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η-Secretase processing of APP inhibits neuronal activity in the hippocampus

Nature volume 526, pages 443–447 (2015)Cite this article

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Abstract

Alzheimer disease (AD) is characterized by the accumulation of amyloid plaques, which are predominantly composed of amyloid-β peptide1. Two principal physiological pathways either prevent or promote amyloid-β generation from its precursor, β-amyloid precursor protein (APP), in a competitive manner1. Although APP processing has been studied in great detail, unknown proteolytic events seem to hinder stoichiometric analyses of APP metabolism in vivo2. Here we describe a new physiological APP processing pathway, which generates proteolytic fragments capable of inhibiting neuronal activity within the hippocampus. We identify higher molecular mass carboxy-terminal fragments (CTFs) of APP, termed CTF-η, in addition to the long-known CTF-α and CTF-β fragments generated by the α- and β-secretases ADAM10 (a disintegrin and metalloproteinase 10) and BACE1 (β-site APP cleaving enzyme 1), respectively. CTF-η generation is mediated in part by membrane-bound matrix metalloproteinases such as MT5-MMP, referred to as η-secretase activity. η-Secretase cleavage occurs primarily at amino acids 504–505 of APP695, releasing a truncated ectodomain. After shedding of this ectodomain, CTF-η is further processed by ADAM10 and BACE1 to release long and short Aη peptides (termed Aη-α and Aη-β). CTFs produced by η-secretase are enriched in dystrophic neurites in an AD mouse model and in human AD brains. Genetic and pharmacological inhibition of BACE1 activity results in robust accumulation of CTF-η and Aη-α. In mice treated with a potent BACE1 inhibitor, hippocampal long-term potentiation was reduced. Notably, when recombinant or synthetic Aη-α was applied on hippocampal slices ex vivo, long-term potentiation was lowered. Furthermore, in vivo single-cell two-photon calcium imaging showed that hippocampal neuronal activity was attenuated by Aη-α. These findings not only demonstrate a major functionally relevant APP processing pathway, but may also indicate potential translational relevance for therapeutic strategies targeting APP processing.

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Figure 1: A novel APP proteolytic processing pathway.
Figure 2: Inhibition of BACE1 results in increased levels of CTF-η and Aη-α.
Figure 3: Aη-α impairs hippocampal LTP.
Figure 4: Aη-α reduces neuronal activity in vivo.

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Acknowledgements

The authors thank S. Lammich, N. Exner and H. Steiner for critical comments. We thank A. Sülzen, N. Astola, S. Diederich, E. Grießinger and J. Gobbert for technical help. The APPPS1-21 colony was established from a breeding pair provided by M. Jucker. MT1-MMP−/− mouse brains were obtained from Z. Zhou. MT5-MMP−/− mouse brains were obtained from I. Farinas. We thank H. Jacobsen for the BACE1 inhibitor RO5508887. This work was supported by the European Research Council under the European Union’s Seventh Framework Program (FP7/2007–2013)/ERC grant agreement no. 321366-Amyloid (advanced grant to C.H.). The work of D.R.T. was supported by AFI (grant 13803). The research leading to these results has received funding (F.M. and D.H.) from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 318987 [TOPAG]. We thank J. Cox and M. Mann for critical discussions and the mass spectrometry infrastructure. We also acknowledge support by grants from Deutsche Forschungsgemeinschaft (MU 1457/9-1, 9-2 to U.M.) and the ERA-Net Neuron (01EW1305A to U.M.). Further support came from the ATIP/AVENIR program (Centre national de la recherche scientifique, CNRS) to H.M.; the French Fondation pour la Coopération Scientifique – Plan Alzheimer (Senior Innovative Grant 2010) to M.C. and H.M., and the French Government (National Research Agency, ANR) through the “Investments for the Future” LABEX SIGNALIFE: program reference ANR-11-LABX-0028-01 to S.K. M.A.B. was supported by the Langmatz Stiftung. F.J.L. is a Wellcome Trust Investigator. In vivo BACE1 inhibition experiments with APP transgenic mice were performed together with reMYND (Bio-Incubator, 3001 Leuven-Heverlee, Belgium).

Author information

Authors and Affiliations

  1. Biomedical Center (BMC), Ludwig-Maximilians-University Munich, Munich, 81377, Germany

    Michael Willem, Anna Daria, Heike Hampel, Veronika Müller, Camilla Giudici, Brigitte Nuscher & Christian Haass

  2. German Center for Neurodegenerative Diseases (DZNE) Munich, Munich, 81377, Germany

    Sabina Tahirovic, Saak V. Ovsepian, Andrea Wenninger-Weinzierl, Elisabeth Kremmer, Jochen Herms & Christian Haass

  3. Department of Psychiatry and Psychotherapy, Technische Universität München, Munich, 81675, Germany

    Marc Aurel Busche

  4. Institute of Neuroscience, Technische Universität München, Munich, 80802, Germany

    Marc Aurel Busche & Arthur Konnerth

  5. Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University Munich, Munich, 81377, Germany

    Marc Aurel Busche, Elisabeth Kremmer, Arthur Konnerth & Christian Haass

  6. Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Centre National de la Recherche Scientifique (CNRS), Université de Nice Sophia Antipolis, UMR 7275, Valbonne, 06560, France

    Magda Chafai, Scherazad Kootar & Hélène Marie

  7. Max Planck Institute of Biochemistry, Martinsried, 82152, Germany

    Daniel Hornburg & Felix Meissner

  8. Cambridge Stem Cell Institute & Department of Biochemistry, Gurdon Institute, University of Cambridge, Cambridge, CB2 1QN, UK

    Lewis D. B. Evans, Steven Moore & Frederick J. Livesey

  9. Institute of Molecular Immunology, German Research Center for Environmental Health, Munich, 81377, Germany

    Elisabeth Kremmer

  10. Department of Neurology, Clinical Neuroscience Unit, University of Bonn, Bonn, 53127, Germany

    Michael T. Heneka

  11. German Center for Neurodegenerative Diseases (DZNE) Bonn, Bonn, 53175, Germany

    Michael T. Heneka

  12. Institute of Pathology - Laboratory for Neuropathology, University of Ulm, Ulm, 89081, Germany

    Dietmar R. Thal

  13. Department of Public Health/Geriatrics, Uppsala University, Uppsala, 751 85, Sweden

    Vilmantas Giedraitis & Lars Lannfelt

  14. Institute for Pharmacy and Molecular Biotechnology IPMB, Functional Genomics, University of Heidelberg, Heidelberg, 69120, Germany

    Ulrike Müller

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  1. Michael Willem
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Contributions

M.W. and C.H. designed the study and interpreted the results. M.W. generated all biochemical data together with H.H., V.M., B.N. and C.G. S.T., supported by A.W.-W., provided primary neuronal cultures, performed and analysed immunohistological stainings, and together with A.D. performed LCM. D.R.T. provided and analysed human brain sections. M.T.H. provided CSF samples. U.M. provided APP-knockout mice. E.K. produced new monoclonal antibodies. D.H. and F.M. designed and conducted mass spectrometry and data analysis. L.D.B.E., S.M. and F.J.L. carried out BACE1 inhibition of human neurons. H.M. together with M.C. and S.K. performed all electrophysiological recordings (LTP) in vitro and analysis in relation to application of peptides. S.V.O. and J.H. performed all electrophysiological recordings (LTP) in vitro in relation to the BACE1 inhibitor tests. M.A.B and A.K. performed all Ca2+-imaging experiments in vivo and analysis. M.W. and C.H. wrote the manuscript with input from the other authors. Correspondence and requests for materials should be addressed to M.W. and C.H.

Corresponding authors

Correspondence to Michael Willem or Christian Haass.

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Competing interests

M.W. and C.H. have filed a patent for analytical and therapeutic use of Aη peptides.

Extended data figures and tables

Extended Data Figure 1 Schematic presentation of the η-secretase processing pathway.

Schematic representation of the η-secretase pathway (left) as compared to the amyloidogenic pathway (right). Antibodies used in this study are indicated.

Extended Data Figure 2 η-Secretase cleavage at Met505 of APP695.

MMP proteins can cleave human APP695 at the indicated position (arrow) between amino acids N504 and M505 in the N-terminal domain. The epitope for the neo-epitope-specific antibody 10A8 is indicated (grey line). sAPP-η was specifically detected in diethylamine (DEA; 0.2% diethylamine in 50 mM NaCl, pH 10) extracts of P10 wild-type mouse brain using antibody 10A8, but was absent in APP-knockout brains. Of note, antibody 10A8 failed to detect sAPP-α/β, confirming its selectivity for the η-cleavage site.

Extended Data Figure 3 Mass spectrometry analysis of Aη peptides.

a, b, After removal of sAPP-α from conditioned media of CHO 7PA2 cells using appropriate centricon filters, the flow-through was used to isolate Aη peptides by immunoprecipitation. Synthetic peptides (1 ng per lane) were loaded to indicate the respective sizes of Aη-α and Aη-β. Aη peptides were captured with antibodies 9476M and 9478D directed against the putative N-terminal epitopes (Supplementary Table 1), 2E9 against a middle domain of Aη and 2D8 (which also immunoprecipitates amyloid-β). 2D8 detection (a) revealed that all antibodies captured peptides positive for the N-terminal part of the amyloid-β domain in a molecular mass range of synthetic Aη-α between 12 and 16 kDa. The same samples analysed with 2E9 (b) confirmed the presence of Aη in all samples, with the lowest levels when precipitated with 9476M. Note that unlike 2D8 antibody, 2E9 allows the additional detection of Aη-β. (Asterisks denote IgG.) c, A heat map of peptides identified after analytic proteolysis by mass spectrometry analysis, with 7PA2 supernatants immunoprecipitated with 2D8, 2E9, 9476M and 9478D antibodies. Arrows indicate peptides that start exactly with the amino acid sequence C-terminal of the respective cleavage sites of the η-secretase, β-secretase or α-secretase site. Chy, chymotrypsin; LysC, protease LysC; tryp, trypsin.

Extended Data Figure 4 MT5-MMP displays η-secretase activity in brain.

a–c, MT1-MMP−/− and MT5-MMP−/− (also known as Mmp14−/− and Mmp24−/−, respectively) mice were analysed for changes in η-secretase activity. Membrane and soluble proteins from P10 mouse brains were analysed. a, In RIPA lysates, no changes in APP and BACE1 levels were detected in knockout brains. MT1- and MT5-MMP were selectively knocked out as shown by the lack of signals in western blots. Calnexin served as a loading control. Soluble Aη levels, detected by antibodies 9478D and M3.2 (Aη-α) were unchanged in MT1-MMP−/− mouse brains (b), but reduced in MT5-MMP−/− mouse brains (c). Total levels of secreted APP (22C11), sAPP-α or sAPP-β were unchanged (b, c).

Extended Data Figure 5 Accumulation of CTF-η in dystrophic neurites.

a, Immunohistological stainings of cortical sections of 6-month-old APPPS1-21 transgenic mice (n = 3) revealed 6E10-positive amyloid-β plaque cores (encircled) surrounded by dystrophic neurites positive for 2E9 (white arrowheads, top) and 9476M (white arrowheads, middle). Y188 (bottom panel) staining co-localized with 2E9-positive signal (yellow arrowheads, bottom). Nuclei were counterstained with DAPI. Scale bar, 10 μm. b, Accumulation of CTF-η fragment in dystrophic neurites of 14-month and 24-month-old APPPS1-21 mice. Western blot analysis of material obtained by LCM of APPPS1-21 brain sections (n = 5) bearing thioflavin-S-positive amyloid-β plaque core (P) and the surrounding amyloid-β plaque halo (H). As a control (C), brain areas devoid of plaques were used. While amyloid-β was readily detected by antibody 2D8 in lysates containing plaque-enriched material and halo regions (fractions P and H; bottom), CTF-η was selectively detected in the lysates prepared from the region enriched in dystrophic neurites (H; top), but not detected in plaque or control regions (P or C). As expected, CTF-β/α species are also enriched in dystrophic neurites (H).

Extended Data Figure 6 Dystrophic neurites in AD brains are positive for Aη-epitope antibodies.

a–f, Immunohistochemistry with 22C11 (a, b), 9478D (c, d) and 9476M (e, f) antibodies in the human hippocampus (CA1-subiculum region) of a control case (a, c, e) and an AD case (b, d, f). Immuno-positive signals were observed with 22C11 (a), 9478D (c) and 9476M (e) antibodies in the somata and neuropils of a normal and AD brain. In AD brains, these antibodies decorate dystrophic neurites (b, d, f, denoted by arrowheads). Scale bar, 30 μm. NP, neuritic plaque.

Extended Data Figure 7 Increased Aη levels after acute treatment with a BACE1 inhibitor.

a, In membrane lysates of brains obtained from animals treated with BACE1 inhibitor (BI, 100 mg kg−1 SCH1682496), an increase in CTF-η was observed, which was paralleled by a strong reduction of CTF-β, while CTF-α was unchanged. APP-FL and BACE1 signals remained unchanged (asterisk indicates background band). Calnexin served as a loading control. b, In the soluble fraction, BACE1 inhibition resulted in enhanced Aη-α levels and reduced sAPP-β levels, indicating efficient BACE1 inhibition. c, Production of Aη-α species, which was detected by antibody M3.2, revealed a 95.4% increase after BACE1 inhibition. n = 3; P < 0.01, Student’s t-test.

Extended Data Figure 8 Aη-α and Aη-β derived from CHO cells did not influence baseline activity at the hippocampal CA3–CA1 synapse.

Soluble Aη-α and Aη-β peptides were expressed in CHO cells and collected in OPTIMEM medium. a, b, SEC fractions containing Aη were diluted (1:15) in ACSF for the treatment of hippocampal slices and LTP measurements. Aη-α or Αη-β SEC fractions were perfused over mouse hippocampal slices after obtaining a 15-min stable baseline of a fEPSP at the CA3–CA1 synapse. The baseline remained unchanged for another 15 min when slices were incubated with CHO-cell-derived recombinant Aη-α (a) or Aη-β (b).

Extended Data Figure 9 AβS26C dimers and synthetic Aη-α impair hippocampal LTP.

a, In line with previous findings23, AβS26C cross-linked dimers (containing cysteine instead of serine at residue 26; 100 nM final; JPT Peptide Technologies; diluted in 25 ml re-circulating ACSF) reduced LTP as compared to interleaved control LTP recordings in 25 ml re-circulating ACSF. b, Illustrated is the average LTP magnitude (at 45–60 min after LTP induction) normalized to pre-LTP baseline values (100%) in untreated and treated conditions (***P < 0.001; Student’s t-test). c, Treatment with synthetic Aη-α (100 nM final; Peptide Speciality Laboratories; diluted in 25 ml re-circulating ACSF) reduced LTP as compared to interleaved control LTP recordings in 25 ml re-circulating ACSF. d, Illustrated is the average LTP magnitude (at 45–60 min post-LTP induction) normalized to pre-LTP baseline values (100%) in treated and untreated conditions (*P < 0.05; Student’s t-test).

Extended Data Figure 10 Aη-α decreases the frequencies of neuronal calcium transients in vivo.

a–f, Histograms showing in each panel the corresponding distributions of calcium transients before (control) and during subsequent exposure of Aη peptides (b, c, e, f) and controls (a, d).

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Willem, M., Tahirovic, S., Busche, M. et al. η-Secretase processing of APP inhibits neuronal activity in the hippocampus. Nature 526, 443–447 (2015). https://doi.org/10.1038/nature14864

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