University of Zurich
Zurich Open Repository and Archive
Site-specific blockade of RAGE-Vd prevents amyloid-beta
Sturchler, Emmanuel; Galichet, Arnaud; Weibel, Mirjam; Leclerc, Estelle; Heizmann,
Sturchler, Emmanuel; Galichet, Arnaud; Weibel, Mirjam; Leclerc, Estelle; Heizmann, Claus W (2008). Site-specific
blockade of RAGE-Vd prevents amyloid-beta oligomer neurotoxicity. Journal of Neuroscience, 28(20):5149-5158.
Postprint available at:
Posted at the Zurich Open Repository and Archive, University of Zurich.
Originally published at:
Journal of Neuroscience 2008, 28(20):5149-5158.
Site-specific blockade of RAGE-Vd prevents amyloid-beta
In the genesis of Alzheimer's disease (AD), converging lines of evidence suggest that amyloid-beta
peptide (Abeta) triggers a pathogenic cascade leading to neuronal loss. It was long assumed that Abeta
had to be assembled into extracellular amyloid fibrils or aggregates to exert its cytotoxic effects. Over
the past decade, characterization of soluble oligomeric Abeta species in the brains of AD patients and in
transgenic models has raised the possibility that different conformations of Abeta may contribute to AD
pathology via different mechanisms. The receptor for advanced glycation end products (RAGE), a
member of the Ig superfamily, is a cellular binding site for Abeta. Here, we investigate the role of
RAGE in apoptosis induced by distinct well characterized Abeta conformations: Abeta oligomers
(AbetaOs), Abeta fibrils (AbetaFs), and Abeta aggregates (AbetaAs). In our in vitro system, treatment
with polyclonal anti-RAGE antibodies significantly improves SHSY-5Y cell and neuronal survival
exposed to either AbetaOs or AbetaAs but does not affect AbetaF toxicity. Interestingly, using
site-specific antibodies, we demonstrate that targeting of the V(d) domain of RAGE attenuates
AbetaO-induced toxicity in both SHSY-5Y cells and rat cortical neurons, whereas inhibition of
AbetaA-induced apoptosis requires the neutralization of the C(1d) domain of the receptor. Thus, our
data indicate that distinct regions of RAGE are involved in Abeta-induced cellular and neuronal toxicity
with respect to the Abeta aggregation state, and they suggest the blockage of particular sites of the
receptor as a potential therapeutic strategy to attenuate neuronal death.
In the genesis of Alzheimer’s disease (AD), converging lines of evidence suggest that amyloid-? peptide (A?) triggers a pathogenic
models has raised the possibility that different conformations of A? may contribute to AD pathology via different mechanisms. The
receptor for advanced glycation end products (RAGE), a member of the Ig superfamily, is a cellular binding site for A?. Here, we
investigate the role of RAGE in apoptosis induced by distinct well characterized A? conformations: A? oligomers (A?Os), A? fibrils
(A?Fs), and A? aggregates (A?As). In our in vitro system, treatment with polyclonal anti-RAGE antibodies significantly improves
SHSY-5Y cell and neuronal survival exposed to either A?Os or A?As but does not affect A?F toxicity. Interestingly, using site-specific
antibodies, we demonstrate that targeting of the Vddomain of RAGE attenuates A?O-induced toxicity in both SHSY-5Y cells and rat
The concept that cerebral accumulation of amyloid-? peptide
evidence that a particular A? species induces neuronal death.
Early evidence suggested that A?-induced neurotoxicity in cell
culture and in vivo was associated with insoluble fibrillar (A?F)
et al., 1997; McLean et al., 1999; Naslund et al., 2000). In these
studies, the A? neurotoxic effect persisted while aggregation was
ongoing but diminished as the process of aggregation neared
completion. Studies in human and transgenic mice revealed a
weak correlation between amyloid plaque load, neuronal loss,
Moechars et al., 1996; Irizarry et al., 1997a,b; Westerman et al.,
progressive dementia dependent on insoluble A?-induced neu-
ronal death and indicate that other species may underlie neuro-
the amyloid cascade hypothesis was modified to include soluble
oligomers (A?Os). Although they differ in structure, A?Os in-
clude dimers, trimers, dodecamers, and higher-molecular-
weight complexes and possess a variety of biological activities,
including the ability to disrupt cognitive function in vivo (Walsh
et al., 2002; Cleary et al., 2005; Lesne et al., 2006; Lacor et al.,
2007) and to induce neuronal apoptosis in vitro (Chong et al.,
2006; Malaplate-Armand et al., 2006).
Several mechanisms could potentially target and concentrate
glycation end products (RAGE) was identified as one of the cell-
surface binding sites for A? (Yan et al., 1996). RAGE is a multi-
ligand receptor composed of three extracellular Ig-like domains
(Vd, C1d, C2d), a single transmembrane domain, and a short cy-
Sasaki et al., 2001; Deane et al., 2003). Previous experiments in-
dicate that RAGE mediates A?-induced oxidative stress and nu-
clear factor-?B activation (Yan et al., 1996) as well as neuronal
expression of macrophage colony-stimulating factor (Du Yan et
al., 1997), mitogen-activated protein (MAP) kinases signaling
The current study dissects the role of the distinct Ig-like do-
RAGE-expressing SHSY-5Y cells and rat cortical neurons
Bierhaus and Prof. P. Nawroth for providing RAGE?/?mice, L. Allen for proofreading, T. Ballard for help with
Correspondence should be addressed to Dr. Claus W. Heizmann, Department of Pediatrics, Division of Clinical
Chemistry and Biochemistry, University of Zurich, Steinwiesstrasse 75, 8032 Zurich, Switzerland. E-mail:
TheJournalofNeuroscience,May14,2008 • 28(20):5149–5158 • 5149
system, simultaneous application of polyclonal anti-RAGE anti-
bodies effectively prevented apoptosis induced by A?Os and
A?As. In contrast, this treatment did not affect A?F-induced
SHSY-5Y cell death. Furthermore, using site-specific antibodies,
we showed that attenuation of RAGE-mediated A?O- and A?A-
induced toxicity required the blockage of specific and distinct
Ig-like domains of the receptor, the Vdand C1ddomains, respec-
tively. Our data provide the first evidence that RAGE mediates
A?-induced cellular and neuronal apoptotic events by mecha-
aggregation state. In addition, our data support the view that
effects especially with respect to preventing neuronal apoptosis
early in the disease process.
Preparation and analysis of A?(1–40)conditioned media. Synthetic A?(1–
water at 1 mM and adjusted to 10 ?M with either RPMI-1640 (supple-
mented with 2 mM L-glutamine, 100 IU/ml penicillin, and 100 ?g/ml
streptomycin) or Neurobasal (supplemented with B27, 2 mM
L-glutamine, 100 IU/ml penicillin, and 100 ?g/ml streptomycin) me-
liquid N2. The relative proportions of soluble and fibrillar A? present in
both media were determined by Congo red assay and transmission elec-
tron microscopy (TEM) at 0, 1, 3, 4, 6.5, 8, 10.5, 12, 15, and 24 h after
peptide addition. According to Klunk et al. (1999), the absorbance of
?-sheet structures, was recorded at 540 nm with an Anthos Labtec In-
struments (Eugendorf, Austria) plate reader. A? structures were imaged
using TEM. Briefly, samples were added (1 min) to 400-mesh copper
grids, washed once with H2O, and negatively stained for 1 min with 2%
uranyle acetate. Grids were air dried and examined on a Philips (Eind-
hoven, The Netherlands) CM12 electron microscope. Data analysis
showed that both media predominantly contained spherical vesicles of
A? with diameters of ?5 nm, similar to previously described oligomers
(Mastrangelo et al., 2006; Moore et al., 2007) at 0–8 h of incubation. Up
to 12 h after peptide addition, Congo red assay revealed the presence of
?-sheet-containing assemblies exhibiting typical fibril structures as im-
aged by TEM. Therefore, aliquots of A?-containing media were snap
frozen in liquid N21 h after incubation at 37°C to generate A?Os. To
generate A?F preparations, A?-containing medium was centrifuged
(14,000 ? g; 10 min) 12 h after incubation at 37°C, and the pellet con-
taining the fibrils was resuspended in equal amounts of medium and
2 h incubation at room temperature (RT), aggregates were collected by
RPMI or Neurobasal medium. The presence and stability of the aggre-
Dot blot assay with A11 and 6E10. Dot blot assay was performed as
described previously (Kayed et al., 2003). Briefly, 25 ?l of 10 ?M A?
samples were dripped onto 0.2 ?M nitrocellulose membrane (Bio-Rad,
in 10% milk in TBST (Tris-buffered saline with 0.01% Tween 20) for at
least 1 h. The blots were washed three times in TBST before and after
incubation with a 1:10,000 dilution of rabbit anti-oligomer antibody
(A11; BioSource, Camarillo, CA) and goat anti-rabbit horseradish per-
oxidase (Sigma, St. Louis, MO) in 5% milk in TBST. The blots were
developed using the SuperSignal West Dura System (Pierce, Rockford,
IL). Blots were stripped and reprobed with mouse monoclonal anti-
human A? 6E10 (1:5000; Signet, Dedham, MA).
SHSY-5Y cells and rat primary neuronal cultures. Human neuroblas-
were grown in RPMI supplemented with 10% fetal calf serum (FCS), 2
air and 5% CO2.
For the RCN cultures, the frontal cortices of three rat embryos (em-
bryonic day 18) were dissected and washed with PBS containing 5.5 mM
with 0.5 mg/ml papain (Sigma), 10 mM glucose, 1 mg/ml BSA, and 10
?g/ml DNAaseI (Roche Diagnostics, Mannheim, Germany) for 15 min
at 37°C. Cells were washed with DMEM and mechanically dissociated in
DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml
(in 5% CO2) onto poly-L-lysine (100 ?g/ml)-coated multiwells or cov-
erslips, medium was removed and replaced with Neurobasal medium
centage of glial cells (?10%) as assessed by immunofluorescence using
anti-PGP 9.5 and anti-glial fibrillary acidic protein (data not shown).
Cell culture treatments. SHSY-5Y cells were serum deprived for 24 h
before treatment. Rat cortical neurons were plated at a density of 30,000
cells/cm2and kept in serum-free medium for ?8 d. Respective condi-
tioned media were added for 24 h to SHSY-5Y cells or to rat neuronal
cultures 8 d after plating [8 d in vitro (8 DIV)]. A?O conditioned media
conditioned media were exchanged every 12 h to avoid generation of
contaminating aggregates. Control cultures underwent similar medium
changes. To investigate the role of RAGE in A?-induced cell death, the
soluble RAGE (sRAGE; 50 ?g/ml), containing the three extracellular
Ig-like domains of RAGE, the recombinant Vddomain of RAGE (recVd;
18.5 ?g/ml), and the different polyclonal antibodies (25 ?g/ml) were
added to the different conditioned media, and apoptosis was assessed
Vd(recVd) were expressed and purified as described previously (Osten-
dorp et al., 2006; Dattilo et al., 2007). The polyclonal goat anti-human
sRAGE antibody (anti-RAGE) was obtained from R & D Systems (Min-
specific antibodies (anti-C1), and RAGE-C2d-specific antibodies (anti-
C2) were produced in rabbit as described previously (Ostendorp et al.,
2006). Residues 54–70, 158–179, and 272–293 of human RAGE were
selected for the generation of the anti-Vd, anti-C1, and anti-C2antibod-
A column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) ac-
cording to the manufacturer’s protocol. The IgG concentrations of the
antisera were determined by the BCA method (Pierce). The RAGE-
C1dC2d-specific antibodies (anti-C1C2) and RAGE-VdC1dC2d-specific
antibodies (anti-VdC1C2) were generated by mixing equal amounts of
anti-Vd, anti-C1, and anti-C2. In control experiments, we used nonspe-
cific IgG (R & D Systems) with respect to the species used in the treat-
ment. The nonspecific antibodies had no effect on cell survival either in
the presence or absence of A? (data not shown).
Cell viability assays. Cell death was determined by fluorescence-
activated cell sorting (FACS) using the cycleTEST Plus DNA kit (Becton
Dickinson, Mountain View, CA) and a FACSCalibur flow cytometer. A
total of 104cells was analyzed for each condition, and data from three
separate experiments were pooled. Apoptosis was scored by terminal
deoxynucleotidyltransferase-mediated dUTP biotin nick end labeling
(TUNEL) assay according to the manufacturer’s protocol (Roche Diag-
SHSY-5Y cells and RCNs were counted on coverslips, and at least 10
fields per culture in triplicate cultures were analyzed per individual ex-
quantified using the Caspase-Glo 3/7 kit (Promega, Madison, WI). Each
experiment was repeated four times.
Immunofluorescence. Cortical rat neurons were fixed in 4% parafor-
maldehyde for 1 h at RT, permeabilized with 0.2% Triton X-100 in PBS,
5150 • J.Neurosci.,May14,2008 • 28(20):5149–5158Sturchleretal.•RAGEMediatesApoptosisInducedbyDistinctA?Conformations
and blocked for 1 h in 5% horse serum/PBS. Cultures were incubated
with rabbit anti-Vd(1:1000), mouse anti-PGP 9.5 (1:500; Abcam, Cam-
bridge, MA), mouse anti-synaptophysin (1:500; Calbiochem, La Jolla,
vitrogen, Eugene, OR). Omission of the primary antibody resulted in
complete loss of specific labeling. The fluorescence signals were visual-
ized using a Leica (Nussloch, Germany) SP2 confocal laser microscope.
Immunoblotting. SHSY-5Y cells, rat neuronal cultures, mouse neuro-
pH 7.5, 300 mM NaCl, 1% Triton X-100, 10 nM NaF, and 1 mM Na3VO4
supplemented with complete proteinase inhibitor cocktail (Roche Diag-
nostics) at the indicated time points. Protein concentration of the sam-
ples was measured using the BCA method
(Pierce). Equal amounts of protein (50 ?g)
were separated by 10% PAGE, blotted onto ni-
trocellulose membrane, and probed with anti-
regulated kinase 1/2 (ERK1/2), anti-ERK1/2,
anti-phosphorylated c-Jun N-terminal kinase
(JNK), and anti-JNK (1:1000; Cell Signaling
Technology, Beverly, MA). The blots were in-
cubated with a secondary antibody conjugated
bands were visualized using ECL solution (GE
Healthcare). Densitometric values from gels
800 and analyzed with Bio-Rad Quantity One
software. The amounts of phosphorylated ERK
and JNK were normalized to the total amount
of ERK and JNK, respectively.
as mean ? SEM and were analyzed using one-
way ANOVA followed by Bonferroni’s post hoc
The mechanism by which A? aggregates is
not fully understood, although it has been
ization process and fibril conversion (Isaacs
et al., 2006; Ha et al., 2007). In the present
study, we generated two conditioned media
(RPMI and Neurobasal) containing 10 ?M
analogs of soluble A?Os, A?Fs, or amor-
phous A?As. Under our experimental con-
ditions, the formation of fibrils containing
?-sheet structures started 9 h after the addi-
dia, as revealed by enhanced absorbance in
absorbance was obtained after 12 h and
thereafter slowly decreased during an addi-
tional 12 h (Fig. 1A). Electron microscopy
confirmed the presence of typical 100–200
ysis performed at 0–8 h of incubation
showed small spherical A? assemblies that
resembled previously described oligomers
(Fig. 1B, A?Os) (Losic et al., 2006; Mas-
trangelo et al., 2006; Moore et al., 2007).
These observations conform to previous
ber of intermediate structural forms referred to as oligomers (Ari-
mon et al., 2005; Shahi et al., 2007). Although the precise oligomer
stoichiometry remains unclear, the preparations were predomi-
nantly free of protofibrils and entirely free of fibrils as indicated by
period of time (Fig. 1A). A?As were prepared as described previ-
ously (Lorenzo and Yankner, 1994) by dissolving A?(1–40)(1 mM)
directly into PBS before adjusting the concentration to 10 ?M in
RPMI or Neurobasal medium. The prevalence and stability of the
Sturchleretal.•RAGEMediatesApoptosisInducedbyDistinctA?ConformationsJ.Neurosci.,May14,2008 • 28(20):5149–5158 • 5151
variation in Congo red binding confirmed
that A?As did not coexist with ?-sheet-
containing structures (Fig. 1A). Based on
media enriched in A?Os, A?Fs, or A?As
(see Materials and Methods). The A11 anti-
body, which reacts well with the soluble oli-
gomers but not with soluble monomers or
mature amyloid fibers (Kayed et al., 2003),
was used to further characterize the distinct
A? preparations. In accord with our previ-
body detected A? in A?O conditioned me-
dia but did not react with A? in A?F and
A?A preparations (Fig. 1C). The mouse
monoclonal antibody 6E10, which recog-
nizes A? independently of its conforma-
tional state, confirmed the presence of A?
peptide in the different preparations (Fig.
We subsequently investigated the tox-
icity of the distinct A? preparations on
RAGE-expressing SHSY-5Y cells (Sajithlal
et al., 2002). For this purpose, neuroblas-
ing 10 ?M A?Os, A?Fs, or A?As for 24 h,
and the cell death was measured by FACS.
To avoid A?O conversion into fibrils dur-
ing the time course of the experiment,
twice (every 8 h). Similarly, A?F condi-
tioned medium was changed after 12 h
(the same protocols were used in the following cell death exper-
iments). FACS analysis revealed that the distinct A? conforma-
tions significantly increased cell death in our in vitro system (Fig.
1D). Chronic exposure to A?Os caused massive cell death, and
after 24 h, ?60% of the cells were dead (Fig. 1D, A?Os). In
contrast, addition of A?F and A?A conditioned media resulted
in moderate effects with ?30 and 15% cell death, respectively
(Fig. 1D, A?Fs and A?As). When A?F and A?A preparations
the insoluble A? fraction was toxic, whereas the supernatant did
not elicit any toxicity, indicating that small amounts of contam-
inating soluble A?Os were not responsible for A?F and A?A
toxicity (data not shown). Moreover, A?Os induced apoptotic
features including disintegration of processes, swelling of cell
produced dystrophic effects on neuroblastoma cell processes
(Fig. 1E, A?Fs). These results corroborate work done previously
(Grace and Busciglio, 2003; Deshpande et al., 2006). In contrast,
A?As (Fig. 1E, A?As) did not induce degenerative morphology
when compared with control cells (Fig. 1E, control). These data
indicate that A?O, A?F, and A?A conditioned media consis-
tently trigger SHSY-5Y cell death with a difference in toxicity
correlating with the A? aggregation state.
Extracellular A? may induce neurotoxicity by interacting with
al., 1997; Yaar et al., 1997; Yao et al., 2005; St. John, 2007). Pre-
vious work provided strong evidence that RAGE interacts with
direct link between RAGE and A?-induced cell death has not yet
been demonstrated. We therefore investigated whether RAGE
directly contributes to A?-induced cell death in our cellular sys-
tem. For this purpose, SHSY-5Y cells were exposed for 24 h to
ize the three extracellular Ig-like domains (Vd, C1d, C2d) of the
receptor. Treated cultures and control cells were processed for
two parameters associated with apoptosis: DNA fragmentation
and the activation of caspase 3/7 pathways. In accordance with
our FACS studies, A?Os induced massive cell death with a five-
fold increase in the mean percentage of TUNEL-positive cells
(Fig. 2A) and a 380% increase in caspase activity (Fig. 2B). Anti-
in SH-SY5Y cells exposed to either A?O or A?A preparations as
indicated by a decrease in cells undergoing DNA fragmentation
(Fig. 2A) and a significant reduction in A?O- or A?A-induced
caspase activation (Fig. 2B). These effects were specific because
treatment of cells with a control isotype IgG did not affect A?-
induced cell death (data not shown). In contrast, treatment with
anti-RAGE neither affected DNA fragmentation (Fig. 2A) nor
Thus, our data indicate that RAGE is implicated, at least in part,
in A?O- and A?A-induced apoptosis.
RAGE is a multivalent receptor that binds several other ligands
besides A?. These include advanced glycation end products
significantly reduces the toxic effect of A?Os and A?As as measured by TUNEL, but it did not influence A?F-induced DNA
Effect of anti-RAGE antibody on A?-induced apoptosis. A, Simultaneous application of anti-RAGE (25 ?g/ml)
5152 • J.Neurosci.,May14,2008 • 28(20):5149–5158Sturchleretal.•RAGEMediatesApoptosisInducedbyDistinctA?Conformations
(Schmidt et al., 1992), the chromatin-binding protein HMGB1
(Huttunen et al., 2000; Tian et al., 2007), as well as several mem-
leading to either a trophic or a toxic cellular effect. We recently
showed that S100B and S100A6, two structurally closely related
activate distinct signaling pathways suggesting that the cellular
effects triggered by RAGE might be specific for each ligand
(Leclerc et al., 2007). We therefore hypothesized that RAGE-
mediated A?O- and A?A-induced apoptosis could involve dis-
tinct domains of the receptor. To investigate this hypothesis, we
exposed SHSY-5Y cells to A?O or A?A conditioned media for
24 h in the presence or absence of site-specific antibodies target-
ing particular epitopes within the Vd(anti-Vd), the C1d(anti-
C1), or the C1dand the C2d(anti-C1C2) domain of the receptor.
Here again, A?O conditioned medium was exchanged every 8 h
to avoid the formation of fibrils. In the
presence of A?Os, we observed a signifi-
cant decrease in cells undergoing DNA
fragmentation and a reduction in caspase
activity when the cultures were treated
with anti-Vd(Fig. 3A), whereas anti-C1or
anti-C1C2treatment did not affect A?O-
induced apoptotic events (Fig. 3B,C). In
contrast, A?A-induced cell death was un-
by TUNEL (Fig. 3A), whereas anti-C1or
anti-C1C2treatment significantly blocked
DNA fragmentation as well as caspase ac-
tivation induced by A?As (Fig. 3B,C).
Thus, our data indicate that blockage of
the Vd of RAGE effectively protects
SHSY-5Y cells from A?O-induced cell
death, whereas attenuation of A?A toxic-
suggesting that A?Os and A?As interact
with distinct sites of RAGE.
The truncated isoform of RAGE
lar domains only of the receptor, has been
suggested to function as a decoy, abrogat-
ing RAGE-mediated cellular activation by
To confirm the involvement of RAGE in
SHSY-5Y cells to either A?Os or A?As in
the presence or absence of recombinant
sRAGE or the recombinant form of the Vd
with sRAGE significantly decreased A?O-
and A?A-induced caspase activation (Fig.
3D). In addition, recVdtreatment also at-
In contrast, the addition of recVddid not
affect the increase in caspase activity in
cells exposed to A?As (Fig. 3D). These re-
sults are in accordance with our previous
observations suggesting that RAGE medi-
mechanisms involving distinct sites of the
in human neuroblastoma cells could be reproduced in a more
investigated the effect of the anti-Vdand anti-C1antibodies on
A?O- and A?A-induced apoptosis in RCNs. Initial tests were
performed to validate the experimental model and the effective-
ness of RAGE–A? interactions to induce cellular responses.
Western blot analysis using anti-VdC1C2revealed a band of ?50
able to differentially glycosylated RAGE. These observation had
previously been described in AD brains (Sasaki et al., 2001). We
antibodies as indicated by the absence of immunoreactive bands
in brain extracts of RAGE?/?mice (Fig. 4A). Interestingly,
Sturchleretal.•RAGEMediatesApoptosisInducedbyDistinctA?Conformations J.Neurosci.,May14,2008 • 28(20):5149–5158 • 5153
RAGE expression was more prominent at
8 DIV in RCNs (Fig. 4A), whereas mouse
tor at this stage (Fig. 4A). Thus, we de-
cided to perform subsequent experiments
using 8 DIV RCNs.
The ability of RAGE to colocalize with
A?Os in our model was evaluated by im-
munofluorescence. RAGE immunoreac-
tivity (Fig. 4B, red) was detected in cell
by PGP 9.5 colabeling (Fig. 4B, merge).
Higher-magnification images showed a
prominent RAGE immunoreactivity at
a clear punctuated staining defining
submicrometer-sized subdomains along
neuronal processes (Fig. 4B, arrowheads).
Double immunolabeling of RCN cultures
exposed to A?Os for 2 h revealed that a
fraction of A?Os colocalizes with RAGE
along neuronal processes (Fig. 4C, arrow-
heads) suggesting that RAGE might inter-
synaptic targeting of soluble A? species in
rat hippocampal and human cortical neu-
rons (Lacor et al., 2004; Deshpande et al.,
2006). To determine whether RAGE is lo-
calized at synaptic sites, we performed
multiple fluorescence labeling, and syn-
apses were defined by using the presynap-
tic marker synaptophysin. We found a
with the synaptic marker along sections of
processes exhibiting poor synaptophysin
immunoreactivity, whereas some other
tain the receptor (Fig. 4D).
A?O conditioned medium for 24 h in the
presence or absence of either anti-Vdor
recVd. Control and treated cells were pro-
cessed for TUNEL and caspase activity.
A?Os induced a significant increase in
neuronal cell death as indicated by an in-
crease in both TUNEL-positive cells and
the human neuroblastoma cells, neuronal
apoptosis induced by A?Os could be at-
tenuated significantly by anti-Vdtreat-
ment as indicated by a reduction in DNA
4E). In contrast, recVdtreatment did not
affect DNA fragmentation nor caspase ac-
tivity in RCNs exposed to A?Os (Fig. 4E).
RCN cultures were also exposed to A?As
in the presence or absence of anti-C1.
However, under our experimental condi-
tions, the A? aggregates failed to signifi-
cantly induce neuronal death (Fig. 4F).
Thus, these data support the hypothesis
of the boxed area in the merged image shows that RAGE is present at en passant synapses (bottom right, arrow) and at
The merged image shows the colocalization of RAGE and A?Os along neuronal processes (arrows). Scale bar, 2 ?m. D,
5154 • J.Neurosci.,May14,2008 • 28(20):5149–5158Sturchleretal.•RAGEMediatesApoptosisInducedbyDistinctA?Conformations
that RAGE might participate in A?O-induced neuronal apopto-
sis and confirm that specific neutralization of the Vdis sufficient
to significantly promote RCN survival.
RAGE-mediated A?O-induced cell death. Previous studies have
indicated that RAGE–ligand interactions modulate MAP kinase
pathways (Arancio et al., 2004; Monteiro et al., 2006). Further-
more, it has been suggested that defects in both ERK and JNK
signaling underlie neuronal dysfunction such as caspase activa-
2004; Chong et al., 2006; Ma et al., 2007; Townsend et al., 2007;
Yan and Wang, 2007). Therefore, using our models, we investi-
gated whether RAGE could be involved in A?O-induced ERK
and/or JNK signaling defects. In our experimental conditions,
Western blot analysis of neuroblastoma cell extracts exposed to
A?Os for 8 h revealed an increase in ERK activation (Fig. 5A).
Densitometric analysis of gels from separate experiments dem-
130% increase in phosphorylated ERK in SHSY-5Y cells exposed
to A?Os as compared with control cells (Fig. 5A). Interestingly,
simultaneous application of either anti-Vdor recVdconsistently
suppressed A?O-induced activation of ERK as indicated by the
absence of significant variation in control and treated SHSY-5Y
exposed to A?Os for 8 h revealed a downregulation of the phos-
phorylated form of ERK (Fig. 5C). Densitometric analysis of gels
form of ERK, normalized to the total amount of ERK, decreased
to 70% of the control value in the presence of A?Os (Fig. 5C). A
comparable reduction in ERK phosphorylation was observed at
24 h, whereas the addition of A?Os for 1, 2, and 4 h had no
significant effect on the basal activity of ERK (data not shown).
However, anti-Vd treatment consistently suppressed A?O-
induced hypophosphorylation of ERK in RCNs as indicated by
(Fig. 5C). In contrast, similar experiments revealed no change in
phosphorylated JNK immunoreactivity in control and treated
defects in human SHSY-5Y cells and RCNs.
A? is thought to be the instigator of the neuronal death driving
fibrils, and amorphous aggregates have been found in AD brains
(Lorenzo and Yankner, 1994; McLean et al., 1999; Naslund et al.,
2000). The aim of the present study was to rigorously investigate
the contribution of RAGE in apoptosis induced by distinct well
imental conditions allowing the reproducible generation of par-
A?(1–40)because A?(1–42)is much more prone to aggregation
that routinely required micromolar concentration of A? to in-
controlled by both Congo red binding assays and TEM. Under
our conditions using multiple lots of synthetic peptides, we ob-
tained consistent and reproducible results for the distinctly gen-
lished human neuroblastoma cell line (SHSY-5Y) expressing
of neurons (RCNs) as experimental paradigms. In our in vitro
tinct and reproducible patterns of toxicity that differed from ag-
gregated preparations of the peptide as determined by FACS
analysis (Fig. 1). Consistently, the different preparations pro-
moted distinct morphological alterations in SHSY-5Y cells as re-
vealed by light microscopy analysis (Fig. 1). A?Os were found to
be the most toxic conformation promoting SHSY-5Y cell death
fold more than A?As. Similarly, we showed that A?O prepara-
and RCNs. Cell cultures were incubated with A?Os for 8 h in the presence or absence of the
(*p ? 0.01), and the bottom panels show representative immunoblots. Error bars indicate
Sturchleretal.•RAGEMediatesApoptosisInducedbyDistinctA?ConformationsJ.Neurosci.,May14,2008 • 28(20):5149–5158 • 5155
2006; Malaplate-Armand et al., 2006), we found that A?O-,
tion of apoptotic pathways as revealed by TUNEL and caspase
activity assays (Figs. 2–4). With respect to our FACS data, A?Os
in SHSY-5Y cells and RCNs compared with A?F or A?A effects.
In accordance with recent studies using natural and synthetic
A?(1–42)oligomers (Chong et al., 2006; Townsend et al., 2006,
2007), our distinct A?O(1–40)preparations were found to affect
the pattern of ERK activation, indicating that cellular homeosta-
sis is challenged (Fig. 5). In our experimental paradigm, A?Os
In contrast, ERK phosphorylation was suppressed by A?O treat-
ment in RCNs (Fig. 5C). Conflicting results with the stimulatory
vivo have previously been reported (Chong et al., 2006; Ma et al.,
2007; Townsend et al., 2007). Furthermore, soluble oligomers
have been shown to initially stimulate, but later downregulate,
ERK in hippocampal slice cultures (Bell et al., 2004), and studies
activation followed by loss of active ERK (Dineley et al., 2001;
soluble oligomers on either SHSY-5Y neuroblastoma cells
(Frasca et al., 2004, 2008) or RCNs (Tong et al., 2004; Florent et
al., 2006) observed the same alterations of the ERK signaling
activation and downregulation of the ERK survival-promoting
pathway are associated with susceptibility to cell death (Dineley
Webster et al., 2006; Ma et al., 2007; Townsend et al., 2007). In
contrast, A?As did not affect the ERK phosphorylation state in
kinase pathway recruitment has been shown to be dependent on
the A? conformational state (Bell et al., 2004; Echeverria et al.,
2005). Our data are thus in good agreement with these and other
reports (Deshpande et al., 2006; St. John, 2007) suggesting that
A? exhibits specific and distinct toxic effects depending on a
particular A? aggregation.
We next rigorously characterized the role of RAGE in medi-
ating apoptosis induced by the different conformations of A?.
Our study revealed that RAGE is involved in A?O- and A?A-
induced apoptosis because simultaneous application of a poly-
clonal anti-RAGE antibody prevented both caspase activation
nificant (?50–60%) but not absolute prevention of A?O-
induced neuronal and cell death. These findings are consistent
with previous reports showing that other receptors/mechanisms
may also participate in A? toxicity (Wogulis et al., 2005; Wright
et al., 2007). In addition, anti-Vd-specific antibodies prevented
A?O toxicity in RCNs (Fig. 4), supporting the specificity of
RAGE contribution in A? signaling. Interestingly, in contrast to
A?Os and A?As, we showed that apoptosis induced by mature
A?Fs was not RAGE dependent. In this regard, previous reports
indicated that A? toxicity occurs through distinct pathways de-
pande et al., 2006). Thus, our results suggest that A?O and A?A
but not A?F signal, at least in part, through RAGE to induce
In an additional step, we aimed to map more precisely the
domain(s) of RAGE involved in A?-induced apoptosis. For this
as well as the recombinant form of the Vddomain (recVd). We
found that attenuation of RAGE-mediated A?A-induced apo-
ptosis required the specific antagonism of the C1dof the receptor
ies (anti-Vd) or the recVditself was necessary and sufficient to
prevent A?O-induced SHSY-5Y cell death (Fig. 3A,D). Impor-
in RCNs, providing evidence of the specificity and the relevance
of the treatment (Fig. 4E). In accordance with these data, previ-
ous reports (Chaney et al., 2005; Mruthinti et al., 2007) demon-
in the Vddomain. Unexpectedly, recVdtreatment did not affect
A?O-induced neuronal apoptosis (Fig. 4E). Consistently,
Mruthinti et al. (2007) reported that soluble A?(1–42)and
RAGE(23–54) form a toxic complex for neuronal cells. In accor-
dance with our previous observations, anti-Vdand recVdtreat-
ments, which inhibited caspase activation and DNA fragmenta-
tion (Fig. 3), were also found to block A?O-induced ERK
signaling perturbations in neuroblastoma cells and RCNs (Fig.
5), highlighting the involvement of RAGE as a signal transduc-
tion receptor mediating the effects of A?Os. Chronic ERK per-
turbation might be an early and sustained signaling amplifier of
A?O-induced cytotoxicity ultimately leading to the activation of
have revealed that RAGE-dependent activation of MAP kinases
proceeds via an oxidant-sensitive mechanism involving p21ras
(Lander et al., 1997), and more recently, the RAGE intracellular
al., 2003). However, detailed mechanisms linking occupancy of
RAGE to ERK modulation remain to be elucidated.
sites of RAGE are involved in A? toxicity with respect to a par-
ticular A? conformational state. In this regard, previous work
2007; Xie et al., 2007), S100A12 (Dattilo et al., 2007; Xie et al.,
2007), and S100A6 (Leclerc et al., 2007), which possess high
structural homology, also interact with different Ig-like domains
effects of A? monomers, dimers, trimers, and higher-order oli-
gomers, it allows us to determine the involvement of RAGE in
apoptosis induced by distinct well defined A? species. At the
aggregates, fibrils, or soluble oligomers represent the sole molec-
ular pathogen in AD; indeed, various A? species may play rele-
vant roles in neurotoxicity (Haass and Selkoe, 2007). Our find-
ings provide a new insight into how multiple A? species may
contribute to neurodegeneration. Furthermore, in AD patho-
with A?As at later stages of the disease. In addition, Yan et al.
AD brain, particularly in neurons associated with aggregated de-
posits. Because RAGE expression increases and remains elevated
as long as ligands are present, RAGE may be important in initi-
ating and perpetuating A? neuronal toxicity “amplification
loops.” These observations provide an interesting parallel with
the A?-induced changes in RAGE expression observed recently
in rat hippocampus (Minogue et al., 2007).
can act as a receptor exacerbating critical effects of A? on several
signaling molecules involved in the apoptotic pathway. Further-
5156 • J.Neurosci.,May14,2008 • 28(20):5149–5158 Sturchleretal.•RAGEMediatesApoptosisInducedbyDistinctA?Conformations
more, these studies establish that RAGE mediates A?O- and
the engagement of distinct nonoverlapping regions of the recep-
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tion. Although RAGE–ligand interactions support normal cellu-
lar functions and homeostasis, our results suggest that the
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