Roles of Heparan Sulfate in Amyloid-β Pathology and Hypoxia

Till familjerna Hjertström och Sandwall.
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
O’Callaghan P, Sandwall E, Li JP, Yu H, Ravid R, Guan ZZ,
van Kuppevelt TH, Nilsson L.N.G, Ingelsson M, Hyman BT,
Kalimo H, Lindahl U, Lannfelt L, Zhang X. (2008) Heparan
sulfate accumulation with Aβ deposits in Alzheimer’s disease
and Tg2576 mice is contributed by glial cells. Brain Pathology,
18(4):548-61
II
Sandwall E*, O’Callaghan P*, Zhang X, Lindahl U, Lannfelt
L, Li JP. (2010) Heparan sulfate mediates amyloid-beta internalization and cytotoxicity. Glycobiology, 20(5):533-41
III
Sandwall E, Bodevin S, Nasser NJ, Nevo E, Avivi A, Vlodavsky I, Li JP. (2009) Molecular structure of heparan sulfate
from Spalax. Implications of heparanase and hypoxia. Journal
of Biological Chemistry, 6;284(6):3814-22
*These authors contributed equally to the work.
Reprints were made with permission from the respective publishers.
Contents
Introduction.....................................................................................................9
Background ...................................................................................................10
Heparan sulfate proteoglycans .................................................................10
Nomenclature.......................................................................................10
Heparan sulfate biosynthesis ...............................................................10
Core proteins........................................................................................12
Interactions between heparan sulfate and proteins ..............................15
Heparanase...........................................................................................16
Heparan sulfate in pathological conditions ..............................................19
Inflammation .......................................................................................19
Amyloid diseases .................................................................................19
Alzheimer’s disease .............................................................................20
Tumor progression and metastasis.......................................................23
Heparan sulfate as a potential therapeutic target .................................25
Present investigations....................................................................................27
Aims of the study .....................................................................................27
Results and discussion..............................................................................28
Heparan sulfate accumulation with Aβ deposits in Alzheimer's disease
and Tg2576 mice is contributed by glial cells (Paper I) ......................28
Heparan sulfate mediates amyloid-beta internalization and cytotoxicity
(Paper II)..............................................................................................29
Molecular structure of heparan sulfate from Spalax. Implications of
heparanase and hypoxia (Paper III) .....................................................30
Concluding remarks and future perspectives ................................................32
Populärvetenskaplig sammanfattning ...........................................................35
Acknowledgments.........................................................................................36
References.....................................................................................................39
Abbreviations
Aβ
AD
AICD
APP
BACE1
CS
CTF
DS
ECM
ERK
EXT
FGF
GAG
GlcA
GlcN
GlcNAc
GlcNS
HIF
HS
HSPGs
IdoA
IL
MAPK
MIP
MMP
NA
NDST
NS
PG
PS
TGF-β
VEGF
Amyloid-β
Alzheimer’s disease
APP intracellular domain
Amyloid precursor protein
β-site APP-cleaving enzyme
Chondroitin sulfate
C-terminal fragment
Dermatan sulfate
Extracellular matrix
Extracellular signal-regulated kinases
Exostosin
Fibroblast growth factor
Glycosaminoglycan
Glucuronic acid
Glucosamine
N-acetylglucosamine
N-sulfated glucosamine
Hypoxia-inducible factor
Heparan sulfate
Heparan sulfate proteoglycans
Iduronic acid
Interleukin
Mitogen-activated protein kinase
Macrophage inflammatory protein
Matrix metalloproteinase
N-acetylated
N-deacetylase/N-sulfotransferase
N-sulfated
Proteoglycan
Presenilin
Transforming growth factor-β
Vascular endothelial growth factor
Introduction
Heparan sulfate (HS), found at cell surfaces and in the extracellular matrix
(ECM), is highly anionic due to the presence of sulfate groups along the
saccharide chain. The HS-binding proteins include morphogens, cytokines,
growth factors and receptors, which are crucial for fundamental biological
processes such as development and homeostasis. In addition, HS is implicated in several pathological conditions including inflammation, infection,
cancer and amyloid diseases such as Alzheimer’s disease (AD). The aims of
this thesis were to gain deeper insights into AD and cancer progression by
elucidating the roles of HS in Aβ pathology and hypoxia.
The first part of the thesis concerns the role of HS in amyloid-β (Aβ) pathology. The toxic Aβ peptide has a central role in AD where it aggregates
and forms the characteristic amyloid plaques present in AD brain, of which
HS is an established component. However, the role of HS in these amyloid
deposits and how the Aβ peptide exerts its toxic effects are not fully understood.
The last part of this thesis focuses on the role of HS in hypoxia and its
implications for tumor progression. Heparanase is an endo-glucuronidase
that specifically cleaves HS chains. Although heparanase activity strongly
correlates with the metastatic potential of tumor cells, there is limited
knowledge regarding its regulation during cancer progression.
9
Background
Heparan sulfate proteoglycans
Nomenclature
Proteoglycans (PGs) are a family of proteins that carry covalently bound,
unbranched glycosaminoglycan (GAG) chains. In heparan sulfate proteoglycans (HSPGs) the GAG chains are of heparan sulfate (HS)/heparin type. In
other PGs the GAG chains can be chondroitin sulfate (CS)/dermatan sulfate
(DS) or keratan sulfate. The GAG chain is composed of repeating disaccharide units where an amino sugar alternates with a uronic acid or galactose
residue. HS/heparin is composed of glucosamine (GlcN) residues that can be
N-acetylated (GlcNAc), N-sulfated (GlcNS) or in rare cases N-unsubstituted
and glucuronic acid (GlcA) or iduronic acid (IdoA). CS/DS contains Nacetylgalactosamine and GlcA or IdoA whereas keratan sulfate is composed
of GlcNAc and galactose residues. All of these GAGs contain sulfate substituents in various positions (Lindahl and Kjellen 1991).
Heparan sulfate biosynthesis
One of the main functions of HS is to bind to a wide array of proteins and
thereby affect their functions. The binding depends on the presence of sulfate groups along the saccharide chain, which make the HS structure highly
anionic. The complex and heterogenic structure of HS is generated through
an elaborate biosynthetic process (Figure 1). The identification of tissuespecific structures of HS by structural analysis and specific antibodies
against different HS-epitopes indicates that HS synthesis is well regulated
(Maccarana et al. 1996; van Kuppevelt et al. 1998; Ledin et al. 2004; Kurup
et al. 2007). Studies of mice and other model organisms emphasize the importance of HS biosynthetic enzymes for animal development and disease
(Bulow and Hobert 2006).
10
Figure 1. The major steps in HS biosynthesis, polymerization and modifications
(epimerization, N- and O-sulfation). The structures to the left represent examples of
disaccharide structures from the different steps. The final structure can be divided
into highly N-sulfated domains (NS), alternating N-acetylated/N-sulfated domains
(NA/NS) and N-acetylated domains (NA).
The HS chain is synthesized in the Golgi apparatus on a primer tetrasaccharide sequence that starts with a xylose residue linked to a serine residue
in the HSPG core protein, followed by two galactose residues and one GlcA
residue. Galactosyltransferase I catalyzes the formation of the β1,4 glycosidic bond between the galactose and xylose residues. Defects in this enzyme
are linked to progeroid Ehlers-Danlos syndrome that is characterized by
mental retardation and defects in the connective tissue (Quentin et al. 1990).
The addition of GlcNAc by EXTL2/EXTL3 (belonging to the exostosin enzymes (EXT)) determines that the GAG chain will be of HS/heparin type
(Kim et al. 2001; Busse et al. 2007). A heterodimeric complex of EXT1 and
EXT2 then adds alternating GlcA and GlcNAc residues (McCormick et al.
2000). Mutations in the EXT1 and EXT2 genes are associated with hereditary multiple exostoses; a dominantly inherited bone disorder (Cook et al.
1993; Wu et al. 1994).
The HS chain subsequently undergoes further modifications catalyzed by
specific enzymes utilizing the sulfate donor PAPS. HS is N-deacetylated and
N-sulfated by the NDST (N-deacetylase/N-sulfotransferase) enzymes that
convert GlcNAc into GlcNS residues. PAPS may have a regulatory effect on
NDST action, in addition to providing one of the substrates, since access to
PAPS generates extended N-sulfated domains that are formed in a processive
manner (Carlsson et al. 2008). Vertebrates have four NDSTs; NDST1 and
NDST2 are expressed in most tissues while NDST3 and NDST4 are expressed during development and in the adult brain (Aikawa et al. 2001).
Mice deficient in NDST1 die neonatally due to respiratory failure while mice
11
deficient in NDST2 have severe defects in mast cells due to the abnormal
structure of heparin (Forsberg et al. 1999; Ringvall et al. 2000).
C5-epimerase is the enzyme that catalyzes the conversion of GlcA into
IdoA. Epimerase knockout in mice results in a lethal phenotype with renal
agenesis, lung defects and skeletal malformations (Li et al. 2003). 2-Osulfotransferase is responsible for 2-O-sulfation of IdoA and GlcA, although
with preference for IdoA (Rong et al. 2001). Knockout of 2-Osulfotransferase in mice shows similar defects as the epimerase knockout
mice (Bullock et al. 1998).
6-O- and 3-O-sulfation of glucosamine residues are catalyzed by specific
6-O-sulfotranferases (three isoforms and one alternatively spliced form) and
3-O-sulfotransferases (six isoforms). Deficiency in 6-O-sulfotransferase-1
leads to defective HS biosynthesis and late embryonic/perinatal lethality
(Habuchi et al. 2007). 3-O-Sulfation of heparin/HS is crucial for the synthesis of sequences that specifically interact with the anticoagulant protein antithrombin (Lindahl et al. 1980). Unexpectedly, 3-O-sulfotransferase-1
knockout mice did not exhibit any procoagulant phenotype (HajMohammadi
et al. 2003). The synthesized chain can undergo further modification at the
cell surface and in the ECM by two 6-O-endosulfatases, which remove 6-Osulfate groups, preferentially from trisulfated disaccharides in the HS chain
(Ai et al. 2006).
The final HS product is composed of three different types of domains defined by their degree of sulfation. N-sulfated domains (NS) are the most
modified sequences with the highest sulfation degree, N-acetylated domains
(NA) are unmodified whereas N-acetylated/N-sulfated domains (NA/NS) are
a mixture of the two (Figure 1) (Maccarana et al. 1996).
Core proteins
Depending on the type of core protein that carries HS chains, HSPGs are
present on the cell surface, in the ECM or intracellularly. The cell membrane-bound HSPGs are the syndecans (four members) and the glypicans
(six members) (Lindahl and Li 2009). Perlecan, agrin, and collagen XVIII
are extracellular PGs with functions, in particular, in basement membranes
(Iozzo et al. 2009). The only intracellular HSPG is serglycin (Kolset and
Tveit 2008).
Syndecans
The transmembrane syndecans consist of a large ectodomain to which GAG
chains are attached, a transmembrane region and a short cytoplasmic tail.
There are usually at least three HS chains attached close to the N-terminus
(Kirn-Safran et al. 2009; Multhaupt et al. 2009). The expression of the different syndecan members is cell- and tissue-specific and varies during development (Kim et al. 1994). They are generally described as co-receptors
12
that have key roles in signal transduction and can for instance act as coreceptors for fibroblast growth factors (FGFs) and transforming growth factor-beta (TGF-β) (Coutts and Gallagher 1995; Chen et al. 2004). Syndecans
regulate integrin activity in different cell models, and integrins and syndecans are believed to provide a physical link between the ECM and the cytoskeleton (McQuade et al. 2006; Morgan et al. 2007). Clustering of syndecans and phosphorylation of the cytoplasmic tail are proposed to be important for recruiting cytoplasmic cytoskeleton and signaling molecules
(Yoneda and Couchman 2003). Shedding of syndecan ectodomains by matrix metalloproteinases (MMPs) regulates signal transduction and adhesion
(Manon-Jensen et al. 2010).
Syndecan-1, present on stromal and epithelial cells, carries both CS and
HS chains (Kirn-Safran et al. 2009). Syndecan-1 knockout mice have no
major developmental abnormalities. However, both knockout and overexpression of syndecan-1 lead to defect wound healing (Stepp et al. 2002;
Elenius et al. 2004).
Syndecan-2, found on fibroblasts, has three conserved sites for HS attachment (Kirn-Safran et al. 2009). This isoform is implicated in angiogenesis (Fears et al. 2006) and is found to be upregulated in human colon cancer
(Ryu et al. 2009).
Syndecan-3 has a large ectodomain with six potential sites for GAG attachment (both HS and CS) and is expressed in the nervous system, the adrenal gland and the spleen (Kirn-Safran et al. 2009). Mice that lack syndecan-3 have impaired hippocampus-dependent memory and show resistance
to obesity under high fat diet (Kaksonen et al. 2002; Strader et al. 2004).
Syndecan-4, with three HS attachment sites, is found in most tissues but
seems to be less abundant (Kim et al. 1994; Kirn-Safran et al. 2009). Its importance in angiogenesis and wound healing was demonstrated in syndecan4 null mice showing delayed wound repair and impaired angiogenesis
(Echtermeyer et al. 2001). However, the most crucial role of syndecan-4
seems to be in the formation of focal adhesions (Woods and Couchman
1994).
Glypicans
Glypicans, attached to the cell membrane through a glycosylphosphatidylinositol anchor, are characterized by cysteine-rich globular ectodomains with
two to four HS chains attached close to the plasma membrane. Glypicans can
regulate signaling of Wnts, Hedgehogs, FGFs and bone morphogenic proteins, either promoting signaling by stabilizing the interaction between
growth factor and receptor or inhibiting signaling by sequestering growth
factors, thereby competing with the growth factor receptor (Fransson et al.
2004; Filmus et al. 2008).
Glypican-1 is expressed in the central nervous system during development and in many other tissues in the adult, while glypican-2 expression is
13
limited to axons and growth cones in the developing brain (Fransson et al.
2004). Patients with brachydactyly E (mental retardation and limb abnormalities) were found to lack glypican-1 (Syrrou et al. 2002). Glypican-3 is
expressed widely in the embryo (Pellegrini et al. 1998). Mutations in the
glypican-3 gene are linked to the Simpson-Golabi-Behmel syndrome that is
characterized by pre- and postnatal overgrowth (Pilia et al. 1996). Glypican3 deficient mice display many of the abnormalities associated with this human disease (Cano-Gauci et al. 1999). Glypican-4, -5 and -6 are expressed in
various organs in the embryo with restricted expression in adults (Fransson
et al. 2004).
Perlecan
The perlecan core protein carries up to four HS chains. The large size of
perlecan makes it a perfect linker molecule between the ECM and cell surface receptors (Iozzo et al. 2009). Indeed, perlecan mediates FGF signaling
and suppression of perlecan therefore reduces growth factor signaling leading to decreased tumor growth and angiogenesis (Aviezer et al. 1994;
Sharma et al. 1998). The importance of perlecan in developmental angiogenesis in zebrafish has been reported (Zoeller et al. 2008). On the other
hand, an anti-angiogenic fragment, endorepellin is derived from the Cterminus of perlecan (Mongiat et al. 2003). Null mutations in the perlecan
gene are lethal and result in severe developmental brain defects and skeletal
abnormalities (Arikawa-Hirasawa et al. 1999).
Agrin
Agrin is mainly expressed in the brain, lung and kidney (Gesemann et al.
1998) and is involved in clustering of acetylcholine receptors (Nitkin et al.
1987). Homozygous agrin-deficient mutant mice form defective neuromuscular synapses (Gautam et al. 1996).
Collagen XVIII
Expression of collagen XVIII is prominent in the vascular and epithelial
basement membranes of human and mouse tissues. Studies in mice have
shown that collagen XVIII is critical for normal blood vessel formation in
the eye. This may explain the eye defects seen in patients with Knobloch
syndrome (with loss-of-function mutations in collagen XVIII) (Fukai et al.
2002). Collagen XVIII is proposed to play a negative regulatory role in angiogenesis presumably due to release of its C-terminal anti-angiogenic fragment endostatin (O'Reilly et al. 1997).
Serglycin
Serglycin, the only known intracellular PG, is found in endothelial and hematopoietic cells and can bear heparin (highly sulfated HS species) as well
as CS chains (Kolset and Tveit 2008). Many studies have focused on sergly14
cin found in mast cells where it is involved in granulopoiesis and protease
storage (Henningsson et al. 2002; Abrink et al. 2004).
Interactions between heparan sulfate and proteins
As mentioned above, the negative charge of HS makes it prone to interact
with various proteins. HS-binding proteins include enzymes, cytokines,
morphogens, growth factors, matrix proteins, lipoproteins and disease associated proteins such as amyloid proteins (Lindahl and Li 2009).
Many studies of HS-protein interactions are performed with heparin, used
in clinics worldwide as an anticoagulant. A specific pentasaccharide sequence in heparin, responsible for binding to the anticoagulant protein antithrombin, was identified 30 years ago (Lindahl et al. 1980). However, this
type of specific HS-protein interaction is rare, rather HS domain organization and levels of sulfation appear the most important parameters for HSprotein interactions (Kreuger et al. 2006; Lindahl 2007).
HS is often described as a co-receptor for FGFs that is required for effective activation of the FGF receptor (Yayon et al. 1991; Rapraeger 1993).
Downstream FGF signaling includes the Ras-mitogen-activated pathway
kinase (MAPK) pathway where the extracellularly signal-regulated kinases
(ERK1/2) are considered responsible for the mitogenic response (Dailey et
al. 2005). Most studies have employed heparin oligosaccharides to study the
interaction between polysaccharide, FGFs and FGF receptors. Two different
crystallographic models have illustrated how this interaction occurs, one
symmetric model that contains two heparin molecules stabilizing the complex (Schlessinger et al. 2000) and one asymmetric model with one heparin
molecule that has a bridging function (Pellegrini et al. 2000). Highly sulfated
HS chains appear important for efficient formation of ternary complexes and
sustained downstream signaling (Jastrebova et al. 2006; Jastrebova et al.
2010). 2-O-Sulfated IdoA was recently shown to be critical for FGF2 mediated signaling which may explain the observed phenotype in the epimerase
and the 2-O-sulfotransferase null mice. However, it is not clear if the poor
response to FGF is directly due to lack of IdoA or indirectly due to disturbed
domain organization along the HS chain (Bullock et al. 1998; Li et al. 2003;
Jia et al. 2009).
The expression of the potent angiogenic vascular endothelial growth factor (VEGF) is induced by hypoxia, oncogenes and different cytokines.
HSPGs can mediate angiogenesis through binding to VEGF (except
VEGF121 that lacks the HS-binding domain) (Houck et al. 1992; Stringer
2006). Furthermore, HS polarizes and guides sprouting endothelial cells to
initiate vascular branch formation (Ruhrberg et al. 2002). The HS structure,
important for VEGF binding, includes NS domains divided by a NA/NS
domain (Robinson et al. 2006).
15
Heparanase
Functions
Heparanase was first identified in mouse mastocytoma, as an endo-β-Dglucuronidase capable of fragmenting macromolecular heparin (Ogren and
Lindahl 1975). Subsequently, an enzyme was identified that could similarly
cleave also HS chains (Oldberg et al. 1980), and more recent work pointed to
a single, common enzyme species (Gong et al. 2003).
Heparanase is expressed in all tissues with significantly varied levels. The
cleavage of HS chains generates fragments of 10-15 sugar units (4–7 kDa) in
size (Fux et al. 2009b). Heparanase is not essential for animal development
(Zcharia et al. 2009) but the enzyme is involved in embryonic implantation
and development, wound repair, HS turnover, tissue remodeling and hair
growth (Zcharia et al. 2004; Zcharia et al. 2005a; Zcharia et al. 2005b;
Escobar Galvis et al. 2007). On the other hand, heparanase cleavage of HS
directly influences the many HS-dependent biological functions (Li and
Vlodavsky 2009). Transgenic mice that overexpress heparanase appear normal, are fertile and have a normal life span (Zcharia et al. 2004). Nonetheless, these mice show several physiological changes such as reduced fat
deposition (Karlsson-Lindahl, personal communication), accelerated hair
growth, excess branching and duct-widening of the mammary glands associated with enhanced neovascularization and disruption of the epithelial basement membrane compared with control mice. Further studies revealed that
the mice have elevated wound angiogenesis (Zcharia et al. 2005b). The role
of heparanase in angiogenesis is linked to ECM degradation and endothelial
cell migration (vascular sprouting) as well as release of active FGF and
VEGF from the ECM (Elkin et al. 2001). Furthermore, heparanase cleaves
HS to release fragments that potentiate FGF receptor dimerization and formation of ternary complexes (Escobar Galvis et al. 2007; Nasser 2008).
Heparanase also influences angiogenesis through a non-enzymatic mechanism, involving regulation of the VEGF gene (Zetser et al. 2006). The impact of heparanase function was further demonstrated in heparanase knockout mice (Zcharia et al. 2009). These mice do not show obvious phenotypes,
are fertile and have a normal lifespan but show elevated expressions of
MMP-2, -9 and -14. Partially overlapping functions of MMPs and heparanase may explain the mild phenotype in the mice.
Only one gene (HPSE) encodes for a protein with heparanase activity. A
second gene was found coding for heparanase-2, a protein that shares 40%
sequence identity with heparanase (McKenzie et al. 2000). Interestingly, a
recent report showed that heparanase-2 is devoid of enzymatic activity but
has a high affinity for HS and heparin and thereby has potential to actually
inhibit heparanase activity (Levy-Adam et al. 2010).
16
Molecular properties
Heparanase is synthesized as a 65 kDa pro-enzyme and has a predicted TIMbarrel fold. Two acidic residues, a proton donor at Glu 225 and a nucleophile
at Glu 343 are involved in the catalytic activity of heparanase and the enzymatic pH optimum is between 5.0 and 6.0 (Hulett et al. 2000). Pre-proheparanase is targeted to the ER via its own signal peptide, proteolytic
cleavage at the N-terminus cleaves off the signal peptide and the 65-kDa
latent heparanase is then shuttled to the Golgi. Heparanase is secreted via
vesicles that bud off from the Golgi and interacts with cell surface HSPGs.
Two HS binding domains have been identified in heparanase; Lys 158-Asn
162 and Gln 270-Lys 280 (Levy-Adam et al. 2005). The heparanase-HSPG
complex is rapidly endocytosed and ends up in the lysosomes where the
proteolytic processing occurs. A 6 kDa linker segment is cleaved off by
cathepsin-L and the heterodimeric, active form of heparanase that consists of
a 50 kDa and an 8 kDa subunit, participates in HS degradation (Levy-Adam
et al. 2003; McKenzie et al. 2003; Gingis-Velitski et al. 2004b; Zetser et al.
2004; Abboud-Jarrous et al. 2008). Lipoprotein receptor-related protein and
mannose 6-phosphate receptors can also mediate binding to the secreted
heparanase precursor and convey it to the intracellular sites for processing
and activation (Vreys et al. 2005). These high affinity receptors are used at
low heparanase concentrations, while the low affinity HSPGs receptors are
important at higher heparanase concentrations (Ben-Zaken et al. 2008). The
active heparanase is stably stored in the lysosomal compartment, which may
be a way to regulate its secretion and extracellular activities (Goldshmidt et
al. 2002). Indeed, active heparanase can be secreted after stimulation with
physiological concentrations of ADP and ATP (Shafat et al. 2006). The finding that heparanase can be secreted in response to a stimulus indicates that
the extracellular levels of heparanase are tightly regulated. Furthermore, the
gene expression of heparanase can be regulated by early growth response
transcription factor, p53 and inflammatory cytokines (Li and Vlodavsky
2009). A newly identified COOH-terminal domain in heparanase seems to
play roles in heparanase secretion and activity but also in facilitating Aktphosphorylation, cell proliferation and tumor xenograft progression (Fux et
al. 2009a).
Substrate specificity
Heparanase cleaves the glycosidic bond between a GlcA and a GlcNS in
HS/heparin chains. Several groups have attempted to identify the optimal
substrate for heparanase cleavage. A detailed study of heparanase substrate
specificity used variously modified capsular polysaccharide generated from
Echerichia coli, K5, as target (Pikas et al. 1998). The unmodified backbone
that consists of GlcA and GlcNAc residues was not cleavable. Similarly
resistant was a product obtained after modification of the backbone by Ndeacetylation/N-sulfation and partial C5-epimerization of GlcA to IdoA.
17
Instead, structures that remained N-acetylated but were chemically (presumably randomly) O-sulfated were susceptible to heparanase cleavage. A
2-O-sulfated hexuronic acid, two monosaccharide units away from the
cleavage site towards the reducing end seemed to be crucial for heparanase
recognition (Figure 2). Other investigators, using purified recombinant
heparanase and structurally defined oligosaccharides isolated from porcine
intestine or bovine kidney, found that highly sulfated structures located on
both sides of the actual cleavage site are important (Okada et al. 2002) (Figure 2). Recently, heparanase specificity was studied using purified recombinant heparanase and polysaccharide substrates that were modified by recombinant HS biosynthetic enzymes (Peterson and Liu 2010). These investigators suggested that heparanase cleaves the linkage between GlcA and a 3-Oor 6-O-sulfated GlcNS. Cleavage was claimed to be precluded when the
hexuronic acid unit toward the reducing end from the cleavage site was a
sulfated IdoA. However, our recent investigation shows that a HS substrate
lacking IdoA residues is a significantly poorer substrate than a HS substrate
with GlcA residues (Li et al, unpublished data).
Figure 2. Proposed substrate sequence for heparanase cleavage (Pikas et al. 1998;
Okada et al. 2002; Gong et al. 2003).
18
Heparan sulfate in pathological conditions
Inflammation
HS modulates several steps in the inflammatory process. Close to the inflammatory site, P-selectins and E-selectins are expressed by endothelial
cells. Glycosylated ligands on leukocytes bind weakly to the selectins and
the leukocytes initiate their rolling along the vessel wall. HS in the vessel
wall can interact with L-selectin on the rolling leukocytes and facilitate the
rolling process. In the activation phase, chemokines are presented, bound to
endothelial HS and induce leukocyte integrins to bind adhesion molecules on
endothelial cells, which results in leukocyte arrest and transmigration
through the endothelial cell layer (Ley et al. 2007). Heparin acts as an antiinflammatory agent in asthma, ulcerative colitis and burns probably by competitive binding to chemokines (Young 2008) as well as P- and L-selectins
(Wang et al. 2002). Indeed, HS interacts with several chemokines and cytokines including macrophage inflammatory protein (MIP)-1α, RANTES, interleukin (IL)-2, IL-8 and IL-10 (Najjam et al. 1998; Spillmann et al. 1998;
Salek-Ardakani et al. 2000; Vives et al. 2002; Stringer et al. 2003). Our recent study demonstrates that an intravascular MIP-2 chemokine gradient on
endothelial HS directs crawling of leukocytes toward transmigration sites
(Massena et al. 2010).
Heparanase is upregulated in several inflammatory conditions and may
potentially affect inflammatory reactions in different ways. HS degradation
by heparanase can affect the architecture of the ECM and thereby facilitate
migration of inflammatory cells (Li and Vlodavsky 2009). Furthermore,
increased heparanase activity results in shorter HS chains (Escobar Galvis et
al. 2007). Fragmented endotheial HS may have impaired ability to interact
with chemokines and L-selectin, which would result in a reduced inflammatory response.
Amyloid diseases
HSPGs are present in all kinds of amyloid deposits, regardless of the type of
amyloidogenic protein that is present (van Horssen et al. 2003). HSPGs colocalize with amyloid in the prion diseases, including Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome and scrapie (Snow et al. 1990).
HSPGs are also present within islet amyloid deposits in type 2 diabetes mellitus (Young et al. 1992). HS binds to the N-terminal of the pro-islet amyloid
polypeptide and stimulates amyloid formation (Abedini et al. 2006). Heparanase overexpression makes mice resistant to induced AA-amyloidosis, which
suggests that the HS chains are involved in AA amyloid deposition (Li et al.
2005).
19
Alzheimer’s disease
The amyloid disorder AD is the most common form of dementia in the aging
population. The patients suffer from cognitive deterioration, memory loss
and behavioral changes. Senile plaques, neurofibrillary tangles (accumulation of hyperphosphorylated tau), cerebral amyloid angiopathy and inflammatory reactions are pathological characteristics of AD. Aβ is the major
protein component of the senile plaques and has a central role in the pathology of AD. The known mutations in the gene coding for the amyloid precursor protein (APP) increase the production and/or deposition of Aβ in the
brain, also seen in patients with sporadic AD. Current treatments for AD
may relieve symptoms but do not alter the course of the disease. Attempts to
delay disease progress include measures aiming at reducing Aβ production,
inhibiting Aβ aggregation, enhancing Aβ-clearance but also immunotherapy
(Selkoe 2001; Citron 2010; Crews and Masliah 2010).
Generation of Aβ
Aβ is formed by proteolytic cleavage of APP, a type 1 integral membrane
glycoprotein expressed in all human tissues and is localized to the plasma
membrane, ER, Golgi apparatus and mitochondria. APP is suggested to be
involved in cell adhesion, synaptic function and neural plasticity, although
its exact biological functions are unclear. Alternative splicing gives rise to
several isoforms, of which the APP695 isoform is mainly expressed in neurons (Young and Bennett 2010). The human APP gene is located on chromosome 21 and over 20 mutations in this gene have been implicated with the
hereditary form of familial AD and the related disease hereditary cerebral
amyloid angiopathy. All these mutations are amino acid substitutions within
or close to the Aβ domain (Brouwers et al. 2008).
The processing of APP that generates amyloid plaques can be referred to
as the amyloidogenic pathway and requires release of the Aβ domain from
APP by the β- and γ-secretases (Figure 3A). BACE1 (β-site APP-cleaving
enzyme or β-secretase) is a transmembrane aspartyl protease. It cleaves APP
giving rise to a soluble fragment called APPsβ and a remaining C-terminal
fragment (β-CTF, C99). β-CTF is further cleaved by γ-secretase to yield Aβ
peptides that range from 38 to 43 amino acids and an APP intracellular domain (AICD). γ-Secretase is a large protein complex comprised of presenilins-1 and -2 (PS-1 and PS-2), nicastrin, presenilin enhancer 2 and anterior
pharynx-defective phenotype-1. PS-1 knockout mice have reduced Aβ levels
and present altered skeletal and brain development. In humans, mutations in
PS-1 are responsible for over 50% of familial AD cases. Mutations in PS-1
and PS-2 lead to increased Aβ42 production over Aβ40, possibly by inducing conformational changes in the γ-secretase complex. The complex is also
responsible for processing Notch. Non-specific γ-secretase inhibitors inter-
20
fere with Notch cleaving and signaling and affect the gastrointestinal tract,
thymus and spleen.
An alternative way to process APP is the non-amyloidogenic pathway
(Figure 3A), which involves α-secretase (also called ADAM9, ADAM10,
ADAM17/TACE) cleavage of APP at Leu 17. This cleavage releases a soluble APPsα and leaves a remaining peptide (α-CTF, C83) that is further
processed by γ-secretase to yield a p3 fragment and AICD (Thinakaran and
Koo 2008; Young and Bennett 2010).
A strongly confirmed genetic risk factor for early and late-onset AD is the
ε4 allele of apolipoprotein E. This molecule functions as a ligand in receptor-mediated endocytosis of lipoprotein particles in normal physiology
whereas in AD it is believed to affect Aβ aggregation and clearance (Kim et
al. 2009).
Most of the transgenic mouse models used for investigation of AD are
based on the familial AD mutations and overexpress APP, PS-1 or PS-2
alone or together. The Tg2576 mouse model of AD, carrying the Swedish
double mutation K670N/M671L in APP, develops plaques as well as vascular deposits at 9-12 months of age. If a mutation in the PS-1 gene is introduced as well, the plaques develop as early as at four months of age (Crews
and Masliah 2010).
Figure 3. Schematic illustration of APP processing. (A) APP processing via the nonamyloidogenic pathway (α- and γ-secretases) and the amyloidogenic pathway (βand γ-secretases). (B) Sequence of Aβ, enlarged letters highlight the HHQK domain
that interacts with HS.
21
Aβ toxicity
Aβ can spontaneously aggregate into β-sheet-rich fibrils in vitro which are
similar to the aggregates found in amyloid plaques. These fibrils were long
believed to cause neurodegeneration in AD but emerging data suggest that
prefibrillar, soluble forms of Aβ are the key components in AD pathogenesis
(Caughey and Lansbury 2003; Sakono and Zako 2010). This is supported by
data showing that the number of plaques in a particular region of the brain
does not correlate with neuron death, synaptic loss or cognitive impairment
(Terry 2000). In addition, soluble Aβ oligomers are cytotoxic, cause synaptic
loss, impaire cognitive functions, destabilize neuronal networks and inhibit
long-term potentiation (Selkoe 2008; Palop and Mucke 2010; Sakono and
Zako 2010). The mechanisms underlying the toxic effects are not clear but
possible scenarios include formation of membrane pores, mitochondrial and
lysosomal dysfunction as well as signaling pathway failure (Crews and
Masliah 2010).
Inflammation in Alzheimer’s disease
The knowledge regarding the contribution of inflammation to AD pathogenesis is limited. It is known that activated microglia associate with senile
plaques and produce pro-inflammatory cytokines such as IL-1 and tumor
necrosis factor-α. According to the original view, this inflammatory reaction
was neurotoxic and believed to accelerate the disease. However, it is now
suggested that the inflammatory response actually can be beneficial and
hence promote neuronal survival. The role of circulating monocytes infitraing the brain in the immune reaction is currently being investigated
(Cameron and Landreth 2009; Schwartz and Shechter 2010).
Involvement of heparan sulfate in Alzheimer’s disease
More than 20 years ago, HS was found deposited in amyloid plaques of AD
(Snow et al. 1988). Direct interaction between Aβ and HS was demonstrated
to involve a motif in Aβ, HHQK (Figure 3B) (Giulian et al. 1998). The interaction between Aβ and HS also affects the aggregation state of the peptide, the sulfate moities promoting fibril formation (Castillo et al. 1999;
McLaurin et al. 1999). In vivo and in vitro data have shown that heparin can
attenuate Aβ toxicity in cell cultures as well as reduce plaque burden in a
mouse model of AD (Bergamaschini et al. 2002; Bergamaschini et al. 2004).
HS, CS and DS as well as a number of sulfated compounds and dyes have
been demonstrated to attenuate Aβ toxicity in cell cultures (Pollack et al.
1995; Woods et al. 1995). In addition, glypican has been suggested to mediate Aβ toxicity in PC12 cells (Schulz et al. 1998). There are divergent results
regarding the effect of HS/heparins on BACE1 activity. HS was shown to
inhibit BACE1 activity in neuroblastoma cells (Scholefield et al. 2003) while
22
other groups reported that heparin activates BACE1 (Leveugle et al. 1997;
Beckman et al. 2006).
Perlecan, agrin, glypicans, syndecans and collagen XVIII have all been
associated with senile plaques. Agrin and glypican-1 are found in both nonfibrillar and fibrillar senile plaque, whereas the syndecans are only associated with fibrillar senile plaques (Castillo et al. 1997; Cotman et al. 2000;
van Horssen et al. 2002; van Horssen et al. 2003). Furthermore, HS is found
associated with neurofibrillary tangles (Perry et al. 1991). Interestingly, a
recent study reported that neurofibrillary tangles are present in the brains of
patients with mucopolysaccharidosis IIIB (an autosomal recessive disease
where HS is accumulated in the lysosomes) (Ohmi et al. 2009).
Tumor progression and metastasis
HS, HSPGs and heparanase are widely associated with different types of
cancers. HSPGs are crucial to the integrity of the basement membrane, proliferation of cells and angiogenesis, all key factors in tumor progression and
metastasis (Li 2008).
Expression of heparan sulfate proteoglycans
Syndecan-1 is upregulated in different types of human cancers and the soluble ectodomain of syndecan-1 is present in the serum of several malignancies. Shed syndecan-1 is biologically active and can promote tumor growth
and metastasis (Yang et al. 2002). Expression and shedding of syndecan-1
are proposed to be induced through heparanase-mediated stimulation of
phosphorylated ERK, which in turn can activate MMP-9 (Mahtouk et al.
2007; Purushothaman et al. 2008). Both glypican-1 and -3 have been implicated in different cancers (Filmus and Selleck 2001). For example, glypican1 is upregulated in breast and pancreatic cancer and presumably promotes
growth factor signaling in cancer cells (Kleeff et al. 1998; Matsuda et al.
2001). Glypican-3 on the other hand is downregulated in breast and ovarian
cancers while upregulated in hepatocellular carcinoma and neuroblastoma
(Theocharis et al. 2010). Agrin is strongly associated with tumor angiogenesis in the liver (Iozzo et al. 2009).
Heparanase activity
Increased heparanase expression in human tumors is linked to metastasis,
tumor angiogenesis and reduced postoperative survival of cancer patients
(Vlodavsky et al. 2008; Fux et al. 2009b). High heparanase expression in
tumors may be ascribed to mutational inactivation of the tumor suppressor
p53 during cancer development; this mutated form activates the heparanase
promotor and hence heparanase expression (Baraz et al. 2006). Heparanase
activity can induce angiogenesis by releasing growth factors such as FGF
and VEGF upon heparanase cleavage (Vlodavsky et al. 1987; Folkman et al.
23
1988; Vlodavsky et al. 1996) or by increasing shedding of syndecan-1 with
bound VEGF (Purushothaman et al. 2010). Lymph angiogenesis is stimulated by heparanase-induced expression of VEGF-C (Cohen-Kaplan et al.
2008). Furthermore, HS sulfation is increased as a consequence of heparanase overexpression, which promotes formation of ternary complexes with
FGF1 or FGF2 and FGF receptor-1 (Escobar Galvis et al. 2007).
Heparanase promotes Akt-dependent endothelial cell invasion and
upregulation of VEGF through a non-enzymatic mechanism involving Src
activation (Gingis-Velitski et al. 2004a). Furthermore, a newly identified
splice variant of heparanase, T5, devoid of enzymatic activity has protumorigenic properties (Barash et al. 2010).
Hypoxia
During tumor progression the interior of the tumor becomes hypoxic due to
insufficient oxygen supply. Hypoxic conditions in tumors are associated
with increased stability and activity of the hypoxia-inducible factor-1α (HIF1α), a transcription factor inducing genes involved in cell invasiveness and
angiogenesis. HIF consists of two sub-units, HIF-1α and HIF-1β; the HIF1α subunit is degraded under normoxic conditions but stabilized under hypoxic conditions. This subunit binds to hypoxia responsive elements present
on over 100 genes involved in erythropoiesis, angiogenesis, glucose metabolism and apoptosis and promotes their transcription (Ke and Costa 2006;
Liao and Johnson 2007; Rademakers et al. 2008; Finger and Giaccia 2010).
Few studies have evaluated the role of HS in hypoxia. Yet, HIF-1α is
suggested to augment the FGF-2 response of endothelial cells by increasing
the gene expression of HS biosynthetic enzymes (Li et al. 2002). Another
example of hypoxia-induced change in HS structure is increased synthesis of
antithrombin-binding sequences (Karlinsky et al. 1992). Furthermore, increase of perlecan and syndecan-2 expression in cells in response to hypoxia
was recently reported (Asplund et al. 2009; Kaur et al. 2009). Endorepellin,
the anti-angiogenic peptide derived from the C-terminus of perlecan, decreases tumor angiogenesis and increases tumor hypoxia (Bix et al. 2006).
Hypoxia can also induce heparanase expression in adult rat hippocampus
(Navarro et al. 2008).
It is worth mentioning that several of the HIF-1α induced genes such as
VEGF, insulin growth factor 2, fibronectin, angiopoetin-like 4 and plateletderived growth factor-BB are also HS-binding proteins highly implicated in
cancer (Bernfield et al. 1999; Cazes et al. 2006; Ke and Costa 2006;
Abramsson et al. 2007; Semenza 2010).
The Spalax animal model
Spalax, a subterranean mole rat, has through natural selection developed
physiological changes to deal with hypoxic stress. Since many of the
changes are identical to those found in human tumors, this animal is an ex24
cellent mammalian model organism for studying hypoxia tolerance and tumor development. Spalax tolerates low oxygen levels for long periods of
time; under laboratory conditions Spalax can survive at 3% oxygen for at
least 11 hours without any deleterious effects or behavioral changes, while
Rattus is near death after 2.5 hours (Avivi et al. 1999).
Many genes and proteins that are involved in increasing oxygen supply to
tissues are preferentially expressed in Spalax. HIF-1α is expressed at high
levels. Erythropoietin gene expression is regulated by HIF. Unlike Rattus,
Spalax can respond to hypoxia by rapid and sustained expression of
Erythropoietin presumably reflecting the need for erythrocyte production
during drastic fall in oxygen supply (Shams et al. 2004). Already at the fetal
stage, Spalax liver and kidney express higher levels of erythropoietin mRNA
than Rattus indicating that fetal erythropoiesis is important for adaptation to
hypoxic stress (Shams et al. 2005).
Spalax has a high expression of VEGF and an increased density of blood
vessels although VEGF expression is not increased during hypoxic stress
(Avivi et al. 2005b). This suggests that sufficient vascularization balances
the effects of hypoxia. Similarly, the expression of the VEGF receptor 2 was
higher in Spalax muscle than Rattus muscle under normoxia, but did not
change after hypoxia was induced (Band et al. 2008).
The tumor suppressor gene p53 controls cellular responses to DNA damage and hypoxia. Upregulation of wild-type p53 during hypoxia results in
apoptosis. p53 mutations are the most common genetic alteration in human
cancers. Intriguingly, mutations in the Spalax p53 were reported to be identical to those found in human cancers. A mutation in the DNA binding domain
in the Spalax p53 gene exchanges Arg for Lys at position 174, which appears to enhance transcription of cell cycle arrest and p53 stabilization/homeostasis genes, but reduce transcription of apoptosis genes (AshurFabian et al. 2004; Avivi et al. 2005a; Avivi et al. 2007).
Heparanase is highly expressed in Spalax compared to its scarce expression in normal human tissues (Nasser et al. 2005). The heparanase splice
variants that have been identified in Spalax may reflect the need for regulation of heparanase activity in response to hypoxic stress. One of the splice
variants has a dominant negative effect on the wild-type enzyme and inhibits
HS degradation, tumor growth and metastasis (Nasser et al. 2009) whereas
another splice variant lacks enzymatic activity but enhances tumor growth
(Nasser et al. 2005).
Heparan sulfate as a potential therapeutic target
Interfering with HS function is a chosen strategy for developing drugs aimed
at treating cancer and amyloid diseases such as AD. Variants of heparin and
HS mimetics are used for the purpose of binding HS ligands, thus competing
with endogenous HS.
25
The low molecular-weight heparin Neuroparin penetrates the blood-brain
barrier and prevents abnormal tau protein formation seen in AD (Dudas et al.
2002). The low molecular-weight heparin, Enoxaparin, attenuates neurotoxicity and proinflammatory activity of Aβ and is under evaluation in preclinical studies (Bergamaschini et al. 2009). However, the HS mimetic tramiprosate (Alzhemed™) failed to show significant results in Phase III clinical
trials (Sabbagh 2009). HS mimetics are efficient inhibitors of AA amyloidogenesis and have been reported to reduce the rate of progression of AA amyloid renal failure in clinical trials (Kisilevsky et al. 2007).
Low molecular weight heparins can prolong survival of cancer patients.
The mechanisms behind this effect are not fully explored but are likely to
include competition with endogenous HS for binding to various ligands involved in tumor progression and metastasis (Casu et al. 2008). Heparin reduces the number of lung metastases in rodents injected with melanoma,
sarcoma, breast and colonic tumor cells (Mousa and Petersen 2009). Heparin
can possibly mediate anti-metastatic effects by inhibiting P- and L-selectins,
known to facilitate the interaction between tumor cells, platelets and leukocytes (Stevenson et al. 2007).
Inhibition of heparanase by blocking the active site and the heparin binding domains holds promise for inhibition of both angiogenesis and metastasis (Vlodavsky et al. 2007). To avoid the anticoagulant activities of heparin,
N-acetylated and glycol-split heparins have been tested for inhibition of
myeloma growth in mice with promising results (Naggi et al. 2005). These
heparin variants effectively inhibit heparanase activity and fail to release
ECM-bound growth factors, and thus are potent antiangiogenic and antimetastatic agents. HS mimetics include the heparanase inhibitor PI-88, which
inhibits both angiogenesis and metastasis and is currently in clinical trials.
Other HS mimetics (tetra- and pentasaccharides) are under development and
their ability to inhibit heparanase, bind to growth factors and affect blood
coagulation is evaluated. Some of these compounds have shown potent antitumor effect in a mouse model of melanoma (Johnstone et al. 2010).
26
Present investigations
HS and the HS-degrading enzyme heparanase are implicated in many different pathological conditions. In AD pathology, HS plays roles in both amyloid deposition and Aβ toxicity. In cancer, HS and heparanase are involved
in several of the steps that facilitate tumor progression and metastasis. The
principal aims of this thesis were to gain deeper insights into AD and cancer
progression by elucidating the roles of HS in Aβ pathology and hypoxia.
Aims of the study
•
To investigate the roles of HS in Aβ deposition and Aβ toxicity
(Papers I and II)
•
To investigate the role of HS in hypoxia (Paper III)
27
Results and discussion
Heparan sulfate accumulation with Aβ deposits in Alzheimer's
disease and Tg2576 mice is contributed by glial cells (Paper I)
Amyloid plaques are frequently observed in AD and can be divided into two
morphological categories, neuritic and diffuse (Selkoe 2001). Various types
of HSPGs are expressed in both kinds of plaques (van Horssen et al. 2003)
and HS is believed to catalyze fibrillization and facilitate early deposition of
Aβ (McLaurin et al. 1999). It is suggested that diffuse plaques represent an
early stage of amyloid deposition, which subsequently evolve into neuritic
plaques with dense cores. Another possibility is that the different types of
plaques form independently of each other (Armstrong 1998; Selkoe 2001).
To gain deeper understanding of the role of HSPGs in Aβ deposition we
examined different types of plaques in cases of sporadic and familial AD and
in a mouse model of AD (Tg2576) by immunohistochemistry.
Image analysis of sporadic AD revealed that HS had a 10-fold higher tendency to accumulate with Aβ40 neuritic plaques than Aβ42 diffuse plaques.
In the case of Swedish APP mutation AD, we found that HS associated with
all investigated Aβ40 neuritic plaques and occupied 60% of the plaque area.
30% of the Aβ42 diffuse plaques were positive for HS and occupied less
than 5% of the plaque area. In both sporadic AD and Swedish APP mutation
AD we found that the cores of the neuritic plaques associated with expression of glypican-1, syndecan-3 and GFAP (an astrocyte marker).
Tg2576, a mouse model of the Swedish mutation where the Aβ load is
high, had a distinct plaque morphology, referred to as “doughnut-shaped”
deposits. The plaque capsules stained positive for both Aβ40 and Aβ42,
glypican-1 and syndecan-3. GFAP positive cells surrounded the plaques
while CD11b-positive microglia cells were detected throughout the plaque
body.
In the patient with PS-1 Δ9 AD, diffuse “cotton wool” plaques were
found with almost exclusive staining for Aβ42. HS was associated with
Aβ42 in 20% of the plaques but occupied only 3% of the total area of these
plaques. We did not detect any expression of glypican-1, syndecan-3 or
GFAP within these plaques.
Because glial cells were identified as the cellular source of HS, we
wanted to evaluate the effect of Aβ exposure on HSPG expression. We isolated murine primary glial cells and stimulated them with Aβ. Both glypican1 and syndecan-3 levels were increased upon Aβ exposure as demonstrated
by Western blotting.
From these studies, we suggest that Aβ accumulation in amyloid deposits
results in elevated levels of the cell surface HSPGs glypican-1 and syndecan3 on glial cells. Associated HS chains accumulated, predominantly with
28
Aβ40, in neuritic plaques. The lack of a glial response in diffuse plaques
explains the absence of HSPGs and the low HS content of these plaques. The
diffuse plaques have been candidate precursors of neuritic plaques
(Armstrong 1998). The absence of HS in diffuse plaques suggests that HS is
not initiating plaque formation and implies that different types of plaques
develop independently of each other. An alternative interpretation is that the
shift from a diffuse plaque to a neuritic plaque depends on recruitment of
glial cells with subsequent HSPG upregulation. Indeed, preliminary data
from our heparanase overexpressing mouse imply that HS is important for
mediating an inflammatory response to Aβ, whereas heparanase overexpression attenuates such response (Zhang et al, unpublished data). It is suggested
that microglia participate in the formation of amyloid plaques by internalization of Aβ but also by driving fibrillogenesis within the plaque (Nagele et al.
2004). Although this needs further studies, HS expressed on glial cells is
likely to participate in these processes.
Heparan sulfate mediates amyloid-beta internalization and
cytotoxicity (Paper II)
Previous studies suggested that heparin and other GAGs may protect cells
from Aβ toxicity (Pollack et al. 1995; Woods et al. 1995; Bergamaschini et
al. 2002). In this study, we further investigated the role of HS in Aβ toxicity.
We used the well-established cell line psgD-677 that lacks HS due to a
mutated EXT1 gene (Lidholt et al. 1992; Wei et al. 2000). Aβ exposure induced cell death in wild-type cells, while the HS-deficient cells were resistant to the treatment. Moreover, only the wild-type cells were able to efficiently internalize the Aβ peptide. The pgsD-677 cells express three-fold
higher levels of CS (Lidholt et al. 1992), suggesting that CS is not capable of
mediating Aβ internalization and cytotoxicity. A recent study has demonstrated that plaque formation is initiated by cellular internalization of Aβ into
multivesicular bodies, where it starts to form fibrils and eventually causes
cell death (Friedrich et al. 2010). This internalization is dependent on endocytosis or phagocytosis, processes known to involve HS (Ji et al. 1993; Poon
et al. 2010). Our data clearly demonstrate the essential role of HS in Aβ internalization.
We proceeded to test the importance of cell surface HS chains for Aβ interaction by using HEK293 cells that stably express heparanase and thus
carry shorter HS chains. Incubation with Aβ had a modest toxic effect on
heparanase overexpressing cells compared with HEK293 wild-type cells.
This difference can possibly be explained by the release of HS chains with
bound Aβ from the cell surface upon heparanase cleavage. Alternatively, the
truncated HS chains generated by heparanase cleavage may bind weaker to
Aβ than wild-type chains. Yet another possibility is that heparanase overex29
pression results in accelerated turnover of HSPGs, as shown for heparanase
overexpressing mice (Escobar Galvis et al. 2007), which in turn would affect
Aβ uptake.
The importance of cell surface HS in mediating Aβ uptake and toxicity
was further illustrated in a third set of experiments using an endothelial cell
line. Cell death was evaluated by flow cytometry after Aβ exposure in the
presence or absence of heparin using annexin V and propidium iodide staining for apoptosis/necrosis. Added heparin clearly increased the amount of
viable cells. Similar results were obtained if the cells were pretreated with
heparin or if heparin was added together with Aβ. Furthermore, heparin prevented internalization of Aβ, presumably through inhibiting Aβ interaction
with cell-surface HS. Notably, the chain length of heparin is crucial to increase cell survival; heparin oligomers of 6- and 12-mers were ineffective
while 18-mers and full-length heparin (corresponding to ∼50 sugar units)
efficiently protected the cells. This size requirement is an important consideration in development of HS-based drugs aimed at ameliorating AD pathology. The protective effect of heparin on endothelial cells is potentially significant in view of the accumulation of Aβ in cerebral vessels that contributes to cerebral amyloid angiopathy (Thal et al. 2008).
In the above experiments, three different preparations of Aβ40 were used
at various states of aggregation. The non-aggregated preparation (na-Aβ) and
the aggregated preparation (a-Aβ) were toxic to the cells and were detected
intracellularly. These preparations contain oligomeric, soluble forms of Aβ.
The third preparation, agitated and aggregated Aβ (aa-Aβ) was toxic to the
cells but could not be detected intracellularly. It is possible that small
amounts of toxic oligomeric Aβ were present also in this preparation but at
levels below the threshold for detection by immunocytochemistry. In agreement with our results, current notion favors the oligomeric species of Aβ as
the toxic element (Sakono and Zako 2010).
Molecular structure of heparan sulfate from Spalax. Implications
of heparanase and hypoxia (Paper III)
In this study, we analyzed HS from the subterranean Spalax mole rat. HS
chain length, domain structure and disaccharide composition from the animal indicated a high activity of heparanase. Previous findings have demonstrated a high expression of heparanase in several Spalax tissues (Nasser et
al. 2005). Our structural data verified that the high expression could be correlated with high enzymatic activity. Spalax has been extensively studied for
its striking physiological adaptations to a hypoxic environment. We hypothesized that the high expression of heparanase could be a hypoxic adaptation, resulting for example in stimulated angiogenesis and release of growth
factors bound to HS. Spalax HS was highly sulfated and shorter HS chains
30
were detected in the kidney and liver compared with the same tissues in
mice. These observations are in line with previous findings of HS changes in
heparanase overexpressing mice and cancer tissues (Escobar Galvis et al.
2007). To test if such changes could indeed be ascribed to hypoxia, we used
HEK293 cells, mock-transfected and transfected with Spalax heparanase.
The Spalax heparanase transfected cells showed extensively shorter chains
than the mock-transfected cells and the HS domain structure was modulated
toward patterns typically seen in HS from Spalax organs. Furthermore,
phosphorylation of ERK was increased in the heparanase-transfected cells,
indicating that high heparanase activity stimulates release of growth factors
and subsequent down-stream signaling. Interestingly, HS from the mock
cells cultured in hypoxic condition showed a HS domain structure that indicated increased heparanase activity and had upregulated heparanase mRNA.
Our findings imply that increased heparanase expression and activity result
in increased MAPK/ERK activity as a response to the hypoxic environment.
Indeed, high heparanase activity has recently been reported to increase phosphorylation of ERK that in turn results in increased MMP-9 expression and
syndecan shedding, implicated with invasion and angiogenesis of myeloma
cells (Purushothaman et al. 2008; Purushothaman et al. 2010). Whether
MMP expression and syndecan shedding are enhanced in Spalax remains to
be elucidated.
31
Concluding remarks and future perspectives
HS is variously involved in physiological as well as pathological processes.
Diseases such as multiple hereditary exostoses, Simpson-Golabi-Behmel
syndrome, brachydactyly E and Knobloch syndrome are directly linked to
mutations in HS biosynthetic enzymes or in HS core proteins. Further, HS
affects inflammatory conditions, amyloid diseases and cancer, by interacting
with key proteins, such as chemokines, amyloidogenic peptides and growth
factors. The HS-cleaving enzyme heparanase also influences HS functions,
for example by degrading basement membranes and releasing HS fragments
with bound growth factors.
In AD, HS codeposits with the amyloidogenic peptide Aβ within amyloid
plaques found in the brain of AD patients. We have further investigated the
role of HS in these amyloid plaques and established that HS found in the
plaques originates from glial cells (Paper I). This indicates that HS is involved in inflammation in AD, which is supported by our recent data showing that heparanase overexpression attenuates the inflammatory response to
Aβ (Zhang et al, unpublished data). Signaling pathways such as the
MAPK/ERK and TGF-β Smad2/3 pathways have previously been implicated
in the inflammatory response in AD (Webster et al. 2006; Town et al. 2008).
Whether changes in expression of HS and heparanase contribute to alterations in these pathways in AD remains to be elucidated.
HS and heparin have previously been implicated in Aβ cytotoxicity
(Bergamaschini et al. 2002). We demonstrated that HS mediates internalization and cytotoxicity of Aβ (Paper II). Cells that completely lack HS failed
to internalize the peptide and resisted Aβ toxicity. Moreover, truncated HS
chains due to heparanase overexpression, or addition of heparin together
with Aβ protected the cells from Aβ toxicity (Figure 4A). In order to elucidate if Aβ internalization and toxicity are dependent on specific HS structures, additional cell models that differ with regard to HS structure could be
employed. Cell-surface HS might also play a role in phagocytosis by glial
cells of Aβ and necrotic cells. Indeed, HS has previously been reported to be
involved in phagocytosis of necrotic cells (Poon et al. 2010).
Upregulated expression of heparanase in tumor tissues results (at least in
selected studied cases) in increased HS sulfation, which may facilitate formation of ternary complexes with growth factors and receptors (Escobar
Galvis et al. 2007). This is in agreement with our study (Paper III) of an
animal model for hypoxic tolerance, the Spalax mole rat, with a constitutive
32
high expression and activity of heparanase and associated highly sulfated
HS. In fact, we showed that heparanase expression leads to increased
MAPK/ERK activity, presumably a result of augmented cell-surface signaling. We demonstrated that heparanase expression is induced by hypoxia and
hypothesize that a similar condition occurs during tumor progression, associated with oxygen deficiency. The frequent and high heparanase expression
observed in human tumors may thus conceivably be ascribed to hypoxia.
Subsequent increase in MAPK/ERK activity would promote angiogenesis
and tumor growth (Figure 4B). Our study emphasizes the importance of developing heparanase inhibitors for anti-cancer therapy. Targeting of the
heparanase C-domain would possibly affect several heparanase functions
including secretion, activity, Akt phosphorylation, cell proliferation and
tumor progression (Fux et al. 2009a).
Changes in HS structure and expression, as well as heparanase activity
occur in different pathological processes without necessarily being the primary cause of the particular diseases. The changes are perhaps an attempt to
maintain homeostasis when the fine balance of molecular events in organs is
disturbed during disease. Earlier reports have implicated HS in both Aβ pathology and hypoxia and we have identified new important roles of HS in
these conditions. More detailed studies are needed to understand the mechanisms by which HS contributes to different pathological conditions, for example, how changes in HS structure affect different signaling pathways. We
showed that hypoxia can induce expression and activity of heparanase and as
a result change HS structure. Identifying other factors that can regulate
heparanase expression and activity is important for further understanding
heparanase functions. The complex and well-regulated HS biosynthesis indicates that changes within this system have the potential to affect both biological and pathological processes; a deeper knowledge regarding regulation
of HS biosynthesis is therefore needed.
33
Figure 4. Model of the different roles of HS in Aβ pathology and hypoxia. (A) Aβ
interacts with cell surface HSPGs mediating Aβ internalization and toxicity. Cells
that lack HS, overexpress heparanase, or are treated with heparin fail to internalize
Aβ and are resistant to Aβ toxicity. (B) (1) Hypoxia in tumor cells induces the expression of heparanase, which is secreted, and acts on the HS chains of HSPGs (2)
to release growth factors that through receptor interactions result in increased
MAPK/ERK activity (3). MAPK/ERK activity can induce expression of MMP-9 and
syndecan shedding and further stimulate tumor growth and angiogenesis (4).
34
Populärvetenskaplig sammanfattning
Heparansulfat (HS) är en kolhydrat som återfinns på cellytan och utanför
cellerna i kroppens alla vävnader. HS är starkt negativt laddat och binder till
en mängd proteiner som är viktiga för grundläggande biologiska processer. I
studier på möss har det visat sig att avsaknad av HS leder till att embryonalutvecklingen avstannar och embryot dör på ett mycket tidigt stadium. Dessutom verkar HS vara inblandat i flera olika sjukdomstillstånd så som cancer
och amyloida sjukdomar (sjukdomar där olika proteiner klumpar ihop sig
och förstör organens funktion).
Alzheimers sjukdom är en amyloidsjukdom som karaktäriseras av amyloida inlagringar i hjärnan. De amyloida inlagringarna består till stor del av
molekylen amyloid-β (Aβ), som också orsakar celldöd. Vi har undersökt hur
HS ansamlas i dessa amyloidinlagringar och hur HS påverkar Aβ-toxicitet.
Vi har även studerat hur HS påverkas av låga syrenivåer, hypoxi, något som
är vanligt i tumörer.
Studierna i denna avhandling har visat att HS finns utryckt på immunceller kring amyloidinlagringarna hos Alzheimerspatienter och att HS som finns
på cellernas yta möjligör för Aβ att ta sig in i cellerna och orsaka celldöd. Vi
har dessutom visat att hypoxi leder till att enzymet heparanas, som bryter ner
HS, ökar i mängd och aktivitet. Detta kan vara viktigt för att frigöra tillväxtfaktorer som behövs för bildning av nya blodkärl och därmed ökning av
syretillförseln.
Sammanfattningsvis så har studierna i denna avhandling gett nya inblickar i hur HS påverkar olika sjukdomsprocesser. Detta är viktig kunskap som
behövs för att utveckla läkemedel mot olika sjukdomstillstånd där HS deltar.
35
Acknowledgments
During my years as a PhD student at IMBIM I have met fantastic researchers
and wonderful colleagues. I would like to express my deepest gratitude to all
of you who supported me and made my PhD time full of happy memories.
A very inspiring lecture regarding heparan sulfate in amyloid diseases was
given by Ulf Lindahl during an evening course in 2004. A few months later,
I also had the luck to meet Jin-Ping Li, my main supervisor, and a very
helpful, passionate and talented scientist who trained me both in research
and how to make the most delicious dumplings. My exciting PhD projects
were planned by Jin-Ping and Ulf, my co-supervisor who always has excellent suggestions regarding projects, presentations and writing. Arriving to
the B9:3 corridor I was first seated in an office together with an intelligent
and wise French girl, Sabrina Bodevin, who also is a wonderful and very
fun friend that unfortunately nowadays lives in Malmö with her Robert.
Sabrina and I had too much fun and I was moved to a new office sitting next
to an extremely sweet girl named Juan Jia and a very nice, helping and
healthy old man; Anh-Tri Do. I also had the luck to meet Eva Gottfridsson,
a wonderful person and friend with invaluable knowledge about life and
isolating heparan sulfate. In the same corridor, Rebecka Hjelm, a kind and
caring person, taught me everything worth knowing about weddings! I also
had the chance to collaborate with two Alzheimer’s experts; Xiao Zhang
and Paul O’Callaghan, two exceptionally nice and helpful men who work
in the group of Lars Lannfelt together with my other collaborators and coauthors; Lars Nilsson and Martin Ingelsson. Fredrik Noborn joined JinPings group in 2006 and finally the “Sugarbabes” team had a fair chance to
win the “BMC-stafett”! Many helpful and pleasant colleagues were present
in the B9:3 corridor when I was there; Marta Busse, Sindhu Kurup, Nadja
Jastrebova, Emanuel Smeds, Elisabeth Roman, Martha Escobar Galvis,
Maarten Vanwildemeerch, Wei Sun, Sophia Schedin Weiss, Celestine
Chi, Per Jemth, Eva Hjertson, Gunilla Pettersson, Helena Grundberg
and Lena Nylund. However, in the end of 2007 it was not that many people
left in the corridor. During a conference in Rio De Janeiro I had very much
fun with Pernilla Carlsson; a PhD student in the lab of my examiner; Lena
Kjellén. Lena is a great scientist with a wonderful personality, sitting in the
D9:4 corridor, to which our group moved in 2008. The people in the new
corridor hated us for putting old heparan sulfate-analyzing equipment eve36
rywhere but they soon realized that heparan sulfate is the most fascinating
molecule. I was placed in an office together with Christoffer
Taaaaammmmmm, who I was supposed to marry?! He was constantly
arguing with the crazy and fun Irmeli Barkefors, maybe she was allergic to
him? When Anna Eriksson, a smart person and a fantastic friend, joined the
corridor, my life was divided into the time before and after Anna. Anna
started in the group of the helpful, skilled and sporty researcher Dorothe
Spillmann, and the atmosphere in the new corridor was now very nice!
Jimmy Larsson also contributed to the nice atmosphere even though not for
that long (he didn’t have the time). Thanks Tobias Bergström, Inger Eriksson, Peder Fredlund Fuchs, Johan Heldin, Cecilia Annéren, Maud
Forsberg, Xiao-qun Zhang, Ulrika Wallenquist, Karin Forsberg Nilsson,
Sebastian Le Jan, Johan Kreuger, Ludwig Peterson, Pär Gerwins, Lars
Lundin and Dagmar Sandbäck Pikas for contributing to the nice feeling in
the lab! We were all very pleased to have the amyloid experts, Robert Kisilevsky and John Ancsin from Canada as well as the nice researchers, Bo
Wang and Jian Wang from China visiting the lab. Starting from November
2009, I spent 10 months with my beloved son Valter and came back to the
lab to write this thesis. This time I was put in the quiet-room shared with
several nice persons; Katarina Holmborn Garpenstrand, Anders Dagälv,
Beata Filipek Gorniok and Zsolt Kasza. New pleasant persons had arrived
to our corridor; Audrey Deligny, Sara Pijuan Galitó, Ramesh Namburi,
Sara Thorslund and Natalia Strid while Rashmi Ramachandra and Juan
were away from the lab taking care of Lilly and Maanvi. During these years
I have had excellent technical and administrative help from Erika Engström, Kerstin Lidholt, Rehné Åkerblom, Barbro Lowisin, Marianne
Wigenius and Olav Nordli. I have shared many nice dinners and laughs
with my dear friend Kerstin Ahlgren during the 10 years we have been
studying in Uppsala. I have also shared many discussions about life with my
warm and caring friend Jenny Sågetorp during these years.
Jag vill även rikta ett stort tack till mina nära och kära utanför BMC.
Tack mina älskade vänner från Finspång, Jenny och Linda med familjer för
alla timmar i telefonen och för att vi alltid har så otroligt kul tillsammans!
Tack Sabina och Hanna med familjer i Göteborg för roliga midsommarfiranden och tjejträffar!
Tack till er som jag lärt känna genom Martin: Barndomsvännerna i Norrköping: för att ni är så sjukt roliga! FestU gänget och Fam All (för skojiga
fester i Göteborg och på Chalmers!
37
Tack älskade Farmor Maria och Mormor Gun för att ni är så otroligt snälla och omtänksamma!
Tack moster Ann-Marie och Benny, Fam Bengtsson och Fam Siegers för
att ni gör det så kul att träffa släkten!
Tack till mina svärföräldrar Leif och Annelie för all hjälp och för att jag
alltid känt mig så välkommen hos er!
Tack Madeleine och Bruce, jag har en otrolig tur som fått chansen att lära
känna er! Ni är underbara!
Tack älskade syster Moa för att det aldrig blir tråkigt när du är med och för
att du har samma sjuka humor som jag.
Tack mina älskade, underbara föräldrar Britt-Marie och Hans, för att ni gett
mig den bästa uppväxt man kan tänka sig och för att ni alltid ställer upp och
hjälper till med allt!
Tack älskade Martin, för att du är en så otroligt fin person. Tack älskade
lilla Valter, för att du gör mig så lycklig. Jag har världens bästa lilla familj.
38
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