief in the field that Cdk5/p25 is actually hyperactive,” said John Lew, senior author on the PNAS study with first author Dylan Peterson and colleagues. A 2002 in vitro analysis by a group in Tokyo, Japan, appeared to support this theory; the researchers found differential enzyme kinetics between the p25 and p35 forms (Hashiguchi et al., 2002).

However, Peterson and Lew doubted those results. When they repeated in vitro analysis of the enzyme kinetics, collecting more extensive data, they found that p25 and p35 were statistically the same on every parameter. “This is done by direct comparison of the two enzymes, purified, with nothing else interfering,” Lew said. Kinetic parameters and phosphorylation sites, determined by nuclear magnetic resonance, matched with both histone H1, Cdk5’s best-known substrate, and tau. Why the difference from the 2002 results? “There is really no good explanation,” Lew said. “I think it is just a matter of different hands.” He noted that he cannot rule out cellular factors, not present in the in vitro experiments, that could influence p25 activity.

Researchers must now think of another reason that p25 causes trouble, Lew said. “P25 is a much more stable protein, with a longer half-life,” noted Li-Huei Tsai of MIT, but Lew doubts protein levels are the answer, and said p25 and p35 steady-state levels are similar. Localization could also make a difference: While p35 resides in the cell membrane, p25 is free in the cytoplasm, where it is likely to encounter different substrates. One important substrate, Tsai suggested, is histone deacetylase 1 (HDAC1). Cdk5/p25 inhibits HDAC1, leading to DNA breaks, cell cycle disruption, and neurodegeneration (see ARF related news story on Kim et al., 2008).

“In a way, I am not surprised at all,” Tsai said. “This happens all the time in science.” Together, these two papers show the importance of keeping an open mind. “When nature throws hints in your path, you should pay attention,” Kopan said.—Amber Dance

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Comments on News and Primary Papers

  1. Zhao et al. present a novel and interesting set of findings that shed light on one of the most intriguing features of the γ-secretase enzyme: its dependence on the formation of a high-molecular-weight complex. This study provides further support for the involvement of nicastrin in assembly, maturation, and stabilization of the γ-secretase complex rather than in substrate recognition or as a crucial component for catalytic activity of the complex. The results presented in this article are in accordance with our previously published study (Chavez-Gutierrez et al., 2008) and our current working hypothesis for γ-secretase.

    Remarkably, this report shows that a nicastrin-less γ-secretase complex, consisting of PS1/APH1a/PEN2, displays indistinguishable catalytic properties relative to the mature γ-secretase (containing nicastrin). Moreover, this nicastrin-less complex also requires ectodomain shedding of the substrate prior to catalysis, demonstrating that nicastrin does not participate in the recognition of the short substrate amino terminus. Interestingly, the signal peptide peptidase (SPP), an intramembrane protease that is most closely related to PS, does not need a cofactor protein for catalytic activity but does require prior substrate shedding, suggesting that recognition/binding of the substrate amino terminus by the γ-secretase complex may be carried out by presenilin, although the participation of Aph1 and/or Pen2 cannot be ruled out.

    Additionally, given that the extremely unstable “nicastrin-less” γ-secretase complex maintains approximately 50 percent of the wild-type enzyme activity, this result strongly suggests that nicastrin is not crucial for substrate recognition/binding. Nevertheless, our data and the findings presented in this article cannot exclude another contribution of nicastrin to γ-secretase activity and/or specificity, although no biochemical data have been published to support this assumption.

    The model of nicastrin as the substrate receptor for the γ-secretase complex is exclusively supported by the putative interaction between the Glu333 and the N-terminus of the substrate. In contrast, our published data has shown that mutations in the putative binding pocket, including Glu332 (Glu333 in human), affect the levels of the mature γ-secretase complex in the mutant cell lines, but they do not have an effect on the specific activity of the complex. Therefore, our results exclude the participation of the Glu332 in the activity of the complex and indicate that the observed effects could be due to a problem in the assembly or maturation (stability) of the mutant complexes. Importantly, the results presented by Zhao et al. elegantly corroborate our findings and present a “simplified” version of the γ-secretase complex that advances our understanding of its structure-function.

    γ-secretase has been considered as a potential drug target for Alzheimer disease. The proposal of nicastrin as a gatekeeper of the enzyme complex presented the opportunity to approach the γ-secretase “drug targetability” from a different angle. However, the fact that two independent studies present data that do not support this model not only reopens the discussion about the function of nicastrin in the γ-secretase complex, but also adds a question mark to this drug-targeting strategy.

    References:

    . Glu(332) in the Nicastrin ectodomain is essential for gamma-secretase complex maturation but not for its activity. J Biol Chem. 2008 Jul 18;283(29):20096-105. PubMed.

    View all comments by Lucia Chavez-Gutierrez

References

News Citations

  1. Nicastrin—A DAPper Role in γ-Secretase
  2. Overworked HDACs Leave Transcriptional Posts to Push DNA Repair

Paper Citations

  1. . Nicastrin functions as a gamma-secretase-substrate receptor. Cell. 2005 Aug 12;122(3):435-47. PubMed.
  2. . Glu(332) in the Nicastrin ectodomain is essential for gamma-secretase complex maturation but not for its activity. J Biol Chem. 2008 Jul 18;283(29):20096-105. PubMed.
  3. . Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature. 1999 Dec 9;402(6762):615-22. PubMed.
  4. . Cyclin-dependent kinase 5 is a mediator of dopaminergic neuron loss in a mouse model of Parkinson's disease. Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13650-5. PubMed.
  5. . Deregulation of Cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions. Neuron. 2001 Apr;30(1):135-47. PubMed.
  6. . Truncation of CDK5 activator p35 induces intensive phosphorylation of Ser202/Thr205 of human tau. J Biol Chem. 2002 Nov 15;277(46):44525-30. PubMed.
  7. . Deregulation of HDAC1 by p25/Cdk5 in neurotoxicity. Neuron. 2008 Dec 10;60(5):803-17. PubMed.

Further Reading

Papers

  1. . Neurodegeneration in an Abeta-induced model of Alzheimer's disease: the role of Cdk5. Aging Cell. 2010 Feb;9(1):64-77. PubMed.
  2. . Expression of p25, an aberrant cyclin-dependent kinase 5 activator, stimulates basal secretion in PC12 cells. Mol Cells. 2010 Jan;29(1):51-6. PubMed.
  3. . Analysis of the gamma-secretase interactome and validation of its association with tetraspanin-enriched microdomains. Nat Cell Biol. 2009 Nov;11(11):1340-6. PubMed.
  4. . Glu-333 of nicastrin directly participates in gamma-secretase activity. J Biol Chem. 2009 Oct 23;284(43):29714-24. PubMed.
  5. . Single chain variable fragment against nicastrin inhibits the gamma-secretase activity. J Biol Chem. 2009 Oct 9;284(41):27838-47. PubMed.
  6. . Targeting Cdk5 activity in neuronal degeneration and regeneration. Cell Mol Neurobiol. 2009 Dec;29(8):1073-80. PubMed.

Primary Papers

  1. . Gamma-secretase composed of PS1/Pen2/Aph1a can cleave notch and amyloid precursor protein in the absence of nicastrin. J Neurosci. 2010 Feb 3;30(5):1648-56. PubMed.
  2. . No difference in kinetics of tau or histone phosphorylation by CDK5/p25 versus CDK5/p35 in vitro. Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):2884-9. PubMed.