Top

Published in:

Open Access 01-12-2016 | Research article

Seizure protein 6 and its homolog seizure 6-like protein are physiological substrates of BACE1 in neurons

Authors: Martina Pigoni, Johanna Wanngren, Peer-Hendrik Kuhn, Kathryn M. Munro, Jenny M. Gunnersen, Hiroshi Takeshima, Regina Feederle, Iryna Voytyuk, Bart De Strooper, Mikail D. Levasseur, Brian J. Hrupka, Stephan A. Müller, Stefan F. Lichtenthaler

Published in: Molecular Neurodegeneration | Issue 1/2016

Abstract

Background

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.

Methods and results

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.

Conclusions

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.

Available only for authorised users

Vassar R, Kuhn PH, Haass C, Kennedy ME, Rajendran L, Wong PC, Lichtenthaler SF. Function, therapeutic potential and cell biology of BACE proteases: current status and future prospects. J Neurochem. 2014;130:4–28.CrossRefPubMedPubMedCentral

Hussain I, Powell D, Howlett DR, Tew DG, Meek TD, Chapman C, Gloger IS, Murphy KE, Southan CD, Ryan DM, et al. Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol Cell Neurosci. 1999;14:419–27.CrossRefPubMed

Sinha S, Anderson JP, Barbour R, Basi GS, Caccavello R, Davis D, Doan M, Dovey HF, Frigon N, Hong J, et al. Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature. 1999;402:537–40.CrossRefPubMed

Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, et al. Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science. 1999;286:735–41.CrossRefPubMed

Yan R, Bienkowski MJ, Shuck ME, Miao H, Tory MC, Pauley AM, Brashier JR, Stratman NC, Mathews WR, Buhl AE, et al. Membrane-anchored aspartyl protease with Alzheimer’s disease beta-secretase activity. Nature. 1999;402:533–7.CrossRefPubMed

Selkoe DJ, Hardy J. The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol Med. 2016;8:595–608.CrossRefPubMed

Willem M, Garratt AN, Novak B, Citron M, Kaufmann S, Rittger A, DeStrooper B, Saftig P, Birchmeier C, Haass C. Control of peripheral nerve myelination by the beta-secretase BACE1. Science. 2006;314:664–6.CrossRefPubMed

Hu X, Hicks CW, He W, Wong P, Macklin WB, Trapp BD, Yan R. Bace1 modulates myelination in the central and peripheral nervous system. Nat Neurosci. 2006;9:1520–5.CrossRefPubMed

Fleck D, van Bebber F, Colombo A, Galante C, Schwenk BM, Rabe L, Hampel H, Novak B, Kremmer E, Tahirovic S, et al. Dual cleavage of neuregulin 1 type III by BACE1 and ADAM17 liberates its EGF-like domain and allows paracrine signaling. J Neurosci. 2013;33:7856–69.CrossRefPubMed

10.

Cheret C, Willem M, Fricker FR, Wende H, Wulf-Goldenberg A, Tahirovic S, Nave KA, Saftig P, Haass C, Garratt AN, et al. Bace1 and Neuregulin-1 cooperate to control formation and maintenance of muscle spindles. Embo J. 2013;32:2015–28.CrossRefPubMedPubMedCentral

11.

Hitt B, Riordan SM, Kukreja L, Eimer WA, Rajapaksha TW, Vassar R. beta-Site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1)-deficient mice exhibit a close homolog of L1 (CHL1) loss-of-function phenotype involving axon guidance defects. J Biol Chem. 2012;287:38408–25.CrossRefPubMedPubMedCentral

12.

Barao S, Gartner A, Leyva-Diaz E, Demyanenko G, Munck S, Vanhoutvin T, Zhou L, Schachner M, Lopez-Bendito G, Maness PF, De Strooper B. Antagonistic effects of BACE1 and APH1B-gamma-secretase control axonal guidance by regulating growth cone collapse. Cell Rep. 2015;12:1367–76.CrossRefPubMedPubMedCentral

13.

Barao S, Moechars D, Lichtenthaler SF, De Strooper B. BACE1 physiological functions may limit its use as therapeutic target for alzheimer’s disease. Trends Neurosci. 2016;39:158–69.CrossRefPubMed

14.

Dislich B, Wohlrab F, Bachhuber T, Müller SA, Kuhn P-H, Hogl S, Meyer-Luehmann M, Lichtenthaler SF. Label-free quantitative proteomics of mouse cerebrospinal fluid detects β-Site APP Cleaving Enzyme (BACE1) protease substrates in vivo. Mol Cell Proteomics. 2015;14:2550–63.CrossRefPubMedPubMedCentral

15.

Kuhn PH, Koroniak K, Hogl S, Colombo A, Zeitschel U, Willem M, Volbracht C, Schepers U, Imhof A, Hoffmeister A, et al. Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons. Embo J. 2012;31:3157–68.CrossRefPubMedPubMedCentral

16.

Zhou L, Barao S, Laga M, Bockstael K, Borgers M, Gijsen H, Annaert W, Moechars D, Mercken M, Gevaert K, De Strooper B. The neural cell adhesion molecules L1 and CHL1 are cleaved by BACE1 protease in vivo. J Biol Chem. 2012;287:25927–40.CrossRefPubMedPubMedCentral

17.

Stutzer I, Selevsek N, Esterhazy D, Schmidt A, Aebersold R, Stoffel M. Systematic proteomic analysis identifies beta-site amyloid precursor protein cleaving enzyme 2 and 1 (BACE2 and BACE1) substrates in pancreatic beta-cells. J Biol Chem. 2013;288:10536–47.CrossRefPubMedPubMedCentral

18.

Gunnersen JM, Kim MH, Fuller SJ, De Silva M, Britto JM, Hammond VE, Davies PJ, Petrou S, Faber ES, Sah P, Tan SS. Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons. Neuron. 2007;56:621–39.CrossRefPubMed

19.

Miyazaki T, Hashimoto K, Uda A, Sakagami H, Nakamura Y, Saito SY, Nishi M, Kume H, Tohgo A, Kaneko I, et al. Disturbance of cerebellar synaptic maturation in mutant mice lacking BSRPs, a novel brain-specific receptor-like protein family. FEBS Lett. 2006;580:4057–64.CrossRefPubMed

20.

Dominguez D, Tournoy J, Hartmann D, Huth T, Cryns K, Deforce S, Serneels L, Camacho IE, Marjaux E, Craessaerts K, et al. Phenotypic and biochemical analyses of BACE1- and BACE2-deficient mice. J Biol Chem. 2005;280:30797–806.CrossRefPubMed

21.

Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256:495–7.CrossRefPubMed

22.

Li MZ, Elledge SJ. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods. 2007;4:251–6.CrossRefPubMed

23.

Brodney MA, Beck EM, Butler CR, Barreiro G, Johnson EF, Riddell D, Parris K, Nolan CE, Fan Y, Atchison K, et al. Utilizing structures of CYP2D6 and BACE1 complexes to reduce risk of drug-drug interactions with a novel series of centrally efficacious BACE1 inhibitors. J Med Chem. 2015;58:3223–52.CrossRefPubMedPubMedCentral

24.

Jeppsson F, Eketjall S, Janson J, Karlstrom S, Gustavsson S, Olsson LL, Radesater AC, Ploeger B, Cebers G, Kolmodin K, et al. Discovery of AZD3839, a potent and selective BACE1 inhibitor clinical candidate for the treatment of Alzheimer disease. J Biol Chem. 2012;287:41245–57.CrossRefPubMedPubMedCentral

25.

Malamas MS, Barnes K, Johnson M, Hui Y, Zhou P, Turner J, Hu Y, Wagner E, Fan K, Chopra R, et al. Di-substituted pyridinyl aminohydantoins as potent and highly selective human beta-secretase (BACE1) inhibitors. Bioorg Med Chem. 2010;18:630–9.CrossRefPubMed

26.

Mitterreiter S, Page RM, Kamp F, Hopson J, Winkler E, Ha HR, Hamid R, Herms J, Mayer TU, Nelson DJ, et al. Bepridil and amiodarone simultaneously target the Alzheimer’s disease beta- and gamma-secretase via distinct mechanisms. J Neurosci. 2010;30:8974–83.CrossRefPubMed

27.

Liu L, Duff K. A technique for serial collection of cerebrospinal fluid from the cisterna magna in mouse. J Vis Exp. 2008. doi:10.3791/960.PubMedPubMedCentral

28.

Schagger H. Tricine-SDS-PAGE. Nat Protoc. 2006;1:16–22.CrossRefPubMed

29.

Lichtenthaler SF, Dominguez DI, Westmeyer GG, Reiss K, Haass C, Saftig P, De Strooper B, Seed B. The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1. J Biol Chem. 2003;278:48713–9.CrossRefPubMed

30.

Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics. 2014;13:2513–26.CrossRefPubMedPubMedCentral

31.

Rappsilber J, Ishihama Y, Mann M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem. 2003;75:663–70.CrossRefPubMed

32.

Shimizu-Nishikawa K, Kajiwara K, Kimura M, Katsuki M, Sugaya E. Cloning and expression of SEZ-6, a brain-specific and seizure-related cDNA. Brain Res Mol Brain Res. 1995;28:201–10.CrossRefPubMed

33.

Halim A, Ruetschi U, Larson G, Nilsson J. LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins. J Proteome Res. 2013;12:573–84.CrossRefPubMed

34.

Allen Mouse Brain Atlas [http://mouse.brain-map.org/]

35.

Stachel SJ, Coburn CA, Steele TG, Jones KG, Loutzenhiser EF, Gregro AR, Rajapakse HA, Lai MT, Crouthamel MC, Xu M, et al. Structure-based design of potent and selective cell-permeable inhibitors of human beta-secretase (BACE-1). J Med Chem. 2004;47:6447–50.CrossRefPubMed

36.

Colombo A, Wang H, Kuhn PH, Page R, Kremmer E, Dempsey PJ, Crawford HC, Lichtenthaler SF. Constitutive alpha- and beta-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines. Neurobiol Dis. 2013;49:137–47.CrossRefPubMed

37.

Reiss K, Cornelsen I, Husmann M, Gimpl G, Bhakdi S. Unsaturated fatty acids drive disintegrin and metalloproteinase (ADAM)-dependent cell adhesion, proliferation, and migration by modulating membrane fluidity. J Biol Chem. 2011;286:26931–42.CrossRefPubMedPubMedCentral

38.

Kuhn PH, Colombo AV, Schusser B, Dreymueller D, Wetzel S, Schepers U, Herber J, Ludwig A, Kremmer E, Montag D, et al. Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function. Elife. 2016; 5, doi: 10.7554/eLife.12748.

39.

Jorissen E, Prox J, Bernreuther C, Weber S, Schwanbeck R, Serneels L, Snellinx A, Craessaerts K, Thathiah A, Tesseur I, et al. The disintegrin/metalloproteinase ADAM10 is essential for the establishment of the brain cortex. J Neurosci. 2010;30:4833–44.CrossRefPubMedPubMedCentral

40.

Gruninger-Leitch F, Schlatter D, Kung E, Nelbock P, Dobeli H. Substrate and inhibitor profile of BACE (beta-secretase) and comparison with other mammalian aspartic proteases. J Biol Chem. 2002;277:4687–93.CrossRefPubMed

41.

Lichtenthaler SF, Haass C, Steiner H. Regulated intramembrane proteolysis--lessons from amyloid precursor protein processing. J Neurochem. 2011;117:779–96.CrossRefPubMed

42.

Hemming ML, Elias JE, Gygi SP, Selkoe DJ. Proteomic profiling of gamma-secretase substrates and mapping of substrate requirements. PLoS Biol. 2008;6:e257.CrossRefPubMedPubMedCentral

43.

Lin Y, Lin H, Liu Z, Wang K, Yan Y. Improvement of a sample preparation method assisted by sodium deoxycholate for mass-spectrometry-based shotgun membrane proteomics†. J Sep Sci. 2014;37:3321–9.CrossRefPubMed

44.

Lin Y, Wang K, Liu Z, Lin H, Yu L. Enhanced SDC-assisted digestion coupled with lipid chromatography-tandem mass spectrometry for shotgun analysis of membrane proteome. J Chromatogr B. 2015;1002:144–51.CrossRef

45.

Sharma K, Schmitt S, Bergner CG, Tyanova S, Kannaiyan N, Manrique-Hoyos N, Kongi K, Cantuti L, Hanisch U-K, Philips M-A, et al. Cell type- and brain region-resolved mouse brain proteome. Nat Neurosci. 2015;18:1819–31.CrossRefPubMed

46.

Ivankov DN, Bogatyreva NS, Hönigschmid P, Dislich B, Hogl S, Kuhn P-H, Frishman D, Lichtenthaler SF. QARIP: a web server for quantitative proteomic analysis of regulated intramembrane proteolysis. Nucleic Acids Res. 2013;41:W459–64.CrossRefPubMedPubMedCentral

47.

May PC, Dean RA, Lowe SL, Martenyi F, Sheehan SM, Boggs LN, Monk SA, Mathes BM, Mergott DJ, Watson BM, et al. Robust central reduction of amyloid-beta in humans with an orally available, non-peptidic beta-secretase inhibitor. J Neurosci. 2011;31:16507–16.CrossRefPubMed

48.

Ring S, Weyer SW, Kilian SB, Waldron E, Pietrzik CU, Filippov MA, Herms J, Buchholz C, Eckman CB, Korte M, et al. The secreted beta-amyloid precursor protein ectodomain APPs alpha is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice. J Neurosci. 2007;27:7817–26.CrossRefPubMed

49.

Li H, Wang B, Wang Z, Guo Q, Tabuchi K, Hammer RE, Sudhof TC, Zheng H. Soluble amyloid precursor protein (APP) regulates transthyretin and Klotho gene expression without rescuing the essential function of APP. Proc Natl Acad Sci U S A. 2010;107:17362–7.CrossRefPubMedPubMedCentral

50.

Mulley JC, Iona X, Hodgson B, Heron SE, Berkovic SF, Scheffer IE, Dibbens LM. The role of seizure-related SEZ6 as a susceptibility gene in febrile seizures. Neurol Res Int. 2011;2011:917565.CrossRefPubMedPubMedCentral

51.

Yu ZL, Jiang JM, Wu DH, Xie HJ, Jiang JJ, Zhou L, Peng L, Bao GS. Febrile seizures are associated with mutation of seizure-related (SEZ) 6, a brain-specific gene. J Neurosci Res. 2007;85:166–72.CrossRefPubMed

52.

Xu C, Mullersman JE, Wang L, Bin Su B, Mao C, Posada Y, Camarillo C, Mao Y, Escamilla MA, Wang KS. Polymorphisms in seizure 6-like gene are associated with bipolar disorder I: evidence of gene x gender interaction. J Affect Disord. 2013;145:95–9.CrossRefPubMed

53.

Gautam V, D’Avanzo C, Hebisch M, Kovacs DM, Kim DY. BACE1 activity regulates cell surface contactin-2 levels. Mol Neurodegener. 2014;9:4.CrossRefPubMedPubMedCentral

54.

Ohno M, Sametsky EA, Younkin LH, Oakley H, Younkin SG, Citron M, Vassar R, Disterhoft JF. BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer’s disease. Neuron. 2004;41:27–33.CrossRefPubMed

55.

Ohno M, Chang L, Tseng W, Oakley H, Citron M, Klein WL, Vassar R, Disterhoft JF. Temporal memory deficits in Alzheimer’s mouse models: rescue by genetic deletion of BACE1. Eur J Neurosci. 2006;23:251–60.CrossRefPubMed

56.

Laird FM, Cai H, Savonenko AV, Farah MH, He K, Melnikova T, Wen H, Chiang HC, Xu G, Koliatsos VE, et al. BACE1, a major determinant of selective vulnerability of the brain to amyloid-beta amyloidogenesis, is essential for cognitive, emotional, and synaptic functions. J Neurosci. 2005;25:11693–709.CrossRefPubMedPubMedCentral

57.

Kobayashi D, Zeller M, Cole T, Buttini M, McConlogue L, Sinha S, Freedman S, Morris RG, Chen KS. BACE1 gene deletion: impact on behavioral function in a model of Alzheimer’s disease. Neurobiol Aging. 2008;29:861–73.CrossRefPubMed

58.

Savonenko AV, Melnikova T, Laird FM, Stewart KA, Price DL, Wong PC. Alteration of BACE1-dependent NRG1/ErbB4 signaling and schizophrenia-like phenotypes in BACE1-null mice. Proc Natl Acad Sci U S A. 2008;105:5585–90.CrossRefPubMedPubMedCentral

Title: Seizure protein 6 and its homolog seizure 6-like protein are physiological substrates of BACE1 in neurons
Authors: Martina Pigoni
Johanna Wanngren
Peer-Hendrik Kuhn
Kathryn M. Munro
Jenny M. Gunnersen
Hiroshi Takeshima
Regina Feederle
Iryna Voytyuk
Bart De Strooper
Mikail D. Levasseur
Brian J. Hrupka
Stephan A. Müller
Stefan F. Lichtenthaler
Publication date: 01-12-2016
Publisher: BioMed Central
Published in: Molecular Neurodegeneration / Issue 1/2016
Electronic ISSN: 1750-1326
DOI: https://doi.org/10.1186/s13024-016-0134-z

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Seizure protein 6 and its homolog seizure 6-like protein are physiological substrates of BACE1 in neurons

Abstract

Background

Methods and results

Conclusions

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Abstract

Background

Methods and results

Conclusions

Please log in to get access to this content

Other articles of this Issue 1/2016

Affinity of Tau antibodies for solubilized pathological Tau species but not their immunogen or insoluble Tau aggregates predicts in vivo and ex vivo efficacy

ULK1-mediated phosphorylation of ATG14 promotes autophagy and is impaired in Huntington’s disease models

Induced pluripotent stem cells in Alzheimer’s disease: applications for disease modeling and cell-replacement therapy

Network-driven plasma proteomics expose molecular changes in the Alzheimer’s brain

Pim1 inhibition as a novel therapeutic strategy for Alzheimer’s disease

Alzheimer’s disease-like APP processing in wild-type mice identifies synaptic defects as initial steps of disease progression