Effects of Dietary Supplementation of Carnosine on
Mitochondrial Dysfunction, Amyloid Pathology, and
Cognitive Deficits in 3xTg-AD Mice
Carlo Corona1,2, Valerio Frazzini1,2, Elena Silvestri3, Rossano Lattanzio4, Rossana La Sorda4, Mauro
Piantelli4, Lorella M. T. Canzoniero3, Domenico Ciavardelli1, Enrico Rizzarelli5, Stefano L. Sensi1,2,6*
1Molecular Neurology Unit, Center of Excellence on Aging (Ce.S.I.), University ‘‘G. d’Annunzio’’, Chieti-Pescara, Italy, 2Department of Neuroscience and Imaging,
University ‘‘G. d’Annunzio’’, Chieti-Pescara, Italy, 3Department of Biological and Environmental Science, University of Sannio, Benevento, Italy, 4Department of Oncology
and Neuroscience, University ‘‘G. d’Annunzio’’, Chieti-Pescara, Italy, 5Department of Chemistry, University of Catania, Catania, Italy, 6Department of Neurology, University
of California Irvine, Irvine, California, United States of America
Background: The pathogenic road map leading to Alzheimer’s disease (AD) is still not completely understood; however, a
large body of studies in the last few years supports the idea that beside the classic hallmarks of the disease, namely the
accumulation of amyloid-b (Ab) and neurofibrillary tangles, other factors significantly contribute to the initiation and the
progression of the disease. Among them, mitochondria failure, an unbalanced neuronal redox state, and the
dyshomeostasis of endogenous metals like copper, iron, and zinc have all been reported to play an important role in
exacerbating AD pathology. Given these factors, the endogenous peptide carnosine may be potentially beneficial in the
treatment of AD because of its free-radical scavenger and metal chelating properties.
Methodology: In this study, we explored the effect of L-carnosine supplementation in the 3xTg-AD mouse, an animal model
of AD that shows both Ab- and tau-dependent pathology.
Principal Findings: We found that carnosine supplementation in 3xTg-AD mice promotes a strong reduction in the
hippocampal intraneuronal accumulation of Ab and completely rescues AD and aging-related mitochondrial dysfunctions.
No effects were found on tau pathology and we only observed a trend toward the amelioration of cognitive deficits.
Conclusions and Significance: Our data indicate that carnosine can be part of a combined therapeutic approach for the
treatment of AD.
Citation: Corona C, Frazzini V, Silvestri E, Lattanzio R, La Sorda R, et al. (2011) Effects of Dietary Supplementation of Carnosine on Mitochondrial Dysfunction,
Amyloid Pathology, and Cognitive Deficits in 3xTg-AD Mice. PLoS ONE 6(3): e17971. doi:10.1371/journal.pone.0017971
Editor: Mel Feany, Brigham and Women’s Hospital, Harvard Medical School, United States of America
Received January 8, 2011; Accepted February 16, 2011; Published March 15, 2011
Copyright: ? 2011 Corona et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: SLS and ER are supported by funds from the Italian Department of Education [Fondo per gli Investimenti della Ricerca di Base (FIRB) 2003; Progetti
Ricerca Interesse Nazionale (PRIN) 2006 and PRIN 2008]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Mitochondria-driven overproduction of reactive oxygen species
(ROS) and imbalanced homeostasis for endogenous metals and
zinc (Zn2+) in particular, are important co-factors in the
development and progression of several neurological disorders,
including Alzheimer’s Disease (AD; [1,2]). Furthermore, a growing
body of evidence indicates that mitochondrial failure is an early
event in AD, suggesting that, along with deposits of amyloid-b (Ab)
and hyperphosphorylated tau (h-tau) protein, the malfunctioning
of the organelles plays a synergistic role in triggering the neuronal
death and cognitive decline associated with the disease (Reviewed
in ). For instance, enhanced ROS generation of mitochondrial
origin [4,5] can greatly interfere with homeostatic mechanisms
regulating levels of intracellular free Zn2+([Zn2+]i), thereby
producing intraneuronal [Zn2+]irises that generate a vicious loop
leading to enhanced Zn2+-dependent formation of ROS as well as
Ab oligomerization [2,6,7,8].
The rationale for addressing Zn2+dyshomeostasis in AD is
substantial . In vitro, Zn2+induces the aggregation and
oligomerization of Ab  and, in AD transgenic animals, in vivo
release of pre-synaptic Zn2+
promotes amyloid plaque formation . Moreover, recent
findings in cultured neurons from triple-transgenic AD mice, the
3xTg-AD, overexpressing mutant amyloid precursor protein
(APP), presenilin 1 (PS1), and h-tau indicate that the pro-AD
environment fostered by these mutants enhances ROS generation
and ROS-mediated [Zn2+]imobilization from intracellular Zn2+-
binding proteins, thereby providing a potential mechanism for the
initiation of the intraneuronal aggregation of Ab [11,12].
Finally, treatment with Zn2+and Cu2+chelators like clioquinol
(CQ) and its derivative, PBT2, shows efficacy in reducing amyloid
from glutamatergic terminals
PLoS ONE | www.plosone.org1March 2011 | Volume 6 | Issue 3 | e17971
plaques and counteracts cognitive deficits in AD transgenic mice
[13,14]. Furthermore, recent clinical trials employing CQ or
PBT2 have found that the compounds are also effective in
protecting against the development and progression of cognitive
deficits in AD patients [15,16].
Carnosine (b-alanyl-L-histidine) is a dipeptide found at high
concentrations in glial and neuronal cells throughout the brain
. The functional role of carnosine is still not completely
understood; however, several studies indicate that the dipeptide
exerts protective actions against metal- and or Ab-mediated
toxicity by acting as anti-oxidant and free-radical scavenger
[18,19,20,21]. Moreover, because of its enrichment in histidine
residues, carnosine has also been proposed as a chelator for
divalent cations like Cu2+and Zn2+. Furthermore, most likely
because of its chelating properties, carnosine can relieve the Zn2+-
mediated inhibition of NMDA and GABA receptors and therefore
modulate neuronal excitability . Carnosine also exerts anti-
aging activities by neutralizing injurious glycated proteins and
aldehydic products of lipids peroxydation (i.e., acetaldehyde,
malondialdehyde, and hydroxynonenal), thereby attenuating
the toxicity of these bioproducts and preventing the cross-
linking of glycoxidised proteins to physiological macromolecules
[23,24,25,26]. In the context of AD, carnosine has been suggested
as therapeutic agent given its capability to act as a metal chelator,
free radical scavenger as well as an inhibitor of Ab toxicity .
However, one possible limitation in employing carnosine as an
anti-AD drug is that brain carnosine is rapidly inactivated by the
activity of three different isoforms of the carnosine degrading
enzyme, carnosinase. Increased carnosinase activity has in fact
been found in AD patients as well as in aging individuals [27,28]
and decreased plasmatic levels of carnosine have been reported in
AD patients .
In this study we investigated the potential beneficial effects of
dietary carnosine supplementation (10 mM in drinking water) in
3xTg-AD mice, an AD animal model that develops amyloid- and
tau-dependent pathology as well as AD-related cognitive deficits
Carnosine chelates intracellular Zn2+
Carnosine has been described to form complexes with Zn2+in
aqueous solution [31,32]; however, to date, no ‘‘in situ’’
experiments have demonstrated whether the dipeptide can chelate
Zn2+in cellular models. Thus, to evaluate the, in vitro, chelating
properties of the peptide, we loaded cultured cortical glial cells
with the Zn2+-sensitive fluorescent probe, Newport green (Kdfor
Zn2+=1?1026M), and studied whether the addition of carnosine
would decrease [Zn2+]i rises triggered by oxidative stress. We
chose to use a relatively high carnosine concentration (20 mM)
because of the nature of the employed paradigm where we
triggered acute and intense [Zn2+]i rises. In the first set of
experiments, Newport Green loaded cells were, after baseline
evaluation, exposed for 20 min to the disulfide oxidizing agent
2,29-dithiodipyridine [(DTDP; 100 mM), a compound that pro-
motes [Zn2+]irelease from Zn2+-binding proteins like metallothio-
neins (MTs; [33,34]). [Zn2+]irises were evaluated over a period of
20 min (Figure 1). As expected, DTDP exposure caused a
sustained increase of [Zn2+]i levels. However, when the same
experiment was repeated in cultures treated with 20 mM
carnosine during the basal period as well as during the DTDP
exposure, ROS-driven cytosolic [Zn2+]i rises were largely
attenuated, confirming the idea that carnosine is an effective cell
permeable [Zn2+]ichelator (Figure 1).
Carnosine shows sub-maximal effects in rescuing long-
term memory deficits in treated 3xTg-AD mice
3xTg-AD mice have been reported to show age-dependent
spatial memory decline as early as 5–6 months of age (m.o.a.;
[11,30]). We designed our study to test the effect of carnosine
supplementation on AD-like memory deficits of 3xTg-AD mice.
We chose to use 10 mM carnosine, moving from the assumption
that the AD-related Zn2+dyshomeostasis is a chronic process that
is likely to be associated with less intense rises in cation levels
compared to what we have described in the Zn2+imaging
Figure 1. Carnosine chelates [Zn2+]i rises mobilized from
intracellular sites. (A) Time course of DTDP-mediated [Zn2+]irises in
cortical glial cells. Newport Green loaded astrocytes (white circle) were
imaged upon a 20 min incubation in a physiological buffer, HCSS, and
during a 20 min exposure to DTDP (100 mM), a compound that
mobilizes Zn2+from intracellular Zn2+-binding proteins. In parallel
experiments, Newport Green loaded glial cultures (black circles) were
incubated in HCSS plus carnosine (20 mM) for 20 min and subsequently
exposed to DTDP plus carnosine for other 20 min. The graph shows the
time course of DTDP-induced Newport Green fluorescence changes
(expressed as ratio of FX/F0) in carnosine treated and untreated glial
cultures. Traces show mean (6 SEM) fluorescence changes deriving
from 3 different experiments for each condition. (B) Bar graph depicts
the overall cytosolic [Zn2+]irise expressed as area under the curve after
the DTDP exposure. (*) indicates differences between control and
carnosine treated astrocytes (p,0.001).
Carnosine Supplementation in 3xTg-AD Mice
PLoS ONE | www.plosone.org2March 2011 | Volume 6 | Issue 3 | e17971
experiments). To evaluate the cognitive performance of treated
and control mice, we employed the Morris Water Maze (MWM)
test. Animals were subjected to 3 consecutive training days for the
hidden-platform version of the MWM, a task that measures
hippocampus-dependent spatial memory . At first, we assessed
the integrity of learning processes and found no differences in task
acquisition (data not shown), indicating that all groups learned the
same way to find a submerged platform using intra- and extra-
maze visible cues. After the last training trial, spatial reference
memory probe trials were conducted at 1.5 and 24 h in order to
evaluate short- and long-term memory performances, respectively.
As expected, compared to control (PS1KI) animals , 3xTg-AD
mice showed no impairment in short-term memory while they
manifested long-term memory deficits as indicated by the
statistically significant increase in the time spent to find the
platform (Figure 2). Carnosine supplementation was not able to
completely rescue long-term memory deficits in treated 3xTg-AD
mice. We observed a positive trend toward a better cognitive
performance as indicated by the decreased latency to find the
platform. Untreated 3xTg-AD mice showed a statically significant
worst performance (p,0.05), no differences were found when the
comparison was made between treated 3xTg-AD and control mice
(p,0.1); however, when treated and untreated 3xTg-AD mice
were compared, the difference failed to reach statistical signif-
icance (Figure 2).
Carnosine supplementation reduces intraneuronal Ab
deposition but is ineffective on tau pathology in the
hippocampus of 3xTg-AD mice
After cognitive evaluation, mice were killed and neuropathology
assessed. 3xTg-AD mice have been reported to undergo a
progressive intraneuronal accumulation of Abin AD-relevant
regions starting at 4 m.o.a. . To investigate whether carnosine
supplementation can decrease the brain Ab load, immunohisto-
chemistry was performed with the anti-Ab DE2B4 primary
antibody and analysis of this assay showed a significant decrease
of intraneuronal Ab in the hippocampus of treated 3xTg-AD mice
At about 12 m.o.a., 3xTg-AD mice also develop extensive
neurofibrillary tangles, first in the hippocampus (in particular
within pyramidal neurons of the CA1 subfield) and then in the
cortex . To investigate the effect of carnosine on the
development of tau pathology we employed an anti-tau AT180
primary antibody that specifically detects tau phosphorylation at
the thr231/ser235 site. Diffuse phospho-tau immunoreactivity was
found in the CA1 subfield of untreated 3xTg-AD mice
(Figure 3E,G). Interestingly, although carnosine was able to
reduce the amyloid load, the treatment produced no decreases of
phospho-tau immunoreactivity in the 3xTg-AD CA1 subfield
(Figure 3E-H), indicating that the peptide has no effect on tau
Carnosine supplementation counteracts age-dependent
Previous studies have shown that brain mitochondria of 3xTg-
AD mice show deficits in mitochondrial respiration . We have
also found a similar age-dependent decrease in the activity of
complex I (NADH-dehydrogenase), II (succinate-dehydrogenase),
and IV (COX) in mitochondria isolated from the cortex and
hippocampus of 3xTg-AD animals . To test the effect of
carnosine on such age-dependent mitochondrial deficits, treated
and untreated 3xTg-AD mice were investigated for the activity of
mitochondrial complexes I, II, and IV by employing a
combination of blue-native poliacrylammide gel electrophoresis
(BN-PAGE) and subsequent histochemical in-gel staining of
isolated mitochondria from the hippocampus and cerebral cortex.
Data from these experiments indicate that, compared to control
mice, hippocampal mitochondria of untreated 3xTg-AD mice
show a strong deregulation in the activity of complexes I, II, and
IV (Fig. 4A–B). Interestingly, carnosine-fed 3xTg-AD mice
exhibited a complete recovery of all these deficits and in the case
of complexes II and IV the activity was actually significantly
higher compared to mitochondria of control animals (Figure 4A–
B). Analysis of mitochondrial activity in the cortex revealed that
untreated 3xTg-AD mice showed a dramatic decline in the activity
of complex I and, to a lesser extent, of complex IV (Figure 4C–D).
Similarly to what was found in the hippocampus, carnosine
treatment promoted a complete recovery of all these cortical
Carnosine is an endogenous dipeptide highly expressed
throughout the brain that has been suggested as a therapeutic
tool in the treatment of AD, because the compound can act as an
endogenous anti-oxidant, free radical- and metal ion chelator, and
also has neuroprotective activity against in vitro Ab-induced
toxicity [20,32,37]. Thus, the major aim of this study was to
evaluate the effect of dietary carnosine supplementation in a model
of AD that develops an age-related neurodegenerative phenotype
that is driven by intraneuronal deposition of Ab and accumulation
of h-tau . We chose to treat only male 3xTg-AD mice as
female hormones are known to negatively influence the activity of
Zn2+transporters  and differently affect the disease progres-
sion , therefore producing confounding effects. We chose to
use PS1-KI animals as control group as these mice overexpress
Figure 2. Carnosine treatment has sub-maximal effect in
counteracting memory deficits in 3xTg-AD mice. Carnosine-
treated (n=9) and untreated 3xTg-AD (n=13) as well as control mice
(n=11) were tested for the spatial memory version of the MWM. Mice
were given a memory probe trial where the platform was removed 1.5
and 24 h after the last training trial to evaluate short-term and long-
term memory, respectively. All the three groups showed no short-term
memory impairment as assessed by MWM performance at 1.5 h. When
tested for long-term memory deficits at 24 h, untreated 3xTg-AD mice
exhibited a significant deficit in their performance as assessed by the
marked increase in the time (latency) they employed to reach the point
where the platform used to be. Carnosine-fed mice showed a trend
toward decreased long-term memory deficit. Error bars are shown
mean (6 SEM); (*) indicates p,0.05.
Carnosine Supplementation in 3xTg-AD Mice
PLoS ONE | www.plosone.org3 March 2011 | Volume 6 | Issue 3 | e17971
mutant Presenilin-1 gene (M146V substitution) but, by lacking the
expression of mutant APP and h-tau, do not show Ab or tau-
dependent pathology nor AD-related cognitive deficits .
Carnosine has been shown to protect against Zn2+-mediated
toxicity in cell cultures and that activity has been linked to the
chelating properties of the compound. The peptide has indeed
been shown to complex Zn2+in acqueous solution but, to date,
there were no experimental data demonstrating its chelating
capability in biological systems. We tested this hypothesis in
cultured glial cells and show that the peptide is in fact able to
chelate [Zn2+]i(Figure 1).
In our study, we also found a very potent effect of carnosine in
rescuing mitochondrial dysfunctions in aged 3xTg-AD mice. As
discussed above, mitochondrial deficits are emerging as key players
in AD   and, in line with observations indicating that Ab
and tau synergistically impair OXPHOS complexes , we found
signs of potent deregulation of mitochondrial respiration in our
AD mice. In 3xTg-AD mice at 12–14 m.o.a., we observed a
reduction in the activity of complex I, II and IV in the
hippocampus as well as of complex I and IV in the cortex.
Interestingly, carnosine supplementation not only prevented such
deficits but, in the hippocampus, we found that complexes II and
IV activity of carnosine-fed AD mice actually increased over
baseline (Figure 4). Such results can be linked to the antioxidant
activity of the dipeptide, a property that can prevent ROS-
dependent mobilization of [Zn2+]i. Such activity can block Zn2+-
dependent mitochondrial dysfunction , and inhibit the
overproduction of nitric oxide , a process strongly potentiated
by Zn2+that eventually contributes to a self perpetuating
mobilization of the cation. In addition, carnosine may have a
direct effect on Ab deposition and mitochondrial function by
acting as an osmolyte as shown in the case of its action on
methylene blue and cytochrome c oxidase .
When we analyzed the effects of carnosine on amyloid and tau
pathology we found that the peptide is very effective in decreasing
intraneuronal Ab deposition in the hippocampus but does not
affect the development of tau pathology.
Analysis of the effect of carnosine supplementation on cognitive
deficits of 3xTg-AD mice showed a positive trend, indicating that
it might have a beneficial role in preventing long-term memory
deficits, although, this effect did not reach statistical significance.
The sub-maximal effect we observed could be related to the fact
that carnosine is able to greatly inhibit the Ab load but not the
appearance of tau pathology and these two molecular components
Figure 3. Carnosine supplementation reduces intraneuronal Ab but not tau accumulation in the hippocampus of 3xTg-AD mice.
Immunohistochemistry was employed to detect deposits of intraneuronal Ab (A–D) and h-tau (E–H) in brain slices from treated (n=3) and untreated
(n=3) 3xTg-AD mice (left column: 56magnification, scale bar 200 mm; right column: 406magnification of the hippocampal CA1 subregion, scale bar
20 mm). Compared to untreated 3xTg-AD mice (A,B), immunohistochemical staining shows a strong decrease of intraneuronal Ab immunoreactivity in
the hippocampus of carnosine treated 3xTg-AD mice (C,D). (I) Quantification of intraneuronal Ab load as shown in B and D. Untreated 3xTg-AD mice
(E,F) show comparable intraneuronal h-tau deposits in the hippocampus compared to treated mice (G,H). (J) Quantification of h-tau levels as shown
in F and H. Error bars are shown as mean (6 SEM); (*) indicates p,0.05.
Carnosine Supplementation in 3xTg-AD Mice
PLoS ONE | www.plosone.org4 March 2011 | Volume 6 | Issue 3 | e17971
are definitely acting synergistically in the development of the
cognitive decline [43,44,45].
In the last few years a growing body of evidence is supporting
the intriguing hypothesis that the alteration in the equilibrium of
brain Zn2+levels can be a significant contributing factor for AD
. Interestingly, both excess as well as deficit of brain Zn2+can
favour AD-like pathology in AD animal models [36,46] suggesting
the existence of a finely tuned Zn2+set point. Such hypothesis has
been substantiated by a recent study indicating that deficits of
negatively interfering with glutamatergic and BDNF signalling
. Thus, in such scenario, a decreased Zn2+bioavailability
induced by carnosine supplementation may in part exert negative
effects on the neurotransmission and neurotrophic signalling that
modulate cognitive functions, and in doing so, counteracts the
positive activity on amyloid deposition and mitochondrial
functioning. In that respect, it could be interesting to verify the
possibility that more effective synergistic activity can be achieved
promotes AD-like cognitive impairment by
Figure 4. Carnosine supplementation rescues mitochondria deficits in 3xTg-AD mice. BN-PAGE was employed to assess the activity of
mitochondrial complexes I, II, and IV in isolated mitochondria obtained from the hippocampus and cerebral cortex of control (PS1KI), untreated, and
carnosine-treated 3xTg-AD (n=3 to 5) mice at 12–14 m.o.a. (A,B) When compared with age-matched untreated mice, activity of mitochondrial
complexes I and IV are found decreased in the hippocampus of 3xTg-AD mice while carnosine supplementation completely prevents the deficits.
(C,D) When comparing complex activities of mitochondria obtained from the cortex of 3xTg-AD vs age-matched control mice, complex I and IV are
found strongly compromised and carnosine treatment rescued these deficits. Error bars are expressed as mean (6 SEM); (*) indicates p,0.05; (**)
Carnosine Supplementation in 3xTg-AD Mice
PLoS ONE | www.plosone.org5 March 2011 | Volume 6 | Issue 3 | e17971
when carnosine and Zn2+are administered together in a combined
form similarly to what has been described for the compound
named Polaprezinc, a preparation that combines Zn2+and L-
carnosine [48,49]. In theory, this compound, given the different
Kd for Zn2+of carnosine and Ab can act as an homeostatic
molecule that sequesters Zn2+from Ab but then releases a
sufficient amount of the cation in the synaptic cleft to exert
Another possibility that could explain the subthreshold effect on
cognition is associated with changes in carnosinase activity as the
enzyme has been found to undergo an age-dependent enhanced
activity in brains of aging individuals and AD patients [28,29].
Finally, it is also possible that a more robust effect could be
revealed by extending these behavioural studies to a larger cohort
In summary, carnosine has a strong effect in restoring
mitochondrial functioning and in counteracting amyloid pathol-
ogy but these activities do not translate in a robust effect on
cognition. These results suggest that, at least in complex AD
animal models, addressing mitochondrial dysfunction and Ab
aggregation without a parallel intervention on h-tau deposition is
not sufficient to promote major beneficial cognitive effects.
Supporting this idea, recent reports have in fact indicated that
therapeutic measures addressing Ab overloads but unable to
reduce the development of tau pathology do not prevent the
development of cognitive deficits in 3xTg AD mice [44,50].
Materials and Methods
Newport Green and pluronic acid were purchased from
Molecular Probe (Invitrogen). L-Carnosine and DTDP (2,29-
dithiodipyridine) were obtained from Sigma Aldrich. Tissue
culture media and serum were purchased from Gibco (Invitrogen).
Glial cell cultures
Murine cortical glial cultures were prepared from CD-1 mice as
previously described . Briefly, neocortices from 1–3 day pups
were dissociated and plated in 35 mm glass bottom dishes in a
MEM medium in the presence of 10% horse serum and 10% fetal
bovine serum. Cells were maintained in a humidified atmosphere
containing 5% CO2and used for the experiments 10 days after
Zn2+imaging was performed using an inverted microscope
(Nikon Eclipse TE 300) equipped with a xenon lamp, a filter wheel
(Lambda shutter 10–2, Sutter Instruments), and a 40X epifluor-
escence oil immersion objective. Glial cells were loaded in the dark
with the DCF diacetate form of the Zn2+-sensitive probe Newport
Green (5 mM + 0.2% of Pluronic Acid), in a HEPES-buffered
medium (HCSS) whose composition was (in mM): 120 NaCl, 5.4
KCl, 0.8 MgCl2, 20 HEPES, 15 glucose, 1.8 CaCl2, 10 NaOH,
pH 7.4 for 30 min at 25uC. Cultures were then washed in HCSS
and kept in the dark for an additional 30 min. Excitation was at
490 nm, with emission at 510 nm as previously described .
Images were acquired every 30 sec during all the experimental
session. To compensate for cell-to-cell variability in dye loading,
after background subtraction from a cell-free region of the dish,
Newport Green fluorescence measurements for each cell (Fx) were
normalized to the fluorescence intensity for that cell at the
beginning of the experiment (F0). Drugs were applied by bath
application and removed through a rapid flow exchange system.
Images were acquired with a 12-bit digital CCD camera (Orca,
Hamamatsu) and analyzed with Metafluor 6.0 software (Invitro-
gen). Values are reported as mean 6 SEM of FluoZin-3 Fx/F0
Animals and treatment paradigm
Procedures involving animals and their care were approved by
the institutional Ethics Committee (CeSI protocol #: AD-301) and
conducted in conformity with the institutional guidelines that are
in compliance with national (D.L. n. 116, G.U., suppl. 40, 18
February 1992) and international laws and policies. All efforts
were made to minimize the number of animals used and their
suffering. Transgenic mice were characterized and described by
Oddo et al.  and generously provided by Frank Laferla. One
month old male 3xTg-AD mice (n=9) were treated with 10 mM
L-Carnosine (Sigma-Aldrich) in standard tap water for a period of
11–13 months. Control groups [3xTg-AD (n=13) and control
PS1-KI (n=11)] mice were given just tap water.
Morris water maze
The Morris water maze (MWM) apparatus consisted of a circular
plastictank filled with water(1.3 m diameter).The mazewaslocated
in a room containing several intra and extra-maze visual cues. Mice
were trained to swim on a (12613 cm) rectangular platform
submerged 2 cm beneath the surface of the water and invisible to
the animals while swimming. To reduce stress, mice were placed on
the platform 10 s prior to the first training trial. They were allowed
to find an escape by climbing on the submerged platform; if the
mouse failed in finding the platform within 90 s, it was manually
guided to the platform and allowed to remain there for 10 s. After
that period, each mouse was placed into a holding cage under a
warming fan for 20 minutes, until the start of the next training trial.
Mice were given 4 trials per day for 3 consecutive days with an inter-
trial time of 20 min. Retention of the spatial memory was assessed
1.5 and 24 hours after the end of the last training trial. Both probe
trials consisted of a 60 s free swim in the pool without the platform.
Mice were monitored by a digital camera mounted on the ceiling of
the room directly above the pool and all trials stored for subsequent
analysis. The parameter employed to evaluate memory skills was the
time (latency) to reach and cross the platform location.
Carnoy-fixed and paraffin embedded brains were sagittaly
sectioned (n=3 per group). Antigen retrieval was performed by
microwave treatment at 750 W for 10 min in a 10 mM sodium
citrate buffer (pH 6.0). After blocking the endogenous mouse IgG
antibodies (Biocare Medical), sections were incubated overnight
with the primary antibody. Slices were then incubated with the
secondary anti-mouse (HRP-polymer, EnVision kit, Dako),
counterstained with Mayer’s hematoxylin, and the reaction
visualized using diamminobenzidine as chromogen. The number
of stained pyramidal neurons and neurofibrillary tangles was
measured using Photoshop 8.0 (Adobe Systems Incorporated) by
using the Photoshop Lasso Tool and pixel numbers obtained from
the resulting histogram. After this first step, we used the Magic
Wand tool to select a representative positive cell signal. All the
immunostained cells were automatically selected and the total
pixel number recorded. Pixel counts were normalized to a
hippocampal area of 1 mm2.
The following antibodies were used: anti-Ab, clone DE2B4,
diluted 1:400 (Abcam); anti-phosphoTau,clone AT180, 1:400
Carnosine Supplementation in 3xTg-AD Mice
PLoS ONE | www.plosone.org6March 2011 | Volume 6 | Issue 3 | e17971
Mitochondrial sample preparation
Samples for Blue Native-Poly-Acrylamide Gel Electrophoresis
(BN-PAGE) were prepared as described in detail by Scha ¨gger with
minor modifications . AD and control mice were killed by
decapitation. Brains were quickly dissected on ice; cortex and
hyppocampus were immediately frozen on dry ice and stored at
280uC until use. Brain tissues (10 mg; wet weight) were
homogenized in BN-sample buffer 1 (250 mM Sucrose, 30 mM
morpholine-propane sulfonate buffer, 0.2 mM phenylmethylsulfo-
nyl fluoride, pH 7.2) using a homogenizer with a tight-fitting
Teflon pestle (1 min at 500 rpm) and kept cold by immersing the
homogenizer in an ice-filled beaker. The homogenates were
centrifuged at 20,000 g for 20 min at 4uC and the supernatants
were discarded. To solubilize mitochondrial membranes, mito-
chondria-rich pellets were vigorously pipetted in BN-sample buffer
2 (1 M aminocaproic acid, 50 mM Bis-Tris-HCl buffer, pH 7) and
homogenized by twirling with a tiny spatula. Next, freshly
prepared 10% dodecyl maltoside was added to mitochondria
containing sediment to solubilize individual respiratory chain
complexes. The homogenates were centrifuged at 20,000 g for 1 h
at 4uC. The supernatants were collected and transferred intto a
new tube. The gel loading mixture was prepared by adding 5%
Coomassie Blue Brilliant G-250 dissolved in 1 M aminocaproic
acid to the sample at a ratio of 1:6 (dye:sample volume).
After solubilization of mitochondrial membranes by dodecyl
maltoside, BN-PAGE, staining and densitometric quantification of
oxidative phosphorylation complexes were performed essentially
as described by Zerbetto . Briefly, samples were applied to a
6–13% gradient acrylamide gel with a 4% polyacrylamide stacking
gel. To assure the same conditions, gels were made simultaneously
using Mini-Protein II Multi-gel casting chamber (Bio-Rad).
Twenty micrograms of each sample were loaded at 4uC into
561.5 mm gel wells and were at first run until they have entered
the stacking gel, typically 80 V for 30–40 min, after which the
voltage was increased to 100 V and the blue cathode buffer was
replaced by an uncoloured buffer. Electrophoresis continued until
the blue dye front reached the end of the gel. Immediately after
electrophoresis, the gels were fixed in 40% methanol and stained
with a Coomassie Blue Solution (Brillant Blue G and methanol
used at a ratio of 1:4 respectively) overnight for measuring the
amount of proteins. The next day, the gels were destained with
10% acetic acid and 25% methanol for 1–2 min and then washed
different times with 25% methanol. For the histochemical
evaluation of BN-PAGE of each set of gels, one gel was stained
with Coomassie Blue (as described above), other gels were used for
determining specific enzyme activity by the following reactions.
Complex I (NADH-Dehydrogenase) activity was determined by
incubating the gel with 0.1 M Tris–HCl, 768 mM glycine,
0.1 mM b-NADH and 0.04% Nitro Blue Tetrazolium (NTB)
pH 7.4 at room temperature (RT). As a measure for complex II
activity, Succinate Dehydrogenase (SDH) activity was determined
as follows. The gel was incubated in 0.1 M Tris–HCl, 100 mM
glycine, 10 mM succinic acid and 1 mg/ml NTB pH 7.4 at RT.
Complex IV (COX) activity was estimated by incubating the gels
with 5 mg of 3,39–diaminobenzidine tetrahydrochloride (DAB)
dissolved in 9 ml phosphate buffer (0.05 M pH 7.4), 1 ml catalase
(20 mg/ml), 10 mg cytocrome C and 750 mg sucrose. Violet
colored complex I and complex II bands and red-stained complex
IV were measured using the Bio-Rad Imaging Densitometer
(Quantity One Analysis Software, BioRad) with a blue filter
inserted to minimize interference from the residual Coomassie
Blue. The band intensities were expressed as absolute values
(arbitrary units; AU). Optical densities (OD) of the bands from
each loading amount were plotted against the respective value
determined in Coomassie Blue gel.
Statistical differences were determined with the Student’s t-test
for unpaired data. For analysis of the MWM latencies and BN-
PAGE experiments, the homogeneity of the variances was
determined by the Bartlett test (90% confidence level) and a
one-way ANOVA was performed followed by a post hoc
Bonferroni’s correction. For immunohistochemistry experiments,
Mann-Whitney test was employed. All data are expressed as mean
6 SEM and the threshold for statistically significant differences
was set at P,0.05.
We thank Mary Evangeline Oberschlake for helping with the editing of the
Conceived and designed the experiments: CC ER SLS. Performed the
experiments: CC VF ES RL RLS MP LMTC DC SLS. Analyzed the data:
CC VF ES RL RLS MP LMTC DC ER SLS. Contributed reagents/
materials/analysis tools: ER MP LMTC SLS. Wrote the paper: CC SLS.
1. Querfurth HW, LaFerla FM (2010) Alzheimer’s disease. N Engl J Med 362:
2. Sensi SL, Paoletti P, Bush AI, Sekler I (2009) Zinc in the physiology and
pathology of the CNS. Nat Rev Neurosci 10: 780–791.
3. Crouch PJ, Cimdins K, Duce JA, Bush AI, Trounce IA (2007) Mitochondria in
aging and Alzheimer’s disease. Rejuvenation Res 10: 349–357.
4. Rhein V, Song X, Wiesner A, Ittner LM, Baysang G, et al. (2009) Amyloid-beta
and tau synergistically impair the oxidative phosphorylation system in triple
transgenic Alzheimer’s disease mice. Proc Natl Acad Sci U S A 106:
5. Yao J, Irwin RW, Zhao L, Nilsen J, Hamilton RT, et al. (2009) Mito-
chondrial bioenergetic deficit precedes Alzheimer’s pathology in female
mouse model of Alzheimer’s disease. Proc Natl Acad Sci U S A 106:
6. Bush AI, Pettingell WH, Multhaup G, d Paradis M, Vonsattel JP, et al. (1994)
Rapid induction of Alzheimer A beta amyloid formation by zinc. Science 265:
7. Gazaryan IG, Krasinskaya IP, Kristal BS, Brown AM (2007) Zinc
irreversibly damages major enzymes of energy production and antioxidant
defense prior to mitochondrial permeability transition. J Biol Chem 282:
8. Sensi SL, Yin HZ, Carriedo SG, Rao SS, Weiss JH (1999) Preferential Zn2+
influx through Ca2+-permeable AMPA/kainate channels triggers prolonged
mitochondrial superoxide production. Proc Natl Acad Sci U S A 96: 2414–2419.
9. Adlard PA, Bush AI (2006) Metals and Alzheimer’s disease. J Alzheimers Dis 10:
10. Lee JY, Cole TB, Palmiter RD, Suh SW, Koh JY (2002) Contribution by
synaptic zinc to the gender-disparate plaque formation in human Swedish
mutant APP transgenic mice. Proc Natl Acad Sci U S A 99: 7705–7710.
11. Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, et al. (2003) Triple-
transgenic model of Alzheimer’s disease with plaques and tangles: intracellular
Abeta and synaptic dysfunction. Neuron 39: 409–421.
12. Sensi SL, Rapposelli IG, Frazzini V, Mascetra N (2008) Altered oxidant-
mediated intraneuronal zinc mobilization in a triple transgenic mouse model of
Alzheimer’s disease. Exp Gerontol 43: 488–492.
13. Adlard PA, Cherny RA, Finkelstein DI, Gautier E, Robb E, et al. (2008) Rapid
restoration of cognition in Alzheimer’s transgenic mice with 8-hydroxy quinoline
analogs is associated with decreased interstitial Abeta. Neuron 59: 43–55.
14. Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, et al. (2001)
Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-
amyloid accumulation in Alzheimer’s disease transgenic mice. Neuron 30:
Carnosine Supplementation in 3xTg-AD Mice
PLoS ONE | www.plosone.org7 March 2011 | Volume 6 | Issue 3 | e17971
15. Lannfelt L, Blennow K, Zetterberg H, Batsman S, Ames D, et al. (2008) Safety, Download full-text
efficacy, and biomarker findings of PBT2 in targeting Abeta as a modifying
therapy for Alzheimer’s disease: a phase IIa, double-blind, randomised, placebo-
controlled trial. Lancet Neurol 7: 779–786.
16. Ritchie CW, Bush AI, Mackinnon A, Macfarlane S, Mastwyk M, et al. (2003)
Metal-protein attenuation with iodochlorhydroxyquin (clioquinol) targeting
Abeta amyloid deposition and toxicity in Alzheimer disease: a pilot phase 2
clinical trial. Arch Neurol 60: 1685–1691.
17. Quinn PJ, Boldyrev AA, Formazuyk VE (1992) Carnosine: its properties,
functions and potential therapeutic applications. Mol Aspects Med 13: 379–444.
18. Horning MS, Blakemore LJ, Trombley PQ (2000) Endogenous mechanisms of
neuroprotection: role of zinc, copper, and carnosine. Brain Res 852: 56–61.
19. Kohen R, Yamamoto Y, Cundy KC, Ames BN (1988) Antioxidant activity of
carnosine, homocarnosine, and anserine present in muscle and brain. Proc Natl
Acad Sci U S A 85: 3175–3179.
20. Preston JE, Hipkiss AR, Himsworth DT, Romero IA, Abbott JN (1998) Toxic
effects of beta-amyloid(25-35) on immortalised rat brain endothelial cell:
protection by carnosine, homocarnosine and beta-alanine. Neurosci Lett 242:
21. Trombley PQ, Horning MS, Blakemore LJ (2000) Interactions between
carnosine and zinc and copper: implications for neuromodulation and
neuroprotection. Biochemistry (Mosc) 65: 807–816.
22. Trombley PQ, Horning MS, Blakemore LJ (1998) Carnosine modulates zinc
and copper effects on amino acid receptors and synaptic transmission.
Neuroreport 9: 3503–3507.
23. Brownson C, Hipkiss AR (2000) Carnosine reacts with a glycated protein. Free
Radic Biol Med 28: 1564–1570.
24. Hipkiss AR, Brownson C, Carrier MJ (2001) Carnosine, the anti-ageing, anti-
oxidant dipeptide, may react with protein carbonyl groups. Mech Ageing Dev
25. Hipkiss AR, Michaelis J, Syrris P (1995) Non-enzymatic glycosylation of the
dipeptide L-carnosine, a potential anti-protein-cross-linking agent. FEBS Lett
26. Hipkiss AR, Worthington VC, Himsworth DT, Herwig W (1998) Protective
effects of carnosine against protein modification mediated by malondialdehyde
and hypochlorite. Biochim Biophys Acta 1380: 46–54.
27. Balion CM, Benson C, Raina PS, Papaioannou A, Patterson C, et al. (2007)
Brain type carnosinase in dementia: a pilot study. BMC Neurol 7: 38.
28. Bellia F, Calabrese V, Guarino F, Cavallaro M, Cornelius C, et al. (2009)
Carnosinase levels in aging brain: redox state induction and cellular stress
response. Antioxid Redox Signal 11: 2759–2775.
29. Fonteh AN, Harrington RJ, Tsai A, Liao P, Harrington MG (2007) Free amino
acid and dipeptide changes in the body fluids from Alzheimer’s disease subjects.
Amino Acids 32: 213–224.
30. Billings LM, Oddo S, Green KN, McGaugh JL, LaFerla FM (2005)
Intraneuronal Abeta causes the onset of early Alzheimer’s disease-related
cognitive deficits in transgenic mice. Neuron 45: 675–688.
31. Baran EJ (2000) Metal complexes of carnosine. Biochemistry (Mosc) 65:
32. Mineo P, Vitalini D, La Mendola D, Rizzarelli E, Scamporrino E, et al. (2002)
Electrospray mass spectrometric studies of L-carnosine (beta-alanyl-L-histidine)
complexes with copper(II) or zinc ions in aqueous solution. Rapid Commun
Mass Spectrom 16: 722–729.
33. Aizenman E, Stout AK, Hartnett KA, Dineley KE, McLaughlin B, et al. (2000)
Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular
zinc release. J Neurochem 75: 1878–1888.
34. Sensi SL, Ton-That D, Sullivan PG, Jonas EA, Gee KR, et al. (2003)
Modulation of mitochondrial function by endogenous Zn2+ pools. Proc Natl
Acad Sci U S A 100: 6157–6162.
35. Sutherland RJ, McDonald RJ (1990) Hippocampus, amygdala, and memory
deficits in rats. Behav Brain Res 37: 57–79.
36. Corona C, Masciopinto F, Silvestri E, Del Viscovo A, Lattanzio R, et al. (2010)
Dietary zinc supplementation of 3xTg-AD mice increases BDNF levels and
prevents cognitive deficits as well as mitochondrial dysfunction. Cell Death and
Dis 1: e91.
37. Boldyrev AA (1994) Carnosine and free-radical defence mechanisms. Trends
Neurosci 17: 468.
38. Lee JY, Kim JH, Hong SH, Lee JY, Cherny RA, et al. (2004) Estrogen decreases
zinc transporter 3 expression and synaptic vesicle zinc levels in mouse brain.
J Biol Chem 279: 8602–8607.
39. Hirata-Fukae C, Li HF, Hoe HS, Gray AJ, Minami SS, et al. (2008) Females
exhibit more extensive amyloid, but not tau, pathology in an Alzheimer
transgenic model. Brain Res 1216: 92–103.
40. Ittner LM, Gotz J (2011) Amyloid-beta and tau - a toxic pas de deux in
Alzheimer’s disease. Nat Rev Neurosci 12: 65–72.
41. Calabrese V, Colombrita C, Guagliano E, Sapienza M, Ravagna A, et al. (2005)
Protective effect of carnosine during nitrosative stress in astroglial cell cultures.
Neurochem Res 30: 797–807.
42. Atamna H, Kumar R (2010) Protective role of methylene blue in Alzheimer’s
disease via mitochondria and cytochrome c oxidase. J Alzheimers Dis 20(Suppl
43. Brunden KR, Trojanowski JQ, Lee VM (2009) Advances in tau-focused drug
discovery for Alzheimer’s disease and related tauopathies. Nat Rev Drug Discov
44. Clinton LK, Blurton-Jones M, Myczek K, Trojanowski JQ, LaFerla FM (2010)
Synergistic Interactions between Abeta, tau, and alpha-synuclein: acceleration of
neuropathology and cognitive decline. J Neurosci 30: 7281–7289.
45. Oddo S, Vasilevko V, Caccamo A, Kitazawa M, Cribbs DH, et al. (2006)
Reduction of soluble Abeta and tau, but not soluble Abeta alone, ameliorates
cognitive decline in transgenic mice with plaques and tangles. J Biol Chem 281:
46. Stoltenberg M, Bush AI, Bach G, Smidt K, Larsen A, et al. (2007) Amyloid
plaques arise from zinc-enriched cortical layers in APP/PS1 transgenic mice and
are paradoxically enlarged with dietary zinc deficiency. Neuroscience 150:
47. Adlard PA, Parncutt JM, Finkelstein DI, Bush AI (2010) Cognitive loss in zinc
transporter-3 knock-out mice: a phenocopy for the synaptic and memory deficits
of Alzheimer’s disease? J Neurosci 30: 1631–1636.
48. Odashima M, Otaka M, Jin M, Konishi N, Sato T, et al. (2002) Induction of a
72-kDa heat-shock protein in cultured rat gastric mucosal cells and rat gastric
mucosa by zinc L-carnosine. Dig Dis Sci 47: 2799–2804.
49. Ueda K, Ueyama T, Oka M, Ito T, Tsuruo Y, et al. (2009) Polaprezinc (Zinc L-
carnosine) is a potent inducer of anti-oxidative stress enzyme, heme oxygenase
(HO)-1 - a new mechanism of gastric mucosal protection. J Pharmacol Sci 110:
50. Caccamo A, Maldonado MA, Bokov AF, Majumder S, Oddo S (2010) CBP
gene transfer increases BDNF levels and ameliorates learning and memory
deficits in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci U S A 107:
51. Sensi SL, Canzoniero LM, Yu SP, Ying HS, Koh JY, et al. (1997) Measurement
of intracellular free zinc in living cortical neurons: routes of entry. J Neurosci 17:
52. Schagger H (1996) Electrophoretic techniques for isolation and quantification of
oxidative phosphorylation complexes from human tissues. Methods Enzymol
53. Zerbetto E, Vergani L, Dabbeni-Sala F (1997) Quantification of muscle
mitochondrial oxidative phosphorylation enzymes via histochemical staining of
blue native polyacrylamide gels. Electrophoresis 18: 2059–2064.
Carnosine Supplementation in 3xTg-AD Mice
PLoS ONE | www.plosone.org8 March 2011 | Volume 6 | Issue 3 | e17971