Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, 3, 361-370
361
Proteoglycans and Amyloidogenic Proteins in Peripheral Amyloidosis
Francine Gervais*, Céline Morissette and Xianqi Kong
Neurochem Inc., 1375, autoroute transcanadienne, Bureau 530, Dorval (Québec), Canada H9P
2W8
Abstract: Amyloidogenic proteins have the characteristic of adopting a β-sheet conformation and
assembling into fibrils. Although similar in fibrillar appearance, each type of peripheral amyloid
deposits differs in the nature of the amyloidogenic protein forming fibrils. Other elements, known
as the common structural elements of the amyloid deposits, also contribute to amyloidogenic
process in vivo. Among these elements, heparan sulfate proteoglycans (HSPGs) have been shown
to bind to different types of amyloidogenic proteins and to promote the formation of β-sheet
secondary structure. Once fibrils are formed, HSPGs protect the fibrils from proteolytic
degradation, which lead to the accumulation of the deposits in the targeted organs. Understanding the regulation of protein
folding by proteoglycans can lead to the development of low molecular weight compounds, which bind to the
amyloidogenic proteins prior to their organization as fibrils. Such binding would interfere with the natural association of
amyloidogenic protein with HSPGs and maintain the amyloid protein in a non-fibrillar structure (either random coil or a
mix of α-helix and β-sheet structure). It would also favor their clearance, and thereby inhibit or completely block the
formation of amyloid deposits. Since HSPGs interact with several types of amyloidogenic proteins, such an approach may
be beneficial for the treatment of systemic and localized types of amyloidosis.
INTRODUCTION
Amyloid deposits are associated with a number of
seemingly unrelated disorders. These deposits are either
found in several organs (systemic amyloidosis) or localized
to one specific organ (e.g. brain, pancreas). Amyloidosis has
been associated with disorders such as chronic inflammatory
and infectious diseases (e.g. systemic AA amyloidosis),
multiple myelomas (systemic AL amyloidosis), Alzheimer’s
disease (AD) (Aβ brain amyloidosis), and type 2 diabetes
(e.g. islet associated polypeptide (IAPP) amyloidosis). More
than 20 amyloidotic disorders have been reported.
The proteinaceous fibrillar deposits found in the various
amyloidotic diseases are structurally and morphologically
similar, even though the proteins forming the deposits are
specific for each amyloid disease and do not share any
sequence homology with one another. They all possess the
same amyloidogenic properties: they can adopt a β-sheet
conformation, organize as protofilaments, and form
unbranched fibrils of various lengths with a 70-120 Å
diameter [1]. The similarity in their structure suggests that
they undergo a common fibrillogenic process.
The fibrillar protein components of amyloid deposits,
which are specific to each disorder, associate with other
constituents that are present in all types of amyloidotic
deposits and are thus referred to as “common elements” of
amyloid deposits. These common elements consist of serum
amyloid P (SAP), apolipoprotein E (apoE), complement
component C1q, and proteoglycans (PGs). Their presence in
*Address correspondence to this author at the Neurochem Inc., 1375,
autoroute transcanadienne, Bureau 530, Dorval (Québec), Canada H9P
2W8; Tel: (514) 337-4646; Fax: (514) 684-7972;
E-mail: [email protected]
1568-0134/03 $41.00+.00
all types of amyloid disease suggests that they could play a
major active role in the fibrillogenic process.
PGs in particular have been the subject of numerous
studies on their role in fibril formation. This amyloid
interaction was first suggested by Oddi in 1894 [2] and was
extensively studied by several groups in the 1980-1990s [313]. It has been proposed that the sulfated glycosaminoglycan (GAG) moiety of the PGs plays an active role in the
fibrillogenic process, influencing the structural shift of the
amyloidogenic protein toward β-sheet conformation and
protecting the fibrillar protein from proteolysis [14-17].
Studies using animal models of inflammation-induced
secondary amyloidosis have clearly demonstrated the
involvement of PGs during the amyloidogenic process. The
formation of amyloid fibrils in vivo was always accompanied
by the presence of heparan sulfate (HS) proteoglycans
(HSPGs) [18-21]. GAGs also promote fibrillogenesis of the
Aβ protein, which is associated with the development of
brain amyloidosis in patients with AD, cerebral amyloid
angiopathy (CAA), and Down’s syndrome (DS).
Furthermore, GAGs were localized in both diffuse and senile
plaques from brain sections of these patients [8,14,15,17,2224]. In brains of DS patients of various ages, HS were found
to co-accumulate with Aβ in the extracellular matrix (ECM),
reinforcing an active role that GAGs might play in the brain
amyloid deposition process [25,26]. Several other types of
amyloid deposits have also been examined for their
association with GAGs. All types of amyloids have been
reported to be closely associated with different types of PGs;
for example: IAPP amyloid, amyloid associated with prion
diseases, hemodialysis-associated β2-microglobulin amyloid,
and primary AL amyloid [27-32]. All these studies strongly
suggest that GAGs, after binding to an amyloidogenic
© 2003 Bentham Science Publishers Ltd.
362
Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4
protein, promote a shift in protein structure towards β-sheet
fibrils and protect fibrils from proteolytic degradation.
GAGs can thus be regarded as a relevant target for the
development of anti-amyloid therapeutics. Understanding the
interaction of GAGs with specific amyloid proteins can lead
to the development of low molecular weight (LMW) organic
compounds capable of interfering with the fibrillogenesis
process of these diverse amyloidogenic proteins.
Gervais et al.
most abundant disaccharide unit of heparin is the trisulfated
β-L-IdoA(2S)-(1→4)-D-GlcNS(6S)
(Fig.
(2)).
The
combination of carboxyl and sulfo groups in heparin gives it
the highest negatively-charged density of any known
biological macromolecule under physiological conditions
[35]. In HS, there is one sulfo group per disaccharide unit
compared to 2.7 groups in heparin.
1. Proteoglycans and Amyloidogenic Proteins
The predominant class of PGs found associated with all
types of amyloid deposits is HSPGs and their highly sulfated
heparin analogues [33]. HS and heparin are linear
polysaccharides consisting of uronic acid-(1→4)-Dglucosamine repeating disaccharide subunits. The sequence
is complex due to the variable patterns of substitution of the
disaccharide subunits with O-sulfo, N-sulfo, and N-acetyl
groups [34]. The disaccharide units are constructed from the
same monosaccharide building blocks with different
substitutions: α-L-iduronic acid, β-D-glucuronic acid, and Nsulfated and/or N-acetylated α-D-glucosamine (Fig. (1)). The
OH
HO
HO
OR
OR
OR
O
O
OR'
OH
OH
HO
HO
NHSO3H
OH
NHAc
N-Sulfo-α-D-Glucosamine
N-Acetyl-α-D-Glucosamine
R = H or SO 3H; R' = H or SO3H
Fig. (1). The primary building blocks of heparin and HS are all
monosaccharide units. The repeating disaccharide subunits of HS
and heparin are constructed from these 4 monosaccharide building
blocks with different substitutions.
OR
O
CO2H
OH
OR
β-D-Glucuronic acid
α-L-Iduronic acid
OSO3H
O O
O OH
CO2H
OH
PGs are macromolecules of various sizes and structures
that are distributed almost everywhere in the body. They can
be found in the intracellular compartment, on the surface of
cells, and as part of the ECM. The basic structure of all PGs
comprises of a core protein and at least one, but frequently
more, polysaccharide chains (GAGs) attached covalently to
the core protein. Many different GAGs have been discovered
including chondroitin sulfate, dermatan sulfate, keratan
sulfate, heparin, HS, and hyaluronan.
A
CO2H
O OH
O
CO2H
OH
NHSO3H
O
OR
O O
O
O
NHR'
OH
O
OSO3H
OR
R = H or SO 3H, R' = Ac, SO3 H or H
Main sequence
Variable sequence
OR
OH
B
O
O
CO2H
CO2H
OH
O O
O O
O
NHAc
OH
OR
O
NHR'
OH
O
O
OH
Main sequence
OR
R = H or SO 3H, R' = Ac, SO3 H or H
Variable sequence
Fig. (2). (A) The most abundant disaccharide unit of heparin is the tri-sulfated β-L-IdoA(2S)-(1→4)-D-GlcNS(6S). (B) The disaccharide unit
in HS presents a lesser degree of sulfation with about one sulfo group per disaccharide unit in HS compared to 2.7 units in heparin.
Proteoglycans and Amyloidogenic Proteins
The polyanionic characteristic of heparin and HS results
from the large number of carboxyl and sulfo groups in their
linear polysaccharide chain. Although the monosaccharide
building blocks are the same in heparin and HS, the partially
sulfated disaccharide unit found in the variable sequence
leads to a much lower sulfation degree in HS than in heparin
(Fig. (2B)). The binding of proteins to the HS chains of
HSPG or to heparin is primarily through electrostatic
interactions between the anionic sites of the polysaccharides
and the cationic sites of the basic side-chain functional
groups of the peptides or proteins.
The wide range of proteins capable of binding to heparin
suggests that a common structural feature is required for
such binding patterns. In 1989, Cardin and Weintraub
demonstrated that the basic residues of heparin–binding
peptide sequences tend to be arranged on one side of an αhelix with the pattern XBBBXXBX (X, non-basic residue;
B, a basic residue) [36]. A second pattern, XBBXBX, was
proposed to align the basic residues on one side of a βstrand. In 1992, Sobel and co-workers proposed another
pattern, XBBXXBBBXXBBX [37]. Subsequently, Margalit
and co-workers suggested that the common feature of
heparin-binding sequences consists of the presence of outer
basic residues, which are always 20 Å apart [38]. However,
since many heparin/HS-binding protein sequences lack these
consensus patterns, it is clear that other patterns have yet to
be defined.
Ancsin and Kisilevsky studied the HS-serum amyloid A
(apoSAA) binding sites for different isoforms of apoSAA
[39]. Both mouse and human apoSAA (m-apoSAA and hapoSAA, respectively) possess specific binding sites for
heparin and HS. Mouse apoSAA1 and apoSAA2 isoforms
contain a XBBXBX pattern as a putative GAG binding site
at residues 82-87 (NRHGRS). This sequence lies within a Cterminal 27mer peptide (SAA 27mer) generated by cyano
bromide (CNBr) cleavage of the full-length apoSAA protein.
Studies performed with this C-terminal 27mer peptide,
which includes the XBBXBX pattern, showed significant
changes in the chemical shifts of H1-NMR upon incubation
with HS and/or heparin1, suggesting that the GAG binding
site of apoSAA is located within residues 82-87. However,
His-93 in h-SAA 27mer (which lies outside the XBBXBX
region) also interacts strongly with heparin, as indicated by a
chemical shift change of H-2 in the immidazolino moiety of
about 0.21 ppm. This change in chemical shift indicates a
fairly strong interaction of h-SAA 27mer with heparin
through or around residue 93 (histidine). These results
suggest that the primary sequence alone cannot define the
heparin/HS-binding site [39]. Protein conformation may also
play an important role in placing critical basic residues into
favorable positions for interaction with the anionic groups on
the GAG chain. For example, if h-SAA 27mer adopted a βstrand conformation, then Arg-86 and His-93 would be
spaced correctly to allow binding to heparin according to the
hypothesis of Margalit and co-workers [38].
1
Wu, X.; Aman, A.; Rodionova, L.; Martineau, E.; Valade, I.; Szarek, W. A.; Kong,
X. 224th ACS National Meeting, Boston, MA, 2002; Part 2, MEDI 308.
Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 363
Another amyloidogenic peptide having a well defined
XBBXBX pattern is Aβ. Amino acid residues at position 1217, VHHQKL, have been identified as a GAG binding site of
Aβ [40]. The kinetics of Aβ fibril formation in vitro depends
on the Aβ concentration, pH, and temperature, as well as on
the presence of promoters such as heparin [41].
Understanding the interaction of amyloidogenic proteins
with GAGs is definitely an asset in the design of antiamyloid compounds as new therapeutic agents for the
treatment of amyloidotic diseases. Interrupting the
interaction between an amyloidogenic protein and GAGs
using anionic compounds, which interfere in the kinetics of
amyloid formation, is a potential therapeutic approach that
can be applied to a wide range of amyloid-related diseases
[41-44]. GAG mimetics are classes of LMW compounds
having negative charge(s) at physiological pH. The anionic
center of the mimetic could interact with the positive
charge(s) of the amyloidogenic protein and thereby inhibit
the GAG-protein interaction, leading to a reduction or an
inhibition of amyloid fibril formation in vivo.
Considering the anionic characteristics and the sugar
building blocks of GAGs, sulfated mono-, di-, or oligosaccharides of the GAG backbone would be the closest mimetics of a GAG and may find therapeutic application in the
treatment of amyloidosis. Fraser et al. showed that chondroitin sulfate-derived mono- and disaccharides compete with
the intact chondroitin sulfate and heparin GAGs for Aβ binding [45]. Other sugar sulfates such as trehalose octasulfate
and sucrose octasulfate also bind to amyloidogenic peptides1.
Another approach to interfere in the GAG-amyloid interaction consists in altering HS properties to minimize their
binding to amyloid proteins. Recently, the effect of altering
the GAG structure during biosynthesis on amyloidogenesis
in vivo was studied [46-49]. The termination of HS elongation in primary hepatocyte cultures was obtained using 4deoxy analogues of N-acetylglucosamine as sugar precursors. One of the 4-deoxy sugars, 2-acetamido-1,3,6-tri-Oacetyl-2,4-dideoxy-α-D-xylo-hexopyranose, administered in
mice undergoing AA-amyloid induction, inhibited splenic
AA amyloid deposition by 50% and 85% in the spleen and
liver, respectively. However, development of therapeutics
aimed at interfering in the normal HS biosynthesis may be
hindered by severe side effects.
2. GAG Mimetics: Anionic Low Molecular Weight
Compounds
Small sulfonated molecules, which mimic the anionic
elements of GAGs, may compete with naturally occurring
GAGs for binding to specific sites on amyloidogenic
proteins and thereby interfere in the fibrillogenic process
normally promoted by these GAGs. In 1995, Kisilevsky and
co-workers showed that a series of small-molecule alkanesulfonates and alkyl sulfates inhibited the development of
amyloidosis in an inflammation-induced animal model of
AA amyloidosis [42]. Three of these compounds, poly
(vinylsulfonic acid), 1,2-ethanediol disulfate, and 1,3propanediol disulfate (all in their sodium salt form) when
given at a 50 mM concentration in drinking solution, reduced
splenic amyloid deposition by 98%, 92%, and 89%,
respectively. Another compound, 1,3-propane disulfonate
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Gervais et al.
(1,3-PD) has been extensively studied for its anti-amyloid
activity in acute models of AA amyloidosis. Fig. (3) shows
the dose-response curve of 1,3-PD when mice were treated
orally with 15, 30, and 50 mg/ml for 5 consecutive days. In
this study, an aggressive model of AA amyloidosis was used.
Animals were injected intravenously (i.v.) with a nucleating
agent, the amyloid enhancing factor (AEF), and subcutaneously (s.c.) with a strong inflammatory stimulus (AgNO3)
a day prior to the initiation of treatment with 1,3-PD.
Additionally, this class of compounds was also shown
previously to block the deposition process in animals in
which the amyloid deposits had already accumulated. Using
the AEF + AgNO3 model, it was shown that a 2-week
poly(vinylsulphonate) treatment (40 mM) administered to
mice having well established amyloid deposition, promptly
arrested amyloid deposition [42]. These sets of results
suggest that anionic LMW compounds can prevent as well as
arrest the deposition of amyloid in animals.
A significant reduction in amyloid deposition was
obtained when mice were treated with a drinking solution of
30 and 50 mg/ml of 1,3-PD (Fig. (3) and (4)). The antiamyloid activity of 1,3-PD was also determined using a
milder inflammatory stimulus (i.e. casein). The latter triggers
a lower SAA production compared to that seen with AgNO3
stimulation. Animals were first injected with casein (3
injections over 6 days) to stabilize SAA production. On day
6, mice were concomitantly injected with casein and AEF.
1,3-PD treatment was initiated one day later (i.e. on day 7)
and for 5 consecutive days. In this case, significant reduction
of amyloid deposition was seen at a lower dose than that
seen with the AEF + AgNO3 model. Indeed, animals treated
with a 15 mg/ml drinking solution showed an ~ 70% reduction in amyloid deposition (data not shown), suggesting that
the 1,3-PD effective dose is inversely correlated with serum
SAA levels.
This in vivo proof of concept of the anti-amyloid activity
of GAG mimetics is pivotal to the consideration of GAG
mimetics as a therapeutically relevant approach for
amyloidotic diseases such as AA amyloidosis, AD and IAPP
amyloidosis.
3. GAG Mimetics and Their Binding Affinity to Amyloid
Proteins
GAG mimetics need to bind to amyloid proteins to exert
their activity at inhibiting amyloidogenesis. We postulated
that GAG mimetics bind to the soluble form of amyloid
proteins in order to inhibit the amyloidogenic process. We
therefore determined the ability of a series of sulfonated
small molecules to bind to two different soluble or fibrillar
amyloid proteins: Aβ and IAPP. This study allowed us to
Fig. (3). AA amyloidosis was induced with injections of AEF (i.v.) + AgNO3 (s.c.) on day 0 as previously described [27]. Animals were
treated with 1,3-PD at different doses (15, 30, and 50 mg/ml in drinking water) for 5 consecutive days starting on day 1. The percent (%) area
of spleen section occupied by amyloid was determined by image analysis following Congo red staining of sections. Both 30 and 50 mg/ml of
1,3-PD in drinking solution significantly reduced amyloid burden in the spleen. Linear regression analysis shows the dose-response of 1,3PD efficacy. Points represent data obtained from individual mice and dashed lines represent the prediction and confidence intervals.
Proteoglycans and Amyloidogenic Proteins
Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 365
Fig. (4). Spleen histological sections obtained after 5 days from control (A) and 1,3-PD-treated animals (30 mg/ml in drinking solution for 5
days) (B). Significant reduction of amyloid burden is seen in the treated animals.
determine the binding ability and specificity of a number of
GAG mimetics.
To this end, we developed an electrospray ionization
mass spectrometry (ESI-MS) assay, to determine the ability
of small molecules to bind to the soluble form of Aβ or
IAPP. Aβ preparations were verified for the presence or
absence of fibrils by electron microscopy as previously
described [41]. Fig. (5) illustrates the binding pattern of two
different molecules that form a complex with Aβ and IAPP.
Compounds were found to usually bind preferentially to one
amyloidogenic protein. As example, Fig. (5) shows that one
compound, N-benzyloxycarbonyl-3-aminopropanesulfonic
acid (compound A), binds more to IAPP than to Aβ while
(+)-3-[(S)-1-hydroxy-2-butyl]amino-1-propanesulfonic acid
(compound B) shows opposite binding properties i.e.
stronger binding to Aβ.
Compounds were also tested for their ability to bind
fibrillar Aβ. Following incubation of compounds with the
fibrillar form of Aβ1-40, the amount of unbound (free)
compound remaining in solution was determined by UV
detection or mass spectrometry. Table 1 shows the binding
affinity of a series of anionic compounds to fibrillar Aβ.
These results demonstrate that GAG mimetics such as
sulfonated and sulfated small molecules can bind to amyloid
proteins with specificity, i.e. they preferably bind to only one
type and one form (soluble vs. fibrillar) of amyloid protein.
Therefore, the binding ability can be used as a primary
screening marker to identify potential anti-amyloid GAGmimetic compounds that are specific for a particular type of
amyloid protein.
4. GAG and Amyloid-Induced Cellular Changes
Amyloid proteins have been shown to affect cellular
functions. Aβ protein can be toxic to neuronal and smooth
muscle vascular cells and pericytes, and can also induce a
microglial inflammatory response. Similarly, IAPP is toxic
to islet cells in culture. Polysulfated GAGs as well as sulfatecontaining compounds such as Congo red and its derivatives
have been shown to attenuate the neurotoxic effects of Aβ
[50,51]. It has been suggested that the presence of sulfate in
these molecules is necessary for the prevention of Aβinduced neurotoxicity. By maintaining Aβ in a non-fibrillar
form, these molecules may prevent toxicity induced by the
fibrillar form of amyloid.
However, these compounds may also protect neuronal
and microglial cells by preventing soluble, oligomeric Aβ
from binding to the cell surface, thereby precluding any Aβinduced cellular changes. The soluble, oligomeric form of
Aβ has been shown to be toxic to neuronal cells [52]. We
have studied the effect of GAGs on the interactions between
either soluble or fibrillar Aβ and the cell surface using an
Aβ1-42–induced microglial inflammatory response assay
system. The Aβ1-42 conformation of monomer and fibrillar
preparations was verified by electron microscopy analysis as
previously described [41]. Both monomerized and fibrillar
Aβ1-42 induced nitric oxide production in IFN-γ-primed
microglial cells (Fig. (6 A, B)). The addition of low (3,000)
or high (17,000-19,000) molecular weight heparin (LMWH
and HMWH, respectively) as a source of GAGs to primary
microglial cell cultures, which were concomitantly treated
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Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4
Gervais et al.
Fig. (5). ESI-MS spectra (acquired in positive mode using a ZQ 4000 spectrometer) of 20 µM Aβ1-40 or 20 µM IAPP at pH
7.4±0.2: (A) Aβ1-40 alone, cluster showing the peptide molecule associated with different number of sodium ions and the
species having + 5 charge; (B) IAPP alone, cluster showing the peptide molecule associated with sodium ions and the species
having + 4 charge; (C) Aβ1-40 with 100 µM compound A (3-benzyloxycarbonylamino-1-propanesulfonic acid); (D) IAPP with
100 µM compound A; (E) Aβ1-40 with 100 µM compound B ((+)-3-((S)-1-hydroxy-2-butylamino)-1-propanesulfonic acid); and
(F) IAPP with 100 µM compound B. The percentage in the panel indicates the ratio of the peak intensity of compound-peptide
complex to that of free peptide.
Proteoglycans and Amyloidogenic Proteins
Table 1.
Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 367
Compound Binding Properties to Soluble and Fibrillar Aβ1-40.
Binding to Soluble
Binding to Fibrillar
Name and Structure
Rating
%a
Rating
%b
Strong
67
None
<3
None
<3
Strong
94
Weak
5
Strong
61
3-(2-(1,2,3,4-tetrahydro-9H-pyido(3,4-b)indolyl)-1-propanesulfonic acid, sodium salt
N
N
SO3Na
H
Thiazol Yellow G
N
N
H3 C
S
S
SO3Na
N
N
CH3
SO3Na
N
H
Chicago Sky Blue 6B
SO3Na
OCH3 N
N
NH 2
NaO3 S
OH
SO3Na
OH
NH 2
N
N
OCH3
SO3Na
a
The percentage represents the ratio of binding-complex to non-bound peptide at a concentration of 40 µM of the peptide and 200 or 400 µM of the testing compound.
The percentage is calculated from the amount of peptide-bound compound and the original amount of compound added to the mixture; the experiment was performed in duplicates
at a concentration of 50 µM of fibrillar Aβ and 20 µM of the testing compound.
b
with monomerized Aβ1-42, resulted in a significant dosedependent reduction of nitrite production when compared to
that seen with Aβ1-42 alone (Fig. (7)). Similar levels of
reduction of nitrite production were seen with both LMWH
and HMWH preparation (data not shown). In contrast, the
addition of Congo red to the assay system did not block the
nitrite production by microglia exposed to a soluble Aβ1-42
preparation. These results combined with a microscopic
evaluation of the degree of adherence of Aβ to the microglial
cell surface (immunochemistry using 6E10 antibody),
suggest that the binding of GAGs to soluble Aβ1-42 decreases
the ability of Aβ to interact with the microglial cell surface
and thereby reduces the magnitude of the Aβ-induced
inflammatory response (data not shown). On the other hand,
Congo red did not affect the amount of Aβ binding to the
cell surface. These findings emphasize the need for a
therapeutic agent that binds to the soluble form of Aβ to
favor a protective effect on neuronal as well as on microglial
cells. Interaction of molecules such as the sulfonated,
sulfated GAG-mimetic compounds with the soluble form of
Aβ may therefore have several consequences: their binding
to Aβ would maintain Aβ in a soluble form and diminish its
interaction with the cell surface, leading to a reduction in
soluble Aβ-induced neurotoxicity and a lower Aβ-induced
microglial inflammatory response.
LMWHs have recently been evaluated both in vitro and in
vivo for their anti-amyloid therapeutic ability. Zhu et al. [53]
showed that clinically relevant doses of LMWH interfere in
the HS-stimulated fibrillogenic process of SAA in vitro.
Furthermore they reported that LMWHs inhibit AA amyloid
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Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4
deposition following AgNO3+AEF induction. LMWHs have
also been suggested for the treatment of AD. These have
been shown to interact with Aβ in vitro and to attenuate the
behavioral symptoms of AD in rodent models [54-59].
However, the long-term effect of treatment with LMWHs is
unknown. GAG mimetics may present a better pharmacokinetic and pharmacodynamic profile as they can be administered orally and do not interfere in coagulation process.
Fig. (6). Rat microglial cells were treated with or without 5 U/ml of
IFN-γ and increasing concentrations (1-15 µM) of monomeric (A)
or 24-hour aggregated Aβ1-42 (B). Production of nitrite was
determined in the supernatant after 48 hours. Levels of NO2-, a
breakdown product of NO, were measured in the supernatant with a
fluorescent method using 2,3-diaminonaphthalene (DAN). Both
forms of Aβ1-42 were found to induce comparable levels of nitrite
production by IFN-γ-treated microglial cells.
CONCLUSION
Intervention at the level of amyloid protein assembly
may lead to a disease-modifying therapeutic approach to
amyloid-related disease. PGs interact with amyloidogenic
Gervais et al.
proteins by promoting fibrillogenesis and by stabilizing the
interaction between amyloid proteins and the cell surface.
This activity makes PGs a relevant target for developing
approaches that would interfere with their ability to promote
amyloid formation. Targeting the association between PGs
and amyloidogenic proteins could preclude amyloid
formation as well as decrease the cellular damage caused by
the interaction of amyloid proteins with specific cells.
Fig. (7). Rat microglial cells were treated with 5 U/ml of IFN-γ and
5 µM of monomeric Aβ1-42 in presence of increasing concentrations
of heparin (0.1 to 100 µM) (LMW) (A) or Congo red (0.1 to 10
µM) (B). Production of nitrite was determined in the supernatant
after 48 hours. Levels of NO2-, a breakdown product of NO, were
measured in the supernatant with a fluorescent method using 2,3diaminonaphthalene (DAN). Symbols represent the mean of
duplicates from 6 independent experiments. Congo red did not
block the nitrite production by microglia exposed to soluble Aβ1-42.
LMW compounds having properties similar to GAGs,
with regards to their interaction with amyloid proteins, may
be considered a potential approach for therapeutic
intervention in amyloid diseases. These LMW molecules
bind to specific amyloid proteins and have been shown to
have in vitro and in vivo anti-amyloid activity with at least 2
different amyloid proteins, AA and Aβ [41,42]. Pre-clinical
Proteoglycans and Amyloidogenic Proteins
toxicity studies conducted with one molecule of this class of
sulfonated compound, 1,3-PD, further supports the
development of this type of anti-amyloid approach, since this
compound was well-tolerated and safe at doses higher than
the anticipated clinically effective dose.
ACKNOWLEDGEMENTS
We thank Marie Boulé for the preparation of primary rat
microglial cultures and the conduction of the assay for the
Aβ1-42-induced microglial activation, Dr. Robert Kisilevsky
for his previous work on secondary amyloidosis, and Dr.
Diane Lacombe for the preparation and the revision of the
manuscript.
ABBREVIATIONS
Aβ
= β-amyloid peptide.
AD
AEF
apoSAA
apoE
=
=
=
=
APP
CAA
DS
= β-amyloid precursor protein.
= Cerebral amyloid angiopathy.
= Down’s syndrome.
Alzheimer’s disease.
Amyloid enhancing factor.
Apolipoprotein serum amyloid A.
Apolipoprotein E.
ECM
= Extracellular matrix.
ESI-MS = Electrospray ionization mass spectrometry.
GAG
= Glycosaminoglycan.
Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 369
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
HMWH = High molecular weight heparin.
[30]
[31]
HS
= Heparan sulfate.
[32]
HSPG
= Heparan sulfate proteoglycan.
[33]
i.v.
= Intravenously.
IAPP
= Islet amyloid polypeptide.
LMW
= Low molecular weight.
LMWH
= Low molecular weight heparin.
PG
= Proteoglycan.
SAA
= Serum amyloid A.
SAP
= Serum amyloid P.
[39]
[40]
[41]
s.c.
= Subcutaneously.
[42]
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
McLaurin, J.; Fraser, P. E. Eur. J. Biochem., 2000, 267, 6353.
Cohen, A. S. Int. Rev. Exp. Pathol., 1965, 4, 159.
Gupta-Bansal, R.; Brunden, K. R. J. Neurochem., 1998, 70, 292.
Snow, A. D.; Willmer, J.; Kisilevsky, R. Lab. Invest., 1987, 57,
687.
Kisilevsky, R.; Fraser, P. Ciba. Found. Symp., 1996, 199, 58.
McLaurin, J.; Franklin, T.; Kuhns, W. J.; Fraser, P. E. Amyloid,
1999, 6, 233.
Stenstad, T.; Magnus, J. H.; Kolset, S. O.; Husby, G. Scand. J.
Rheumatol., 1991, 20, 1.
Young, I. D.; Willmer, J. P.; Kisilevsky, R. Acta. Neuropathol.
(Berl), 1989, 78, 202.
Kisilevsky, R. J. Intern. Med., 1992, 232, 515.
Magnus, J. H.; Kolset, S. O.; Husby, G. Ann. Rheum. Dis., 1991,
50, 562.
[34]
[35]
[36]
[37]
[38]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
Leveugle, B.; Scanameo, A.; Ding, W.; Fillit, H. NeuroReport,
1994, 5, 1389.
Fraser, P. E.; Nguyen, J. T.; Chin, D. T.; Kirschner, D. A. J.
Neurochem., 1992, 59, 1531.
Shirahama, T. Neurobiol. Aging, 1989, 10, 508.
Kroger, S.; Schroder, J. E. News Physiol. Sci., 2002, 17, 207.
Cotman, S. L.; Halfter, W.; Cole, G. J. Mol. Cell. NeuroSci., 2000,
15, 183.
Gupta-Bansal, R.; Frederickson, R. C.; Brunden, K. R. J. Biol.
Chem., 1995, 270, 18666.
Verbeek, J. S.; Otte-Holler, I.; van den Born, J.; van den Heuvel, L.
P. W. J.; David, G.; Wesseling, P.; de Wall, R. M. W. Am.. J..
Pathol., 1999, 155, 2115.
Snow, A. D.; Bramson, R.; Mar, H.; Wight, T. N.; Kisilevsky, R. J.
Histochem. CytoChem., 1991, 39, 1321.
Snow, A. D.; Kisilevsky, R. Lab. Invest., 1985, 53, 37.
Snow, A. D.; Kisilevsky, R.; Stephens, C.; Anastassiades, T. Lab.
Invest., 1987, 56, 665.
Norling, B.; Westermark, G. T.; Westermark, P. Clin. Exp.
Immunol., 1988, 73, 333.
Snow, A. D.; Mar, H.; Nochlin, D.; Kimata, K.; Kato, M.; Suzuki,
S.; Hassell, J.; Wight, T. N. Am. J. Pathol., 1988, 133, 456.
Berzin, T. M.; Zipser, B. D.; Rafii, M. S.; Kuo-Leblanc, V.;
Yancopoulos, G. D.; Glass, D. J.; Fallon, J. R.; Stopa, E. G.
Neurobiol. Aging, 2000, 21, 349.
Donahue, J. E.; Berzin, T. M.; Rafii, M. S.; Glass, D. J.;
Yancopoulos, G. D.; Fallon, J. R.; Stopa, E. G. Proc. Natl. Acad.
Sci. USA, 1999, 96, 6468.
Snow, A. D.; Castillo, G. M. Amyloid, 1997, 4, 135.
Snow, A. D.; Mar, H.; Nochlin, D.; Sekiguchi, R. T.; Kimata, K.;
Koike, Y.; Wight, T. N. Am. J. Pathol., 1990, 137, 1253.
Magnus, J. H.; Stenstad, T.; Kolset, S. O.; Husby, G. Scand. J.
Immunol., 1991, 34, 63.
McBride, P. A.; Wilson, M. I.; Eikelenboom, P.; Tunstall, A.;
Bruce, M. E. Exp. Neurol., 1998, 149, 447.
Snow, A. D.; Kisilevsky, R.; Willmer, J.; Prusiner, S. B.;
DeArmond, S. J. Acta. Neuropathol. (Berl), 1989, 77, 337.
Park, K.; Verchere, C. B. J. Biol. Chem., 2001, 276, 16611.
Nelson, S. R.; Lyon, M.; Gallagher, J. T.; Johnson, E. A.; Pepys,
M. B. Biochem. J, 1991, 275, 67.
Young, I. D.; Ailles, L.; Narindrasorasak, S.; Tan, R.; Kisilevsky,
R. Arch. Pathol. Lab. Med., 1992, 116, 951.
Snow, A. D.; Willmer, J.; Kisilevsky, R. Lab. Invest., 1987, 56,
120.
Rabenstein, D. L. Nat. Prod.Rep., 2002, 19, 312.
Capila, I.; Linhardt, R. J. Angew. Chem. Int. Ed. Engl., 2002, 41,
391.
Cardin, A. D.; Weintraub, H. J. Arteriosclerosis, 1989, 9, 21.
Sobel, M.; Soler, D. F.; Kermode, J. C.; Harris, R. B. J. Biol.
Chem., 1992, 267, 8857.
Margalit, H.; Fischer, N.; Ben-Sasson, S. A. J. Biol. Chem., 1993,
268, 19228.
Ancsin, J. B.; Kisilevsky, R. J. Biol. Chem., 1999, 274, 7172.
Kisilevsky, R. Neurobiol. Aging, 1989, 10, 499.
Gervais, F.; Chalifour, R.; Garceau, D.; Kong, X.; Laurin, J.;
McLaughlin, R.; Morissette, C.; Paquette, J. Amyloid, 2001, 8, 28.
Kisilevsky, R.; Lemieux, L. J.; Fraser, P. E.; Kong, X.; Hultin, P.
G.; Szarek, W. A. Nat. Med., 1995, 1, 143.
Serpell, L. C.; Kong, X.; Szarek, W. A.; Kisilevsky, R.; Fraser, P.
E. In Amyloid. and. Amyloidosis; Kyle, R. A., Gertz, M. A., Eds.;
Parthenon: New York, 1998; pp 20.
Gervais, F.; Chalifour, R.; Kong, X.; Morissette, C.; Paquette, J. In
Amyloid. and. Amyloidosis; Bély, M., Apáthy, A., Eds.; David
Apathy: Hungary, 2001; pp 589.
Fraser, P. E.; Darabie, A. A.; McLaurin, J. A. J. Biol. Chem., 2001,
276, 6412.
Kisilevsky, R.; Szarek, W. A. J. Mol. NeuroSci., 2002, 19, 45.
Berkin, A.; Szarek, W. A.; Kisilevsky, R. Carbohydr. Res. , 2002,
337, 37.
Berkin, A.; Szarek, W. A.; Kisilevsky, R. Carbohydr. Res. , 2000,
326, 250.
Berkin, A.; Szarek, M. A.; Plenkiewicz, J.; Szarek, W. A.;
Kisilevsky, R. Carbohydr. Res., 2000, 325, 30.
Woods, A. G.; Cribbs, D. H.; Whittemore, E. R.; Cotman, C. W.
Brain. Res., 1995, 697, 53.
370
[51]
[52]
[53]
[54]
Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4
Pollack, S. J.; Sadler, I. I.; Hawtin, S. R.; Tailor, V. J.; Shearman,
M. S. Neurosci. Lett., 1995, 184, 113.
Lambert, M. P.; Barlow, A. K.; Chromy, B. A.; Edwards, C.;
Freed, R.; Liosatos, M.; Morgan, T. E.; Rozovsky, I.; Trommer, B.;
Viola, K. L.; Wals, P.; Zhang, C.; Finch, C. E.; Krafft, G. A.;
Klein, W. L. Proc. Natl. Acad. Sci. USA, 1998, 95, 6448.
Zhu, H.; Yu, J.; Kindy, M. S. Mol. Med., 2001, 7, 517.
Ma, Q.; Dudas, B.; Daud, A.; Iqbal, O.; Hoppensteadt, D.; Jeske,
W.; Cornelli, U.; Lee, J.; Lorens, S.; Mervis, R.; Hanin, I.; Capila,
I.; Linhardt, R.; Fareed, J. Thromb. Res., 2002, 105, 303.
Gervais et al.
[55]
[56]
[57]
[58]
[59]
Cornelli, U. In Non-anticoagulant. action. of. glycosaminoglycans;
Harenberg, J., Casu, B., Eds.; Plenum: New York, 1996; pp 249.
Passeri, M.; Cucinotta, D. Mod. Probl. Pharmacopsychiatry, 1989,
23, 85.
Santini, V. Mod. Probl. Pharmacopsychiatry, 1989, 23, 95.
Lorens, S. A.; Guschwan, M.; Hata, N.; van de Kar, L. D.;
Walenga, J. M.; Fareed, J. Semin. Thromb. Hemost., 1991, 17, 164.
Conti, L.; Re, F.; Lazzerini, F.; Morey, L. C.; Ban, T. A.; Santini,
V.; Modafferi, A.; Postiglione, A. Prog. Neuropsychopharmacol.
Biol. Psychiatry, 1989, 13, 977.