The Amyloid-Β Precursor Protein (App)-Binding Protein Fe65 and App Processing

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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 proteolysis 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 characterized 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 regulator together with the APP intracellular domain (AICD), generated after 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 identified 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 localization similarly, indicating an important role of α-secretase in regulating 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 sAPPα. These results, together with the observation that APP Ser675

phosphorylation occur in AD brains, suggest that Ser675 phosphorylation could contribute to AD pathology by decreasing α-secretase processing 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 suggested 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 on regulated intramembrane proteolysis (RIP) ...... 53 4.3 Regulation of APP processing upon APP Ser675 phosphorylation (Paper IV) ...... 54 4.3.1 APP-Ser675 phosphorylation decreases sAPPα secretion without affecting APP cell surface localization ...... 55 4.3.2 APP-Ser675 phosphorylation generates, through α-secretase processing, a slower migrating APP-CTF band...... 56 4.3.3 Regulation of APP-Fe65 interaction upon APP Ser675 phosphorylation ...... 57 4.3.4 How does APP Ser675 phosphorylation affect APP processing? ... 58

5 Conclusions and outlook ...... 61

6 Populärvetenskaplig sammanfattning på svenska ...... 64

7 Acknowledgement ...... 67

8 References ...... 69

Abbreviations

Aβ amyloid-β CSF cerebrospinal fluid AD Alzheimer’s disease CTF C-terminal fragment ADAM a disintergrin and metalloprotease CuBD Copper binding domain AFT APP/Fe65/Tip60 Dab1 disabled-1 Ago2 argonaute protein 2 DAPT N-[N-(3,5-Difluorophenacetyl-L- AICD APP intracellular domain alanyl)]-(S)-phenylglycine t-butyl AP-3 adaptor protein complex 3 ester APH-1 anterior pharynx defective-1 DAT dopamine transporter APL-1 APP like-1 DNA deoxyribonucleic acid APLP1 APP like protein 1 dsDNA double stranded DNA APLP2 APP like protein 2 DTT dithiothreitol ApoE apolipoprotein E EGFR epidermal growth factor receptor ApoEr2 apolipoprotein E receptor 2 EO-FAD early-onset familiar AD APP amyloid-β precursor protein ER endoplasmic reticulum APPL amyloid-beta-like protein precur ERK1/2 extracellular signal-regulated sor protein kinase 1 and 2 ATM ataxia telangiectasia mutated ERα estrogen receptor α ATR ataxia telangiectasia and Rad3 Fe65L1 Fe65-like 1 related Fe65L2 Fe65-like 2 BACE β-site APP cleaving enzyme FEH-1 Fe65 homolog-1 BCA bicinchoninic acid GFLD growth factor like domain BCa breast cancer GFP green fluorescent protein BLM bloom syndrome protein Grb2 growth factor receptor-bound C83 83 amino acids long APP CTF protein C99 99 amino acids long APP CTF GSK-3β glycogen synthase kinase 3β c-Abl Abelson murine leukemia viral GWAS genome-wide association studie oncogene homolog 1 HEK293 human embryonic kidney cells CBP calmodulin binding protein hESNSCs human embryonic stem cell- CCD charge-coupled device derived neural stem cells Cdc2 cell division cycle protein 2 HRP horse radish peroxidase Cdk5 cyclin-dependent kinase 5 IF immunofluorescence cDNA complementary DNA IR infrared ChAT choline acetyltransferase JIP 1 JNK interacting protein CNS central nervous system JNK c-Jun N-terminal kinase

KI knock-in PTM posttranslational modification KO knock-out RA retinoic acid KPI Kunitz protease inhibitor RAR retinoic acid receptor LBP1 lipopolysaccharide binding protein RIP regulated intramembrane proteol 1 ysis LOAD late-onset Alzheimer’s disease RIPA radioimmunoprecipitation assay LRP low-density lipoprotein receptor- RISC RNA-induced silencing complex related protein RNA ribonucleic acid LSF late SV40 factor SAPK1b stress activated protein kinase 1b LTP long term potentiation sAPPα secreted APPα MAPK mitogen-activated protein kinase sAPPβ secreted APPβ MEFs mouse embryonic fibroblasts SDS-PAGE sodium dodecyl sulfate poly Mena mammalian-enabled acrylamide gel electrophoresis MMP matrix metalloproteinase SGK-1 serum/glucocorticoid regulated mRNA messenger RNA kinase 1 N2a neuro2a SH2 src homology 2 NGF nerve growth factor ShcA Src homology/collagen NICD Notch intracellulardomain siRNA short interfering RNA NLS nuclear localization signal TACE TNFα converting enzyme NMJ neuro muscular junction TAF-1β template-activating factor-1β NFT neuro fibrillary tangles TAP-tag tandem affinity purification PAT-1 protein interacting with APP tail 1 TEV tobacco etch virus PC12 pheochromocytoma 12 TGN trans golgi network PC6.3 pheochromocytoma 6.3 TH tyrosine hydroxylase PCR polymerase chain reaction Tip60 Tat interactive protein 60 PEN-2 presenilin enhancer TM transmembrane Pin1 peptidyl-prolyl cis/trans isomerase TNFα tumor necrosis factor α NIMA interacting-1 TS thymidylate synthase PKC protein kinase C UV ultraviolet PLC phospholipase C VLDLR very low-density lipoprotein PMA phorbol 12-myristate 13-acetate receptor PS1 presenilin 1 WW proline-proline PS2 presenilin 2 ZnBD zinc binding domain PTB phosphotyrosine binding

1 Introduction

1.1 Alzheimer´s Disease Alzheimer´s disease (AD) is a progressive neurodegenerative disease affecting the brain, and the most common form of dementia [1]. AD is characterized by the loss of cognitive and executive functions which include impairments in memory, language, computational skills, rea- soning, judgement, and social behavior. Interestingly, functional abili- ties that were acquired during the latest stages of adolescent development are lost during the earliest stages of AD. Thus, the cognitive developmental theory [2] has been used to explain the systematic order in which cognitive functions are lost, and as a foundation for predicting cognitive skills of patients at certain stages of the disease. Neuropathologically, AD is characterized by extracellular deposits, so called senile plaques composed of amyloid β-peptides (Aβ) [3], and intracellular neurofibrillary tangles (NFTs), composed of paired helical filaments of hyperphosphorylated tau protein [4]. The amyloid cascade hypothesis, first proposed by Hardy and Hig- gins in 1992, [5, 6], has been the dominant model for AD pathogenesis, and has been leading the development of potential treatments. The amyloid cascade hypothesis states that abnormal processing of APP causes the overproduction of toxic Aβ fragments, leading to the formation of senile plaques, and then to NFTs, neuronal cell death and ultimately dementia. On the other hand, the tau hypothesis identifies tau hyper- phosphorylation and the formation of NFTs as the primary cause of AD. This is based on the observations that tau tangle pathology occurs prior to Aβ plaque formation [7], and NFT load correlates more closely with AD disease progression and severity than plaque load [8]. Whether senile plaques or NFTs is the cause or the consequence of AD pathology

1 is still controversial. However, the work described in this thesis only focus on Aβ, and hence the contribution of the Aβ-peptide and its precursor, amyloid β-precursor protein (APP), in AD. The APP protein can be processed by at least three different proteases termed α-, β-, and γ-secretases (see section 1.2.2). APP processing can occur through two different pathways; the non-amyloidogenic (α- secretase) and the amyloidogenic (β-secretase) pathway. The neurotoxic Aβ peptides are derived through the sequential cleavage of APP by β- and γ-secretases. Genetic research has identified two distinct forms of AD; the early onset form of familial AD (EO-FAD), and late onset AD (LOAD). Mu- tations in three genes, APP, PS1 and PS2 (presenilin 1 and presenilin 2; PS is the catalytic component of the γ-secretase complex) [9-11] have been found to cause the autosomal dominant EO-FAD. EO-FAD has shown to affect people younger than age 65, and to represent approx. 5% of all AD cases. Of these, PS1 account for most of EO-FAD, while APP and PS2 are rarer. However, without exception, the mutations increase the generation of all Aβ isoforms (total Aβ) or the relative proportion of the 42-amino acid isoform (Aβ42) that is more neurotoxic. Pathological mutations in one of these genes, virtually guarantees that one will develop early onset AD. Late onset AD (LOAD) is the most common form of the disease accounting for approx. 90% of all AD cases. While EO-FAD is certainly genetically based, there are no specific gene mutations that are associated with inheritance of the disease in LOAD. Most likely, etiological factors are involved in the development of LOAD, including epigenetic, metabolic and environmental factors. However, one major genetic risk factor identified for LOAD, is the apolipoprotein E (ApoE) ε4 allele [12]. Human ApoE exists as three major alleles, designated ε2, ε3, and ε4 [13]. Carrying one copy of the ε4 allele increases the risk of developing AD 3-4 times, whereas homo- zygous ε4/ε4 carriers have an 8-12 times increased risk of developing AD compared with ε2, which has a protective effect [14, 15]. However, inheriting an ApoE ε4 allele does not mean that a person will develop AD. ApoE has a key role in lipid metabolism [16], and studies have suggested that ApoE4 can increase Aβ peptide aggregation and impair Aβ clearance in the brain (reviewed in [17]), which acts as a driving

2 force for pathogenesis of AD. In addition to ApoE, genome-wide association studies (GWAS) have led to the identification of 11 additional candidate genes involved in LOAD (reviewed in [18]). Interestingly, three main pathways can be identified, with regard of function for these genes in AD pathogenesis: 1) lipid metabolism, 2) innate immunity, and 3) cellular signaling. Currently there is no cure for AD, and this will become an even greater social problem in the future, as the aging population increases. Increasing age is considered as one of the major risk factors for AD. To date, only symptomatic treatments exists for AD. Treatments capable of stopping, or at least effectively modifying the course of AD, are still under extensive research. Inhibition of the β- and γ-secretase enzymes (see section 1.2.2.2 and 1.2.2.3, respectively) have been the prime therapeutic strategy for the development of drugs for the treatment of AD. The limitations of new therapeutics to reach clinical trials may be due to the animal models currently available, which are based on the rare EO-FAD forms of the disease, and the heterogeneity of the LOAD. Thus, to block the progression of the disease, several pathogenic steps responsible for the clinical symptoms must be considered, including deposition of extracellular Aβ plaques, intracellular NTFs, inflammation and cholesterol metabolism. Therefore, to uncover the mosaic mechanisms underlying the pathogenesis of AD is of vast importance. Consequently, in this thesis, I try to bring some more light upon cellular functions of an APP binding adaptor protein, Fe65, proposed to be involved in APP processing, hence in AD pathogenesis. In addition, the role of APP phosphorylation at its C terminus, in APP processing is investigated.

1.2 The Amyloid-β Precursor Protein (APP) APP is the precursor to the Aβ peptide, the major constituent of the extracellular amyloid plaques. The physiological roles of APP are not fully understood, and remains one of the enigmatic issues in this field. Purification and sequencing of the Aβ peptide led to the cloning of APP in 1987 [19]. The APP gene is located on chromosome 21, and, interestingly, individuals suffering from Down´s syndrome (trisomy

3 21) with an extra copy of this chromosome develops an EO-FAD [20, 21]. This, and the mutations in APP, PS1 and PS2, have served as a fertile ground for the amyloid cascade hypothesis. APP is classified as a type-I transmembrane glycoprotein with a large N-terminal extracellular domain, separated by a single transmembrane domain from a short C-terminal cytoplasmic tale (reviewed in [22]). Alternative splicing of the APP transcript generates at least 8 isoforms of which the 695 (APP695), 751 (APP751) and 770 (APP770) amino acid forms are the most common [23](reviewed in [24]). The specific APP isoforms are expressed in a cell-specific manner, with APP695 being predominantly expressed in neurons, whereas APP751 and APP770 are expressed in most tissues examined [25]. The three isoforms differ mainly by the absence (APP695) or presence (APP751/APP770) of a Kunitz protease inhibitor (KPI) domain. This domain is located towards the N- terminus of the protein, and exhibit protease inhibitory activity [26]. The large extracellular portion of APP contains E1 and E2 domains of which the E1 domain can be further divided into a heparin-binding/growth factor-like domain (GFLD), and a metal (copper (CuBD) and zinc (ZnBD)) [27] binding domain. The E2 region contains a second heparin binding domain, and a RERMS motif implicated in trophic functions [28-32]. The E1 and E2 domains are separated by an acidic region and the KPI domain (absent in APP695). In addition, APP770 contains an OX-2 related domain [33, 34]. Several studies have suggested that the large N-terminal extracellular portion of APP possesses neurotrophic properties [32, 35-44]. How- ever, most of the neuroprotective properties have been assigned to sAPPα, a soluble cleavage product generated through α-secretase processing of APP (see section 1.2.2.1). Interestingly, APP has shown to form dimers in both cis- and trans orientation (within the same cell or at adjacent cells, respectively) at the cell surface [45, 46] (reviewed in [47]). cis-dimerization affects APP processing [48-50], whereas interaction in trans has been reported to promote cell adhesion [45, 48, 51- 53]. The dimerization has shown to be dependent on the E1 domain with a possible contribution from the E2 domain in the presence of heparin [54, 55]. In addition, binding of copper to the CuBD and GFLD

4 promotes APP dimerization in both cis- and trans-cellular manner, affecting APP physiological function at the synapse [56]. Hence, APP has been shown to localize at the pre- and postsynaptic sites, where it can form membrane tethers across the synapse [52]. The transmembrane (TM) domain, and the juxtamembrane region of APP, contains GXXXG motifs critical for TM protein interactions [57]. In addition, the Aβ sequence is situated between the extracellular juxtamembrane region and the TM domain. Interestingly, several of the familiar APP mutations of AD are located in the TM domain and juxtamembrane region [58-60]. Some of the pathogenic mutations on APP are believed to affect APP dimerization, changing the conformation and/or stability of the dimer, leading to the increase of the Aβ42:Aβ40 ratio, hence leading to the accumulation of the more neurotoxic Aβ42 peptide [57, 59, 61]. The short intracellular C-terminal domain of APP contains at least three functionally important motifs, which can be phosphorylated at several residues (discussed further in section 1.2.3), and are able to mediate interaction with different adaptor proteins (see section 1.2.4 and 653 656 1.3) [62]. The YTSI motif (according to APP695 numbering) is a basolateral sorting signal which binds the microtubule binding protein PAT1 (protein interacting with APP tail 1) mediating APP intracellular transport through the secretory pathway [63]. In a recent study, mutation of Tyr653 to alanine disrupted the transit of APP from the Golgi to the lysosome [64]. In addition, the 653YTSI656 motif was shown to be responsible for the interaction of APP with the adaptor protein complex-3 (AP-3), previously linked to the trafficking of APP from the Golgi to the lysosome [65]. The APP/AP-3 interaction was disrupted by Ser655 phosphorylation, and decreased lysosomal transport of APP from the Golgi was observed [64]. The APP C-terminal 667VTPEER672 motif forms a type I β-turn and N-terminal helix-capping box structure stabilizing APPs C-terminal helix structure [66, 67]. The 667VTPEER672 motif contains a phosphorylation site at Thr668 which has shown to be phosphorylated by several different kinases [68-72], and is believed to be involved in neuron-specific aspects of APP metabolism and function [73]. Thr668 is one of seven amino acid residues shown to be phosphorylated in AD brain [62].

5 Phosphorylation of Thr668 has shown to induce a significant conformational change in the cytoplasmic region of APP, for instance affecting its interaction with a neuron-specific adaptor protein Fe65 [74] (discussed further in section 1.2.4 and 1.3). The 682YENPTY687 motif of APP is responsible for the efficient internalization of APP, in clathrin coated vesicles, to early endosomes [75]. Deletion or mutation of this motif leads to impaired endocytosis of APP and significantly reduced Aβ production [76, 77]. Interestingly, transgenic mice expressing an Y682G mutation in APP was shown to produce higher sAPPα and CTFα levels, suggesting a shift towards the non-amyloidogenic pathway [78, 79]. The 682YENPTY687 motif is also recognized by proteins containing a phosphotyrosine binding domain (PTB) [80-82] (see section 1.2.4), such as the Fe65 family of proteins (discussed further in section 1.3) [83-86]. APP is synthesized in the endoplasmic reticulum (ER) and is subject to posttranslational modifications (PTMs), such as N- and O-linked gly- cosylation, phosphorylation, and tyrosine sulphation in the Golgi before the mature APP is transported to the cell surface via the secretory pathway (reviewed in [87]). Still, only 10% of trafficked APP reaches the plasma membrane [88]. The transport of APP from ER to plasma membrane may differ in non-polarized and polarized cells, such as neurons, since soma, dendrites, and axons play different functions and have different sets of proteins and lipids that regulate protein trafficking [88]. In addition, at the cell surface or in the endocytic compartment, APP is subject to regulate intramembrane proteolysis (RIP) [89] (see section 1.2.2) which leads to the release of different biologically active fragments. Further, APP expression is developmentally regulated [90-92], suggesting an important role for APP in development. Hence, APP has shown to have a role in neuronal migration in early embryogenesis [93]. A role for APP in synapse formation and maintenance have been suggested, since APP levels have shown to peak in the second postnatal week, and coincides with the timing of brain maturation and completion of synaptic connections [90]. Indeed, APP KO mice display reduced spine number [94], altered long term potentiation (LTP) response [95], and reduced brain mass [96], while increased spine density is observed

6 in APP overexpressing transgenic mice [97]. In neurons, APP can be found in presynaptic and postsynaptic compartments [98, 99] where APP processing can be regulated by synaptic activity [100].

1.2.1 APP-like proteins Two mammalian APP homologues, APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2), share similar structure and membrane to- pology as APP [101-103]. APLP2 is expressed ubiquitously [104], while APLP1 is only reported to be expressed in the nervous system [105, 106]. Interestingly, the E1- and E2-domains are highly conserved between APP and the APP-like proteins [101, 102, 106]. The formation of homo- and heterodimers of the APP-family proteins has been shown to affect e.g. cell-cell adhesion [51] and their proteolytic processing [61]. The APLPs undergo proteolytic processing like APP, however the Aβ domain is unique for APP, hence Aβ peptides can only be generated from the processing of APP. Single knockouts (KOs) of the APP-family members only cause mi- nor abnormalities [107-109], whereas triple KO mice show a 100% lethal phenotype [110]. However, studies made of combined KO mice have shown that the APP family of proteins are essential for survival and possesses partially redundant functions; APLP1-/-/APP-/- mice are viable and apparently normal, whereas APLP2-/-/APP-/- and APLP2-/- /APLP1-/- were shown to be prenatally lethal [107]. Genetic redundancy of the APP family members in synapse formation have been suggested, since no synaptic deficits are present in APLP2 KO mice [41, 111], whereas APLP2-/-/APP-/- and APLP2-/-/APLP1-/- mice exhibit deficits in neuromuscular junction (NMJ) formation and incorrect apposition of pre- and postsynaptic sites [53, 107, 108, 112]. Important clues to the function of APP family proteins in development have come out from genetic analyses in Caenorhabditis elegans and Drosophila melanogaster, two species shown to have APP-like orthologues, APL-1 [113], and APPL [114], respectively. The human APLP1 occurs to be the closest homolog of the invertebrate APP-like proteins (APL-1 and APPL) [115], based on direct sequence compari-

7 son. However, prokaryotes, plants and yeasts seem not to possess members of the APP family of proteins. In C. elegans, KO of APL-1 induces lethal molting defects [116], whereas KO of APPL in Drosophila melanogaster is not lethal, but leads to subtle behavioral deficits [117], deficiency in maintenance of synaptic boutons at the NMJ [118], and to increased vulnerability to brain injury [119].

1.2.2 Regulated intramembrane proteolysis (RIP) of APP APP can be processed by at least three different proteases, termed α- , β-, and γ-secretases, in a process called regulated intramembrane proteolysis, RIP. Simply put, RIP occurs when a transmembrane protein is initially cleaved (shedded) close to the transmembrane domain, by α- or β-secretases, resulting in shedding of an ectodomain, and subsequently cleaved within the membrane region by γ-secretase, releasing a cytoplasmic fragment that can translocate into the nucleus, and activate gene expression [89, 120]. RIP has shown to have two fundamentally different functions, namely signaling and degradation of membrane proteins. Regulation of RIP processes have shown to mostly occur at the shedding level (reviewed in [121]). The proteolytic processing of APP can be divided into two different pathways, the non-amyloidogenic and the amyloidogenic. The processing results in the production of several different bioactive APP fragments, and may therefore lead to different functional consequences in the cell. The major pathway is the non-amyloidogenic pathway precluding the formation of Aβ due to the cleavage by α-secretases within the Aβ sequence. In the amyloidogenic pathway, Aβ is generated from the sequential cleavage of APP by β-secretase, and then γ-secretase. APP processing by secretases occurs in different cellular compartments. The α-secretase processing of APP occurs preferentially at the plasma membrane [122-124] (see section 1.2.2.1), whereas β-secretase cleavage predominantly occurs in endosomes [125, 126] (see section 1.2.2.2). The subsequent γ-secretase cleavage has shown to take place at the plasma membrane or in the endosomal/lysosomal system [127- 129] (see section 1.2.2.3). Whether APP is processed by α- or β-secre-

8 tase is highly dependent on the colocalization of APP with its processing enzymes, thus the transport and cleavage of APP are intimately related processes. Hence, increased β-secretase-mediated processing of APP can be observed when APP trafficking to the cell surface is impaired [88, 130], while reducing APP internalization, or enhancing APP routing to the cell surface facilitates α-secretase-mediated processing [131]. Cholesterol and lipid metabolism may also play role in determining where and how APP is processed [132, 133]. It is hypothesized that APP within lipid rafts undergo amyloidogenic processing, whereas APP outside lipid rafts is subjected to non-amyloidogenic processing [134].

1.2.2.1 α-secretase processing of APP The α-secretase cleavage of APP produces a large N-terminal secreted ectodomain, sAPPα, and a C-terminal membrane tethered fragment, C83. The C83 fragment is further processed by the γ-secretase complex (see section 1.2.2.3) leading to the production of a small secreted peptide, p3, and the APP intracellular domain, AICD. The sAPPα fragment has shown to be sufficient to rescue the phenotype of APP deficient mice, and have been reported to have neurotrophic and neuroprotective functions [37, 135-137]. APP fragments, including the N- terminal sAPPα, has shown to be concentrated at process endings [138], and may play a role in synaptic function [135]. The α-secretase cleavage is located at position 16 within the Aβ sequence, precluding the generation of Aβ [139]. α-secretase and β-secretase may compete for APP as a substrate, hence, the increase in sAPPα secretion, when overexpressing the α-secretase ADAM10, was shown to be accompanied by a reduction in β-secretase processing and Aβ production [140]. On the contrary, reduced α-secretase cleavage was observed when β-secretase cleavage was strongly increased upon overexpression of BACE1 [126, 141]. Therefore, an increase of APP processing by α-secretase has been considered as a therapeutic approach for AD [142], as it is assumed to reduce Aβ generation. The α-secretase is a type-I transmembrane zinc metalloproteinase, and several members of the a disintegrin and metalloprotease (ADAM)

9 family, like ADAM9, ADAM10, and tumor necrosis factor α converting enzyme (TACE)/ADAM17 [122, 143-145], acts as α-secretases. However, ADAM10 is believed to be the α-secretase involved in the constitutive cleavage of APP in neurons [141, 146], whereas ADAM17 was shown to cleave APP upon stimulation with the phorbol ester PMA [144]. The role of ADAM17 in APP shedding was further confirmed when increased ADAM17 expression, and increased levels of sAPPα, was observed in rat neocortex upon induction of hypoxia [147]. Still, ADAM10 has a more coordinated expression with APP in human brain compared with ADAM17 [148]. There are several studies showing that reduction of ADAM10 expression or activity is linked to AD pathology. ADAM10 mRNA levels are lowered in moderate to severe AD [148], and α-secretase activity is reduced in temporal cortex homogenates from AD patients [149]. In addition, AD patients display lower protein levels of both ADAM10 and sAPPα in platelets, and lower levels of sAPPα in the cerebrospinal fluid [150, 151]. Moreover, forebrain-specific conditional ADAM10 KO [152], and impaired ADAM10 trafficking in adult mice [153], was shown to shift APP processing towards Aβ production, suggesting that reduced sAPPα generation by ADAM10 may contribute to early stages of sporadic AD. Intriguingly, two rare mutation in the prodomain of ADAM10, was suggested to cause LOAD by attenuating α-secretase activity and reducing adult hippocampal neurogenesis [154, 155]. In addition, a mutation adjacent to the α-secretase site (K687N in APP770) have shown to make this specific APP mutant protein a poor α-secretase substrate and lead to EO-FAD [47, 156]. Besides APP, ADAM10 has several other substrates, including Notch [157], and is involved in a wide range of cellular functions such as cell migration [158], proliferation [159-161], and cell signaling [162]. Constitutive and conditional ADAM10-KO mice both exhibit embryonic lethality at early stages, most likely because of deficient processing of Notch and its ligands [146, 163].

1.2.2.2 β-secretase processing of APP β-secretase cleavage of APP generates sAPPβ and a membrane tethered C-terminal fragment (CTF), C99 [164], which is subsequently

10 cleaved by γ-secretase (see section 1.2.2.3) giving rise to the Aβ peptide and AICD. Hence, the processing of APP by both β-and γ-secretase is necessary for the generation of Aβ, and β-secretase cleavage of APP is the rate limiting step in Aβ production [126]. Intriguingly, it has been shown that, in neuronal cell lines, sAPPβ, Aβ, and AICD are preferentially formed from the neuronal APP695 isoform, whereas the longer APP isoforms (APP751 and APP770), harboring the KPI domain, are more prone to localize to the cell surface and are preferentially cleaved by the non-amyloidogenic pathway [165-167]. In addition, despite that the secreted sAPPβ fragment only differ by 16 amino acids from sAPPα, it lacks all the neuroprotective properties associated with sAPPα [136, 168]. The APP cleaving β-secretase, β-site cleaving enzyme 1 (BACE1) [126, 169-173], is a type I aspartic protease, mainly localized to acidic compartments such as endosomes and the Golgi apparatus [126, 174- 176], providing a low pH environment, which is more favorable for BACE1 activity. Highest β-secretase activity was observed in neural tissue and neuronal cell lines [177], hence BACE1 is predominantly expressed in brain and abundantly in neurons [126, 170, 178-180]. Ele- vated levels of BACE1 have been directly correlated with Aβ induced pathology in AD brains [181-184]. Additionally, BACE1 has shown to initiate the generation of Aβ at the synaptic terminals [178], and to ac- cumulate around normal and dystrophic presynaptic terminals surrounding amyloid plaques in brains of AD mouse models and patients [178-180]. This accumulation likely results in a menacing cycle by increasing Aβ production near synapses. KO of BACE1 drastically reduces AD-related symptoms in AD mouse models [179, 185, 186], probably due to reduced Aβ production [187-189], and therefore reducing the amyloid plaque load. Interestingly, the disease causing Swedish mutation (K670N;M671 L) [190] and A673V [191] mutation in APP make the cleavage of APP by the β-secretase more efficient, generating greater amounts of C99 and Aβ, whereas A673T mutation in APP has shown to be protective [192]. Hence, inhibition or modulation of β- secretase in the brain should decrease Aβ levels and be beneficial for AD. Mice deficient in BACE1 show relative mild phenotypes [193, 194], however, they do display deficits in both synaptic transmission

11 and plasticity at hippocampal synapses [179, 193, 195]. Interestingly, physiological concentrations, in pM range, of Aβ have been shown to facilitate synaptic plasticity [196]. Since BACE1 is the rate limiting step in the production of Aβ, it is considered as the most promising drug target for AD therapy. However, caution should be taken considering the role of BACE1 in synaptic plasticity, hence, loss of physiological levels of Aβ may lead to synaptic deficits. In addition, APP is not the only substrate for BACE1 cleavage; BACE1 also cleaves neuregulin-1 [197], voltage gated sodium channels [198], P-selectin glycoprotein lig- and 1 [199], type II α 2,6 sialytransferase [200], and interleukin-1 receptor type II [201] .

1.2.2.3 γ-secretase processing of APP In both the non-amyloidogenic and the amyloidogenic pathway, ectodomain shedding of APP by α- or β-secretase is followed by γ-secretase processing of the membrane associated APP-CTFs, C83 and C99, respectively. The γ-secretase cleavage releases the p3 or Aβ fragment, respectively, and an APP intracellular fragment, AICD. To date no clear biological function has been established for the p3 fragment, and it has generally been considered as non-amyloidogenic. However, the p3 fragment have shown to be deposited in brains of AD and Down syndrome patients [202-204], leading to the assumption that p3 might be neurotoxic [205, 206]. The γ-secretase cleavage of APP, following β- secretase processing, varies, and Aβ peptides ranging from 34-50 amino acids have been identified (reviewed in [207]). Hence, γ-secretase cleavage shows flexibility in its site of C-terminal cleavage of APP, and AICD is generated by prior cleavage of APP at the so-called ε-site following initial release of its ectodomain [208]. In addition to AICD, γ- secretase cleavage at the ε-site has shown to release Aβ49 or Aβ48, which are by cleavage after every third C-terminal amino acid, further processed by γ-secretase to Aβ40 or Aβ42, respectively [208]. The major fragment generated is Aβ40 [209], whereas the Aβ induced neurotoxicity is thought to result from the Aβ42 form, which has proven to be more hydrophobic and amyloidogenic than shorter Aβ forms [210].

12 γ-secretase is an aspartyl protease and a transmembrane multiprotein complex composed of presenilin 1 (PS1) or presenilin 2 (PS2), nicastrin, anterior pharynx defective 1 (APH-1), and PS enhancer 2 (PEN-2) [211]. Presenilin (PS) contains the two essential aspartyl residues which are critical for the activity of the aspartyl protease complex [212]. Dom- inant missense gene mutations causing EO-FAD are found in both PS1 and PS2, and have shown to affect the conformation of the PS proteins [11, 213-215]. Moreover, these mutations lead to increased Aβ42:Aβ40 ratio, and overall increased aggregation tendency of the Aβ peptide [216-219]. In the γ-secretase complex, nicastrin is thought to function as a size selecting substrate receptor [220, 221], and PEN-2 has shown to facilitate endoproteolysis of PS to its heterodimeric active form, and to stabilize PS within the complex [222, 223]. APH-1 may act as a scaffold for the initial binding of nicastrin and the assembly of the complex [224]. A small fraction of the γ-secretase complex is trafficked through the secretory pathway to the cell surface, the rest is mainly localized at the ER, Golgi/trans golgi network (TGN), and endosomes [127-129, 225- 229]. APP proteolysis by γ-secretase has been proposed to occur in all places, from the Golgi apparatus, to the cell membrane, and endocytic vesicles [219, 230-232]. The γ-secretase complex has also been called the “proteasome of the membrane”, since APP is not the only substrate for the γ-secretase complex (reviewed in [233]). Hence, in addition to its essential role in cell signaling pathways, one other function may be to clear out membrane protein stubs that remain after ectodomain release by sheddases. One important γ-secretase substrate is the Notch family of receptors [234, 235]. Notch is an evolutionally conserved type I transmembrane receptor regulating neuronal development and controlling processes in the adult brain such as neurogenesis, neurite outgrowth, synaptic plasticity, and LTP [236-238]. Notch receptors are activated upon ligand binding on the adjacent cells, and undergo γ-secretase-dependent cleavage to generate the Notch intracellular domain (NICD). NICD is subsequently

13 translocated into the nucleus where it can modulate transcription by interacting with specific transcription factors [239-241]. As previously mentioned, γ-secretase processing of APP/C83 or APP/C99 not only liberates Aβ, but also the APP intracellular domain, AICD [242-245], analogous with NICD. AICD has been reported to be present in the nucleus [246-251], where it might act as a transcription factor [252, 253]. It has been proposed that the transcriptionally active AICD is mainly generated through the β-secretase pathway [165, 247, 254], however, in one study the absence of the β-secretase was shown to not affect AICD production [255]. Interestingly, FAD-linked mutations in APP primarily affect gamma-secretase-mediated Aβ42 formation, and not AICD signaling, which remains unaltered [256]. Still, studies have shown that the levels of AICD are increased in brains of AD patients and murine models reproducing the disease [257], proposing a toxic role of AICD in addition to Aβ. AICD was shown to form a complex with the adaptor protein Fe65 and the histone acetyl transferase Tip60, which stimulated gene transcription in a heterologous reporter system [258] (discussed in more detail in section 1.3).

1.2.3 APP phosphorylation Phosphorylation of the APP C-terminus has shown to be important for the binding of cytosolic interacting proteins, subcellular trafficking, and processing of APP. APP is constitutively phosphorylated in brain tissues and neuronal cells [60, 69, 70]. However, altered phosphorylation of APP has shown to be closely related to AD, hence, hyperphosphorylated APP has been observed in the brains of AD patients [62]. Of eight possible phosphorylation sites in the C terminus of APP, seven was found to be phosphorylated in AD brains (Tyr653, Ser655, Thr668, Ser675, Tyr682, Thr686, and Tyr687) [62]. Among them, phosphorylation of Thr668 has been the most studied. Phosphorylation of Thr668 was detected on mature (N- and O-glycosylated), but not immature APP [69], suggesting that phosphorylation of Thr668 may regulate APP maturation in the early secretory pathway. However, several of the identified kinases, such as glycogen synthase kinase 3β (GSK3β), cell division cycle protein 2 (Cdc2), cyclin-dependent kinas 5 (Cdk5), and stress activated

14 protein kinase 1b/c-Jun N-terminal kinase 3 (SAPK1b/JNK3), responsible for APP Thr668 phosphorylation, are associated with neurotoxicity and implicated in neurodegenerative diseases [60, 68, 69, 71]. Hence, APP phosphorylated at Thr668 was shown to be upregulated in AD brains, and to colocalize with BACE1 in enlarged endosomes [62]. Consequently, the APP-C99-CTF, but not APP-C83-CTF was shown to be preferentially phosphorylated at Thr668 [62]. In addition, overexpression of an APP T668A mutant significantly reduced Aβ levels in neuronal cultures [62]. Moreover, in AD mouse models, Thr668 phosphorylation was shown to be required for the development of neuronal pathology [259-263]. Phosphorylation of Thr668 have also shown to reduce Fe65 binding to APP [264] (see section 1.3.2.1), and to control the interaction between APP and the prolyl isomerase Pin1 [265-267]. Phosphorylation of Thr668 induces a conformational change of the APP intracellular domain, from trans to cis. Pin1 binds to Thr668-phosphorylated APP and catalyzes its isomerization, hence regulating the intracellular domain between the two conformations, from cis to trans [265]. Interestingly, Pin1 overexpression has shown to decrease Aβ secretion in cell culture, whereas KO of Pin1 in mice have shown to increases β- secretase processing of APP leading to elevated levels of Aβ. However, Thr668-phosphorylated APP has also been found in neurite endings [69, 73], suggesting that phosphorylation is involved in regulating APP transport into neurites. Low levels of Thr654 and Ser655 phosphorylation have been observed in healthy rat brain and cultured cells [70, 268]. These amino acids are found in the 653YTSI656 motif, functioning as a basolateral sorting signal of APP [63, 269]. Ser655 was shown to be phosphorylated by protein kinase C (PKC) [270]. Phosphorylation of Ser655 has shown to enhance APP secretory trafficking, and increase production of sAPPα [271]. In a recent study, Tam and coworkers [64] demonstrated that phosphory- 655 lation of Ser decreased the production of Aβ42. In addition, decreased APP trafficking to lysosomes was observed. Particularly, tyrosine phosphorylation of APP has been proposed to regulate APP processing. Interestingly, APP-C99-CTFs, but not APP- C83-CTFs appear to be phosphorylated on Tyr residues from AD brains [272]. Specifically, phosphorylation of the 682YENPTY687 motif has

15 shown to regulate the binding of specific adapter proteins. For instance, phosphorylation of Tyr682 has shown to inhibit binding of the Fe65 family of proteins [273, 274], whereas the Src homology/collagen A (ShcA) and growth factor receptor-bound protein 2 (Grb2) requires phosphorylation of Tyr682 for interaction [275, 276]. Intriguingly, knock-in (KI) of the T682G mutation, leads to a marked increase of the non-amyloidogenic pathway in the brains of mice, with increased levels of sAPPα and decreased levels of Aβ [78]. However, the mutation still induces synaptic loss, neuronal degeneration, and cognitive and learning deficits, despite the absence of Aβ [78, 79]. It is most likely that the T682G mutation prohibits adaptor protein interactions, which are either dependent on the phosphorylated Tyr residue, or by the Tyr residue per se, and attenuates proper APP trafficking. Mutation of the second Tyr residue in the YENPTY motif, Tyr687, to glutamic acid (APP-Y687E) was shown to target APP to the membrane, and dramatically decreased Aβ production, whereas the non-phosphorylated form (APP-Y687F) was endocytosed similar to wild type and favored by β-secretase cleavage [277]. In addition, an APP-Y687A mutant was shown to decrease the levels of APP at the cell surface, and to generate less APP-C83-CTFs compared with APP wild type [278], suggesting that Tyr687 phosphorylation is also involved in regulating APP trafficking and processing.

1.2.4 APP interacting proteins APP interacts with adaptor proteins through its C-terminal domain, however, it is not known whether these interactions occur exclusively with full-length APP, CTFs or AICD. Several PTB or Src Homology 2 (SH2) domain containing proteins bind the intracellular 682YENPTY687 motif of APP in a phosphorylation-dependent manner. SH2-domain containing proteins preferentially bind to the 682YENPTY687 motif of APP, when the first tyrosine (Tyr682) in this motif is phosphorylated [275, 279], whereas some PTB domain containing proteins show decreased interaction upon this same phosphorylation [279]. Several binding partners for APP have been identified (reviewed in [280]), however only a few of them will be discussed in this thesis. In

16 addition, one of the most studied APP interacting proteins, the brain enriched adapter protein Fe65, will be exclusively discussed in section 1.3. Two APP interacting proteins requiring phosphorylation of Tyr682, are Grb2 [276] and ShcA [275]. However, different domains are involved in the interaction with APP, the SH2 domain of Grb2 and PTB domain of ShcA. Interestingly, increased levels of ShcA and Grb2 bound to tyrosine phosphorylated APP-CTFs was observed in AD brains, however the interaction was confined to activated astroglial cells only [272]. In addition, ShcA/APP-CTF interaction was detected in cultured astrocytes upon thrombin treatment, whereas the complexes were undetectable in neuronal cells, suggesting that APP/ShcA is involved in AD related inflammation and gliosis [272]. In addition, the X11 protein has shown to require phosphorylation of the 682YENPTY687 motif, in order to bind APP via its PTB domain [273]. The binding of X11 to APP has shown to inhibit Aβ production in transfected non-neuronal cells [281], and in brains of double APPswe/X11 transgenic mice [282]. The X11-family of proteins have been shown to be important in neuronal synaptic functions [283]. The interaction of Dab1 with APP has shown to increase APP cell surface levels, and to increase α-secretases processing of APP. Dab1 has shown to interact with APP through its PTB domain, in a phosphorylation independent manner [284-286]. Dab1 is an adaptor protein involved in Reelin signaling, triggering an intracellular kinase cascade that inhibits for instance GSK3β [287]. Interestingly, enhanced Reelin expression was observed in AD brains, however Reelin biological activity was shown to be hindered by its interaction with Aβ aggregates [288]. Anterograde APP transport has been shown to be mediated by the scaffold protein JNK-interacting protein (JIP) 1, and the interaction of JIP 1 with APP has shown to occur through the PTB domain of JIP 1 [289-291]. Interestingly, JIP 1 preferentially interacts with Thr668 phosphorylated APP. In addition, in neurons, JIP 1 colocalization with vesicles containing Thr668 phosphorylated APP was favored. Hence, APP phosphorylation was shown to regulate the transport of APP/JIP 1 complexes in neurites [291].

17 APP has also been shown to bind to the Notch inhibitor Numb, and AICD can repress Notch nuclear activity [292]. Numb has been suggested to function as an endocytic adapter protein [293]. Interestingly, an isoform specific effect of Numb on APP transport and processing was observed [294]. While Numb with a shorter PTB was shown to increase Aβ production, Numb with a longer PTB was shown to sup- press Aβ formation [294]. According to aforementioned observations, neuronal degeneration and death could be due to variations in APP binding to specific adaptor proteins, which are presumably involved in triggering APP miss-sorting in neurons. To inhibit, or to promote, specific APP adaptor protein interactions could be a way to manipulate APP trafficking and processing, hence, be a target for the development of drugs for the treatment of AD in the future.

1.3 The adapter protein Fe65 Fe65 is a brain-enriched adaptor protein shown to be important for brain development [295]. Using a library from rat brain, Duilio and coworkers first isolated the Fe65 cDNA in 1991 [296]. In 1995, Fiore and coworkers then identified APP as a binding partner for Fe65 [297], suggesting that Fe65 may play a role in the pathogenesis of AD. In addition to Fe65, the Fe65 protein family consist of two closely related homologues, Fe65L1 (Fe65-like 1) [85] and Fe65L2 (Fe65-like 2) [84]. The Fe65 gene was mapped to the distal end of chromosome 11 [83], whereas Fe65L1 and Fe65L2 are encoded by chromosomes 4 and 5, respectively [298, 299]. The expression of Fe65 is enriched in the brain, whereas both F65L1 and Fe65L2 are more widely expressed [83- 86, 295, 300]. All three Fe65 family members comprises an identical multidomain structure, with three different protein-protein interacting domains, a WW domain and two consecutive PTB domains (reviewed in [301]) . All three members have shown to interact with the APP 682YENPTY687 motif, through their second PTB (PTB2) domain, in a tyrosine phosphorylation-independent manner [83-85, 273, 297, 300, 302]. In addition, Fe65 and Fe65L2 was reported to interact with the two other APP-family members, APLP1 and APLP2 [84, 85, 300, 303],

18 whereas Fe65L1 was shown to interact with APLP2, but not with APLP1 [85]. All three family members have also been shown to localize into the nucleus [86, 304]. Fe65 KO mice exhibit no obvious phenotypes, probably due to the expression of the two homologues, Fe65L1 and Fe65L2. Hence, KO mice deficient of both Fe65 and Fe65L1 show cortical dysplasia [295], similar to the triple mutant mice lacking all three APP family members [110]. In addition, Fe65/Fe65L1 KO mice exhibit cataract in adult mouse eyes, and limb muscle weakness [305]. However, despite the high homology level of the Fe65-family proteins, only cellular functions of Fe65 will be further addressed in this thesis. Fe65 has shown to exist as two cell-type specific isoforms, generated through alternative splicing of exon 9 (a 6 bp miniexon). This alternative splicing generates two additional amino acids, Arg and Glu, located in the PTB1 domain of the neuronal isoform. In the brain, the exon 9- inclusive isoform is exclusively expressed in neurons [296, 306], whereas the exon 9-exclusive isoform is only expressed in non-neuronal cells, including those within peripheral tissues, and the central nervous system (CNS) [306]. Interestingly, in the temporal and frontal cortex of AD brains, reduced levels of the neuronal form, and increased levels of the non-neuronal form was observed [307], possibly reflecting reduced number of neurons and/or increased number of non-neuronal cells in AD. Fe65 is primarily found in the CNS and is highly expressed in neurons of the hippocampus, cerebellum, thalamus, and brain stem nuclei in the adult mouse brain [308]. Fe65 gene expression is developmentally regulated [309, 310]. In the rat developing nervous system, Fe65 mRNA was detected as early as embryonic day 10 (E10) [310], and was followed by the appearance of the Fe65 transcript in the spinal cord, enchephalic and spinal ganglia, nerve structures of the sense organs, the autonomous nervous system and brain. However, Fe65 expression was shown to peak at day E12.5 [310], and then reduce until P0 [308, 310], before the expression increases again from P10 to 3-mont-old adults [308, 309]. Interestingly, this pattern of Fe65 expression corresponds to that of APP [90], suggesting a function for Fe65 and APP together in

19 the developing nervous system, hence, in cell migration and synaptogenesis. One Fe65 orthologues gene is present in C. elegans, namely feh-1. Single alignment of Fe65 homologue-1 (FEH-1) with the three mammalian Fe65-family members suggests that FEH-1 resembles Fe65 and Fe65L1 the most. FEH-1 was shown to bind to the cytoplasmic domain of the APP homologue APL-1 [113] and be expressed in the pharynx and nervous system of C. elegans. KO of feh-1 leads to lethality during the early stages of embryo development, or at the larva stage L1 [311]. Impaired feeding ability, due to affected rate of pharyngeal contraction, is observed in the arrested larvae, a phenotype similar to that caused by the suppression of the nematode APP orthologue apl-1 [311]. Interest- ingly, decreasing the expression of FEH-1 or APL-1 by RNA interference leads to changes in pharyngeal contractions, due to variations in the expression of two acetylcholinesterase genes [312]. The observations in C. elegans indicate that Fe65 may be needed for survival, and that the Fe65/APP interaction may be involved in the regulation of physiological processes such as the maintenance of neuromuscular junctions.

1.3.1 Fe65 interacting proteins Due to its multidomain structure, Fe65 can interact with a wide range of proteins, and, hence, modulate diverse cellular processes in the nucleus and cytoplasm. To date, over 20 Fe65-interacting proteins have been identified (reviewed in [313]), however, only a few of them will be discussed in this thesis. Nevertheless, the functional outcomes of the many different interacting proteins include Fe65-dependent gene transactivation, DNA repair, trafficking and processing of cell surface receptors (APP, Notch, lipoprotein receptors), and cytoskeletal regulation, including neurite outgrowth.

1.3.1.1 The WW-domain of Fe65 Fe65 has shown to interact through its WW domain with mammalian enabled (Mena) [314, 315], and Abelson murine leukemia viral oncogene homolog 1 (c-Abl) kinase [316, 317]. Mena is an actin-regulatory

20 protein that functions in cell motility and adhesion [318]. Fe65 was shown to colocalize and interact with Mena and APP in integrin-based focal complexes in lamellipodia [315]. In addition, Fe65 and APP overexpression has shown to cause the acceleration of cell migration [315], which suggests that a complex composed of Fe65/APP/Mena could be involved in the regulation of cell movement. Interestingly, Fe65 and APP have shown to be present in actin rich mobile structures of growth cones and synapses [319], even further supporting the role of Fe65 and APP in cell migration. Moreover, during cortical development, Fe65/Fe65L1 double KO mice show impairment in neuronal position- ing and in the establishment of normal axonal projections [295], which resembles the phenotype observed for mutant mice lacking all three APP family members [110], or the Mena/Vasp protein family members [320]. Further, targeted mutation in the 682YENPTY687 motif of APP abrogate the rescue effect of full-length APP in a neuronal migration study [93], which is in line with observations that Fe65 interaction seems to be required for migration. Upon Fe65 binding, an active form of c-Abl kinase was found to phosphorylate both APP at Tyr682 [317] and Fe65 at Tyr547 [316]. The active c-Abl was also shown to form a stable complex with the tyrosine phosphorylated APP, possibly through the phosphorylated p682YENPTY687 motif and the SH2 domain of c-Abl [317]. Further- more, c-Abl mediated phosphorylation of Fe65 at Tyr547, which resides within the APP-binding PTB2 domain, was shown to stimulate APP/F65 transcriptional activity, without affecting the APP/Fe65 interaction [316]. However, the phosphorylation of APP at Tyr682 has shown to abolish the interaction with Fe65 [321], suggesting that c-Abl induced phosphorylation of APP at Tyr682 and Fe65 Tyr547 are two sepa- rate events, and that APP Tyr682 is not involved in the c-Abl induced APP/Fe65 dependent gene transcription.

1.3.1.2 The PTB1-domain The first PTB domain (PTB1) of Fe65 was shown to interact with ApoE receptors, such as the the low-density lipoprotein receptor-related protein (LRP) [322-325], apolipoprotein E receptor 2 (ApoEr2) [285],

21 and very low density lipoprotein receptor (VLDLR) [326], via the receptors NPXY motifs. Fe65 was shown to function as a linker between APP and all three ApoE receptors [285, 324-326]. Overexpression of Fe65 was shown to increase cell surface levels of both ApoEr2 and VLDLR [285, 326], and to modulate processing of ApoEr2 [285]. In addition, nuclear localization of both LRP1-ICD [323] and VLDLR- CTF [326] were observed upon Fe65 co-expression, of which the LRP1-ICD was shown to have an inhibitory effect on APP/Fe65 dependent transcriptional activity [323]. Interestingly, mice lacking VLDLR or ApoEr2 display deficits in neuronal migration [327, 328], LTP, learning and memory [329, 330]. In addition, ApoEr2 has shown to be involved in dendritic spine formation [330]. Through its PTB1 domain, Fe65 was also found to interact with the transcription factor CP2/LSF/LBP1 [331]. Interestingly, a polymorphism in the CP2/LSF/LBP1 gene is associated with a modified risk of LOAD [332]. In fact, a GbA polymorphism in the 3´-UTR of the gene is significantly associated with AD susceptibility, while the A allele have a protective effect. Moreover, CP2/LSF/LBP1 has been implicated in the regulation of GSK3β expression [333], a kinase shown to have a crucial role in AD pathogenesis [334]. GSK3β can phosphorylate APP at Thr668 [68] and Fe65 at Thr579 [335], as well as the Tau protein, the main constituent of the neurotoxic NFT [336]. Intriguingly, Fe65 has been found to be associated with NFTs in AD [337], and has shown to interact with Tau in neuronal cells [338]. Fe65/Tau interaction was shown to occur through the PTB1 domain, and to be modulated by the phosphorylation status of Tau. Thus, Fe65 could be an essential feature connecting APP and Tau in AD pathogenesis.

1.3.1.3 The PTB2-domain APP was the first Fe65 binding partner identified [297], and was shown to bind the second PTB (PTB2) domain in Fe65. The interaction between Fe65 and APP will be discussed in more detail in sections 1.3.2 and 1.3.3. In addition to APP, the PTB2 domain of Fe65 has also been shown to bind Dexras1 [339], the estrogen receptor α (ERα) [246, 340], and to mediate Fe65 homodimers formation [341]. Dexras1 is a mem-

22 ber of the Ras family of small G proteins. The protein is widely expressed in the brain, and is activated by glucocorticoids [342]. Interest- ingly, the interaction of Fe65 with Dexras1 connects Fe65, and APP, to the glucocorticoid hypothesis of AD [343], which proposes that chronic exposure to glucocorticoids, for instance due to psychological stress, promotes hippocampal aging and AD. An interaction between Fe65, APP, and ERα has been shown to occur in both cell lines and in mouse brain. Fe65 interacts with ERα through its PTB2 domain, and 17-β estradiol was shown to inhibit APP/Fe65 dependent transcriptional activity in neurons [246]. How- ever, in contrast to in neuronal cells, Fe65 promoted ERα dependent transcriptional activity in breast cancer (BCa) cells [340]. It was suggested that estrogens, together with Fe65, are involved in protecting neuronal cells from toxicity induced by APP cleavage products, whereas Fe65 binding to ERα in BCa cells stimulate cell growth. Inter- estingly, studies have reported a bidirectional inverse association between cancer and AD, such that cancer risk was lower in AD patients, and vice versa [344]. Estrogens have been shown to be neuroprotective, and their decrease after menopause is believed to contribute to the development of AD, whereas increased estrogen levels have shown to promote breast cancer [345]. Recently, Feilen and coworkers revealed that the Fe65-PTB2 domain can form homodimers in the absence of APP [341]. Fe65-PTB2 dimerization was shown to mimic AICD-interaction, and was suggested to compete with AICD binding. The dimerization was proposed to be a way of regulating Fe65 function, by keeping it in a closed, protected, conformation before its activation at the membrane by APP, or another membrane bound protein.

1.3.2 Fe65 nuclear functions How nuclear localization of Fe65 is regulated is still not completely clear. Since Fe65 by its own do not possess nuclear localization signal (NLS), its entry into the nucleus has to be regulated by other means.

23 However, at least two different APP dependent mechanisms are involved in the regulation of Fe65 nuclear localization; phosphorylation of APP at Thr668 and/or APP processing by α-, β-, and γ-secretases.

1.3.2.1 In APP dependent transcriptional regulation In 2001, Minopoli and coworkers reported that both endogenously and exogenously expressed Fe65 was able to localize into the nucleus, and APP was shown to anchor Fe65 in the cytosol preventing Fe65 from entering the nucleus [346]. In addition, the N-terminal WW domain containing region was proposed to be responsible for Fe65 nuclear localization [346, 347]. In 2001, Cao and Südhof reported that Fe65 stimulated APP dependent transcription, and that all three domains of Fe65 were essential for this stimulation [258]. In the same study, Fe65 and APP was also shown to interacts with the histone acetyl transferase Tip60, forming a trimeric stable complex later referred to as the AFT (APP/Fe65/Tip60) complex. The Fe65 interaction with Tip60 was shown to occur through the PTB1 domain of Fe65. The transcriptional activity of APP was dedicated to AICD, the cytoplasmic tale of APP released by γ-secretase cleavage of C83 or C99 (see section 1.2.2.3). Fe65 was proposed to stabilize AICD and to promote it nuclear localization [248, 348]. Since its discovery, the trimeric AFT complex has been shown to be recruited to different promoters. For instance, was binding to the KAI1 promoter shown to increase KAI1 expression, and γ-secretase inhibition was show to abolish this effect [250, 349, 350]. The KAI1 gene codes for the pro-apoptotic tetraspanin KAI1 protein, also called CD82. In addition, a member of the inhibitor of histone acetyl transferase (INHAT) complex, SET/template-activating factor-1 (SET/TAF1-β) was shown to bind to a region of Fe65 overlapping the WW domain [351]. SET was shown to be necessary for KAI1 gene expression induced through the Fe65/APP/SET complex. In another study, Fe65 was shown to simultaneously recruit SET and a transcription factor, Teashirt3, bound to histone deacetylases, generating a gene silencing complex [352]. The Fe65/Teahirt3 complex showed direct interaction with the promoter region of CASP4. Interestingly, decreased expression of Teashirt3 was shown to increase the expression of caspase-4, a protease implicated in the progression of AD.

24 In addition, the AICD/Fe65 complex has been reported to regulate the expression of GSK3β [250, 353], Tip60 [250], LRP1 [354], the epidermal growth factor receptor (EGFR) [355], tumor suppressor p53 [356], the Aβ degrading enzyme neprilysin [249, 357-359], BACE1 [250], and APP itself [250]. Hence, the AICD/Fe65 complex has been proposed to regulate the expression of several proteins which are involved in the regulation of their own functions, such as APP processing, trafficking and cellular clearance, in addition to Fe65 dependent transactivation. Nevertheless, there are reports with contradicting data stating that the AICD/Fe65 complex did not produce changes in the expression of some of these genes [360, 361]. Thus, the potential nuclear signaling mechanism of AICD together with Fe65 is controversially discussed, and Fe65 has shown to transactivate different promoters independent of AICD co-expression [361]. In addition, a study by Yang and coworkers revealed that Fe65 by itself induced transcriptional activity in several different cell lines, including HEK293 and the neuroblastoma cell line SK- N-SH, but not in COS cells [362]. In 2004, Cao and Südhof suggested that AICD nuclear localization is not necessary for APP/Fe65 dependent transactivation. They proposed that membrane tethered APP/AICD recruited Fe65 to the plasma membrane and mediated the activation of bound Fe65, which was then released by γ-secretase cleavage of APP and subsequently entered the nucleus [363]. A model where Fe65 was kept in a closed inactive state through an intramolecular interaction of the WW domain with the PTB2 domains, was also proposed. The intramolecular interaction was proposed to inhibit Fe65 transactivation, and was proposed to be released by binding of APP, or an yet unknown membrane-associated factor, which interferes with the WW/PTB2 interaction, opening the closed conformation. In addition, APP Thr668 phosphorylation has shown to decrease APP/Fe65 interaction, due to a conformational change of the APP C- terminal domain [74]. Higher levels of nuclear Fe65 was observed upon APP Thr668 phosphorylation [364, 365]. This lead to the conclusion that Fe65 might translocate independent of AICD to the nucleus, since APP

25 Thr668 phosphorylation releases Fe65 for nuclear localization [364]. In- triguingly, APP Thr668 phosphorylation is increased in AD [62, 366], hence this may also lead to higher nuclear levels of Fe65 in AD. Fe65 has shown to play a role in Bloom syndrome protein (BLM) subcellular localization. This protein is involved in DNA replication and repair [367], and Fe65 KO in cells was characterized by lower levels of proliferation and DNA replication. In this study, elevated or diminished nuclear Fe65 levels was proposed to contribute to neuronal cell cycle re-entry, a phenomena proposed to occur in AD [368, 369], and result in neuronal cell death [370].

1.3.2.2 In response to DNA damage Fe65 overexpression was shown to block cell growth of both mouse fibroblasts and of PC12 cells, by inhibiting the LSF-mediated activation of the thymidylate synthase (TS) gene [304]. TS is a key enzyme in the synthesis of 2´-deoxythymidine-5´-monophosphate, an essential precursor for DNA biosynthesis, and is a critical target in cancer chemotherapy [371]. Interestingly, exogenous thymidine addition counteracted Fe65-induced cell cycle arrest only in non-neuronal cells [304]. These results are intriguing, since it indicates cell type specific mechanisms of Fe65. In addition, Fe65 is proposed to be involved in cell proliferation and DNA damage, since an abundance of proteins involved in DNA replication and repair are significantly reduced upon Fe65 knockdown [367]. Moreover, Fe65 can interact with chromosomes during both non-stress and stress conditions [372]. Ablation of the Fe65 gene was shown to be associated with increased sensitivity to DNA damaging agents. Mouse embryo fibroblasts (MEFs) isolated from Fe65 KO mice, treated with H2O2 or irradiation, showed higher levels of DNA damage and accumulation of γ-H2AX (phosphorylated histone H2AX), compared to wild type MEFs [373]. In addition, sorbitol treatment of neuroblastoma N2a cells revealed increased nuclear levels of Fe65 prior to γ-H2AX induction, suggesting that γ-H2AX might be induced by Fe65 nuclear localization [364]. Interestingly, H2AX is phosphorylated by the ataxia telangiectasia-mutated kinase (ATM) [374], which has previously been shown to phosphorylate Fe65 upon IR and

26 UV induced DNA damage [375], and to regulate Fe65 dependent transcription (see section 1.3.4). In addition, Tip60, one of the binding partners of Fe65, has been implicated in the activation of ATM in response to DNA breakage [376, 377]. Hence, binding of Fe65 to Tip60 may activate Tip60s histone acetyltransferase function to produce a locally relaxed chromatin conformation, a requirement for further steps of transcription.

1.3.3 The effect of Fe65 on APP processing The regulation of APP processing is of substantial significance, since APP fragments, including Aβ, have shown to have important functions and pathological roles. To date, studies on the effect of Fe65 on APP processing have thus provided seemingly contradictory results. Over- expression of Fe65 was shown to increase cell surface levels of APP, and to increase the levels of both sAPPα and Aβ [378]. However, the increase in Aβ secretion was more dramatic, compared with sAPPα secretion, suggesting that Fe65 binding is involved in β-secretase processing of APP. This was supported by a study where reduced Aβ levels were observed upon Fe65 knock-down [379]. However, Fe65 knockdown also increased the levels of both APP-C83-CTF and APP-C99- CTF, indicating that Fe65 might be involved in APP trafficking/endocytosis. Surprisingly, Ando and coworkers showed in a study that coexpression of Fe65 together with APP, lowered the maturation of APP, and suppressed the secretion of both sAPP and Aβ [74]. Further, Fe65 interacted preferentially with the immature, rather than the mature form of APP. In Fe65/Fe65L1 KO mice decreased levels of Aβ was observed in hippocampal neurons [380], and lower Aβ was also found in neuronal cultures, established from Tg2576 mice lacking Fe65 [381]. In addition, APP-KI mice carrying an Y682G mutation in the 682YENPTY687 motif, show decreased levels of Aβ and a massive increase in secreted sAPPα [78]. The Y682G mutation has shown to inhibit Fe65 binding. On the other hand, brains of transgenic mice overexpressing Fe65, have shown to contain reduced deposits of Aβ [382]. Interesting in this regard is also that the full-length APP with the Swedish mutation (K670N;M671 L) [190] is a very poor Fe65 interactor [274]. The mutation have shown to

27 increase the total levels of secreted Aβ by making APP a better substrate for β-secretase cleavage [156]. However, the mutation resides within the juxtamembrane region of APP. Hence, the mutations do not reside within the Fe65 interacting domain of the protein. Strong deficits in hippocampus-dependent learning and memory was observed in p97Fe65 specific KO mice, which show upregulation of a shorter, N-terminally truncated Fe65 isoform, p60Fe65 [381]. p60Fe65 demonstrated higher affinity for APP, and an attenuated transcriptional activity, compared to p97Fe65 [383]. In addition, an N-terminally truncated Fe65 cleavage product, designated p65Fe65, demonstrated higher affinity for APP, and suppressed sAPPα secretion compared with p97Fe65 [384]. The endoproteolytic cleavage event of Fe65 was shown to be activated by high cell density, and was proposed to be a regulator of α-secretase processing of APP. DNA damage have also shown to induce caspase cleavage of Fe65 to an N-terminally truncated p65Fe65 fragment [385]. Hence, the increased interaction with APP, and the decrease in sAPPα secretion, due to the lack of the N-terminus of Fe65, suggests that “truncation” of Fe65 is a way of regulating Fe65 cellular function, including APP processing. It is intriguing to speculate that increased Fe65 interaction with APP might be involved in AD pathology. Indeed, a minor Fe65 allele in humans that codes for a Fe65 isoform with weak binding to APP is associated with a reduced risk for developing a very late onset AD [386-388]. However, since age seems to be the major risk factor for development of AD pathology, it is intriguing that Fe65 expression has shown decrease during aging in mice [381].

1.3.4 Phosphorylation of Fe65 Phosphorylation of Fe65 can affect Fe65 protein interactions and nuclear localization. Several phosphorylation sites have been identified, and in SDS-PAGE, Fe65 migrates as at least three major species [331, 389], of which the slower migrating species disappear upon treatment with λ protein phosphatase [331, 389]. DNA damage causes Fe65 to undergo a rapid gel mobility shift, consistent with protein phosphorylation [372]. Fe65 Ser228 was identified

28 as a novel phosphorylation site targeted by ATM and ATR protein kinases after IR and UV, respectively [375]. Mutation of Fe65 Ser228 to alanine (Fe65-S228A) did not interfere with APP/Fe65 interaction, however, APP/Fe65 dependent transcription was significantly increased in untreated cells. Phosphomimetic mutants of Fe65 Ser228, on the other hand, decreased APP/Fe65 dependent activity. Interestingly, Ser228 is adjacent to the WW domain proposed to harbor the transactivating capacity of Fe65. As mentioned previously, a dominant active mutant of c-Abl was shown to phosphorylate Fe65 at Tyr547. This phosphorylation event was shown to stimulate APP/Fe65 dependent transcription [316]. The APP/Fe65 interaction was not affected by this event, which is surprising considering that PTB2 also mediates APP interaction. However, as Tyr547 localize within the most distal N-terminal end of the motif [264], this phosphorylation may not interfere with APP/Fe65 interaction. On the contrary, phosphorylation of Fe65 Tyr547 decreased the interaction between Fe65 and Dexras1, another Fe65-PTB2 domain binding protein [339]. Interestingly, APP and Dexras1 was shown to bind simultaneously to Fe65, creating a tripartite complex, and to translocate into the nucleus. However, Dexras1 interaction was shown to inhibit APP/Fe65 dependent transcription. Hence, phosphorylation of Fe65 Tyr547 could be a way to regulate the transcriptional inhibition of Dex- ras1. Fe65 Ser610 lies within the binding interface of Fe65 and APP [264]. Phosphorylation of this residue by serum/glucocorticoid regulated kinase-1 (SGK-1) was suggested to diminish the interaction with APP, and lead to nuclear localization of Fe65. Phosphorylation of Fe65 Ser610 also affected APP processing by decreasing the levels of secreted Aβ [390]. Hence, stimulation of SGK-1 was suggested to be a possible way to ameliorate AD pathology. Fe65 was previously shown to interact through its WW domain with GSK3β [391]. In a recent study [335], the phosphorylation of Fe65 at Thr579 by GSK3β was shown to potentiate Fe65/APP interaction, enhance APP processing and Aβ production. In addition, phosphorylation of Thr579 by GSK3β was proposed to disrupt dimerization of Fe65 PTB2

29 domains [341]. Disruption of Fe65 PTB2 dimers was suggested to regulate cellular levels of monomeric Fe65, for the formation APP/Fe65 complexes, hence promoting amyloidogenic processing of APP. Fe65 can also be targeted for phosphorylation by extracellular signal- regulated protein kinase 1/2 (ERK1/2) [389]. However, the amino acid(s) targeted for phosphorylation by ERK1/2 was never identified [389], although the phosphorylation was proposed to occur on the N- terminal part of Fe65 protein.

30 2 Methodological considerations

More general aspects of the methods used are discussed below. For more detailed description of the methods the reader is referred to the papers included in this thesis.

2.1 Cell lines Primary cells are believed to be more biologically relevant tools than cell lines, however, cell cultures provide a more controlled manipula- tion of cell functions and processes. In addition, the ability of cell lines to proliferate virtually indefinitely, have made cell lines a cost-effective tool used in basic research. However, care should be taken when using cell lines, since serial passaging over time can cause genotypic and phenotypic variations. Moreover, recent estimates suggest that 20-36% of cell lines used in scientific research are contaminated or misidentified [392]. Hence, caution should be taken when analyzing, and drawing conclusions from a designed experiment in cell lines. Throughout this thesis, we have used three different cell lines, SH- SY5Y, SK-N-AS and PC6.3. SH-SY5Y and SK-N-AS cells are human derived, whereas PC6.3 is derived from rat. The advantage of using human derived cells, such as SH-SY5Y and SK-N-AS cells, as a model system when studying human neurological disorders such as AD, is that you do not have to consider species-specific differences in, such as, gene expression, and metabolic processing. However, using a rodent derived neuronal cell line, such as the PC6.3 cell line, to replicate your experiments, can be a cost-effective way to confirm/validate your data, instead/before using primary cells.

31 2.1.1 SH-SY5Y SH-SY5Y is a human neuroblastoma cell line used frequently in in vitro studies. SH-SY5Y is a third-generation subline from SK-N-SH, originally isolated from a metastatic bone tumor biopsy from a four year old girl in 1971 [393]. SH-SY5Y cells are of N-type (neuroblastic) and can be differentiated into a more adult neuronlike phenotype including cholinergic, adrenergic, or dopaminergic, depending on methods used (see section 2.2). Following differentiation, SH-SY5Y cells become morphologically more like primary neurons with long, exquisite processes [394]. However, undifferentiated SH-SY5Y cells are most rem- iniscent of immature catecholaminergic neurons [395, 396]. Both undifferentiated and differentiated SH-SY5Y cells have been utilized for in vitro studies requiring neuronal-like cells. SH-SY5Y cells have also been shown to endogenously express Fe65, and all three family members of the APP family of proteins, as well as the APP processing secretases, making it a suitable cell model for AD research.

2.1.2 PC6.3 PC6.3 pheochromocytoma cells originate from PC12 cells [397]. PC12 cells are derived from a pheochromocytoma of rat adrenal me- dulla. Additionally, PC12 cells have shown to express the neuron-specific Fe65 mRNA [296].

2.1.3 SK-N-AS Human neuroblastoma SK-N-AS cell line is of S-type (substrate ad- herent) and is derived from the bone marrow of an eight year old girl. The original site of cancer was in the adrenal glands from which it me- tastasized to the bone marrow. S-type neuroblastoma cells do not exhibit neuronal activities. SK-N-AS cells are easier to transfect than SH- SY5Y cells, and do not express detectable levels of APP695.

2.2 Cell treatments Immortalized cell lines of neuronal origin can be used to study neuronal properties, such as processes that occur during differentiation in

32 neurons. In our studies, we have differentiated SH-SY5Y and PC6.3 cells into a more neuronal phenotype, to be able to study the expression of a specific protein during neuronal differentiation. In addition, to inhibit the action of different secretases in our studies, we have used small molecule inhibitors instead of siRNA. Small molecule inhibitors provide several advantages, such as they can be applied directly to cells, they are usually membrane permeable, hence, diffuse very rapidly inside the cell, and may usually only targets one protein or enzyme. siRNA on the other hand may take some time to induce an effect.

2.2.1 Retinoic acid (RA) One of the most commonly used methods to induce differentiation of SH-SY5Y cells is the addition of RA to the cell medium. RA is an active metabolite of vitamin A (all-trans-retinol) and is an essential morpho- gen in the development and differentiation of the nervous system. RA has also shown to induce differentiation and cell growth arrest of a variety of neuroblastoma cell lines, including SH-SY5Y cells [394, 398, 399]. RA controls cellular differentiation processes by modulating the expression of several RA-responsive genes by the activation of retinoic acid/retinoid nuclear receptors [400]. In response to RA, SH-SY5Y cells differentiate primarily to a cholinergic neuron phenotype indicated by the increased activity of choline acetyltransferase (ChAT) [401]. However, RA-induced differentiation of SH-SY5Y cells have also been shown to increase the expression of dopaminergic markers, such as the tyrosine hydroxylase (TH), and the dopamine transporter (DAT), when RA administration is followed by treatment with phorbol esters [402]. Since RA-induced differentiation of SH-SY5Y cells has shown to significantly increase the expression of APP and its processing enzymes [403-405], RA differentiated SH-SY5Y cells is a suitable model for studying AD.

33 2.2.2 PMA (Phorbol 12-myristate 13-acetate) PMA treatment of SH-SY5Y cells increases the cellular noradrena- line content up to 200-fold compared with a fourfold induction of nor- adrenalin upon RA treatment [394]. Thus, PMA differentiation of SH- SY5Y cells produces a predominantly adrenergic cellular phenotype. PMA is a phorbol ester structurally analogous to diacylglycerol and is a specific activator of PKC. Former studies have shown that phorbol esters enhances the α-secretase cleavage of APP, hence the secretion of the neuroprotective sAPPα [406].

2.2.3 NGF Nerve growth factor (NGF) has shown to prevent PC12 cells from dividing, and to induce differentiation of PC12 cells to resemble sympathetic ganglion neurons. Therefore, the PC12 and PC6.3 (see section 2.1.2) cell lines are often used as model system for neuronal differentiation [407]. NGF is a small neuropeptide involved in the regulation of growth and differentiation of the sympathetic and certain sensory neurons [408]. The binding of NGF to its transmembrane receptor, TrkA, induces receptor homodimerization, and the intracellular part of the receptor becomes autophosphorylated. This leads to the activation of number of pathways through mitogen-activated protein kinase (MAPK), AKT, and phospholipase-γ (PLCγ) [409-411].

2.2.4 DAPT (Difluorophenylacetyl-alanyl-phenylglycin-t- butyl-ester) DAPT, is a widely used γ-secretase inhibitor, and can be used to inhibit cleavage of several γ-secretase substrates, including Notch and APP. DAPT is a non-transition state analogue believed to bind to the C-terminalt fragment of presenilin (PS) of the γ-secretase complex [412]. The binding site of DAPT is pharmacologically distinct from the catalytic site and the substrate binding site of the γ-secretase. Interest- ingly, DAPT-mediated inhibition of the Notch response has shown to promote differentiation of, among others, human embryonic stem-cell derived neural stem cells (hESNSCs) [413].

34 2.2.5 ADAM10 inhibitor (GI254023X) GI254023X is a selective inhibitor of ADAM10 and have a 100-fold selectivity for ADAM10 over ADAM17 (also known as TACE, tumor necrosis factor-alpha-converting enzyme), another α-secretase shown to be involved in APP processing[144, 414, 415]. GI254023X binds to the zinc ion in the active site of ADAM10, thereby inhibiting the action of the α-secretase.

2.2.6 Batimastat (BB-94) Batimastat is a broad-spectrum matrix metalloproteinase (MMP) inhibitor shown to inhibit not only ADAM10 but also ADAM-9 and ADAM17/TACE. Batimastat has shown to possess anticancer activity in vivo [416]. Batimastat binds covalently to the zinc ion in the active site of MMPs, thereby inhibiting the action of MMPs.

2.3 Tandem Affinity Purification (TAP)-tag method Pull-down assays are used to determine a physical interaction between two or more proteins, and can be used as an initial screening assay for identifying previously unknown protein-protein interactions, in addition to confirming the existence of a protein-protein interaction pre- dicted by other research techniques. The TAP-tag method is a two-step affinity purification procedure that can be used for native purification of protein complexes. The puri- fied complexes can be used for protein identification, functional, or structural studies. The TAP method involves the fusion of a TAP-tag to the target protein and the expression of the construct in a host cell. The TAP-tag can be incorporated C- or N-terminally to the target protein. The TAP-tag consists of an IgG binding domain (ProtA) and a calmodulin binding peptide (CBP) separated by a TEV (tobacco etch virus) protease cleavage site. ProtA binds tightly to an IgG matrix, and is eluted using a TEV protease. The eluate is then incubated with calmodulin coated beads in the presence of calcium. The calmodulin bound

35 protein complexes are then released with EGTA. However, in our studies, only the first purification step utilizing the ProtA domain followed by TEV protease cleavage was employed.

2.4 siRNA gene silencing Short, or small, interfering RNA (siRNA) is a tool for inducing short term silencing of protein coding genes. siRNA is a synthetic RNA duplex designed to specifically target messenger RNA (mRNA) for degradation. The siRNA duplex consists of a sense and an antisense strand, 20 to 25 base pairs in length, with 3’ dinucleotide overhangs. siRNA requires transfection of cells using a lipid-based transfection reagent. In the cell, the siRNA duplex binds to RNA-induced silencing complex (RISC) and is unwound by helicase. The single stranded RNA in the RISC complex, complementary to target mRNA, recognizes and binds the target mRNA. Argonaute protein 2 (Ago2) cleaves the bound mRNA, and complete degradation of the target mRNA is carried out by ribonuclease activity. However, despite that siRNA is designed to silence the expression of a specific target mRNA, siRNA may lead to the downregulation of un- intended, unpredicted targets, resulting in off-target effects. Other factors that siRNA gene silencing depend on is the transfection efficiency of cells, cost, and the transcription rate of the selected gene, hence, high transcription rate may lead to incompleteness of knockdown.

2.5 Site directed mutagenesis Site directed mutagenesis has become an important tool to modify genes, and to study how changes of specific amino acid on the protein expressed by these genes may affect its structural and functional properties. Site directed mutagenesis is a polymerase chane reaction (PCR)- based method to create specific, targeted, changes in double stranded plasmid DNA (dsDNA). Substitutions, insertions or deletions can be incorporated into dsDNA using specifically designed forward and re- verse primers. During PCR, the mutation is incorporated into the am- plicon replacing the original sequence. Briefly, the primers are extended

36 during temperature cycling by DNA polymerase, without primer dis- placement. Extension of the primers generates a mutated plasmid containing staggered nicks. Following temperature cycling, the product is treated with an endonuclease, specific for methylated and hemimethyl- ated DNA, to digest the parental DNA template. The nicked vector, containing the desired mutation, is then transformed into competent cells for nick repair and amplification.

2.6 Phosphatase inhibition and alkaline phosphatase treatment Phosphatase inhibitors were used during lysis of cells to inhibit the removal of possible phosphate groups from proteins. Three different phosphatase inhibitors were used during lysis: 1) PmSF which targets Ser phosphatases, 2) Na2VO4 acts on Tyr phosphatases, and 3) NaF is a general inhibitor of Ser and Thr phosphatases. Cell lysates were incubated with alkaline phosphatase (FastAP, Thermo Scientific), to further confirm protein phosphorylation. Alka- line phosphatase is an enzyme usually used in cloning. The enzyme catalyzes the release of 5´- and 3´-phosphate groups from DNA, RNA and nucleotides to prevent relegation of linearized plasmid DNA. However, alkaline phosphatase can also catalyze the removal of phosphate groups from proteins.

2.7 Biotinylation assay Cell surface biotinylation assay provides a method by which the levels of a particular protein at the cell surface can be measured. The levels of proteins on the cell surface is regulated by the trafficking of newly synthesized proteins to the cell surface, endocytosis, and recycling of proteins back to the cell surface. Sulfo-NHS-SS-biotin is a cell imper- meable, cleavable biotinylation reagent which labels exposed primary amines of proteins on the surface of intact cells. After labeling with sulfo-NHS-SS-biotin, cells are lysed, and the isolation of biotin-labeled proteins is carried out by a streptavidin pull-down. The streptavidin bound biotin-labeled proteins are then released by the reduction of the

37 disulfide bond by dithiothreitol (DTT), or the addition of sample buffer to the streptavidin beads followed by boiling. Cell surface target proteins can then be detected by western blot.

2.8 Harvesting of cells Cells were harvested as described in the individual papers, using different protocols, dependent on what was analyzed during each study. In general, cells were collected, and lysed by salts and detergents. In paper I and IV, SH-SY5Y and SK-N-AS cells were lysed in RIPA buffer when analyzing total cellular levels of Fe65 and APP, respectively. In paper II, III, and IV, SK-N-AS cells were lysed in NP-40 buffer when analyzing the interaction between TAP-tagged Fe65 and APP, and in paper I when APP cell surface levels were investigated. In paper II and III, to study Fe65 nuclear localization, subcellular fractionation was utilized, separating the cytoplasmic and nuclear fraction. The cytoplasmic fraction was isolated by first swelling the cells in a hypotonic buffer with the addition of NP-40 for complete lysis, followed by the isolation of nuclear proteins in a high salt buffer containing TritonX-100.

2.9 Protein concentration measurement Bicinchoninic acid (BCA) protein assay was used for determining the protein concentration throughout all papers included in this thesis. The BCA assay combines the reduction of copper in alkaline medium with the colorimetric detection of cuprous cations by BCA. In the first step copper (cupric ions) chelates with proteins forming a colored che- late complex. In step two BCA reacts with the cuprous cation from step one. The chelation of two molecules of BCA with one cuprous ion produces an intense purple colored reaction product. The BCA/copper complex exhibits a strong linear absorbance at 562 nm with increasing protein concentration. Four amino acid residues of the protein (Cys or cystine, Tyr and Trp) strongly influences the reaction that leads to BCA color formation as does the universal peptide backbone, helping to min- imize variability causing by protein compositional differences.

38 2.10 Western blot Western blot is a frequently used analytical method to analyze the expression levels of a specific protein in a given sample of mixed proteins. Proteins are separated according to their size in an electric field in an acrylamide gel after the protein samples have been boiled together with sodium dodecyl sulfate (SDS). SDS denatures the proteins and gives them a negative net charge in proportion to their size. After the separation in the gel, proteins are transferred to a membrane. The membrane is first blocked to prevent unspecific binding before a primary antibody against the protein of interest is applied to the membrane. A secondary antibody, which binds specifically to the primary antibody, is applied to visualize the specific target proteins. In our case the secondary antibody is labeled with horse radish peroxidase (HRP). An HRP substrate is applied to the membrane to generate chemilumines- cent light as a product of an enzymatic reaction, which can be detected using CCD (charge-coupled device) imaging. Western blot is a very sensitive and selective analysis method, due its ability to detect very low levels of protein in a sample (in the range of ng) by specific antibodies, and the size sorting by gel electrophoresis. However, a false positive can result when an antibody reacts with an un-intended protein, leading to multiple bands, and to results hard to interpret. The western blot method is also time consuming, and hard to master.

2.11 Immunofluorescence (IF) imaging IF can be used on tissue sections, cultured cells or individual cells that are fixed by a variety of methods. Antibodies can be used in IF to analyze the subcellular distribution of proteins. There are two classes of IF techniques when using antibodies for protein detection; direct (primary) and indirect (secondary) IF. Direct IF uses a single antibody that is conjugated directly to a fluorescent dye. The antibody recognizes the target molecule, binds to it and the conjugated fluorescent dye can be detected by the microscope. In indirect IF, two antibodies are used; a primary antibody that recognizes the target molecule, and a secondary antibody conjugated to a fluorescent dye, that bind specifically to the

39 primary antibody. Multiple secondary antibodies can bind to a primary antibody, amplifying the signal. In our papers we utilized direct IF, however, using only a secondary antibody conjugated to a fluorophore, which recognized the IgG sequence (ProtA) of the TAP-tagged Fe65 protein. IF has shown to be one of the most important and effective methods in analyzing subcellular localization of molecules. Several targets can be stained and detected in the same sample. However, nonspecific background signal from numerous sources, such as antibody cross-reactivity and the fixation method, has to be taken into account when using this technique.

40 3 Aim

The amyloid-β precursor protein (APP) has been in the limelight of AD research, as the processing of APP generates the neurotoxic Aβ- peptide, the main constituent of amyloid plaques in the brains of AD patients. However, processing of APP also generates several other biologically active fragments, including the neuroprotective sAPPα. The processing of APP has shown to be tightly regulated by several different factors, such as posttranslational modifications, subcellular localization, and the binding of adaptor proteins. A malfunction in one or several of these mechanisms may lead to increased levels of the neurotoxic Aβ, and less of the neuroprotective sAPPα. The brain enriched adaptor protein, Fe65, has shown to bind to APP, and affect APP processing. In addition, Fe65 has shown to act as an APP dependent transcriptional regulator. However, the effect of Fe65 on APP processing, and Fe65 nuclear function remains controversial. Hence, dysfunction or aberrant regulation of APP processing by Fe65 may alter APP metabolism, suggesting a possible cause for AD. Therefore, the aim of this thesis was to elucidate the mechanisms behind APP/Fe65 interaction, as well as to Fe65 nuclear localization, and to investigate how phosphorylation is involved in regulating APP processing. The more specific aims of this thesis were to: o Investigate how Fe65 is regulated during neuronal differentiation of SH-SY5Y and PC6.3 cells (Paper I) o Determine the role of different Fe65 domains in Fe65 nuclear localization (Paper II) o Study how α- and γ-secretase processing affects Fe65 nuclear localization in undifferentiated and differentiated SH-SY5Y cells (Paper II and III)

41 o Analyze the role of Fe65 Ser228 phosphorylation in Fe65 nuclear localization and APP/Fe65 interaction (Paper III) o Investigate how phosphorylation of APP at Ser675 affects APP processing and APP/Fe65 interaction (Paper IV)

42 4 Results and discussion

4.1 Regulation of Fe65 phosphorylation and expression upon neuronal differentiation of SH-SY5Y cells (Pa- per I and III)

4.1.1 Neuronal differentiation of SH-SY5Y and PC6.3 cells induce Fe65 phosphorylation SH-SY5Y cells differentiated by RA or PMA obtain more neuron- like properties, including morphological changes and neurite outgrowth [394]. Our previous studies have shown that RA-induced neuronal differentiation of SH-SY5Y cells leads to increased expression levels of APP and APP processing enzymes [405], as well as increased non-amyloidogenic processing of APP [404]. Since Fe65 has been proposed to affect APP metabolism, specifically increasing the levels of Aβ, we hypothesized that neuronal differentiation of SH-SY5Y cells could also influence Fe65 expression. We therefore compared the expression of Fe65 in undifferentiated and differentiated SH-SY5Y and PC6.3 cells. Western blot analysis of whole cell lysates (Paper I: Fig 1 and Fig S2) and cytoplasmic fractions (Paper III: Fig 1 and 4) from undifferentiated SH-SY5Y and PC6.3 cells revealed the expression of detectable levels of endogenous Fe65. Endogenous Fe65 was shown to migrate as one band of approximately 100kDa, as well as a band(s) with apparently higher molecular weight of 105kDa. siRNA knock down of Fe65 in undifferentiated SH-SY5Y cells confirmed that all observed bands on western blot were generated by Fe65 expression (Paper I: Fig 1). Interestingly, in our study, differentiation of SH-SY5Y cells with RA, PMA and DAPT, as well as differentiation of PC6.3 cells with

43 NGF, induced an electrophoretic mobility shift of the Fe65 bands, increasing the intensity of the upper slower migrating band(s) (Paper I and Paper III). The Fe65 105kDa/100kDa ratio was increased two-fold. As discussed in section 1.3, Fe65 exists as two isoforms, neuronal and non-neuronal. The neuronal isoform of Fe65 differs from the non-neuronal isoform by having only two additional amino acids [296, 386]. Therefore, the expression of the two isoforms of Fe65 is less likely the cause of the multitude of bands observed for Fe65 in our study. How- ever, as also discussed in section 1.3, Fe65 possesses several putative phosphorylation sites, and former studies have shown that Fe65 migrates as several bands on SDS-PAGE due to phosphorylation [331, 389]. Consistent with this, subsequent alkaline phosphatase treatment of cell lysates abolished the shift towards the slower migrating band(s), leading to increased intensity of the Fe65 100kDa band in both undifferentiated and differentiated SH-SY5Y and PC6.3 cells (Paper I: Fig 3 and S2, Paper III: Fig 6). Our results hence clearly indicate that the multitude of Fe65 bands observed is due to phosphorylation rather than some other posttranslational modifications, and that increased phosphorylation of Fe65 seems to occur during neuronal differentiation. Hence, our obtained results raise the question: what is the cellular consequence of increased Fe65 phosphorylation during differentiation? Fe65 has been reported to be a phosphoprotein, however the functional significance of Fe65 phosphorylation is largely obscure. Fe65 has shown to be targeted for phosphorylation by ERK1/2 [389], c-Abl tyrosine kinase [316, 339], SGK1 [417], ATM/ATR [375, 418] and GSK3β [335]. IR and UV induced DNA-damage was shown to affected N-terminal phosphorylation leading to the regulation of Fe65 transcriptional activity [375]. Whereas, in unstimulated/untreated cells, phosphorylation(s) of Fe65 in the APP-binding PTB2 domain has shown to mediate APP/Fe65 dependent transcription, affect APP metabolism, and Fe65 nuclear localization [316, 335, 390]. Interestingly, ERK1/2 has shown to have various roles in the development of the nervous system [419], and has shown to be involved in proliferation, differentiation and cell survival of cell lines and primary neuronal cultures [420-423]. RA has also been shown to increase ERK1/2 activity in SH-SY5Y cells [424]. Thus, phosphorylation seems to be crucial in the regulation of

44 Fe65 function. Further work on identifying the Fe65 phosphorylation sites, and the responsible kinases, during neuronal differentiation, is therefore of vast importance.

4.1.2 Expression of Fe65 during neuronal differentiation of SH- SY5Y and PC6.3 cells differs dependent on differentiating-agent used When analyzing the total expression levels of Fe65 during neuronal differentiation, our results revealed that total Fe65 protein levels were not changed upon 3-day RA- or PMA-induced differentiation, nor during 24 h treatment with DAPT (Paper I: Fig 2). Additionally, 6-day treatment of SH-SY5Y cells with PMA did not reveal any significant change of Fe65 protein levels in the cytoplasmic fraction following subcellular fractionation (Paper III: Fig 5). However, 6-day RA-treatment of SH-SY5Y cells (cytoplasmic fraction, Paper III: Fig 4) and 6-day NGF-treatment of PC6.3 cells (whole cell lysate, Paper I: Fig S2) decreased the protein levels of Fe65 with approximately 40%. This we found surprising, since RA has shown to increase the transcription of the Fe65 interacting protein APP [403], and the APP processing enzymes, ADAM10 and BACE1 [405], in SH-SY5Y cells. In addition, Fe65 mRNA levels was shown to be increased several fold in the tetra- carcinoma cell line NTERA-2 upon RA treatment [310]. However, interesting in this regard is that RA- and NGF-induced differentiation of cells gives rise to a more cholinergic phenotype, whereas PMA generates a more adrenergic phenotype of cells [394]. Fe65 has shown to be developmentally regulated [308, 310] and the expression of Fe65 mRNA and protein levels have shown to differ dependent on brain areas investigated [308, 310]. Hence, the molecular mechanisms by which the expression of the Fe65 protein is regulated seems to differ between specific cells during development.

4.2 Regulation of Fe65 nuclear localization (Paper II and III)

45 4.2.1 Fe65 nuclear localization is dependent on the PTB2 domain Several mechanisms have been proposed for how Fe65 nuclear localization may be regulated. Hence, in this study, we wanted to further investigate the nuclear localization of Fe65, since this has shown to be essential for Fe65/APP dependent transcriptional activity. Subcellular fractionations of SH-SY5Y cells showed that endogenously expressed Fe65 was present both in the cytoplasmic and nuclear fractions (Paper II: Fig 1, Paper: III: Fig 1). We estimated the nuclear fractions to contain approx. 4% of the total cellular Fe65. Thus, the low nuclear levels of endogenous Fe65 indicates strongly that Fe65 plays a more dominant role in the cytoplasm, at least in SH-SY5Y cells. Several studies have indicated that the WW domain of Fe65 has a dominant role in Fe65 nuclear translocalization, hence in Fe65/AICD dependent transcriptional activity [258, 346]. In addition, an N-terminally truncated form of Fe65 designated p60Fe65 lacking part of the WW domain was shown to localize into the nucleus when overexpressed in COS cells [383]. Next, we therefore investigated how an N-terminally truncated form of Fe65, p60Fe65, and the deletion of the total WW domain, or the APP binding PTB2 domain affected the nuclear translocation of Fe65 in SH- SY5Y cells. For this, full-length Fe65 (p97Fe65, amino acids 1-710) the N-terminally truncated 60kDa isoform (p60Fe65, amino acids 261- 710), a deletion mutant lacking the N-terminus including the WW domain (ΔWWFe65, amino acids 328-710), and a deletion mutant lacking the C-terminus including the PTB2 domain (ΔPTB2Fe65, amino acids 1-520) was fused to a TAP-tag (Paper II: Fig 1). The TAP-tag is originally designed for isolation and analysis of protein complexes (see section 2.3 and 4.3), however, the TAP-tag can also be used as a tag to discriminate between endogenous and overexpressed proteins on western blot analysis. Overexpression of the different cDNA constructs in SH-SY5Y cells followed by subcellular fractionation and western blot analysis revealed, surprisingly, that only ΔPTB2Fe65 was excluded from the nuclear fractions (Paper II: Fig 1). The same results were obtained using another neuroblastoma cell line, SK-N-AS (Paper II: Fig 1), in which verification with immunofluorescence was also performed

46 (Paper II: Fig 2). Moreover, the TAP-tag was removed from ΔWWFe65 and ΔPTB2Fe65 by introducing a STOP codon before the start of the TAP tag, to rule out that the large TAP tag was involved in Fe65 nuclear localization (Paper II: Fig 1). Despite the removal of the TAP-tag, ΔWWFe65 was still detected in in the nuclear fractions of both SH- SY5Y and SK-N-AS cells, whereas ΔPTB2Fe65 was not (Paper II: Fig 1). We found these results intriguing. Hence, in SH-SY5Y and SK-N- AS cells, the PTB2 domain seems to be more important in Fe65 nuclear localization than the WW domain. Since Fe65 lacks a nuclear localization signal (NLS), it seems that Fe65 nuclear localization is dependent on the PTB2 domain, possibly through anchoring of Fe65 to APP (or another still unidentified protein) at the plasma membrane, and the recruitment of Fe65 interacting proteins harboring potential nuclear localization and export signals. One potential candidate for this task is the histone acetyl transferase Tip60. Nuclear Fe65 has been shown to be transcriptionally active together with AICD and Tip60 [249, 250, 258, 349, 353]. Hass and Yankner have proposed a scenario where APP re- cruits Tip60 to the membrane and induces complex formation with Fe65, followed by Fe65 nuclear translocation to the nucleus together with Tip60 [425]. It might be possible that in the absence of the Fe65- PTB2 domain the APP/Fe65/Tip60 complex formation is disturbed.

4.2.2 Phosphorylation of Fe65 generating an electrophoretic mobility shift resides between residues 192 and 260 Interestingly, when overexpressing the different TAP-tagged deletion mutants (see section 4.2.1), we discovered that two of the mutants, p60Fe65 (amino acids 261-710) and ΔWWFe65 (amino acids 328-710), migrated as one band, whereas p97Fe65 (amino acids 1-710) and ΔPTB2Fe65 (amino acids 1-520) migrated as several bands according to western blot analysis (Paper II: Fig 1). Alkaline phosphatase treatment did not have any effect on the bands corresponding to p60Fe65 and ΔWWFe65, suggesting that these forms of Fe65 are not phosphorylated (Paper II: Fig 3), whereas alkaline phosphatase treatment abolished the slower migrating p97Fe65 and ΔPTB2Fe65 bands. Thus, our results indicate that phosphorylation(s) within the first N-terminal 261

47 amino acids of Fe65 gives rise to the observed electrophoretic mobility shift, at least in our cell models used. Previously, Zambrano and coworkers showed in a mutational study that removal of the first 191 N-terminal amino acids of Fe65 do not have any effect on the heterogeneity of the Fe65 bands. However, deleting the N-terminal 356 amino acids, which includes the WW domain, resulted in the appearance of only a single band [331]. Thus, together with our results, this suggests that the phosphorylation of Fe65 causing the shift occurs within the amino acid sequence 192 and 260. However, it should also be noted that phosphorylation of at least three sites within the PTB2 domain has been associated with regulation of nuclear localization and/or transcriptional activity of Fe65, namely Ser597, Ser610 and Tyr547 [316, 335, 390]. Phos- phorylation at these sites may not occur to a large extent in the cells used in our study, or may not cause a detectable mobility shift on western blot. The amino acid sequence between 192 and 260 has indeed shown to contain several putative phosphorylation sites. Interestingly, phosphorylation of Fe65 at residue Ser228 has been reported to inhibit APP-Fe65 mediated gene transcription [375]. In a follow-up study, we therefore investigated the nuclear localization of Fe65 upon Fe65 Ser228 mutation to alanine (Fe65-S228A) or glutamic acid (Fe65-S228E) (see section 4.2.3), Both mutants induced an electrophoretic mobility shift of Fe65 bands on western blot (Paper III: Fig 1 and Fig 3). The Fe65-Ser228A 228 mutant migrated slightly faster than Fe65WT, whereas Fe65-Ser E mutant slightly slower, suggesting that Ser228 phosphorylation could contribute to the observed electrophoretic mobility shift of Fe65. However, since both Fe65 Ser228 mutants still migrated as several bands, phosphorylation of other residues still seems to occur on Fe65, and contribute to the observed electrophoretic mobility shift of Fe65.

4.2.3 Phosphorylated forms of endogenously expressed Fe65 are preferentially localized outside the nucleus As discussed previously, we discovered that endogenous Fe65 migrates as several bands due to phosphorylation (see section 4.1.1), and that phosphorylation of Fe65 is upregulated in differentiated SH-SY5Y

48 cells (see section 4.1.1). Additionally, we discovered that RA- and PMA-induced differentiation of SH-SY5Y cells increased Fe65 nuclear levels 5- and 3-fold, respectively, compared to undifferentiated control cells (Paper III: Fig 4 and 5). Hence, the observed increase in Fe65 phosphorylation together with the increase in nuclear localization of Fe65 upon differentiation, indicated that Fe65 nuclear localization may be regulated by phosphorylation. Since several studies have shown that Fe65 nuclear localization and transcriptional regulation is dependent on Fe65 phosphorylation [316, 335, 375, 390], we further investigated which of the observed Fe65 bands could be translocated into the nucleus. Therefore, both cytoplasmic and nuclear fractions were subject to enzymatic dephosphorylation. In contrast to the cytoplasmic fractions, treatment of the nuclear fractions with alkaline phosphatase did not give rise to any significant mobility shift of the Fe65 bands, regard- less of whether the cells were differentiated or not (Paper II: Fig 3, Pa- per III: Fig 6). This indicates that the phosphorylated forms of endogenous Fe65 are preferentially localized in the cytoplasm of SH-SY5Y cells, and phosphorylation could thus be involved in inhibiting Fe65 from entering the nucleus. Moreover, phosphorylation indeed seems to regulate Fe65 nuclear localization, at least in SH-SY5Y cells. Interestingly, we measured the increase of nuclear Fe65 upon RA treatment to be approx. 100%, whereas the cytoplasmic fraction showed 40% lower levels of Fe65 protein (Paper III: Fig 4). Hence, Fe65 seems to be localized to a greater extent into the nucleus during RA-induced neuronal differentiation of SH-SY5Y cells compared with undifferentiated control cells. Consequently, the fact that only the slower migrating, phosphorylated Fe65 is observed in the cytoplasm upon RA induced differentiation could be explained by the localization of the unphosphorylated, lower migrating form of Fe65 into the nucleus. Thus, more of the unphosphorylated form of Fe65 can be observed in the cytoplasmic fraction of the undifferentiated SH-SY5Y cells (Paper III: Fig 4). Hence, these results are proposing that unphosphorylated Fe65 may have different roles in undifferentiated and differentiated SH-SY5Y cells. Intriguingly, Fe65 was shown to be endogenously expressed in BCa cells and was shown to act as an ERα transcriptional coregulator by the

49 recruitment to the promoters of estrogen target genes by 17β-estradiol. Deletion analysis mapped the ERα binding domain to the PTB2 domain of Fe65 [340]. ERα and the retinoic acid receptors (RAR) belong to the same superfamily of steroid/thyroid nuclear receptors (reviewed by [426]). It could be possible that the increased nuclear levels of Fe65 upon RA-induced differentiation is due to the recruitment of Fe65 to the promoters of RA target genes, acting as a transcriptional coregulator in the same manner as for ERα. PMA treatment was also able to induce an increase of nuclear Fe65 protein, but however only to approx. 50%, and without any significant change of Fe65 levels in the cytoplasmic fraction. These varying results on Fe65 nuclear localization, dependent on the differentiation method used, strongly indicates that different signaling mechanism are involved in Fe65 nuclear localization. As discussed in section 4.1.2, our results suggest that different molecular mechanisms may be involved in regulating Fe65 expression, and Fe65 nuclear localization, between specific cells, for instance during development. Note that overexpressed nuclear TAP-tagged Fe65 behaves slightly differently compared with endogenous nuclear Fe65 (Paper II: Fig 1, Paper III: Fig 1). The overexpressed Fe65 still migrates as several bands in the nuclear fractions, indicating that phosphorylated forms of Fe65 can be found both in the nucleus and cytoplasm. Studies have shown that overexpressed Fe65 is found primarily in the nucleus, whereas coexpression with APP changes its subcellular localization to the cytoplasm [258, 335, 346]. It has been proposed that APP functions as an anchor for Fe65, retaining it in the cytoplasm [346]. However, no APP is coexpressed together with Fe65 in our studies. Hence, the endogenous levels of APP may not be sufficient to retain both the endogenous and overexpressed Fe65 in the cytoplasm. Moreover, since ΔPTB2Fe65 lacking the TAP-tag was excluded from nuclear fractions (Paper II: Fig 1 and Fig 2), we could rule out the influence of the large TAP-tag on Fe65 nuclear localization. However, our results revealing that ΔPTB2Fe65 is excluded from the nucleus, despite that it is not coexpressed together with APP, validates our results that the PTB2 domain is more important in Fe65 nuclear localization rather than the WW domain.

50 4.2.4 Regulation of Fe65 nuclear localization and Fe65-APP interaction upon Fe65 Ser228 phosphorylation 228 228 By mutating TAP-tagged Fe65WT on Ser to alanine (Fe65-S A) or glutamic acid (Fe65-S228E), mimicking unphosphorylated and phosphorylated states of Ser228 in the Fe65 protein respectively, we investigated if phosphorylation of Fe65 on Ser228 could be involved in Fe65 nuclear localization. Interestingly, Jowsey and coworkers showed that mutation of Fe65 on Ser228 to alanine (Fe65-S228A) caused a significant increase in Fe65-APP-mediated transcriptional activity, whereas mutation of Ser228 to aspartic acid (Fe65-S228D) or glutamic acid (Fe65- S228E) decreased this activity [375]. According to our results (see section 4.2.3), one possible reason to the decreased activity of the Fe65 phosphomutants, Fe65-S228E and Fe65-S228D, could be the inhibition of Fe65 nuclear localization due to phosphorylation. Surprisingly, our results from subcellular fractionation followed by western blot, showed that mimicking phosphorylation of Fe65 Ser228 does not exclude a nuclear localization of Fe65 in SK-N-AS cells (Paper III: Fig 1). The results were confirmed with immunofluorescence studies in the same cell line (Paper III: Fig 2). However, according to our results we can not rule out that phosphorylation of Fe65 Ser228 still may influence Fe65 nuclear localization, since overexpressed Fe65, as discussed in section 4.2.3., is mainly localized in the nucleus if not coexpressed together with APP. Considering that phosphorylation per se of Fe65 Ser228 is not enough to retain Fe65 in the cytoplasm, and since the increase in transcriptional activity of Fe65 Ser228 mutated to alanine (Fe65-S228A) was shown to be dependent on coexpression of APP [375], we wanted to investigate if Ser228 phosphorylation affected the interaction between Fe65 and APP. Hence APP has been proposed to act as an anchor for Fe65, inhibiting Fe65 from translocating into the nucleus [346]. Here we utilized the TAP-tag method for interaction studies between Fe65 and APP. Each TAP-tagged Fe65 construct was overexpressed in SK-N-AS cells together with APPwt, followed by TAP-tag pulldown of cell lysates. To our surprise, we observed that both the Fe65 S228A and S228E mutation significantly increased the interaction with APPwt by approx.

51 100% (Paper III: Fig 3). In conclusion, our obtained results indicate that phosphorylation of Fe65 Ser228 is mainly involved in regulating Fe65 transcriptional activity. However, since the coexpression of APP was shown to increase the transcriptional activity of Fe65 Ser228A, it is possible that APP has the function to recruit other Fe65 interacting proteins to the plasma membrane involved in the proposed transcriptional activity. A note should be made of that increased Fe65 Ser228 phosphorylation was shown to occur during UV treatment (to induce DNA damage) of HEK293 cells overexpressing GFP-Fe65, whereas the luciferase-based reporter gene assay, with overexpressed pMST-APP and GFP-Fe65, was conducted in untreated HEK293 cells in the study by Jowsey and coworkers [375]. In addition, the expression levels of endogenous APP were concomitantly reduced upon UV induced DNA damage in the study, whereas GFP-Fe65 Ser228 phosphorylation was increased. There- fore, it is most difficult to conclude if the observed decrease in APP dependent transcriptional activity upon Fe65 Ser228 phosphorylation, is something that occur normally within healthy cells. On the other hand, the increased interaction between Fe65 and APP upon Fe65 Ser228 mutations detected in our study is most fascinating and surprising, as the interaction between Fe65 and APP has shown to occur through the Fe65 C-terminal PTB2-domain [83, 314]. Interest- ingly, truncation of the N-terminal domain of Fe65, right before the WW-domain by a still unknown protease, has shown to result in higher affinity to APP, reduced transcriptional activity, and decrease the secretion of sAPPα [384]. Additionally, a shorter 60kDa Fe65 isoform (p60Fe65), lacking the N-terminal domain, including a part of the WW- domain, was suggested to possess higher binding affinity to AICD than p97Fe65. Moreover, p60Fe65 localized into the nucleus, but was however, shown to have attenuated transcriptional activity [381, 383]. Our data indicate that it is most possible that mutation of Fe65 Ser228 dis- turbs the conformation of the N-terminal domain of Fe65, leading to disrupted interactions with other proteins, or itself [363], hence increasing Fe65 binding to APP. The increased interaction between Fe65 Ser228 mutants and APP is intriguing, and suggests that modulation of Fe65 at its N-terminus may be involved in the regulation of APP processing.

52 4.2.5 Fe65 nuclear localization is dependent on regulated intramembrane proteolysis (RIP) Due to our results showing that Fe65 nuclear localization is PTB2- domain dependent (see section 4.2.1), and since Fe65 has been shown to be involved in transcriptional regulation, specifically together with AICD [258], we wanted to further investigate how the inhibition of α- and γ-secretase processing affects the nuclear localization of Fe65 in SH-SY5Y cells. Both undifferentiated, and RA- or PMA-differentiated SH-SY5Y cells were treated with an ADAM10 specific inhibitor GI254023X (A10inh), a broad-spectrum metalloprotease inhibitor Bati- mastat, or a γ-secretase inhibitor DAPT. As expected, from previous studies [247, 254], γ-secretase inhibition by DAPT decreased the nuclear localization of Fe65 in both undifferentiated and differentiated SH-SY5Y cells (Paper II: Fig 4, Paper III: Fig 4 and 5). Most interestingly, α-secretase inhibition by both A10inh and Batimastat resulted in a similar decrease in nuclear Fe65 to a similar degree as DAPT in undifferentiated SH-SY5Y cells (Paper II: Fig 4). This indicate that blocking the α-secretase activity strongly affected Fe65 nuclear localization, which is surprising as previous studies indicate that APP/AICD dependent transcription occurs predominantly through the β-secretase pathway [247, 254]. However, it has been proposed that AICD and Fe65 can translocate into the nucleus independently [364]. A most fascinating thought is that Fe65 released from the membrane by the α- secretase pathway may not be linked to APP dependent transcription at all, as APP is not the only Fe65 interacting protein that can undergo RIP. Fe65 has also shown to interact with two of the other members of the APP-family, APLP1 and APLP2 [83], LRP1 [324], VLDLR [326], ApoEr2 [285], and Notch [427]. In addition, treatment with DAPT and Batimastat significantly, and to the same extent, decreased the levels of nuclear Fe65 in both RA and PMA differentiated SH-SY5Y cells (Paper III: Fig 4 and 5). A10inh, on the other hand, had less pronounced effect on Fe65 nuclear localization in RA differentiated cells (Paper III: Fig 4), and, surprisingly, no significant effect in PMA differentiated cells at all (Paper III: Fig 5). Since Batimastat decreased the levels of nuclear Fe65 to a greater extent than

53 A10inh in differentiated cells, but the effect was the same in undifferentiated cells, other α-secretases along with ADAM10, seems to play large role in the regulation of Fe65 nuclear localization during differentiation. The broad-spectrum metalloprotease (MMP) inhibitor Batimastat has also been shown to inhibit TACE and ADAM9, two other α-secretases thought to be involved in the non-amyloidogenic APP processing [163]. Phorbol esters has shown to increase TACE activity [428], and TACE has shown to be involved in the regulated α-secretase cleavage of APP upon PMA treatment [144]. Taken together, our results suggest that ADAM10 is preferentially the α-secretase mediating Fe65 nuclear localization in undifferentiated SH-SY5Y cells, whereas TACE is most likely the α-secretase involved in Fe65 nuclear localization during PMA induced differentiation. On the other hand, the identity of the α-secretase involved in Fe65 nuclear localization in RA differentiated SH- SY5Y cells seems more complex. It is possible that both ADAM10 and TACE may be responsible for Fe65 nuclear localization, hence RA has shown to increase protein levels of both ADAM10 and TACE in SH- SY5Y cells [405]. Furthermore, in a recent study, Chen and coworkers [429] identified a novel interaction between α- and γ-secretase, proposing a model in which α- and γ-secretases reside in a complex for efficient sequential processing of substrates. Our results showing that the γ-secretase inhibitor DAPT inhibits Fe65 nuclear localization to the same extent as the broad-spectrum MMP Batimastat in both undifferentiated and differentiated SH-SY5Y cells implicates that the nuclear localization of Fe65 is indeed regulated by RIP, and preferentially by the non-amyloidogenic pathway.

4.3 Regulation of APP processing upon APP Ser675 phosphorylation (Paper IV) In AD brain , phosphorylation of APP at its C-terminal domain has shown to occur at seven different residues, including APP Ser675 [62]. Phosphorylation of APP Ser675 was not, on the other hand, detected in

54 recombinant APP expressing CAD cells, indicating that phosphorylation of APP Ser675 may be related to AD pathology. In this thesis we have therefore for the first time tried to characterized the role of APP Ser675 phosphorylation in APP processing, trafficking and protein-protein interaction.

4.3.1 APP-Ser675 phosphorylation decreases sAPPα secretion without affecting APP cell surface localization The initial step in the non-amyloidogenic, and neuroprotective, APP processing pathway is α-secretase cleavage of APP and the secretion of the neuroprotective ectodomain sAPPα. To investigate if APP Ser675 phosphorylation might affect α-secretase processing of APP, APP695 (APPwt) was mutated on APP Ser675 to alanine (APP-S675A) or glutamic acid (APP-S675E), to mimic the non-phosphorylated and phosphorylated forms of APP, respectively. The secretion of sAPPα from cells expressing the different APP versions was then measured. Inter- estingly, sAPPα secreted from SK-N-AS cells overexpressing the phosphomutant APP-Ser675E was decreased to approx. 50% compared to APPwt and the non-phosphorylated form, APP-S675A (Paper IV: Fig 1). We found these results interesting, since reduced sAPPα levels in CSF have been reported in AD patients [430-432], and decreased α-secretase processing is suggested to contribute to AD pathogenesis, Several studies have also shown that phosphorylation of APP may be involved in APP trafficking to the cell surface. Thus, hypo-phosphorylation of APP may be involved in aberrant subcellular localization, hence deviant processing of APP. The α-secretase processing of APP has mainly shown to occur at the cell surface [123]. Therefore, we next focused on analyzing APP levels at this cellular compartment upon APP Ser675 phosphorylation. Since we observed decreased levels of sAPPα secretion upon APP-S675E expression, we expected to see decreased levels of APP at the cell surface. Surprisingly, a biotinylation assay revealed that there were no significant differences in total cell surface levels of APP-S675E compared with APPwt or APP-S675A (Pa- per IV: Fig 3), indicating that phosphorylation of APP Ser675 is not involved in regulating APP trafficking, but most likely influences α-

55 secretase processing at the plasma membrane. Nevertheless, it has been proposed that α-secretase and β-secretase may compete for APP as a substrate. However, in contrast to α-secretase cleavage, β-secretase cleavage of APP occurs mainly in the endosome and to a lower extent in the trans-Golgi network [126, 175]. Could APP Ser675 phosphorylation possibly be involved in increased β-secretase processing of APP? Thus, the 50% decrease in sAPPα secretion of APP-S675E indicates that APP-S675E could be a better substrate for β-secretase processing than APP-S675A. However, on the other hand, unaltered cell surface levels of both APP-S675A and APP-S675E demonstrate that APP Ser675 phosphorylation may not be involved in increased β-secretase processing of APP, but rather in some other events inhibiting α-secretase processing of APP at the cell surface.

4.3.2 APP-Ser675 phosphorylation generates, through α- secretase processing, a slower migrating APP-CTF band A decrease in α-secretase processing of APP should generate lower levels of the APP-C83-CTF, and, if β-secretase competes with α-secretase for APP as a substrate, increased levels of APP-C99-CTF should be observed. To investigate if this applies for APP-S675E, SK-N-AS cells overexpressing APPwt, APP-S675A or APP-S675E were treated with a γ-secretase inhibitor, DAPT, to increase the levels of the other- wise barely detectable APP-CTFs. Two APP-CTF bands were detected for all three APP cDNA constructs on western blot, however, the APP- S675E phosphomutant gave rise to a prominent shift of the CTF bands, increasing the intensity of the upper slower migrating band by three- fold (Paper IV: Fig 1). On the other hand, there were no significant change in the total levels of APP-S675E-CTFs compared with APPwt and APP-S675A, supporting the fact that APP Ser675 phosphorylation is involved in the regulation of APP processing. Also, the induced shift of APP-CTF bands, and the observed decrease in sAPPα secretion in deed supported the hypothesis that APP Ser675 phosphorylation may be involved in shifting the processing of APP towards the β-secretase pathway. To reveal if the induced shift of APP-CTF bands generated upon APP Ser675 phosphorylation, was due to β-secretase cleavage and the

56 accumulation of APP-C99-CTF fragments, we investigated if the APP- CTFs generated by APP-S675E were resistant to α-secretase inhibition. To our surprise, we observed that both the levels of the slower migrating APP-CTFs and the total levels of APP-CTFs generated by APP-S675E were decreased to the same extent as for APPwt and APP-S675A upon α-secretase inhibition (Paper IV: Fig 2). No APP-C99-CTFs were detected in our experimental setup. Hence, the three-fold shift of APP- CTF bands observed for APP-S675E cannot be due to increased β-secretase processing. Interestingly, no competition between α- and β-secretase cleavage have been observed in cell lines, such as HEK293, CHO and SH-SY5Y, probably due to lower expression levels of the β-secretase BACE1 in cell lines compared with neurons [141, 433]. Consider- ing the decreased levels of secreted sAPPα and the unchanged levels of CTFs in SK-N-AS cells, it is most likely that APP Ser675 phosphorylation increases APP-C83-CTF stability, specifically the stability of the upper slower migrating APP-CTF, probably through inhibiting APP- CTFs from degradation. Phosphorylation of APP-CTF fragments has shown to induce a shift of bands on western blot [144, 434]. The increased levels of the slower migrating C83 band generated upon APP- S675E could be due to additional phosphorylation of APP-S675E. Addi- tionally, APP Ser675 phosphorylation might be involved in stabilizing the α-secretase cleaved APP-C83-CTF fragment protecting it from proteasomal degradation. Thus, proteasome inhibition has shown to increase the levels of APP-CTFs [435-437].

4.3.3 Regulation of APP-Fe65 interaction upon APP Ser675 phosphorylation Since APP Ser675 resides between Thr668 and the 684NPTY687 motif of APP, within an α-helix shown to stabilize the APP-Fe65 interaction [264], we wanted to study how phosphorylation of APP Ser675 may affect this interaction. Here we utilized the TAP-tag method for interaction studies between Fe65 and APP (see section 2.3 for a more detailed description of the method). Each APP cDNA construct, APPWT, APP- S675A and APP-S675E, was overexpressed in SK-N-AS cells together with Fe65WT-TAP, followed by TAP-tag pulldown of cell lysates. We

57 observed, to our surprise that both mutants, APP-S675A and APP-S675E, significantly increased the interaction with Fe65 by approx. 60 and 75%, respectively (Paper IV: Fig 4), indicating that Fe65 interacts with APP irrespective of APP Ser675 phosphorylation. However, our results demonstrate that a modification of APP Ser675 per se seems to stabilize the APP-Fe65 interaction. This finding was surprising and to validate our pull-down method we therefore also studied some additional APP mutants Thr668 and Tyr687. Phosphorylation of Thr668 has shown to induce a significant conformational change of the cytoplasmic domain of APP, decreasing the interaction with Fe65 [74], and to affect APP processing [62]. Mutation of N684PTY687 to 687NATA687 has also shown to abolish the interaction between Fe65 and APP [258]. Our obtained data revealed decreased interaction between APP-Thr668A and TAP-tagged Fe65 as expected [74, 264], whereas APP-Tyr687A abolished the interaction with TAP- tagged Fe65 entirely (Paper IV: Fig 4). These results rule out that the increased interaction of both APP Ser675 mutants with the TAP-tagged Fe65 is a discrepancy of the pull-down method used. Interestingly, increased binding of Fe65 to APP, specifically of an N-terminally truncated Fe65, has shown to decrease the secretion of sAPPα [384]. The increased APP/Fe65 interaction upon S675A or S675E mutation could thus be involved in the altered APP processing.

4.3.4 How does APP Ser675 phosphorylation affect APP processing? The observed decrease in sAPPα secretion of APP-S675E, unaltered levels of APP-S675E at the cell-surface, and the increase of the slower migrating APP-C83-CTF band of the APP Ser675E mutant, indicates that APP-Ser675 phosphorylation most likely influences α-secretase processing of APP at the plasma membrane, rather than β-secretase processing. Since α-secretase processing of APP is an essential step in decreasing the generation of Aβ, understanding how proteolysis of phosphorylated APP Ser675 by α-secretase is regulated, may be of great help when elucidating the intrinsic signaling network regulating the generation of Aβ. So, how could APP Ser675 phosphorylation be involved in

58 the regulation of α-secretase processing of APP at the cell surface? A few plausible explanations exist, which are shortly discussed below. The observed increase of the slower migrating APP-C83-CTF band, generated upon APP-S675E mutation, could be explained by additional phosphorylation(s) of APP-S675E, specifically of APP-Thr668. Phos- phorylation of APP-Thr668 has shown to be elevated in AD brain samples, as has phosphorylation of APP-Ser675 [62]. In addition, phosphorylation of the APP-T668-P669 motif has shown to induce a conformational change of APP [67, 74], hence changing the conformation of APP from trans to cis. Isomerization of the phosphorylated APP T668-P669 motif could be involved in the recruitment of isomer specific binding partners influencing the processing of APP, hence decreasing the secretion of sAPPα. Proline directed phosphorylation by CDK5 [62, 438, 439], and GSK3β [440] of APP-Thr668 has shown to increase amyloid pathology. However, increased APP-Thr668 phosphorylation, and the subsequent change of conformation, have also been shown to decrease the APP-Fe65 interaction [74]. Our results showing increased Fe interaction with APP-Ser675E are though conflicting in this regard. Still, the previous APP-Fe65 interaction studies have been conducted during conditions where APP Ser675 may not be phosphorylated, since APP Ser675 phosphorylation was not detected in recombinant APP expressing CAD cells. In our study, APP-S675A behaves as APPWT, suggesting that APP Ser675 phosphorylation indeed occur to a limited extent during normal cellular conditions. Thus, phosphorylation of APP Ser675 may be a neuron specific event, specifically occurring during neurodegeneration. Intriguingly, APP processing has also shown to be regulated by APP dimerization. APP can form homo- and heterodimers at the cell surface, through its extracellular domain, as well as transmembrane GXXXG motifs [51]. However, low levels of APP dimerization seem to occur during normal cellular conditions, since dimerization was estimated to approx. 5% [49]. Studies have shown that dimerization of APP modulates Aβ production [49, 441], as well as reduces the levels of both sAPPα and sAPPβ [441]. Interestingly, dimerization was also shown to inhibit γ-secretase processing of APP [49]. Hence, our observed decrease in sAPPα secretion upon APP-S675E mutation, and the observed

59 shift of APP-C83-CTFs, could be the consequence of increased dimerization upon APP Ser675 phosphorylation, and a decrease in γ-secretase processing generating the higher migrating APP-C83-CTF fragment. However, induced APP dimerization was shown to decrease APP localization to the cell surface, nevertheless APP internalization rate was not shown to be affected by this event [441]. Interestingly, in a recent study, Decock and coworkers [442] showed that the deletion of the APP C- terminal region increased APP dimerization, indicating that the APP C- terminus is involved in regulating APP dimerization. However, sAPPα secretion was increased, whereas sAPPβ levels were reduced upon the deletion of the APP C terminus in this study. The deletion of the APP C terminus, hence the canonical endocytic signal for membrane-associated receptors, the Y682ENPTY687 motif, has previously shown to inhibit APP internalization, increase APP cell surface localization, and increase sAPPα secretion [75]. In addition, mutations of Y682, N684 and P685 in the Y682ENPTY687 has shown to inhibit the internalization of APP and decrease the generation of secreted sAPP and Aβ [76, 443]. Therefore, Decock and coworker´s observed effects on APP processing, upon deletion of the C terminus of APP, are more likely dependent on reduced endocytosis, than increased dimerization. Thus, could mutation of APP Ser675 to glutamic acid, hence phosphorylation of APP at Ser675, also be involved in disturbing the endocytic signal of APP, retaining APP at the cell surface? Since APP has shown to anchor Fe65 to the plasma membrane, the increased interaction between Fe65 and APP- S675E could therefore simply be explained by a decrease in endocytosis of APP-S675E. Moreover, the increased interaction between Fe65 and APP-S675E could be due to the observed decrease in α-secretase processing, which we have shown to influence Fe65 nuclear localization (see section 4.2.5.).

60 5 Conclusions and outlook

We found that:

o Phosphorylation of Fe65 results in generation of an electrophoretic mobility shift, and Fe65 migrating as multiple bands on western blot. Phosphorylation of Ser228 could contribute to this shift.

o Phosphorylation of Fe65 results in a preferentially cytoplasmic Fe65 localization

o Nuclear translocation of Fe65 is dependent on the PTB2 domain in Fe65, and regulated by α-secretase processing. In fact, we found that inhibition of α-secretases could inhibit nuclear entry of Fe65 to the same extent as γ-secretase inhibition. Taken together, this suggests that binding to a membrane tethered, PTB2 interacting protein, like APP, is required before Fe65 can enter the nucleus. o Neuronal differentiation affects the phosphorylation and subcellular localization of Fe65, indicating that Fe65 might play a role in differentiation. Upon differentiation, the total cytoplasmic level of Fe65 decreased, however the ratio of phosphorylated/nonphosphorylated Fe65 was increased in the cytoplasm and this was accompanied by an increase in nonphsophorylated Fe65 in the nucleus. Differentiation also affected which α-secretases that can mediate Fe65 nuclear translocation. While ADAM10 played a considerable role in undifferentiated cells, other α-secretases appear to take a more prominent part in releasing Fe65 from the plasma membrane in differentiated cells. How- ever, some differences in the regulation of Fe65 in response to

61 differentiation could be observed depending on differentiation method used, suggesting that Fe65 regulation can vary depending on cell phenotype. o Mutation of Ser228 in Fe65 increases the APP-Fe65 interaction with approximately 100%, suggesting that the N-terminal domain of Fe65 influence this interaction and could thus also regulate APP processing. o Phosphorylation of APP at Ser675 alters the α-secretase processing of APP at the plasma membrane, resulting in decreased levels of neuroprotective sAPPα and increased levels of an alternative APP-C83-CTF fragment. In addition, we found that that mutation of Ser675 to alanine or glutamic acid increases the Fe65- APP interaction. As APP Ser675 phosphorylation is observed in AD brains and α-secretase processing of APP is an essential step in decreasing the generation of Aβ, these finding suggests that APP Ser675 phosphorylation could contribute to AD pathology.

The regulation of Fe65 nuclear localization by α-secretase processing is intriguing, since it indicates that Fe65 nuclear function is not exclusively dependent on its interaction with APP/AICD. α-secretases cleave a myriad of proteins, and are involved in a subset of cellular functions, such as cell adhesion, migration and signaling. Identification of α-secretase substrates, other than APP, interacting with Fe65, may contribute to understanding of Fe65 functions, both cytosolic and nuclear, during both normal and pathological conditions in the brain. In addition, our results clearly demonstrate that both Fe65 and APP cellular functions are regulated by phosphorylation. Interestingly, APP has shown to be hyper-phosphorylated in AD brain. Moreover, Fe65 has shown to be phosphorylated by kinases, also involved in APP phosphorylation. However, the effect of Fe65 phosphorylation on APP/Fe65 interaction, APP processing and trafficking, and the nuclear function of Fe65, is controversial. To identify possible phosphorylation sites on

62 Fe65, and the responsible kinases involved in the act, is of great importance, since inhibiting or enhancing Fe65 phosphorylation could be a way by which APP processing could be regulated. Previous studies have shown that increased phosphorylation of APP at its C-terminus influences its trafficking, processing and interaction with adaptor proteins. The proposed decrease in sAPPα, upon APP Ser675 phosphorylation, is intriguing, since phosphorylated APP Ser675 was primarily observed in AD brains. Hence, APP Ser675 phosphorylation is most likely related to AD pathology. In this regard, it would be highly relevant to identify the kinase(s) responsible for APP Ser675 phosphorylation, and specifically to reveal the mechanisms behind how APP C-terminal phosphorylation is involved in regulating ectodomain shedding of APP.

63 6 Populärvetenskaplig sammanfattning på svenska

Alzheimers sjukdom (Alzheimers) är den vanligaste förekommande demenssjukdomen idag. I Sverige idag lever ca 100 000 personer med Alzheimers och ca 15 000 är beräknade att insjukna årligen. Vanligtvis drabbas människor äldre än 65 år, men vid mycket sällsynta fall kan man även drabbas av sjukdomen redan vid 35 års ålder. De vanligaste riskfaktorerna är hög ålder och ärftlighet. Orsaken till Alzheimers är inte känd, men sjukdomen gör att hjärn- vävnaden gradvis förstörs då nervceller förtvinar och dör i onormal om- fattning, speciellt i de regioner där minnet behandlas. Flera kognitiva funktioner blir påverkade, som minne, tankeförmåga, orienteringsför- måga och språk. Oro och ångest hört till de vanliga symptomen, samt hallucinationer, vanföreställningar och beteendeförändringar. Sympto- men kan dock variera från person till person. Sjukdomen kommer smy- gandes, och har ett utdraget förlopp. Vissa av symptomen kan i ett tidigt skede lindras, men sjukdomsförloppet går ej att hejda, dvs det finns inget känt botemedel mot Alzheimers sjukdom idag. En av flera bakomliggande orsaker till att hjärnvävnaden gradvis för- störs vid Alzheimers är onaturliga proteininlagringar, s.k. senila plack som bildas i hjärncellerna. Senila plack består av peptiden amyloid-beta (Aβ), ett ämne som även förekommer i friska hjärnor. Aβ bildas genom att ett större protein, amyloid-β precursor protein (APP), klyvs av s.k sekretasenzymer, β- och γ-secretaser, till kortare fragment i nervceller i hjärnan. Aβ frisätts till vätskan utanför nervcellerna och kan i för hög koncentration klumpa ihop sig till senila plack. I friska nervceller klyvs APP vanligtvis av α- och γ-sekretaser, som hindrar att Aβ genereras. Istället frisätts ett fragment, sAPPα, som visat sig ha ett skyddande ef- fekt på nervceller. I Alzheimers är balansen mellan frisättningen av

64 sAPPα och Aβ från nervceller förändrar. Lägre nivåer av sAPPα och högre nivåer av Aβ tros bidra till att nervcellerna dör. Att selektivt kunna öka nivåerna av sAPPα, och att minska nivåerna av Aβ, i behand- ling av Alzheimers är därför önskvärt. I vår forskning som presenteras i denna avhandling har vi bl.a under- sökt hur APP klyvningen kan påverkas av specifika faktorer, så som fosforylering, samt försökt karakterisera egenskaperna hos ett APP-in- teragerande adapterprotein kallat Fe65. Fosforylering av proteiner är ett vanligt sätt för cellen att ändra ett proteins egenskaper, men för lite eller för mycket fosforylering av ett protein kan leda till oönskade cellulära effekter. Speciellt i Alzheimers har man sett att fosforyleringen av proteiner ökar och leder till oönskade konsekvenser, som att nervceller dör. ” Ensam är ingen stark” gäller även för proteiner. För att en signal ska kunna genereras t.ex. från utsidan av en cell till dess kärna, krävs det att proteiner binder till varandra och ”skickar vidare” signalen inne i cellen tills den når sitt mål. Fe65 proteinet är ett s.k. adapterprotein, som sam- manlänkar proteiner som inte direkt kan interagera med varandra, och kan på detta sätt förmedla signalen vidare inne i cellen. Blir det fel i sammanlänkningen, blir det fel i signalen. Våra resultat indikerar på bl.a att ökad fosforylering av en icke tidigare undersökt aminosyra på APP, Ser675, minskar frisättningen av det beskyddande fragmentet sAPPα. Denna aminosyra har tidigare visats vara fosforylerad i hjärnor från Alzheimers patienter, men ingen har un- dersökt dess effekter på APP klyvningen tidigare. Våra resultat visar även att Fe65s funktioner är reglerad av fosforylering, samt att de APP klyvande sekretaserna, α- och γ-sekretaserna, är involverade i hur Fe65 kan translokeras in i cellkärnan. Sammantaget har våra studier bidragit till förståelsen av både APP-klyvningen och samspelet mellan Fe65 och APP, och kan förhoppningsvis i framtiden leda till lösningen till en del av gåtorna som omger Alzheimers sjukdom.

A healthy and a diseased brain half, freely interpreted by NK.

66 7 Acknowledgement

Blir vi inte alltid mållösa i livets största stunder? Med känslan av både lättnad och vemod skriver jag mina sista rader i denna avhandling. Det har varit en omtumlande tid. De planera fem åren blev till långa åtta år. Detta har medfört i sin tur att min väg har korsats av många fina människor som jag skulle vilja tacka med några ord. Inledningsvis vill jag tacka Kerstin Iverfeldt, min handledare och men- tor, som gav mig möjligheten att få utvecklas till en självständig forskare i det fängslande ämnesområdet neurokemi. Hon var en fantastisk handledare och en sann förebild. Hon har lämnat ett stort tomrum efter sig. ”I hjärtan finns du evigt, i minnen finns du kvar, där slocknar aldrig livet, där finns du osår- bar” Anna-Lena Ström, vad ska jag säga? Min kära handledare, utan att blinka dök du ner i APP och Fe65-träsket, och drog upp mig till ytan när det behövdes som mest. Det går inte med ord beskriva den tacksamhet jag hyser för dig, för den stöd du gett mig, både som handledare och som vän. Ditt brinnande engagemang för ämnet neurokemi, och studenternas välbefinnande, är inspirerande. Jag hoppas att den lågan får fortsätta brinna långt i i framtiden. Ett stort tack till er alla lärare här på institutionen; Einar Hallberg, ”the perfect prefect”, hoppas du får långa, och mer avslappnade, somrar framöver på sjön, Ülo Langel, Say no more: You are one of a kind, Anders Undén, tack för en inspirerande C-kurs, den fick mig att stanna kvar på institutionen, Anna Forsby, en kvinnlig förebild i mångt och mycket, Bengt Mannervik, tack för alla bra kommentater under tisdags-seminarierna och Henrietta Ni- elsen, ditt engagemang och kunskap både imponerar och inspirerar. Marie-louise, jag säger som alla andra: Vad vore denna institution utan dig? Du ÄR dess ryggrad. Sylvia, tack för alla trevliga pratstuder i köket och hjälp kring de byrokratiska ekonomiska bitarna. Ett stort tack vill jag även rikta till KI-gruppens alla medlemmar som jag fått nöjet att lära känna och arbeta med: Sofia, tack för att du tog mig an som ex-jobbare, det blev några publikationer på Fe65 till slut, Tom, svårt att inte ryckas med av din energi och glädje, Veronica R, du var en toppentjej även att dela kontor med, Kristin J, du är saknad både som arbetskamrat och vän, Linda, finare och omtänksammare person är svårt att hitta, saknar dig massor, Ylva, hoppas du kommer tillbaka snart och löser O-GlcNacyleringens gåta. Ett speciellt tack vill jag rikta till mina medförfattare, Preeti, Elena och Anna E. Tack för all praktisk och teoretisk hjälp runt artiklar och manuskript.

67 Preeti, you are now the new Fe65-expert, I wish you the best of luck with the two manuscripts! Elena, I hope everything turns out fine for you, regarding both finishing your PhD and becoming a boring Swede … Anna E, jag tror dina nya project passar dig som handen i handsken. Stort lycka till! I would like to thank you all PhD students, postdocs and researchers at this department, since you are the main reason to why this departments is such great place to come to every day. It has been an honor to work with you, and specifically to take part of your exciting research. Special thanks to: Min bästaste bästa rumskompis Veronica… Undrar om vågskålen med gråt väger lika tungt som den med skratt inne på vårt kontor? Jag kommer att sakna våra ”icke vetenskapliga” diskussioner. Och du...! Kämpa på, du har målsnöret inom räckhåll! Finaste fina Kristina och Jessica... Vad hade jag gjort utan er de här sista åren? Ni är mina jobb-bästisar! Alltid där för att lyssna, stötta, lugna, glädja, trösta. Och tack Kristina, för alla timmar i gymmet, jag saknar verkligen de stunderna. Jessica, en finare, varmare och goare person får man leta efter. Alla borde ha en Jessica i sina liv... Ja vill även rikta ett tack till mina studenter jag handlett under åren: Mi- chael, Hanin, Anton, Smaranda, Stelia, Anna V och Elliot. Ett speciellt tack till min medförfattare Smaranda: You are amazing! Till mina underbara vänner utanför akademien, Maarit, Minna och Nina (med respektive Joakim, Conny och Henrik) vill jag bara säga NO NIIN! Och KIITOS för att ni ser till att det inte går för lång tid mellan gångerna vi träffas. Jag saknar er oerhört, fast jag är både dålig på att säga det och visa det, samt göra någonting åt det. Jag är otroligt tacksam över att ni finns i mitt liv! <3 Paula och Lasse, tack för de trevliga traditionsenliga midsommarfes- terna, som ses fram emot med glädje årligen! Även ett extra stort tack till dig Paula för att du introducerade mig till löpningens tjusning. Birgitta och Anders, det är svårt att hitta mer positiva och lättsammare människor att umgås med än ni! Tack för alla middagar ni anordnat. Jag ser fram emot fler middagar och nöjesparksutflykter med er! Jag vill tacka min(a) familj(er) för allt stöd under årens lopp. Ett speciellt tack vill jag rikta till Åke och Birgitta för att vi i så många år fått utnyttja Gryt, ett litet paradis för både stora och små. Conrad och Cleo, det finns inget liv utan er! Ni är det bästa som hänt mig. Till sist, den som fått utstå mest, men fått ut av det minst, min älskade Andreas. Det skulle inte finnas en avhandling, eller en disputation, utan dig och ditt stöd. Du är min klippa, och borgen där jag finner räddning. Ingenting av det det här skulle var möjligt utan dig. Tack!

68 8 References

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