[en] Alzheimer disease (AD) is characterized by the accumulation of amyloid plaques, which are predominantly composed of amyloid-β peptide. Two principal physiological pathways either prevent or promote amyloid-β generation from its precursor, β-amyloid precursor protein (APP), in a competitive manner. Although APP processing has been studied in great detail, unknown proteolytic events seem to hinder stoichiometric analyses of APP metabolism in vivo. 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.
Disciplines :
Neurology
Author, co-author :
Willem, Michael; Biomedical Center (BMC), Ludwig-Maximilians-University Munich, 81377 Munich, Germany
Tahirovic, Sabina; German Center for Neurodegenerative Diseases (DZNE) Munich, 81377 Munich, Germany
Busche, Marc Aurel; Department of Psychiatry and Psychotherapy, Technische Universität München, 81675 Munich, Germany ; Institute of Neuroscience, Technische Universität München, 80802 Munich, Germany ; Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University Munich, 81377 Munich, Germany
Ovsepian, Saak V; German Center for Neurodegenerative Diseases (DZNE) Munich, 81377 Munich, Germany
Chafai, Magda; Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Centre National de la Recherche Scientifique (CNRS), Université de Nice Sophia Antipolis, UMR 7275, 06560 Valbonne, France
Kootar, Scherazad; Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Centre National de la Recherche Scientifique (CNRS), Université de Nice Sophia Antipolis, UMR 7275, 06560 Valbonne, France
Hornburg, Daniel; Max Planck Institute of Biochemistry, Martinsried 82152, Germany
Evans, Lewis D B; Gurdon Institute, Cambridge Stem Cell Institute &Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, UK
Moore, Steven; Gurdon Institute, Cambridge Stem Cell Institute &Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, UK
Daria, Anna; Biomedical Center (BMC), Ludwig-Maximilians-University Munich, 81377 Munich, Germany
Hampel, Heike; Biomedical Center (BMC), Ludwig-Maximilians-University Munich, 81377 Munich, Germany
Müller, Veronika; Biomedical Center (BMC), Ludwig-Maximilians-University Munich, 81377 Munich, Germany
Giudici, Camilla; Biomedical Center (BMC), Ludwig-Maximilians-University Munich, 81377 Munich, Germany
Nuscher, Brigitte; Biomedical Center (BMC), Ludwig-Maximilians-University Munich, 81377 Munich, Germany
Wenninger-Weinzierl, Andrea; German Center for Neurodegenerative Diseases (DZNE) Munich, 81377 Munich, Germany
Kremmer, Elisabeth; German Center for Neurodegenerative Diseases (DZNE) Munich, 81377 Munich, Germany ; Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University Munich, 81377 Munich, Germany ; Institute of Molecular Immunology, German Research Center for Environmental Health, 81377 Munich, Germany
HENEKA, Michael ; Department of Neurology, Clinical Neuroscience Unit, University of Bonn, 53127 Bonn, Germany ; German Center for Neurodegenerative Diseases (DZNE) Bonn, 53175 Bonn, Germany
Thal, Dietmar R; Institute of Pathology - Laboratory for Neuropathology, University of Ulm, 89081 Ulm, Germany
Giedraitis, Vilmantas; Department of Public Health/Geriatrics, Uppsala University, 751 85 Uppsala, Sweden
Lannfelt, Lars; Department of Public Health/Geriatrics, Uppsala University, 751 85 Uppsala, Sweden
Müller, Ulrike; Institute for Pharmacy and Molecular Biotechnology IPMB, Functional Genomics, University of Heidelberg, 69120 Heidelberg, Germany
Livesey, Frederick J; Gurdon Institute, Cambridge Stem Cell Institute &Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, UK
Meissner, Felix; Max Planck Institute of Biochemistry, Martinsried 82152, Germany
Herms, Jochen; German Center for Neurodegenerative Diseases (DZNE) Munich, 81377 Munich, Germany
Konnerth, Arthur; Institute of Neuroscience, Technische Universität München, 80802 Munich, Germany ; Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University Munich, 81377 Munich, Germany
Marie, Hélène; Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Centre National de la Recherche Scientifique (CNRS), Université de Nice Sophia Antipolis, UMR 7275, 06560 Valbonne, France
Haass, Christian; Biomedical Center (BMC), Ludwig-Maximilians-University Munich, 81377 Munich, Germany ; German Center for Neurodegenerative Diseases (DZNE) Munich, 81377 Munich, Germany ; Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University Munich, 81377 Munich, Germany
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-MMP2/2 mouse brains were obtained from Z. Zhou. MT5-MMP2/2 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).
Haass, C. & Selkoe, D. J. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid b-peptide. Nature Rev. Mol. Cell Biol. 8, 101-112 (2007).
Dobrowolska, J. A. et al. CNS amyloid-b, soluble APP - A and-b kinetics duringBACE inhibition. J. Neurosci. 34, 8336-8346 (2014).
Haass, C., Koo, E. H., Mellon, A., Hung, A. Y. & Selkoe, D. J. Targeting of cell-surface b-amyloid precursor protein to lysosomes: alternative processing into amyloidbearing fragments. Nature 357, 500-503 (1992).
Estus, S. et al. Potentially amyloidogenic, carboxyl-terminal derivatives of the amyloid protein precursor. Science 255, 726-728 (1992).
Haass, C. et al. b-Amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms. J. Biol. Chem. 268, 3021-3024 (1993).
Mü ller, U. et al. Behavioral and anatomical deficits in mice homozygous for a modified b-amyloid precursor protein gene. Cell 79, 755-765 (1994).
Nikolaev, A., McLaughlin, T., O'Leary, D. D. & Tessier-Lavigne,M. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature 457, 981-989 (2009).
Jefferson, T. et al.MetalloproteasemeprinbgeneratesnontoxicN-terminalamyloid precursor protein fragments in vivo. J. Biol. Chem. 286, 27741-27750 (2011).
Vella, L. J. & Cappai, R. Identification of a novel amyloid precursor protein processing pathway that generates secreted N-terminal fragments. FASEB J. 26, 2930-2940 (2012).
Radde, R. et al. Ab42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology. EMBO Rep. 7, 940-946 (2006).
Portelius, E. et al. Mass spectrometric characterization of amyloid-b species in the 7PA2 cell model of Alzheimer's disease. J. Alzheimers Dis. 33, 85-93 (2013).
Welzel, A. T. et al. Secreted amyloid b-proteins in a cell culture model include N-terminally extended peptides that impair synaptic plasticity. Biochemistry 53, 3908-3921 (2014).
Higashi, S.&Miyazaki,K.Novel processingof b-amyloidprecursor protein catalyzed bymembrane type 1 matrixmetalloproteinase releases a fragment lacking the inhibitor domain against gelatinase A. Biochemistry 42, 6514-6526 (2003).
Ahmad, M. et al. Cleavage of amyloid-b precursor protein (APP) by membranetype matrix metalloproteinases. J. Biochem. 139, 517-526 (2006).
Folgueras, A. R. et al. Metalloproteinase MT5-MMP is an essential modulator of neuro-immune interactions in thermal pain stimulation. Proc. Natl Acad. Sci. USA 106, 16451-16456 (2009).
Zhou, Z. et al. Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I. Proc. Natl Acad. Sci. USA 97, 4052-4057 (2000).
Shi, Y., Kirwan,P.&Livesey, F. J. Directed differentiation ofhuman pluripotent stem cells to cerebral cortex neurons and neural networks. Nature Protocols 7, 1836-1846 (2012).
Moechars, D. et al. Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain. J. Biol. Chem. 274, 6483-6492 (1999).
Cai, H. et al. BACE1 is the major b-secretase for generation of Ab peptides by neurons. Nature Neurosci. 4, 233-234 (2001).
Radde, R. et al. Ab42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology. EMBO Rep. 7, 940-946 (2006).
Walsh,D.M. et al.Naturally secreted oligomers ofamyloid b protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535-539 (2002).
Shankar, G. M. et al. Natural oligomers of the Alzheimer amyloid-b protein induce reversible synapse loss by modulating an NMDA-type glutamate receptordependent signaling pathway. J. Neurosci. 27, 2866-2875 (2007).
Shankar, G. M. et al. Amyloid-b protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nature Med. 14, 837-842 (2008).
Busche, M. A. et al. Clusters of hyperactive neurons near amyloid plaques in a mouse model of Alzheimer's disease. Science 321, 1686-1689 (2008).
Busche, M. A. et al. Critical role of soluble amyloid-b for early hippocampal hyperactivity in a mouse model of Alzheimer's disease. Proc. Natl Acad. Sci. USA 109, 8740-8745 (2012).
Haass, C. Take five-BACE and the c-secretase quartet conduct Alzheimer's amyloid b-peptide generation. EMBO J. 23, 483-488 (2004).
Sekine-Aizawa, Y. et al. Matrix metalloproteinase (MMP) system in brain: identification and characterization of brain-specific MMP highly expressed in cerebellum. Eur. J. Neurosci. 13, 935-948 (2001).
Jonsson, T. et al. Amutation in APP protects against Alzheimer's disease and agerelated cognitive decline. Nature 488, 96-99 (2012).
Kuhn, P. H. et al. Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons. EMBO J. 31, 3157-3168 (2012).
Zhou, L. et al. The neural cell adhesion molecules L1 and CHL1 are cleaved by BACE1 protease in vivo. J. Biol. Chem. 287, 25927-25940 (2012).
Podlisny, M. B. et al. Aggregation of secreted amyloid b-protein into sodium dodecyl sulfate-stable oligomers in cell-culture. J. Biol. Chem. 270, 9564-9570 (1995).
Kaech, S. & Banker, G. Culturing hippocampal neurons. Nature Protocols 1, 2406-2415 (2006).
Israel, M. A. et al. Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells. Nature 482, 216-220 (2012).
Shi, Y., Kirwan, P., Smith, J., Robinson, H. P. & Livesey, F. J. Human cerebral cortex development frompluripotent stemcells to functional excitatory synapses. Nature Neurosci. 15, 477-486 (2012).
Jacobsen, H. et al. Combined treatmentwithaBACEinhibitor and anti-Ab antibody gantenerumab enhances amyloid reduction in APPLondon mice. J. Neurosci. 34, 11621-11630 (2014).
Nolan, R. L.& Teller, J. K.Diethylamine extraction of proteins and peptides isolated with a mono-phasic solution of phenol and guanidine isothiocyanate. J. Biochem. Biophys. Methods 68, 127-131 (2006).
Westmeyer, G. G. et al. Dimerization of b-site b-amyloid precursor protein-cleaving enzyme. J. Biol. Chem. 279, 53205-53212 (2004).
Rappsilber, J.,Mann, M.&Ishihama, Y. Protocol formicro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nature Protocols 2, 1896-1906 (2007).
Scheltema,R. A. et al. TheQExactiveHF, aBenchtopmass spectrometer with a prefilter, high-performancequadrupole and anultra-high-field Orbitrap analyzer.Mol. Cell. Proteomics 13, 3698-3708 (2014).
Olsen, J. V. et al. Higher-energy C-trap dissociation for peptide modification analysis. Nature Methods 4, 709-712 (2007).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnol. 26, 1367-1372 (2008).
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794-1805 (2011).
R Development Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, http://www.R-project.org/(2014).
Haass, C. et al. The Swedish mutation causes early-onset Alzheimer's-disease by b-secretase cleavage within the secretory pathway. Nature Med. 1, 1291-1296 (1995).
McKhann, G. et al. Clinical diagnosis of Alzheimer's disease: report of theNINCDSADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology 34, 939-944 (1984).
Lannfelt, L. et al. Amyloid precursor protein mutation causes Alzheimer's disease in a Swedish family. Neurosci. Lett. 168, 254-256 (1994).
Lannfelt, L. et al. Amyloid b-peptide in cerebrospinal fluid in individuals with the Swedish Alzheimer amyloid precursor protein mutation. Neurosci. Lett. 199, 203-206 (1995).
Braak, H. & Braak, E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol. 82, 239-259 (1991).
Mirra, S. S. et al. The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease. Neurology 41, 479-486 (1991).
Hyman, B. T. et al. National Institute on Aging-Alzheime's Association guidelines for the neuropathologic assessment of Alzheimer's disease. Alzheimers Dement. 8, 1-13 (2012).
Liao, L. et al. Proteomic characterization of postmortem amyloid plaques isolated by laser capture microdissection. J. Biol. Chem. 279, 37061-37068 (2004).
Houeland, G. et al. Transgenicmice with chronic NGF deprivation and Alzheimer's disease-like pathology display hippocampal region-specific impairments in shortand long-term plasticities. J. Neurosci. 30, 13089-13094 (2010).
Townsend, M., Shankar, G. M., Mehta, T., Walsh, D. M. & Selkoe, D. J. Effects of secreted oligomers of amyloid beta-protein on hippocampal synaptic plasticity: a potent role for trimers. J. Physiol. (Lond.) 572, 477-492 (2006).
Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl Acad. Sci. USA 100, 7319-7324 (2003).
