! !"#$ #"%"" & '( ) ' *#+,% - . /0 - 1 2 /20 %2 3 2 /02 4 % 5 ' 64 ' 6% 7 1 +8 % +8 ' - /790' 4 %5 ': +8 ' 79: +8 1 %; +8 +<8 % = +8% ' +8 / 70' +8 - / 770% ' ;,! +8' == ' - +8/ 770%7 ' !!$ +8 : +8 / 7770%; ' 64 +8 - ' 6 +8 - / 777770%> ' 7? +<8 ' 6%; ' +<8 ' +<8 6 2% 7 +8 ' % ! !"#$ @:: %% : A B @@ @ @ #CDD"+ 7,D<$D#<<D<##!! 7,D<$D#<<D<##ED " # '#"+D# THE AMYLOID-Β PRECURSOR PROTEIN (APP)-BINDING PROTEIN FE65 AND APP PROCESSING Niina Koistinen The amyloid-β precursor protein (APP)-binding protein Fe65 and APP processing Niina Koistinen ©Niina Koistinen, Stockholm University 2018 ISBN print 978-91-7797-112-2 ISBN PDF 978-91-7797-113-9 Cover: An healthy and AD diseased brain half freely interpreted by Conrad, aged 9, and Cleo, aged 6 Printed in Sweden by Universitetsservice US-AB, Stockholm 2017 Distributor: Department of Neurochemistry, Stockholm University Till min familj List of Publications I. Koistinen NA, Bacanu S, Iverfeldt K Phosporylation of Fe65 amyloid precursor protein-binding protein in respons to neuronal differentiation Neuroscience Letters, 2016, Feb2;613:54-9 II. Koistinen NA, Edlund AK, Menon PK, Ivanova EV, Bacanu S, Iverfeldt K Nuclear localization of amyloid-β precursor protein-binding protein Fe65 is dependent on regulated intramembrane pro- teolysis PLoS One, 2017, Mars 21;12(3) III. Koistinen NA, Menon PK, Ivanova E, Kumchu ME, Iverfeldt K, Ström AL ADAM10 dependent nuclear localization of the amyloid-β precursor protein-binding protein Fe65 is attenuated in neu- ronally differentiated SH-SY5Y cells Manuscript IV. Koistinen NA, Menon PK, Iverfeldt K, Ström AL APP Ser675 phosphorylation affects α-secretase processing in decreased secretion of the neuroprotective ectodomain sAPPα Manuscript Minor parts of the work presented in this thesis have been previously published in my Licentiate thesis: Koistinen N The adaptor protein Fe65 and APP processing (2014) ISBN 978-91-7447-863-1 Abstract Alzheimer’s disease (AD) is a neurodegenerative disorder character- ized by abnormal deposition of neurotoxic amyloid-β (Aβ) peptide. Aβ is generated by sequential cleavage of the amyloid-β precursor protein (APP) by β- and then γ-secretase. However, APP can also be processed by α- and γ-secretase, instead resulting in generation of neuroprotective sAPPα. Increased APP phosphorylation and altered expression levels of the brain enriched Fe65 protein have been observed in the brains of AD patients. Fe65 can not only interact with membrane tethered APP, but can also localized into the nucleus and act as a transcriptional regu- lator together with the APP intracellular domain (AICD), generated af- ter γ-secretase processing. How APP processing, APP/Fe65 interaction, and the nuclear AICD/Fe65 complex is regulated has not yet been fully understood. The aim of this thesis was therefore to further elucidate how Fe65 is regulated and how APP Ser675 phosphorylation affects APP processing. We could identify several factors regulating Fe65. First, we identi- fied that neuronal differentiation induces Fe65 phosphorylation (paper I), and that phosphorylated forms of Fe65 were preferentially localized outside the nucleus (paper II). Second, we found that the APP binding PTB2 domain of Fe65, rather than the previously proposed N-terminal WW domain, is important for the nuclear localization of Fe65 (paper II). In addition, we surprisingly found that mutation of Ser228 in the Fe65 N-terminus could increase the APP/Fe65 interaction (paper III). Third, both α- and γ-secretase inhibitors decreased Fe65 nuclear local- ization similarly, indicating an important role of α-secretase in regulat- ing Fe65 nuclear localization (paper III). Lastly, we could in paper IV for the first time show that phosphorylation of APP at Ser675 regulates APP processing at the plasma membrane, resulting in reduced levels of 675 sAPPα. These results, together with the observation that APP Ser phosphorylation occur in AD brains, suggest that Ser675 phosphoryla- tion could contribute to AD pathology by decreasing α-secretase pro- cessing and instead increasing the levels of Aβ. In summary these studies have contributed to the understanding of APP processing and the interplay between Fe65 and APP, two sug- gested key players in AD. Contents 1 Introduction ......................................................................................... 1 1.1 Alzheimer´s Disease ...................................................................................... 1 1.2 The Amyloid-β Precursor Protein (APP) ...................................................... 3 1.2.1 APP-like proteins .................................................................................. 7 1.2.2 Regulated intramembrane proteolysis (RIP) of APP ...................... 8 1.2.3 APP phosphorylation .......................................................................... 14 1.2.4 APP interacting proteins .................................................................... 16 1.3 The adapter protein Fe65 ............................................................................ 18 1.3.1 Fe65 interacting proteins .................................................................. 20 1.3.2 Fe65 nuclear functions ...................................................................... 23 1.3.3 The effect of Fe65 on APP processing ............................................ 27 1.3.4 Phosphorylation of Fe65 ................................................................... 28 2 Methodological considerations ....................................................... 31 2.1 Cell lines ......................................................................................................... 31 2.1.1 SH-SY5Y ............................................................................................... 32 2.1.2 PC6.3 .................................................................................................... 32 2.1.3 SK-N-AS ............................................................................................... 32 2.2 Cell treatments .............................................................................................. 32 2.2.1 Retinoic acid (RA) .............................................................................. 33 2.2.2 PMA (Phorbol 12-myristate 13-acetate) ........................................ 34 2.2.3 NGF ....................................................................................................... 34 2.2.4 DAPT (Difluorophenylacetyl-alanyl-phenylglycin-t-butyl-ester) 34 2.2.5 ADAM10 inhibitor (GI254023X) ....................................................... 35 2.2.6 Batimastat (BB-94) ............................................................................ 35 2.3 Tandem Affinity Purification (TAP)-tag method ...................................... 35 2.4 siRNA gene silencing .................................................................................... 36 2.5 Site directed mutagenesis ........................................................................... 36 2.6 Phosphatase inhibition and alkaline phosphatase treatment................ 37 2.7 Biotinylation assay ........................................................................................ 37 2.8 Harvesting of cells ........................................................................................ 38 2.9 Protein concentration measurement ......................................................... 38 2.10 Western blot .................................................................................................. 39 2.11 Immunofluorescence (IF) imaging ............................................................ 39 3 Aim ...................................................................................................... 41 4 Results and discussion .................................................................... 43 4.1 Regulation of Fe65 phosphorylation and expression upon neuronal differentiation of SH-SY5Y cells (Paper I and III) ............................................. 43 4.1.1 Neuronal differentiation of SH-SY5Y and PC6.3 cells induce Fe65 phosphorylation .................................................................................................. 43 4.1.2 Expression of Fe65 during neuronal differentiation of SH-SY5Y and PC6.3 cells differs dependent on differentiating-agent used .............. 45 4.2 Regulation of Fe65 nuclear localization (Paper II and III) .................... 45 4.2.1 Fe65 nuclear localization is dependent on the PTB2 domain ..... 46 4.2.2 Phosphorylation of Fe65 generating an electrophoretic mobility shift resides between residues 192 and 260 ................................................. 47 4.2.3 Phosphorylated forms of endogenously expressed Fe65 are preferentially localized outside the nucleus .................................................. 48 4.2.4 Regulation of Fe65 nuclear localization and Fe65-APP interaction upon Fe65 Ser228 phosphorylation .................................................................. 51 4.2.5 Fe65 nuclear localization is dependent