α-Secretase processing of the Alzheimer amyloid-β precursor protein and its homolog APLP2 Kristin Jacobsen 1 Doctoral dissertation, 2013 Department of Neurochemistry Arrhenius Laboratories for Natural Sciences Stockholm University Cover: Schematic illustration of APP and APLP2 processing during stimulated conditions ©Kristin T. Jacobsen, Stockholm University 2013 ISBN 978-91-7447-732-0 Printed in Sweden by Universitetsservice US-AB, Stockholm 2013 Distributor: Department of Neurochemistry, Stockholm University 2 To my family 3 4 List of Publications I. Jacobsen KT, Adlerz L, Multhaup G and Iverfeldt K. IGF-1-induced processing of amyloid-β precursor protein and APP-like protein 2 is mediated by different enzymes Journal of Biological Chemistry. 2010; 285 (14): 10223-10231 II. Holback S, Adlerz L, Gatsinzi T, Jacobsen KT and Iverfeldt K PI3-K- and PKC-dependent up-regulation of APP processing enzymes by retinoic acid Biochemical and Biophysical Research Communications. 2008; 365(2): 298-303 III. Jacobsen KT and Iverfeldt K. O-GlcNAcylation increases non-amyloidogenic processing of the amyloid-β precursor protein (APP) Biochemical and Biophysical Research Communications. 2011; 404(3): 882-886 IV. Jacobsen KT, Strååt Y, Koistinen N and Iverfeldt K. O-GlcNAcylation of the α-secretase ADAM10 selectively affects APP processing in neuron-like cells Manuscript V. Jacobsen KT and Iverfeldt K The E1 domain of APP and APLP2 determines α-secretase specificity Manuscript 5 Additional publications . Jacobsen KT and Iverfeldt K Amyloid precursor protein and its homologues: a family of proteolysis-dependent receptors Cellular and Molecular Life Science. 2009; 66: 2299-2318 (Review) . Tracy, LM, Bergqvist F, Ivanova EV, Jacobsen KT and Iverfeldt K Exposure to the saturated free fatty acid palmitate alters BV-2 microglia inflammatory response Journal of Molecular Neuroscience. 2013; 51(3): 805-812 6 Abstract The amyloid-β precursor protein (APP) has been widely studied due to its role in Alzheimer´s disease (AD). When APP is sequentially cleaved by β- and γ-secretase, amyloid-β (Aβ) is formed. Aβ is prone to aggregate and is toxic to neurons. However, the main processing pathway for APP involves initial cleavage at the α-site, within the Aβ region, instead generating a neu- roprotective soluble fragment, sAPPα. APP is a member of a protein family, also including the proteins APLP1 and APLP2, which are processed in a similar way as APP. In addition, knock-out studies in mice have shown that the three proteins have overlapping functions where APLP2 play a key phys- iological role. The aim of this thesis was to study mechanisms regulating the α-secretase processing of APP and APLP2. We have used the human neuro- blastoma cell line SH-SY5Y as a model system and have stimulated α- secretase processing with insulin-like growth factor-1 (IGF-1) or retinoic acid (RA). Our results show that the stimulated α-site cleavage of APP and APLP2 is regulated by different signaling pathways and that the cleavage is mediated by different enzymes. APP was shown to be cleaved by ADAM10 in a PI3K-dependent manner, whereas APLP2 was cleaved by TACE in a PKC-dependent manner. We further show that protein levels and maturation of ADAM10 and TACE is increased in response to RA, mediated by a PI3K- or PKC-dependent signaling pathway, respectively. Another focus of our research has been O-GlcNAcylation, a dynamic post-translational modifica- tion regulated by the enzymes O-GlcNAc transferase and O-GlcNAcase (OGA). We show that decreased OGA activity stimulates sAPPα secretion, without affecting APLP2 processing. We further show that ADAM10 is O- GlcNAcylated. Lastly, we show that APP can be manipulated to be cleaved in a similar way as APLP2 during IGF-1 stimulation by substituting the E1 domain in APP with the E1 domain from APLP2. Together our results show distinct α-site processing mechanisms of APP and APLP2. 7 Contents 1. Introduction ........................................................................................ 13 1.1 The role of APP in Alzheimer’s disease ......................................................... 13 1.2 The APP family .................................................................................................. 14 1.2.1 Structure ................................................................................................... 14 1.2.2 Proteolytic processing ............................................................................. 17 1.2.3 Biological functions ................................................................................. 19 1.3 Processing enzymes ......................................................................................... 21 1.3.1 α-Secretase .............................................................................................. 21 1.3.1.1 Substrate selectivity of TACE and ADAM10 ............................... 23 1.3.2 β-Secretase .............................................................................................. 24 1.3.3 γ-Secretase............................................................................................... 25 1.4 Signaling pathways affecting APP family processing ................................. 26 1.4.1 IGF-1 .......................................................................................................... 26 1.4.2 Retinoic acid ............................................................................................. 27 1.5 Regulation of APP family α-site processing ................................................. 28 1.5.1 Trafficking of enzyme and substrate .................................................... 28 1.5.2 Post-translational modifications of enzyme and substrate .............. 30 1.5.2.1 Post-translational modifications of ADAM10 and TACE............ 31 1.5.2.2 Post-translational modifications of the APP family ................... 32 2. Methodological considerations ..................................................... 34 2.1 Cell lines ............................................................................................................. 34 2.2 Cell treatments ................................................................................................. 35 2.2.1 Retinoic acid and IGF-1 .......................................................................... 35 2.2.2 Pharmacological inhibitors ..................................................................... 35 2.3 siRNA gene silencing ........................................................................................ 36 2.4 BCA assay .......................................................................................................... 36 2.5 Western blot ...................................................................................................... 37 2.6 ELISA .................................................................................................................. 37 2.7 32P-labeling and immunoprecipitation .......................................................... 38 2.8 sWGA precipitation ........................................................................................... 39 2.9 Biotinylation assay ........................................................................................... 39 2.10 Design and cloning of the APP/APLP2 chimer ........................................... 39 3. Aims ....................................................................................................... 41 4. Results and discussion .................................................................... 42 8 4.1 Stimulated α-secretase processing of APP is mediated by ADAM10 in a PI3K-dependent manner (Paper I and II) ........................................................... 42 4.2 Stimulated α-secretase processing of APLP2 is mediated by TACE in a PKC-dependent manner (Paper I and II) ............................................................ 44 4.3 O-GlcNAcylation induces α-secretase processing of APP but not of APLP2 (Paper III and IV) .................................................................................................... 45 4.3.1 O-GlcNAcylation selectively enhances α-secretase processing of APP in neuron-like cells (Paper IV) ................................................................. 46 4.3.2 ADAM10 is O-GlcNAcylated (Paper IV) ................................................ 47 4.4 The difference in regulation of α-secretase processing between APP and APLP2 is determined by their E1 domain (Paper III) ........................................ 48 4.5 What about APLP1? (Paper I and IV) ........................................................... 50 5. Conclusions ......................................................................................... 51 6. Populärvetenskaplig sammanfattning på svenska ............... 53 7. Acknowledgements .......................................................................... 55 8. References ........................................................................................... 57 9 Abbreviations Aβ Amyloid-β AD Alzheimer’s Disease ADAM A disintegrin and metalloprotease AICD APP intracellular domain ALID APP-like intracellular domain ANOVA Analysis of variance APH-1 Anterior pharynx defective-1 APLP APP-like protein APP Amyloid-β precursor protein BACE β-site cleaving enzyme BCA Bicinchoninic acid BDNF Brain-derived neurotrophic factor C83 APP C-terminal stub of 83 amino acids C99 APP C-terminal stub of 99 amino acids CNS Central nervous system CRABs Cellular retinoic