α-Secretase processing of the Alzheimer amyloid-β precursor protein and its homolog APLP2

α-Secretase processing of the
Alzheimer amyloid-β precursor
protein and its homolog APLP2
Kristin Jacobsen
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Doctoral dissertation, 2013
Department of Neurochemistry
Arrhenius Laboratories for Natural Sciences
Stockholm University
Cover: Schematic illustration of APP and APLP2 processing during stimulated conditions
©Kristin T. Jacobsen, Stockholm University 2013
ISBN 978-91-7447-732-0
Printed in Sweden by Universitetsservice US-AB, Stockholm 2013
Distributor: Department of Neurochemistry, Stockholm University
2
To my family
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List of Publications
I.
Jacobsen KT, Adlerz L, Multhaup G and Iverfeldt K.
IGF-1-induced processing of amyloid-β precursor protein and
APP-like protein 2 is mediated by different enzymes
Journal of Biological Chemistry. 2010; 285 (14): 10223-10231
II.
Holback S, Adlerz L, Gatsinzi T, Jacobsen KT and Iverfeldt K
PI3-K- and PKC-dependent up-regulation of APP processing
enzymes by retinoic acid
Biochemical and Biophysical Research Communications. 2008;
365(2): 298-303
III.
Jacobsen KT and Iverfeldt K.
O-GlcNAcylation increases non-amyloidogenic processing of the
amyloid-β precursor protein (APP)
Biochemical and Biophysical Research Communications. 2011;
404(3): 882-886
IV.
Jacobsen KT, Strååt Y, Koistinen N and Iverfeldt K.
O-GlcNAcylation of the α-secretase ADAM10 selectively affects
APP processing in neuron-like cells
Manuscript
V.
Jacobsen KT and Iverfeldt K
The E1 domain of APP and APLP2 determines α-secretase
specificity
Manuscript
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Additional publications
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
Jacobsen KT and Iverfeldt K
Amyloid precursor protein and its homologues: a family of
proteolysis-dependent receptors
Cellular and Molecular Life Science. 2009; 66: 2299-2318 (Review)

Tracy, LM, Bergqvist F, Ivanova EV, Jacobsen KT and Iverfeldt K
Exposure to the saturated free fatty acid palmitate alters BV-2
microglia inflammatory response
Journal of Molecular Neuroscience. 2013; 51(3): 805-812
Abstract
The amyloid-β precursor protein (APP) has been widely studied due to its
role in Alzheimer´s disease (AD). When APP is sequentially cleaved by βand γ-secretase, amyloid-β (Aβ) is formed. Aβ is prone to aggregate and is
toxic to neurons. However, the main processing pathway for APP involves
initial cleavage at the α-site, within the Aβ region, instead generating a neuroprotective soluble fragment, sAPPα. APP is a member of a protein family,
also including the proteins APLP1 and APLP2, which are processed in a
similar way as APP. In addition, knock-out studies in mice have shown that
the three proteins have overlapping functions where APLP2 play a key physiological role. The aim of this thesis was to study mechanisms regulating the
α-secretase processing of APP and APLP2. We have used the human neuroblastoma cell line SH-SY5Y as a model system and have stimulated αsecretase processing with insulin-like growth factor-1 (IGF-1) or retinoic
acid (RA). Our results show that the stimulated α-site cleavage of APP and
APLP2 is regulated by different signaling pathways and that the cleavage is
mediated by different enzymes. APP was shown to be cleaved by ADAM10
in a PI3K-dependent manner, whereas APLP2 was cleaved by TACE in a
PKC-dependent manner. We further show that protein levels and maturation
of ADAM10 and TACE is increased in response to RA, mediated by a PI3Kor PKC-dependent signaling pathway, respectively. Another focus of our
research has been O-GlcNAcylation, a dynamic post-translational modification regulated by the enzymes O-GlcNAc transferase and O-GlcNAcase
(OGA). We show that decreased OGA activity stimulates sAPPα secretion,
without affecting APLP2 processing. We further show that ADAM10 is OGlcNAcylated. Lastly, we show that APP can be manipulated to be cleaved
in a similar way as APLP2 during IGF-1 stimulation by substituting the E1
domain in APP with the E1 domain from APLP2. Together our results show
distinct α-site processing mechanisms of APP and APLP2.
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Contents
1. Introduction ........................................................................................ 13
1.1 The role of APP in Alzheimer’s disease ......................................................... 13
1.2 The APP family .................................................................................................. 14
1.2.1 Structure ................................................................................................... 14
1.2.2 Proteolytic processing ............................................................................. 17
1.2.3 Biological functions ................................................................................. 19
1.3 Processing enzymes ......................................................................................... 21
1.3.1 α-Secretase .............................................................................................. 21
1.3.1.1 Substrate selectivity of TACE and ADAM10 ............................... 23
1.3.2 β-Secretase .............................................................................................. 24
1.3.3 γ-Secretase............................................................................................... 25
1.4 Signaling pathways affecting APP family processing ................................. 26
1.4.1 IGF-1.......................................................................................................... 26
1.4.2 Retinoic acid ............................................................................................. 27
1.5 Regulation of APP family α-site processing ................................................. 28
1.5.1 Trafficking of enzyme and substrate.................................................... 28
1.5.2 Post-translational modifications of enzyme and substrate .............. 30
1.5.2.1 Post-translational modifications of ADAM10 and TACE............ 31
1.5.2.2 Post-translational modifications of the APP family ................... 32
2. Methodological considerations ..................................................... 34
2.1 Cell lines ............................................................................................................. 34
2.2 Cell treatments ................................................................................................. 35
2.2.1 Retinoic acid and IGF-1 .......................................................................... 35
2.2.2 Pharmacological inhibitors ..................................................................... 35
2.3 siRNA gene silencing ........................................................................................ 36
2.4 BCA assay .......................................................................................................... 36
2.5 Western blot ...................................................................................................... 37
2.6 ELISA .................................................................................................................. 37
2.7
32
P-labeling and immunoprecipitation .......................................................... 38
2.8 sWGA precipitation ........................................................................................... 39
2.9 Biotinylation assay ........................................................................................... 39
2.10 Design and cloning of the APP/APLP2 chimer ........................................... 39
3. Aims ....................................................................................................... 41
4. Results and discussion .................................................................... 42
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4.1 Stimulated α-secretase processing of APP is mediated by ADAM10 in a
PI3K-dependent manner (Paper I and II)........................................................... 42
4.2 Stimulated α-secretase processing of APLP2 is mediated by TACE in a
PKC-dependent manner (Paper I and II) ............................................................ 44
4.3 O-GlcNAcylation induces α-secretase processing of APP but not of APLP2
(Paper III and IV).................................................................................................... 45
4.3.1 O-GlcNAcylation selectively enhances α-secretase processing of
APP in neuron-like cells (Paper IV) ................................................................. 46
4.3.2 ADAM10 is O-GlcNAcylated (Paper IV) ................................................ 47
4.4 The difference in regulation of α-secretase processing between APP and
APLP2 is determined by their E1 domain (Paper III) ........................................ 48
4.5 What about APLP1? (Paper I and IV)........................................................... 50
5. Conclusions ......................................................................................... 51
6. Populärvetenskaplig sammanfattning på svenska ............... 53
7. Acknowledgements .......................................................................... 55
8. References ........................................................................................... 57
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Abbreviations
Aβ
AD
ADAM
AICD
ALID
ANOVA
APH-1
APLP
APP
BACE
BCA
BDNF
C83
C99
CNS
CRABs
CSF
CS GAG
CTF
CuBD
DAG
DTT
ECL
ELISA
ER
ERK
GFLD
HBD
HRP
IGF-1
IGF-1R
IP
IP3
IRS
KI
K/O
10
Amyloid-β
Alzheimer’s Disease
A disintegrin and metalloprotease
APP intracellular domain
APP-like intracellular domain
Analysis of variance
Anterior pharynx defective-1
APP-like protein
Amyloid-β precursor protein
β-site cleaving enzyme
Bicinchoninic acid
Brain-derived neurotrophic factor
APP C-terminal stub of 83 amino acids
APP C-terminal stub of 99 amino acids
Central nervous system
Cellular retinoic acid binding proteins
Cerebrospinal fluid
Chondroitin sulphate glycosaminoglycan
C-terminal fragment
Copper binding domain
Diacylglycerol
Dithiothreitol
Enhanced chemiluminescence
Enzyme-linked immunosorbent assay
Endoplasmatic reticulum
Extracellular signal-regulated kinase
Growth factor-like domain
Heparin binding domain
Horseradish peroxidase
Insulin-like growth factor
IGF-1 receptor
Immunoprecipitation
Inositol 1,4,5-triphosphate
Insulin receptor substrate
Knock-in
Knock-out
KPI
LRP
LTP
MAPK
NMDA
NMR
OGA
O-GlcNAc
OGT
PC
PEN2
PI3K
PIP2
PKC
PLC
PNS
PS
RA
RAR
RARE
RBP
RIP
RISC
RNA
RT-PCR
RXR
sAPP
SAP97
SDS
Shc
siRNA
SorLA
TGF
TGN
TIMPs
TM
TMB
TNF
Tspan
Wt
ZnBD
Kunitz protease inhibitor
Low-density lipoprotein receptor-related protein
Long term potentiation
Mitogen activated protein kinase
N-methyl D-aspartate
Nuclear magnetic resonance
O-GlcNAcase
O-linked β-N-acetylglucosamine
O-GlcNAc transferase
Pro-hormone convertase
Presenilin enhancer 2
Phosphatidyl inositol-3 kinase
Phosphatidylinositol 4,5-bisphosphate
Protein kinase C
Phospholipase C
Peripheral nervous system
Presenilin
Retinoic acid
Retinoic acid receptor
Retinoic acid responsive element
Retinol binding protein
Regulated intramembrane proteolysis
RNA-induced silencing complex
Ribonucleic acid
Reverse-transcriptase polymerase chain reaction
Retinoid X receptor
Secreted APP
Synapse associated protein 97
Sodium dodecyl sulphate
Src homology collagen
Short interfering RNA
Sorting-related receptor with A-type repeats
Transforming growth factor
Trans Golgi network
Tissue inhibitors of metalloproteases
Transmembrane
Tetramethylbenzidine
Tumor necrosis factor
Tetraspanin
Wild type
Zinc binding domain
11
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1. Introduction
Proteolytic processing of the amyloid-β precursor protein (APP) has been
extensively studied due to the link between the APP cleavage product, amyloid-β (Aβ) and Alzheimer’s disease (AD). However, this thesis is focused
on mechanisms involved in APP α-secretase processing, i.e., the processing
pathway that excludes Aβ formation. There are clear evidences that APP
together with its homolog APP-like protein-2 (APLP2) have important functions in the brain. In our studies we aimed to elucidate how α-secretase processing of APP differs from the processing of its homolog APP-like protein2 (APLP2). α-Secretases are also involved in regulated intramembrane proteolysis (RIP), an important novel form of signal transduction. In addition, it
has lately been shown that dysregulation of the α-secretase pathway may
cause AD, and that enhancing this cleavage of APP constitutes a potential
therapeutic strategy.
1.1 The role of APP in Alzheimer’s disease
APP was, as the name suggests, identified as being the precursor of the Aβ
peptide (Kang et al., 1987). Aβ is a secreted peptide generated through sequential cleavage of APP and its accumulation in the brain is today considered the main cause of the widespread neurodegeneration seen in AD
(Hardy, 2009, Hardy and Higgins, 1992), although other mechanisms contribute. The neurodegeneration in turn leads to clinical manifestations such
as problem with speech, disorientation and progressive loss of memory and
cognitive function (Querfurth and LaFerla, 2010).
Aβ is highly hydrophobic and aggregates into several different conformations, including low molecular weight species such as dimers and trimers
as well as oligomers and larger aggregates such as fibrils and plaques (reviewed in Finder and Glockshuber, 2007). The presence of plaques in AD
post-mortem brain was described already in 1907 by the German physician
Alois Alzheimer and was initially believed to be the inducer of cell death.
However, later studies have demonstrated that it rather is Aβ oligomers that
mediate the main neurotoxicity (Klyubin et al., 2005, Shankar et al., 2007,
Walsh et al., 2002). The exact mechanism of Aβ neurotoxicity is not yet
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established, but several studies indicate that Aβ may 1) bind to various cell
surface receptors causing aberrant signaling 2) interact with the membrane
and be inserted as pores causing calcium influx or 3) internalize and cause
many cellular dysfunctions through direct interactions, including mitochondrial dysfunction (reviewed in Kayed and Lasagna-Reeves, 2013). In addition to its direct harmful effects, Aβ may also induce hyperphosphorylation
of the microtubule-associated protein tau, causing it to aggregate intracellularly into neurotoxic tangles (Chabrier et al., 2012). Both Aβ and tau aggregates may cause chronic activation of microglia, the immune cells of the
brain. This can result in increased production of inflammatory agents in the
brain amplifying the degree of neurodegeneration. This line of events triggered by Aβ is called the amyloid cascade hypothesis (Hardy and Higgins,
1992). It is supported by the fact that several mutations in APP, and in an
enzyme component that cleaves APP to release Aβ, causes familial forms of
AD (reviewed in Duyckaerts et al., 2009).
1.2 The APP family
Soon after its discovery, APP was found to be evolutionary highly conserved. In addition, two mammalian genes encoding the homologous proteins, APP-like protein-1 and -2 (APLP1 and APLP2) were identified
(Wasco et al., 1992, Wasco et al., 1993). APP shares 42% amino acid sequence homology with APLP1 (64% similar) and 52% homology with
APLP2 (71% similar). However, only APP generates Aβ. Homologues in
other species also include the APP-like proteins APPL (Rosen et al., 1989)
and APL-1 (Daigle and Li, 1993) in fruit fly (Drosophila melanogaster) and
nematode (Caenorhabditis elegans), respectively. Here, the structure, function and the proteolytically processing of the mammalian APP family proteins will be discussed.
1.2.1 Structure
There are three major isoforms of mammalian APP; APP695, APP751 and
APP770. Alternative splicing of APLP2 also produces multiple protein
isoforms, the major ones being 763 and 707 amino acids long, while only
one form of APLP1 has been detected. The main difference between the
APP isoforms is the presence or absence of the Kunitz protease inhibitor
(KPI) domain and a chondroitin sulfate glycosaminoglycan (CS GAG) attachment site. This is also the case for the APLP2 isoforms. APLP1 consists
of 650 amino acids and lacks both the KPI domain and the CS GAG attachment site (Paliga et al., 1997, Wasco, et al., 1992). APP and APLP2 are
ubiquitously expressed. However, the different isoforms can be preferentially expressed in different cell types, such as the 695 amino acid long isoform
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of APP, which is mainly found in cells of neuronal origin. APLP1 expression has been reported to be restricted to the nervous system (Lorent et al.,
1995, Slunt et al., 1994).
All APP family members are type 1 integral membrane proteins, with a single membrane-spanning domain, a large ectoplasmic N-terminal region and
a shorter cytoplasmic C-terminal region (c.f., (Kang, et al., 1987, Dyrks et
al., 1988). The APP sequence can be divided into multiple distinct domains
(Fig. 1). The ectoplasmic region of APP, which constitutes the major part of
the protein, can be divided into the E1 and E2 domains (reviewed in Gralle
and Ferreira, 2007). The E1 domain can be further divided into a number of
subdomains, including a heparin-binding/growth-factor-like domain
(HBD/GFLD) and a copper binding domain (CuBD). The E1 domain is followed by an acidic region rich in aspartic acid and glutamic acid and a KPI
domain (not present in APP695). The cytoplasmic region of APP contains a
protein interaction motif, namely the YENPTY sequence (including the
NPXY internalization signal), which is conserved in all APP homologues.
The sequences of APLP1 and APLP2 can be divided into similar domain
structures as APP.
Figure 1. Schematic illustration of the domain structure of
APP. The structure can be divided into distinct domains,
including the ectoplasmic E1 and E2 domains. The E1
domain can be further divided into a heparinbinding/growth factor-like domain (HBD/GFLD) and a
cupper-binding domain (CuBD). The E1 domain is linked
to the E2 domain via an acidic region, and in some APP
isoforms also a Kunitz protease inhibitor domain (KPI).
APP also has a cytoplasmic/intracellular domain (ICD),
which contains the YENPTY protein interaction motif.
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There is no complete crystal structure of APP, but the structure of several
domains has been studied separately. The major part of the protein is as previously mentioned comprised of the ectoplasmic domain, which has several
independent folding structures. The most N-terminal part of APP, the
HBD/GFLD has been described to 1.8Å (Rossjohn et al., 1999), showing
that this part forms one α-helix and nine β-sheets that are mostly comprised
of very short strands of amino acids. Three disulfide bridges bring the domain into a compact, globular shape creating a positively charged surface
and a hydrophobic region. The basic surface was suggested to interact with
glycosaminoglycans and the hydrophobic surface to be important for ligand
binding. The disulfide bridge between Cys98 and Cys105 (in the APP695
isoform) creates a loop which contains several positively charged amino
acids. The corresponding loop section in APLP2 also contains positively
charged residues, whereas in APLP1 these mainly are uncharged.
The CuBD has been shown to contain one α-helix and three β-sheets using
nuclear magnetic resonance (NMR) (Barnham et al., 2003). The CuBD also
contains three disulfide bridges dividing the domain into one positive and
one negative region. There is also a small hydrophobic region within the
CuBD. Recently, the entire E1 structure was solved at 2.7Å resolution,
showing that the GFLD and CuBD interacted with each other forming one
functional unit stabilized by a salt bridge and several hydrogen bond networks (Dahms et al., 2010). The KPI domain, which is not present in all
APP isoforms, contains approximately 60 residues with six cysteines arranged in three disulfide bonds with little secondary structure. The E2 domain has been described to 2.0Å (Lee et al., 2011, Wang and Ha, 2004).
This part of the protein has the most complex secondary structure including
six α-helices, which form two coiled coil antiparallel substructures (e.g. parts
are folded over each other with the N-terminal of one helix aligned with the
C-terminal of the other helix). Recently, a metal binding site was discovered
within the E2 domain of APP containing 4 highly conserved histidines
(His314, His382, His432 and His436). The site was shown to bind copper
and zinc thereby inducing large conformational changes of the E2 domain
(Lee, et al., 2011).
Several studies indicate that the APP family proteins are able to form homoand heterodimers in both cis (within the same cell) and trans (between two
adjacent cells), thereby affecting their function and processing (Eggert et al.,
2009, Kaden et al., 2008, Soba et al., 2005). Three regions have been implicated in dimerization, namely the E1, the E2 and the transmembrane domains. Co-immunoprecipitation studies with overexpressed APP/APLPs
constructs where ectoplasmic domains were deleted showed that the E1 domain was the main interaction interface for dimerization (Soba, et al., 2005).
In another study, deletion of the E1 domain in APP or APLP2 was shown to
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diminish homo-dimerization completely whereas the deletion of the E1 domain in APLP1 only reduced its homo-dimerization by 50% (Kaden et al.,
2009). It has further been shown that heparin is required for APP E1 homodimerization and that this complex formation was initiated by about 10 sugar
rings in heparin that span the positively charged surface area made up by two
GFLDs bridging them together (Dahms, et al., 2010). Other studies indicate
that also the E2 domain can dimerize. X-ray spectroscopy revealed an antiparallel orientation of the two dimeric E2 domains (Wang and Ha, 2004).
The same mode of E2 dimerization has also been shown for APLP1 (Lee, et
al., 2011, Xue et al., 2011). As for E1 dimerization, E2 dimerization has also
been shown to be induced by heparin (Lee, et al., 2011). The transmembrane
(TM) domain was shown by NMR to be a flexible curved α-helix (Barrett et
al., 2012). A GxxxG motif is found within the TM domain of APP. This
motif is known to mediate TM helix-helix interactions within the membrane
(reviewed in Russ and Engelman, 2000), which has been shown to occur
also for APP (Munter et al., 2007). Notably, there is no GxxxG motif in
APLP-1 or -2.
1.2.2 Proteolytic processing
The proteolytic processing of APP can be divided into two different pathways, the amyloidogenic pathway, which leads to generation of Aβ, and the
non-amyloidogenic pathway (Fig. 2). Both pathways include several cleavage events. The amyloidogenic processing of APP is initiated through cleavage by β-secretase, which leads to secretion of the large N-terminal ectodomain, sAPPβ. The remaining C-terminal fragment (CTF) of 99 amino acids
(C99) can then be further processed by γ-secretase, generating Aβ and a
soluble APP intracellular domain (AICD). The γ-secretase processing of C99
occurs at multiple sites. APP is first cleaved at the ε-site at the Thr645Leu646 bond or at the Leu646-Val647 bond, generating soluble AICD50 or
AICD49, respectively (Takami et al., 2009, Weidemann et al., 2002, Yu et
al., 2001). Next, the remaining membrane-bound fragment is cleaved stepwise at every third residue from the ε-site towards the γ-site (Qi-Takahara et
al., 2005, Takami, et al., 2009), generating the Aβ49Aβ46Aβ43Aβ40
or Aβ48Aβ45Aβ42Aβ38 product lines.
In the non-amyloidogenic pathway, generation of Aβ is precluded since APP
is initially cleaved by α-secretase within the Aβ sequence near the ectoplasmic side of the plasma membrane (Esch et al., 1990, Sisodia et al., 1990).
The α-secretase cleavage of APP releases the N-terminal ectodomain, sAPPα
from the cell surface leaving an 83 amino acid long C-terminal membranebound fragment (C83). The remaining C83 fragment can be further processed by γ-secretase in a similar way as in the amyloidogenic pathway,
giving rise to the small peptide p3 and AICD.
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Figure 2. Schematic illustration of APP processing. In the non-amyloidogenic pathway, APP is initially cleaved by α-secretase, generating the secreted sAPPα fragment. In the amyloidogenic pathway APP is instead cleaved by β-secretase, generating sAPPβ. The remaining C-terminal membrane-bound stubs, C83 and C99, are
subsequently cleaved by γ-secretase, releasing the APP intracellular domain (AICD)
and p3 or Aβ. γ-Secretase cleaves APP first at the ε-site and then stepwise at every
third residue (indicated by arrowheads) towards the γ-site.
The amyloidogenic and the non-amyloidogenic pathways have been proposed to compete with each other, since enhanced α-secretase activity in
animal models of AD or in cultured cells can significantly lower Aβ generation (Nitsch et al., 1992, Postina et al., 2004). In addition to the two major
processing pathways, APP has been shown to undergo N-terminal processing, generating several soluble fragments varying in size between 12 to
30 kDa (Vella and Cappai, 2012). This processing was regulated by protein
kinase C (PKC) in an α- and β-secretase-independent manner. APP may also
be cleaved by caspases (6 or 8) in the C-terminal at Asp664, generating a
cytotoxic C31 fragment (Gervais et al., 1999, Lu et al., 2000).
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Although the amino acid sequences in APP where the secretases cleave are
very different in the two mammalian homologs, both APLP1 and APLP2 are
believed to be proteolytically processed in a similar way as APP. The first
indication that APLPs were cleaved by γ-secretase was the detection of elevated levels of an APLP1 membrane-bound C-terminal fragment in brain
tissues from animals deficient in the γ-secretase component, presenilin-1
(Naruse et al., 1998). Cleavage of APLPs by γ-secretase was further shown
to generate fragments corresponding to AICDs, denoted ALID1 and 2 (APPlike intracellular domain 1 and 2) (Gu et al., 2001). Furthermore, β-secretase
processing of the APLPs was demonstrated in a study by Li et al. where coexpression of the identified β-secretase BACE1 (β-site APP cleaving enzyme
1) with APLP-1 or -2 induced production of CTFs slightly larger in size than
those seen without BACE1 overexpression (Li and Südhof, 2004). βsecretase processing of APLP2 has also been demonstrated in vivo
(Pastorino et al., 2004), whereas this processing event of APLP1 has been
questioned (Eggert et al., 2004). Additionally, the production of secreted
fragments and CTFs in APLP1 or APLP2 transfected cells were decreased
by the metalloproteinase inhibitor batimastat (known to inhibit α-site processing of APP) (Eggert, et al., 2004), indicating α-secretase processing of
the two homologs. Recently, the α-, β- and γ-secretase cleavage sites inAPLP2 were determined by mass spectrometry (Hogl et al., 2011). The αsecretase cleavage site was mapped to the Arg670-Val671 bond (in the
APLP2 753 isoform), the β-cleavage was shown to occur at the Leu659Asp660 bond and the γ-cleavage between Ala694 and Val700. Notably, the
α-site in APP is at a distance of 12 amino acids from the membrane, whereas
the corresponding site in APLP2 is located 22 amino acids from the membrane. The exact cleavage sites in APLP1 have not yet been determined.
1.2.3 Biological functions
APP has mainly been studied in relation to AD, but several physiological
functions in both the adult and developing nervous system have been suggested, including roles in neuronal migration, cell adhesion, axonal transport,
dendritic outgrowth, synapse formation and learning and memory. Even
though Aβ is considered the main cause of AD pathogenesis, APP loss of
function might also play a role, though this is less investigated. The functions of APP may be mediated by the full-length protein as well as secreted
and intracellular fragments.
Full-length APP has been shown to participate in cell-adhesion through direct contacts with extracellular matrix molecules, such as laminin and heparin sulphate proteoglycans and also through trans-dimerization
(Narindrasorasak et al., 1992, Soba, et al., 2005). Several studies have also
suggested a role of APP, and especially sAPPα in neurite outgrowth
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(Milward et al., 1992, Perez et al., 1997, Small et al., 1994). The mechanism
was further elucidated in a study by Young-Pearce et al., showing that
sAPPα interfered with the interaction between APP and integrin β1, thereby
inducing integrin β1-mediated neurite elongation (Young-Pearse et al.,
2008). The same phenomenon was observed for APLP1 and APLP2 as well.
sAPPα has also been shown to have several neuroprotective properties. For
example, pre-treatment of sAPPα prevented neuronal death in human cortical cell cultures deprived of glucose or exposed to excitotoxins (Mattson et
al., 1993). Further, sAPPα has even been shown to protect against Aβ toxicity (Goodman and Mattson, 1994). Survival of rat hippocampal neurons was
reduced to 40% after a four-day exposure to Aβ, whereas 75% of the neurons survived the same exposure when co-treated with sAPPα.
APP knock-out (K/O) mice were generated in order to further study the
function of APP in vivo. These mice were viable and fertile, but with minor
defects including reduced grip strength, increased copper levels in the cerebral cortex and liver and impaired long term potentiation (LTP) in aged mice
paralleled by impaired performance in memory-related behavioral tasks
(Dawson et al., 1999, Muller et al., 1994, Seabrook et al., 1999, White et al.,
1999). Interestingly, these deficits in LTP in aged APP K/O mice could be
rescued by sAPPα knock-in (KI), indicating that sAPPα plays a pivotal role
in synaptic plasticity (Ring et al., 2007). About 60% of APP K/O mice die
within 48 h after induced cerebral ischemic injury and display reduced cerebral blood flow in response to hypoxia. The same response is seen in
BACE1 K/O mice, suggesting a role for APP as a stress response protein
mediated by the action of sAPPβ and/or Aβ (Koike et al., 2012).
APLP1 K/O mice were viable and fertile but with reduced body weight, and
APLP2 K/O mice had no apparent phenotype neither in young nor in aged
mice (Heber et al., 2000, von Koch et al., 1997). However, triple K/O leads
to perinatal lethality with a 100% penetrance, clearly demonstrating essential
functions for the APP family members during development and also illustrating a functional redundancy amongst the three homologs (Herms et al.,
2004). Furthermore, APP/APLP1 K/O mice were viable and fertile without
additional defects compared to single disruptions, whereas APP/APLP2 K/O
as well as APLP1/APLP2 K/O mice died within 24 h after birth (Heber, et
al., 2000), probably due to pre- and postsynaptic defects at the neuromuscular junction and at central synapses and widening of the endplate (Wang et
al., 2005, Wang et al., 2009). These studies indicate that APLP2 has a key
physiological role amongst the APP family, being the only member capable
of compensating for simultaneous loss of both other family members.
Interestingly, sAPPα-KI, rescued the perinatal lethality of APP/APLP2 (DM)
deletion in about 50% of the animals, however, the surviving sAPPα-DM
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mice displayed a complex phenotype with severe deficits both in synaptic
transmission in the adult PNS and CNS and in learning and memory (Weyer
et al., 2011). This indicates that although sAPPα is essential for development, other APP or APLP2 derived fragments and/or full-length APP or
APLP2 have important functions in the adult nervous system. In contrast to
sAPPα, sAPPβ-KI was not able to rescue the perinatal lethality of
APP/APLP2 K/O in mice (Li et al., 2010). In addition, sAPPβ has been
shown to be further processed and thereby inducing apoptosis through interaction with the death receptor 6 (Nikolaev et al., 2009).
As discussed previously, APP is sequentially processed. This type of proteolytic processing is also called regulated intramembrane proteolysis (RIP).
Several other transmembrane proteins, including Notch, a key player in developmental processes, are cleaved in the same way, generating a soluble
intracellular domain (ICD) which can translocate to the nucleus and induce
gene transcription (reviewed in Lichtenthaler et al., 2011). AICD has also
been shown to regulate gene transcription, though this effect is dependent on
its interaction with the adaptor protein Fe65 and the histone acetyltransferase
Tip60 (Cao and Südhof, 2001). AICD has been shown to regulate transcription of neprilysin, p53, APP, Fe65 and Tip60 (reviewed in Müller et al.,
2008). Furthermore, AICD overexpression has also been shown to induce
neuron-specific apoptosis (Kinoshita et al., 2002, Nakayama et al., 2008).
Recently, several studies have shown that a functional AICD is only generated from the β-pathway (Belyaev et al., 2010, Flammang et al., 2012), since
α-pathway generated AICD is quickly degraded in the cytosol.
1.3 Processing enzymes
The identity of the APP processing enzymes was initially unknown and they
were therefore referred to as α-, β-, and γ-secretases. Today, much more is
known about these enzymes, which like the APP family have been shown to
be highly conserved. The focus of most research has been on γ-secretase in
the hope to develop an inhibitor of Aβ generation. In this thesis, the focus is
on regulation of α-secretase processing, and therefore a more detailed description of α-secretase will be presented as compared to β-, and γ-secretase.
1.3.1 α-Secretase
α-Secretase cleavage of APP was early determined to occur at the cell surface, suggesting that α-secretase was a plasma membrane-bound protease
(Sisodia, 1992). It was soon characterized as a zinc metalloproteases that
cleaved APP at the Lys613-Leu614 bond (Roberts et al., 1994). Several
21
members of the ADAM (a disintegrin and metalloprotease) family were suggested as α-secretase candidates, but today the consensus is that ADAM10
and ADAM17, also known as TACE (tumor necrosis factor-α (TNFα) converting enzyme) cleaves APP at the α-site. It should be noted that ADAM9
was previously considered to have α-secretase activity (Koike et al., 1999),
though it has since then been shown that ADAM9 regulates APP processing
through modulation of ADAM10 activity (Cisse et al., 2005, Moss et al.,
2011).
ADAMs are type 1 integral membrane glycoproteins with a multi-domain
structure, including signal peptide, pro-domain, catalytic metalloprotease
domain, disintegrin/cysteine-rich domain, transmembrane domain and a
short cytoplasmic domain (Howard et al., 1996). The pro-domain is cleaved
off by a cysteine switch mechanism in order to activate the protease. A major function of ADAM proteases is the ectoplasmic cleavage of cell membrane proteins, also called ectodomain shedding. Many functionally diverse
proteins, such as cytokines, growth factors and their receptors, and celladhesion proteins are initially synthesized as membrane-anchored proteins,
which are released upon proteolysis mediated by ADAMs (reviewed in
Edwards et al., 2008).
TACE was identified as an α-secretase candidate since it was shown that it
could cleave an α-site spanning peptide (Buxbaum et al., 1998). Several
other studies has suggested a role for TACE in α-site cleavage of APP based
on the fact that overexpression of TACE or that certain stimuli induces
sAPPα secretion in a TACE-dependent manner (Buxbaum, et al., 1998, Merlos-Suarez et al., 2001, Slack et al., 2001). The relevance of TACE overexpression is discussed further in section 1.3.1.1. Interestingly, no study has
shown that TACE cleaves endogenous APP in response to a physiological
stimulus. Therefore, the physiological importance of TACE processing of
APP during endogenous conditions still remains to be investigated. Lately,
ADAM10 was identified as the constitutive α-secretase for APP since
knock-down of ADAM10, but not TACE completely blocked sAPPα generation in primary murine neurons (Kuhn et al., 2010), which was in agreement
with our previous findings (see section 4.1).
Overexpression of either ADAM10 or TACE has been shown to increase the
secretion of sAPLP2 in cultured cells (Endres et al., 2005). Lately, ADAM10
was shown to be the constitutive α-secretase for APLP2 in primary murine
neurons, as previously shown for APP (Hogl, et al., 2011, Kuhn, et al.,
2010). However, due to the redundant nature of ADAM10 and TACE (discussed below), these studies do not exclude the possibility that APP and
APLP2 may be cleaved by TACE in response to certain stimuli.
22
1.3.1.1 Substrate selectivity of TACE and ADAM10
TACE and ADAM10 have a higher sequence homology to each other (39%
identity), than to any other mammalian ADAMs (Black et al., 1997). Like all
members of the ADAM family, they both have broad substrate specificity
that seems to somewhat overlap. However, some substrates are considered to
be preferentially cleaved by ADAM10 or TACE, e.g., TGFα (transforming
growth factor α) is cleaved by TACE (Peschon et al., 1998) and E-cadherin
by ADAM10 (Maretzky et al., 2005). The partial overlap of substrates
makes it difficult to understand the factors important for their selectivity.
The amino acid sequence of the cleavage site is usually not an important
factor for substrate recognition (Deuss et al., 2008, Tsakadze et al., 2006).
Instead, it seems like the membrane proximity and the length of the stalk
(i.e., the susceptible membrane-proximal region between the ectoplasmic
globular domain and the transmembrane domain) is more important. The
substrate recognition of TACE and ADAM10 was further studied by Caescu
and colleagues (Caescu et al., 2009). They demonstrated by peptide library
screening and analysis of individual substrates that TACE and ADAM10
have distinct amino acid preferences at multiple positions surrounding the
substrate cleavage site. Furthermore, they performed mutation studies of the
active sites of the enzymes and found that certain amino acids in the active
site could partially explain why some substrates are selective for either
ADAM10 or TACE. Stawikowska et al. have recently shown that the secondary structure of the substrate regulates its interaction with non-catalytic
domains of ADAM10 and TACE, thereby differently modifying the enzymes’ activity. For example, inducing α-helical conformation of a TNFα
based sequence dramatically increased TACE activity, whereas the effect on
ADAM10 activity was not as significant (Stawikowska et al., 2013).
Interestingly, even though certain substrates are thought to be selective for
either ADAM10 or TACE, Le Gall and colleagues demonstrated that their
substrate selectivity can be manipulated (Le Gall et al., 2009). The group had
previously demonstrated that TACE was required for constitutive and phorbol ester-induced TGFα shedding (Peschon, et al., 1998), and were surprised
to find shedding activity in TACE-/- cells upon ionomycin (a Ca2+ ionophore)
stimulation. Acute treatment of wild type (wt) cells with a selective TACE
inhibitor showed that TACE was the main sheddase for TNFα and TGFα.
However, upon chronic inhibition of TACE, ADAM10 rescued the shedding
of these substrates. This indicates that many studies in which APP and/or
ADAM10/TACE have been overexpressed or knocked-out may not be of
physiological significance since ADAM10 and TACE clearly can be triggered to cleave substrates that they under physiological conditions do not
cleave. As further discussed in section 4.1 the latent promiscuity of the enzymes makes it difficult to get a complete understanding of APP α-secretase
23
processing. Clearly, there is a sensitive balance in the selectivity between
ADAM10 and TACE. In agreement with LeGall et al. another study showed
that even though TACE was the major sheddase for TNFα in most cell types
investigated (Hikita et al., 2009), ADAM10 was shown to be the major
sheddase in some cell types (e.g. the mouse embryo fibroblast NIH3T3 cell
line). The significance of ADAM10 and TACE as sheddases for TNFα could
be correlated to the expression levels, but also to the abundance of different
TIMPs (tissue inhibitors of metalloproteases) which specifically inhibit either ADAM10 or TACE.
In some cases the overlapping substrate specificity may reflect a selective
use of ADAM10 and TACE as sheddases, depending on the stimulus. Both
TACE and ADAM10 have been shown to cleave Notch1. However, in a
study by Bozulak et al., the role of the two proteases was further investigated. It was found that ADAM10 was required for Notch1 signaling induced
by ligand binding (delta-like-1), whereas ligand-independent signaling required TACE (Bozkulak and Weinmaster, 2009). TACE was not able to
rescue ADAM10 processing of Notch1 in ADAM10-/- mouse embryonic
fibroblasts.
1.3.2 β-Secretase
β-Secretase is an aspartyl protease that cleaves APP at the Met597-Asp598
bond, which constitutes the first and rate-limiting step towards Aβ production. Two β-secretases have been identified; BACE1 (β-site APP-cleaving
enzyme) and BACE2 (Hussain et al., 1999, Sinha et al., 1999, Vassar et al.,
1999, Yan et al., 1999). BACE1 is more widely expressed in the brain and
BACE1 K/O animals do not produce any detectable levels of Aβ (Luo et al.,
2001, Roberds et al., 2001). BACE1 is a single domain integral protein with
its active site located on the ectoplasmic side of the membrane (Hussain, et
al., 1999, Vassar, et al., 1999). It has an N-terminal prodomain that is
cleaved by a furin-like protease or through autoproteolysis generating a mature enzyme. The optimal pH of BACE1 activity is approximately 4.5, indicating that the -site cleavage of APP takes place in more acidic cellular
compartments, such as the endosomes (Vassar, et al., 1999). BACE1 cleavage has also been suggested to occur in lipid rafts (Ehehalt et al., 2003), but
this has been challenged since inhibition of BACE1 palmitoylation, a prerequisite for raft localization has no effect on Aβ production (Vetrivel et al.,
2009). A recent study by Das et al. demonstrated that BACE1 was mainly
localized to recycling endosomes in cultured hippocampal neurons expressing low levels of fluorescent-labeled BACE1 and APP. The study further
showed that increased neuronal activity induced clathrin-dependent endocytosis of APP, in turn leading to co-localization of APP and BACE1 in recycling endosomes and subsequent β-site cleavage (Das et al., 2013). BACE1
24
was initially thought to be rather specific for the APP family, but today it is
known to cleave many substrates. Importantly, it regulates the function of
neuregulin-1, which is involved in myelination of Schwann cells in the peripheral nervous system (Willem et al., 2006).
1.3.3 γ-Secretase
γ-Secretase is an aspartyl protease that cleaves APP stepwise within the
transmembrane domain. The enzyme is a protein complex that consists of
anterior pharynx defective 1 (APH-1), nicastrin, presenilin-1 or -2 (PS1 or
PS2), and presenilin enhancer-2 (PEN-2) (Edbauer et al., 2003, Francis et al.,
2002, Goutte et al., 2000, Yu et al., 2000). Although other proteins are
known to interact with the complex, these four components are sufficient for
its activity. PS1 is a 9 TM protein and has been shown to harbor the active
site of the enzyme (Yu et al., 1998). Two highly conserved aspartate residues
(Asp257 and Asp385 in human PS1) within TM6 and TM7 constitute the
core of the catalytic site (Wolfe et al., 1999). Both presenilins undergoes
endoproteolysis within the cytosolic loop between TM6 and TM7, generating a C- and an N-terminal fragment. The two fragments form a stable heterodimer, which constitutes the active conformation of presenilin. Interestingly, most mutations causing familial AD are found in the PS1 gene.
Nicastrin has one TM and a large ectodomain, proposed to function as a
gatekeeper to the PS active site (Shah et al., 2005). Nicastrin also participates in binding of the substrate and is important for the assembly process of
the γ-secretase complex. The functions of APH-1 and PEN-2 are not yet
fully understood. However, PEN-2 has been shown to stabilize the final
complex and APH1 plays an important part during assembly of the complex
and has further been implicated in substrate recognition (Chen et al., 2010,
Wolfe, 2006). There are two APH-1 homologs that each can undergo alternative splicing generating either a short or a long isoform. The presence of
different isoforms in the γ-secretase complex seems to affect its substrate
specificity (reviewed in De Strooper et al., 2012). Mature, proteolytically
active γ-secretase is mainly localized at the plasma membrane and in the
endosomal/lysosomal system (Dries and Yu, 2008). The subcellular localization of the enzyme complex has been shown to affect the ε-cleavage of APP,
generating longer AICDs at the plasma membrane than in endosomes
(Fukumori et al., 2006). In addition to APP, γ-secretase has more than 90
identified substrates, including Notch, N-cadherin and ephrin B, and the list
is continuously growing. Most of the substrates are type I transmembrane
proteins that initially is cleaved in the ectodomain by another protease (reviewed in McCarthy et al., 2009).
25
1.4 Signaling pathways affecting APP family processing
Direct activation of PKC by phorbol ester stimulation was one of the earliest
α-secretase activating stimuli identified (Buxbaum et al., 1993, Caporaso et
al., 1992). Like APP, APLP1 and APLP2 processing have been demonstrated to be induced by phorbol esters (Eggert, et al., 2004, Xu et al., 2001).
Since then, processing of APP by the amyloidogenic and the nonamyloidogenic pathway has been shown to be differentially modified by
activation of certain cell surface receptors such as the 5-hydroxytryptamin
(5-HT4) receptor, metabotropic glutamate receptors, muscarinic acetylcholine receptors, synaptic NMDA receptors and many growth factor receptors.
Activation of these receptors induces downstream signaling events, including activation of several kinases, small GTPases, cAMP production and regulation of cytosolic calcium levels (reviewed in Allinson et al., 2003). In our
studies we have mainly used the neurotrophic factors retinoic acid (RA) and
insulin-like growth factor-1 (IGF-1) to induce α-secretase processing. Therefore, a more detailed description of IGF-1 and RA signaling is presented
below. It should be noted that both RA and IGF-1 are physiological relevant
stimuli that in addition are decreased in AD brains (Corcoran et al., 2004,
Steen et al., 2005). Furthermore, previous studies in our group have shown
that RA and IGF-1 stimulates α-secretase processing of all three APP family
proteins. The mechanisms and signaling involved during this stimulated αsecretase processing will be discussed in section 4.
1.4.1 IGF-1
Insulin-like growth factor-1 (IGF-1) is a hormone that regulates cell survival
and differentiation. In the brain, IGF-1 plays important roles in neuroprotection, regulation of energy and has also been implicated in modulation of LTP
(reviewed in Carro and Torres-Aleman, 2004). IGF-1 mainly signals through
the IGF-1 receptor (IGF-1R), which belongs to the receptor tyrosine kinase
family. However, since IGF-1 is structurally related to insulin, it can also
bind to the insulin receptor; although with 100-fold lower affinity than to
IGF-1R (Navarro et al., 1999). The IGF-1R has a tetrameric structure composed of two extracellular subunits and two transmembrane subunits covalently linked by disulfide bridges. Upon IGF-1 binding, the receptor undergoes conformational changes causing autophosphorylation of cytoplasmic
tyrosine residues. This in turn leads to recruitment of the adaptor proteins
insulin receptor substrate (IRS) and Shc (Src homology/collagen), which
then become phosphorylated enabling them to activate the mitogen activated
kinase (MAPK) pathway and the phosphatidyl inositol-3 kinase (PI3K)/Akt
pathway. Some studies also indicate that the IGF-1R can signal through
phospholipase C-γ (PLCγ) (Chattopadhyay and Carpenter, 2002), which
hydrolyses phosphatidyl-inositol 4,5-bisphosphate (PIP2) to produce inositol
26
1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 promotes release of
calcium from the endoplasmatic reticulum (ER) and DAG activates PKC.
Impaired insulin and IGF-1 signaling has been suggested to be a part of AD
pathology, since AD patients have been shown to have lower insulin and
IGF-1 levels in the cerebrospinal fluid (CSF) and higher plasma insulin levels than non-AD individuals (Steen, et al., 2005). Furthermore, increased
serum IGF-1 levels, through chronic subcutaneous infusion for ten weeks
have been demonstrated to decrease Aβ levels in the brain of aging rats
(Carro et al., 2002), and in AD transgenic mice (Carro et al., 2006). In addition, IGF-1 has been shown to induce a shift in the processing of APP, increasing the levels of sAPPα (Adlerz et al., 2007), which will be discussed
further in the results section.
1.4.2 Retinoic acid
Retinoic acid (RA) is a vitamin A derivate and a physiological signaling
molecule that is involved in neuronal differentiation, axon outgrowth and
neuronal plasticity (reviewed in Maden, 2007). Retinol is taken up through
the diet and is transported into the cells by retinol binding proteins (RBPs),
which interact with membrane receptors. In the cytoplasm, retinol is metabolized to RA, which can leave the cell and signal in an autocrine or paracrine
fashion. RA exerts its effect by translocating to the nucleus, assisted by cellular RA-binding proteins (CRABs). RA then binds to the RA receptor
(RAR) or the retinoic X receptor (RXR), which both are ligand-activated
nuclear receptors. RAR and RXR function as heterodimers, and bind to RA
response elements (RAREs) in the target genes. Binding of RA to the receptor complex induces gene transcription of the target genes, e.g., various transcription factors, cell signaling molecules, structural proteins etc. RA in
complex with RAR can also signal through rapid non-transcriptional pathways, through direct interaction and subsequent activation of PKC (Hoyos et
al., 2000), PI3K and MAPK (Masia et al., 2007).
Interestingly, a connection between RA signaling and AD has been observed, since vitamin A deprived mice displayed Aβ accumulation and RAR
down-regulation (Corcoran, et al., 2004). Furthermore, RA has been shown
to regulate the expression of APP, ADAM10, TACE and BACE1 (see section 4.2), as well as to induce a shift in the APP processing toward the αsecretase pathway (Adlerz et al., 2003, Holback et al., 2007, Holback et al.,
2005).
27
1.5 Regulation of APP family α-site processing
As mentioned above, activation through signaling pathways can alter processing of APP. However, the mechanism behind this is not well characterized. One important determinant is co-localization of enzyme and substrate
in subcellular compartments. Moreover, post-translational modifications,
such as phosphorylation or O-GlcNAcylation of substrate, enzyme or interacting protein could affect localization, enzyme activity or substrate conformation resulting in effects on α-secretase processing.
1.5.1 Trafficking of enzyme and substrate
Since α- and β-secretase processing of APP is spatially segregated; ADAMs
are mainly active at the cell surface whereas BACE1 is active in endosomes,
it is clear that APP trafficking plays a pivotal role in regulating its processing. APP is synthesized in the ER and is then targeted into the secretory
pathway and transported to the cell surface. During the transit through the
Golgi, APP is glycosylated and sulfated. Only 10% of APP is located at the
plasma membrane, based on overexpression of APP in cultured cells, whereas the majority of APP is localized to the Golgi and trans-Golgi network
(TGN). In neurons, APP is rapidly transported to axons and dendrites in
post-Golgi transport vesicles mediated by kinesin-1 (Kins et al., 2006).
APP is not cleaved at the cell surface is rapidly internalized, due to the presence of the YENTPY internalization motif in the C-terminal (Lai et al.,
1995), followed by delivery to endosomes. Blockage of APP endocytosis
through deletion of the internalization motif or by overexpression of a dominant-negative dynamin mutant impairs Aβ production (Carey et al., 2005,
Chyung and Selkoe, 2003). A fraction of endocytosed APP is then recycled
to the cell surface and some is degraded in lysosomes (Haass et al., 1992).
APP may also be shuttled (in both directions) between the TGN and lysosomes. APLP2 has been shown to undergo similar trafficking, whereas
APLP1 is present at the cell surface to a much higher degree (Kaden, et al.,
2009). Although the main site for APP α-secretase cleavage is the cell surface, some studies also implicate the TGN as a site for this cleavage event
(Skovronsky et al., 2000).
There are many proteins known to regulate the trafficking of APP. The
YENPTY motif in APP serves as a binding site for several cytosolic proteins, including Fe65, Fe65-like proteins-1 and -2, Mint/X11 1-3, Dab1 and
Shc (reviewed in Jacobsen and Iverfeldt, 2009). Fe65 and Mint/Xll are the
most well studied APP adaptor proteins and have been shown to interact
with both APLPs. Overexpression of Fe65 or X11 in AD transgenic mice
leads to decreased Aβ generation, possibly through regulation of APP traf28
ficking to the cell surface (Ando et al., 2001, Borg et al., 1998, Lee et al.,
2003a, McLoughlin and Miller, 2008). In addition to APP and APLPs, Fe65
can also interact with the endocytotic receptor LRP1 (low-density lipoprotein (LDL) receptor-related protein 1), forming a trimeric complex with APP
(Pietrzik et al., 2004) and thereby accelerating internalization of APP (Cam
et al., 2005). APP trafficking has also been shown to be regulated by the
neuronal trafficking receptor SorLA (sorting-related receptor with A-type
repeats) (Andersen et al., 2005). SorLA has been suggested to interact with
the N-terminal of APP in early endosomes, where retromer, an adaptor complex in the endosome-to-Golgi retrieval pathway, induces retrograde sorting
of the SorLA-APP complex to the TGN (Fjorback et al., 2012). This results
in decreased levels of APP at the plasma membrane and subsequently less αand β-secretase cleavage. Another receptor in the same family (the Vps10domain receptor family), namely Sortilin has also been shown to affect APP
processing (Gustafsen et al., 2013). Sortilin interacts with the E2 domain of
APP in neurites of hippocampal neurons and promotes α-secretase processing of APP without affecting subcellular localization of APP.
There is limited information concerning how TACE and ADAM10 trafficking is regulated and how it affects APP processing. Similar to APP, both
TACE and ADAM10 are synthesized in the ER and are then targeted into the
secretory pathway and transported to the cell surface. Immunohistochemical
studies suggest that most active TACE is localized in the cellular perinuclear
region, with a small amount present at the plasma membrane (Schlondorff et
al., 2000). However, as it was pointed out by a group who analyzed TACE
trafficking, overexpressed TACE is processed differently than its endogenous counterpart, which should be considered when analyzing trafficking of
overexpressed TACE (Borroto et al., 2003). TACE trafficking has been reported to be regulated by iRhom2, a proteolytically inactive member of the
rhomboid family through direct interaction with TACE, thereby promoting
exit from the ER. It was further shown by siRNA down-regulation of iRhom2, that the protein was essential for lipopolysaccharide (LPS)-induced
TNFα shedding (Adrain et al., 2012). TACE has further been shown to interact with several binding partners, but nothing is known about how they affect TACE trafficking (reviewed in Gooz, 2010). TACE trafficking is affected by phorbol ester stimulation, which will be further discussed in section
1.5.2.
ADAM10 trafficking was shown in a study by Dornier and colleagues to be
regulated by tetraspanins of the TspanC8 subfamily (Dornier et al., 2012).
Tetraspanins are integral membrane proteins implicated in several cellular
processes, including membrane compartmentalization (Berditchevski and
Odintsova, 2007). Confocal microscopy analysis demonstrated that TspanC8
expression led to redistribution of ADAM10 from the ER to the cell surface
29
in HeLa cells, which have low TspanC8 endogenous expression and also low
endogenous cell surface ADAM10 levels. ADAM10 retention in the ER had
previously been shown to be dependent on a mechanism involving an arginine-based motif in the C-terminal of ADAM10 (Marcello et al., 2010), that
probably is masked by complex formation with tetraspanins, thereby allowing ER exit. Similar to APP, ADAM10 endocytosis has been shown to be
inhibited by expression of a dominant negative form of dynamin, resulting in
increased cell surface levels of both mature and immature ADAM10 and
subsequent increase of sAPPα (Carey et al., 2011). Neither phorbol esters
nor muscarinic receptor M3 activation, known to increase sAPPα secretion
was shown to affect ADAM10 cell surface localization. SAP97 (synapse
associated protein 97), which traffics proteins to the excitatory synapse has
been shown to interact with ADAM10 (Marcello et al., 2007). NMDA receptor activation increased the interaction between SAP97 and ADAM10, leading to ADAM10 trafficking from dendritic shaft to spine-like structures. In
vivo experiments have further demonstrated that disruption of the interaction
between ADAM10 and SAP97 in adult mice, leads to decreased sAPPα secretion and increased Aβ production as well as tau hyperphosphorylation
after only two weeks (Epis et al., 2010). Furthermore, ADAM10/SAP97
interaction was shown to be reduced in the brain of AD patients.
1.5.2 Post-translational modifications of enzyme and substrate
The post-translational modification we have focused on in our studies is OGlcNAcylation. The reason for our interest in O-GlcNAcylation is firstly
that already in 1995 APP was reported to be O-GlcNAc modified (Griffith et
al., 1995) and secondly that during recent years O-GlcNAcylation has
emerged as an important type of post-translational modification playing roles
in synaptic function and plasticity (Hart et al., 2011, Tallent et al., 2009). OGlcNAcylation has also been directly linked to AD since the level of protein
O-GlcNAcylation has been shown to be decreased by approximately 20% in
AD brains (Liu et al., 2004b). Furthermore, it has been demonstrated that
decreased O-GlcNAcylation of tau is correlated to tau hyperphosphorylation
and thereby increased formation of neurofibrillary tangles.
O-GlcNAcylation is the attachment of the monosaccharide β-Nacetylglucosamine (GlcNAc) to serine and threonine residues of target proteins. The modification, unlike other glycosylations is dynamic and can cycle rapidly on and off the target protein similar to phosphorylation. In contrast to phosphorylation, which is regulated by several different enzymes, OGlcNAcylation is regulated by only two enzymes, O-GlcNAc transferase
(OGT) and O-GlcNAcase (OGA), which catalyzes the attachment and removal of O-GlcNAc, respectively (Dong and Hart, 1994, Haltiwanger et al.,
1992). OGT utilizes UDP-GlcNAc produced in the hexosamine biosynthetic
30
pathway. OGA K/O in mice results in developmental delay (Yang et al.,
2012), and OGT K/O is lethal (Shafi et al., 2000). The levels of O-GlcNAc
seem to be regulated by the expression of OGT and OGA, the concentration
of UDP-GlcNAc and availability of protein substrates (Groves et al., 2013).
The cycling of O-GlcNAc is influenced by both intracellular and extracellular stimuli, including insulin, nutrient levels, cellular stress, cell cycle changes and development. NMR studies have shown that O-GlcNAcylation of
proteins may directly influence the conformation of the target protein
(Simanek et al., 1998), which may affect interactions with other proteins. OGlcNAcylation has also been shown to affect cellular localization. In this
thesis, we have investigated O-GlcNAcylation in relation to effects on αsecretase processing of the APP family.
In addition to O-GlcNAcylation, other post-translational modification of
both substrate (the members of the APP family) and the enzyme (ADAM10
and/or TACE) might be important in regulating α-site processing, possibly
through the effect on enzyme activity or interactions with other proteins
thereby inducing changes in subcellular localization. Here, some of these
modifications, mostly glycosylation and phosphorylation are discussed in
relation to APP family processing.
1.5.2.1 Post-translational modifications of ADAM10 and TACE
The most important post-translational modification of ADAM10 and TACE
is the pro-domain removal, which occurs mainly in the TGN. Furin, as well
as several other pro-hormone convertases (PCs) catalyzes the pro-domain
removal of ADAMs. PC7 has been specifically implicated in maturation of
ADAM10, whereas PC1-6 seems to be important for TACE (Anders et al.,
2001, Srour et al., 2003). In addition to inhibiting ADAM activity during
biosynthesis, the pro-domain has also been suggested to possess chaperone
functions to ensure correct folding of the enzyme (Anders, et al., 2001). Interestingly, mutations in the pro-domain (Glu170His and Arg181Gly) have
been shown to impair this chaperone function of the pro-domain, without
affecting ADAM10 trafficking, thereby decreasing sAPPα production and
increasing Aβ formation (Suh et al., 2013). These mutations have been
linked to late onset AD.
TACE has been shown to be phosphorylated, and stimulation with phorbol
esters, which enhances the shedding activity of TACE, was reported by
Soond et al. to increase phosphorylation of pro-TACE at Thr735 in the intracellular domain (Soond et al., 2005). The phosphorylation was extracellular
signal-regulated kinase (ERK)-dependent, and increased both maturation of
TACE and its translocation from ER to the cell surface. TACE has further
been shown to co-localize to the plasma membrane with one of its substrates,
31
CD44, within 10 min after phorbol ester stimulation (Nagano et al., 2004).
These studies suggest that phosphorylation of TACE affects the subcellular
localization, leading to increased substrate shedding. In a study by Alfa Cissé and colleagues it was demonstrated that mutations at Thr735 abolished the
carbachol (an acetylcholine receptor agonist)-induced increase of TACE
phosphorylation as well as the induced processing of the cellular prion protein (Alfa Cisse et al., 2007). This study demonstrates a clear and direct correlation between TACE phosphorylation and activity induced by a physiological stimulus. However, in one study the cytoplasmic domain of TACE
was shown not to be required for phorbol ester stimulated shedding of TNFα
(Reddy et al., 2000), which have made the significance of TACE phosphorylation a debated area. TACE has also been shown to be N-glycosylated
(Peiretti et al., 2003), but there are no data how this affects its activity or
trafficking.
ADAM10 is N-glycosylated at four sites, three located in the metalloprotease domain and one in the disintegrin domain. Mutation of the N278 site in
the metalloprotease domain of ADAM10 resulted in accumulation in ER as
the unprocessed immature pro-form. Mutations of the three other Nglycosylation sites (N257, N439 and N551) all lead to cell surface localization similar to wt ADAM10. However, the mutated constructs showed
strongly reduced ability to cleave the natural substrate L1 cell adhesion molecule, as compared to wt ADAM10. The authors speculate that the lack of
glycosylation does not directly interfere with substrate binding, but instead
affects the conformation of the protease, causing a decreased activity
(Escrevente et al., 2008).
1.5.2.2 Post-translational modifications of the APP family
In addition to proteolytic processing, APP is known to undergo several different kinds of post-translational modifications, including sulfation, sumoylation, siaylation and addition of CS GAG (chondroitin sulphate glycosaminoglycan). The two major modifications of APP, glycosylation and phosphorylation, and their relation to APP processing will be discussed here.
There are two consensus sites for N-glycosylation in APP, N467 and N496,
but only N467, which also is highly conserved within the family, has been
shown to be modified by complex glycans (Pahlsson et al., 1992). Three Oglycosylation sites have been identified by mass spectrometry, namely
Thr291, Thr292 and Thr576 (Perdivara et al., 2009). There are contradictory
results concerning whether or not O-glycosylation is required for ectodomain
shedding of APP (Pahlsson and Spitalnik, 1996, Tomita et al., 1998). Several
reports show that inhibition of N-glycosylation diminishes sAPP secretion
(McFarlane et al., 1999, Pahlsson and Spitalnik, 1996). However, when mu32
tating the N-glycosylation sites in APP, secreted fragments were still detected in the cell medium of transfected cells, indicating that N-glycosylation of
APP is not essential for secretion. The rate of the secretion was however
reduced (Pahlsson and Spitalnik, 1996, Yazaki et al., 1996). Instead, Nglycosylation of another, yet unidentified protein seems to be necessary for
APP shedding. Another study by Eggert et al. show that only APLP1, and
not APP or APLP2 processing is affected by tunicamycin (N-glycosylation
inhibitor), leading to production of α-, and γ-generated fragments that were
not seen in absence of tunicamycin (Eggert, et al., 2004).
APP has several sites in its intracellular domain which can be phosphorylated in vitro (Gandy et al., 1988, Knops et al., 1993, Suzuki et al., 1994),
and many of these sites have also been found to be phosphorylated in postmortem brain tissue of patients with AD (Lee et al., 2003b). Several studies
have shown that Thr668 phosphorylation affects APP processing, mostly
indicating increased Aβ production, but there are contradictory reports (reviewed in Jacobsen and Iverfeldt, 2009). It was later shown that the peptidylprolyl cis/trans isomerase Pin1 binds to Thr668 phosphorylated APP both in
vitro and in vivo, thereby promoting large conformational changes in the Cterminal by accelerating the production of the trans conformation of Pro669
(Pastorino et al., 2006). It was further demonstrated that overexpression or
knockout of Pin1, reduced or increased the secretion of Aβ, respectively.
Pin1 K/O mice were further shown to display age-dependent neuropathy
(Liou et al., 2003). In post-mitotic neurons both cyclin-dependent kinase 5
(cdk5) and glycogen synthase kinase-3β (GSK-3β) have been implicated to
phosphorylate APP at Thr668 (Aplin et al., 1996, Iijima et al., 2000). Other
phosphorylation sites besides Thr668 have also been implicated in the regulation of APP processing, e.g., Tyr687, since overexpression of the mutant
Tyr687Ala decreased the level of the C83 fragment (Takahashi et al., 2008).
The mechanism of altered APP processing upon phosphorylation may include altered interactions with intracellular adaptor proteins, such as Fe65
and X11/Mint.
Synthetic peptides corresponding to the cytoplasmic domain of APLP1 and
APLP2 have been shown to be phosphorylated by PKC (Suzuki et al., 1997),
as previously shown for APP (Gandy, et al., 1988). Alignment of the APP
family members shows that the APP Thr668 phosphorylation site is conserved in all APP homologues; APLP1 (Thr624) and APLP2 (Thr636).
APLP2, as well as APP was observed in a study by Taru and Suzuki to be
phosphorylated at this site in response to cellular stress (Taru and Suzuki,
2004). Furthermore, Pro669 in APP, that undergoes isomerization following
Thr668 phosphorylation is conserved in APLP2 (Pro737) but not in APLP1.
Four other phosphorylation sites in APP, Tyr653, Tyr682, Thr686 and
Tyr687, are also conserved in all APP homologues.
33
2. Methodological considerations
2.1 Cell lines
We have used human neuroblastoma SH-SY5Y cells as a model to study
APP family processing. This cell line is used in all papers of this thesis and
is a commonly used cell line serving as an in vitro model for many types of
studies especially concerning neuronal function and differentiation. The SHSY5Y cell line is a third generation clone originating from SK-N-SH, which
was isolated from a metastatic tumor in the bone marrow of a four year old
girl in 1971 (Biedler et al., 1978). SH-SY5Y cells can be differentiated into a
neuron-like phenotype by treatment with retinoic acid (RA) (Pahlman et al.,
1984). During RA-differentiation, SH-SY5Y cells express TrkB receptors
(Encinas et al., 2000), which can be activated by BDNF and thereby inducing further differentiation of SH-SY5Y cells into an even more neuron-like
phenotype. SH-SY5Y cells have been shown to endogenously express all
members of the APP protein family (Adlerz, et al., 2003, Beckman and Iverfeldt, 1997), and are commonly used to study APP processing. SH-SY5Y
cells also express insulin and IGF-1 receptors. It should be noted that in contrast to what is seen in primary cortical neurons, α- and β-secretase processing of APP was recently found not to be coupled in SH-SY5Y cells
(Colombo et al., 2012). This is however not in agreement with our studies
using SH-SY5Y cells, were we consistently have seen an inverse coupling of
sAPPα and Aβ secretion into the cell medium.
In paper V, we also used human neuroblastoma SK-N-AS cells and human
embryonic kidney HEK293 cells to compare effects on APP processing in
different cell lines. SK-N-AS is derived from the bone marrow of an eight
year old boy. The cancer cells had metastasized from adrenal glands to the
bone marrow. The HEK293 cell line was generated by adenovirus transformation of cultured human kidney cells derived from a single healthy fetus
legally aborted under Dutch law (Graham et al., 1977).
34
2.2 Cell treatments
During this work, we have used RA and IGF-1 in combination with several
different kinase inhibitors to analyze signaling pathways involved in APP
family processing. Both RA and IGF-1 had previously been shown by our
group to induce a shift in APP processing towards the α-secretase pathway
(Adlerz, et al., 2007, Holback, et al., 2005). In addition, inhibitors targeting
OGT and OGA have been used to analyze effects of O-GlcNAcylation.
2.2.1 Retinoic acid and IGF-1
RA was used in paper I and II. SH-SY5Y cells were treated for 6 days with
10 µM RA in serum-containing medium or 1 µM RA in serum-free medium.
In paper II, we also treated the cells with RA concomitantly with 50 ng/ml
BDNF to analyze additional effects on expression levels of ADAM10,
TACE and BACE1, since it previously was shown in the group that BDNF
further increase RA-induced α-secretase processing (Holback, et al., 2005).
We used IGF-1 to stimulate α-secretase processing in paper I and V. We
treated the cells for 18 h with 10 nM IGF-1 in serum-free medium supplemented with N2 (transferrin, insulin, progesterone, selenite and putrescine).
Longer treatments of IGF-1 were avoided to minimize the effects of IGF-1
on the expression levels of the APP family members.
2.2.2 Pharmacological inhibitors
The effect of various kinases during IGF-1 and RA treatment was investigated in paper I, II and V by co-treating the cells with pharmacological inhibitors for the last 18 h of the treatment. To inhibit PKC we used bisindolylmaleimide XI (BIM11), which is a staurosporine derivate that is cell-permeable
and blocks PKC activity by competitively inhibiting the binding of ATP by
the kinase. BIM11 inhibits all PKC isoforms, but with varying potency with
a 3- to 10-fold preference for the PKCα isoform. It should be noted that an
independent screen of various kinase inhibitors revealed that the related
BIM10 also inhibited Msk-1 and MAPKAP-K2 to a similar potency as PKC
(Davies et al., 2000). These kinases are further “downstream” in signaling
pathways than PKC, and are mainly involved in phosphorylation of the transcription factor CREB at Ser133. In paper I, we also used the natural compound curcumin as an unselective PKC inhibitor. However, curcumin is
known to also inhibit c-Jun kinase (JNK) as well as the transcription factors
NFκB and AP-1. LY29002 (LY) is a reversible, cell-permeable inhibitor of
PI3K that acts through competitive binding to the ATP-binding site of the
catalytic subunit of the kinase. According to the manufacturer (Merck) LY
does not affect the activity of MAPK, PKC or PI4K at low concentrations.
However, in the previously mentioned independent screen, LY was shown to
35
also inhibit casein kinase 2, which is involved in cell cycle control and DNA
repair. PD098059 (PD) was used to inhibit MEK1/2. It interacts with the
dephosphorylated form of MEK1, thereby preventing its activation. PD also
works as a weak inhibitor of MEK2. PD was one of the most selective kinase
inhibitor in the independent screen.
To raise the level of cellular O-GlcNAcylation in paper III, IV and V we
used the OGA inhibitor PUGNAc. PUGNAc is a 1,5-hydroximolactone,
proposed to be a transition-state analogue. Lately, it has been shown that
PUGNAc, in addition to its effect on OGA, acts as an inhibitor of a variety
of N-acetylhexosaminidases. In paper III, we also used siRNA downregulation of OGA to confirm that the effects on APP processing stimulated
by PUGNAc indeed were mediated through decreased O-GlcNAcylation and
not by off-target effects.
2.3 siRNA gene silencing
In paper I we wanted to investigate the effect of TACE on APP and APLP2
processing, and since no specific pharmacological inhibitor was available on
the market at the time, we chose to knock-down the expression of TACE
with short interfering RNA (siRNA). We also used siRNA targeted against
OGA in paper III to further strengthen the effects seen with the inhibitor
PUGNAc. siRNA, like the name suggests, are small RNA sequences which
interfere with the expression of a specific gene. siRNA incorporates into the
RNA-induced silencing complex (RISC), which cleaves the target mRNA
strand complementary to the bound siRNA, leading to post-transcriptional
gene silencing. Two strategies were applied to knock-down the expression of
the target protein. SH-SY5Y cells were transfected with either 100 nM of a
mix of 4 different siRNA sequences, or with 5-50 nM of one single sequence
(two different sequences were used separately). The use of different sequences makes it possible to determine the specificity of the knock-down,
and to exclude off-target effects. In addition, a negative control composed of
non-targeting siRNA was used.
2.4 BCA assay
The protein content in the cell lysate and cell medium was determined using
bicinchoninic acid (BCA) assay. The principle of the BCA assay is similar to
the Lowry procedure. However, in the BCA assay the mechanism can be
divided into two reactions. First, cysteines, tryptophans, tyrosines, and the
peptide bond will reduce Cu2+ from the cupric sulfate present in the BCA
stock solution, to Cu1+. Then, Cu1+ will form a purple-blue complex, by che36
lating with two BCA molecules. This complex has a strong absorbance at
562 nm. The amount of protein present in a solution can be quantified by
measuring the absorption spectra and comparing with protein solutions, e.g.,
bovine serum albumin (BSA) with known concentrations. The BCA assay is
more sensitive than Bradford and Lowry, and is also less susceptible to detergents.
2.5 Western blot
Western blot is a frequently used method to analyze the expression levels of
a specific protein. The protein sample is first boiled together with sodium
dodecyl sulphate (SDS), which denaturates the proteins and gives them a
negative net charge in proportion to their size. This difference in charge can
then be used to separate proteins according to size on a polyacrylamide gel
that is run in an electric field. The separated proteins in the gel are then
transferred to a membrane with the help of an electric current. The protein of
interest can now be probed for by using a specific antibody directed against
the target protein. A secondary antibody, coupled to horseradish peroxidase
(HRP) that binds to the primary antibody is added in order to visualize the
protein by enhanced chemiluminescence (ECL). When adding a solution
containing hydrogen peroxide (H2O2) and luminol, HRP will catalyze the
oxidation of luminol by H2O2, generating acridium ester intermediates,
which in turn will react with peroxide and generate an excited state that
emits light as it decays to a lower energy level. This signal is captured on a
film or by a CCD camera. It is important to make sure that the exposure time
is not too long, i.e., the signal is saturated. The relative abundance of the
protein is quantified by densiometric analysis. Both the specificity and sensitivity of the method mainly relies on the nature of the antibody. If there is
cross reactivity of the antibody, the results may be difficult to interpret and
the specificity of the band should be confirmed using either overexpression
or knock-down of the target protein. It is also important to make sure that a
correct concentration of the antibody is used to ensure quantitative measurements.
2.6 ELISA
Enzyme-linked immunosorbant assay (ELISA) was carried out in paper I
and III to analyze the levels of secreted Aβ40, since this is a more sensitive
method compared to western blot. The method, like western blot is based on
the binding of an antigen (in this case Aβ40) to a specific antibody. We used
sandwich ELISA, in which a capture antibody directed against the Nterminal of Aβ40 is immobilized on the bottom surface of the wells in a 96
37
well plate. The cell medium sample was then added to the wells, resulting in
Aβ40 binding to the antibody. Without the first layer of capture antibody, any
proteins in the sample may competitively adsorb to the plate surface. A detection antibody directed against the C-terminal Aβ40 and a secondary, HRPlinked antibody was added before colorimetric detection. For colorimetric
detection, the antibody-antigen-antibody-antibody/HRP complex (or sandwich) is incubated with a chromogen, in this case tetramethylbenzidine
(TMB). TMB is oxidized during the enzymatic degradation of H2O2 by HRP.
The oxidized product of TMB has a deep blue color, but after addition of an
acidic stop solution, a clear yellow color is formed. The optical density of
the yellow color is measured at 450 nm, and the intensity reflects the amount
of Aβ40. In order to translate intensity to concentration, standard samples
containing known concentrations of Aβ40 are run alongside the cell medium
samples. This is a highly sensitive method, detecting Aβ40 concentrations as
low as 6 pg/ml. However, when treating SH-SY5Y cells with either IGF-1 or
PUGNAc, the Aβ levels decrease close to the detection limit. Previous studies in the group have shown that the Aβ42 levels follow the same trend as
Aβ40 during similar conditions as used in the studies in this thesis.
2.7 32P-labeling and immunoprecipitation
In paper II we wanted to study if TACE was phosphorylated in response to
IGF-1. A common method to study phosphorylation of proteins is 32Plabeling. Cells are incubated with 32P-orthophosphate and during this incubation the 32P will be taken up by the cells and incorporated into ATP. In this
study, we labeled phospho-proteins in cells in the presence or absence of a
protein kinase C inhibitor. After the labeling, the cells were treated with
IGF-1 before harvesting. To isolate TACE from the cell lysate we performed
immunoprecipitation (IP) with an antibody directed against the C-terminal of
TACE. IP is based on specific binding of an antibody against the target protein and immobilization on sepharose or agarose beads. The complex consisting of sepharose/agarose beads, antibody and target protein can then be
precipitated by centrifugation. The precipitated protein is finally released
from the beads by boiling in SDS-sample buffer. The immunoprecipitated
TACE sample was resolved by SDS-PAGE and the levels of phosphorylated
TACE was then visualized by exposure to a phosphoimager screen. The
signal to noise ratio is often low in these kinds of experiments due to the
difficulty to enrich large amounts of phosphoproteins. Immunoprecipitation
was also used in paper III and IV. High background is a common problem
when only using immunoprecipitation followed by western blot, since secondary antibodies also will detect the denatured heavy and light chain of the
immunoglobulin.
38
2.8 sWGA precipitation
Succinylated weat germ agglutin (sWGA) is a lectin that specifically binds
GlcNAc. Before succinylation, WGA will recognize both silaic acid and
GlcNAc. In paper IV we used sWGA coupled to agarose beads to isolate and
precipitate O-GlcNAcylated proteins in the cell lysate. Following incubation
of the cell lysate with the sWGA-bead slurry, O-GlcNAcylated proteins are
precipitated by centrifugation and released from the beads by boiling in
SDS-sample buffer. The levels of APP or ADAM10 that precipitated with
sWGA beads were then analyzed by western blot. In paper III, we used immunoprecipitation with an antibody targeted against O-GlcNAc for the same
purpose. As discussed in paper IV and in the results section of this thesis, the
fact that a protein is precipitated with sWGA or an O-GlcNAc antibody does
not necessary mean that the protein is O-GlcNAcylated. The precipitated
protein may instead be in complex with a protein that is O-GlcNAcylated. In
our study, this is further investigated by pre-treating the cell lysate with
SDS, thereby breaking all complexes, before sWGA precipitation.
2.9 Biotinylation assay
In paper IV we wanted to study APP trafficking in response to increased OGlcNAcylation. Since the cell surface is the main site for α-secretase cleavage, we used a biotinylation assay to analyze APP levels on the cell surface.
This assay makes use of sulfo-NHS-SS-biotin, which is a non-membrane
permeable compound that reacts with amines. The cells are incubated in PBS
containing sulfo-NHS-SS-biotin at 4oC (to inhibit endocytosis), resulting in
selective biotin labeling of all cell surface proteins via a disulfide bond. Cells
are then lysed and biotin-labeled proteins are isolated by their selective binding to streptavidine that has been conjugated to agarose beads. The proteins
are then released from the streptavidine-bound biotin label through dithiothreitol (DTT) reduction of the disulfide bond. Cell surface APP can then be
detected using western blot. To verify membrane integrity during the biotinylation protocol and that only surface proteins have been biotinylated, the
western blot membrane can be stripped and re-blotted against intracellular
proteins such as tubulin or microtubule-associated protein 1a (MAP1a).
2.10 Design and cloning of the APP/APLP2 chimer
The initial strategy of this project was to first create APP/APLP2 chimers
where large parts of the proteins had been replaced. After analyzing the effects of these substitutions on ADAM10/TACE specificity during IGF-1
stimulation, the domain that was found to be determining the specificity
39
would be further investigated by narrowing down the substituted part. We
decided to focus on the ectoplasmic part of APP, since this is the part that is
cleaved. Two chimeric constructs were created, substituting either the E1 or
E2 domain in APP with the corresponding part of APLP2. The constructs
were created by inserting unique restriction sites flanking the domain to be
replaced. The sites were inserted using Pfu Ultra mediated site-directed mutagenesis. The same restriction sites were also incorporated, using PCR in
the 5’- and 3’-end of the specific APLP2 domain, enabling ligation (after
restriction site cleavage) into the mutated vector. Unfortunately, we later
realized that the E2 domain in APLP2 that we inserted into APP contained a
KPI domain. Since this domain is not present in the APP695 isoform, it
would be preferable to replace the domain with the corresponding domain in
an APLP2 isoform that also does not contain a KPI domain (e.g., the 707
isoform). This APP/E2/APLP2 construct was successfully expressed in SHSY5Y cells but the secretion was not stimulated by IGF-1 and displayed
broad “smeared” bands on the western blot using an anti-FLAG antibody
(data not shown). A new E2 chimer is currently being constructed.
Since our focus was to analyze secretion of the constructs, we wanted an Nterminal tag. However, APP is normally expressed with a signal sequence,
directing it to the plasma membrane, which is then cleaved off. To avoid
removal of the N-terminal tag, the signal sequence in APP was deleted. Furthermore we used the pCMV8 vector (Sigma Aldrich) which contains the
preprotrypsin leader sequence and a 3xFLAG-tag preceding the multiple
cloning site. A drawback of using this vector in our study is the CMV promoter, which is a very strong promoter causing high degree of expression.
This is further discussed in the results section and paper V.
40
3. Aims
sAPPα levels in the brain are reduced in both familial and sporadic AD
(Lannfelt et al., 1995), and several studies have shown that α-secretase processing is negatively correlated with Aβ production. Furthermore, mutations
in ADAM10 have been linked to late onset AD (Suh, et al., 2013). Together,
this illustrates the importance of studying the regulation of α-site cleavage of
APP, since enhancers of this processing pathway clearly constitutes a potential therapeutic strategy. The aim of this thesis was to elucidate signaling
pathways involved in stimulated APP α-site processing. In addition, we
wanted to further investigate the previously shown difference in regulation
of APP and APLP2 processing (Adlerz, et al., 2007).
The more specific aims of this thesis were to:




Determine the signaling pathways involved in IGF-1- and RAinduced processing of APP and APLP2.
Determine if the same α-secretase cleaves both APP and APLP2.
Investigate the role of O-GlcNAcylation on APP and APLP2 processing.
Investigate which part of APP and APLP2 that determines their αsecretase selectivity.
41
4. Results and discussion
All five papers are focused on the α-secretase processing of the APP family
during stimulated conditions, and more specifically the distinct mechanisms
regulating processing of APP and APLP2. The most important results from
these articles are discussed below.
4.1
Stimulated α-secretase processing of APP is
mediated by ADAM10 in a PI3K-dependent
manner (Paper I and II)
It has previously been shown that RA and IGF-1 induce α-secretase processing of all three members of the APP family in SH-SY5Y cells (Adlerz,
et al., 2007, Holback, et al., 2005). Furthermore, the signaling pathway behind IGF-1-induced APP processing was investigated and was found to be
completely dependent on PI3K (Adlerz, et al., 2007). Based on these studies
we aimed to determine if also the RA-induced APP processing was dependent on the same signaling pathway. We used an antibody that recognizes the
first 17 amino acids in the Aβ region, making it possible to specifically detect sAPPα in conditioned cell medium from RA-differentiated SH-SY5Y
cells. Our results show that inhibition of PI3K reduced the RA-induced
sAPPα secretion by ~50% (Paper I: Fig. 1B). So why is the RA-induced APP
ectodomain shedding not completely blocked by the PI3K inhibitor, as seen
for the IGF-1-induced processing? It should be pointed out that RA was added for 6 days, as compared to 18 h with IGF-1, and the PI3K inhibitor was
only present the last 18 h. RA can mediate its effects both by transcriptional
effects through activation of nuclear retinoid receptors as well as direct effects on protein kinases (see section 1.4.2). The PI3K-independent effects on
RA-induced APP processing are probably a result of transcriptional effects,
such as up-regulation of α-secretases, which have been induced during several days.
Next, the involvement of PKC during RA- and IGF-1-induced APP ectodomain shedding was investigated using a PKC inhibitor. The RA-induced
processing was found to be completely unaffected by PKC inhibition where42
shown to be strongly affected by PI3K inhibition (Paper II: Fig. 2). This
suggests that PI3K is involved in stabilizing mature ADAM10 and not involved in the transcriptional effects of RA on ADAM10. This stabilization
of mature ADAM10 is subsequently important for the increased APP α-site
processing seen during stimulated conditions.
4.2
Stimulated α-secretase processing of APLP2 is
mediated by TACE in a PKC-dependent manner
(Paper I and II)
In contrast to APP, which previously was shown to be processed in a PI3Kdependent manner during IGF-1 stimulation, APLP2 processing was unaffected by blockage of this signaling pathway during the same conditions
(Adlerz, et al., 2007). To elaborate this work, we used a PKC inhibitor to
determine if signaling pathways including this kinase was important for
stimulated APLP2 processing. Activation of PKC by phorbol esters has previously been shown to induce both APP and APLP2 processing (Caporaso,
et al., 1992, Xu, et al., 2001). Our results demonstrate that the IGF-1-induced
APLP2 processing was completely blocked by PKC inhibition (Paper I: Fig.
2). Furthermore, the RA-induced sAPLP2 secretion was also shown to be
completely dependent on PKC (Paper 1: Fig. 1B). Our results suggest that
IGF-1 induces activation of a pathway involving PKC that is independent of
PI3K, probably through receptor activation of PLCγ. This is not a consensus
signaling pathway for IGF-1R, but has been established in some cell lines
(c.f., (Chattopadhyay and Carpenter, 2002). The signaling behind RAinduced APLP2 processing might involve up-regulation of PKC or direct
interaction and thereby activation of PKC (see section 1.4.2). Together our
data suggest that stimulated APLP2 processing is PKC-dependent during
these conditions.
Based on the difference in signaling behind stimulated APP and APLP2
processing, we speculated that the two proteins were cleaved by different
enzymes. Indeed, knocking down the expression of TACE using siRNA we
found a strong correlation between the degree of TACE down-regulation and
the degree of IGF-1-induced sAPLP2 secretion (Paper I: Fig. 4). On the other hand, inhibition of ADAM10 had no effect on the IGF-1-induced processing of APLP2 (Paper I: Fig. 3). The RA-induced sAPLP2 secretion was
also blocked by TACE knock-down. Interestingly, we could also detect an
increased phosphorylation of TACE in response to IGF-1 using 32Pphosphate labeling followed by immunoprecipitation with a TACE antibody.
This increased phosphorylation was also shown to be dependent on PKC,
since it was blocked using the PKC inhibitor (Paper I: Fig. 5). There are
44
contradictory reports regarding the connection between TACE activity and
phosphorylation (see section 1.5.2.1), but based on our results we speculate
that the increased TACE activity during IGF-1 stimulation is dependent on
TACE phosphorylation.
In addition to TACE phosphorylation, we also wanted to study the maturation of TACE (i.e., its processing) during stimulated conditions. Similar to
ADAM10, the protein levels of TACE were also shown to be up-regulated in
response to 6 days exposure of RA (Paper II: Fig. 1). However, it was mainly the immature form of TACE that was increased, although the mature form
was increased as well, but not to the same extent. The PI3K inhibitor had no
effect on the level of neither immature nor mature TACE. Inhibition of PKC
had a small effect on the immature level of TACE, but more importantly it
strongly reduced the level of mature TACE (Paper II: Fig. 2 and table 1).
Our result suggest that PKC is involved in stabilizing the mature form of
TACE in response to RA, perhaps through regulation of TACE phosphorylation, leading to increased APLP2 processing.
In conclusion, we have used two physiological stimuli, RA and IGF-1 to
induce α-secretase processing of APP and APLP2 in a human neuroblastoma
cell line. Our results suggest that stimulated APP processing is PI3Kdependent and is mediated by ADAM10, whereas APLP2 processing is
PKC-dependent and mediated by TACE during these relevant conditions.
4.3
O-GlcNAcylation induces α-secretase processing
of APP but not of APLP2 (Paper III and IV)
There are many studies on APP phosphorylation, but the reported effects on
APP processing are contradictory. Many studies have introduced a mutation
to evaluate the effect of phosphorylation at a specific site. However, what
has not been considered in these studies is the possibility that another posttranslational modification, namely O-GlcNAcylation also can occur at the
same site as phosphorylation. O-GlcNAcylation is also a rapid modification
that cycle on and off the protein (see section 1.5.2). Many cytosolic and nuclear proteins have been shown to be O-GlcNAcylated, thereby affecting
their activity and/or localization. APP was the first membrane protein reported to be O-GlcNAcylated (Griffith, et al., 1995), but the consequence of this
has not been investigated. We aimed to determine if APP localization was
affected by O-GlcNAcylation, and since our research mainly concerns the αsecretase processing we focused on the effect on plasma membrane localization where the main part of α-site processing occurs. We treated SH-SY5Y
cells with PUGNAc, an inhibitor of OGA-the enzyme that catalyzes the re45
moval of O-GlcNAc from target protein, thereby increasing the amount of
O-GlcNAcylated proteins in the cell. Immunoprecipitation followed by
western blot using antibodies against APP demonstrated that the level of
APP that precipitated with an O-GlcNAc antibody increased in response to
PUGNAc (Paper III: Fig. 1). The levels of APP at the cell surface were analyzed by a biotinylation assay followed by western blot. The results show
that cell surface localization of mature APP was greatly enhanced in response to PUGNAc without affecting the total level of APP (Paper IV: Fig.
2). This indicates that O-GlcNAcylation affects the trafficking of APP targeting it to the cell surface.
Based on the observed PUGNAc-induced cell surface localization of APP,
we speculated that the degree of its α-secretase processing was affected. To
test this we treated cells with PUGNAc and analyzed by western blot the
secretion of sAPPα in conditioned cell medium. Indeed, PUGNAc increased
the sAPPα secretion with approximately 150% and reduced the Aβ secretion
(Paper III: Fig. 2), suggesting that O-GlcNAcylation induces a shift in APP
processing towards increased α-secretase processing through enhanced cell
surface localization. Interestingly, when analyzing the conditioned cell medium, we found that the sAPLP2 secretion was unaffected by PUGNAc (Paper IV: Fig. 1). Thus, our result shows that it is possible to induce αsecretase processing of APP without affecting the processing of APLP2.
In addition to the effects on α-secretase processing, it has further been shown
that O-GlcNAcylation of the γ-secretase component nicastrin regulates Aβ
production and inhibition of OGA reduced plaque formation and rescued
memory impairment in an AD mouse model (Kim et al., 2013). OGA and
OGT are both enriched and highly active in the brain, and most abundant in
the hippocampus, a region important for learning and memory. Interestingly,
LTP was shown to be enhanced in vivo upon inhibition of OGA (Tallent, et
al., 2009). The study further demonstrated that the effect was meditated
through O-GlcNAcylation of several pre-synaptic proteins. OGlcNAcylation has also been directly linked to AD since the level of protein
O-GlcNAcylation is decreased by approximately 20% in AD brains (Liu et
al., 2004a). Furthermore, it has been demonstrated that decreased OGlcNAcylation of tau is correlated to tau hyperphosphorylation and thereby
increased formation of neurofibrillary tangles (Liu, et al., 2004a).
4.3.1 O-GlcNAcylation selectively enhances α-secretase
processing of APP in neuron-like cells (Paper IV)
We also analyzed the effect of O-GlcNAcylation on APP processing in different cell lines. In addition to SH-SY5Y cells, we used the human neuro-
46
regarding the enzyme selectivity between APP and APLP2 and the fact that
only APP and not APLP2 processing was affected by O-GlcNAcylation, we
speculated that perhaps ADAM10 was O-GlcNAcylated. Indeed, we could
demonstrate that the level of ADAM10 that was precipitated with sWGA or
with an O-GlcNAc antibody was increased in response to PUGNAc. When
pre-treating the cell lysate with SDS (breaking up complexes), ADAM10 but
not APP is still precipitated with the O-GlcNAc antibody (Paper IV: Fig. 5),
suggesting that ADAM10 is in fact O-GlcNAcylated whereas APP is not.
This will be further investigated using mass spectrometry. Together with our
previous results we now speculated that O-GlcNAcylation is a neuronspecific modification of ADAM10 resulting in increased interaction and
subsequent α-site cleavage of APP. Further studies showed however that
also this hypothesis had its faults, since ADAM10 was shown to be OGlcNAcylated in HEK293 cells as well (Paper IV: Fig 6), which do not result in increased α-site processing. In addition, the level of cell surface APP
was unchanged in HEK293 cells in response to PUGNAc (Paper IV: Fig. 6).
At this point, the mechanism of O-GlcNAc-regulated APP processing is still
unknown, but our results indicate that it involves a neuron-specific protein
that either stabilizes APP at the cell surface or increases it transport to the
cell surface.
Together our result suggest that although APP is not O-GlcNAcylated, an
increase of O-GlcNAcylation will affect its trafficking towards the cell surface where it will be cleaved at the α-site, resulting in a shift in its processing
generating more sAPPα and less Aβ. ADAM10 is O-GlcNAcylated, but this
is not sufficient for increased α-secretase processing of APP. We also show
that O-GlcNAcylation only affects APP processing in neuron-like cells
without affecting APLP2 processing, which is important in the search for
therapeutic treatment aimed to selectively modify APP processing in the
brain.
4.4
The difference in regulation of α-secretase
processing between APP and APLP2 is
determined by their E1 domain (Paper III)
Having established a clear difference in the regulation of α-site processing
between APP and APLP2, including cleavage by different enzymes, we
sought to determine which part of these two homologous proteins defines
their selectivity for either ADAM10 or TACE, respectively. We started by
constructing an APP/E1/APLP2 chimer, in which the entire E1 domain in
APP was substituted with the E1 domain from APLP2. We then analyzed the
48
processing of the chimeric protein in response to IGF-1. The secretion of the
chimeric protein was enhanced upon IGF-1 stimulation (Paper V: Fig. I), as
seen for both APP and APLP2. However, in contrast to APP, this increased
secretion of sAPP/E1/APLP2 was unaffected by PI3K inhibition (Paper V:
Fig. 2), as previously shown for APLP2. We further investigated the IGF-1induced processing of APP/E1/APLP2 showing that it was PKC-dependent
(Paper V: Fig. 3), as seen for APLP2. It should be noted that this experiment
only has been performed once and needs to be repeated to draw any statistically reliable conclusions. Our results indicate that the chimer is cleaved by
TACE, and not by ADAM10. Since our results show that the chimeric protein behaves more like APLP2 during IGF-1 stimulation, we wanted to investigate if this would be the case during increased O-GlcNAcylation. We
treated transfected cells with PUGNAc and in accordance to what was
shown for IGF-1, the processing of the chimeric protein was similar to
APLP2 processing; i.e., it was unaffected (Paper V: Fig. 4). Also this experiment needs to be repeated. Our results indicate that differences in the APP
and APLP2 E1 domain determine their selectivity for ADAM10 and TACE,
respectively. It should be noted that the E1 domain of APP and APLP2 are
62% identical on amino acid level and 82% similar, i.e., they are very homologous. It has previously been shown that the secondary structure of the
substrate is important for ADAM10 and TACE recognition and plays a role
in the substrate selectivity of the two enzymes (Stawikowska, et al., 2013).
We therefore speculate that the E1 domain of APP might differ in its secondary structure, targeting them to either enzyme. Another possibility could
be a difference in the loop sequence in the proteins. This loop in APP, created by a disulfide bridge between Cys98 and Cys105 (Cys116 and Cys123 in
APLP2) contains mostly positively charged amino acids, except one glycine
(Fig. 5). In the corresponding loop in APLP2, there is an Asp instead of the
glycine, introducing a negative charge, which we speculate might be important for the selective TACE interaction and subsequent cleavage. It would
be interesting to perform a point mutation at this site and analyze the stimulated processing.
APP
50
SDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNW
CKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLH
C+R +KQCK+
DP+GTK+C +TKE +LQYCQE+YPELQITNV+EANQ V+I NW
APLP2
68
PDPTGTKSCFETKEEVLQYCQEMYPELQITNVMEANQRVSIDNW
CRRDKKQCKS—
FV P++CLVGEFVSD LLVP+KC+F H+ERM+VCE H H
RFVTPFKCLVGEFVSDVLLVPEKCQFFHKERMEVCENHQH
Figure 5. Alignment of a sequence in the E1 domain of APP695 with the corresponding
sequence of APLP2 763. The cysteine loop is indicated with enlarged characters.
49
5. Conclusions
In our studies, we have demonstrated that;
 Both RA- and IGF-1-induced processing of APLP2 is mediated by
TACE in a PKC-dependent manner, while the stimulated processing
of APP is mediated by ADAM10 in a PI3K-dependent manner. We
further show that RA up-regulated the mRNA and protein levels of
ADAM10 and TACE, as previously seen for the APP family
(Beckman and Iverfeldt, 1997). The maturation of ADAM10 and
TACE during RA treatment was PI3K and PKC dependent, respectively.
 Enhancing cellular O-GlcNAcylation through pharmacological inhibition or siRNA knock-down of OGA increases the α-site processing of APP, concomitantly with decreased Aβ production. In
addition, we show that APLP2 processing is unaffected by increased O-GlcNAcylation. We have further shown that APP is not
O-GlcNAcylated and that the mechanism rather involves OGlcNAcylation of ADAM10 as well as increased cell surface localization of APP, possibly mediated through O-GlcNAcylation of an
unknown neuron-specific protein.
 The E1 domains determine the difference in α-secretase processing
between APP and APLP2 during certain stimulated conditions. This
was demonstrated by creating a chimeric APP/APLP2 construct.
Insulin receptor signaling has been shown to involve PI3K-dependent OGT
translocation from the nucleus to the plasma membrane, where it O-GlcNAcmodifies Akt and IRS-1, thereby regulating their phosphorylation status,
resulting in attenuated insulin signaling (Yang et al., 2008). Based on this, it
is not unlikely that the effect of IGF-1 on APP processing is dependent on
OGT and subsequent O-GlcNAcylation of ADAM10 and the unknown neuron-specific protein that regulates APP trafficking. In addition to regulating
OGT translocation, we also show that PI3K regulates ADAM10 maturation.
Although the mechanism behind this is still unclear, it does not seem to involve O-GlcNAcylation. APLP2 processing is, however, independent of
PI3K and O-GlcNAcylation. This mechanism rather involves PKC, leading
51
to phosphorylation of TACE (not yet investigated during RA stimulation),
which possibly affects its subcellular localization and/or maturation. Although ADAM10 today is considered the constitutive α-secretase for APP
and APLP2 (Hogl, et al., 2011, Kuhn, et al., 2010), little is known about the
mechanism involved during stimulated conditions. Our studies indicate that
TACE preferably cleaves APLP2 during conditions when the activity of both
enzymes clearly is increased. This could be due to many different factors,
including the possibilities that APLP2 and TACE are spatially segregated
during non-stimulated conditions, or that APLP2 during certain stimulations
undergoes conformational changes (in the E1 domain) making it a better
substrate for TACE.
The overall conclusion of our studies presented in this thesis is that the αsecretase processing of APP and APLP2 display clear divergences during
stimulated conditions (Fig. 7). This includes different signaling pathways
and processing enzymes. Moreover, our results provide insight to what could
determine these differences in regulation. These finding may be important
for a general understanding of the regulation of ectodomain shedding; the
initial and rate-limiting step of RIP signaling (se section 1.2.3). Many proteins, including growth factors, cytokines, cell adhesion proteins and receptors undergo RIP. Regulation of this form of signal transduction thereby
controls several important cellular functions. However, little is known about
substrate recognition and specificity of RIP proteases, which are important
factors when aiming to selectively modify the processing of a specific protein. More specifically our results contribute to the understanding of the
normal function of APP/APLP2 and how their processing may be regulated.
This might be useful in the devolvement of selective enhancers of APP αsecretase processing that does not affect the processing of the physiologically important homolog APLP2.
Figure 7. Schematic illustration concluding the results presented in this
thesis. During stimulated conditions a
PI3K- and a PKC-dependent pathway
is activated. The PI3K pathway results
in OGT mediated ADAM10 OGlcNAcylation and APP cleavage,
generating sAPPα. The PKC pathway
leads to TACE phosphorylation and
APLP2 cleavage, generating sAPLP2.
52
6. Populärvetenskaplig sammanfattning på
svenska
Alzheimers sjukdom är en demenssjukdom som ca 70 000 människor lider
av i Sverige. Sjukdomen innebär att nervceller i specifika delar av hjärnan
dör, vilket resulterar i symptom som t.ex. problem med minnesfunktioner,
nedsatt uppmärksamhetsförmåga och språkförståelse, svårare att uttrycka
sig, sämre lokalsinne samt svårare att utföra ändamålsenliga kroppsrörelser.
Det är ännu inte känt vad som orsakar Alzheimers sjukdom, men hög ålder
är den i särklass mest bidragande riskfaktorn. Det som dödar nervcellerna i
hjärnan är framförallt ett ämne som kallas amyloid-β (Aβ). Aβ finns normalt
i hjärnan och bildas genom att ett större protein, APP klyvs av specifika
enzymer. Normalt sätt så klyvs APP till största del av ett annat enzym, som
hindrar bildningen av Aβ. Denna klyvning kallas α-klyvning och de enzymer
som medierar detta kallas α-secretaser. Vid Alzheimers sjukdom bildas av
någon anledning Aβ till mycket större grad och α-klyvningen minskar.
Förutom dess bidragande roll i Alzheimer, så har även APP många viktiga
funktioner i hjärnan, vilket inte har studerats lika väl. APP ingår i en s.k
familj utav liknande proteiner som även innefattar de två homologa
proteinerna APLP1 och APLP2. Forskning har visat att det finns ett samspel
mellan dessa tre proteiner där APLP2 spelar en särskilt stor roll. En möjlig
strategi för att utveckla ett läkemedel som skulle skydda mot Alzheimers
sjukdom är att stimulera α-klyvningen av APP. För att minimera oönskade
effekter på APLP2, som klyvs på ett likande sätt som APP, vore det att
föredra om man selektivt kunde påverka α-klyvningen av APP.
I vår forskning som presenteras i denna avhandling har vi studerat αklyvningen av APP och APLP2 i en human neuroblastoma cell-linje (SHSY5Y). I våra studier har vi behandlat cellerna med IGF-1 och RA, två
ämnen som tidigare visats öka α-klyvningen av både APP och APLP2 och
som det dessutom är brist på vid Alzheimers sjukdom. Vi har sett att den
stimulerade α-klyvningen av APP och APLP2 är reglerad via olika
signaleringsvägar i cellen och att de även klyvs av olika α-secretaser. APP
klyvs av ADAM10 och APLP2 av TACE. Våra studier visar även att
nivåerna av ADAM10 och TACE är beroende av olika signaleringsvägar.
53
En annan del av vår forskning har fokuserat på O-GlcNAcylering, en process
där ett sorts socker kopplas på proteiner vilket påverkar deras funktion. Även
O-GlcNAcylering har visats minska i hjärnan hos patienter med Alzheimers
sjukdom. Vi har sett att om man ökar O-GlcNAcyleringen i vårt
modellsystem, så ökar α-klyvningen av APP, men påverkar inte klyvningen
av APLP2. Vi visar även att det är ADAM10 och inte APP som OGlcNAcyleras.
För att förstå varför dessa två liknande proteiner, APP och APLP2 uppvisar
en skillnad i hur och när de klyvs vid deras α-site har vi klonat fram ett
chimert protein där en del av APP ersatts av den motsvarande delen av
APLP2. Poängen var att få APP α-klyvningen att bete sig som α-klyvningen
för APLP2. Det visade sig vara den mest N-terminala delen av proteinerna,
den s.k. E1 domänen som avgjorde skillnanden i α-klyvning. Tillsammans
visar dessa studier att α-klyvningen av APP och APLP2 är reglerad genom
distinkta mekanismer och därmed kan särskiljas.
54
7. Acknowledgements
Det har gått nästan 6 år sen jag började här och nu helt plötsligt så är den här
tiden nästan över. Det är sjukt hur snabbt tiden går. När jag började tänkte
jag att 5 år var hur lång tid som helst, och jag skulle vara 30 år när jag var
klar-alltså jätte gammal! Nu blev det inte så, jag är ännu äldre. Vet inte hur
många gånger jag funderat på att sluta, speciellt nu i slutet för att undvika
disputationen  Anledningen till att man stannat kvar har till stor del varit
en massa härliga männsikor som jag härmed vill tacka:
Först och främst och mest av allt, ett stort tack till Kerstin Iverfeldt. Tack
för att du antog mig som doktorand och för att du trott på mig under alla
dessa år. Tack för alla givande och roliga diskussioner och för att du lyssnar
och hjälper när det inte går så bra eller man inte mår så bra. Du är en
fantastisk handledare som gett mig frihet men även stöd och hjälp när jag
behövt det. Tack för att du alltid tar dig tid, även när du har fullt upp.
Ett stort tack även till alla i gruppen. Tack Sofia Holback för handledning
redan som D-kursare men även under min första tid här. Du har alltid varit
en inspirationskälla. Tack Tom Gatsinzi för att du är en så himla glad och
nyfiken kille, det har varit kul att retas med dig. Hoppas vi kan jobba
tillsammans igen snart. Linda Tracy min vapendragare under alla dessa år
som jag delat många tårar och glädjestunder med. Hade inte klarat det här
utan dig. Vi började samma dag, och slutar nästan samma dag. Tack för att
du alltid lyssnar och bryr dig. Det har varit så himla roligt att dela kontor och
lab med dig. Tack även till min vän och medförfattare Niina Koistinen du är
så mycket bättre än du någonsin kommer våga tro. Tack för alla tidiga
frukostar, träningspass, pratstunder och hjälp med projekten. Jag kommer
verkligen sakna att jobba med dig. Thank you Elena Ivanova your passion
for science is inspiring! Ylva Strååt min O-GlcNAc kollega; det har varit
roligt att jobba och bolla idéer med dig. Anna Edlund det känns jätte roligt
att du börjat i gruppen.
Ett vamt tack går såklart även till alla mina fina rumskamrater i
”tjejrummet”. Christin Svensson som nog har skrattat och gråtit lika mycket
som jag inne på kontoret och Marie Danielsson som var en lugn punkt i
stormen-jag har saknat er! Tack Jessica Lundqvist för att du är en sån
fantastisk människa som alltid ställer upp. Tack även för moraliskt stöd
55
under skrivandet av avhandlingen och all hjälp med korrekturläsningen.
Kristina Attoff som har samma humor som jag-jätte rolig alltså! Det har
varit så roligt och mysigt att dela kontor med er alla.
Thank you to all PhD students at the department; it has been a joy to work
with all of you. Thanks for all the fun Christmas parties, summer barbeques
and other festivities that we have had at the department during these years.
You are an amazing bunch of people and it feels great that so many of you
are dedicated not only to your own research but also for making this department an even better place to work at. A special thanks to:
Kristin Webling för att du är världens gulligaste, Staffan Lindberg för alla
dryga kommentarer , Andrés Muñoz-Alarcón för att du alltid är glad och
för att du kämpar för oss doktorander i din roll som doktorandordförande,
Rania Abdo för att du en trevlig tjej med skinn på näsan, Henrik Helmfors
för att du aldrig skrattar åt mina ”problem” med datorn och för att du är en
himla snäll kille, Veronica Larsson för att du är en väldigt snäll och stark
tjej, Santhosh Gudise for alway being so happy, Xin Yu for your
friendship-it is amazing everything that you have overcomed and
accomplished in your life.
Ett stort tack till alla lärare, Ülo Langel, Anders Undén, Anna Forsby,
Einar Hallberg and Anna-Lena Ström, för att ni engagerar er i alla oss
doktorander och hjälper oss på vägen. Ett speciellt tack till Anna-Lena för att
du styrde upp Tisdags seminarierna och för att du korrekturläste min
avhandling. Tack även till Bengt Mannervik för att du tar dig tid att
kommenterar ens insatser på seminarierna.
Det är även många utanför institutionens väggar som bidragit, framförallt
med moraliskt stöd under dessa år. Tack till mina kära föräldrar som alltit
har trott på mig, jag skulle aldrig ens påbörjat det här utan ert stöd. Mina
älskade systrar för att ni alltid finns där för mig, även fast ni alltid är så
långt borta. Mina vänner, Monique, Katti, Märta, Jenny, Camilla och
Linda, för er villkorslösa vänskap och för alla trevliga tjejmiddagar. Alla
klätterkompisar, Lollo, Erik och Anton, som jag tyvärr inte träffar så ofta
längre men som ändå betytt mycket under åren. Tack även till min nya
familj-Molindrarna; Lars, Britt och Tomas för att ni alltid ställer upp när
det behövs. Sist av allt, ett enormt tack till min man Anders, ord går inte att
beskriva hur mycket jag uppskattar allt stöd och all kärlek som du gett mig
denna tid. Jag skulle verkligen inte klarat det här utan dig. Jag älskar dig.
Tack för att du gett mig en liten busunge!
56
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