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Abstract

The urea cycle is strongly implicated in the pathogenesis of Alzheimer’s disease (AD). Arginase-I (ARGI) accumulation at sites of amyloid-beta (Aβ) deposition is associated with L-arginine deprivation and neurodegeneration. An interaction between the arginase II (ARGII) and mTOR-ribosomal protein S6 kinase β-1 (S6K1) pathways promotes inflammation and oxidative stress. In this study, we treated triple-transgenic (3×Tg) mice exhibiting increased S6K1 activity and wild-type (WT) mice with L-norvaline, which inhibits both arginase and S6K1. The acquisition of spatial memory was significantly improved in the treated 3×Tg mice, and the improvement was associated with a substantial reduction in microgliosis. In these mice, increases in the density of dendritic spines and expression levels of neuroplasticity-related proteins were followed by a decline in the levels of Aβ toxic oligomeric and fibrillar species in the hippocampus. The findings point to an association of local Aβ-driven and immune-mediated responses with altered L-arginine metabolism, and they suggest that arginase and S6K1 inhibition by L-norvaline may delay the progression of AD. Electronic supplementary material The online version of this article (10.1007/s13311-018-0669-5) contains supplementary material, which is available to authorized users.
ORIGINAL ARTICLE
L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine
Model of Alzheimers Disease
Baruh Polis
1,2
&Kolluru D. Srikanth
2,3
&Evan Elliott
3
&Hava Gil-Henn
2
&Abraham O. Samson
1
Published online: 4 October 2018
#
Abstract
The urea cycle is strongly implicated in the pathogenesis of Alzheimers disease (AD). Arginase-I (ARGI) accumulation at sites
of amyloid-beta (Aβ) deposition is associated with L-arginine deprivation and neurodegeneration. An interaction between the
arginase II (ARGII) and mTOR-ribosomal protein S6 kinase β-1 (S6K1) pathways promotes inflammation and oxidative stress.
In this study, we treated triple-transgenic (3×Tg) mice exhibiting increased S6K1 activity and wild-type (WT) mice with L-
norvaline, which inhibits both arginase and S6K1. The acquisition of spatial memory was significantly improved in the treated
3×Tg mice, and the improvement was associated with a substantial reduction in microgliosis. In these mice, increases in the
density of dendritic spines and expression levels of neuroplasticity-related proteins were followed by a decline in the levels of Aβ
toxic oligomeric and fibrillar species in the hippocampus. The findings point to an association of local Aβ-driven and immune-
mediated responses with altered L-arginine metabolism, and they suggest that arginase and S6K1 inhibition by L-norvaline may
delay the progression of AD.
Key Words Alzheimers disease .L-norvaline .L-arginine .arginase .ribosomal protein S6 kinase β-1 .mTOR.
Introduction
Alzheimers disease (AD) is a slowly progressive neurodegen-
erative disorder, with an insidious onset. Advanced age is a
prominent risk factor for AD, atherosclerosis, and metabolic
disorders, such as type II diabetes. Their causal mechanisms
are multifaceted and not fully interpreted [1]. Recent clinical
and experimental data have shown that neurodegenerative
disorders often coexist with metabolic dysfunction [2].
According to 1 recent hypothesis, impaired bioenergetic me-
tabolism may play a key role in the pathogenesis of AD [3].
This hypothesis proposes that AD is characterized by a
combination of several interrelating pathological events,
which include bioenergetic, metabolic, neurovascular, and in-
flammatory processes. A recent study provided evidence that
brain hypo-metabolism occurs decades before clinical mani-
festations of AD, further suggesting that metabolic dysfunc-
tion is a causal factor in AD [4].
New objective lipidomics and metabolomics approaches
have been applied to analyze changes in postmortem brains
of AD patients [5]. These approaches have strongly implicated
the L-arginine metabolic pathway in the development of AD
[6]. Similar results pointing to dysregulation of L-arginine
metabolism were acquired in a rodent model of AD [7].
In initial clinical studies on L-arginine and its derivatives in
patients with various neurological disorders, L-arginine ad-
ministration within 30 min of a stroke significantly decreased
the frequency and severity of stroke-like symptoms [8].
Supplementation with 1.6 g/day of L-arginine for 3 months
in patients with senile dementia increased cognitive function
by about 40% [9]. A recent study also established the benefit
of L-arginine administration in a rodent model of AD [10].
In healthy individuals, L-arginine is transported from the
circulating blood into the brain via a Na
+
-independent cationic
amino acid transporter 1 (CAT1), which is expressed at the
blood-brain barrier (BBB) and functions as a supply pathway
*Baruh Polis
baruhpolis@gmail.com
1
Drug Discovery Laboratory, The Azrieli Faculty of Medicine,
Bar-Ilan University, 1311502 Safed, Israel
2
Laboratory of Cell Migration and Invasion, The Azrieli Faculty of
Medicine, Bar-Ilan University, 1311502 Safed, Israel
3
Laboratory of Molecular and Behavioral Neuroscience, The Azrieli
Faculty of Medicine, Bar-Ilan University, 8th Henrietta Szold Street,
P.O. Box 1589, 1311502 Safed, Israel
Neurotherapeutics (2018) 15:10361054
https://doi.org/10.1007/s13311-018-0669-5
The Author(s) 2018
for L-arginine to the brain [11,12]. In mammals, L-arginine is
derived mostly from renal de novo synthesis and dietary in-
take. Thus, despite the capability of the CAT1 to pass through
the BBB, its capacity as a transporter of L-arginine is limited
[13]. As a result, oral administration is not an option toinduce
the potential neurotrophic benefits of L-arginine. However,
pharmacological targeting of enzymes that metabolize L-
arginine may be a beneficial method to treat neurological dis-
orders, such as AD.
Recent research has pointed to a role for arginase, the en-
zyme that converts L-arginine to urea and L-ornithine, in AD,
cardiovascular diseases, and metabolic disorders [14]. One
study detected significantly decreased levels of L-arginine in
thecorticesofADpatients[15]. In addition, the activity of
arginase was significantly higher in the hippocampi of AD
patients [16]. Consequently, a hypothesis linking L-arginine
brain deprivation and the development of AD cognitive defi-
ciency was proposed [17].
There are 2 arginase enzymes, arginase I (ARGI) and argi-
nase II (ARGII). ARGI and ARGII are both expressed in the
rodent brain, especially in hippocampal neurons [18].
Previous research demonstrated that ARGII was the predom-
inant isoform in the human frontal cortex [19]. The expression
of both isoforms of arginase can be induced in different tissues
via exposure to a variety of cytokines and catecholamines
[20]. Stimuli that induce ARGII and lead to its translocation
from mitochondria to the cytosol include lipopolysaccharides,
tumor necrosis factor-α, oxidized low-density lipoprotein, and
hypoxia [21,22].
A recent study provided evidence for increased ARGII
gene expression in AD brains [23]. Studies also demonstrated
that ARGII deficiency reduced the level of hyperoxia-
mediated retinal neurodegeneration [24] and suggested that
arginase may be involved in the pathogenesis of neuronal
degeneration via excessive activation of excitotoxic N-meth-
yl-D-aspartate receptors (NMDARs) [25]. Therefore,
targeting ARGII has been proposed as a potential treatment
for decelerating age-related diseases [26].
Another way in which L-arginine may confer neuroprotec-
tion is through its effects on the nitric oxide (NO) pathway.
Nitric oxide synthase (NOS) utilizes L-arginine as a substrate
to produce NO and L-citrulline [27]. Accordingly, the bio-
availability of L-arginine influences NO synthesis [28]. A
growing body of evidence indicates that NO induces neuro-
protection by triggering vasodilation and increasing the blood
supply to neurons, thereby reducing their susceptibility to ox-
idative stress [29]. Moreover, NO regulates Ca
2+
influx into
neurons and provides neuroprotection against excitotoxicity
[30]. A previous study demonstrated reduced NOS activity
in AD brains, with a decrease in the levels of NOS1 and
NOS3 proteins [16].
Previously, an APP/NOS2
/
murine model was developed
to investigate the role of NO in the development of AD [31].
These mice exhibit behavioral deficits and biochemical hall-
marks of AD during aging. In this murine model, ARGI was
highly expressed in regions of amyloid-beta (Aβ)accumula-
tion [17]. Pharmacological disruption of the arginine utiliza-
tion pathway by irreversible inhibition of ornithine decarbox-
ylase with α-difluoromethylornithine (DFMO) protected the
mice from AD-like pathology and reversed memory loss. The
authors of the study suggested that L-arginine depletion was
responsible for neuronal cell death and cognitive deficits in the
course of AD development.
In another study, the arginase inhibitor L-norvaline ampli-
fied the level of NO production and reduced urea production
[31]. L-norvaline, which exerts its activity via negative feed-
back regulation, was later used successfully to treat artificial
metabolic syndrome in a rat model [32]. Research also
showed that L-norvaline inhibited the activity of ribosomal
protein S6 kinase β-1(S6K1)andthatitpossessedanti-
inflammatory properties [33], indicating that L-norvaline
could be extremely effective in AD.
Based on the literature, we hypothesized that upregulation
of arginase activity and consequent L-arginine and NO defi-
ciencies in the brain might contribute to the manifestations of
AD pathology and that targeting arginase might ameliorate the
symptoms of the disease.
In this study, we treated triple-transgenic (3×Tg) mice with
L-norvaline dissolved in water. Animals from a control group
displayed significant memory deficiencies as compared with
those in the treated (experimental) group, as evident from 2
paradigms.The cognitive improvements observed in the treat-
ed group were associated with reduced levels of beta-amyloid-
osis, microgliosis, and astrodegeneration. The L-norvaline
treatment also reversed the decline in dendritic spine density
in the hippocampi and amplified the expression levels of pre-
and postsynaptic proteins.
Methods
Strains of Mice and Treatment
Homozygous 3×Tg mice harboring a mutant APP (KM670/
671NL), which is a human mutant PS1 (M146V) knock-in,
and tau (P301L) transgenes (B6;129-Psen1
tm1Mpm
Tg(APPSwe, tauP301L)1Lfa/J) were obtained from Jackson
Laboratory (Bar Harbor, ME) and bred in our animal facility.
These mice exhibit a synaptic deficiency, with both plaque
and tangle pathology [34]. Four-month-old homozygous
3×Tg mice and age-matched male C57BL/6 mice were used
as nontransgenic controls (non-Tg) in all the experiments.
C57BL/6 mice are commonly used as non-Tg controls of
3×Tg mice [35]. The average weights of the 3×Tg and non-
Tg animals were marginally different at the beginning of the
experiment. However, the difference was not statistically
L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine Model of AlzheimersDisease 1037
significantly different, and the difference was negligible at the
end of the experiment.
Randomly chosen animals were divided into 4 groups (14
15 mice in each group). The animals were housed in cages and
provided with water and food ad libitum. The dose adminis-
tered was about 4050 mg/kg/day, which was similar to that
employed in a previous study in which L-norvaline was used
to treat artificial metabolic syndrome in a rat model [32]. In the
earlier study, the animals received norvaline dissolved in wa-
ter at a dose of 50 mg/kg/day for 6 week via force gavage
feeding. In the present study, L-norvaline (Sigma) was dis-
solved in the animalswater (250 mg/L) supplied in the ani-
malscages. For 3×Tg mice aged 5 months with an average
weight of about 30 g (33 g by the end of the experiment), the
dose was about 1.5 mg/day, taking into account that the aver-
age consumption of water by mice under laboratory condi-
tions equals about 56mL/day[36].
The mice underwent appropriate treatment. The experi-
mental design is presented in Fig. 1. The weights of the ani-
mals were measured every week during the experiment, and
no significant treatment-related effects on the weights were
observed.
All animal housing and procedures were performed in
compliance with the guidelines established by the Israeli
Ministry of Healths Council for Experimentation on
Animals and with Bar-Ilan University guidelines for the
use and care of laboratory animals in research. The exper-
imental protocol was approved by the ethics committee
for animal experiments of Bar-Ilan University (Permit
Number: 82-102017).
Behavioral Tests
Morris Water Maze
The Morris water maze (MWM) task was conducted to mea-
sure long-term learning and memory function as described
previously [37]. Briefly, a black plastic pool with a diameter
of 120 cm and a height of 50 cm was filled with water (24 ±
2 °C) and rendered opaque by the addition of skim milk pow-
der (Sigma). A circular dark colored platform with a diameter
of 10 cm was placed 0.3 cm under the water surface in 1 of the
quadrants. It remained in this quadrant throughout the tests.
Training was conducted on consecutive days for 7 days, with
2trialseachday.
The test consisted of 3 separate procedures. On the first
day, the platform was visible during the test, and a flag was
placed on the platform to increase its visibility [38]. The vis-
ible platform test aimed to identify gross visual deficits caused
by the treatment that could confuse subsequent interpretations
of the results obtained in the MWM experiment [37]. The
mice underwent 2 trials with 90-s cutoffs each and a 15-min
interval between the trials. On days 26, the tests were con-
ducted with the water platform hidden and no flag. Each an-
imal was placed in the pool at 1 of 4 locations (poles). The
mice were required to find the platform within 90 s, and they
were allowed to remain on the platform for 15 s. If a mouse
failed to locate the platform during the time limit, it was di-
rected gently toward the platform. A probe test without the
platform was performed on the 7th day. In this test, the mice
were released into the center of the maze and subjected to a
single trial with free swimming for 90 s.
Spontaneous Alternation Y-Maze
Short-termworkingmemorywasassessedinaY-maze
consisting of 3 arms (each 35 cm long, 25 cm high, and
10 cm wide) at a 120° angle from each other. After an intro-
duction to the center of the maze, each animal was allowed to
freely explore the maze for 8 min. The maze was cleanedwith
a 10% aqueous ethanol solution between each trial.
Spontaneous alternation was defined as successive entry into
3 different arms on overlapping triplet sets. An arm entry was
counted when the hind paws of the mouse were entirely within
the arm [39].
Video Tracking
All the behavioral experiments were recorded using a
Panasonic WV-CL930 camera with a Ganz IR 50/50 infrared
panel. The recorded video files were analyzed using
Ethovision XT 10 software (Noldus Information
Technology, Wageningen, Netherlands) by an individual
blinded to the treatment schedule.
Tissue Sampling
After the behavioral tests, the mice were deeply anesthetized
with sodium pentobarbital (60 mg/kg) administered intraperi-
toneally and decapitated. Their brains (4 from each group)
were sliced (0.5 mm thick) immediately in a mouse brain
slicer matrix. Sections between 1.7 and 2.2 mm posterior to
bregma according to the atlas of Franklin and Paxinos were
used for sampling [40]. The hippocampi were punched in the
Fig. 1 Experimental design
1038 B. Polis et al.
region of the dentate gyrus in each hemisphere using a 13-
gauge microdissection needle, frozen, and stored at 80 °C.
Antibody Microarray
The assessment of Bhit^proteinsexpression was performed
using a Kinex KAM-1150E antibody microarray (Kinexus
Bioinformatics) in accordance with the manufacturers in-
structions. The analyses were done with hippocampal lysates
as described on Kinexusweb page (www.kinexus.ca).
Briefly, lysate protein from each sample (100 μg) was labeled
covalently with a fluorescent dye combination. Free dye mol-
ecules were then removed via gel filtration. After blocking
nonspecific binding sites on the array, an incubation chamber
was mounted onto the microarray to permit the loading of the
samples. After incubation, unbound proteins were washed
away. Two 16-bit images from each array were then captured
using a ScanArray Reader (Perkin-Elmer). An antibody array
was performed in parallel on 4 different chips. The output of
the array consisted of the average normalized net signals (i.e.,
the average of4 normalized net signal values of each antibody
on the microarray).
The standard deviation and percent standard deviation of 4
separate measurements of globally normalized signal intensity
values for each different antibody on the microarray were
calculated. The data are presented as change from control
(CFC%). A positive value corresponded to an increase in sig-
nal intensity in response to the treatment, with a value of
100% corresponding to a 2-fold increment in signal intensity.
A negative CFC value indicated the degree of reduction in
signal intensity from that of the control.
Each parameter has its Studentsttest pvalue, which is the
probability (p) value that there is no difference between the
control and test samples. A pvalue determined with N=4
measurements in each set, which were paired and 2-tailed in
distribution. A pvalue of 0.05 was accepted as statistically
significant.
Golgi Staining Procedure
For Golgi staining, 3 mice in each group were perfused
transcardially with 0.1 M phosphate-buffered saline (pH
7.4), and their brains were processed using a superGolgi Kit
according to the manufacturers protocol (Bioenno
Lifesciences, Santa Ana, CA, USA). Briefly, the brains were
immersed in impregnation solution for 11 days, followed by
incubationfor 2 days in a postimpregnation solution. Once the
impregnation of neurons was complete, the brain samples
were coronally sliced (150 μm) using a vibrating microtome
(Campden Instruments, Lafayette, IN) and collected serially in
a mounting buffer. They were then mounted upon 1% gelatin-
coated glass slides, stained with a staining solution, and
coverslipped using Permount (Fisher Scientific, Houston,
TX, USA). Two corresponding sections between 1.7 and
2.0 mm posterior to bregma according to the atlas of
Franklin and Paxinos per animal were chosen for stereological
analysis [40].
An upright ApoTome (Quorum Technologies, Lewes, UK)
microscope was used for imaging. To assess dendritic mor-
phology, low magnification (× 10/0.3, × 20/0.8, and × 40/0.75
lens) images (Z-stack with 0.5 μm intervals) of pyramidal
neurons, with cell bodies located in the CA1 region of the
hippocampus, were captured using an ORCA-Flash4.0 V3
camera. High-magnification (× 100/1.4 oil objective with dig-
ital zoom 3) images (Z-stack with 0.25 μm intervals) were
also obtained.
Dendritic Spine Density Measurement
The captured images were coded, and an investigator blinded
to the experimental conditions measured the numbers of
spines per dendritic length using Zen Blue 2.5 (Zeiss,
Oberkochen, Germany) and Neurolucida 360, version
2018.1.1 (MBF Bioscience, Williston, VT, USA). software,
which provides an automatic unbiased quantitative 3D analy-
sis of identified neurons [41]. The spine detection threshold
was set an outer range of 2.5 μm, the minimal height was
0.3 μm, the sensitivity was 100%, and the minimum count
was 10 voxels.
The spine density was analyzed in each hemisphere of each
murine brain. Two hippocampal pyramidal cells, with soma
located in the center of 150-μm corresponding sections, were
selected for the analysis (24 neurons per experimental group).
The spine density on a secondary oblique dendritic branch
localized in the stratum radiatum of the CA1 hippocampal
pyramidal neurons was also quantified.
Immunostaining
Seven animals from each group were deeply anesthetized and
transcardially perfused with 30 mL of phosphate-buffered sa-
line (PBS), followed by 50 mL of chilled paraformaldehyde
4% in PBS. The brains were carefully removed and fixed in
4% paraformaldehyde for 24 h. The brains from 3 mice per
group were thentransferred to 30% sucrose in PBS at 4 °C for
24 h and frozen at 80 °C. The brains from another 4 mice
(including non-Tg mice) in each group were dehydrated and
paraffin embedded.
β-Amyloid, 1-16 (6E10) Antibody Staining
The brains were sliced on a Leica (Wetzlar, Germany)
CM3050 S cryostat to produce 30-μm floating sections. For
β-amyloid, 1-16 (6E10) antibody staining, corresponding sec-
tions (between 1.7 and 2.2 mm posterior to bregma) were
blocked and incubated overnight with primary purified
L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine Model of AlzheimersDisease 1039
anti-β-amyloid, 1-16 antibody 6E10 (1:100, Biolegend) at
4 °C. This was followed by washing and incubation with goat
anti-mouse Immunoglobulin G secondary antibody Alexa555
(1:200, Invitrogen) at room temperature for 1 h and Hoechst
(1:5000, Sigma) for 3 min.
Quantitative Histochemical Analysis of Microglia,
Astrocytes, ARGI, and ARGII
The paraffin-embedded tissue blocks were chilled on ice and
sliced on a Leica (Leica Biosystems, Nussloch, Germany)
RM2235 manual rotary microtome at a thickness of 4 μm.
The sections were mounted onto gelatin-coated slides, dried
overnight at room temperature, and stored at 4 °C inside stor-
age boxes.
For the quantitative histochemical analysis of microglia
(Iba1), astrocytes (GFAP), ARGI, and ARGII
immunopositivity, 5 brain coronal sections per mouse (1.9
2.0 mm posterior to bregma) were used. Five serial sections at
a thickness of 4 μm were cut at 20-μm intervals throughout
the brain. Immunohistochemistry (IHC) was carried out onthe
plane-matched coronal sections.
Staining was performed on a fully automated Leica Bond III
system (Leica Biosystems Newcastle Ltd., UK). The tissues
were pretreated with an epitope-retrieval solution (Leica
Biosystems Newcastle Ltd., UK), followed by incubation with
primary antibodies for 30 min. The Iba1 antibody (Novus
Biologicals, #NB100-1028), GFAP (Biolegend, #835301),
ARGI (GeneTex, GTX113131), and ARGII (GeneTex,
#GTX104036) dilutions were 1:500, 1:1000, 1:500, and 1:60,
respectively. A Leica Refine-HRP kit (Leica Biosystems
Newcastle Ltd., UK) was used for detection and
counterstaining with hematoxylin. The omission of Iba1,
GFAP, ARGI, and ARGII primary antibodies served as a neg-
ative control (Supplementary Fig. S1). For a positive control of
ARGI, murine liver tissue and kidney were stained for ARGII.
Imaging and Quantification
The sections were mounted and viewed under an Axio
Scan.Z1 (Zeiss, Oberkochen, Germany) fluorescent and
bright-field slide scanner with a × 40/0.95 objective. Images
were taken with a Z-stack of 0.5 μm. Also, an Axio Imager 2
Upright ApoTome microscope was used to capture images
with a × 100/1.4 oil immersion objective. Immunolabeling
was performed in the corresponding hippocampal areas, and
image analysis was carried out using Zen Blue 2.5 (Zeiss,
Oberkochen, Germany) and Image-Pro® 10 (Media
Cybernetics, Rockville, MD) software with a fixed back-
ground intensity threshold for all sections representing a sin-
gle type of staining.
Morphometric Cell Analysis
Resting microglia exhibit a ramified phenotype and adopt dif-
ferent morphologies, ranging from a highly ramified to an
amoeboid-like phenotype upon activation [42]. To quantify
morphological changes in resting microglia, we used morpho-
logical parameters that are accepted in the literature and ex-
pected to capture the shift from resting to activated phenotype
[43]. The parameters included the cell perimeter length,
Ferets diameter, soma size, and sphericity (4π×area/perime-
ter
2
). To assess the staining density, the integrated optical den-
sity (IOD), which was defined as the mean density per pixel
area (lum/pix
2
), was calculated [44]. The IOD values of the
Iba1-stained microglia from CA3 regions with a surface area
of 0.1 mm
2
were calculated as log
10
of the values acquired
using Image-pro® 10. Two appropriate sections (bregma 1.8
and 2.0 mm) from each mouse were included in the analyses
(N= 125 cells in the untreated groups; N= 90 cells in the L-
norvaline-treated groups).
Western Blotting
To determine the level of β-amyloidosis, the immunoreactiv-
ity of the A11 antibody and anti-amyloid fibril OC antibody in
hippocampal lysates was examined. The A11 antibody detects
the conformation of amyloid oligomers, irrespective of their
amino acid sequence [45], and the anti-amyloid fibril OC an-
tibody recognizes fibrillary forms.
In addition, the relative differences in the expression levels
of various neuroplasticity-related proteins that showed signif-
icant changes in the antibody microarray assay were analyzed
and validated. The setup and the antibody nomenclature are
presented in Supplementary Table 1.
The protein concentration was determined using the
Bradford assay. Hippocampal lysates (15 μg) obtained from
brain samples of the 3×Tg mice treated with L-norvaline or
vehicle (4 tissue punches from each group) were analyzed
using a KinetworksCustom Multi-Antibody screen 1.0
(Kinexus Bioinformatics, Vancouver, Canada) in accordance
with the instructions of the manufacturer. Briefly, the
Kinetworksanalysis involves resolution of a single lysate
sample by sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis and subsequent immunoblotting using validated an-
tibodies. Antibodies bound to their target antigen on the nitro-
cellulose membrane were detected using an enhanced chemi-
luminescence detection system.
Statistical Analysis
Statistical analyses were conducted using SPSS version 22
(IBM, Armonk, NY, USA) for Windows. The significance
was set at 95% of confidence. All the results are presented
as mean with standard error. The ShapiroWilk test showed
1040 B. Polis et al.
that the data fit a normal distribution, and Levenestestwas
performed to confirm equal variance between the groups be-
ing compared.
For the Y-maze, visible platform test, and probe trials
(MWM), the means were compared between groups using a
one-way analysis of variance (ANOVA), with Tukeysmulti-
ple comparison test used for post hoc analyses. For the hidden
platform test, the escape latencies in the 2 trials were averaged
for each mouse each day and then analyzed across the 5 days
of testing [37]. An ANOVA was applied, with day as the
repeated measure and latency as the dependent variable. For
comparisons between groups, Tukeys multiple comparison
test was conducted when the main analysis revealed signifi-
cance. The Student ttest was performed to compare the means
between 2 groups. All data are presented as mean values.
Throughout the text and in bar plots, the variability is indicat-
ed by the standard error of the mean (SEM). In the figures with
box and whisker graphs, the boxes extend from the 25th to
75th percentiles. The line in the middle of the box is plotted at
the median. The whiskers denote the smallest and largest
values.
Results
L-Norvaline Ameliorated Memory Deficits in the 3×Tg
Mice
To investigate whether L-norvaline treatment affected learn-
ing and memory in AD pathogenesis, a set of behavioral tests
was administered following L-norvaline treatment for
2 months, beginning when the mice were aged 4 months.
Short-term working memory was assessed in a Y-maze
spontaneous alternation test. The results of the one-way
ANOVA test revealed a significant treatment-related effect on
the Bpercentage of alternations^(F(3,54) = 4.525, p= 0.0067).
The post hoc Tukey multiple comparison test indicated that the
percentage of alterations in the control 3×Tg mice was lower
than that in the L-norvaline-treated mice and wild-type (WT)
controls (p< 0.05) (Fig. 2a). The L-norvaline treatment had no
significant effect on the alternative behavior of the non-Tg
mice. The treatment also did not affect the total distance trav-
eled by the animals during the test (p= 0.86) (Fig. 2b).
The MWM was used to determine the effect of L-norvaline
upon spatial memory. In the visible platform test, there were
no significant between-group differences (p= 0.99), which
indicated that the treatment had no influence on murine mo-
tility or vision (Fig. 2c).
In the hidden platform swimming test, there were signifi-
cant differences between the groups according to the repeated
measures ANOVA (F(3,12) = 7.725, p= 0.0039). Tukeys
multiple comparison test revealed a significant (p<0.01)
effect of the treatment on memory acquisition in the 3×Tg
group (Fig. 2d).
In the probe trial on the last day of testing, the platformwas
removed. The relative time spent by the mice in the target
quadrant, where the hidden platform had been previously
placed, was significantly longer in the L-norvaline-treated
group as compared with that of the control group (34.8 ±
8.06 s vs 24.5 ± 7.88 s; p<0.05;Fig.2e).To visualize spatial
preferences in the probe test, a heat map was used (Fig.
2g). The plots demonstrated that the treated 3×Tg animals
spentmoretimethanthecontrolmicedidinthetarget
quadrant. The treatment did not affect the swim speed of
the animals (Fig. 2f).
Overall, the results of the behavioral experiments sug-
gested that L-norvaline improved spatial memory acquisition
in 3×Tg mice.
The L-Norvaline Treatment Reduced the Quantities
of AβFibrils and Prefibrillar Oligomers
in the Hippocampi of the 3×Tg Mice
Recent evidence pointed to a role for soluble amyloid oligo-
meric and fibrillar species in synaptic dysfunction, neuronal
apoptosis,and brain damage [46]. To investigate the impact of
L-norvaline on the amount of toxic prefibrillar Aβoligomers
and fibrillary forms, we examined A11 and OC immunoreac-
tivity of hippocampal lysates. Pooled equal amounts (10 μg)
of totallysates from the hippocampi of each group of mice
were run on sodium dodecyl sulfatepolyacrylamide gels
and incubated with appropriate antibodies (Supplementary
Table 1and Supplementary Fig. S2). The L-norvaline
treatment resulted in a substantial (about 30%) reduction
in the levels of A11-reactive oligomers and OC-reactive
fibrillar species (Table 1).
L-Norvaline Decreased the Level of β-Amyloidosis
in the Cortex of the 3×Tg Mice
To examine the effect of the L-norvaline treatment on the
total amyloid burden in the brains of the 3×Tg mice, cor-
onal brain sections were stained with human APP/Aβ-
specificantibody.Bythetime they were 6 months old,
the 3×Tg mice exhibited enhanced intracellular deposition
of Aβin the IVV layers of the prefrontal cortex (PFC)
(Fig. 3ac) and hippocampus (Fig. 3df).
We did not detect significant differences in the level of Aβ
deposition in the hippocampi of the 2 groups. However, there
was a general trend toward a slight reduction in 6E10 positiv-
ity in the treated group. There was a significant (p=0.032)
reduction in the level of 6E10 positivity in the cortex of the
3×Tg mice treated with L-norvaline (Fig. 3g). These data
pointed to an effect of L-norvaline on the level of β-
amyloidosis in the 3×Tg mice.
L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine Model of AlzheimersDisease 1041
Fig. 2 The effects of L-norvaline on animal behavior. The Y-maze
spontaneous alternation test. (a) Percentage of alternation. (b) The total
distance traveled during a trial. (c) (MWM) The visible platform test
(averaged escape latencies of 2 trials). (d) The hidden platform test
(averaged escape latencies of 2 trials/day). (e) The time spent by the
mice in the target quadrant in the probe trial. (f) The average swimming
speed in the probe test. (g) Heat maps showing the search intensity during
the probe trials, where a dashed circle indicates the platform. A highdwell
time across the pool area is indicated by colors close to red, whereas
colors close to blue indicate lower dwell time (arbitrary scale). Data are
shown as means ± SEM. (n= 15 in the non-TG groups, n=14 in the
3×Tg groups). *p<0.05,**p<0.01
1042 B. Polis et al.
L-Norvaline Increased Pyramidal Cell Dendritic Spine
Density in the Hippocampus of the 3×Tg Mice
Dendritic spines are specialized structures on neuronal processes,
which are involved in neuronal plasticity [47]. A strong
correlation between dendritic spine density and memory acqui-
sition was demonstrated in rodents using different behavioral
paradigms [48]. Furthermore, 3×Tg mice showed progressive
dendritic spine deficiency as compared with WT mice [49],
and this deficiency can be reversed by the treatments [50].
Table 1 L-Norvaline reduced the level offibrillar amyloid and prefibrillar oligomers in hippocampal brain lysates of the 3×Tg mice (blotssummary).
Western blot, showing β-actin normalized trace quantities of Aβfibrils (a) and prefibrillar oligomers (b)
(a) Actin normalized trace quantity (b) Actin normalized trace quantity
MW (kda) Control Norvaline Change (%) MW (kda) Control Norvaline Change (%) Structure
135.938 514 265 48 85.776 787 141 82
89.692 428 518 + 21 61.237 1622 1288 22
79.654 574 308 46 48.587 8723 6127 30 Dodecamers
68.494 590 521 12 40.324 150 30 80 Octamers
62.231 725 593 18 36.076 393 456 + 16
50.635 1703 1206 30 33.865 406 462 + 14
43.94 1799 1185 34 27.314 2784 1502 46 Hexamers
37.763 1150 1289 + 12 24.216 1218 1245 + 2
25.766 4254 3229 24
Sum 11,737 9114 22 Sum 16,084 11,251 30
Fig. 3 The impact of L-norvaline on the total amyloid burden.
Quantification of the area of Aβimmunoreactivity in brain sections
from the 3×Tg mice treated with vehicle (control) or L-norvaline.
Representative × 20 magnification view of PFC from the control (a)
and L-norvaline-treated mice (b) with apparent deposits in layers IVV.
The insets (c,f) represent × 100 magnification of double staining with
DAPI. Intense Aβdeposition in the dentate gyrus (df) of the 3×Tg
control mice, (f, scale bar 20 μm). Bar chart of the immunoreactive area
in the hilus and PFC (g). The Student ttest was used to compare the
means between 2 groups, *p<0.05(n= 15, 3 mice per group)
L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine Model of AlzheimersDisease 1043
In the present study, dendritic spines were examined by
Golgi staining in the vehicle- and L-norvaline-treated groups.
Dendritic spine density was quantified on a secondary oblique
dendritic branch localized in the stratum radiatum of the CA1
hippocampal pyramidal neurons (Fig. 4a, c, d). The results of
the one-way ANOVA test revealed a significant effect of the
treatment on dendritic spine density (F(3,92) = 3.338, p=
0.0173). The post hoc Tukey multiple comparison test pointed
to a significantly lower spine density inthe control 3×Tg mice
as compared with that in the L-norvaline-treated mice
(p< 0.05) (Fig. 4b). The average increase following the treat-
ment was about 20% (1.326 ±0.059 vs 1.10767 ± 0.06 spines
per micrometer). The treatment did not affect dendritic spine
density in the non-Tg groups.
L-Norvaline Amplified the Expression Levels
of Proteins Related to Synaptic Plasticity in the 3×Tg
Mice
To gain additional molecular insight into the behavioral phe-
notype, the effect of the L-norvaline treatment onthe levels of
neuroplasticity-associated proteins in the 3×Tg mice was in-
vestigatedusing a set of proteomics assays. First, we tested the
protein levels of 12 known important neuroplasticity-related
proteins in the hippocampus by a Western blot analysis
(Supplementary Table 1). Second, we performed an anti-
body array analysis to detect changes in protein levels at
the whole throughput level (approximately 1000 pro-
teins). The Western blot analysis revealed an increase
in synaptic- and neuroplasticity-related proteins in the
L-norvaline-treated group (Fig. 5a). Remarkably, the ex-
pression of vesicular glutamate transporters 1 and 3 in
the brain increased by 458% and 349% respectively, po-
tentially increasing vesicular glutamate levels.
The protein microarray analysis revealed elevated levels of
other neuroplasticity-related proteins following the L-
norvaline treatment. The expression level of postsynaptic pro-
tein neuroligin-1, which mediates the formation and mainte-
nance of synapses, was amplified by 252% following the
treatment. However, the pvalue of the change detected by
the antibody microarray was 0.07. Furthermore, the levels of
synaptophysin, which is a synaptic vesicle glycoprotein, in-
creased by 50% in the hippocampi of the 3×Tg mice treated
with L-norvaline, and this finding was statistically significant
(p=0.039).
Notably, the expression of endothelial NOS increased by
68% (p= 0.041). Moreover, there was a substantial significant
(p= 0.015) increase in cyclin E protein, which is a central
component of the cell cycle machinery and known to regulate
synaptic plasticity and memory formation [51].
Fig. 4 Photomicrographs of Golgi-stained hippocampal neurons from a
coronal slice. (a) A representative Golgi-impregnated × 40 image of the
CA1 region of non-Tg mice, showing 2 pyramidal cells. The inset is a ×
100 image of a secondary apical oblique dendrite in the stratumradiatum.
Automated quantitation of spine density using Neurolucida software was
subjectedto a statistical analysis by a one-way ANOVA. (b) Spine density
of hippocampal neurons. *p<0.05. The data are mean ± SEM, n=24for
hippocampal cells (3 mice per group). Representative dendritic CA1
segments of 3×Tg control (c) and 3×Tg L-norvaline-treated mice (d).
The scale bar is 5 μm
1044 B. Polis et al.
L-Norvaline Reduced Microgliosis in the 3×Tg Mice
Microglia are the first line of active immune defense in the
brain, and an increase in microglial density is indicative of
elevated pathogenic insults [52]. Previous studies reported
increased microglial density in the hippocampi of AD patients
[53] and hippocampi of 3×Tg mice [54]. Microglia densities
in the CA3 area (0.2 mm
2
) of the 3×Tg (Fig. 6a, b) and non-Tg
(Fig. 6c, d) mice were analyzed using image-processing soft-
ware and a manually set threshold. To guarantee the selection
of only cells present in the acquired field, for Iba1 staining,
only cells with an area greater than 25 μm
2
were considered in
the analysis, as widely accepted in the literature [55].
The results of the one-way ANOVA test revealed a significant
effect of the treatment on microglial density and the BIba1-posi-
tive area fraction^(F(3,52) = 4.64, p= 0.006 and F(3,52) = 4.098,
p= 0.01, respectively). The post hoc Tukey multiple comparison
test indicated a significant reduction (p< 0.05) in microglial den-
sity in the area of interest in the L-norvaline-treated 3×Tg group
(63.84 ± 7.43 vs 83.66 ± 5.11 cells/mm
2
, on average) (Fig. 6e)
and a significant (p< 0.05) decrease in the Iba1-positive area
fraction (0.48 ± 0.04 vs 0.67 ± 0.05%) (Fig. 6f).
The Microglial Morphology of Iba1-Positive Cells
in the Hippocampus of the L-Norvaline-Treated 3×Tg
Mice Pointed to a Prevalence of the Resting
(Ramified) Phenotype
Microglial morphology and function are closely related.
Microglia are sensitive to the surrounding microenvironment,
and various stimuli regulate their morphology [56].
Neuroinflammationischaracterizednotonlybyanincreasein
microglial density but by diverse morphological changes, in-
cluding de-ramification [57]. To address this issue, several mor-
phological features of stained microglia were analyzed using
image analysis software Image-Pro® 10 (Media Cybernetics,
Rockville, USA) and ZEN 2.5 (Zeiss, Oberkochen, Germany).
Iba1 is known to be expressed weakly by ramified microglia
but upregulated and strongly expressed by activated microglia
[58]. We used the IOD index to characterize Iba1 staining in-
tensity [59]. The results revealed a significant treatment-related
effect on the microglial IOD (Fig. 7a). In the treated group, this
parameter was reduced by 30%.
A detailed analysis of microglial somata revealed several
other treatment-related effects. First, the average size (surface
area) of the measured cells in the treated group contracted by
17% (50.92 ± 1.69 μm
2
in the treatment group vs 60.93 ±
1.06 μm
2
in the untreated group; p< 0.0001) (Fig. 7c).
Second, the roundness or sphericity of the somata (4π
area/perimeter
2
), which reflects microglial reactivity, in-
creased significantly in the L-norvaline-treated group (0.763
± 0.012 in the treatment group vs 0.71 ±0.005 in the untreated
group; p<0.0037) (Fig. 7d). Additionally, we measured the
Feret diameter or maxmin caliper. The analysis revealed a
significant reduction in Ferets diameter in the treated group
(6.82 ± 0.182 μmvs 8.61 ± 0.26 μm; p< 0.0001) (Fig. 7b).
Furthermore, the distribution analyses revealed a left-
shifted distribution of soma size and right-shifted distribution
of circularity in the treated group (Fig. 7e, f). Together, these
findings suggested that the number of microglia with a small,
round cell body (resting microglia) increased after the treat-
ment. The cells in the control group had a bigger and more
Fig. 5 L-Norvaline increased the
expression levels of
neuroplasticity-related proteins in
the hippocampus of the 3×Tg
mice. Results of the Western blot
with β-actin normalized trace
quantities (a) and antibody array
selected data (b). CFC = change
from control
L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine Model of AlzheimersDisease 1045
irregular soma shape (activated microglia) as compared with
those in the treated group. The observations herein were in
accord with the common classification of microglia morphol-
ogy, in which resting cells are considered small, round cells
with elaborate ramifications, and activated cells are consid-
ered amoeboid, with retracted processes [60].
L-Norvaline Reversed Astrocyte Degeneration in Brain
Areas of the 3×Tg Mice, with Pronounced
β-Amyloidosis
Astrocytes modulate and control synaptic activity [61].
GFAP is highly expressed in astrocytes [62]. We stained
the brains and observed GFAP immunoreactive cells with
a typical stellate shape (Fig. 8e, f). Of note, the shape of
the astrocytes in brain sections from the 3×Tg mice dif-
fered from those in non-Tg animals, with the astrocytes of
the non-TG mice displaying a highly ramified star-shaped
configuration (Supplementary Fig. S4). The astroglia in
brain sections from the control 3×Tg mice showed fewer
processes and a reduced volume (Fig. 8a, c) as compared
with astroglia in brain sections from the animals treated
with L-norvaline (Fig. 8b, d). We quantified the levels of
GFAP immunoreactivity (reflected by the volume density)
within the hilus area. The results of a comparative analy-
sis using a one-way ANOVA (F(3,51) = 9.263,
Fig. 6 L-Norvaline significantly decreased the microglial density in the
3×Tg mice. Visualization and quantification of microglia with Iba1
staining of the hippocampus of the 3×Tg mice (a,b) and non-Tg mice
(c,d). Representative hippocampal ×20 bright-field micrographs of the
CA3 areas of the control (a,c) and L-norvaline-treated mice (b,d).The
boxplots show the area density of microglia in the hippocampal CA3 area
of the 3×Tg mice and non-Tg mice treated with vehicle or L-norvaline (e).
A significant reduction in the area fraction (%) of Iba1 immunoreactivity
in the hippocampal CA3 areas of the 3×Tg mice treated with L-norvaline
(f).*p<0.05versus controls using a one-way ANOVA with Tukeyspost
hoc test, n=20, 4 mice per group
1046 B. Polis et al.
p<0.0001),followedbyTukeys multiple comparison
tests between the experimental groups, revealed signifi-
cantly increased (p< 0.001) glial somatic volumes in the
3×Tg animals treated with L-norvaline (Fig. 8g). The
analysis did not reveal any significant changes in the
number and surface area of GFAP-positive objects in the
non-Tg mice following the treatment.
In addition, we measured the astrocyte density within the
hilus. According to the literature, the average surface area of
an astrocyte is about 250 μm
2
[63]. To count the cells, we
filtered out all GFAP-positive objects less than 70 and more
than 500 μm
2
. The results revealed no significant differences
(p= 0.55) in the density of GFAP-positive astrocytes between
the control and experimental groups (Fig. 8h).
Fig. 7 Quantitative
characterization of microglial
morphology in the CA3
hippocampal area with S=
0.2 mm
2
. Five sections per mice
were included in the analysis. (a)
IOD, (b) Ferets diameter, (c) cell
surface area, and (d) sphericity
index. A frequency histogram of
all microglial cells sampled in the
CA3 area with S=0.2mm
2
. Data
are presented via box andwhisker
graphs. The boxes extend from
the25thto75thpercentiles.The
line in the middle of the box is
plotted at the median. The
whiskers denote the smallest and
largest values. (e) The frequency
distribution analysis of the soma
area showed a shift from large to
smaller cell body sizes after the
treatment, and (f) the distribution
analysis of circularity indicated
that control cells were more likely
to possess a more irregular shape
(i.e., to have a lower sphericity
index)
L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine Model of AlzheimersDisease 1047
ARGI Was Distinctly Expressed in Areas
with Pronounced Amyloidosis, and the L-Norvaline
Treatment Effectively Reduced ARGI Expression
We examined ARGI protein spatial expression within the hip-
pocampi of the 3×Tg mice and its spatial relationship with Aβ
deposition using immunocytochemistry (Fig. 9a, b). ARGI
preferentially accumulated in the CA1CA4 areas of the hip-
pocampus (Fig. 9cf) and was mainly detected inside the cells
(Fig. 9g, h). The L-norvaline treatment led to a significant
(p= 0.0073) reduction (more than 2-fold) in the overall
ARGI-positive cell surface area (Fig. 9i) and IOD (p=
0.0004) (Fig. 9j), which reflected the decrease in the levels
of the ARGI protein in the brains of the treated animals.
Fig. 8 The L-norvaline did not lead to changes in GFAP-positive
astroglial density in the 3×Tg mice but led to a significant increase in
the volume of somata of astrocytes. Representative hippocampal bright-
field × 20 micrographs of the hippocampi from the 3×Tg control (a) and
L-norvaline-treated mice (b). The hilus area of the control (c) and treated
(d) 3×Tg mice. Representative × 40 images of astrocytes in theCA4 area
of the control (e) and treated (f) mice. (g) Bar charts show a significant
increase in the area fraction (%) of GFAP-positive cells in the
hippocampal hilus area of the 3×Tg mice treated with L-norvaline. (h)
GFAP-positive cell density in the hilus. n= 20, 4 mice per group.
***p<0.001
1048 B. Polis et al.
In the non-Tg mice, hippocampal pyramidal neurons
expressed a significantly lower level of the ARGI protein as
compared with that in hippocampal pyramidal neurons of the
3×Tg mice (Supplementary Fig. S5). No differences in the
level of immunopositivity were detected in the treated and
control groups using the same software and the same thresh-
old. Of note, several ARGI-positive star-shaped cells with
dark nuclei were seen in the hippocampus (Fig. S5 insets).
Mitochondrial ARGII Was Expressed in the Cytosol
of CA2CA3 Hippocampal Cells
We detected augmented ARGII immunopositivity of cells in
the CA2 hippocampal area of the 3×Tg mice (Fig. 10ad) but
not in that of the non-Tg animals (Supplementary Fig. S6).
The same pattern of staining was observed in the treated and
control groups. At × 40 and higher magnification, mitochon-
dria were observed, most clearly in the dentate gyrus, without
significant differences in the numbers and intensity between
the groups (Fig. 10e, f). We did not detect any significant
effect of the treatment on the BARGII-positive objects surface
area^(Fig. 10g). However, the IOD index was significantly
reduced (p< 0.05) in the L-norvaline-treated 3×Tg mice
(Fig. 10h).
Discussion
In this study, we treated 3×Tg mice with a nonproteinogenic
unbranched-chain amino acid L-norvaline, which possesses
arginase-inhibiting properties [64]. The results indicated that
L-norvaline was well tolerated by the mice and that long-term
treatment with L-norvaline did not lead to detectable behav-
ioral changes or weight loss in WT mice. The 3×Tg mice in
the L-norvaline-treated group not only exhibited improved
spatial learning and memory but also a reduction in the cere-
bral Aβburden, together with a substantial decrease of Aβ
toxic fibrillary and oligomeric species. Moreover, microglial
density was decreased in the treated 3×Tg mice, and the acti-
vated microglial phenotype shifted to a resting phenotype.
Blocking L-arginine depletion and reversal of its depriva-
tion, thereby halting memory loss and reducing other AD
symptoms, has been explored previously. For example, in a
murine model of AD, Kan et al. [17] used DFMO, a relatively
toxic, mostly irreversible, inhibitor of ornithine decarboxylase
and putrescine in AD. In the present study, we used L-
norvaline to inhibit arginase. L-Norvaline is structurally sim-
ilar to ornithine and acts via a negative feedback inhibition
mechanism. Accordingly, we did not administer polyamines
to avoid the undesirable side effects of DFMO. Furthermore,
we dissolved L-norvaline in water, and the animals received
the agent via water in their home cages. This mode of admin-
istration is much less invasive than forced gavage feeding.
Furthermore, we utilized a universally recognized animal
model of AD.
The mice in the L-norvaline-treated group showed a very
strong phenotype in 2 different behavioral paradigms, which
supports the effect of the treatment on short- and long-term
spatial memory acquisition. Moreover, we found a significant
reduction in the level of 6E10 positivity in the cortices of the
3×Tg mice treated with L-norvaline. Also, we found evidence
that the hippocampi of the 3×Tg-treated mice at age 6
7 months harbored significantly lower levels of Aβoligomeric
and fibrillary conformers as compared with hippocampi of the
untreated animals. A previous study demonstrated that soluble
low-weight Aβoligomers and intermediate aggregates were
the most neurotoxic forms of Aβand that these triggered
synaptic dysfunction and neuronal damage, which manifested
in behavioral attributes of AD, such as memory deficits [65].
Furthermore, in the present study, we detected a substantial
increase in dendritic spine density in the hippocampi of the
3×Tg mice treated with L-norvaline. In addition, we evi-
denced a significant rise in the levels of pre- and postsynaptic
proteins in the experimental group. We attributed this rise to
an increase in spine density.
In this study, ARGI expression was significantly increased in
areas with pronounced Aβdeposition. There is a consensus in
the literature that various stimuli can induce the expression of the
2isoformsofarginase[66]. In an AD murine model, ARGI was
localized not only in brain cells but also distributed in extracel-
lular spaces [17]. In the hippocampus, ARGI displayed a spatial
correlation with Aβdeposition and Iba1 expression [17]. The
activation of ARGII was shown to be associated with transloca-
tion from the mitochondria to the cytosol [22].
The immunohistochemistry approaches applied herein re-
vealed significantly increased ARGI protein levels in the
CA2CA4 hippocampal areas of the 3×Tg mice, where we
detected the most distinct intracellular Aβdeposition. These
findings are in accordance with those in the literature [17]. Of
note, in the present study, ARGI staining was primarily local-
ized inside the cells. The discord between these findings and
those of the earlier study may be attributed to differences in
the murine models of AD. Furthermore, the L-norvaline treat-
ment significantly reduced the level of ARGI expression in the
hippocampus.
Additionally, we observed augmented ARGII
immunopositivity in cells in the CA2 area of the 3×Tg mice.
This finding pointed to the translocation of ARGII from the
mitochondria to the cytoplasm, presumably by the same
mechanism described previously [22]. However, we did not
detect between-group differences in the relative surface area
of ARGII-positive objects located in the CA2 area, although
the optical density of the objects was significantly reduced in
the treated mice.
Chronic neuroinflammation is a prominent feature of AD
pathogenesis [67]. Microglial proliferation is increased in AD
L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine Model of AlzheimersDisease 1049
Fig. 9 L-Norvaline significantly decreased ARGI immunopositivity in
the 3×Tg mice. Representative hippocampal bright-field × 20
micrographs of the control (a) and L-norvaline-treated 3×Tg mice (b).
CA4 area of the control (c) and treated (d) Tg mice. CA3 area of the
control (e) and treated (f) mice, with × 40 insets (g) and (h), respectively.
(i) The boxplots show a significant reduction in the ARGI-positive cell
surface area in the CA3 areas (S=0.2 mm
2
)and(j) the IOD of ARGI-
positive cells in the CA3 area in the 3×Tg mice treated with L-norvaline.
Studentsttest (n= 24, with 4 mice pergroup), **p< 0.01, ***p<0.001
1050 B. Polis et al.
patients, as well as in AD animal models, and it is positively
correlated with disease severity [68]. It has been hypothesized
that activated microglia cause synaptic and wiring dysfunction
by pruning synaptic connections [69] and that targeting the
microgliasynapse pathways might prevent AD symptoms
[70]. In the present study, we observed an increase of more
than 30% in the density of Iba1 immunopositive cells in the
CA3 area of the hippocampi of 7-month-old 3×Tg mice as
compared with that in age- and sex-matched non-Tg controls.
This finding is in accordance with the literature [71].
Moreover, we confirmed that cognitive function improve-
ments in the mice treated with L-norvaline were associated
with a significant reduction in hippocampal microgliosis.
In parallel with microgliosis and astrogliosis [72], the de-
velopment of AD is associated with astrodegeneration [73].
Astrocytes are essential for neurotransmission, and
Fig. 10 Representative hippocampal CA2CA3 bright-field × 20
micrographs of the control (a) and L-norvaline-treated 3×Tg mice (b).
CA2 × 40 insets of the control (c) and treated (d) mice. (e) A
representative × 20 image of the dentate gyrus, with mitochondria
stained with ARGII antibody (f) and a × 40 zoomed-in inset clearly
showing a mitochondrion. (g) ARGII-positive cell surface area in the
CA2 (S=0.2 mm
2
). (h) The IOD of ARGII-positive cells in the CA2
area. Studentsttest, *p<0.05,n= 12, with 4 mice per group
L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine Model of AlzheimersDisease 1051
astrodegeneration leads to synaptic strength deficits and con-
tributes to the decline in the number of active synapses ob-
served in the early stages of AD [74]. Atrophy of astrocytes
was previously detected in the hippocampi and cortices of
3×Tg mice [75]. Astrocytic atrophy was characterized by a
reduction in cell volume and a decrease in the number of
processes [76]. Interestingly, the density of astrocytes
remained relatively stable in the hippocampus and cortices of
3×Tg mice for more than 1 year and was comparable to that
observed in non-Tg controls [77]. Olabarria et al. [78]reported
an apparent reduction in the astrocytic somatic volume in the
dentate gyrus of 6-month-old 3×Tg mice and a significant
decrease at 12 months. The results of our study, which re-
vealed astrodegeneration in areas with intense Aβdeposition,
are in accordance with the data presented in the literature.
Moreover, the L-norvaline treatment led to a significant
(75%) increase in the GFAP-positive surface area in the hilus.
L-Norvaline possesses multiple mechanisms of activity. Ming
et al. [33] demonstrated that L-norvaline inhibited endothelial
inflammation induced by tumor necrosis factor-αindependently
of NOS and arginase activity. The authors utilized RNA interfer-
ence technology to prove that the anti-inflammatory properties of
L-norvaline were attributed to its ability to inhibit the S6K1
kinase, which is involved in the regulation of protein synthesis.
Subsequently, Caccamo et al. provided evidence that the activity
of S6K1 was higher in 3×Tg mice as compared with that in WT
mice [79]. The authors also showed that removing 1 copy of the
S6K1 gene from the 3×Tg mice was sufficient to rescue long-
term potentiation (LTP) deficits. They speculated that the im-
provement in LTP corresponded to observed changes in the ex-
pression of the synaptic marker synaptophysin, which was re-
duced in 3×Tg mice but increased dramatically in S6K1-
deficient mice. In the present study, the levels of synaptophysin
in the hippocampi of the 3×Tg mice treated with L-norvaline
increased by 50%. This finding agrees with the data presented
in the aforementioned literature.
The observation of a significant (p=0.038) reduction by
53% in RAC-αprotein-serine/threonine kinase (Akt1) levels
in the treated group is noteworthy (Supplementary Table 2).
This kinase is a key modulator of the AKTmTOR signaling
pathway and regulates many processes, including metabolism,
proliferation, and cell survival [80]. As it is located upstream
of mTOR and S6K1, downregulation of Akt1 can negatively
influence S6K1 activity. Upregulation of mTOR signaling
pathway is known to play a central role in major pathological
processes of AD [81]. Consequently, inhibition of mTOR is a
novel therapeutic target for AD [82].
Overall, our results provide compelling evidence that L-
norvaline is a multifunctional agent and a potential drug for
the treatment of AD. It appears to be a promising candidate for
clinical development. The functional effects of L-norvaline
are diverse, and much work remains to be done to disclose
its full potential and precise mechanisms of action.
Acknowledgments 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. We gratefully acknowledge
Mr. Basem Hijazi for his valuable advice in the statistical analysis and Dr.
Zohar Gavish for his help with immunohistochemistry.
Required Author Forms Disclosure forms provided by the authors are
available with the online version of this article.
Author Contributions Baruh Polis and Abraham Samson designed the
experiments. Baruh Polis and Kolluru Devi Dutt Srikanth performed the
experiments and analyzed the data. Hava Gil-Henn advised and super-
vised the experiments. Evan Elliott supervised parts of the experiments.
Baruh Polis wrote the manuscript, and Abraham Samson, Hava Gil-
Henn, and Evan Elliott edited the manuscript.
Compliance with Ethical Standards
All animal housing and procedures were performed in compliance with
the guidelines established by the Israeli Ministry of Healths Council for
Experimentation on Animals and with Bar-Ilan University guidelines for
the use and care of laboratory animals in research. The experimental
protocol was approved by the ethics committee for animal experiments
of Bar-Ilan University (Permit Number: 82-102017).
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
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... Moreover, plant-derived compounds such as polyphenols "chlorogenic acid" [38], glycoside derivative "Piceatannol-3′-O-β-D-glucopyranoside" [39], flavonoids "(2S)-5,2′5′-trihydroxy-7,8-dimethoxy" [40], and cinnamide derivatives [41] are also investigated for developing potent arginase inhibitor. In addition, arginase inhibition using L-norvaline reverses neurodegeneration in a murine model of Alzheimer's disease [42]. However, one potential concern is the lack of selectivity of the drugs for ARG1 and ARG2. ...
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... It was also 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 [209]. As a result, the bioavailability of arginine is a regulatory factor for the synthesis of several proteins essential for neuronal survival. ...
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Amyloid β(Aβ) accumulates in the brains of patients with Alzheimer’s disease. Based on the amyloid hypothesis and the oligomeric hypothesis that evolved from it, Aβ-aggregated substances are considered to be the etiology. In addition, gene mutations found in familial Alzheimer’s disease (FAD) indicate that increased Aβ production and aggregation are at risk for AD. Immunotherapy for Aβ and suppression of aggregation are under development as anti-AD therapeutic agents.
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Microglia, resident macrophages that act as the brain’s innate immune cells, play a key role in initiating a defense response to the infection or neuroinflammation of the host. Once a broad spectrum of dangers is confronted, microglia get triggered and transform their role against immune stimuli. Recent studies have shown that remarkable metabolic changes present in activated microglia affect their immune function. Given that the important role of microglia in the progression of neurodegeneration is widely recognized, it is crucial to know whether metabolic reprogramming of microglia also presents in neurodegeneration and how this may influence their role in neurodegeneration progression. This paper provides an overview of the metabolic reprogramming of microglia, the major pathways involved in recent advances in five major neurodegenerative diseases of aging (NDAs), including Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD), etc. And then we elucidated their impacts on the disease progression of neurodegeneration. Furthermore, growing evidence suggests that microbiota-derived metabolites, including acetate, N⁶-carboxymethyllysine (CML), and isoamylamine (IAA), regulate metabolic pathways and functions of microglia, and play a crucial role in cellular homeostasis. We shed light on this topic and concluded these metabolites are potential therapeutic targets for NDAs.
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Background “Subjective cognitive decline plus” (SCD plus) increases the risk of Alzheimer's disease (AD), and this may be an early stage of AD that precedes amnestic mild cognitive impairment (aMCI). We examined alterations of serum metabolites and metabolic pathways in SCD plus subjects using ¹H-magnetic resonance spectroscopy (¹H NMR) metabolomics. Methods Serum samples from subjects with SCD plus (n = 32), aMCI (n = 33), and elderly controls (ECs, n = 41) were analyzed using an 800 MHz NMR spectrometer. Multivariate analyses were used to identify serum metabolites, and two machine-learning methods were used to evaluate the diagnostic power of these metabolites in distinguishing SCD plus subjects, aMCI subjects, and ECs. Results Eight metabolites differentiated SCD plus from EC subjects. A random forest (RF) model discriminated SCD plus from EC subjects with an accuracy of 0.883 and an area under the receiver operating characteristic curve (AUROC) of 0.951. A support vector machine (SVM) model had an accuracy of 0.857 and an AUROC of 0.946. Nine other metabolites distinguished SCD plus from aMCI subjects. An RF model discriminated SCD plus from aMCI subjects (accuracy: 0.975, AUROC: 0.998) and an SVM model also discriminated these two groups (accuracy: 0.955, AUROC: 0.991). Disturbances of glucose and branched-chain amino acid (BCAA) metabolism were the most striking features of SCD plus subjects, and valine was positively correlated with Auditory Verbal Learning Test delayed-recall score. Conclusions Serum metabolomics using ¹H NMR provided noninvasive identification of perturbations in glucose and BCAA metabolism in subjects with SCD plus.
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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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Background: There are few tissue-based biomarkers that can accurately predict prostate cancer (PCa) progression and aggressiveness. We sought to evaluate the clinical utility of prostate and breast overexpressed 1 (PBOV1) as a potential PCa biomarker. Methods: Patient tumor samples were designated by Grade Groups using the 2014 Gleason grading system. Primary radical prostatectomy tumors were obtained from 48 patients and evaluated for PBOV1 levels using Western blot analysis in matched cancer and benign cancer-adjacent regions. Immunohistochemical evaluation of PBOV1 was subsequently performed in 80 cancer and 80 benign cancer-adjacent patient samples across two tissue microarrays (TMAs) to verify protein levels in epithelial tissue and to assess correlation between PBOV1 proteins and nuclear architectural changes in PCa cells. Digital histomorphometric analysis was used to track 22 parameters that characterized nuclear changes in PBOV1-stained cells. Using a training and test set for validation, multivariate logistic regression (MLR) models were used to identify significant nuclear parameters that distinguish Grade Group 3 and above PCa from Grade Group 1 and 2 PCa regions. Results: PBOV1 protein levels were increased in tumors from Grade Group 3 and above (GS 4 + 3 and ≥ 8) regions versus Grade Groups 1 and 2 (GS 3 + 3 and 3 + 4) regions (P = 0.005) as assessed by densitometry of immunoblots. Additionally, by immunoblotting, PBOV1 protein levels differed significantly between Grade Group 2 (GS 3 + 4) and Grade Group 3 (GS 4 + 3) PCa samples (P = 0.028). In the immunohistochemical analysis, measures of PBOV1 staining intensity strongly correlated with nuclear alterations in cancer cells. An MLR model retaining eight parameters describing PBOV1 staining intensity and nuclear architecture discriminated Grade Group 3 and above PCa from Grade Group 1 and 2 PCa and benign cancer-adjacent regions with a ROC-AUC of 0.90 and 0.80, respectively, in training and test sets. Conclusions: Our study demonstrates that the PBOV1 protein could be used to discriminate Grade Group 3 and above PCa. Additionally, the PBOV1 protein could be involved in modulating changes to the nuclear architecture of PCa cells. Confirmatory studies are warranted in an independent population for further validation.
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Progress toward the development of efficacious therapies for Alzheimer’s disease (AD) is halted by a lack of understanding early underlying pathological mechanisms. Systems biology encompasses several techniques including genomics, epigenomics, transcriptomics, proteomics, and metabolomics. Metabolomics is the newest omics platform that offers great potential for the diagnosis and prognosis of neurodegenerative diseases as an individual’s metabolome reflects alterations in genetic, transcript, and protein profiles and influences from the environment. Advancements in the field of metabolomics have demonstrated the complexity of dynamic changes associated with AD progression underscoring challenges with the development of efficacious therapeutic interventions. Defining systems-level alterations in AD could provide insights into disease mechanisms, reveal sex-specific changes, advance the development of biomarker panels, and aid in monitoring therapeutic efficacy, which should advance individualized medicine. Since metabolic pathways are largely conserved between species, metabolomics could improve the translation of preclinical research conducted in animal models of AD into humans. A summary of recent developments in the application of metabolomics to advance the AD field is provided below.
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Over the past few decades, research on Alzheimer’s disease (AD) has focused on pathomechanisms linked to two of the major pathological hallmarks of extracellular deposition of beta-amyloid peptides and intra-neuronal formation of neurofibrils. Recently, a third disease component, the neuroinflammatory reaction mediated by cerebral innate immune cells, has entered the spotlight, prompted by findings from genetic, pre-clinical, and clinical studies. Various proteins that arise during neurodegeneration, including beta-amyloid, tau, heat shock proteins, and chromogranin, among others, act as danger-associated molecular patterns, that—upon engagement of pattern recognition receptors—induce inflammatory signaling pathways and ultimately lead to the production and release of immune mediators. These may have beneficial effects but ultimately compromise neuronal function and cause cell death. The current review, assembled by participants of the Chiclana Summer School on Neuroinflammation 2016, provides an overview of our current understanding of AD-related immune processes. We describe the principal cellular and molecular players in inflammation as they pertain to AD, examine modifying factors, and discuss potential future therapeutic targets.
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Brain aging is central to late-onset Alzheimer's disease (LOAD), although the mechanisms by which it occurs at protein or cellular levels are not fully understood. Alzheimer's disease is the most common proteopathy and is characterized by two unique pathologies: senile plaques and neurofibrillary tangles, the former accumulating earlier than the latter. Aging alters the proteostasis of amyloid-β peptides and microtubule-associated protein tau, which are regulated in both autonomous and non-autonomous manners. Microglia, the resident phagocytes of the central nervous system, play a major role in the non-autonomous clearance of protein aggregates. Their function is significantly altered by aging and neurodegeneration. This is genetically supported by the association of microglia-specific genes, TREM2 and CD33, and late onset Alzheimer's disease. Here, we propose that the functional characterization of microglia, and their contribution to proteopathy, will lead to a new therapeutic direction in Alzheimer's disease research.
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Body-wide changes in bioenergetics, i.e., energy metabolism, occur in normal aging and disturbed bioenergetics may be an important contributing mechanism underlying late-onset Alzheimer’s disease (LOAD). We investigated the bioenergetic profiles of fibroblasts from LOAD patients and healthy controls, as a function of age and disease. LOAD cells exhibited an impaired mitochondrial metabolic potential and an abnormal redox potential, associated with reduced nicotinamide adenine dinucleotide metabolism and altered citric acid cycle activity, but not with disease-specific changes in mitochondrial mass, production of reactive oxygen species, transmembrane instability, or DNA deletions. LOAD fibroblasts demonstrated a shift in energy production to glycolysis, despite an inability to increase glucose uptake in response to IGF-1. The increase of glycolysis and the abnormal mitochondrial metabolic potential in LOAD appeared to be inherent, as they were disease- and not age-specific. Our findings support the hypothesis that impairment in multiple interacting components of bioenergetic metabolism may be a key mechanism contributing to the risk and pathophysiology of LOAD.
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The mitochondrial arginase type II (Arg-II) has been shown to interact with ribosomal protein S6 kinase 1 (S6K1) and mitochondrial p66Shc and to promote cell senescence, apoptosis and inflammation under pathological conditions. However, the impact of Arg-II on organismal lifespan is not known. In this study, we demonstrate a significant lifespan extension in mice with Arg-II gene deficiency (Arg-II−/−) as compared to wild type (WT) control animals. This effect is more pronounced in the females than in the males. The gender difference is associated with higher Arg-II expression levels in the females than in the males in skin and heart at both young and old age. Ablation of Arg-II gene significantly reduces the aging marker p16INK4a levels in these tissues of old female mice, whereas in the male mice this effect of Arg-II deficiency is weaker. In line with this observation, age-associated increases in S6K1 signaling and p66Shc levels in heart are significantly attenuated in the female Arg-II−/− mice. In the male mice, only p66Shc but not S6K1 signaling is reduced. In summary, our study demonstrates that Arg-II may play an important role in the acceleration of aging in mice. Genetic disruption of Arg-II in mouse extends lifespan predominantly in females, which relates to inhibition of S6K1, p66Shc, and p16INK4a. Thus, Arg-II may represent a promising target to decelerate aging process and extend lifespan as well as to treat age-related diseases.
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It is known that microglia morphology and function are closely related, but only few studies have objectively described different morphological subtypes. To address this issue, morphological parameters of microglial cells were analyzed in a rat model of aseptic neuroinflammation. After the injection of a single dose of the enzyme neuraminidase (NA) within the lateral ventricle (LV) an acute inflammatory process occurs. Sections from NA-injected animals and sham controls were immunolabeled with the microglial marker IBA1, which highlights ramifications and features of the cell shape. Using images obtained by section scanning, individual microglial cells were sampled from various regions (septofimbrial nucleus, hippocampus and hypothalamus) at different times post-injection (2, 4 and 12 h). Each cell yielded a set of 15 morphological parameters by means of image analysis software. Five initial parameters (including fractal measures) were statistically different in cells from NA-injected rats (most of them IL-1β positive, i.e., M1-state) compared to those from control animals (none of them IL-1β positive, i.e., surveillant state). However, additional multimodal parameters were revealed more suitable for hierarchical cluster analysis (HCA). This method pointed out the classification of microglia population in four clusters. Furthermore, a linear discriminant analysis (LDA) suggested three specific parameters to objectively classify any microglia by a decision tree. In addition, a principal components analysis (PCA) revealed two extra valuable variables that allowed to further classifying microglia in a total of eight sub-clusters or types. The spatio-temporal distribution of these different morphotypes in our rat inflammation model allowed to relate specific morphotypes with microglial activation status and brain location. An objective method for microglia classification based on morphological parameters is proposed. Main pointsMicroglia undergo a quantifiable morphological change upon neuraminidase induced inflammation. Hierarchical cluster and principal components analysis allow morphological classification of microglia. Brain location of microglia is a relevant factor.
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Using the most well-studied behavioral analyses of animal subjects to promote a better understanding of the effects of disease and the effects of new therapeutic treatments on human cognition, Methods of Behavior Analysis in Neuroscience provides a reference manual for molecular and cellular research scientists in both academia and the pharmaceutical industry. The materials presented draw from the scholarly works of recognized experts in several fields of cognitive and behavioral neuroscience. Each contributor describes the most frequently used and most widely accepted version of the model, and each chapter includes: (1) a well-referenced introduction that covers the theory behind and the utility of the model; (2) a detailed and step-by-step methodology; and (3) an approach to the interpretation of the data presented. Many chapters also provide examples of actual experiments that use the method. This text eliminates the guesswork from the process of designing the methodology for many of the animal behavioral models that are used to study brain disorders, drug abuse, toxicology, and cognitive drug development. It will also significantly reduce the time spent assessing literature and developing models for studying animal subjects. Overall, Methods of Behavioral Analysis in Neuroscience is an invaluable reference source for the professional who seeks to comprehend the effects of disease on human cognition.
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In recent years, neuropathological and epidemiological studies have indicated an association between Alzheimer's disease (AD) and several cardiovascular risk factors. In this study, the cardio-protective effects of folic acid (FA) in early stage AD was elucidated using a triple-transgenic (3xTg) Alzheimer's mouse model. Eleven-month-old C57BL/6 mice and 3xTg mice were assigned to five groups. During the four-month treatment period, the low-FA treatment group received FA through their diet, and the high-FA treatment groups received 3 mg/dl folate in drinking water and were also gastric-fed 1.2 mg/kg folate every day. In the C57B1/6J mice, treatment with high doses of FA (HFA) did not show any considerable effect compared to the control group or the low-dose dietary FA treatment group. However, Alzheimer's mice treated with HFA showed enhanced cardio-protection. Western blot analysis revealed that FA treatment restored SIRT1 expression, which was suppressed in 3xTg mice, through enhanced AMPK expression. FA significantly enhanced the IGF1 receptor survival mechanism in the hearts of the 3xTg mice and suppressed the expression-intrinsic and extrinsic apoptosis-associated proteins. The results suggest that FA intake may significantly alleviate cellular pathological events in the heart associated with AD.