Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Total inactivation of γ–secretase activity in presenilin-deficient embryonic stem cells

Nature Cell Biology volume 2, pages 461–462 (2000)Cite this article

The question of how proteins such as the β-amyloid precursor protein (β-APP), Notch-1 and others can be cleaved in the plane of the membrane has challenged cell biologists and researchers into Alzheimer’s disease for some time. Processing of β-APP by γ-secretase is considered an important therapeutic target, as it constitutes the final step in the release of the amyloid β-peptide (Aβ), the principal constituent of the amyloid plaques in the brains of Alhzeimer’s patients. Recently, γ-secretase activity was linked to two proteins, presenilin-1 (PS1) and presenilin-2 (PS2; refs 1, 2). Presenilins are strongly hydrophobic proteins embedded in intracellular membranes, and missense mutations in their genes cause a rare, aggressive form of Alzheimer’s disease3. It has been proposed that two aspartate residues in the transmembrane domains of presenilins constitute the catalytic site of γ-secretase2. One argument against this hypothesis is the important residual γ-secretase activity observed in cells derived from PS1- or PS2-deficient mice1,4. The question is therefore whether cells that are completely devoid of PS1 and PS2 maintain this γ-secretase activity or not.

mNotchΔE is another substrate for PS1-dependent γ-secretase7,8. Processing of this construct mimics that of full-length Notch-1, and results in release of the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates, together with transcription factors of the CBF-1 family, several genes, including HES-1 (ref. 9). We therefore co-transfected the embryonic stem cells with plasmids encoding mNotchΔE and luciferase under the control of the HES-1 promoter (kindly provided by R. Kopan and A. Israel), and measured induction of luciferase activity10. As a control, we also transfected embryonic stem cells with plasmids encoding the NICD. Induction of HES-1 activity with mNotchΔE was observed in PS1+/+PS2–/– cells, but not in PS1–/–PS2–/– cells (Fig. 1d). In contrast, NICD strongly induced luciferase activity, implying that the Notch signalling pathway downstream of the proteolytic processing of mNotchΔE was essentially intact in PS1–/–PS2–/– cells. This was further confirmed by the observation that signalling in PS1–/– PS2–/– cells was also restored after co-transfection with PS1 complementary DNA (data not shown). These observations extend to the molecular level our previous conclusion that the phenotype of PS1–/–PS2–/– embryos is similar to that of Notch-deficient embryos4,11. We further confirmed that proteolysis of mNotchΔE in PS1–/–PS2–/– cells was completely inhibited using a biochemical assay, as before7 (Fig. 1e ). Quantification of the obtained signals by phosphorimaging indicated that NICD production in PS1–/–PS2–/– cells was <1% of that observed in PS1+/+PS2–/– cells. In PS1+/+PS2–/– cells, a weak NICD signal was already observed after 30 min of pulse labelling (Fig. 1f), whereas it was never observed in PS1–/–PS2–/– cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Presenilin-deficient embryonic stem cells lack γ-secretase activity.

References

  1. De Strooper, B. et al. Nature 391, 387–390 (1998).

    Article  CAS  Google Scholar 

  2. Wolfe, M. S. et al. Nature 398, 513–517 (1999).

    Article  CAS  Google Scholar 

  3. Haass, C. & De Strooper, B. Science 286, 916–919 (1999).

    Article  CAS  Google Scholar 

  4. Herreman, A. et al. Proc. Natl Acad. Sci. USA 96, 11872–11877 (1999).

  5. Citron, M. et al. Nature 360, 672–674 (1992).

    Article  CAS  Google Scholar 

  6. Cai, X. D., Golde, T. E. & Younkin, S. G. Science 259, 514– 516 (1993).

    Article  CAS  Google Scholar 

  7. De Strooper, B. et al. Nature 398, 518–522 (1999).

  8. Song, W., Nadeau, P. & Yankner, B. A. Proc. Natl Acad. Sci. USA 96, 6959–6963 (1999).

    Article  CAS  Google Scholar 

  9. Schroeter, E. H., Kisslinger, J. A. & Kopan, R. Nature 393, 382– 386 (1998).

    Article  CAS  Google Scholar 

  10. Jarriault, S. et al. Nature 377, 355–358 (1995).

    Article  CAS  Google Scholar 

  11. Donoviel, D. B. et al. Genes Dev. 13, 2801– 2810 (1999).

    Article  CAS  Google Scholar 

  12. Kimberly, W. T., Xia, W. & Selkoe, D. J. J. Biol.Chem. 275, 3173– 3178 (2000).

    Article  CAS  Google Scholar 

  13. Steiner, H. et al. J. Biol. Chem. 274, 28669– 28673 (1999).

    Article  CAS  Google Scholar 

  14. Brown, M. S., Ye, J., Rawson, R. B. & Goldstein, J. L. Cell 100, 391–398 (2000).

    Article  CAS  Google Scholar 

  15. Annaert, W. G. et al. J. Cell Biol. 147, 277– 294 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Bayer Alzheimer research network (BARN), the Human Frontier of Science Program (HFSP) and the Fund for Scientific Research, Flanders (FWO).

Correspondence and requests for materials should be addressed to B.D.S.

Author information

Authors and Affiliations

  1. Neuronal Cell Biology Laboratory, CME, K.U.Leuven and Flanders Interuniversitary Institute for Biotechnology (VIB4), Herestraat 49, Leuven, 3000, Belgium

    An Herreman, Lutgarde Serneels, Wim Annaert & Bart De Strooper

  2. Thromb–X, NV, Herestraat 49, Leuven, 3000, Belgium

    Desiré Collen & Luc Schoonjans

Authors
  1. An Herreman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lutgarde Serneels
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wim Annaert
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Desiré Collen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Luc Schoonjans
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bart De Strooper
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bart De Strooper.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Herreman, A., Serneels, L., Annaert, W. et al. Total inactivation of γ–secretase activity in presenilin-deficient embryonic stem cells. Nat Cell Biol 2, 461–462 (2000). https://doi.org/10.1038/35017105

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35017105

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing