Neuromol Med (2012) 14:119–130
DOI 10.1007/s12017-012-8176-z
ORIGINAL PAPER
An Aqueous Orally Active Vaccine Targeted Against a RAGE/AB
Complex as a Novel Therapeutic for Alzheimer’s Disease
Scott J. Webster • Shyamala Mruthinti •
William David Hill • Jerry J. Buccafusco •
Alvin V. Terry Jr.
Received: 27 September 2011 / Accepted: 24 February 2012 / Published online: 14 March 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Alzheimer’s disease (AD) is a progressive
neurodegenerative disorder that gradually destroys a per-
son’s memory. Substantial evidence suggests that amyloid
beta (Ab) and the receptor for advanced glycation end-
products (RAGE) play an important and often deleterious
role in the pathogenesis of AD. RAGE facilitates the
translocation of Ab from the periphery into the brain,
mediates the Ab-induced neurotoxicity, and enhances the
release of pro-inflammatory cytokines increasing the
inflammatory response. In addition, soluble forms of
RAGE (sRAGE) and Ab bind together in the periphery
forming high molecular weight complexes that are more
highly immunogenic and less neurotoxic than Ab1-42
alone. We show here that there are elevated anti-RAGE
and anti-Ab titers (in a near 1:1 relationship) in samples
analyzed from human AD patients, aged non-human pri-
mates, and AD transgenic mice (APPSWE-PS1). We show
S. J. Webster  J. J. Buccafusco  A. V. Terry Jr. (&)
Department of Pharmacology and Toxicology, Georgia Health
Sciences University, 1120 15th Street, Augusta, GA 30912, USA
e-mail: Aterry@georgiahealth.edu
S. J. Webster  S. Mruthinti  W. D. Hill 
J. J. Buccafusco  A. V. Terry Jr.
Alzheimer’s Research Center, Georgia Health Sciences
University, Augusta, GA 30912, USA
S. Mruthinti  J. J. Buccafusco
Charlie Norwood VA Medical Center, Augusta, GA 30904, USA
W. D. Hill
Department of Cellular Biology and Anatomy, Georgia Health
Sciences University, Augusta, GA 30912, USA
W. D. Hill
Brain and Behavior Discovery Institute, Georgia Health Sciences
University, Augusta, GA 30912, USA
that an in vitro prepared RAGE/Ab complex induces a
greater immunogenic response (increased anti-Ab1-42 and
anti-RAGE antibody titers) in both human peripheral blood
mononuclear cells (PBMCs) and immunized Balb-C mice
than does either Ab1-42 or RAGE alone. Further, pre-
treatment with endogenous anti-RAGE antibodies isolated
from our transgenic APPSWE-PS1 mice can prevent Ab1-
42-induced neurotoxicity in cultured primary rat cortical
neurons. Finally, we examine the effectiveness of an orally
administered vaccine of either RAGE/Ab complex or Ab1-
42 alone in improving cognitive function in our AD
transgenic mice. Our results to date support the hypothesis
that a protein complex vaccine that targets both RAGE and
Ab1-42 will provide a more effective treatment for AD
than vaccination with Ab1-42 alone.
Keywords Alzheimer’s disease  Amyloid beta 
Receptor for advanced glycation endproducts (RAGE) 
Vaccine  Cognition  APPSWE-PS1 mice 
Alzheimer’s disease therapeutics
Introduction
Alzheimer’s disease (AD), the most common form of
dementia, has a devastating impact not only on memory
and higher cognitive skills but also on the patient’s ability
to perform routine daily activities, such as dressing and
feeding oneself. The symptoms are expressed after the loss
of neurons from specific brain regions that control
thoughts, insight, decision making, impulse control,
memory, and language. AD affects approximately 5 mil-
lion in the United States and nearly 26.6 million worldwide
(Brookmeyer 2007). As the population ages, the prevalence
of AD will increase, potentially becoming the leading
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cause of death in North America by 2050 (Trojanowski
2008). Treatment and long-term care for AD is a major
public health concern (Ziegler-Graham et al. 2008). Cur-
rently, there is no cure for AD. Its causes are still unknown.
Advanced age is the most reliable and potent risk factor for
AD, even compared to genetic or environmental factors.
Therefore, understanding sequential age-related events,
such as the increase in protein expression of the receptor
for advanced glycation endproducts (RAGE) and the
accumulation of amyloid beta in the brain, and the inter-
action between the two could prove vital in understanding
AD pathogenesis. Indeed, the interaction between RAGE
and amyloid beta plays an important role at multiple points
in the AD pathway. RAGE is expressed on the cell surface
of both neural (neurons, astrocytes, and microglia) and
vascular (endothelial cells, pericytes, and smooth muscle
cells) elements in the brain. The expression of RAGE is
increased in AD patients and is colocalized with Ab in
human AD brain tissues, neurons, microglia, and vascular
elements (Yan et al. 1996, 1997). High-affinity binding
properties between RAGE and Ab and its long-term pres-
ence as immunogenic complex antigen in blood circulation
lead to adverse consequences potentially relevant to AD
pathogenesis including potentiation of Ab aggregation,
perturbation of neuronal function, elevation of oxidative
stress, amyloidosis, augmentation of microglial inflamma-
tory responses, vascular dysfunction, and stimulation of
autoimmunity to both Ab and RAGE. (Mruthinti et al.
2004, 2006; Wilson et al. 2009; Fang et al. 2010) The
RAGE–Ab interaction is a suspected cause of brain
inflammation and neuronal death (Yan et al. 1996, 1997;
Mruthinti et al. 2006). It is suggested that ligand-bound
RAGE (RAGE/Ab) may enhance nerve-damaging reactive
oxygen species (ROS), which in turn promote advanced
glycation endproduct generation, inflammation, and ROS
production (Chen et al. 2004; Mruthinti et al. 2006) This
cycle is thought to cause eventual tissue dysfunction and
irreparable neuronal damage (Ramasamy 2005). RAGE
facilitates the accumulation and supports the aggregation of
Ab, resulting in inflammation and cytotoxicity in neuronal
cells (Yan et al. 1997; Lue et al. 2001). Perhaps even more
importantly, RAGE located within cerebral vascular cells
mediates the transport of Ab from the blood to the brain,
thus supporting the buildup of cerebral amyloid levels
(Deane et al. 2003; Tanzi et al. 2004; Herring et al. 2008;
Zhang et al. 2009).
In this study, we examined the immunogenic profile of a
RAGE/Ab complex preparation compared with Ab1-42
alone. We also examined the role of anti-RAGE antibodies
in protecting against Ab1-42-induced neurotoxicity using
primary rat cortical neurons. We examined the effective-
ness of orally administered immunizations of either RAGE/
Ab complex or Ab1-42 alone to decrease the cerebral
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Neuromol Med (2012) 14:119–130
amyloid load and improve the cognitive function in our
transgenic APPSWE-PS1 mouse model of AD. We show
here that the RAGE/Ab complex is highly immunogenic,
producing high titers of anti-Ab1-42 and anti-RAGE anti-
bodies. Further, immunization with the RAGE/Ab complex
(without co-administration of an adjuvant) is more effec-
tive in reducing AD pathology observed in AD mice than is
Ab1-42 alone.
Materials and Methods
Animal Subjects
APPSWE-PS1 transgenic mice (a model of AD) were
obtained from Jackson Laboratories. Plasma samples were
obtained from 49 wild-type and 49 transgenic mice ranging
from 3 to 24 months old. Blood was collected in purple top
EDTA blood collection tubes and spun down to obtain
plasma samples. The animals were grouped four per cage and
housed in Georgia Health Sciences University laboratory
animal facility for at least 4 weeks prior to the experiment.
The animals were allowed access to unlimited food and
water and set on a standard diurnal light cycle. All proce-
dures that included animals were reviewed and approved by
the Georgia Health Sciences University Institutional Animal
Care and Use Committee and are consistent with Association
for Assessment and Accreditation of Laboratory Animal
Care International (AAALAC) guidelines.
Blood was obtained from 41 rhesus monkeys ranging
from 5 to 31 years of age using EDTA collection tubes.
Tubes were immediately spun down to obtain plasma
samples. Monkeys were individually housed at the Animal
Behavior Center of the Medical College of Georgia in
double-tier stainless steel cages composed of multiple
127 9 71 9 66 cm units providing twice the required
housing room. To promote psychological well-being, toys
and foraging tubes were provided routinely. All procedures
were reviewed and approved by the Georgia Health Sci-
ences University Institutional Animal Care and Use Com-
mittee and are consistent with AAALAC guidelines.
Human Subjects
Blood was collected using EDTA collection tubes from
patients diagnosed based on standard criteria for probable
MCI and Alzheimer’s disease. The tubes were immediately
spun down to obtain plasma samples that were placed in
our VA repository and frozen at -80°C. Plasma samples
from 41 Alzheimer’s disease patients, 33 patients with mild
cognitive impairment (MCI), and 37 healthy elderly control
patients were analyzed for the levels of anti-RAGE and
anti-Ab IgGs.
Neuromol Med (2012) 14:119–130
Isolation and Quantification of Autoantibodies
Plasma samples were filtered through a 0.2-l filter and
affinity-purified with a protein A/G ImmunoPure column to
collect the IgG fraction. Ab-specific IgG levels were
determined from the plasma samples by an ELISA assay
where E.I.A/R.I.A 8-well strips were coated with Ab1-42
peptide and incubated overnight at 4°C. ELISA plates were
then washed with PBST and blocked with PBST ?2% milk
for 2 h and rinsed three times with PBST. The IgG fraction
was then added to each plate and maintained at 4°C
overnight. The next day, after washing 3 times with 2%
milk and PBST, an anti-human IgG secondary antibody
was applied to the plates for 3 h at 37°C. After 3 washes,
ready-made TMB substrate was added for color develop-
ment, which was stopped after 15–30 min by adding 1 N
HCl. Plates were read at 450 nm on an ELISA reader.
In Vitro Complex Preparation
Equal molar concentrations of Ab1-42-RAGE 23-54 pep-
tides were mixed with sterile D.I.H20 and incubated for
1 month in accordance with procedures described by
Mruthinti et al. 2007. RAGE 23-54 peptide was used as it
was found to form a less neurotoxic and more immuno-
genic complex with Ab than longer RAGE fragments.
After incubation, samples were centrifuged in 10-kD cut-
off concentrators to remove any unbound peptides. Protein
concentration, dual-antigen specificity, and molecular
weight were then determined by ELISA, dot blot, and
Western blot, respectively.
Primary Cortical Neuronal Cultures
Cortical neurons were harvested from day 0 rat pups and
grown for 6 days in 96-well plates containing neurobasal
media, (Gibco, Inc) B27, L-glutamine, and penicillin/
streptomycin. Media were changed on day three by
removing 90% and replacing it with freshly prepared
media. After cells were grown for 6 days, treatment was
started. In the first experiment, either Ab1-42 alone (1, 10,
100, 250, and 500 nm) or 1-month-old Ab/RAGE complex
(1, 10, 100, 250, and 500 nm) was added to fresh media
and incubated for a 24-h period before subsequent analysis
with a VybrantÒ MTT cell proliferation assay kit (Molec-
ular Probes, Inc). In the second experiment, cells were
grown for 6 days and then incubated with endogenous anti-
RAGE antibodies (anti-RAGE concentrations used here
were based on previous experiments from Takuma et al.
2009) isolated from APPSWE-PS1 mice for 24 h before
the addition of Ab1-42 for an additional 24 h and sub-
sequent analysis with a VybrantÒ MTT cell proliferations
assay kit (Molecular Probes, Inc).
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PBMCs
Blood was drawn from healthy senior citizens (controls)
recruited for participation in this Alzheimer’s study at the
Charlie Norwood VA Medical Center (VAMC). The study
was overseen by the VAMC human IRB committee, and
informed consent was obtained from subjects. Blood
samples were washed twice with PBS and centrifuged at
5,0009g for 5 min and the supernatant was discarded.
Blood pellet was loosened with gentle tapping and incu-
bated with 10 ml of cold & sterile red-blood-cell (RBC)
lysing solution (8.3 g NH4Cl, 1.0 g KHCO3, and 1.8 ml of
5% EDTA). Cells were washed and centrifuged at 59g for
5 min three times. Supernatant was discarded and the
white-blood-cell pellet (WBC) was resuspended in 1 ml of
sterile PBS. Cells were counted and cultured in special
human leukocyte growth media (HLGM) prepared and
established by Dr. Mruthinti et al. (1995), for long-term
sustenance of PBMCs for in vitro experiments. Essentially,
HLGM contains BD-Cell animal component-free media
30 ml, McCoy’s 5A media 30 ml, 20 ml of human mye-
loma cell supernatant culture media, and 20 ml of IgG-
depleted fetal bovine serum (FBS) with the addition of
antibiotic/antimycotic, gentamycin, and fungizone antibi-
otics. PBMCs were counted and plated at a concentration
of 50,000 cells/well and treated with Ab1-42, full-length
recombinant sRAGE and sRAGE/Ab42 in vitro synthe-
sized complex (1 month) following methods described
elsewhere (Mruthinti et al. 2007). Plates were incubated at
37C° in a fully humidified incubator with a constant flow of
C02 (5%) in air. On day 6, culture supernatant was aspi-
rated gently and centrifuged at 59g for 5 min. IgG was
purified using IgG purification steps as described previ-
ously (Mruthinti et al. 2004). IgG subtyping was deter-
mined using a human antibody isotyping kit (from Pierce).
All samples were run in triplicates.
Immunizations
Two-month-old Balb-C mice (N = 10) were divided into
two groups: the first received an oral gavage containing
25 lg of Ab 42 in 100 ll water and the second group
received oral gavage containing 25 lg of 1-month-old
RAGE/Ab 42 complex. Boosters (containing the same
25 lg protein in 100 ll water) were administered
bi-weekly for a total of 6 weeks to each group using the
same gavage method. Plasma was taken bi-weekly before
each immunization or booster was given.
AD transgenic APPswe/PS1 mice (1 week old,
3 months, 9 months, and 15 months old) were split into
groups of 6 animals for each age group; controls received
an oral gavage of 100 ll water, and the treatment group
received 25 lg of 1-month-old RAGE/Ab42 complex in
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100 ll of water by oral gavage. Boosters (containing the
same 100 ll water or the same 25 lg 1-month-old RAGE/
Ab42 complex in 100 ll water) were administered
bi-weekly for a total of 6 weeks to each group using the
same gavage method. Killing age for each group was 3, 6,
12, and 18 months old.
Novel Object Recognition Task
Animals used for the novel object recognition (NOR) task
were divided into groups of N = 6–8 and immunized
bi-weekly starting at 3 months old until the animal reached
7 months of age. Immunizations consisted of control WT
and control TG receiving an oral gavage of 100 ll water.
The TG treatment groups either received 25 lg of
1-month-old RAGE/Ab42 complex in 100 ll of water by
oral gavage or 25 lg of Ab42 with Freund’s adjuvant via
intraperitoneal (IP) injection. Boosters containing the same
25 lg of 1-month-old RAGE/Ab42 complex or 25 lg of
Ab42 were given bi-weekly. The animals began the NOR
behavioral task at 13 months of age. The test apparatus
consisted of an open-field box 15.500 9 3100 diameter, and all
sessions were video-recorded. On day 1, the animal was
allowed to explore the open-field box for a 15-min time
period. The following day the animals were each exposed to
a 10-min information session (i.e., the A/A session with
identical objects present). This information session was then
followed by a 4-h delay where the animals were returned to
their home cages. After the delay, the animals performed a
10-min dissimilar stimuli session (A/B). The objects were
made of glass or hard plastic and had previously been
counterbalanced to control for any object preference bias.
The total amount of time spent with each object was recor-
ded. Time spent was operationally defined as occurring when
an animal directed its nose to the object at a distance of less
than 2.0 cm and/or by the animal touching the object with its
nose or mouth. This is a task of recognition memory where
the animals that can successfully recognize the familiar
object will spend more time exploring the novel object due to
each animal’s innate curiosity/exploratory behavior.
Statistics
Each assay was performed in replicates of three to six
where an individual replicate consisted of a single well
within a concentration series. Each assay (entire plate) was
performed at least three times. All statistical analyses were
performed on raw data. Data were analyzed by the use of a
multifactorial analysis of variance (ANOVA) with SAS,
JMP statistical software. Data analyzed with ANOVA were
followed by an orthogonal multicomparison t test when
comparing individual means. Independent two-sample
t test with equal sample size and equal variance was also
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Neuromol Med (2012) 14:119–130
used where appropriate. For each table/figure (below), error
values denoted by ± indicate the standard error of the
mean. Differences between means from experimental
groups were considered significant at the P \ 0.05 level
(2-sided test). Trends toward significance were considered
at the P \ 0.10 but [0.05.
Results
Baseline Anti-RAGE and Anti-Ab IgGs Titers
Anti-RAGE and anti-Ab antibodies in human subjects,
macaque monkeys, and AD transgenic APPswe/PS1 mice
are presented in Fig. 1. In panel A, the titers of both anti-
RAGE (r = 0.67) and anti-Ab (r = 0.71) antibodies rise as
the age of the animal increases. The regression lines for
both antibodies extrapolate to near 0 at the Y intercept.
Panel B shows the titers of anti-RAGE and anti-Ab plasma
antibodies in AD transgenic APPswe/PS1 mice. The titers
of both anti-RAGE (r = 0.89) and anti-Ab (r = 0.86)
antibodies rise as the age of the animal increases. The
regression lines for both antibodies extrapolate to near 0 at
the Y intercept. Panel C shows the titers of anti-RAGE,
anti-Ab, and anti-RAGE/Ab complex plasma antibodies in
human subjects. Normal elderly controls had the lowest
titers of all antibodies. MCI patients had significant
increase in anti-RAGE titers (P = 0.02), a near significant
increase in anti-Ab titers (P = 0.08), and a significant anti-
RAGE/Ab complex titers (P \ 0.001) compared to elderly
controls. AD patients had a statistically significant increase
in titers for all antibodies (P \ 0.001 for anti-RAGE,
P \ 0.001 for anti-Ab, P \ 0.001 for anti-RAGE/Ab
complex) compared to elderly control and MCI patients
(P \ 0.001 for anti-RAGE, P \ 0.001 for anti-Ab, and
P = 0.03 for anti-RAGE/Ab complex).
Primary Cortical Neuronal Cultures
Rat cortical neurons that were first subjected to a 24-h
exposure of endogenous anti-RAGE antibodies isolated
from APPSWE-PS1 mice (0.1, 1.0, 10, 100, and 1,000 lg/
ml) followed by a subsequent 24-h treatment with 100 nm
Ab1-42 were protected from cell death (Fig. 2). The
100-nm Ab1-42 was sufficient to produce a survivability
rate of 52.9%. Pretreatment with 0.1, 1.0, and 10 lg/ml of
anti-RAGE antibody can increase the survivability rate by
8.1, 20.3, and 27.4%, respectively. Pretreatment with
100 lg/ml anti-RAGE antibody can increase the surviv-
ability rate by 31.4%, while a treatment of 1,000 lg/ml
increased the survivability rate by 41.4%. Pretreatment of
1, 10, 100, and 1,000 lg/ml of anti-RAGE antibodies
produced a statistically significant reversal of the
Neuromol Med (2012) 14:119–130 123

Fig. 1 a Correlation of plasma

anti-RAGE (black) and anti-Ab
(gray) IgGs with age in
macaque monkeys of varying
ages. Note the regression lines
extrapolate to 0 age. b Plasma
anti-RAGE (black) and anti-Ab
(gray) IgGs derived from 49
wild-type (WT) and 49 AD
transgenic (Tg) mice of varting
ages. c Plasma anti-RAGE and
anti-Ab IgGs from study
participants. Study participants:
elderly control (light gray),
MCI–mild cognitive
impairment (gray), and AD
(black)

Ab1-42-induced toxicity. The 1,000 lg/ml of anti-RAGE
antibodies can produce a full reversal with no significant
difference between control neurons and the neurons treated
with 100 nm Ab1-42 ?1,000 lg/ml anti-RAGE antibody.
The neurotoxic profile of Ab1-42 and RAGE/Ab com-
plex was also examined in cortical neuronal cultures. The
RAGE/Ab complex was less neurotoxic than Ab1-42
alone. (Fig. 3) At the 500-nm dose, the neurons treated
with the RAGE/Ab complex survived at a rate of 45.9%.
Only 32.8% of the neurons treated with 500-nm Ab1-42
alone survived. Neurons treated with 1 nm, 10 nm,
100 nm, 250 nm, and 500 nm of the RAGE/Ab complex
survived at a rate of 92.3, 83.8, 66.8, 53.4, and 45.9%,
respectively, while only 93.5, 79.1, 58.5, 42.1, and 32.8%
of the neurons treated with 1, 10, 100, 250, and 500 nm of
Ab1-42 alone survived. For doses of 100, 250, and 500 nm,
the RAGE/Ab complex showed a statistically significant
reduction compared to Ab1-42 alone.
antigens (Ab1-42, sRAGE, and the RAGE/Ab complex),
there was a statistically significant (P \ 0.0001) effect of
treatment, producing anti-RAGE and anti-Ab IgGs. Ab1-
42 and sRAGE antigens produced a moderate but signifi-
cant induction of anti-RAGE and anti-Ab IgGs. Three out
of the four doses of RAGE/Ab complex antigen produced a
statistically significant increase in titer of anti-RAGE IgGs
relative to the sRAGE antigen. All four doses produced a
statistically significant increase in titer of anti-RAGE IgGs
relative to the Ab1-42 antigen. Similarly, all four doses of
RAGE/Ab complex antigen produced a statistically sig-
nificant increase in titer of anti-Ab IgGs relative to the
sRAGE antigen. Three of the four doses of RAGE/Ab
complex antigen produced a statistically significant
increase in titer of anti-Ab IgGs relative to the Ab1-42
antigen. There was no statistical difference between the
doses of 1,000 lg/ml RAGE/Ab complex antigen and
1,000 lg/ml Ab1-42 antigen at producing anti-Ab titers.

RAGE/Ab Complex Immunogenicity in PBMCs RAGE/Ab Complex Immunogenicity in Balb-C Mice

In human peripheral blood mononuclear cells (PBMCs), Balb-C mice immunized with the RAGE/Ab complex
Ab1-42, sRAGE, and the RAGE/Ab complex elicited anti- produce significantly higher antibody titers against both
RAGE and anti-Ab IgGs production (Fig. 4). For all three Ab1-42 (P \ 0.001) and RAGE/Ab complex (P \ 0.001)

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Fig. 2 The effect of incubation with endogenous anti-RAGE anti-
bodies isolated from APPSWE-PS1 mice on survivability of primary
culture rat cortical neuron exposed to 100-nm Ab1-42. The anti-
RAGE antibody can significantly increase in the survivability of the
rat cortical neuron exposed to 100-nm Ab1-42. *P \ 0.05,
**P \ 0.001
Fig. 3 The effects of incubation of Ab1-42 or 1-month-old Ab/
RAGE complex on primary culture rat cortical neuron survivability.
The RAGE/Ab complex shows a less potent neurotoxic profile than
administration of Ab1-42 alone in rat cortical neurons. *P \ 0.001
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Neuromol Med (2012) 14:119–130
Fig. 4 Comparison of the ability potential antigens to elicit the
production of anti-RAGE (panel A) and anti-Ab (panel B) antibodies
from human peripheral blood mononuclear cells (PBMCs) in culture.
The RAGE/Ab complex antigen produced a more rapid and
significantly greater quantity of both IgGs compared to the respective
antigens alone. *P \ 0.05, **P \ 0.01, ***P \ 0.001,  P \ 0.1
than did animals immunized with Ab1-42 alone (Fig. 5).
After just two administrations of immunogens, the RAGE/
Ab complex-immunized animals show more than double
the anti-Ab1-42 and anti-RAGE/Ab complex titer levels
compared to the animals given the Ab1-42 immunizations.
After 2 weeks of immunization with the RAGE/Ab com-
plex, immunized animals had measurably higher anti-Ab
titer levels compared to the Ab1-42 immunized animals.
This trend continued for the rest of immunization with the
4- and 6-week anti-Ab titers remaining statistically sig-
nificant for both.
Similarly, after 2 weeks of immunization with the
RAGE/Ab complex, immunized animals had a statistically
significant increase in titer against RAGE/Ab complex
when compared to the Ab1-42-immunized animals. This
trend continued for the rest of immunization with the
Neuromol Med (2012) 14:119–130
Fig. 5 Plasma titer levels from
2-month-old Balb-C mice
immunized with either Ab1-42
or 1-month-old RAGE/Ab
complex. Plasma was collected
and booster immunizations were
given bi-weekly for a total of
6 weeks to each group.
Antibody titers were isolated by
passage through a protein A/G
column and determined by
ELISA. *P \ 0.05,
***P \ 0.001
4- and 6-week titers against RAGE/Ab complex remaining
significantly higher.
RAGE/Ab Complex Immunization of Transgenic
APPSWE-PS1 Mice
At particular ages, immunization with the RAGE/Ab
complex significantly reduced both plasma and cerebral
Ab1-42 protein levels in APPswe/PS1 mice (Fig. 6).
Plasma levels of Ab1-42 in animals 3 and 6 months old
showed no significant reduction after immunizations.
Plasma levels of Ab1-42 were significantly reduced at the
12- to 18-month time points following immunizations.
Cerebral Ab1-42 levels showed no significant reduction at
the 3-month time point. There was a near statistically
significant effect of immunizations in the 6-month-old
animals, and the reductions were significant in the 12- and
18-month-old animals.
Novel Object Recognition Task
There was a clear preference for novel object exploration
(P = 0.026) during the dissimilar stimuli (A/B) session for
the control wild-type mice compared to the familiar object,
whereas the APPswe/PS1 transgenic mice did not show a
preference (did not spend more time exploring the novel
vs. familiar objects) (Fig. 7). The APPswe/PS1 transgenic
mice that were given the RAGE/Ab complex immuniza-
tions also spent a significantly (P = 0.034) greater amount
125
of time exploring the novel object compared to the familiar
object. The APPswe/PS1 transgenic mice that received
Ab1-42 immunization also appeared to spend more time
exploring the novel object; however, there was only a
strong trend toward statistical significance (P = 0.058).
Discussion
The brain’s immune system has long been implicated in the
pathogenesis of age-dependent neural diseases such as AD.
The brain is no longer viewed as an immunologically
isolated or privileged organ (Marx et al. 1998). Though
brain parenchyma does not possess many of the unique
immunological elements of the periphery, local inflam-
matory conditions can result in the accumulation and
activation of T cells in the brain in AD. T cells are
increased in AD, relative to controls, and they are acti-
vated, though not fully differentiated. It has been proposed
that the inflammatory response found near pathologic
markers in neurodegenerative diseases, including AD,
could result at least in part from the presence of AGE-
modified and highly immunogenic neural proteins (McGeer
and McGeer 2001, 2002). The Ab1-42 peptide and the
RAGE peptide are not particularly immunogenic peptides
by themselves. However, when they complex together,
they become more immunogenic.
Yan et al. (1997) reported that the RAGE–Ab interac-
tion acts as a catalyst for autoimmune responses. Similarly,
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126 Neuromol Med (2012) 14:119–130

Fig. 6 Plasma and brain levels

of Ab1-42 in different aged
APPswe/PS transgenic mice
(a model of AD) after
immunization with vehicle or
RAGE/Ab1-42 complex.
Plasma was collected and
booster immunizations were
given bi-weekly for a total of
6 weeks to each group. Ab
levels were determined by
ELISA assay
(a.u. = absorbance units). For
a 1 a.u. = 1.6 pg/mL and for
b 1 a.u. = 145 pg/g wet weight.
 
P \ 0.1, **P \ 0.01,
***P \ 0.001

(Mruthinti et al. 2003). While aggregation of Ab initially is

Fig. 7 The effects of Ab/RAGE complex vaccination or Ab vacci-
nation alone on behavior in the novel object recognition task. Ab/
RAGE complex vaccination produced a statistically significant
improvement in cognition. A strong trend toward significance was
observed for the Ab-alone vaccination group; however, it did not
quite reach statistical significance (P = 0.058)
mice immunized with AGEs derived from purified human
brain neurofilament protein generated IgGs against a pep-
tide fragment of the receptor for RAGE, and against human
Ab peptide, which suggests that the AGE immunogen
binds to endogenous RAGE- and Ab-like peptides, possi-
bly forming complexes with increased immunogenicity
receptor dependent, it may continue to survive as a
receptor-independent molecule (Bucciarelli et al. 2002).
The presence of circulating Ab-like antibodies in the
peripheral blood of AD patients has been previously
reported (Hyman et al. 1984; Lopez et al. 2001; Nath et al.
2003), although the titers of these antibodies have not
always been correlated specifically with the disease or
disease severity. We were able to detect higher levels in
AD subjects compared to controls by first purifying IgGs
from individual samples derived from AD individuals, then
isolating the specific anti-Ab (Fig. 1) (Mruthinti et al.
2004). AD individuals expressed high levels of anti-RAGE
IgGs as well. We have noted this trend before (Mruthinti
et al. 2006, Mruthinti et al. 2004). This suggests that neural
protein–AGE adducts contribute to the upregulation of
RAGE- and Ab-like proteins that are immunoreactive
species.
Anti-RAGE and anti-Ab antibodies increase with age in
macaque monkeys (Fig. 1), rendering these antibodies a
marker for age. Similarly, anti-RAGE, anti-Ab, and anti-
RAGE/Ab complex antibodies increase with the diseased
state in human AD subjects. We see a graded increase in
the plasma titers of all antibodies in normal elderly con-
trols, MCI, and probable AD, respectively. In our APP-
SWE-PS1 transgenic mice, we also see an increase in both
antibodies as the age and diseased state progress, sug-
gesting that these antibodies may potentially be used as a
marker for disease progression. Taken together, the data
clearly showed that plasma anti-RAGE and anti-Ab

123
Neuromol Med (2012) 14:119–130
antibody titers are related to aging and that in MCI and AD,
the levels are increased.
However, it is unclear whether these increased titers are
beneficial in AD. Increased anti-Ab1-42 antibodies have
been proposed to be beneficial in AD by reducing the
amyloid load and thus reducing the amyloid plaques in the
brain. It has been proposed that this is why there are
increased antibodies present in AD subjects as it is the
body’s way of attempting to clear the excess Ab from the
system. However, the increased antibody titers seen in AD
patients are still insufficient to halt the progression of the
disease. Both passive and active immunotherapy for Alz-
heimer’s disease has been focused around this idea that
producing high amounts of anti-Ab antibodies can more
effectively clear Ab1-42 and thus halt or reverse the
pathology of the disease. Anti-RAGE antibodies may also
be beneficial in AD. RAGE located within cerebral vas-
cular membranes mediates the transport of Ab from the
blood to the brain, thus supporting the buildup of cerebral
amyloid levels (Deane et al. 2003; Tanzi et al. 2004;
Herring et al. 2008). Thus, circulating anti-RAGE anti-
bodies potentially have the ability to block RAGE-medi-
ated Ab transport into the CNS. An increased anti-RAGE
titer could decrease the amount of Ab1-42 that returns to
the brain. RAGE permits the accumulation and supports the
aggregation of Ab, resulting in inflammation and cyto-
toxicity in neuronal cells (Yan et al. 1997; Lue et al. 2001).
Additionally, RAGE located on the neuronal membrane
has been implicated in the transport of Ab1-42 from the cell’s
surface into the intercellular space, increasing the toxicity of
Ab1-42. Increasing anti-RAGE titers has also been shown to
decrease this toxicity by reducing the amount of Ab1-42 that
enters the neurons. (Takuma et al. 2009) Here, we show that
pretreatment of primary rat cortical neurons with endoge-
nous anti-RAGE antibodies isolated from APPSWE-PS1
mice can protect against Ab1-42-induced toxicity (Fig. 2).
Thus, the increase in anti-Ab and anti-RAGE antibodies
could help in AD by the removal of amyloid from the brain
and periphery by decreasing the amount of amyloid that
enters into the brain, and by decreasing the amount of Ab1-
42-induced neurotoxicity.
To determine whether a RAGE/Ab complex vaccine
could potentially be a safe and effective therapeutic option
for AD, we assessed the toxicity, immunogenicity, ability
to decrease amyloid load, and ability to improve the cog-
nition of the RAGE/Ab complex compared to standard
Ab1-42 alone. The RAGE/Ab complex showed a less
potent neurotoxic profile than did the addition of Ab1-42
alone in rat cortical neurons (Fig. 3). This reduced toxicity
of the RAGE/Ab complex would suggest that when used as
a vaccine, it could provide a safer option compared to
standard Ab1-42 vaccines. Further, because of its large
size, it is unlikely that the RAGE/Ab complex can be
127
transported into the brain, whereas vaccines using the small
neurotoxic Ab1-42 peptide can be transported into the
brain causing undesired neurotoxicity.
To test the immunogenicity of the RAGE/Ab complex,
we looked at its ability to produce an immune response in
PBMCs (Fig. 4). The RAGE/Ab complex can produce
significantly greater titers of both anti-RAGE and anti-Ab
antibodies than either sRAGE or Ab1-42 alone. Thus, it
appears that the RAGE/Ab complex antigen is more
immunogenic than either RAGE or Ab alone in our
PBMCs system. Further, the increased anti-RAGE titers we
observed after immunization with the RAGE/Ab complex
could be beneficial by acting to partially block the RAGE
located on the blood–brain barrier and the RAGE located
on the neuronal plasma membrane as well. Next, we
examined the immunogenicity of the RAGE/Ab complex
compared to Ab1-42 in a whole animal model by immu-
nizing Balb-C mice with either the RAGE/Ab complex or
Ab1-42 (Fig. 5). The vastly superior immunogenicity of
the RAGE/Ab complex is first visible by just 2 weeks
following administration. By 4 weeks, the antibody titer of
both anti-RAGE and anti-Ab produced by the RAGE/Ab
complex were more than double what immunizations with
Ab1-42 produced. The ability of the RAGE/Ab complex to
produce significantly greater anti-Ab1-42 antibody titers
than Ab1-42? adjuvant is a promising one for the devel-
opment of using the complex as an oral vaccine. It is also
possible that such a robust titer level could be linked to
some of the negative inflammatory side effects seen in the
failed human Ab-based vaccines clinical trials, as high
antibody titers have been speculated to correlate with
inflammatory side effects. However, other laboratories
have speculated that the side effects are not likely to have
been caused by the overall antibody titer levels (as roughly
28% of the patients who developed these inflammatory side
effects had very low antibody titer levels) but rather the
Q21 adjuvant and polysorbate 80 additive (both have been
shown to produce a proinflammatory Th1(type 1 helper T
cell) response which has been linked to the inflammatory
side effects seen) (Orgogozo et al. 2003, Raghavendra et al.
2004, Coors et al. 2005, Price and Hamilton 2007, Pride
et al. 2008).
A potential limitation to vaccination with the RAGE/Ab
vaccine (or any amyloid beta–based vaccine) used here is
the type of immune response generated; specifically, if
there is a activation of CD4? T-helper cells that lead to a
negative Th1 cellular immunity instead of the targeted Th2
response that leads to a desired humoral immunity, the
balance between levels of Th1- and Th2-helper T cells is
therefore important in regulating the negative autoimmune
inflammatory side effects seen in Ab vaccines. A positive
Th2 response can lead to the robust production of positive
IgG1 and IgG2b antibodies and suppress the negative
123
128
autoimmune encephalitis side effects (Fang et al. 2010). In
future studies, we will characterize the type of humoral
immune response after vaccination with RAGE/Ab by
measuring the production of different classes (IgG1,
IgG2ab, IgG2b, and IgM) of antibodies in order to deter-
mine whether it is working through a Th1 or Th2 response.
Fortunately, the route of administration can play a role in
determining which type of T-helper cell response will be
produced. Here, we used an oral vaccination that employs
the endogenous immune system of the gut, which has been
shown to be highly skewed toward a Th2-helper cell
response (Fang et al. 2010).
Due to the robust immunogenic profile of the RAGE/Ab
complex, we wished to examine whether immunization
using the complex could be successful in reducing plasma
and cerebral Ab1-42 protein levels. Immunization with
RAGE/Ab42 complex can significantly reduce both plasma
and cerebral Ab1-42 protein levels in APPswe/PS1 mice
(Fig. 6). In future studies, we plan to examine the regional
differences in Ab level in the brain using immunohisto-
chemistry to better understand the nature of this observed
Ab reduction. Interestingly, the largest reduction was seen
in 12- to 18-month-old groups, while little to no reduction
was seen in the 3- to 6-month-old groups. In this mouse
model of AD, the mice start to accumulate increased Ab
plaques at 6–7 months of age and cognitive impairment at
10-12 months of age (Savonenko et al. 2005). Hence,
immunization with RAGE/Ab42 complex seems to reduce
Ab load only in diseased states and does not reduce Ab
levels in normal healthy mice. This ability of the RAGE/
Ab complex immunization to reduce plasma and cerebral
Ab1-42 in an already diseased state (rescue strategy) sug-
gests that immunization at an earlier time point (prevention
strategy) may also be beneficial. We examined the effects
of early immunization with the RAGE/Ab complex com-
pared to Ab1-42 immunization alone on cognitive behavior
in the novel object recognition task (Fig. 7). The premise
behind the NOR task is that rodents will explore a novel
object more than a familiar one, but only if they remember
that they have previously been exposed to the familiar
object. The APPswe/PS1 transgenic mice were unable to
discriminate between novel and familiar objects, while the
wild-type controls spent significantly more time with the
novel object. This indicates that the transgenic mice were
cognitively impaired. Immunization using the RAGE/Ab
complex can prevent this observed cognitive impairment in
the APPswe/PS1 transgenic mice. In fact, APPswe/PS1
transgenic mice that were first immunized with the com-
plex closely resembled their wild-type controls spending a
significant more amount of time exploring the novel object.
We have also seen similar cognitive enhancement in the
Radial Arm Water Maze behavioral task after RAGE/Ab
vaccination (data presented in a different manuscript
123
Neuromol Med (2012) 14:119–130
currently submitted and in the review process elsewhere).
The standard Ab1-42 immunization seemed to also show
an improvement (more time spent with the novel object),
although the treatment group did not quite reach statistical
significance.
The RAGE/Ab immunizations here were administered
orally with water unlike adjuvant-based vaccine trials by
others in the past (Buttini et al. 2005). It is our hope that
water may provide a safer alternative compared to harsher
adjuvants used in other vaccine studies. Adjuvants have
long been known to boost inflammation, and this may not
be a desirable thing especially considering that previous
human vaccine studies have been halted due to meningo-
encephalitis and inflammation (Rafii and Aisen 2009). Ab
aggregates found in senile plaques represent end-stage
disease process, indicating that age-related weakening of
the immune system may render the system insufficiently
robust to generate antibodies against active Ab vaccine.
Also, humanized anti-Ab murine mAbs are known to lose
their efficacy and affinity in the humanization process,
suggesting that higher IgG concentrations may be required
for effective treatment. Immunization with the RAGE/Ab
complex would likely not have these shortcomings, as it
produced a much greater antibody titer compared to Ab1-
42 alone. Proposed Ab1-42 vaccine therapy has been
viewed as promising disease-modifying therapy for AD
based on animal experiments. The modus operandi of Ab
vaccination can be explained by the ‘‘peripheral sink’’
hypothesis, which postulates that Ab exists in equilibrium
in the brain and periphery, and thus, anti-Ab antibodies in
systemic circulation can remove the Ab in the periphery,
thereby promoting the efflux of Ab from brain to blood. It
is possible that as insoluble Ab plaques shrink in the cor-
tex, they can release soluble forms of the Ab peptide that
are potentially neurotoxic to neurons. However, this is
usually accompanied by simultaneously higher levels of
soluble Ab in the periphery. Here, we see decreased levels
of soluble amyloid in the periphery after RAGE/Ab vac-
cination (Fig. 6). Thus, it is possible (however still unclear)
that the levels of soluble Ab in the cortex are also
decreased after the administration of the RAGE/Ab-tar-
geted vaccine.
A clinical trial using synthetic Ab peptide (AN-1792) in
the adjuvant vehicle was soon halted as &6% of these
patients developed aseptic meningoencephalitis (Gilman
et al. 2005). Moreover, this vaccine regime resulted in
cerebral microhemorrhage in AD-Tg mice. Thus, there is
an immediate need to develop a safer vaccine therapy.
Some of the alternate vaccine regimens that seem to be
promising include intraperitoneal route of vaccination by
Schenk et al. (1999), which showed a dramatic reduction in
cerebral amyloidosis when injected with Ab1-42 but still
required Freund’s adjuvant to be co-administered, opening
Neuromol Med (2012) 14:119–130
the door to many unwanted inflammatory side effects.
Elimination of these harsh inflammatory adjuvants could
reduce these unwanted side effects. Yet the majority of
vaccines are currently still administered with adjuvants.
Here, we used a novel mechanism of water-based oral
vaccine therapy using in vitro prepared RAGE/Ab active
vaccine. The RAGE/Ab complex used here is highly
immunogenic without the additional use of an accompa-
nying adjuvant. We believe that this strategy could possibly
provide a much safer choice, as compared to standard
immunization methods, by eradicating the problems asso-
ciated with using an adjuvant while still maintaining effi-
cacy at the modulation of amyloid and at improving
cognitive performance.
Acknowledgments This work was supported in part by a Merit
Review award from the Charlie Norwood VA Medical Center funded
to Dr. Mruthinti, P. I, and by Georgia Health Sciences University,
Alzheimer’s Research Center directed by Jerry J. Buccafusco (late).
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