The role of the amyloid precursor protein (APP) in protein homeostasis and neuroprotection Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich 14 der Johann Wolfgang Goethe -Universität in Frankfurt am Main von ARPITA KUNDU aus Kalkutta, Indien Frankfurt 2017 (D 30) vom Fachbereich 14 der Johann Wolfgang Goethe - Universität als Dissertation angenommen. Dekan: Prof. Dr. Michael Karas Gutachter: Prof. Dr. Donat Kögel Prof. Dr. Jochen Klein` Datum der Disputation: TBD To my parents, husband and daughter Table of Contents Table of Contents Abbreviations .................................................................................................................................... ix Tables ............................................................................................................................................... xi Figures .............................................................................................................................................. xi Overview and Summary ..................................................................................................................... 1 Zusammenfassung ............................................................................................................................ 7 1 Introduction .............................................................................................................................. 13 1.1 Alzheimer’s Disease ........................................................................................................ 13 1.1.1 APP family members ............................................................................................... 16 1.1.2 APP structure ........................................................................................................... 17 1.1.3 Biochemical processing of APP ............................................................................... 18 1.1.4 APP function ............................................................................................................ 20 1.2 Cell death ......................................................................................................................... 22 1.2.1 Apoptosis ................................................................................................................. 22 1.2.2 Necrosis ................................................................................................................... 25 1.2.3 Autophagy ................................................................................................................ 26 1.3 BAG1 and BAG3 .............................................................................................................. 27 1.4 Role of stress signaling pathways in neurodegeneration and apoptosis ......................... 29 1.5 Role of APP in survival cascade and stress signaling ..................................................... 31 1.6 Aim of the Thesis ............................................................................................................. 33 2 Materials ................................................................................................................................... 37 2.1 Cell lines ........................................................................................................................... 37 2.2 Animals ............................................................................................................................ 37 2.3 Cell culture medium ......................................................................................................... 38 2.4 Chemicals ........................................................................................................................ 40 2.5 Buffers and Solutions ....................................................................................................... 40 2.6 Antibody ........................................................................................................................... 44 2.7 Oligoneucleotides for cDNA synthesis and qPCR ........................................................... 45 2.8 Enzymes and recombinant proteins ................................................................................ 46 2.9 Inhibitors and drugs ......................................................................................................... 46 2.10 Kits ................................................................................................................................... 47 2.11 Software ........................................................................................................................... 47 2.12 Equipment and Other Instruments ................................................................................... 48 3 Methods ................................................................................................................................... 53 3.1 Cell biological techniques ................................................................................................ 53 3.1.1 Culture conditions .................................................................................................... 53 Table of Contents 3.1.2 Determination of Cell Number and Seeding of Cells ............................................... 53 3.1.3 Cryopreservation and thawing of cells ..................................................................... 53 3.2 Preparation of Hippocampal Neurons .............................................................................. 54 3.3 Transfection of Plasmid .................................................................................................... 55 3.4 Induction of stress ............................................................................................................ 56 3.5 Analysis of proteins .......................................................................................................... 57 3.5.1 Preparation of cell lysates ........................................................................................ 57 3.5.2 Determination of protein concentration .................................................................... 57 3.5.3 SDS-PAGE ............................................................................................................... 58 3.5.4 Western Blot ............................................................................................................. 58 3.5.5 Coomassie staining of polyacrylamide gels ............................................................. 59 3.5.6 Immunodetection ...................................................................................................... 59 3.6 Immunostaining ................................................................................................................ 59 3.7 Nucleic acid techniques ................................................................................................... 60 3.7.1 Isolation of RNA ....................................................................................................... 60 3.7.2 Determination of RNA concentration........................................................................ 60 3.7.3 cDNA synthesis ........................................................................................................ 60 3.7.4 Quantitative real-time polymerase chain reaction (qPCR) ....................................... 61 3.8 SUC-LLVY-AMC Assay .................................................................................................... 61 3.9 FACS Analysis ................................................................................................................. 62 3.10 Subcellular fractionation and immunodetection of cytochrome c ..................................... 63 3.11 Purification of sAPPα, sAPPβ and E1 from yeast P. pastoris .......................................... 64 4 Results ..................................................................................................................................... 67 4.1 Purification of sAPPα, sAPPβ and E1 from P. pastoris ................................................... 67 4.2 Neuroprotective properties of sAPPα ............................................................................... 69 4.3 Recombinant sAPPα and APP-E1 domain activate the PI3K/Akt survival pathway ........ 73 4.4 sAPPα alters the expression levels of Akt downstream proteins only in the presence of holo-APP ...................................................................................................................................... 74 4.4.1 sAPPα induces the phosphorylation of FoxO3a and inhibits its nuclear translocation. ............................................................................................................................ 74 4.4.2 sAPPα and E1 decreases Bim expression under serum and glucose deprivation .. 77 4.4.3 sAPPα and E1 increases Bcl-xL and Mcl-1 expression under serum and glucose deprivation ................................................................................................................................ 79 4.4.4 Effect of sAPPα treatment on cytochrome c release from mitochondria ................. 81 4.4.5 Effect of sAPPα and E1 on Bax-Bak activation........................................................ 82 4.5 Modulation of protein homeostasis by sAPPα ................................................................. 83 4.5.1 sAPPα prevents