title: Seizure protein 6 and its homolog seizure 6-like protein are physiological substrates of BACE1 in neurons.
abstract: The protease BACE1 (beta-site APP cleaving enzyme) is a major drug target in Alzheimer's disease. However, BACE1 therapeutic inhibition may cause unwanted adverse effects due to its additional functions in the nervous system, such as in myelination and neuronal connectivity. Additionally, recent proteomic studies investigating BACE1 inhibition in cell lines and cultured murine neurons identified a wider range of neuronal membrane proteins as potential BACE1 substrates, including seizure protein 6 (SEZ6) and its homolog SEZ6L.
  
  
  
  We generated antibodies against SEZ6 and SEZ6L and validated these proteins as BACE1 substrates in vitro and in vivo. Levels of the soluble, BACE1-cleaved ectodomain of both proteins (sSEZ6, sSEZ6L) were strongly reduced upon BACE1 inhibition in primary neurons and also in vivo in brains of BACE1-deficient mice. BACE1 inhibition increased neuronal surface levels of SEZ6 and SEZ6L as shown by cell surface biotinylation, demonstrating that BACE1 controls surface expression of both proteins. Moreover, mass spectrometric analysis revealed that the BACE1 cleavage site in SEZ6 is located in close proximity to the membrane, similar to the corresponding cleavage site in SEZ6L. Finally, an improved method was developed for the proteomic analysis of murine cerebrospinal fluid (CSF) and was applied to CSF from BACE-deficient mice. Hereby, SEZ6 and SEZ6L were validated as BACE1 substrates in vivo by strongly reduced levels in the CSF of BACE1-deficient mice.
  
  
  
  This study demonstrates that SEZ6 and SEZ6L are physiological BACE1 substrates in the murine brain and suggests that sSEZ6 and sSEZ6L levels in CSF are suitable markers to monitor BACE1 inhibition in mice.
authors.affiliation: German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.|Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Pigoni
authors.affiliation: German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.|Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Wanngren
authors.affiliation: Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.|Institute for Advanced Study, Technische Universität München, Munich, Germany.|Institute for Pathology und Pathological Anatomy, Technische Universität München, Munich, Germany.
authors.forename: Peer-Hendrik
authors.initials: PH
authors.lastname: Kuhn
authors.affiliation: Department of Anatomy and Neuroscience, University of Melbourne, Victoria, Australia.
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Munro
authors.affiliation: Department of Anatomy and Neuroscience, University of Melbourne, Victoria, Australia.|The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Victoria, Australia.
authors.forename: Jenny M
authors.initials: JM
authors.lastname: Gunnersen
authors.affiliation: Division of Pharmaceutical Sciences, Graduate School and Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takeshima
authors.affiliation: German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.|Institute for Diabetes and Obesity, Monoclonal Antibody Research Group, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Munich, Germany.|Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
authors.forename: Regina
authors.initials: R
authors.lastname: Feederle
authors.affiliation: VIB Center for the Biology of Disease, Leuven, Belgium.
authors.forename: Iryna
authors.initials: I
authors.lastname: Voytyuk
authors.affiliation: VIB Center for the Biology of Disease, Leuven, Belgium.|Center for Human Genetics, and Leuven Institute for Neurodegenerative Diseases (LIND), University of Leuven (KU Leuven), Leuven, Belgium.|Institute of Neurology, University College London, London, UK.
authors.forename: Bart
authors.initials: B
authors.lastname: De Strooper
authors.affiliation: Department of Neuroscience, Janssen Pharmaceutica NV, Beerse, Belgium.
authors.forename: Mikail D
authors.initials: MD
authors.lastname: Levasseur
authors.affiliation: Department of Neuroscience, Janssen Pharmaceutica NV, Beerse, Belgium.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Hrupka
authors.affiliation: German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.|Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Müller
authors.affiliation: German Center for Neurodegenerative Diseases (DZNE), Munich, Germany. stefan.lichtenthaler@dzne.de.|Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany. stefan.lichtenthaler@dzne.de.|Institute for Advanced Study, Technische Universität München, Munich, Germany. stefan.lichtenthaler@dzne.de.|Munich Cluster for Systems Neurology (SyNergy), Munich, Germany. stefan.lichtenthaler@dzne.de.
authors.forename: Stefan F
authors.initials: SF
authors.lastname: Lichtenthaler
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D015415:Biomarkers / Q000134:cerebrospinal fluid; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D015415:Biomarkers; D009419:Nerve Tissue Proteins; C093131:Sez6 protein, mouse; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509282:Bace1 protein, mouse
keywords: Alzheimer’s disease; BACE1; BACE2; Biomarker; Neuroproteomics; SEZ6; SEZ6L; Secretase