THE PHYSIOLOGICAL FUNCTIONS OF THE AMYLOID PRECURSOR PROTEIN GENE FAMILY EDITED BY : Ulrike C. Müller and Thomas Deller PUBLISHED IN : Frontiers in Molecular Neuroscience 1 January 2018 | APP Physiological Functions Frontiers in Molecular Neuroscience Frontiers Copyright Statement © Copyright 2007-2018 Frontiers Media SA. All rights reserved. All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA (“Frontiers”) or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers. The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. 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Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: researchtopics@frontiersin.org 2 January 2018 | APP Physiological Functions Frontiers in Molecular Neuroscience THE PHYSIOLOGICAL FUNCTIONS OF THE AMYLOID PRECURSOR PROTEIN GENE FAMILY 3D-reconstruction of an EGFP-labeled hippocampal CA1 pyramidal cell. We thank Prof. Dr. Mario Vuksic, Frankfurt/Zagreb for the original image and Annika Mehr for the artwork. Topic Editors: Ulrike C. Müller, Universität Heidelberg, Germany Thomas Deller , Universität Frankfurt, Germany The amyloid precursor protein APP plays a key role in the pathogenesis of Alzheimer’s disease (AD), as proteolytical cleavage of APP gives rise to the A b peptide which is deposited in the brains of Alzheimer patients. Despite this, our knowledge of the normal cell biological and physiological functions of APP and the closely related APLPs is limited. This may have hampered our understanding of AD, since evidence has accumulated that not only the production of the A b peptide but also the loss of APP-mediated functions may contribute to AD pathogenesis. Thus, it appears timely and highly relevant to elucidate the functions of the APP gene family from the molecular level to their role in the intact organism, i.e. in the context of nervous system devel- opment, synapse formation and adult synapse function, as well as neural homeostasis and aging. 3 January 2018 | APP Physiological Functions Frontiers in Molecular Neuroscience Why is our understanding of the APP functions so limited? APP and the APLPs are multi- functional proteins that undergo complex proteolytical processing. They give rise to an almost bewildering array of different fragments that may each subserve specific functions. While A b is aggregation prone and neurotoxic, the large secreted ectodomain APPs α - produced in the non-amyloidogenic α -secretase pathway - has been shown to be neurotrophic, neuroprotective and relevant for synaptic plasticity, learning and memory. Recently, novel APP cleavage pathways and enzymes have been discovered that have gained much attention not only with respect to AD but also regarding their role in normal brain physiology. In addition to the various cleavage products, there is also solid evidence that APP family proteins mediate important functions as transmembrane cell surface molecules, most notably in synaptic adhesion and cell surface signaling. Elucidating in more detail the molecular mechanisms underlying these divers func- tions thus calls for an interdisciplinary approach ranging from the structural level to the analysis in model organisms. Thus, in this research topic of Frontiers we compile reviews and original studies, covering our current knowledge of the physiological functions of this intriguing and medically important protein family. Citation: Müller, C. U., Deller, T., eds. (2018). The Physiological Functions of the Amyloid Precursor Protein Gene Family. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-355-9 4 January 2018 | APP Physiological Functions Frontiers in Molecular Neuroscience Table of Contents Editorial: 06 Editorial: The Physiological Functions of the APP Gene Family Ulrike C. Müller and Thomas Deller Section 1: Secretases – substrates, regulation and functions 08 Proteomic Substrate Identification for Membrane Proteases in the Brain Stephan A. Müller, Simone Scilabra and Stefan F . Lichtenthaler 24 Physiological Functions of the b -Site Amyloid Precursor Protein Cleaving Enzyme 1 and 2 Riqiang Yan 36 The Metalloprotease Meprin b Is an Alternative b -Secretase of APP Christoph Becker-Pauly and Claus U. Pietrzik 47 Regulation of Alpha-Secretase ADAM10 In vitro and In vivo : Genetic, Epigenetic, and Protein-Based Mechanisms Kristina Endres and Thomas Deller 65 The Emerging Role of Tetraspanins in the Proteolytic Processing of the Amyloid Precursor Protein Lisa Seipold and Paul Saftig 72 Corrigendum: The Emerging Role of Tetraspanins in the Proteolytic Processing of the Amyloid Precursor Protein Lisa Seipold and Paul Saftig Section 2: APP/APLP structure and transmembrane signaling 73 Structure and Synaptic Function of Metal Binding to the Amyloid Precursor Protein and its Proteolytic Fragments Klemens Wild, Alexander August, Claus U. Pietrzik and Stefan Kins 85 Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction Lukas P . Feilen, Kevin Haubrich, Paul Strecker, Sabine Probst, Simone Eggert, Gunter Stier, Irmgard Sinning, Uwe Konietzko, Stefan Kins, Bernd Simon and Klemens Wild 97 Role of APP Interactions with Heterotrimeric G Proteins: Physiological Functions and Pathological Consequences Philip F . Copenhaver and Donat Kögel 112 Functional Roles of the Interaction of APP and Lipoprotein Receptors Theresa Pohlkamp, Catherine R. Wasser and Joachim Herz 5 January 2018 | APP Physiological Functions Frontiers in Molecular Neuroscience 134 LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State Uta-Mareike Herr, Paul Strecker, Steffen E. Storck, Carolin Thomas, Verena Rabiej, Anne Junker, Sandra Schilling, Nadine Schmidt, C. Marie Dowds, Simone Eggert, Claus U. Pietrzik and Stefan Kins 151 APP Function and Lipids: A Bidirectional Link Marcus O. W. Grimm, Janine Mett, Heike S. Grimm and Tobias Hartmann Section 3: Functions during development, at the synapse and for neuroprotection 169 Analysis of Amyloid Precursor Protein Function in Drosophila melanogaster Marlène Cassar and Doris Kretzschmar 178 Role of Drosophila Amyloid Precursor Protein in Memory Formation Thomas Preat and Valérie Goguel 185 Physiological Concentrations of Amyloid Beta Regulate Recycling of Synaptic Vesicles via Alpha7 Acetylcholine Receptor and CDK5/Calcineurin Signaling Vesna Lazarevic, Sandra Fie n ́ ko, Maria Andres-Alonso, Daniela Anni, Daniela Ivanova, Carolina Montenegro-Venegas, Eckart D. Gundelfinger, Michael A. Cousin and Anna Fejtova 199 APP—A Novel Player within the Presynaptic Active Zone Proteome Jens Weingarten, Melanie Weingarten, Martin Wegner and Walter Volknandt 205 APP Protein Family Signaling at the Synapse: Insights from Intracellular APP-Binding Proteins Suzanne Guénette, Paul Strecker and Stefan Kins 219 Region-Specific Differences in Amyloid Precursor Protein Expression in the Mouse Hippocampus Domenico Del Turco, Mandy H. Paul, Jessica Schlaudraff, Meike Hick, Kristina Endres, Ulrike C. Müller and Thomas Deller 231 Novel Insights into the Physiological Function of the APP (Gene) Family and Its Proteolytic Fragments in Synaptic Plasticity Susann Ludewig and Martin Korte 246 The Role of APP in Structural Spine Plasticity Elena Montagna, Mario M. Dorostkar and Jochen Herms 253 Amyloid-precursor Like Proteins APLP1 and APLP2 Are Dispensable for Normal Development of the Neonatal Respiratory Network Kang Han, Ulrike C. Müller and Swen Hülsmann 264 APP as a Protective Factor in Acute Neuronal Insults Dimitri Hefter and Andreas Draguhn 280 Therapeutic Potential of Secreted Amyloid Precursor Protein APPs a Bruce G. Mockett, Max Richter, Wickliffe C. Abraham and Ulrike C. Müller EDITORIAL published: 23 October 2017 doi: 10.3389/fnmol.2017.00334 Frontiers in Molecular Neuroscience | www.frontiersin.org October 2017 | Volume 10 | Article 334 | Edited and reviewed by: Nicola Maggio, Sackler Faculty of Medicine, Tel Aviv University, Israel *Correspondence: Ulrike C. Müller u.mueller@urz.uni-heidelberg.de Received: 26 September 2017 Accepted: 02 October 2017 Published: 23 October 2017 Citation: Müller UC and Deller T (2017) Editorial: The Physiological Functions of the APP Gene Family. Front. Mol. Neurosci. 10:334. doi: 10.3389/fnmol.2017.00334 Editorial: The Physiological Functions of the APP Gene Family Ulrike C. Müller 1 * and Thomas Deller 2 1 Department of Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, Universität Heidelberg, Heidelberg, Germany, 2 Institute of Clinical Neuroanatomy, Neuroscience Center, Goethe University Frankfurt, Frankfurt, Germany Keywords: amyloid precursor protein (APP), APP like proteins, synaptic plasticity, development, spine density, neuroprotection and neuronal repair, synaptogenesis, secretases Editorial on the Research Topic The Physiological Functions of the APP Gene Family The amyloid precursor protein APP plays a key role in the pathogenesis of Alzheimer’s disease (AD), as proteolytical cleavage of APP gives rise to the β -amyloid peptide A β , which is deposited in the brains of AD patients (Selkoe and Hardy, 2016). In contrast to this key role in AD, the reviews and original papers in this Special Issue entitled “The physiological functions of the APP gene family” aim to shed some light on the “bright side” of APP, which exhibits important physiological functions during brain development, for adult brain plasticity and protection against injury. This change of perspective is timely, since accumulating evidence suggests that disease symptoms are caused both by an overload of toxic substances, e.g., “too much A β ,” as well as deficits of protective molecules, e.g., “not enough APPs α .” Unraveling APP functions has not been trivial, since APP undergoes complex processing. APP processing is initiated either by α -secretase cleavage within the A β region, or by β -secretase (BACE) cleavage at the N-terminus of A β , leading to the secretion of large soluble ectodomains, termed APPs α and APPs β , respectively. Subsequent processing of the C-terminal fragments (CTF α or CTF β ) by γ -secretase results in the production of A β , p3 and the APP intracellular domain (AICD). This processing—as well as processing along non-canonical pathways (see Müller et al., 2017, for review) results in numerous fragments, which have different and partially opposite functional properties. Furthermore, APP functions are in part shared by APP-like proteins 1 and 2 (APLP1 and 2), which confounds some experimental approaches. Finally, expression changes over time and with aging add additional levels of complexity. In short, understanding APP gene family functions is challenging and this special issue provides a broad overview of the state-of-the art in this field. Several reviews (Seipold and Saftig; Endres and Deller; Yan; Becker-Pauly and Pietrzik) focus on the properties of canonical and non-canonical α -, and β -secretases, their substrates, regulation, and neurobiological functions in health and disease. Müller et al. give a systematic overview over proteomic methods to systematically identify the substrates of membrane proteases. The knowledge of these substrates is crucial to identify the physiological and pathological functions of secretases and to assess potential risks of their pharmacological impairment to treat AD (Endres and Deller; Yan). In addition, there is evidence that the secretases which are transmembrane proteases can form larger complexes with other cell surface proteins that may modulate their activity including members of the tetraspannin family (Seipold and Saftig). APP processing is further modulated by the lipid composition of the plasma membrane and accumulating evidence suggests that A β and the AICD play an important role in regulating lipid homeostasis (Grimm et al.). Likewise, lipoprotein receptors may interact with APP to control developmental processes and synaptic function (Pohlkamp et al.). They have been shown to not only regulate A β uptake and 6 Müller and Deller APP Physiological Functions degradation, but also APP processing and APP trafficking. In this regard, employing live cell imaging in primary neurons Herr et al. demonstrate that low-density lipoprotein receptor- related protein 1 (LRP1) modulates the axonal transport of APP monomers and dimers. There is a large body of evidence indicating that APP family proteins are multimodal proteins that can function as ligands via their secreted fragments, in particular APPs α , or as cell surface proteins mediating signal transduction and synaptic adhesion (as reviewed by Müller et al., 2017). Wild et al. discuss how metal (Cu and Zn) binding affects the structure of the APP extracellular domain and may modulate its role as a synaptic adhesion molecule. As APP family proteins have no enzymatic activities, signal transduction relies on interactions with other membrane proteins and/or adaptors. The role of the Fe65 adaptor family is summarized by Guenette et al. Fe65 binding to the APP C-terminus involves its phosphotyrosine-binding (PTB) domain 2 which can also mediate the formation of cytosolic Fe65 dimers, as shown by X-ray crystallography (Feilen et al.). The importance of heteromeric G-protein interactions with the APP C-terminus for physiological APP signaling and AD pathogenesis is reviewed by Copenhaver and Kogel. Major insight into the in vivo functions of APP family proteins has been obtained from animal models. Drosophila expresses only one APP protein called APP-like (APPL) and two reviews (Cassar and Kretzschmar; Preat and Goguel) deal with APPL functions in flies. In mice the analysis of APP functions is complicated by partially overlapping functions within the gene family and lethality of double and triple knockout mice (Han et al.). To circumvent early postnatal lethality mice with conditional floxed alleles have been generated (Müller et al., 2017). Together, the analysis of engineered mouse models indicated that APP family proteins and their proteolytic fragments are important during nervous system development for neuronal migration, neurite outgrowth, axonal pathfinding, and synaptogenesis (Müller et al., 2017). Proteomic studies, reviewed by Weingarten et al. established APP family proteins as important components of the active zone. Lazarevic et al. demonstrated that low amounts of A β are involved in the regulation of neurotransmitter release. It should be noted, however, that APP family proteins have also been localized at postsynaptic sites including the neuromuscular junction. In addition, APP family proteins have important functions in the adult hippocampus, where they are differentially expressed in all subregions (Del Turco et al.) and regulate synaptic plasticity and memory (Ludewig and Korte). The recently identified function of APP for structural spine plasticity is summarized by Montagna et al. In particular APPs α holds great therapeutic potential for AD as reviewed by Mockett et al. Finally, Hefter and Draguhn highlight the role of APP and APPs α as a protective factor for acute neuronal insults including hypoxia. We thank all contributors for their interesting and informative articles and the reviewers for their constructive and thoughtful suggestions. AUTHOR CONTRIBUTIONS UM and TD wrote the manuscript and both authors approved the final version for publication. FUNDING The authors thank the Deutsche Forschungsgemeinschaft for their support within programme FOR1332. REFERENCES Müller, U. C., Deller, T., and Korte, M. (2017). Not just amyloid: physiological functions of the amyloid precursor protein family. Nat. Rev. Neurosci. 18, 281–298. doi: 10.1038/nrn. 2017.29 Selkoe, D. J., and Hardy, J. (2016). The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol. Med. 8, 595–608. doi: 10.15252/emmm.2016 06210 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2017 Müller and Deller. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Molecular Neuroscience | www.frontiersin.org October 2017 | Volume 10 | Article 334 | 7 REVIEW published: 13 October 2016 doi: 10.3389/fnmol.2016.00096 Proteomic Substrate Identification for Membrane Proteases in the Brain Stephan A. Müller 1,2† , Simone D. Scilabra 1,2† and Stefan F. Lichtenthaler 1,2,3,4 * 1 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany, 2 Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany, 3 Institute for Advanced Study, Technische Universität Munich, Garching, Germany , 4 Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Edited by: Ulrike C. Müller, Heidelberg University, Germany Reviewed by: Robert Vassar, Northwestern University, USA Stefan Kins, Kaiserslautern University of Technology, Germany Ulrich Auf Dem Keller, ETH Zurich, Switzerland *Correspondence: Stefan F. Lichtenthaler stefan.lichtenthaler@dzne.de † These authors have contributed equally to this work. Received: 10 August 2016 Accepted: 21 September 2016 Published: 13 October 2016 Citation: Müller SA, Scilabra SD and Lichtenthaler SF (2016) Proteomic Substrate Identification for Membrane Proteases in the Brain. Front. Mol. Neurosci. 9:96. doi: 10.3389/fnmol.2016.00096 Cell-cell communication in the brain is controlled by multiple mechanisms, including proteolysis. Membrane-bound proteases generate signaling molecules from membrane- bound precursor proteins and control the length and function of cell surface membrane proteins. These proteases belong to different families, including members of the “a disintegrin and metalloprotease” (ADAM), the beta-site amyloid precursor protein cleaving enzymes (BACE), membrane-type matrix metalloproteases (MT-MMP) and rhomboids. Some of these proteases, in particular ADAM10 and BACE1 have been shown to be essential not only for the correct development of the mammalian brain, but also for myelination and maintaining neuronal connections in the adult nervous system. Additionally, these proteases are considered as drug targets for brain diseases, including Alzheimer’s disease (AD), schizophrenia and cancer. Despite their biomedical relevance, the molecular functions of these proteases in the brain have not been explored in much detail, as little was known about their substrates. This has changed with the recent development of novel proteomic methods which allow to identify substrates of membrane-bound proteases from cultured cells, primary neurons and other primary brain cells and even in vivo from minute amounts of mouse cerebrospinal fluid (CSF). This review summarizes the recent advances and highlights the strengths of the individual proteomic methods. Finally, using the example of the Alzheimer-related proteases BACE1, ADAM10 and γ -secretase, as well as ADAM17 and signal peptide peptidase like 3 (SPPL3), we illustrate how substrate identification with novel methods is instrumental in elucidating broad physiological functions of these proteases in the brain and other organs. Keywords: proteomics, degradomics, protease, BACE, ADAM10, ADAM17, Alzheimer’s disease PROTEOLYTIC PROCESSING IN ALZHEIMER’S DISEASE Proteolysis is a biological process playing an essential role in all organisms and tissues, including the brain. For example, proteolysis regulates numerous cell functions, spanning from degradation of faulty proteins to post-translational generation of active signaling molecules, neurite outgrowth and modeling of the extracellular matrix. Therefore, protease activity must be tightly regulated and, conversely, aberrant proteolysis is associated with several pathological conditions ranging from inflammation to cancer and neurodegeneration. A prime example is Alzheimer’s disease (AD), where deregulation of proteolysis leads to neurodegeneration. AD is the most common type of dementia, a syndrome characterized by loss of memory and cognitive decline. AD causes Frontiers in Molecular Neuroscience | www.frontiersin.org October 2016 | Volume 9 | Article 96 | 8 Müller et al. Brain Proteomics a substantial loss of neurons and synapses in the brain, leading to an overall loss in brain weight. Additional neuropathological hallmarks of the disease are the amyloid- β (A β ) plaques, consisting of the mostly 42 amino acid long A β peptide (A β 42), and the intraneuronal accumulation of neurofibrillary tangles, consisting of hyperphosphorylated forms of the microtubule-associated protein tau (Huang and Mucke, 2012). According to the widely accepted amyloid cascade hypothesis (Selkoe and Hardy, 2016), A β forms neurotoxic oligomers, which initiate an inflammatory response involving the activation of microglia and astrocytes. Subsequently tau becomes aberrantly phosphorylated and aggregates in neurofibrillary tangles, leading to synaptic loss, neuronal death, and ultimately dementia (Selkoe and Hardy, 2016). A β derives from the transmembrane protein amyloid precursor protein (APP; Dislich and Lichtenthaler, 2012; Figure 1A ) through sequential cleavage by two proteases, the β - and γ -secretase (Haass and Selkoe, 2007). The β -secretase was identified in 1999 by five independent research groups, and is referred to as β -site APP cleaving enzyme 1 (BACE1; Hussain et al., 1999; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999; Lin et al., 2000). BACE1 cleavage releases a soluble extracellular fragment of APP (sAPP β ) and generates a carboxy (C)-terminal membrane-tethered fragment known as C99 ( Figure 1 ). C99 undergoes a subsequent intramembrane cleavage by γ -secretase, a multi-subunit protease complex comprising four transmembrane proteins: presenilin, nicastrin, Pen2 and Aph1 (De Strooper et al., 2010). The γ -secretase cleavage of C99 generates A β and releases intracellularly the APP intracellular domain (AICD). APP can undergo an alternative cleavage, mediated by a disintegrin and metalloproteinase 10 (ADAM10; Lammich et al., 1999; Kuhn et al., 2010), also known as α -secretase, that releases its soluble ectodomain (sAPP α ) and generates a membrane-tethered fragment, C83 ( Figure 1B ). Importantly, the subsequent cleavage by γ -secretase releases a truncated form of A β , which is non-toxic. Three other proteases emerged to be involved in the processing of APP. Asparagine endopeptidase (AEP), known as the δ - secretase, is a cysteine proteinase that mediates APP processing in an age-dependent manner and is linked to AD pathogenesis (Zhang et al., 2015). Furthermore, the membrane-tethered metalloproteinase (MT5-MMP) cleaves APP at amino acids 504–505, initiating a proteolytic processing that leads to the generation of APP fragments (A η - α ), which lower neuronal activity (Ahmad et al., 2006; Willem et al., 2015; Figure 1C ). Loss of MT5-MMP ameliorates pathology and behavioral deficits in a mouse model of AD (Baranger et al., 2016). A member of the meprin family of metalloproteases, meprin β , was also shown to cleave APP, with the cleavage site being identical to that of the β -secretase or in close proximity to it. This shedding event is followed by the γ -secretase cleavage and leads to the generation of A β or truncated variants of A β (i.e., A β 2–40 ; Bien et al., 2012). Additionally, meprin β can process APP at the N-terminus, releasing two N-terminal fragments of APP of 11 and 22 kDa, namely APP11 and APP22 (Jefferson et al., 2011). REGULATED INTRAMEMBRANE PROTEOLYSIS The proteolytic processing of APP is a prime example for a proteolytic process referred to as regulated intramembrane proteolysis (RIP; Figure 2 ). RIP frequently comprises two proteolytic cleavages, namely shedding and intramembrane proteolysis. Shedding is mediated by membrane-tethered proteases, referred to as ‘‘sheddases’’, which cleave their transmembrane substrates, thereby releasing their soluble ectodomains into the extracellular milieu ( Figure 2 ). Most sheddases cleave their substrates at peptide bonds outside of the membrane, but at a short distance from the lumenal or extracellular membrane surface. Shedding can be followed by a second cleavage within the substrates’ transmembrane domain. This cleavage results in release of the intracellular domain (ICD) into the cytosol and the extracellular secretion of the small remaining peptide. As it occurs for APP, α - and β -secretase function as sheddases, and their activity can be coupled with the action of γ -secretase to perform RIPping of the remaining membrane-tethered protein fragment. Shedding and intramembrane proteolysis initiate a sequence of extracellular and intracellular events that control a broad range of physiological processes in the brain, including cell-cell communication, cell differentiation and development (Murphy et al., 2008; Lichtenthaler et al., 2011; Weber and Saftig, 2012). For instance, the tumor necrosis factor- α (TNF), a proinflammatory cytokine, is generated as a transmembrane protein that needs to be shed by ADAM17 from the cell surface in order to trigger immune responses (Black et al., 1997). Interestingly, the remaining membrane-bound fragment can be further cleaved by SPPL2a or SPPL2b within the membrane, releasing the TNF ICD which acts as an additional signaling molecule (Friedmann et al., 2006). Similarly to TNF, several growth factors, including EGF-like growth factors and neuregulins, are inactive when bound to the membrane and get activated by proteolytic shedding (Blobel, 2005). Sheddases do not only modulate the availability of ligands, but also regulate the activity of signaling receptors. Notch is a clear example of cell surface receptor that requires RIPping to initiate its signaling pathway and control cell-differentiation (Hartmann et al., 2002). For other substrates, RIP is a mechanism to terminate a protein’s function. For example, shedding shuts down the signaling function of TNF receptors (D’Alessio et al., 2012; Deng et al., 2015) or the adhesive functions of cell adhesion proteins (Solanas et al., 2011). SHEDDASES AND INTRAMEMBRANE PROTEASES Members of several different families of proteases have been shown to function as sheddases, including several ADAMs, BACE proteases, membrane-type metalloproteinases (MT- MMPs) and rhomboids ( Figure 2 ; Blobel, 2005; Vassar et al., 2014; Itoh, 2015). In addition, signal peptide peptidase like 3 Frontiers in Molecular Neuroscience | www.frontiersin.org October 2016 | Volume 9 | Article 96 | 9 Müller et al. Brain Proteomics FIGURE 1 | Schematic representation of amyloid precursor protein (APP) processing. (A) A number of proteases can cleave APP at specific sites, including a disintegrin and metalloproteinase 10 (ADAM10; yellow arrow), beta-site APP cleaving enzyme 1 (BACE1; red arrow), γ -secretase (orange arrows), asparagine endopeptidase (AEP; blue arrows), membrane-type matrix metalloproteases (MT5-MMP; fuchsia arrow) and meprin β (green arrows). (B) APP can undergo amyloidogenic processing when cleaved by BACE1. Cleavage of APP by BACE1 results in generation of sAPP β . Subsequent cleavage of the remaining transmembrane domain by γ -secretase releases amyloid- β (A β ). (C) Conversely, cleavage of APP by ADAM10 favors the non-amyloidogenic pathway, releasing sAPP α . Subsequent γ -secretase cleavage releases a non-toxic truncated form of the A β peptide, called p3. (D) In addition, APP can be cleaved by MT5-MMP, which results in the release of sAPP η . Consecutively, C-terminal fragment (CTF)- η can be cleaved by ADAM10 or BACE1 that release A η - α and A η - β , respectively. The recently identified δ -secretase cleaves APP a few amino acids N-terminally to the BACE1 cleavage site (not shown in the figure). Frontiers in Molecular Neuroscience | www.frontiersin.org October 2016 | Volume 9 | Article 96 | 10 Müller et al. Brain Proteomics FIGURE 2 | “Shedding” and “RIPping”. Schematic representation of ectodomain shedding and regulated intramembrane proteolysis (RIP), including a list of protease families known to function as sheddases or intramembrane proteases. (SPPL3) from the SPP family and site-1 protease (S1P) can also act as sheddases (Lenz et al., 2001; Voss et al., 2012). ADAM and BACE proteases cleave substrates in their extracellular domain, at a short distance from the membrane, and need the sequential cleavage of an intramembrane proteinase in order to perform RIPping. Conversely, rhomboids and SPPL3 are intramembrane proteases that cleave their substrates within or close to the transmembrane domain. As a consequence of such cleavage, regardless whether it occurs extracellularly or within the transmembrane domain, the ectodomain of substrates is released into the extracellular milieu. This is of note, as the secreted form of transmembrane proteins can acquire functions different from that of the membrane- bound counterpart. MT-MMPs can act as sheddases. However, compared to the related family of ADAMs, MT-MMPs can cleave their substrates more distantly from the cell surface and on different sites, thereby releasing truncated forms of protein ectodomains or lower molecular weight fragments (Selvais et al., 2011; Fu et al., 2013; Willem et al., 2015). FUNCTION OF PROTEASES IS DETERMINED BY SUBSTRATES Proteases have been well characterized in pathophysiology of disease as key players in the development of several pathological conditions, including neurodegenerative diseases. Thus, protease inhibition has been widely targeted for drug development. Unfortunately, in the vast majority of cases, therapies based on protease inhibition have failed in clinical trials. Indeed, there are critical limitations to the development of therapies targeting proteases. First, distinct members of a protease family share structural features, thus drug-based inhibition of a specific protease can affect the activity of homologs. For example, BACE1 inhibitors have been developed to reduce A β production in the brain and are tested for treatment and prevention of AD. However, they also block the homologous protease BACE2, which has critical functions in pigmentation (Rochin et al., 2013; Neumann et al., 2015). In fact, mice treated with such inhibitors get a gray fur color and patients treated with these drugs need to go for regular dermatology testing (Yan, 2016). More importantly, proteases often do not target a specific substrate, but they can cleave an array of diverse proteins. As a consequence, their inhibition can deregulate a number of cellular processes, and inhibition-based therapies can lead to mechanism-based side effects that often are more pronounced than amelioration of the pathology itself. For instance, due to its central contribution to the pathogenesis of AD, γ -secretase has been extensively targeted for drug development. A number of γ -secretase inhibitors have been generated and tested for their ability to reduce A β production in vitro and in vivo . One of them, called Semagacestat, was terminated in clinical trial Phase III, as it was associated with worsening of patient cognition and with higher incidence of skin cancer (De Strooper, 2014). These mechanism-based side effects were linked to the chronic inhibition of Notch cleavage by γ -secretase. A deep understanding of protease functions and their roles in cell biology is necessary for developing effective therapeutic strategies. As the biological function of proteases depends on their substrate spectrum, the identification of the substrate repertoire is essential to understand the function of a specific Frontiers in Molecular Neuroscience | www.frontiersin.org October 2016 | Volume 9 | Article 96 | 11 Müller et al. Brain Proteomics protease and to predict potential side-effects of their therapeutic inhibition. In the last years, a number of proteomics-based methods have been developed in order to identify the substrate repertoire of specific proteases. In this review, we summarize the most commonly used and other suitable methods and give examples of their applications with a focus on sheddases and intramembrane proteases, in particular on BACE1, ADAM10 and γ -secretase in AD. METHODS FOR MASS SPECTROMETRY BASED SUBSTRATE IDENTIFICATION OF MEMBRANE PROTEASES IN THE BRAIN Mass spectrometry (MS) based proteomics offers powerful methods to identify membrane protease substrate candidates in vitro and in vivo . Especially, non-targeted quantification of protein cleavage products in the secretome of brain-derived primary cells or cell lines, as well as cerebrospinal fluid (CSF) are suitable for protease substrate identification. In this context, the secretome comprises all proteins released by cells into body fluids or into the conditioned medium of cultured cells. For sheddases such as BACE1 and ADAM10, the ectodomain of their substrates is released into the extracellular space. Therefore, usually a loss of function condition, such as protease KO, knockdown (KD), or inhibition, is quantitatively compared with related control conditions to identify substrates. At loss of function conditions, substrate cleavage is fully or partly prevented which leads to a reduced abundance of the related cleavage products in the secretome. Additionally, some substrates accumulate in the cell membrane when the target protease does not cleave them. Therefore, membrane protease substrate candidates might also be identified by quantitative proteomics due to an increased abundance within the cell membrane. Alternatively, also gain of function conditions such as overexpression of the target protease can be used which leads to increased cleavage activity and subsequently to increased abundance of substrate cleavage products in the secretome. Here, we will provide a short overview of the main methods for MS-based protease substrate identification with a focus on methods for sheddase substrate identification. In the first section, methods are described that are used to identify substrates in the secretome or on the cell surface ( Figure 3 ). In the second section, methods are described that also allow protease cleavage site determination ( Figure 4 ). Usually, bottom-up proteomics is used for this purpose. Briefly, in all protocols, secreted or membrane proteins are digested with a protease, usually trypsin, to create proteolytic peptides. In most cases those peptides are separated by C18 reversed phase liquid chromatography (LC) prior to MS analysis. The MS raw data is searched against a protein database to identify proteotypic peptides. Relative peptide and protein quantification can be done by different methods. According to the different protocols for protease substrate identification, label-free and label-based quantification methods are used. A detailed explanation of different quantification methods can be found in several review FIGURE 3 | Workflow of the glyco-capturing and secretome protein enrichment with click sugars (SPECS) method for protease substrate identification. articles (Bantscheff et al., 2007; Schulze and Usadel, 2010; Bakalarski and Kirkpatrick, 2016). Methods for Protease Substrate Identification Glyco-capture Most substrates of sheddases are single-pass transmembrane or GPI-anchored proteins, which are usually glycosylated within their ectodomain. According to UniProt reference database of Homo sapiens (date: 2016-06-30), 92% of all transmembrane type I (1125 out of 1228, term: SL-9905) and 83% of all transmembrane type II (347 out of 420, term: SL-9906) proteins are annotated as glycoproteins (term: KW-0325). Upon membrane protein cleavage, a part of the ectodomain is secreted ( Figure 2 ). Glyco-capturing ( Figure 3 ) facilitates specific enrichment of glycoproteins. In the first step, cis-diol grou