Research Article • DOI: 10.1515/tnsci-2018-0009 • Translational Neuroscience • 9 • 2018 • 43-53
* E-mail: firstname.lastname@example.org
© 2018 Gennadiy Fonar et al., published by De Gruyter.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License.
Alzheimer’s disease is a slowly progressive
neurodegenerative disorder with prevalence
among elderly people and women (Hebert et al.
2013). The AD is characterized morphologically
by diuse neuritic plaques containing Aβ
peptide and neurobrillary tangles, which
are aggregates of hyperphosphorylated tau
protein (Schaeer et al. 2011). The exact cause
of AD is unknown, and several studies have
proposed that the pathogenesis of AD, at least
its sporadic form, is related to inammatory
processes (Heneka and O’Banion 2007),
atherosclerosis, redox stress (Sultana and
Buttereld 2010), and abnormal brain glucose
metabolism (Calsolaro and Edison 2016).
Some studies support the hypothesis that AD
is correlated with dysfunction of metabolic
pathways that are translated into neurological
symptoms (Cai et al. 2012), (Craft 2009).
Targeting these various metabolic pathways
could be used to treat AD.
Current treatment strategies are focusing upon
manipulating with cholinergic and glutamatergic
neurotransmission (Weinstock et al. 2001). All
of them demonstrate a modest clinical success,
and, in fact, no eective treatment is available
to target the principal mechanism of the AD. We
believe that new strategies combining several
approaches and targets for the treatment of
AD have to be proposed. Such approaches
could include an auxiliary intervention into the
metabolic pathways and personalized correction
Arginine as a therapeutic agent
L-Arginine has been used for the treatment of
various diseases. L-arginine has been shown
to stimulate immune responses and promote
wound healing (Barbul A, Lazarou SA, Efron DT,
Wasserkrug HL 1990). In particular, L-arginine
stimulates wound healing and immune
function in the elderly (Kirk et al. 1993). Oral
supplementation with 17 g doses of arginine for
two weeks in the elderly signicantly improves
positive nitrogen balance. Remarkably, arginine
signicantly reduced the levels of total serum
cholesterol and low-density lipoprotein. No
adverse eects were observed at the dosage of
17 g/day of arginine (Hurson et al. 1995). Kirk et
al. showed that elderly patients could tolerate
an even greater arginine dose of 30 g/day (Kirk
et al. 1993).
The L-arginine dietary supplement also
improves performance in elderly male cyclists
and enhances their exercise capacity (S. Chen
et al. 2010). L-arginine dietary supplement
attenuates the increased platelet reactivity in
hypercholesterolemic patients (Wolf et al. 1997)
and prevents atherogenesis (Cooke et al. 1992).
Finally, L-arginine dietary supplement reduces
restenosis after experimental angioplasty in
rabbits (Tarry and Makhoul 1994).
Arginine and its derivatives have also been
used for the treatment of neurological disorders.
L-arginine administration within 30 minutes
of a stroke signicantly decreases frequency
and severity of stroke-like symptoms (Koga
et al. 2005). Remarkably, 1.6 g of L-Arginine
supplemented daily for three months in the
diet of patients with senile dementia increased
cognitive function by about 40% (Ohtsuka and
Nakaya 2000). Remarkably, the eect of the
drug did not last after the end of the treatment.
LARGININE IMPROVES SPATIAL
MEMORY ACQUISITION IN
TRIPLE TRANSGENIC MICE VIA
REDUCTION OF OXIDATIVE
STRESS AND APOPTOSIS
Faculty of Medicine in the Galilee,
Bar Ilan University, Safed, Israel
2 Institute of Higher Nervous Activity and
Neurophysiology, Russian Academy of Sciences,
Abraham O. Samson1*
Arginine is one of the most versatile semi-essential amino acids. Further to the primary role in protein biosynthesis,
arginine is involved in the urea cycle, and it is a precursor of nitric oxide. Arginine deciency is associated with
neurodegenerative diseases such as Parkinson’s, Huntington’s and Alzheimer’s diseases (AD). In this study, we
administer arginine intracerebroventricularly in a murine model of AD and evaluate cognitive functions in a set
of behavioral tests. In addition, the eect of arginine on synaptic plasticity was tested electrophysiologically
by assessment of the hippocampal long-term potentiation (LTP). The eect of arginine on β amyloidosis was
tested immunohistochemically. A role of arginine in the prevention of cytotoxicity and apoptosis was evaluated
in vitro on PC-12 cells. The results indicate that intracerebroventricular administration of arginine improves spatial
memory acquisition in 3xTg-AD mice, however, without signicantly reducing intraneuronal β amyloidosis.
Arginine shows little or no impact on LTP and does not rescue LTP deterioration induced by Aβ. Nevertheless,
arginine possesses neuroprotective and antiapoptotic properties.
Received 25 February 2018
accepted 01 April 2018
• Alzheimer’s disease • L-arginine • spatial memory • amyloid beta • cytotoxicity • apoptosis
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Arginine metabolism as a target for
treatment of AD
Arginine is a conditionally essential α-amino
acid that is used in the biosynthesis of proteins.
Arginine possesses a broad spectrum of
regulatory functions, which are predicated
upon its chemical structure and activity.
An overwhelming review of the possible
physiological eects of the amino acid upon
the development of AD has been done by Yi et
al (Yi et al. 2009).
Quite a lot of vital metabolites, (i.e., NO, urea)
are derived from it, and their cytoprotective
and antioxidant properties are presently well
known. The well-marked cationic properties
of the guanidine group of arginine contribute
to its ability to undergo protonation. It is well
established that arginine derivatives and the
amino acid itself can regulate peroxidation
processes in membranes (Milyutina 1990).
Evidently, arginine reacts directly with the
superoxide anion-radical that may be a basis for
its protective eects under extreme conditions.
Additionally, arginine regulates cell division
and release of hormones, plays a role in the
wounds’ healing and removing of ammonia,
and possesses various immune functions
(Böger and Bode-Böger 2001), (Tapiero et al.
Epidemiological studies indicate that daily
dietary arginine intake inversely correlates
with AD morbidity. The mean intake of
arginine among men is >50% greater than
within women in accordance with the lower
prevalence of AD in men (Mielke, Vemuri, and
Rocca 2014) and the nding that about two-
thirds of the individuals diagnosed with the
AD are women (Hebert et al. 2013). Also, the
elderly consume ~30% less arginine compared
to 20-40-year-olds (King, Mainous, and Geesey
2008). Additionally, a moderate decrease of
arginine level was detected in CSF and plasma
of AD patients in some recent studies (Ibanez et
al. 2012), (Fonteh et al. 2007).
In the healthy individuals, L-Arginine is
transported from the circulating blood into
the brain via Na+-independent cationic amino
acid transporter (CAT1) expressed at the BBB
(O’Kane et al. 2006). It was established that
the L-arginine inux transport at the rat BBB
is saturable with a Michaelis-Menten constant
(Km) value of 56 μM. The physiological serum
concentration of L-arginine is signicantly
higher in the rodents (about 170 μM) and
humans (about 100 μM ) (Stoll, Wadhwani,
and Smith 1993). Therefore, since L-arginine
in mammals is derived mostly from renal de
novo synthesis and dietary intake, CAT1 at the
BBB functions as a sole supply pathway for
L-arginine to the brain (Tachikawa and Hosoya
Despite the capability of arginine to pass
the BBB, the capacity of its transporter is
limited (Shin et al. 1985). The limit makes
oral administration of arginine insucient to
show all of its possible eects. In the present
research, we check the direct eect of arginine
upon cognitive functions in a murine model
of AD. We bypass the BBB by intraventricular
administration of the amino acid and eliminate
the eect of the arginine derivatives, which also
possess neuroprotective qualities and might
be generated in substantial quantities as a
reaction to general administration of arginine.
In the present research, we check the direct
eect of arginine upon cognitive functions in
a murine model of AD. We bypass the BBB by
intraventricular administration of the amino
acid and eliminate the eect of the arginine
derivatives, which also possess neuroprotective
qualities and might be generated in
substantial quantities as a reaction to general
administration of arginine.
Materials and Methods
There are several animal models of the AD
which were created to study the disease. A
triple-transgenic mice model of AD (3xTg-AD)
exhibits synaptic deciency with both plaque
and tangle pathology (Oddo et al. 2003).
3xTg-AD mice were purchased from the
Jackson Laboratory® and bred in our animal
facility. Twenty-four age-matched 6.5 months
old female mice were used for all experiments.
All experimental protocols were performed
in accordance with the instructions of
the Israeli Ministry of Health’s Council for
Experimentation on Animals and with Bar Ilan
University guidelines for the use and care of
laboratory animals in research, supervised
by the institutional animal care and use
committee. The experimental protocol was
approved by the Committee on the Ethics of
Animal Experiments of the Bar Ilan University
(Permit Number: 32 - 08 – 2012).
Before and during the experiment, mice
were socially housed in standard plastic cages
in humidity (30%) and temperature (22°C)
controlled room with a 12-h reverse light/dark
cycle. The animals were provided with water
and food ad libitum.
The animals were randomly divided into two
equal groups (a control group with articial
CSF administration, and an experimental group
with L-arginine solution in ACSF).
Drug administration and surgical
L-arginine and saline control solutions were
directly administered into the brain lateral
ventricles using osmotic minipumps and
cannulae. The animals were anesthetized
with 2% isourane and placed in a stereotaxic
apparatus. Anesthesia was maintained with 1%
isourane for the duration of the surgery. First,
a subcutaneous pocket for the Alzet® pump
capsule was made with a pair of scissors. The
skull of the mice was drilled stereotactically
according to the mouse brain atlas with the
coordinates: -0.2 mm caudal, 0.9 mm lateral
to bregma. Then, a small bent cannula (Alzet®)
was slowly lowered stereotactically into the
hole and cemented to the skull with Loctite
454 (Alzet®). Once in place, the cannula reaches
2.5 mm in the dorsoventral direction (Sanchez-
Mendoza et al. 2016).
An activated during 24 hours in DDW
osmotic minipumps (Alzet, model 1004, 28
days delivery) prelled with 100 μl of L-arginine
(1.148 M, pH 7.3) in ACSF (Ecocyte Bioscience)
or ACSF at pH 7.3 have been connected to the
cannula via a vinyl catheter tube. The pumps
were placed in a spinal subcutaneous pocket
prepared at the earlier stage of the operation
and stapled (Alzet AutoClip). The timeline
of the experiment including treatments and
behavioral tests presented in gure 1.
Rat pheochromocytoma-derived PC12 cells
have similar characteristics of nerve cells and
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are widely used in various studies neurotoxicity
and apoptosis assays (Jung et al. 2008).
It has been commonly used in numerous
investigations of potential neuroprotective
agents to treat neurodegenerative diseases,
such as Huntington’s disease, Parkinson’s
disease, and AD (Scotter et al. 2010), (Wan et al.
The cell line (#88022401) was obtained from
Sigma®. The cells were maintained in DMEM
medium supplemented with 10% fetal bovine
serum, 5% horse serum, 100 U/mL penicillin
and 100 μg/mL streptomycin at 37 °C in a
humidied 95% air/5% CO2 incubator. All cells
were cultured in collagen-coated culture
dishes. The medium was changed every other
day, and cells were plated at an appropriate
density according to each experimental scale.
Apoptotic morphology observation
by Hoechst 33342 staining
Chromosomal condensation and
morphological changes in the nucleus were
observed by using the chromatin dye, Hoechst
33342 (Roche®). Cells with homogeneously
stained nuclei were considered to be viable.
Chromatin condensation or fragmentation
indicated apoptosis. Briey, PC12 cells (1×106)
were plated in 6 well plates and cultured on
cover slips (pretreated with poly-D-lysine)
with three mL of medium in each well. After
24 hours the cells were exposed to 500 µM of
H2O2 with various concentrations of L-arginine
for additional 24 h. After treatments, the cells
were xed with 4% formaldehyde in PBS for
20 min at room temperature and stained
with one μg/mL Hoechst 33342 for 20 min.
Cells were photographed under a uorescent
microscope (Olympus, Tokyo, Japan) and
quantied by ImageJ 1.51 software. In each
image, identical rectangular regions of interest
with the selection tool of ImageJ were selected
randomly, and the average pixel intensity was
measured by the software.
Cell viability assays
Cell viability was assessed by measuring
formazan produced by the reduction of MTT.
The MTT assay is a sensitive measurement of
the normal metabolic status of cells, particularly
those of mitochondria, which reects early
cellular redox changes. Therefore, the amount
of formazan produced is proportional to the
number of viable cells. Briey, the cells were
plated in 96 well poly-d-lysine-coated culture
plates at the density of 1 × 104 cells/well at
37 °C for 24 hours with media containing
various concentrations of L-arginine and were
treated with pre-aggregated during 24 hours
50 μM Aβ(25–35) or 0.5 mM of H2O2. These
concentrations of Aβ and H2O2 have been
used previously in various studies to check
the viability of PC-12 cells (Yao, Drieu, and
Papadopoulos 2001), (Heo and Lee 2005).
Subsequently, MTT reagent (nal concentration,
0.5 mg/mL) was added to each of the wells, and
the plate was incubated for three hours at 37 °C.
At the end of the incubation, the medium with
MTT was removed and 100 μL DMSO was added
to each well. The formazan reduction product
was measured by reading absorbance at 570
nm in a microplate reader (Innite® M1000).
Cell viability was presented as a percentage of
the control culture.
Additionally, trypan blue dye exclusion assay
was used to determine the number of viable
cells present in a cell suspension. Briey, PC-12
cells were treated with varying concentrations
(0-1 mM) of L-Arginine for 24 hours, and after
that, the cells were removed, centrifuged and
resuspended in Dulbecco’s phosphate buer
saline with trypan blue (1 volume per 4 volumes
of the medium (Sigma Chemical Co.)). The
viable and nonviable cells were counted on a
hemocytometer using an inverted microscope
(Olympus, Tokyo, Japan).
Morris water maze (MWM)
The maze consisted of a black circular tank with
of 120 cm diameter and height of 40 cm. Water
was maintained at a temperature of 25°C and
rendered opaque by adding skim milk powder
(Sigma). A platform submerged 1 cm below the
surface was placed in a quadrant of the maze
and kept in the same position during all trials.
Several visual cues were positioned around
the pool. A video camera in conjunction with
the EthoVision XT 10 video tracking software
(Noldus) was used for the measurement of
the time taken to escape to the platform.
Four locations around the pool were labeled
as north, south, east, and west for a test start
The mice were acclimatized to the test room
conditions for 30 min before the experiment.
Each animal was placed in the pool at one of
four locations. The mice were allowed to nd
the platform within 60 seconds and stay on it for
10 seconds. If a mouse could not escape during
the time limit, it was directed gently towards
the platform. Each animal received two learning
sessions per day with a 15 minutes interval
between trials during ve successive days.
The probe test without the platform was
performed on the sixth day. The mice were
subjected to a single trial with free swimming
during 60 seconds. The relative time spent in
the area that was dened by the software as
two platform’s diameters and the number of
times the animals crossed the platform zone
have been analyzed.
Figure 1. The timeline of treatments and tests.
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Spontaneous alternation Y-Maze
Spontaneous alternation behavior reects
spatial working memory, which is a form of short-
term memory. It was assessed by spontaneous
alternation behavior during a single session
in the Y-maze. The Y-maze used in this study
consisted of three arms (each of 35 cm long, 25
cm high, and 10 cm wide). For all animals, the
maze was placed in the same position during
the procedure. The mice were placed at the
center of the maze and allowed to move freely
for 5 min. An arm entry was counted when the
hind paws of the mouse were completely within
the arm. The maze was cleaned with a 10%
aqueous ethanol solution between each trial.
Spontaneous alternation behavior was dened
as the entry into all three arms on consecutive
choices. The number of maximum spontaneous
alternation behaviors was calculated as the
total number of arms entered minus 2. The
percentage of spontaneous alternation was
calculated as [actual alternations] / [total
number of alternations] × 100. A video camera
in conjunction with the EthoVision XT 10
video tracking software (Noldus) was used for
recording the behavior.
Five animals from each group were sacriced
and perfused with ice-cold 4% PFA. The
brain was taken and xated in 4% PFA for
immunohistochemical analysis. Then they
were sliced on a sliding microtome to produce
30-micron oating sections. These sections
were blocked for one hour in blocking solution
containing 10% horse serum, 0.3% triton and
undiluted phosphate-buered saline (PBS).
The sections were then incubated overnight
with primary antibodies 6E10 (1:150 ENCO),
at room temperature, followed by washing
and incubation for an hour with secondary
antibodies Alexa488 (1:200, Thermo Fisher
Scientic), and Hoechst (1:5000, Sigma). Anti-
Aβ antibody (6E10) was purchased from ENCO.
Imaging and quantication
The number of Aβ deposits throughout the
hippocampi (3-6 sections examined per mouse)
was quantied. Imaging was done using a
Nikon Eclipse E600 microscope equipped with a
40× objective and the acquisition software NIS-
Elements AR (Nikon Instruments). Microscopy
images were analyzed using the open source
software ImageJ (http://imagej.nih.gov/ij/) with
the plugin from Wright Cell Imaging Facility.
The average of the mean intensity of the
staining was obtained for each hippocampal
subeld, and the background was subtracted
before calculating the reciprocal values.
Antibody microarray analyses
The evaluation of “hit” proteins’ expression
or phosphorylation of specic residues on
these proteins was performed by the use
of the Kinex KAM-880 Antibody Microarray
(Kinexus Bioinformatics Corp., Vancouver,
B.C.), in accordance with the manufacturer
specication. The analyses were done with
hippocampal lysates of mice treated with
arginine and ASCF as described on Kinexus’
web page (www.kinexus.ca). Briey, lysate
protein from each sample (100 µg) was labeled
covalently with a uorescent dye combination.
Free dye molecules have been then removed
via gel ltration. After blocking non-specic
binding sites on the array, an incubation
chamber was mounted onto the microarray to
permit the loading of 2 samples (one arginine
treated, and one ACSF treated). After the
incubation, unbound proteins were washed
away. Two 16-bit images have been captured
by a ScanArray Reader (Perkin-Elmer) for each
array. Then, microarray data were analyzed
using a web server for functional interpretation
of gene lists (http://biit.cs.ut.ee/gproler),
and a list of priority genes was generated.
Finally, Cytoscape software was applied for the
topological analysis and network visualization
of the priority genes.
Male six months old C57BL/6 mice were
anesthetized with isourane and decapitated.
Brains were quickly removed and submerged in
ice-cold dissection solution (concentrations in
mM: 124 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3,
1.3 CaCl2, 7 MgCl2, and 10 D-glucose, pH
equilibrated, with 95% O2 – 5% CO2). Transverse
hippocampal slices (350µm thick) were
prepared using a vibratome (Leica VT1000S,
Germany) and immediately transferred to a
recording solution (composition as above,
except the CaCl2 and MgCl2 concentrations,
Figure 2. Assessment of long and short-term memory acquisition. (a) MWM test learning curves are showing
escape latencies during ve testing days (n=12 for each group). (b) The percent time spent in the target quadrant
on the probe trial day. The mice injected with arginine spent signicantly more time in the learning phase target
quadrant than the controls. Spontaneous alternation in Y-maze. (c) A total number of alternations. (d) Percentage
of alternation. Values are shown as the mean ± s.e.m. * p < 0.05 (n=12 for each group).
Download Date | 6/2/18 2:47 AM
were adjusted to 2.5 and 1.3, respectively).
Slices were heated to 36°C in water bath for
40 min and then kept at room temperature.
Amyloid β peptide (25-35) was dissolved in mQ
water to 1 mM stock solution and frozen. 24
h before the experiment, the Aβ peptide was
dissolved to 50 nM in ACSF and incubated at
+4°C for oligomerization. Slices were incubated
for 1 hour with control ACSF, one mM or 100
µM of L-arginine or mixture of 50 nM Aβ (25-
35) and L-arginine before transferring to the
recording chamber. During the experiments,
slices were perfused by a continuously owing
(appr. 4 ml/min) recording solution at 32-33°C.
Electrophysiological recordings were carried
out using SliceMaster system (Scientica,
UK). Field excitatory postsynaptic potentials
(fEPSP) were recorded from stratum radiatum
in area CA1 using glass microelectrodes (1-2
MΩ) lled with the recording solution. Baseline
synaptic responses were evoked by paired-
pulse stimulation with 50 ms interval of the
Schaer collaterals at 0.033 Hz with a bipolar
electrode. Test stimulation intensity was
adjusted to evoke fEPSP with amplitude 50%
of maximal and was kept constant throughout
the experiment. LTP was induced with four
100-Hz trains spaced 5 min apart. The data
were recorded and analyzed by Spyke2 and
SigmaPlot. For statistical analysis, the latest
5 minutes (116-120 min after LTP induction)
were used. For baseline responses analysis ber
volley amplitudes and appropriate fEPSP slopes
during test stimulation were evaluated. PPF
ratio was calculated as PPF (S2EPSP/S1EPSP),
where S1EPSP and S2EPSP are the slopes of
EPSP in response to the rst and the second
stimuli with dierent intervals, respectively. PPF
measures were carried out just before and after
Statistical analyses were conducted using SPSS
22.0 for Windows. All results are presented
as mean with standard error. Escape latency
during the training was determined by
repeated-measures ANOVA with the session
as a within-subject factor and treatment as
a between-subjects factor. In the probe trial,
Student’s t-test for latency and two-way ANOVA
with treatment-time in quadrants as between-
subject factors were applied. One-way
ANOVA was used to determine the signicant
dierences between the groups followed by a
Dunnett’s t-test for multiple comparisons. The
results are considered signicant when p <
0.05. All values are expressed as means ± SEM.
Each in vitro experiment consisted of three
separate plates from the same culture which
Figure 3. Immunouorescence and quantication of senile amyloid plaques in the hippocampal areas. a) A typi-
cal 20 x magnication image of the dentate gyrus from treated with L-arginine mouse with visible Aβ deposits.
The inset shows a high magnication (40 x) merged with DAPI view with Aβ deposits. b) A typical 10x magnica-
tion image of the hippocampus from treated with L-arginine mouse. Scarce extracellular Aβ plaques are visible
(arrows show the amyloid plaques in the brain section). No dierences are seen between treated and untreated
mice as shown by quantication of the hilus intraneuronal Aβ loads. c) Quantication of Aβ levels is presented
with bar graphs of mean pixel counts per dentate gyrus ±SEM.
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L-Arginine mitigates hydrogen
peroxide-induced apoptosis in
cultured PC-12 cells
Morphological nuclear changes and rate of
apoptosis in PC12 cells treated with 500 µM of
H2O2 and L-arginine at dierent concentrations
were assessed by use of Hoechst 33258
staining. Alterations of nuclear morphology
characterized by condensed and fragmented
nuclei were considered to be markers of
apoptosis (gure 5). Treatment of PC12 with
H2O2 leads to severe nuclear damage and
alterations of the structure. Chromatin of
the cells cultured with no arginine (gure
5a) appears to be much more condensed
compared to the cells cultured with one mM
of L-arginine(gure 5b). Cells being treated
with H2O2 for 24 h exhibited nuclear chromatin
leads to upregulation of several critical
biological processes, including stress defense,
response to oxygen-containing compounds,
and metabolism of nitrogen compounds
There are 65 regulatory proteins which
demonstrate signicant changes in L-Arginine
treated animals compared to ACSF treated
mice. Among them, 19 are considered as
priority leads with ≥90% change from control
(ACSF treated) and 46 as possible leads with
≥60% change (supplementary table 1).
In order to visualize molecular interaction
networks and biological pathways, Cytoscape was
applied (http://www.cytoscape.org). To link the
dierential changes in protein expression and
the changes in network connectivity to biological
pathways we built a functional map (gure 4b).
were averaged. Each test was replicated for two
more times and the results presented as means
± SEM of three independent experiments.
The statistical analyses were done using one-
way ANOVA followed by Student–Newman–
Keuls multiple comparison tests. Statistical
signicance was drawn at p < 0.05.
Administration of L-arginine during
one month signicantly improved
memory acquisition in 3xTg-AD mice
The learning curve of the mice from the
experimental group reects gradually declining
escape latency pattern across the trials (Figure
2a). On the day with probe tests, the animals
from experimental group spent about 33 %
of the time in the target quadrant, which was
signicantly better than results shown by the
controls (Figure 2b). The mice which were
injected with ACSF did not demonstrate the
results above chance (i.e., 25%), which accords
with the current literature (Niikura et al. 2011).
Figures 2 (c, d) depict spontaneous alternation
results in Y-maze. Both number and percentage
of alternations indicate that mice treated with
L-arginine show signicantly better spatial
working memory than control animals.
Administration of L-arginine has no
signicant impact on the rate of Aβ
Immunohistochemistry was employed to
detect deposits of intra- and extraneuronal Aβ
in the hippocampi of brain slices from treated
(n=5) and untreated (n=5) 3xTg-AD mice.
Multiple intracellular deposits of amyloid beta
and scarce plaques have been detected in the
cortices, amygdala, and hippocampi. There
were no signicant dierences in the amount of
amyloid beta deposition between two groups
L-arginine treatment induces
cellular pathways involved in
neuroprotection including oxidative
stress protection, and defense
KAM 880 antibody microarray analysis reveals
that treatment of 3xTG-AD mice with L-arginine
Figure 4. Highlighted enriched terms and associated dierentially expressed proteins. a) L-Arginine treatment
induces multiple metabolic pathways using DAVID .b) STRING was used to build a physical and functional pro-
tein-protein association network in Cytoscape for dierentially expressed proteins involved in a subset of the
highlighted terms. Node degrees represent the amount of interactions per protein.
Download Date | 6/2/18 2:47 AM
current literature (Todoroki et al. 1998). It was
demonstrated that high concentrations (more
than one mM) of L-Arginine induce severe DNA
damage and arrest at the GI phase. Moreover,
it was approved that ROS are produced in cells
exposed to high (about two mM) of L-arginine.
Our data are in agreement with these results.
Consequently, we show that L-arginine reduces
the cytotoxic eect of 50 µM Aβ(25-35) and 0.5
mM H2O2 in a dose-dependent manner.
L-Arginine does not rescue
impaired by Aβ hippocampal long-
Our results demonstrate that high
concentrations (about one mM) of L-arginine
signicantly deteriorate hippocampal LTP
(gure 6b). Medium containing 50 nM of Aβ
suppresses generation of LTP (gure 6 a,b).
Administration of aggregated Aβ (25-35)
signicantly decreased fEPSP slope and PS
amplitude in Aβ group compared to the control
group; though, did not aect baseline activity
of the neurons.
Inhibition of LTP induction by aged amyloid
beta and particularly Aβ (25--35) without
aecting the basal synaptic transmission and
post-tetanic potentiation was demonstrated
previously in various studies (Q. S. Chen et al.
We evidence that the presence of low
concentrations of L-arginine in the medium
containing Aβ or without it does not inuence
signicantly fEPSP slope and PS amplitude;
however, high concentrations of L-arginine
have a severe deteriorative eect upon LTP
Amino acids play an essential role in neuronal
signaling and energy supply. The correct
balance of amino acids is critical for normal
neuronal functioning. Therefore, deviations
in their metabolism may inuence the
neurodegenerative processes. Postmortem
brains of AD patients demonstrate various
alterations in the level of amino acids, and
a moderate decrease of arginine level was
detected in the CSF and plasma (Ibanez et al.
2012). Therefore, we hypothesize that arginine
Figure 5. L-arginine protected PC12 cells against H2O2-induced apoptosis (apoptotic morphology observation
by Hoechst 33342). Morphological analysis of nuclear chromatin in a) no arginine, b) 1 mM of L-arginine. The
gures are representatives of a set of identical experiments repeated three times. c) Quantication of DAPI uo-
aggregation and apoptotic bodies. Arginine
mitigates these morphological changes of the
nuclei, which was consistent with the MTT
results in Fig. 6.
The rate of cell apoptosis in the plates with
no arginine was signicantly higher than that in
the experimental plates.
L-arginine protects cells against
Aβ(25-35) and Hydrogen peroxide-
induced toxicity in a concentration-
To study the eect of L-arginine on cell viability
in the Aβ(25-35) or H2O2-stressed cultured
PC12 cells, the cells were incubated with
various concentrations of arginine and 50 µM
of Aβ(25-35) or 0.5 mM H2O2. The treatments
induced morphological dose-dependent
alteration which can be observed under the
light microscope (Figure 6d). The relative rate
of induced cell death was evaluated by MTT
and trypan blue assays. As shown in Figure
6b, PC12 cells exposed for 24 hours to 50 µM
of aged Aβ(25-35) in the media containing
L-arginine exhibit signicantly enhanced
viability. Incubation of the cells with 0.5 mM
H2O2 for 24 hours leads to deterioration of
viability rate, which can be reversed by an
increase in arginine concentration up to one
mM. Remarkably, arginine itself demonstrates
cytotoxic properties in concentrations greater
than one mM (gure 6c), which accords with the
Download Date | 6/2/18 2:47 AM
Figure 6. Eects of dierent concentrations of L-arginine in the medium on the viability and morphology of
PC12 cells treated with 50 µM of Aβ(25-35) or 0.5 mM of H2O2. (a) Eect of Aβ(25-35) on the viability of PC12 cells
(exposure to 24 h). (b) Eect of 0.5 mM H2O2 on the viability of PC12 cells (exposure to 24 h). MTT assay demon-
strates that arginine protects cells against Aβ(25-35) and H2O2 toxicity in a concentration-dependent manner;
(c) Cytoprotective and cytotoxic eects of various concentrations of L-Arginine (trypan blue test)(d) treatments
of PC-12 cells with Aβ(25-35) and H2O2 in the media containing dierent concentrations of L-arginine induced
morphological alteration, which seem to be dose-dependent; * p < 0.05, ** p < 0.01, *** p < 0.001.
derivatives by peripheral metabolism, the
substance was administered intraventricularly.
So, we test the direct cognitive eects of
arginine and its metabolites in the brain of 3xTg
Our results elucidate some controversies of
the “arginine paradox,” the term, that has been
used to denote the phenomenon in which
L-arginine administration drives NOS activity
of arginine was motivated by its involvement
in diverse physiological and pathological
processes, including cellular redox metabolism,
inammation, regulation of cerebral blood
ow, and neuroplasticity.
Despite the capability of L-arginine to pass
the BBB, the capacity of its transporter is limited
(Shin et al. 1985). In order to circumvent the
BBB and eliminate possible eects of arginine
plays a role in the pathogenesis of the AD and
may be used to treat the disease.
Neurons are strongly dependent upon
oxidative phosphorylation as an energy source
compared to other cells and highly vulnerable
to oxidative stress (Shetty, Gale, and Turner
2012). As a general rule, the oxidative stress
increases during aging (Finkel and Holbrook
2000). In the course of the progression of age-
related neurodegeneration and, especially,
with the progression of AD, the capacity of
neurons to maintain the redox balance severely
declines, which leads to the mitochondrial
dysfunction, accumulation of free radicals, and
neuronal injury (X. Chen, Guo, and Kong 2012).
Moreover, it was shown in transgenic mouse
models of AD and in ex vivo experiments
using postmortem brain tissue taken from AD
patients that Aβ deposits directly associated
with free-radical generation (Mclellan et al.
2003). Therefore, it was hypothesized that
increased oxidative damage is a primary cause
of AD pathogenesis (Perry et al. 2002).
There is a consensus that L-arginine can
protect neurons against oxidative stress
via exerting its antioxidant potentials.
Furthermore, Kan et al. recently have shown on
a novel rodent model that the development of
AD symptoms is associated with a signicant
reduction of global arginine bioavailability
and an intervention into arginine metabolism
via inhibition of ornithine decarboxylase
protects the animals from AD-like pathology
and improves cognitive functions in mice (Kan
et al. 2015). It was demonstrated on dierent
models that L-arginine enhances resistance
against oxidative stress. The substance extends
the lifespan of C. elegans under both oxidative
and heat stress and possesses free radical
scavenging ability (Ma et al. 2016). Arginine is
the immediate precursor of nitric oxide (NO). It
was shown by way of dierent cell cultures that
NO may serve as an antioxidant agent, which
protects cells from damage caused by ROS.
There are suggestions that the mechanism for
protection by NO is the interception of ROS and
metallo-oxo species generated by NO (Wink et
Our primary intent in the current research
was to study the biochemical roles of
L-arginine in the development of AD. The use
Download Date | 6/2/18 2:47 AM
and improves NO-mediated functions in vivo
even when L-arginine is excessively available
(Kurz and Harrison 1997). The baseline plasma
concentration of arginine is about 25-fold
higher than the Michaelis-Menten constant
(Km) of the isolated eNOS in vitro (Bode-Böger,
Scalera, and Ignarro 2007), however, L-arginine
supplement does aect NOS activity. The
intracellular physiological concentration of
arginine is about several hundred micromoles
per liter, which far exceeds the 5 micromoles Km
for eNOS, nevertheless, the exogenous arginine
still escalates NO production (Dioguardi 2011).
Arginine metabolism is a multifarious
and extremely interregulated physiological
process, which is highly sensitive to the
bioavailability of the amino acid. Exogenous
arginine supplementation has been conrmed
to improve status in a long list of diseases,
particularly, among the elderly people. Our
results validate a signicant improvement of
behavioral function in the 3xTg-AD mouse
model, without altering the amyloid aspects
of AD neuropathology. To our knowledge,
we provide the very rst demonstration that
chronic intraventricular administration of
L-arginine can improve short and long-term
We prove, that the cognitive eect of
L-arginine administration is not related to
the reduction of amyloid plaques formation
or facilitation of neuroplasticity (LTP),
but associated with cytoprotective and
antiapoptotic potentials of arginine. Application
of antibody microarray reveals various cellular
pathways involved in neuroprotection that
were induced by L-arginine treatment.
Amplied response to oxygen-containing
compounds, positive regulation of nitrogen
compound metabolic pathways and defense
response are the most critical, in our opinion,
We have tested our hypothesis in vitro on
the PC-12 cellular model. We evidence the
neuroprotective eect of arginine against H2O2
and Aβ(25-35) induced toxicity on PC12 cells
and demonstrate that arginine itself in high
doses possesses cytotoxic properties.
The results support the conclusion that
L-arginine protects neurons against Aβ
cytotoxicity and reduces the rate of apoptosis.
Our work conrms an intriguing therapeutic
role of L-arginine in the development of AD as a
potent metabolic agent interfering with redox
system and reducing apoptosis. We believe
that our research should aid in the rational
development of therapeutic agents for the
intervention in the course of various relevant
Gennadiy Fonar and Baruh Polis were
involved in all the aspects of the work.
Tomer Meirson performed the bioinformatic
analysis. Alexander Maltsev performed the
electrophysiology experiments. Evan Elliott
supervised parts of the experiments. Avraham
O. Samson conceived and designed the
All experimental protocols were approved by
the Faculty of Medicine, Bar Ilan University
ethics committee, ethics’ protocol number
32 - 08 – 2012. All methods were carried out
in accordance with relevant guidelines and
This research was supported by a Marie Curie
CIG grant 322113, a Leir foundation grant, a
Ginzburg family foundation grant, and a Katz
foundation grant to AOS. Electrophysiological
experiments were supported by Russian
Science Foundation (RSF; grant no. 14-25-
Figure 7. Eect of a) 100 µM and b) 1mM of L-arginine and Aβ(25-35) on LTP induction.
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