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Intracerebroventricular Administration of L-arginine Improves Spatial Memory Acquisition in Triple Transgenic Mice Via Reduction of Oxidative Stress and Apoptosis


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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 deficiency 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 effect of arginine on synaptic plasticity was tested electrophysiologically by assessment of the hippocampal long-term potentiation (LTP). The effect 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 significantly 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.
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Translational Neuroscience
Research Article • DOI: 10.1515/tnsci-2018-0009 • Translational Neuroscience • 9 • 2018 • 43-53
* E-mail:
© 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 diuse neuritic plaques containing Aβ
peptide and neurobrillary tangles, which
are aggregates of hyperphosphorylated tau
protein (Schaeer 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 inammatory
processes (Heneka and O’Banion 2007),
atherosclerosis, redox stress (Sultana and
Buttereld 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 eective 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
of disbalances.
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 signicantly improves
positive nitrogen balance. Remarkably, arginine
signicantly reduced the levels of total serum
cholesterol and low-density lipoprotein. No
adverse eects 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 signicantly 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 eect of the
drug did not last after the end of the treatment.
Faculty of Medicine in the Galilee,
Bar Ilan University, Safed, Israel
2 Institute of Higher Nervous Activity and
Neurophysiology, Russian Academy of Sciences,
Moscow, Russia
Equal contribution
Gennadiy Fonar1‡,
Baruh Polis1‡,
Tomer Meirson1,
Alexander Maltsev2,
Evan Elliott1,
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 deciency 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 eect of arginine on synaptic plasticity was tested electrophysiologically
by assessment of the hippocampal long-term potentiation (LTP). The eect 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 signicantly 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
Download Date | 6/2/18 2:47 AM
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 eects 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 eects 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 inux 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 signicantly
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 insucient to
show all of its possible eects. In the present
research, we check the direct eect 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 eect 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
eect 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 eect 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
Mouse strains
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 deciency 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 articial
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% isourane and placed in a stereotaxic
apparatus. Anesthesia was maintained with 1%
isourane 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) prelled 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.
Cell culture
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
humidied 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. Briey, 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
quantied 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 reects early
cellular redox changes. Therefore, the amount
of formazan produced is proportional to the
number of viable cells. Briey, 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 (Innite® 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. Briey, 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 buer
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).
Behavioral tests
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 dened 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 reects
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 dened
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 sacriced
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-buered 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
Scientic), and Hoechst (1:5000, Sigma). Anti-
Aβ antibody (6E10) was purchased from ENCO.
Imaging and quantication
The number of Aβ deposits throughout the
hippocampi (3-6 sections examined per mouse)
was quantied. 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 ( with
the plugin from Wright Cell Imaging Facility.
The average of the mean intensity of the
staining was obtained for each hippocampal
subeld, and the background was subtracted
before calculating the reciprocal values.
Antibody microarray analyses
The evaluation of “hit” proteins’ expression
or phosphorylation of specic 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
specication. The analyses were done with
hippocampal lysates of mice treated with
arginine and ASCF as described on Kinexus’
web page ( Briey, 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-specic
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 (ler),
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 isourane 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 signicantly 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).
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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 (Scientica,
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
Schaer 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 dierent intervals, respectively. PPF
measures were carried out just before and after
LTP recordings.
Statistical analysis
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 signicant
dierences between the groups followed by a
Dunnett’s t-test for multiple comparisons. The
results are considered signicant 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. Immunouorescence and quantication of senile amyloid plaques in the hippocampal areas. a) A typi-
cal 20 x magnication image of the dentate gyrus from treated with L-arginine mouse with visible Aβ deposits.
The inset shows a high magnication (40 x) merged with DAPI view with Aβ deposits. b) A typical 10x magnica-
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 dierences are seen between treated and untreated
mice as shown by quantication of the hilus intraneuronal Aβ loads. c) Quantication 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 dierent 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
(gure 4a).
There are 65 regulatory proteins which
demonstrate signicant 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 ( To link the
dierential 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
signicance was drawn at p < 0.05.
Administration of L-arginine during
one month signicantly improved
memory acquisition in 3xTg-AD mice
The learning curve of the mice from the
experimental group reects 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
signicantly 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 signicantly better spatial
working memory than control animals.
Administration of L-arginine has no
signicant 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 signicant dierences in the amount of
amyloid beta deposition between two groups
(gure 3).
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 dierentially 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 dierentially expressed proteins involved in a subset of the
highlighted terms. Node degrees represent the amount of interactions per protein.
Translational Neuroscience
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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 eect 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-
term potentiation
Our results demonstrate that high
concentrations (about one mM) of L-arginine
signicantly 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)
signicantly decreased fEPSP slope and PS
amplitude in Aβ group compared to the control
group; though, did not aect baseline activity
of the neurons.
Inhibition of LTP induction by aged amyloid
beta and particularly Aβ (25--35) without
aecting 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 inuence
signicantly fEPSP slope and PS amplitude;
however, high concentrations of L-arginine
have a severe deteriorative eect 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 inuence 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) Quantication of DAPI uo-
rescence intensity.
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 signicantly higher than that in
the experimental plates.
L-arginine protects cells against
Aβ(25-35) and Hydrogen peroxide-
induced toxicity in a concentration-
dependent manner
To study the eect 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 signicantly 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
Translational Neuroscience
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Figure 6. Eects of dierent 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) Eect of Aβ(25-35) on the viability of PC12 cells
(exposure to 24 h). (b) Eect 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 eects 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 dierent 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 eects 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,
inammation, 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 eects 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 signicant
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 dierent
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 dierent 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
al. 1993).
Our primary intent in the current research
was to study the biochemical roles of
L-arginine in the development of AD. The use
Translational Neuroscience
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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 aect 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 conrmed
to improve status in a long list of diseases,
particularly, among the elderly people. Our
results validate a signicant 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
memory acquisition.
We prove, that the cognitive eect 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.
Amplied response to oxygen-containing
compounds, positive regulation of nitrogen
compound metabolic pathways and defense
response are the most critical, in our opinion,
amongst them.
We have tested our hypothesis in vitro on
the PC-12 cellular model. We evidence the
neuroprotective eect 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 conrms 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
human diseases.
Author contributions
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
Study Funding
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. Eect of a) 100 µM and b) 1mM of L-arginine and Aβ(25-35) on LTP induction.
Translational Neuroscience
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Translational Neuroscience
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... Aβ [25][26][27][28][29][30][31][32][33][34][35] (Sigma) was aged following a previously published protocol [36]. Briefly, the peptide was dissolved in mQ water to 1 mM stock solution and frozen. ...
... We suspect mToxin does not pass the blood-brain barrier (BBB); thus, to bypass the barrier, the compound was administered directly into the ventricles using osmotic minipumps and cannulae. We applied the same surgical procedure for cannulation, as described previously [36]. Briefly, the mouse skull was drilled in accordance with the coordinates: −0.2 mm caudal, 0.9 mm lateral to bregma. ...
... Pre-incubation with Aβ peptide (50 nM) resulted in a typical substantial impairment of LTP. These data accord with the current literature [48] and with our previously published results obtained in the same paradigm [36]. Likewise, there was no change in baseline responses within all the tested groups, which is in line with previous studies proving that Aβ has no effect on normal synaptic functioning on a baseline level [49]. ...
Full-text available
Alzheimer’s disease (AD) is the most common cause of senile dementia and one of the greatest medical, social, and economic challenges. According to a dominant theory, amyloid-β (Aβ) peptide is a key AD pathogenic factor. Aβ-soluble species interfere with synaptic functions, aggregate gradually, form plaques, and trigger neurodegeneration. The AD-associated pathology affects numerous systems, though the substantial loss of cholinergic neurons and α7 nicotinic receptors (α7AChR) is critical for the gradual cognitive decline. Aβ binds to α7AChR under various experimental settings; nevertheless, the functional significance of this interaction is ambiguous. Whereas the capability of low Aβ concentrations to activate α7AChR is functionally beneficial, extensive brain exposure to high Aβ concentrations diminishes α7AChR activity, contributes to the cholinergic deficits that characterize AD. Aβ and snake α-neurotoxins competitively bind to α7AChR. Accordingly, we designed a chemically modified α-cobratoxin (mToxin) to inhibit the interaction between Aβ and α7AChR. Subsequently, we examined mToxin in a set of original in silico, in vitro , ex vivo experiments, and in a murine AD model. We report that mToxin reversibly inhibits α7AChR, though it attenuates Aβ-induced synaptic transmission abnormalities, and upregulates pathways supporting long-term potentiation and reducing apoptosis. Remarkably, mToxin demonstrates no toxicity in brain slices and mice. Moreover, its chronic intracerebroventricular administration improves memory in AD-model animals. Our results point to unique mToxin neuroprotective properties, which might be tailored for the treatment of AD. Our methodology bridges the gaps in understanding Aβ-α7AChR interaction and represents a promising direction for further investigations and clinical development.
... Previously, we slow-released Arginine intracerebroventricularly in a 3 × Tg mouse model of AD and demonstrated a significant improvement in spatial memory [28]. Additionally, our laboratory performed a series of experiments to study the effect of arginase inhibition with Norvaline upon AD pathogenesis [29]. ...
... These animals display progressive β-amyloid (Aβ) deposition and NFT formation [33]. The mice have been treated with Norvaline in accordance with the protocol published previously [28]. Briefly, randomly chosen male 4-month-old mice were divided into four groups (3 mice in each group) and housed in individually ventilated cages. ...
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Alzheimer's disease (AD) is an insidious neurodegenerative disorder representing a serious continuously escalating medico-social problem. The AD-associated progressive dementia is followed by gradual formation of amyloid plaques and neurofibrillary tangles in the brain. Though, converging evidence indicates apparent metabolic dysfunctions as key AD characteristic. In particular, late-onset AD possesses a clear metabolic signature. Considerable brain insulin signaling impairment and a decline in glucose metabolism are common AD attributes. Thus, positron emission tomography (PET) with glucose tracers is a reliable non-invasive tool for early AD diagnosis and treatment efficacy monitoring. Various approaches and agents have been trialed to modulate insulin signaling. Accumulating data point to arginase inhibition as a promising direction to treat AD via diverse molecular mechanisms involving, inter alia, the insulin pathway. Here, we use a transgenic AD mouse model, demonstrating age-dependent brain insulin signaling abnormalities, reduced brain insulin receptor levels, and substantial energy metabolism alterations, to evaluate the effects of arginase inhibition with Norvaline on glucose metabolism. We utilize fluorodeoxyglucose whole-body micro-PET to reveal a significant treatment-associated increase in glucose uptake by the brain tissue in-vivo. Additionally, we apply advanced molecular biology and bioinformatics methods to explore the mechanisms underlying the effects of Norvaline on glucose metabolism. We demonstrate that treatment-associated improvement in glucose utilization is followed by significantly elevated levels of insulin receptor and glucose transporter-3 expression in the mice hippocampi. Additionally, Norvaline diminishes the rate of Tau protein phosphorylation. Our results suggest that Norvaline interferes with AD pathogenesis. These findings open new avenues for clinical evaluation and innovative drug development.
... Nitric oxide acts as both an antioxidant and a neurotransmitter and decreased NO transmission in the brain has been implicated in cognitive dysfunction (Zhu et al., 2017, Stephan et al., 2017. It has been shown that drugs replacing NO levels are beneficial in disorders affecting memory and the ability of L-ARG to improve cognition has been attributed to its antioxidant potential as well as its neurotransmitter function (Fonar et al., 2018). ...
... In the AGING PRM rats, L-arginine administration significantly improved memory and reduced anxiety but had no effect on these parameters in the VCD PRM group. The beneficial effect of L-arginine and NO donors on cognition has been well documented (Fonar et al., 2018 whereas the effect on anxiety is controversial (Gulati et al., 2017). The reason for the beneficial effect may be related to the low dose given. ...
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Neuropsychiatric symptoms like cognitive impairment and anxiety are prominent in the perimenopausal period and have been related to increased oxidative stress. Study evaluated effect of L-arginine on these neuropsychiatric symptoms and oxidative stress parameters. Immature female Sprague-Dawley rats were divided into 3 groups; Premenopausal injected with Corn-oil (2.5μl/g) for 15 days; VCD perimenopausal, injected with 4-vinylcyclohexene diepoxide (160mg/kg) diluted in Corn-oil for 15 days; and AGING perimenopausal group. Fourteen weeks after VCD/corn-oil administrations, and 180 days in AGING perimenopausal group, rats were further divided into 2 sub-groups that received L-Arginine (100mg/kg) and distilled water for 30 days. Thereafter, neurobehavioural assessments were carried out in animals at diestrus using Y-maze and elevated plus maze. Animals were humanely sacrificed, hippocampus and frontal lobe were isolated from the brain and homogenized for measurement of oxidative stress parameters. Percentage correct alternation was significantly higher (P< 0.05) in premenopausal compared to VCD and AGING perimenopausal groups. It was significantly lower (P < 0.05) in AGING perimenopausal group administered distilled water compared to AGING perimenopausal group administered L-Arginine with no change in other groups. Close Vs Open arm ratio was significantly lower (P < 0.05) in premenopausal compared to VCD and AGING perimenopausal groups. Similarly, L-Arginine significantly reduced (P < 0.05) Close Vs Open arm ratio in AGING PRM group while it significantly improved (P < 0.05) oxidative stress parameters in all groups. L-arginine improved cognition and anxiety in AGING perimenopausal with no change in premenopausal and VCD perimenopausal rats. (PDF) L-Arginine Administration Improves Cognition and Oxidative Stress Parameters in the Hippocampus and Frontal Lobe of 4-Vinylcyclohexene Diepoxide Perimenopausal Female Rats. Available from: [accessed Sep 02 2020].
... Reductions in urinary arginine levels have been reported in amnestic MCI compared to normal controls, reflecting systemic arginine dysregulation in early cognitive impairment [31]. Arginine is a semi-essential amino acid with numerous biological functions including protein synthesis and formation of vital metabolites (i.e., urea, nitric oxide) [32]. Arginine is a key player in the urea cycle for the disposal of nitrogenous waste [30]; an increase in arginine degradation (and subsequently lower peripheral and central levels) can be a consequence of a greater demand for nitrogen detoxification from increased amino acid metabolism [17]. ...
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Background: Exploration of cerebrospinal fluid (CSF) amino acids and the impact of dietary intake on central levels may provide a comprehensive understanding of the metabolic component of Alzheimer's disease. Objective: The objective of this exploratory study was to investigate the effects of two diets with varied nutrient compositions on change in CSF amino acids levels in adults with mild cognitive impairment (MCI) and normal cognition (NC). Secondary objectives were to assess the correlations between the change in CSF amino acids and change in Alzheimer's disease biomarkers. Methods: In a randomized, parallel, controlled feeding trial, adults (NC, n = 20; MCI, n = 29) consumed a high saturated fat (SFA)/glycemic index (GI) diet [HIGH] or a low SFA/GI diet [LOW] for 4 weeks. Lumbar punctures were performed at baseline and 4 weeks. Results: CSF valine increased and arginine decreased after the HIGH compared to the LOW diet in MCI (ps = 0.03 and 0.04). This pattern was more prominent in MCI versus NC (diet by diagnosis interaction ps = 0.05 and 0.09), as was an increase in isoleucine after the HIGH diet (p = 0.05). Changes in CSF amino acids were correlated with changes in Alzheimer's disease CSF biomarkers Aβ42, total tau, and p-Tau 181, with distinct patterns in the relationships by diet intervention and cognitive status. Conclusion: Dietary intake affects CSF amino acid levels and the response to diet is differentially affected by cognitive status.
... Remarkably, antioxidants have also been shown to provide protection against several comorbid diseases [18]. In the past, we have reported the benefits of several antioxidants in the treatment of AD [19], diabetes [20], and atherosclerosis [21]. Thus, antioxidants present an enormous potential role in treatment of AD and its correlated pathologies. ...
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Alzheimer’s disease (AD) is often comorbid with other pathologies. First, we review shortly the diseases most associated with AD in the clinic. Then we query PubMed citations for the co-occurrence of AD with other diseases, using a list of 400 common pathologies. Significantly, AD is found to be associated with schizophrenia and psychosis, sleep insomnia and apnea, type 2 diabetes, atherosclerosis, hypertension, cardiovascular diseases, obesity, fibrillation, osteoporosis, arthritis, glaucoma, metabolic syndrome, pain, herpes, HIV, alcoholism, heart failure, migraine, pneumonia, dyslipidemia, COPD and asthma, hearing loss, and tobacco smoking. Trivially, AD is also found to be associated with several neurodegenerative diseases, which are disregarded. Notably, our predicted results are consistent with the previously published clinical data and correlate nicely with individual publications. Our results emphasize risk factors and promulgate diseases often associated with AD. Interestingly, the comorbid diseases are often degenerative diseases exacerbated by reactive oxygen species, thus underlining the potential role of antioxidants in the treatment of AD and comorbid diseases.
... Remarkably, administration of arginine in the diet of patients with senile dementia increased cognitive function by about 40% [81], and epidemiological studies indicated that an intake of dietary arginine was inversely correlated with AD morbidity [82]. Finally, an intracerebroventricular injection of arginine improved spatial memory and cognitive functions in transgenic 3xTg mouse model of AD via a reduction of oxidative stress and apoptosis [83]. It has therefore been hypothesized that the upregulation of arginase activity and the resulting arginine and NO deficiency in brain areas characterized by excessive amyloid deposition, contribute to the clinical manifestation of AD and that the improvement of arginine bioavailability, for example, through arginase inhibition and/or the administration of arginine could be explored as a beneficial approach for treating AD [84]. ...
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Alzheimer's disease (AD) leads to the progressive loss of memory and other cognitive functions. It is the most common form of dementia in the elderly and has become a major public health problem due to the increase in life expectancy. Although the detection of AD is based on several neuropsychological tests, imaging and biological analyses, none of these biomarkers allows a clear understanding of the pathophysiological mechanisms involved in the disease, and no efficient treatment is currently available. Metabolomics, which allows the study of biochemical alterations underlying pathological processes, could help to identify these mechanisms, to discover new therapeutic targets and to monitor the therapeutic response and disease progression. In this review, we have summarized and analyzed the results from a number of studies on metabolomics analyses performed in biological samples originated from the central nervous system, in AD subjects and in animal models of this disease. This synthesis revealed modified expression of specific metabolites in pathological conditions which allowed the identification of significantly impacted metabolic pathways both in animals and humans, such as the arginine biosynthesis and the alanine, aspartate and glutamate metabolism. We discuss the potential biochemical mechanisms involved, the extent to which they could impact the specific hallmarks of AD, and the therapeutic approaches which could be proposed as a result.
Objectives: Alzheimer's disease (AD) correlates with the dysfunction of metabolic pathways that translates into neurological symptoms. An arginine deficiency, a precursor of nitric oxide (NO), has been reported for patients with AD. We aimed to evaluate the effect of citrulline oral supplementation on cognitive decline in an AD murine model. Methods: Three-month citrulline or water supplementation was blindly given to male and female wild-type and 3 × Tg mice with AD trained and tested in the Morris water maze. Cerebrospinal fluid and brain tissue were collected. Ultra-performance liquid chromatography was used for arginine determinations and the Griess method for NO. Results: Eight-month-old male 3 × Tg mice with AD supplemented with citrulline performed significantly better in the Morris water maze task. Arginine levels increased in the cerebrospinal fluid although no changes were seen in brain tissue and only a tendency of increase of NO was observed. Conclusions: Citrulline oral administration is a viable treatment for memory improvement in the early stages of AD, pointing to NO as a viable, efficient target for memory dysfunction in AD.
Polybrominated diphenyl ethers (PBDEs) are extensively used as brominated flame retardants in various factory products. As environmental pollutants, the adverse effects of PBDEs on human health have been receiving considerable attention. However, the precise fundamental mechanisms of toxicity induced by PBDEs are still not fully understood. In this study, the mechanism of cytotoxicity induced by 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) was investigated by combining Seahorse XFp analysis and mass spectrometry-based metabolomics and flux approaches in PC12 cells, one of the most widely used neuron-like cell lines for investigating cytotoxic effects. The Seahorse results suggest that BDE-47 significantly attenuated mitochondrial respiration and enhanced glycolysis in PC12 cells. Additionally, metabolomics results revealed the reduction of TCA metabolites such as citrate, succinate, aconitate, malate, fumarate, and glutamate after BDE-47 exposure. Metabolic flux analysis showed that BDE-47 exposure reduced the oxidative metabolic capacity of mitochondria in PC12 cells. Furthermore, various altered metabolites were found in multiple metabolic pathways, especially in glycine-serine-threonine metabolism and glutathione metabolism. A total of 17 metabolic features were determined in order to distinguish potentially disturbed metabolite markers of BDE-47 exposure. Our findings provide possible biomarkers of cytotoxic effects induced by BDE-47 exposure, and elicit a deeper understanding of the intramolecular mechanisms that could be used in further studies to validate the potential neurotoxicity of PBDEs in vivo. Based on our results, therapeutic approaches targeting mitochondrial function and the glycolysis pathway may be a promising direction against PBDE exposure.
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Adult neurogenesis is a complex physiological process, which plays a central role in maintaining cognitive functions, and consists of progenitor cell proliferation, newborn cell migration, and cell maturation. Adult neurogenesis is susceptible to alterations under various physiological and pathological conditions. A substantial decay of neurogenesis has been documented in Alzheimer’s disease (AD) patients and animal AD models; however, several treatment strategies can halt any further decline and even induce neurogenesis. Our previous results indicated a potential effect of arginase inhibition, with norvaline, on various aspects of neurogenesis in triple-transgenic mice. To better evaluate this effect, we chronically administer an arginase inhibitor, norvaline, to triple-transgenic and wild-type mice, and apply an advanced immunohistochemistry approach with several biomarkers and bright-field microscopy. Remarkably, we evidence a significant reduction in the density of neuronal progenitors, which demonstrate a different phenotype in the hippocampi of triple-transgenic mice as compared to wild-type animals. However, norvaline shows no significant effect upon the progenitor cell number and constitution. We demonstrate that norvaline treatment leads to an escalation of the polysialylated neuronal cell adhesion molecule immunopositivity, which suggests an improvement in the newborn neuron survival rate. Additionally, we identify a significant increase in the hippocampal microtubule-associated protein 2 stain intensity. We also explore the molecular mechanisms underlying the effects of norvaline on adult mice neurogenesis and provide insights into their machinery.
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Adult neurogenesis is a complex physiological process, which plays a central role in maintaining cognitive functions, and consists of progenitor cell proliferation, newborn cell migration, and cell maturation. Adult neurogenesis is susceptible to alterations under various physiological and pathological conditions. A substantial decay of neurogenesis has been documented in Alzheimer's disease (AD) patients and animal AD models; however, several treatment strategies can halt any further decline and even induce neurogenesis. Our previous results indicated a potential effect of arginase inhibition, with norvaline, on various aspects of neurogenesis in triple-transgenic mice. To better evaluate this effect, we chronically administered an arginase inhibitor, norvaline, to triple-transgenic and wild-type mice, and applied an advanced immunohistochemistry approach with several biomarkers and bright-field microscopy. Remarkably, we evidenced a significant reduction in the density of neuronal progenitors, which demonstrate a different phenotype in the hippocampi of triple-transgenic mice as compared to wild-type animals. However, norvaline showed no significant effect upon the progenitor cell number and constitution. We demonstrated that norvaline treatment leads to an escalation of the polysialylated neuronal cell adhesion molecule immunopositivity, which suggests an improvement in the newborn neuron survival rate. Additionally, we identified a significant increase in the hippocampal microtubule-associated protein 2 stain intensity. We also explore the molecular mechanisms underlying the effects of norvaline on adult mice neurogenesis and provide insights into their machinery.
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The antioxidant properties of l-arginine (l-Arg) in vivo, and its effect on enhancing resistance to oxidative stress and heat stress in Caenorhabditis elegans were investigated. C. elegans, a worm model popularly used in molecular and developmental biology, was used in the present study. Here, we report that l-Arg, at a concentration of 1 mM, prolonged C. elegans life by 26.98% and 37.02% under oxidative and heat stress, respectively. Further experiments indicated that the longevity-extending effects of l-Arg may be exerted by its free radical scavenging capacity and the upregulation of aging-associated gene expression in worms. This work is important in the context of numerous recent studies that concluded that environment stresses are associated with an increased population death rate.
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Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.
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The pathogenesis of Alzheimer's disease (AD) is a critical unsolved question; and although recent studies have demonstrated a strong association between altered brain immune responses and disease progression, the mechanistic cause of neuronal dysfunction and death is unknown. We have previously described the unique CVN-AD mouse model of AD, in which immune-mediated nitric oxide is lowered to mimic human levels, resulting in a mouse model that demonstrates the cardinal features of AD, including amyloid deposition, hyperphosphorylated and aggregated tau, behavioral changes, and age-dependent hippocampal neuronal loss. Using this mouse model, we studied longitudinal changes in brain immunity in relation to neuronal loss and, contrary to the predominant view that AD pathology is driven by proinflammatory factors, we find that the pathology in CVN-AD mice is driven by local immune suppression. Areas of hippocampal neuronal death are associated with the presence of immunosuppressive CD11c(+) microglia and extracellular arginase, resulting in arginine catabolism and reduced levels of total brain arginine. Pharmacologic disruption of the arginine utilization pathway by an inhibitor of arginase and ornithine decarboxylase protected the mice from AD-like pathology and significantly decreased CD11c expression. Our findings strongly implicate local immune-mediated amino acid catabolism as a novel and potentially critical mechanism mediating the age-dependent and regional loss of neurons in humans with AD. Copyright © 2015 the authors 0270-6474/15/355969-14$15.00/0.
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Reactive oxygen species are constantly produced in aerobic organisms as by-products of normal oxygen metabolism and include free radicals such as superoxide anion (O2 (-)) and hydroxyl radical (OH(-)), and non-radical hydrogen peroxide (H2O2). The mitochondrial respiratory chain and enzymatic reactions by various enzymes are endogenous sources of reactive oxygen species. Exogenous reactive oxygen species -inducing stressors include ionizing radiation, ultraviolet light, and divergent oxidizing chemicals. At low concentrations, reactive oxygen species serve as an important second messenger in cell signaling; however, at higher concentrations and long-term exposure, reactive oxygen species can damage cellular macromolecules such as DNA, proteins, and lipids, which leads to necrotic and apoptotic cell death. Oxidative stress is a condition of imbalance between reactive oxygen species formation and cellular antioxidant capacity due to enhanced ROS generation and/or dysfunction of the antioxidant system. Biochemical alterations in these macromolecular components can lead to various pathological conditions and human diseases, especially neurodegenerative diseases. Neurodegenerative diseases are morphologically featured by progressive cell loss in specific vulnerable neuronal cells, often associated with cytoskeletal protein aggregates forming inclusions in neurons and/or glial cells. Deposition of abnormal aggregated proteins and disruption of metal ions homeostasis are highly associated with oxidative stress. The main aim of this review is to present as much detailed information as possible that is available on various neurodegenerative disorders and their connection with oxidative stress. A variety of therapeutic strategies designed to address these pathological processes are also described. For the future therapeutic direction, one specific pathway that involves the transcription factor nuclear factor erythroid 2-related factor 2 is receiving considerable attention.
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With the aging of the population, the burden of Alzheimer's disease (AD) is rapidly expanding. More than 5 million people in the US alone are affected with AD and this number is expected to triple by 2050. While men may have a higher risk of mild cognitive impairment (MCI), an intermediate stage between normal aging and dementia, women are disproportionally affected with AD. One explanation is that men may die of competing causes of death earlier in life, so that only the most resilient men may survive to older ages. However, many other factors should also be considered to explain the sex differences. In this review, we discuss the differences observed in men versus women in the incidence and prevalence of MCI and AD, in the structure and function of the brain, and in the sex-specific and gender-specific risk and protective factors for AD. In medical research, sex refers to biological differences such as chromosomal differences (eg, XX versus XY chromosomes), gonadal differences, or hormonal differences. In contrast, gender refers to psychosocial and cultural differences between men and women (eg, access to education and occupation). Both factors play an important role in the development and progression of diseases, including AD. Understanding both sex- and gender-specific risk and protective factors for AD is critical for developing individualized interventions for the prevention and treatment of AD.
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The neuropathological correlates of Alzheimer's disease (AD) include amyloid-β (Aβ) plaques and neurofibrillary tangles. To study the interaction between Aβ and tau and their effect on synaptic function, we derived a triple-transgenic model (3×Tg-AD) harboring PS1M146V, APPSwe, and tauP301L transgenes. Rather than crossing independent lines, we microinjected two transgenes into single-cell embryos from homozygous PS1M146V knockin mice, generating mice with the same genetic background. 3×Tg-AD mice progressively develop plaques and tangles. Synaptic dysfunction, including LTP deficits, manifests in an age-related manner, but before plaque and tangle pathology. Deficits in long-term synaptic plasticity correlate with the accumulation of intraneuronal Aβ. These studies suggest a novel pathogenic role for intraneuronal Aβ with regards to synaptic plasticity. The recapitulation of salient features of AD in these mice clarifies the relationships between Aβ, synaptic dysfunction, and tangles and provides a valuable model for evaluating potential AD therapeutics as the impact on both lesions can be assessed.
Background: Alzheimer's disease (AD) is the most frequent type of dementia in people over 65 years of age; type 2 diabetes mellitus is a metabolic condition affecting 382 million of adults worldwide. Glucose is the main substrate for energy for the brain, which consumes 25% of the body's glucose. Glucose metabolism, evaluated using Positron Emission Tomography (PET) scanning using 18F-fluorodeoxyglucose ([18F]-FDG) is commonly used for diagnosis and follow up in AD. Epidemiological and pathophysiological studies showed a link between AD and diabetes, particularly regarding the insulin resistance. Objective: In this paper, we discuss how [18F]FDG is a marker of glucose metabolism, how insulin resistance is related to diabetes, the link between diabetes and AD, and how novel treatments for diabetes could be beneficial in the treatment of Alzheimer's disease. We also review few recent patents in the field, suggesting both diagnostic and therapeutic potential approaches. Methods: The electronic searches were performed in MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials databases. We also manually searched abstract books and reviewed available recent patetns on specific websites. Results: The impaired glucose metabolism in the brain of subject with AD is a widely recognised early feature of the disease; in vivo evaluation with PET is a useful diagnostic tool. The link between diabetes and neurodegeneration is widely recognized and offer a target for novel therapeutic strategies. Conclusions: Early diagnosis and early therapeutic intervention are needed to approach this devastating disease. Important studies targeting different aspect of the disease are currently ongoing.
Background: There is increased complexity of patients’ care in intensive care units that has motivated the involvement of critical care trained physicians as a part of multidisciplinary approach in medical, surgical and neurological ICUs. Multidisciplinary care has been less common in cardiac care units (CCUs). Methods: A formal protocol was implemented starting July 2012 in the CCU of Christiana Hospital whereby a medical intensivist integrated with the multidisciplinary team. This team included a cardiologist, a medical intensivist, a cardiology fellow, internal medicine residents and interns, ICU nurses, ICU pharmacist and respiratory therapists. Responsibilities were delineated in a fashion such that the intensivist primarily managed non-cardiac issues, deferring primary cardiologic issues to the cardiologist. Results: 2475 patients were included with 916 patients admitted in the12 months before and 1516 admitted in the 12 months after intervention. Patients admitted after the intervention had a higher morbidity rate expressed by a higher mean APACHEII (56.26 vs. 53.87; P <0.001), SAPS II (44.49 vs. 41.49; P <0.001). Results showed that the implementation of the intensivist multidisciplinary team model was associated with a significant decrease in both CCU Mortality (3.52 vs. 5.85%; odds ratio OR= 0.59; 95% CI (0.39-0.88); P=0.01) and in-hospital mortality (4.37 vs. 11.13%; OR= 0.37; 95% CI (0.26-0.52); P<0.0001). Furthermore; there was also a significant reduction in both CICU length of stay (LOS) (2.46 ± 3 vs. 2.84± 3 days; P= 0.006), hospital LOS (7.03 ±4.5 vs. 7.53 ±4.5 days; P= 0.006) and days on ventilation for intubated patients (1.96 ±1 vs. 4.32± 2.8; P=0.0001). Comparing Results. Conclusion: The implementation of a multidisciplinary team approach where an intensivist and cardiologist co-manage the critical care of CCU patients is associated with reduced ICU and in-hospital mortality, ICU and in-hospital LOS and duration of mechanical ventilation
In hypoxic/ischemic conditions, neuronal apoptotic events are occurred, resulting in neuronal diseases. Estradiol is a female sex hormone with steroid structure known to provide neuroprotection through multiple mechanisms in the central nervous system. This study was aimed to investigate the signal transduction pathway leading to the inhibitory effects of estradiol against cobalt chloride (CoCl2)-mediated hypoxic death in PC12 cells. Estradiol inhibits CoCl2-induced cell death with genomic DNA fragmentation and morphologic changes such as cell shrinkage and condensed nuclei. Pre-incubation of estradiol prior to CoCl2 treatment attenuated CoCl2-mediated the reactive oxygen species (ROS) production and limited the activities of the caspase cascades, such as caspase-8, -9 and -3. Furthermore, estradiol downregulated the Bax:Bcl-2 ratio and decreased the release of cytochrome c from the mitochondria into the cytosol in CoCl2-treated cells, indicating that estradiol affect on mitochondrial pathway. Estradiol attenuated also CoCl2-induced upregulation of Fas-ligand (Fas-L) and truncated of Bid in sequence of death receptor-mediated pathway. In addition, estradiol increased the phosphorylation of Akt in CoCl2-treated cells, demonstrating that estradiol has no affect on upstream signaling through the PI3K/Akt in inhibition of CoCl2-induced apoptosis in PC12 cells.Taken together, estradiol was found to have a neuroprotective effect against CoCl2-induced apoptosis of PC12 cells by the attenuating ROS production and the modulating apoptotic signal pathway through Bcl-2 family, cytochrome c, Fas/Fas-L as well as PI3K/Akt pathway.