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Pinealon Increases Cell Viability by Suppression of Free Radical Levels and Activating Proliferative Processes


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The synthetic tripeptide pinealon (Glu-Asp-Arg) demonstrates dose-dependent restriction of reactive oxygen species (ROS) accumulation in cerebellar granule cells, neutrophils, and pheochromocytoma (PC12) cells, induced by oxidative stress stimulated by receptor-dependent or -independent processes. At the same time, pinealon decreases necrotic cell death measured by the propidium iodide test. The protective effect of pinealon is accompanied with a delayed time course of ERK 1/2 activation and modification of the cell cycle. Because restriction of ROS accumulation and cell mortality is saturated at lower concentrations, whereas cell cycle modulation continues at higher concentrations of pinealon, one can conclude that besides its known antioxidant activity, pinealon is able to interact directly with the cell genome.
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Pinealon Increases Cell Viability by Suppression of Free
Radical Levels and Activating Proliferative Processes
V. Khavinson,
Y. Ribakova,
K. Kulebiakin,
E. Vladychenskaya,
L. Kozina,
A. Arutjunyan,
and A. Boldyrev
The synthetic tripeptide pinealon (Glu-Asp-Arg) demonstrates dose-dependent restriction of reactive oxygen
species (ROS) accumulation in cerebellar granule cells, neutrophils, and pheochromocytoma (PC12) cells, in-
duced by oxidative stress stimulated by receptor-dependent or -independent processes. At the same time,
pinealon decreases necrotic cell death measured by the propidium iodide test. The protective effect of pinealon is
accompanied with a delayed time course of ERK 1/2 activation and modification of the cell cycle. Because
restriction of ROS accumulation and cell mortality is saturated at lower concentrations, whereas cell cycle
modulation continues at higher concentrations of pinealon, one can conclude that besides its known antioxidant
activity, pinealon is able to interact directly with the cell genome.
Under the influence of environmental factors, emo-
tional stress, and/or progression of age pathologies, the
regulation of body functions is disturbed. The role of regu-
latory neuropeptides in the formation of the adaptive re-
sponse of an organism to stress and the disturbance of
homeostasis is now discussed broadly.
The endogenous
components of living cells, peptide bioregulators, demon-
strate diverse biological effects; they are effective at low
concentrations and show no side effects.
However, their
therapeutic use is limited by their permeability through the
blood–brain barrier, relatively rapid metabolization, and ef-
fect on the immune system. Short synthetic analogs of neu-
ropeptides that preserve their specific activity do not have
these restrictions. Among these short peptides, which are
potential modulators of regulatory functions, is the tripep-
tide pinealon (Glu-Asp-Arg), which has been synthesized
after analysis of amino acid composition of bovine brain
The Glu-Asp-Arg sequence is the most common
motif in a complex peptide from the cerebral cortex called
cortexin that demonstrates neuroprotective properties.
compound was synthesized recently and was found to be
capable of stimulating neuronal regeneration
and protecting
brain neurons from hypoxia.
The aim of present study was
to characterize the effect of pinealon on cell metabolism
under oxidative stress conditions in vitro.
Materials and Methods
Three different kinds of cell preparations were used in
these experiments: (1) Granule cells isolated from cerebel-
lums of 10- to 12-day-old rats, (2) neutrophils isolated from
peripheral blood of intact adult rats, and (3) a commercially
available culture of pheochromocytoma (PC12) cells. Cere-
bellar granule cells represent a standard model for the study
of oxidative stress,
including their induction of the intra-
cellular accumulation of reactive oxygen species (ROS).
contrast, when neutrophils are activated, they generate ROS
in the surrounding medium.
We have used these two
models to estimate the ability of pinealon to diminish the
levels of free radicals both inside and outside the cells. To
estimate the effect of pinealon on cell cycle division, PC12
cells were used because they are characterized precisely in
the literature and the role of ROS in regulation of the PC12
cell cycle is well known.
Experiments using Wistar rats were carried out according
to international rules of working with laboratory animals
Dissociated cerebellar granule cells
Cerebellar granule cells were derived from 7- to 10-day-
old pups. A suspension of surviving neurons was obtained
after treatment of cerebellar slices by dispase, followed by a
wash with standard Tyrode solution (148 mM NaCl, 5 mM
Saint-Petersburg Institute of Bioregulation and Gerontology, and
Research Center of Neurology Russian Academy of Medical Sciences,
St. Petersberg, Russian Federation.
Department of Biochemistry, School of Biology, M.V. Lomonosov Moscow State University, Moscow, Russian Federation.
Volume 14, Number 5, 2011
ªMary Ann Liebert, Inc.
DOI: 10.1089/rej.2011.1172
KCl, mM CaCl
, 1 mM MgCl
, 10 mM glucose, 10 mM
HEPES, pH 7.4) and filtration through a 53-mm nylon filter.
Oxidative stress was induced in the presence or absence of
pinealon by a 30-min exposure of the cells to 100 nM ouabain
or 500 mM homocysteine (HC). Both compounds are known
to be able to induce oxidative stress in cerebellum granule
cells by interacting with specific receptors on the neuronal
membrane. Ouabain affects sodium/potassium–adenosine
triphosphatase (Na/K-ATPase) of the outer cell membrane.
HC is a congener of N-methyl-D-aspartate (NMDA), and it
acts via the glutamate receptor of the NMDA class.
In both
cases, an increase in intracellular ROS levels and activation
of the key enzyme of the mitogen-activated protein kinase
(MAPK) cascade ERK1/2 kinase (extracellular regulated
kinase, isoforms 1 and 2) is observed.
Each ligand was
used in a concentration sufficient to induce oxidative stress
via intracellular accumulation of ROS.
The cell suspen-
sion was incubated with ouabain or HC (in the presence or
absence of pinealon), and then flow cytometry analysis was
Primary culture of cerebellar granule cells
Cerebellums from 7- to 10-day-old rats were washed with
cold Hanks’ solution (PanEko, Russia) and incubated 20 min
with 0.05% trypsin solution (PanEko, Russia). The neurons
were then washed and cultivated for 11 days in Neuroba-
-A cultivation medium, with 2% Supplement B-27 used
as a serum substitute (Invitrogen, USA), GlutaMax (Invitro-
gen, USA), 50 U/mL penicillin, 50 U/mL streptomycin, and
20 mM KCl (at 5% CO
and 378C). Before the following ex-
periment, cells were removed from the substrate with tryp-
sin-EDTA (PanEko, Russia) and exposed to 500 mM HC for
5–30 min in the presence or absence of pinealon. Then cell
suspension was used in western blotting experiments.
Experiments with neutrophils
Before the experiment, adult rats weighing 200–250 grams
were treated with chloral hydrate (500 mg/kg intravenous-
ly). A blood sample was collected from the jugular vein in a
heparin-containing syringe (Spofa, Czech Republic, 50 U/mL
blood). Intact neutrophils were obtained by centrifuging the
blood samples in MonoPoly medium (ICN Biomedicals,
USA) and suspending them in Hanks’ solution. Neutrophils
were activated by adding of 2 mg/mL zymosan to the in-
cubation medium.
To obtain a suspension of neutrophils activated in vivo
(peritoneally induced neutrophils), animals were treated
with 500 mL of zymosan suspension (4.5 mg/mL of Hanks’
solution, intraperitoneally) to induce a local inflammatory
area. Activated neutrophils were isolated from peritoneal
fluid, and a cell suspension with a purity of 95–98% was
obtained. The cells were sedimented by centrifugation, re-
suspended in 1 mL of Hanks’ solution, and stored at 378C for
up to 3 hr.
The functional activity of neutrophils was evaluated by
production of ROS, measured as a chemiluminescence re-
sponse in the presence of 1 mM luminol (Sigma-Aldrich,
Germany) on a SmartLum 5773 chemiluminometer (St.-
Petersburg, Russia). Neutrophils were activated using HC
(Sigma-Aldrich, Germany). In preliminary experiments, we
tested the effect of HC on the chemiluminescence of intact
cells in the range of 5–30 min and found no effect in this time
interval. Thus, for characterization of HC action, we used
30 min of incubation.
PC-12 cell culture
Cells were cultured in 75-mm
flasks in RPMI-1640 me-
dium (PanEko, Russia) supplemented with 10% fetal calf
serum (FCS; PanEko, Russia), 0.2 mg/mL L-glutamine (Pa-
nEko, Russia), and 20 mg/mL gentamicin at 5% CO
378C. Cells were removed from the substrate with trypsin-
EDTA (PanEko, Russia).
Measuring the levels of free radicals
in cerebellar neurons
To determine the intracellular ROS levels, cerebellar gran-
ule cells were loaded with the fluorescent dye 2,7-dihlor-
odihydrofluoresceine-diacetate (DCF-DA; Molecular Probes,
USA) at a final concentration 100 mM.
Cells were removed
from the substrate, washed free of trypsin, and resuspended
in Hanks’ solution. They were then placed in 1.5-mL Eppen-
dorf tubes, allowed to rest for 30min, and incubated for
40 min with DCF-DA. After dye loading, the cells were ex-
posed for 20 min to 1 mM hydrogen peroxide (H
Germany) in the presence or absence of pinealon (Garmonika,
Russia) at concentrations noted in the legends to the figures.
For 1 min before the measurement, the samples were sup-
plemented with 10 mM propidium iodide (PI; Sigma, Ger-
many). Measurements were performed on BD FACSCalibur
flow cytometer (Becton, Dickinson, USA) after gating the cell
population corresponding in size to neuronal cells.
Western blotting
The activity of ERK 1/2, which is tightly connected with
cell viability,
was measured as a ratio between the phos-
phorylated form of ERK 1/2 and an endogenous control
(total actin level). Quantification was performed using
western blotting. Cells were washed with cold Hanks’ so-
lution and were lysed using RIPA buffer (Sigma-Aldrich,
USA). The lysates were then subjected to sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with
10% separating and 6% concentrating gels. A prestained
protein molecular weight marker (Fermentas, Germany) was
run in parallel. After SDS-PAGE, the proteins were electro-
blotted onto polyvinylydine difluoride (PVDF) membranes
(Thermo Scientific, USA). Detection of proteins was per-
formed as described by the manufacturers of the antibodies
anti-ERK 1/2 (Thr202/Tyr204) (1:1,000, Cell Signaling
Technology, USA), anti-phospho-ERK 1/2 (Thr202/Tyr204)
(1:1,000, Cell Signaling Tecnology, USA), and anti-pan-actin
(1:2,000 Cell Signaling Tecnology, USA). Secondary anti-
bodies were conjugated with horseradish peroxidase (HRP;
1:1,000, Cell Signaling Tecnology, USA). Membranes were
visualized via enhanced chemiluminescence (ECL) with Su-
perSignal West Femto solution (Thermo Scientific, USA).
Data were analyzed using TotalLab Quant software (TotalLab
Limited, UK).
Study of the PC12 cell cycle
To measure the parameters of the cell cycle, cells were
stained with PI.
Cells were removed from the substrate,
washed free of trypsin, and then resuspended in phosphate-
buffered saline (PBS; PanEko, Russia); fixation was per-
formed with 70% ethanol (at 208C overnight). The next day,
cells were washed free of ethanol with PBS, and staining was
performed in citrate buffer containing 100 mg/mL RNase A
from bovine pancreas (Sigma, Germany) (40 mg PI per 1
million cells, 40 min in the dark at 378C).
As shown earlier, incubation of neurons with a specific
inhibitor of Na/K-ATPase ouabain results in accumulation
of free radicals and activation of ERK 1/2 kinase,
induces switching on of the so-called early response genes.
Incubation of DCF-DA pre-loaded rat cerebellum granule
cells with 100 nM ouabain causes an increase in their fluo-
rescence, which is a sign of increasing intracellular levels of
free radicals (Fig. 1).
Addition of pinealon to the incubation medium at dif-
ferent concentrations leads to suppression of the level of
free radicals in a dose-dependent manner (Fig. 1), which
corresponds to the earlier described antioxidant action of
A 100 nM concentration of pinealon is enough to
prevent ouabain-induced ROS accumulation completely.
Thus, pinealon in a dose-dependent manner prevents an
increase in the ROS accumulation induced by ouabain.
When cerebellum cells were incubated with 500 mMHC
under the same conditions as above, the stationary ROS
level was increased by 92 5%, and this activation was
abolished in the simultaneous presence of HC and pinealon
(500 nM).
It is known that ROS can act as a secondary messenger in
the cells, triggering cascades of cellular signaling, in partic-
ular, that of MAPK pathway ERK 1/2.
Therefore, we
evaluated the effect of pinealon on the neuronal level of
ERK 1/2 which is activated by HC (Fig. 2). In control sam-
ples containing only HC, activation of ERK 1/2 is observed
within 2.5 min, whereas in the presence of HC and pinealon,
an increase in the level of active forms of ERK 1/2 occurs
20 min later. Thus, pinealon has a suppressing effect on ac-
tivation of ERK 1/2 in rat cerebellar granule cells exposed to
The ability of pinealon to suppress intracellular levels of
free radicals in neurons indicates its possible effects on the
other ROS-generating systems, especially those that generate
them for extracellular use. To this purpose, we investigated
the ability of pinealon to affect the respiratory burst of
neutrophils activated by two different modes—in vitro by
adding zymosan to the reaction medium with freshly
isolated neutrophils, and in vivo by measuring the chemilu-
minescence response of neutrophils isolated from rats after
24-hr of intraperitoneal administration of zymosan, as de-
scribed above. Intraperitoneal administration of zymosan to
intact animals promotes inflammation, which accumulates
intraperitoneally induced neutrophils. Thus, the cells pre-
pared after such a procedure are characteristic of high sta-
tionary levels of ROS production.
Figure 3 shows that under the conditions used both neu-
trophils activated by zymosan in vitro (Fig. 3A) and those
prepared from zymosan-treated animals (Fig. 3B) demon-
strate a similar ability to generate a chemiluminescent signal.
In both models of neutrophil activation, pinealon showed a
dose-dependent ability to inhibit accumulation of ROS, and
its effect was carried out in the same concentration range
(Fig. 3).
In both previous models of oxidative stress, we observed
no cell death. In further experiments on pheochromocytoma
PC12 cell cultures, we used a less physiological, but a
stronger, stress agent, H
, which induced not only ROS
accumulation but also cell death. Exposure of PC12 cells to
1mM H
for 20 min resulted in a five-fold increase in ROS
levels (Fig. 4). Preincubation of the cells with pinealon for
60 min decreased the accumulation of ROS induced by H
(Fig. 4, gray bars). At the same time, pinealon had no sig-
nificant impact on the level of ROS in intact cells (Fig. 4,
white bars).
FIG. 1. Pinealon restricts reactive oxygen species (ROS) accumulation in cerebellar granule cells induced by 30 min of
incubation of cells with 100 nM ouabain. (#) Significant difference in relation to control ( p<0.05; (*) significant difference in
relation to samples with ouabain ( p<0.05). CDF, 2,7-Dihlorodihydrofluoresceine.
Under these conditions, incubation of cells with 1 mM
resulted in a loss of about half of the entire cell pop-
ulation, while increasing concentrations of pinealon pro-
gressively increased the proportion of cells remaining alive,
despite the presence of H
in the medium (Fig. 5). Thus,
pinealon neutralizes effects of toxic compounds that stimu-
late the development of oxidative stress and protects cells
from necrotic death.
It is known that the intracellular ROS level affects distri-
bution of cells between different phases of cell cycle
; i.e.,
FIG. 2. Effect of pinealon (10 nM) on activation of ERK 1/2 in cerebellar granule cells in the presence of 500 mM homo-
cysteine (HC). (A) Results of analysis. (White bars) Activation of ERK 1/2 after incubation of cells in the presence of HC; (gray
bars) the same in the presence of HC and pinealon. (*) Significant difference between groups with p<0.05. (B) Western
blots; rows 1 and 2, samples with HC (correspond to white bars); rows 3 and 4, samples with HC and pinealon (correspond to
grey bars).
FIG. 3. Effect of pinealon on generation of free radicals by rat neutrophils activated in vitro (A, arrows correspond to adding
zymosan), and in vivo (B, arrows correspond to start of measurement).
ROS are capable of regulating mechanisms that promote the
cell cycle from one stage to another.
Taking into account
the ability of pinealon to reduce intracellular levels of free
radicals, we studied its possible effect on the advancement of
cells through the cell cycle. Using flow cytometry, we have
demonstrated that a 24-hr incubation of PC12 cells in pine-
alon-containing medium leads to a distinct redistribution of
cells through the phases of the cell cycle (Fig. 6A and B). This
effect has a clear dose dependence: An increase in the con-
centration of pinealon from 50 to 500 nM leads to a decrease
the number of cells in the G
phase and an increase in the
number of cells in the G
and S phases, indicating the
modulating effect of pinealon on the proliferative activity of
cells (Fig. 6C).
It was shown earlier that pinealon has a pronounced an-
tihypoxic effect on neurons that is explained by restriction of
excitoxicity of NMDA and by inhibition of ROS accumula-
Pinealon also stimulates the activity of the anti-
oxidative enzymes superoxide dismutase and glutathione
peroxidase in the rat brain under hypobaric hypoxia.
data point out the ability of pinealon to diminish oxidative
In this paper, we studied the effects of a synthetic tri-
peptide pinealon on properties of living cells. Using rat cer-
ebellar granule cells, we have demonstrated the ability of
pinealon to reduce stationary ROS levels caused by the ac-
tion of both receptor-dependent (ouabain, HC) and non-
receptor (H
) activators of oxidative stress. On surviving
cultures of neutrophils activated by zymosan, we also
showed the ability of pinealon to reduce ROS production
during the inflammatory response.
ROS take part in a number of physiological processes,
such as inflammation, apoptosis, neoplastic transformation,
and aging. In recent years, the amount of evidence regarding
the functioning of ROS as second messengers has in-
It is known that the redox potential of the cells
changes as they move through several phases of cell cycle.
For example, in the G
phase, ROS control the activity of
cyclin-dependent kinases (CDKs) and phosphorylation of the
retinoblastoma protein (pRb), thereby adjusting the entrance
into the S phase of the cell cycle. Induction of oxidative stress
often leads to cell cycle arrest in G
. Thus, one can assume
that adjusting the ROS level within proliferating cells by
specific substances can affect the cell function.
Comparison of the effect of pinealon on ROS accumulation
and cell death in cultured PC12 cells demonstrated that the
increase in survival roughly corresponded to suppression of
ROS levels. At the same time, it is seen from comparison of
Figs. 4 and 5 that when pinealon concentration reaches from
FIG. 4. Effect of pinealon on intracellular reactive oxygen species (ROS) levels in PC12 cells, as measured in the presence or
absence of 1 mM hydrogen peroxide (H
). (White bars) Sample containing only pinealon at various concentrations as
indicated; (grey bars) samples containing the same concentrations of pinealon and 1 mM H
, the incubation time of 60 min.
(*) Significant difference from the sample containing H
. alone ( p<0.05). DCF, 2,7-Dihlorodihydrofluoresceine.
FIG. 5. Influence of pinealon on cellular death of PC12 cells
after 30 min of exposure to 1 mM hydrogen peroxide (H
(*) Significant decrease from sample with H
100 nM to 500 nM, the ROS level stops decreasing whereas
the number of dead cells continues to decelerate. This ob-
servation suggests some additional points regarding the
pinealon effect.
We have also showed that simultaneously with suppres-
sion of ROS accumulation pinealon delays the time course of
ERK 1/2 activation. Such an effect is in a good correlation
with its action on cell cycle division, suggesting its possible
influence on complex intracellular processes. It is noteworthy
that when pinealon concentration increases from 100 nM to
500 nM (which corresponds to saturating area for ROS ac-
cumulation) both necrotic death prevention and modulation
of cell cycle continue. This observation is in accordance with
data concerning the protective action of pinealon on neuro-
nal survival at oxidative stress levels induced by hypoxia
and suggests some additional mechanisms of its antioxidant
It suggests that, in addition to the antioxidant effect re-
sulting in ROS neutralization, pinealon may act directly with
the cell genome and/or gene expression factors. Our paper is
the first demonstration that pinealon is able to modulate ERK
1/2 activity and thus activate proliferative processes in PC12
cell culture, thus showing pinealon to be a useful tool for
modulating cell metabolism.
The work was partially supported by RFBR Grants, ## 09-
04-00507, 10-04-01461 and 10-04-00906.
Author Disclosure Statement
The authors have no conflict of interests to disclose.
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Address correspondence to:
Alexander Boldyrev
Department of Biochemistry
School of Biology
M.V. Lomonosov Moscow State University
Lenin’s Hills, Building 1/2
119992 Moscow
Received: February 19, 2011
Accepted: April 29, 2011
... The EDR peptide normalized the functional activity of the central nervous system in an experimental prenatal hyperhomocysteinemia model in rats. In cerebellar granule cell cultures, the EDR peptide increased the lag phase of MAP kinase activation and decreased the level of reactive oxygen species (ROS) [13][14][15]. ...
... The EDR peptide decreased ROS synthesis caused by the receptor-dependent (ouabain, homocysteine) and non-receptor (hydrogen peroxide) activators of oxidative stress in gran- ular cells of rat cerebellum. The ability of the tripeptide to reduce the production of ROS during an inflammatory reaction has been demonstrated in zymosan-activated neutrophil cultures [14]. ROS have been established to function as secondary messengers, triggering cascades of cellular signaling-the MAPK-ERK1/2 pathway, in particular [40,41]. ...
... The neuroprotective effect of the EDR peptide is accompanied by a delayed ERK1/2 activation and a change in the onset of the cellular cycle phases. The limitation of ROS accumulation and cell death occurred at lower concentrations of the EDR peptide, while higher concentrations of the EDR peptide resulted in the modulation of the cellular cycle [14]. Thus, the EDR peptide is capable of exerting neuroprotective and antiapoptotic effects through the MAPK/ERK signaling pathway, thus preventing the AD development under oxidative stress conditions ( Figure 2). ...
Full-text available
The EDR peptide (Glu-Asp-Arg) has been previously established to possess neuroprotective properties. It activates gene expression and synthesis of proteins, involved in maintaining the neuronal functional activity, and reduces the intensity of their apoptosis in in vitro and in vivo studies. The EDR peptide interferes with the elimination of dendritic spines in neuronal cultures obtained from mice with Alzheimer’s (AD) and Huntington’s diseases. The tripeptide promotes the activation of the antioxidant enzyme synthesis in the culture of cerebellum neurons in rats. The EDR peptide normalizes behavioral responses in animal studies and improves memory issues in elderly patients. The purpose of this review is to analyze the molecular and genetics aspects of the EDR peptide effect on gene expression and synthesis of proteins involved in the pathogenesis of AD. The EDR peptide is assumed to enter cells and bind to histone proteins and/or ribonucleic acids. Thus, the EDR peptide can change the activity of the MAPK/ERK signaling pathway, the synthesis of proapoptotic proteins (caspase-3, p53), proteins of the antioxidant system (SOD2, GPX1), transcription factors PPARA, PPARG, serotonin, calmodulin. The abovementioned signaling pathway and proteins are the components of pathogenesis in AD. The EDR peptide can be AD.
... In the promoter of the CALM1 gene, binding sites for the EDR peptide have been identified which may determine the neuroprotective effect of the tripeptide in AD models [13]. In addition, it should be noted that, in neurons, the EDR peptide increases the activation of signaling mitogen-activated ERK1/2 kinase, the activity of which is of fundamental importance in the survival of neurons and synaptic plasticity [88]. ...
... .3390/ijms23084259/s1. References [10,13,14,32,33,38,53,63,64,72,78,79,85,88,90,93,99,114] are cited in the supplementary materials. ...
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Epigenetic regulation of gene expression is necessary for maintaining higher-order cognitive functions (learning and memory). The current understanding of the role of epigenetics in the mechanism of Alzheimer’s disease (AD) is focused on DNA methylation, chromatin remodeling, histone modifications, and regulation of non-coding RNAs. The pathogenetic links of this disease are the misfolding and aggregation of tau protein and amyloid peptides, mitochondrial dysfunction, oxidative stress, impaired energy metabolism, destruction of the blood–brain barrier, and neuroinflammation, all of which lead to impaired synaptic plasticity and memory loss. Ultrashort peptides are promising neuroprotective compounds with a broad spectrum of activity and without reported side effects. The main aim of this review is to analyze the possible epigenetic mechanisms of the neuroprotective action of ultrashort peptides in AD. The review highlights the role of short peptides in the AD pathophysiology. We formulate the hypothesis that peptide regulation of gene expression can be mediated by the interaction of short peptides with histone proteins, cis- and transregulatory DNA elements and effector molecules (DNA/RNA-binding proteins and non-coding RNA). The development of therapeutic agents based on ultrashort peptides may offer a promising addition to the multifunctional treatment of AD.
... The MAP kinase activation profile determines which genes associated with adaptation or apoptosis will be expressed in a given period of time. The addition of the tripeptide to cell cultures led to a prolongation of the lag period of MAP kinase activation, which can be considered a defense against the toxic action of homocysteine in [9,10]. ...
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Cortexin is a clinically approved cerebral cortex polypeptide complex in calves. The mechanism of cortexin action is not understood well. Two cortexin derivatives, short peptides EDR and DS with neuroprotective activity, were synthesized. According to the data of molecular modeling, these peptides are able to bind to the histone H1.3 protein. This can affect the conformation of histone H1.3, which leads to a change in the chromatin structure in the loci of some genes, in particular Fkbp1b encoding the FK506-binding protein. Electrophysiological processes associated with the Ca²⁺ exchange are disturbed in the pyramidal neurons of the hippocampus during aging of the brain. The Fkbp1b gene encodes peptidyl-prolyl cis-trans isomerase, regulating the release of calcium ions from the sarcoplasmic and endoplasmic reticulum of neurons. The activation of the Fkbp1b gene transcription under treatment with short peptides can promote the synthesis of its protein product and the activation of the Ca²⁺ release from organelles of the sarcoplasmic and endoplasmic reticulum of neurons, which, in turn, can lead to an increase in the functional activity of neurons.
... The influence of peptide EDR on oxidation process caused by ouabain or hydrogen peroxide in neurons was recently investigated. Peptide EDR reduced ROS in neurons [18]. ...
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Huntington's disease (HD) is a fatal, inherited neurodegenerative disorder. The study in functioning and aging of neurons may give an opportunity to regulate these processes. Previous investigations demonstrated the ability of EDR peptide (Glu-Asp-Arg) to penetrate a cell nucleus and stimulate gene expression. The data obtained prompt EDR peptide capability to restore the morphology of spines in striatum neurons in Huntington’s disease (HD) mouse model. EDR peptide has been shown by us to bind the DNA in solution (absorption spectroscopy and dynamic light scattering) and in a computer model. The proposed model suggests that EDR peptide binds specific binding site oligo (dCG) along the DNA minor groove.
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Experimental hyperhomocysteinemia (HHC) induced in pregnant rats by L-methionine administration was shown to result in an increased content of caspase-3, as well as of a neurotrophic factor neuregulin (NRG1) and of a vesicular monoamine transporter 2 (VMAT2) in the fetal brain tissue analyzed on the 20th day of gestation (E20) while there were no significant changes in catecholamine (noradrenaline and adrenaline) content in blood serum and adrenal glands of pregnant rats. However, on the 20th day of gestation in the placentas of HHC rats the activity of monoamine oxidase as well as the content of neuregulin-1 were significantly decreased compared with the control group. The data obtained suggest that the metabolic changes observed in the embryonic nervous tissue may not only result from the direct effect of L-homocysteine on the developing brain but also be caused by the disturbances of placental functioning caused by increased homocysteine content in the maternal organism.
The interaction of the regulatory biologically active peptide Glu-Asp-Arg (EDR) with DNA is considered by spectral, NMR, viscosimetry, and molecular dynamics methods. It was shown that EDR can partly penetrate into the major groove of DNA and affect the base atoms, mainly the N7 and O6 of guanine. It was observed that Mg2+ ions can promote DNA-EDR interaction due to their effective screening of the negatively charged phosphate groups of DNA. This action of Mg2+ remains in salted solution as well.
GDF11 protein, the growth differentiation factor 11, which belongs to the TGF-β superfamily (transforming growth factor β), shows marked geroprotective effects on the cardiovascular and nervous systems. The cardioprotective and myoprotective effects of the GDF11 protein are associated with its regulation of several signaling molecules, including the MAPK–p38–mioglianin pathway. GDF11 neuroprotective action is associated with the regulation of proliferation and differentiation of brain neurons by means of changing the activity of the p57 (Kip2) and p27 (Kip1) transcription factors. GDF11 may be considered a potential target for geroprotector drugs, as was demonstrated in the case of the Glu-Asp-Arg peptide possessing similar neuroprotective and myoprotective properties as GDF11. For the Glu-Asp-Arg, Ala-Glu-AspGly, and Lys-Glu peptides, binding sites were found in the promoter region of GDF11: the CCTGC, ATTTC, and GCAG motifs, respectively.
During the peptides activity mechanism investigation it was shown that short peptides regulate gene expression and protein synthesis. These peptides stimulated cell proliferation, differentiation and decreased cell apoptosis. This effect is the reason of increasing of various organs function. The peptides injection decreased the frequency of cancerogenesis and increased physiological resource of cells, tissues and organism till 20-42%. Peptides increased vital resource and decreased the mortality rate of elderly people.
The effect of experimental hyperhomocysteinemia on diurnal dynamics of norepinephrine content in the medial preoptic area of the female rat hypothalamus, which is responsible for the synthesis of gonadotropin-releasing hormone (GnRH), has been studied. We investigated plausible neuroprotective effects of the pineal gland peptide pinealon (Glu-Asp-Arg). It has been found that administration of the substance can correct the deficiencies in the normal diurnal dynamics of norepinephrine content in the studied hypothalamic area that were caused by L-methionine loading. The data we obtained testify to the protective effect of pinealon and to its ability to diminish the unfavorable effect of hyperhomocysteinemia on the female rat reproductive function.
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A primary culture of murine cerebellar neurons was used to induce oxidative stress resulting in the accumulation of reactive oxygen species (ROS) and activation of ERK 1/2 kinase. Short-term incubation (15 min) of cerebellar neurons with homocysteine (HC) or N-methyl-D-aspartate (NMDA) induced partial ERK 1/2 phosphorylation thus providing the activation of the enzyme. Inhibitors of NMDA receptors, MK-801 or D-AP5, both prevented the activation of cells by HC or NMDA. Another receptor-dependent means of oxidative stress stimulation is exposure of cells to the cardiac glycoside ouabain, a specific inhibitor of Na/K-ATPase. Ouabain induces ROS accumulation and substantial ERK1/2 activation in neuronal cells at concentrations as low as 1 nM - 1 μM, which corresponds to participation of Na/K-ATPase in intracellular signalling. Neuropeptide carnosine added to the cells 2 hours before oxidative stress prevented both ROS accumulation and ERK1/2 activation. As ERK1/2 kinase plays a key role in gene expression responsible for either cell adaptation or cell death, the model used gives a useful tool to characterize the effect of natural and synthetic anti-cancer drugs on cellular life. The data presented show that carnosine is a natural modulator of oxidative stress in neuronal cells, providing regulation of ERK1/2 activity via buffering intracellular ROS levels.
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We have found that neutrophils begin to express NMDA receptors on their membranes after in vivo activation. These receptors are the target for action of homocysteine (HC). After incubation of activated neutrophils with HC, the degranulation process is stimulated and generation of reactive oxygen species is increased. We conclude that expression of NMDA receptors on neutrophil membrane makes neutrophils sensitive to HC. Thus, hyperhomocysteinemia may induce additional stimulation of immune competent cells.
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Short peptides that have been characterized in previous studies performed in the St. Petersburg Institute of Bioregulation and Gerontology of the Northeastern Division of the Russian Academy of Medical Sciences [1‐3] protect neurons from oxidative stress [4]; however, under in vitro conditions, the peptide action has been observed at high (millimolar) concentrations. The anti-oxidant activity of these peptides is not high; therefore, their biological effect could be mediated by a natural system of antioxidant defense [5]. If this is the case, the peptide protective effect could be observed at almost physiological concentrations under the in vivo conditions. This was aimed at verifying this assumption. The protective effect of short regulatory peptides (vilon, epitalon, pinealon, and vesugen) was studied under the conditions of oxidative stress caused in animals by hypobaric hypoxia. Male Wistar rats weighing 185‐200 g were used in our experiments. Oxidative stress was induced under the conditions of hypobaric hypoxia in an altitude chamber with adjustable airflow, which prevented hypercapnia [6]. By means of a vacuum pump, the pressure in the altitude chamber was reduced to 0.125 atm for 1 min. Under these conditions, the animals were kept until respiration arrest; after this, the animals were returned to the normal-pressure conditions by supplying air into the chamber for 1 min. The following parameters were recorded: the time (s) until the respiration arrest “at the height”; the time (s) of posture recovery, i.e., the period from respiration arrest to the moment when the experimental animal assumed normal posture after “descending from the height” and respiration recovery under normobaric conditions; and the restitution time (s), the total duration of the recovery of physiological activity after hypoxia. To determine the protective effect of the peptides studied, the death rate and coefficient of restitution (the ratio of the restitution time to the time before respiration arrest in the altitude chamber) were estimated. The results obtained were processed statistically using the Mann‐Whitney and Kruskal‐Wallis tests; the differences were assumed to be significant at p < 0.05. The peptides vilon (Lys-Glu), vesugen (Lys-GluAsp), pinealon (Glu-Asp-Arg), and epitalon (Ala-GluAsp-Arg) were injected daily at a dose of 10 µ g/kg body weight intraperitoneously for five days before hypoxia. The rats of the control group were injected with saline according to the same scheme. Each group contained 10 animals.
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The review comprises the results of author's long-term investigation in the mechanisms of aging and a role of peptide bioregulators in prevention of age-related pathology. A number of small peptides have been isolated from different organs and tissues and their analogues (di-, tri-, tetrapeptides) were synthesized from the amino acids. It was shown that long-term treatment with some peptide preparations increased mean life span by 20-40%, slow down the age-related changes in the biomarkers of aging and suppressed development of spontaneous and induced by chemical or radiation carcinogens tumorigenesis in rodents. Possible mechanisms of the biological effects of small peptides are discussed in the paper. The results of clinical applications of peptide preparation during the period of 6-12 years are presented as well.
Biological effects of short regulatory peptides, pinealon, vesugen, vilon and epitalon were studied in model experiments in vitro. These peptides were found not to demonstrate direct antioxidant activity but be able to restrict lipid peroxidation of human lipoproteins by modification of their structure. The short peptides increase stability of red blood cell membranes toward osmotic hemolysis. They also elevate the stationary level of intracellular reactive oxygen species and at the same time decrease (all excepting epitalon) percent of dead cells in neuronal population. The suggestion was made that under in vivo conditions, short peptides may participate in apoptosis/necrosis regulation.
This monograph highlights the gerontological aspects of the peptide regulation of gene expression. It focuses on the mechanisms of the geroprotective action of peptides related to chromatin activation, increase in telomerase enzyme activity, and elongation of telomeres in different cells. A key role in the initiation of the biological activity of peptide is its interaction with DNA which provides genetic stability and a normalization of the age-related metabolic shifts. Study of the genetic mechanisms of peptide action suggests a new concept which most comprehensively reflects on the evolutionary biological role peptide plays in the organism. The impact of peptides on the expression and structure of genes opens a new gate for the prevention of premature aging and age-related pathology which appears to be most promising in pharmacogenomics. This monograph narrates not only the theoretical aspects and experimental data, but also outlines new approaches to the prevention of aging and age-related pathology, thus addressing a wide readership of gerontologists, geneticists, molecular biologists, biochemists, and pharmacologists alike.
Inhibition of rat neuronal Na(+)/K(+)-ATPase alpha3 isoform at low (100 nM) ouabain concentration led to activation of MAP kinase cascade via PKC and PIP(3) kinase. In contrast to ouabain-sensitive alpha3 isoform of Na(+)/K(+)-ATPase, an ouabain-resistant alpha1 isoform (inhibition with 1 mM of ouabain) of Na(+)/K(+)-ATPase regulates MAP kinase via Src kinase dependent reactions. Using of Annexin V-FITC apoptotic test to determine the cells with early apoptotic features allows to conclude that alpha3 isoform stimulates and alpha1 suppresses apoptotic process in cerebellum neurons. These data are the first demonstration showing participation of ouabain-resistant (alpha1) and ouabain-sensitive (alpha3) Na(+)/K(+)-ATPase isoforms in diverse signaling pathways in neuronal cells.
Reactive oxygen species (ROS) regulate the strength and duration of signaling through redox-dependent signal transduction pathways via the cyclic oxidation/reduction of cysteine residues in kinases, phosphatases, and other regulatory factors. Signaling circuits may be segregated in organelles or other subcellular domains with distinct redox states, permitting them to respond independently to changes in the oxidation state of two major thiol reductants, glutathione and thioredoxin. Studies in yeast, and in complex eukaryotes, show that oscillations in oxygen consumption, energy metabolism, and redox state are intimately integrated with cell cycle progression. Because signaling pathways play specific roles in different phases of the cell cycle and the hierarchy of redox-dependent regulatory checkpoints changes during cell cycle progression, the effects of ROS on cell fate vary during the cell cycle. In G1, ROS stimulate mitogenic pathways that control the activity of cyclin-dependent kinases (CDKs) and phosphorylation of the retinoblastoma protein (pRB), thereby regulating S-phase entry. In response to oxidative stress, Nrf2 and Foxo3a promote cell survival by inducing the expression of antioxidant enzymes and factors involved in cell cycle withdrawal, such as the cyclin-dependent kinase inhibitor (CKI) p27. In S phase, ROS induce S-phase arrest via PP2A-dependent dephosphorylation of pRB. In precancerous cells, unconstrained mitogenic signaling by activated oncogenes induces replication stress in S phase, which activates the DNA-damage response and induces cell senescence. A number of studies suggest that interactions of ROS with the G1 CDK/CKI network play a fundamental role in senescence, which is considered a barrier to tumorigenesis. Adaptive responses and loss of checkpoint proteins such as p53 and p16(INK4a) allow tumor cells to tolerate constitutive mitogenic signaling and enhanced production of ROS, leading to altered redox status in many fully transformed cells. Alterations in oxidant and energy metabolism of cancer cells have emerged as fertile ground for new therapeutic targets. The present challenge is to identify redox-dependent targets relevant to each cell cycle phase, to understand how these targets control fate decisions, and to describe the mechanisms that link metabolism to cell cycle progression.
Flow cytometric analysis provides a rapid screen for abnormalities of polymorphonuclear neutrophils (PMN) function and reflect their behavior in vivo more accurately. This review summarizes the major fluorescent probes used to study PMN oxidative burst and apoptosis using flow cytometry (FCM). We also provide examples of FCM studies in physiological and pathological situations, illustrating the advantages of FCM for assessment of PMN oxidative burst and PMN apoptosis. These data point to the role of FCM in detecting primary immunodeficiencies such as IRAK4 deficiency and support the use of the assessment of the PMN oxidative burst for routine testing in patients with bacterial infections. We also demonstrate the utility of whole-blood analysis using FCM for a better understanding of PMN functionality, i.e., tuning PMN responses to inflammatory stimuli. Finally, FCM permits a simultaneous analysis of phenotypic, functional and morphometric parameters assessing whole-blood PMN apoptosis, in particular in response to Toll-like receptor agonists and during simian immunodeficiency virus infection.