ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 8, pp. 1460-1469 © Pleiades Publishing, Ltd., 2026.
1460
Effect of Cu
2+
on the Nucleoli of Cultured Astrocytes
from the Rat Cerebral Cortex
Elizaveta E. Genrikhs
1
, Elena V. Stelmashook
1
, Elena A. Smirnova
2
,
Sergey A. Golyshev
2
, Alina E. Lapieva
1,2
, Olga P. Alexandrova
1
,
Marina R. Kapkaeva
1
, and Nickolay K. Isaev
1,2,a
*
1
Russian Center of Neurology and Neurosciences, 125367 Moscow, Russia
2
Lomonosov Moscow State University, 119991 Moscow, Russia
a
e-mail: nisaev61@mail.ru
Received April 10, 2026
Revised July 20, 2026
Accepted July 23, 2026
Abstract— Cultured astrocytes were incubated with CuCl
2
, which caused dose-dependent cell death (25-200 μM,
24 h). Immunocytochemical detection of the nucleolar protein nucleophosmin/B23 (NPM/B23) demonstrated
that exposure to Cu
2+
(100 μM, 24 h) caused a significant increase in the surface area of NPM/B23 clusters,
which was accompanied by the changes in the nucleolar ultrastructure characteristic of nucleolar stress.
Longer incubation of astrocytes with Cu
2+
(100 μM, 48 h) led to accumulation of the endoplasmic reticu-
lum (ER) stress marker GRP78, which was accompanied by the increase in nucleolar size and migration of
the nucleolar material into the nucleoplasm.
DOI: 10.1134/S0006297926601140
Keywords: astrocytes, nucleolar stress, copper ions, ER stress, nucleolus
* To whom correspondence should be addressed.
INTRODUCTION
Copper is not only one of the most common
variable-valence metals in the body but also is an
essential trace element involved in many import-
ant physiological processes. Discovery of the role of
copper in cellular signaling, autophagy, cell motility,
differentiation, and regulated cell death (cuproptosis)
has significantly expanded the list of its known func-
tions [1]. Both excess copper and copper deficiency
could lead to the cell death. It is worth noting that
deprivation of glucose and amino acids with thiol
groups could lower the threshold for toxic effects of
copper ions (Cu
2+
) [2, 3]. Balance of Cu
2+
ions in the
brain is of particular importance, and its disruption
plays a key role in pathogenesis of many neurode-
generative diseases and pathological conditions, such
as Wilson–Konovalov disease and Alzheimer’s disease
[4-6]. An important function of glial cells such as as-
trocytes is to maintain Cu
2+
homeostasis in the brain,
as they could effectively absorb, accumulate, and ex-
crete copper [7]. Thus, it is extremely important to
understand how Cu
2+
could affect brain astrocytes,
as these cells play a key role in copper metabolism.
Our recent studies have shown that Cu
2+
have a
toxic effect on nucleolus [8]. Nucleolus is an import-
ant nuclear subdomain where ribosome biogenesis
occurs [9, 10]. In turn, disruptions in the ribosome
biogenesis are associated with the significant chang-
es in the structure and function of nucleolus. This
phenomenon, known as nucleolar stress, could lead to
molecular changes, such as activation of the p53 and
other stress signaling pathways, which, in turn, lead
to the changes in cell behavior, cell cycle arrest, or
cell death [11-13]. Previously, we found that at subtox-
ic concentrations of Cu
2+
, astrocytes exhibit signs of
nucleolar stress, such as changes in ultrastructure and
translocation of p53 into the nucleolus. However, not
all of the changes we observed in the nucleolus match
those that characterize nucleolar stress [8]. Inthis re-
gard, we conducted studies aimed at further analyzing
the signs of nucleolar stress in astrocytes under the
action of Cu
2+
. Since the same stimuli could induce
both nucleolar and endoplasmic reticulum(ER) stress,
EFFECT OF Cu
2+
ON ASTROCYTE NUCLEOLI 1461
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
we assessed the ability of Cu
2+
to cause accumulation
of the ER stress marker protein GRP78 in the cells.
MATERIALS AND METHODS
Astrocyte culturing. Dissociated astrocyte cul-
tures were obtained from 1-day-old Wistar rats of
both sexes, purchased from the Stolbovaya nursery.
Enzymatic-mechanical dissociation was performed
using a previously described method [8]. Isolated
cerebral cortex of the rat brains was washed with
a calcium- and magnesium-free phosphate buffer
(PBS, Gibco Life Technologies, USA), minced with a
scalpel, and incubated for 15 min at 37°C in a 0.05%
trypsin solution and 0.02% EDTA solution (Gibco Life
Technologies, USA). Tissue samples were washed in
two changes of phosphate buffer and once with a
medium, and then gradually dissociated mechanical-
ly using a Pasteur pipette. Cell suspension was cen-
trifuged for 3 min at 1000 rpm (centrifuge CM-6M
ELMI Ski Line, Latvia); the pellet was resuspended
in a medium and seeded in a poly-L-lysine coated
flask (Sigma, USA). A culture medium contained 90%
Eagle’s Minimum Essential Medium (MEM, Gibco, UK),
10% fetal bovine serum (HyClone, UK), 2 mM Gluta-
MAX (Gibco, UK), and 10 mM HEPES buffer (Sigma,
USA). The cultures were developed in a CO
2
incuba-
tor (RWD Life Science, China) at 36.5°C and relative
humidity of 98%. After a monolayer formation, the
astrocyte cultures were transplanted into 96-well po-
ly-L-lysine coated plastic plates (Eppendorf, Germany)
or coverslips (22×22 mm). After a monolayer forma-
tion, they were used in further experiments. Live ob-
servations of a culture development were conducted
using phase-contrast microscopy with an inverted
microscope Olympus CKX41 (Olympus, Japan). CuCl
2
was added directly to the culture medium for 24-h
incubation at concentrations of 25, 50, and 100 μM.
Viability test (cytotoxicity test). Cell viability
was assessed using the MTT assay, based on reduction
of the yellow water-soluble tetrazolium dye (3-(4,5-di-
methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-
mide, Sigma-Aldrich, USA) resulting in formation of
a purple formazan by living cells. For this, cultures
were incubated with MTT (0.5 mg/mL, 30 min), next
the medium was removed, and the formed formazan
was dissolved in DMSO (50 μL per well). Optical den-
sity measurements were taken using a microplate
reader (SpectraMax M2, Molecular Devices, USA) at
570 nm [14]. Viability of control cultures was taken
as 100%, and viability of the treated cells was ex-
pressed as a percentage of the control.
Immunofluorescence analysis. Cells were fixed
in 4% formaldehyde (Servicebio, China) in PBS for
10 min, treated with 0.1% Triton X-100 (HiMedia,
India) in PBS for 20 min, and incubated in PBS with
addition of 2.5% BSA and 10% horse serum for1 h to
prevent nonspecific staining. Mouse monoclonal an-
tibodies against fibrillarin (Abcam, ab4566, 1 : 500),
rabbit monoclonal antibodies against nucleophos-
min (NPM/B23, Beyotime, AG2735, 1 : 100), and rab-
bit polyclonal antibodies against GRP78 (Beyotime,
AF0171, 1 : 100) were added as primary antibodies
and incubated overnight at 4°C in a humid cham-
ber. Secondary anti-rabbit antibodies labeled with
Alexa Fluor 594 (Invitrogen, ab21207 1 : 500) and
anti-mouse antibodies labeled with Alexa Fluor 488
(FNSA-0057 FineTest, 1 : 500) were added and incu-
bated for 2 h at room temperature. Preparations were
mounted in a Fluoroshield medium with 4′,6-diamid-
ino-2-phenylindole (DAPI) (Thermo Fisher Scientific,
USA). Cells were photographed using an EVOS M7000
imaging system (Thermo Fisher Scientific). Morpho-
metric analysis of nucleoli was performed using the
Fiji software.
Nucleoli staining and analysis. To stain nucleoli,
cultured astrocytes were fixed in a 4% formaldehyde
solution in PBS for 15-20 min. Cells were washed with
PBS (3 times for 5 min) and treated with a solution
of acridine orange (Fluka, Switzerland, 0.74 mg/mL
in PBS (pH 7.2)) for 10 min. Nucleoli were visualized
using a confocal microscope Olympus IX71 (Japan)
with a spinning disk, ×100 objective, and 488-nm
OBIS laser (USA), controlled by the Coherent Connec-
tion 3 program. Fluorescent images of cells for nu-
cleoli visualization were obtained using an emission
wavelength of 700 ± 75 nm. Morphometric analysis of
nucleoli was performed using the Fiji software.
Electron microscopy. For electron microsco-
py studies, cells were grown on poly-L-lysine-coated
coverslips in 40-mm Petri dishes for 7-8 days in vitro.
Cells were exposed to CuCl
2
for 24 h, then fixed in
an aqueous solution of 2.5% glutaraldehyde (SPI Inc.,
USA), 100 mM sodium cacodylate for 48 h at 4°C.
Fixed cells were washed twice with fresh 100 mM so-
dium cacodylate solution and post-fixed with 1% os-
mium tetroxide dissolved in a 100 mM sodium caco-
dylate solution for 60 min at 4°C. The samples were
next dehydrated in a series of ethanol solutions with
increasing concentrations.
Dehydration included staining with a 2% uranyl
acetate solution in 70% ethanol for 1 h at 4°C, fol-
lowed by ethanol, acetone, and gradually increasing
concentrations of resin-acetone mixtures and two
changes of fresh resin. Finally, the coverslips were
placed in silicone molds filled with fresh Spi-pon 812
resin (SPI Inc.) and polymerized at 70°C for 72 h.
Hardened blocks were trimmed with a razor blade,
and ultrathin 90-nm thickness sections were prepared
using an Ultracut E ultramicrotome (Reichert_Jung,
Austria) equipped with a Diamond Ultra 45 knife
GENRIKHS et al.1462
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
(Diatome, Switzerland). The sections were next trans-
ferred to copper grids coated with formvar and fur-
ther contrasted with a 2% aqueous uranyl acetate
solution for 40 min and lead citrate for 3 min. Sam-
ples were analyzed under transmission electron mi-
croscopes JEOL JEM-1400 (Japan), operating at 100kV
and equipped with a CCD camera Quemesa, and JEOL
JEM-1011 (Japan), operating at 80 kV and equipped
with an ORIUS SC1000W digital camera.
Statistical analysis. The obtained results were
processed using the Statistica 13.3 software (StatSoft,
Inc.). Normality of the data distribution in the ex-
periments was assessed using the Shapiro–Wilk test.
Variations of the studied parameters had normal dis-
tribution and were analyzed using one-way ANOVA
with Dunnett’s post-hoc test or t-test. Data are pre-
sented as mean values and standard error of the
mean (M ± SEM). Differences were considered statis-
tically significant at p < 0.05. The independent unit
in the morphometric analysis was the mean value of
the nucleolus or cluster measurement in a single in-
dependent experiment. Each independent experiment
was repeated at least 5 times with cultures obtained
from different animals. In experiments with nucleoli,
at least 100 nucleoli per 1 culture were examined.
In the experiment with protein cluster measurement,
at least 200 clusters per 1 culture were measured.
The experiment was designed to determine differenc-
es between the samples when assessing differences
between the means with an alternative hypothesis
of superiority at the Type I error probability of 5%
and statistical power of at least 80%. To determine
the sample size, number of the comparison groups,
sample ratio, mean value that could be expected if
the exposure had no effect, standard deviation, and
minimum detectable effect were considered.
RESULTS
Working concentration of copper was select-
ed based on the data previously obtained data for
astrocyte cultures in our study [8]. In this series of
experiments, viability of the cultured cerebral cortex
astrocytes after 24 h incubation under the influence
of 25 μM Cu
2+
did not differ from the control level,
while at Cu
2+
concentrations 50 and 100 μM, it de-
creased to 86 ± 4% and 80 ± 6%, respectively, and at
200 μM, to 59 ± 4%. For further experiments, concen-
trations with the lowest but significant toxic effect –
50 and 100 μM Cu
2+
– were selected (Fig. 1).
Electron microscopy analysis showed that the
astrocyte nucleoli in the control cultures have nor-
mal morphology, with clearly distinguishable granu-
lar component (GC), dense fibrillar component (DFC),
and fibrillar center (FC) (Fig. 2). Cu
2+
at concentra-
tion of 50 μM (24 h) did not cause significant changes
in the astrocyte nucleoli. The GC, DFC, and FC were
well identified. However, at concentration of 100 μM
(24 h), Cu
2+
caused changes in the nucleolar ultra-
structure. We observed redistribution of the DFC to
the periphery of the nucleolus and general disorga-
nization of the nucleolar structure (Fig. 2).
Since the pronounced changes in the nucleoli at
the ultrastructural level were observed at Cu
2+
con-
centration of 100 μM after 24 h incubation, immuno-
cytochemical analysis of the nucleolar components
was performed at this concentration using antibodies
against the nucleolar proteins fibrillarin and NPM/
B23, whose distribution changes under exposures to
stress. Under normal conditions, fibrillarin is local-
ized primarily in the dense fibrillar component of the
nucleolus, where it is involved in the processing of
pre-ribosomal RNA [15, 16]. NPM/B23 in the nucleo-
lus is localized in the granular component, mainly its
outer part, and is involved in biogenesis of ribosom-
al particles [17, 18]. Our images of astrocytes demon-
strate that in the nuclei of the control culture cells,
localization of fibrillarin and NPM/B23 corresponds to
the description presented above (Fig. 3). In contrast,
under the action of Cu
2+
, the NPM/B23 protein was
displaced relative to fibrillarin. Morphometry of the
NPM/B23 clusters showed that in the presence of Cu
2+
(100μM, 24 h), there was increase in the surface area
of NPM/B23 clusters to 3.27 ± 0.4 μm
2
, while in the
control, this indicator was 1.97 ± 0.04 μm
2
(Fig. 4).
Morphometric analysis of the nucleoli in the as-
trocytes stained with acridine orange showed that
treatment with Cu
2+
at concentration of 100 μM for
24 h caused increase in the surface area of nucleoli
from 1.48 ± 0.14 to 2.41 ± 0.27 μm
2
(Fig. 4).
Fig. 1. Dependence of viability of the cultured rat cerebral
cortex astrocytes on the concentration of copper ions (Cu
2+
)
in the medium. * p < 0.05, compared to the control, n = 7
(number of independent experiments). MTT test.
EFFECT OF Cu
2+
ON ASTROCYTE NUCLEOLI 1463
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 2. Ultrastructural changes in the nucleoli under the action of copper ions. Arrows indicate fibrillar centers (FC), dense
fibrillar components (DFC), and granular components (GC). Scale bar: 1 μm.
Fig. 3. Immunocytochemical analysis of distribution of the nucleolar marker proteins nucleophosmin/B23 and fibrillarin in
the control astrocytes and in the astrocytes exposed to the action of Cu
2+
(100μM, 24 h). a)Immunocytochemical detection
of nucleophosmin/B23 and fibrillarin proteins. Scale bar: 10 μm. b) Quantitative assessment of the surface area (S) of the
nucleolar protein nucleophosmin/B23 (NPM/B23) clusters in the cultured rat cerebral cortex astrocytes. White bar– control,
gray bar – Cu
2+
exposure. n = 5, where n is the number of independent experiments.
After 48-h incubation of astrocytes with 100 μM
Cu
2+
, the size of the nucleoli increased even more, so
in this case, we measured not only the area of the
nucleoli but also the maximum and minimum Feret
diameters. In the cells treated with Cu
2+
for 48 h, the
surface area of nucleoli was 3.01 ± 0.39 μm
2
, com-
pared to 1.45 ± 0.09 μm
2
in the control (Fig. 5). The
maximum and minimum Feret diameters of the as-
trocyte nucleoli in the control were 1.51 ± 0.07 and
1.2 ± 0.03 μm, respectively, while in the cells treat-
ed with Cu
2+
for 48 h, these parameters increased to
1.99 ± 0.09 and 1.63 ± 0.1 μm (Fig. 6). In some indi-
vidual cells, the nucleoli lost their round shape, and
the nucleolar material migrated into the nucleoplasm.
Calculation of the ratio of the maximum to minimum
Feret diameters showed significant increase in this pa-
rameter from 1.33 ± 0.01 in the control to 1.48 ± 0.06
in the experiment (Fig. 5), which reflects the change
in the nucleolar shape associated with migration of
the nucleolar material into nucleoplasm.
One of the markers of the ER function disruption
and activation of the adaptive cellular stress response
could be increase in the expression of the ER lumen
resident proteins. To detect it, we performed immu-
nocytochemical detection of the ER resident protein
GRP78 in the astrocytes under normal culturing con-
ditions and under the action of Cu
2+
. Image analysis
did not reveal qualitative differences in the distribu-
tion of this protein in astrocytes after 24-h incubation
with 100 μM Cu
2+
, while after 48-h treatment, fluo-
rescence intensity of the GRP78 protein labeled with
antibodies noticeably increased (Fig. 6).
GENRIKHS et al.1464
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 4. Morphological changes in the nucleolus in the cultured astrocytes exposed to the toxic effect of Cu
2+
(100 μM, 24 h).
a) Cultured astrocytes stained with acridine orange. Nucleoli are indicated by arrows. Scale bar: 10 μm. b) Quantitative
assessment of the nucleolar area in the astrocytes. White bars – control, gray bars – Cu
2+
exposure. n = 5, where n is the
number of independent experiments.
Fig. 5. Morphological changes in the nucleoli in the cultured astrocytes under the toxic effect of Cu
2+
(100 μM, 48 h).
a) Cultured astrocytes stained with acridine orange. Nucleoli are indicated by arrows, disintegrating nucleoli by triangles.
Scale bar: 10μm. b-d)Quantitative assessment of nucleolar sizes (S– surface area of astrocyte nucleoli, Ferr and Ferrmin –
maximum and minimum Feret diameters of astrocyte nucleoli. e)Ferr/Ferrmin ratio shows relative change in the nucleolar
shape. White bars – control, gray bars – Cu
2+
. n = 5, where n is the number of independent experiments.
EFFECT OF Cu
2+
ON ASTROCYTE NUCLEOLI 1465
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 6. Effect of Cu
2+
on distribution of the GRP78 protein in the cultured astrocytes. Immunocytochemical staining with
anti-GRP78 protein antibodies (green staining). Blue – nuclear staining with DAPI. Scale bar: 20 μm.
DISCUSSION
Astrocytes are the first parenchymal cells of the
brain that are exposed to metal ions penetrating
through the blood-brain barrier. The ability of astro-
cytes to absorb and accumulate copper suggests that
these cells perform an important function in regulat-
ing copper homeostasis in the brain. However, incuba-
tion of astrocytes with Cu
2+
for 24 h or more causes
damage to these cells [19], meaning that Cu
2+
have
a negative stressful effect on astrocytes. In the case
of exposure to stress conditions, cells must respond
with a series of changes aimed at protecting the cell
and minimizing the damage. During stress, there is
a strict control of protein metabolism, and there is
continuous exchange of the nucleolar proteins with
the nucleoplasm [20]. Visualization of nucleolus in the
cells could be a promising strategy for studying cellu-
lar stress and death. Earlier, we suggested that copper
ions (Cu
2+
) could affect the nucleolus of astrocytes,
leading to the development of nucleolar stress [8].
Nucleolar stress is characterized by redistribution
of the dense fibrillar component to the periphery
of the nucleolus and general disorganization of the
nucleolar structure [21]. In this study, using electron
microscopy, we showed that the nucleoli of the cul-
tured astrocytes in the control cultures have normal
morphology, while exposure to Cu
2+
(100 μM, 24 h)
causes changes in the nucleolar ultrastructure char-
acteristic of the nucleolar stress. Exposure to lower
concentration of Cu
2+
(50 μM) did not cause signifi-
cant changes in the astrocyte nucleoli during this time
period.
Nucleolus is the center of ribosome biogenesis
and also an important site for regulating the cell cy-
cle, cellular senescence, and response to stress. Nu-
cleolus contains several hundred different proteins
responsible for its multifunctionality [9]. In addi-
tion to its role in the ribosome biogenesis, the nu-
cleolus acts as a central sensor of stress exposures.
GENRIKHS et al.1466
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Disruption of the main function of the nucleolus
triggers a signaling cascade aimed at cell survival or
death. Inhibition of rRNA synthesis leads to the release
of nucleolar proteins, which are normally in a bound
state. Ribosomal proteins (e.g., RPL5, RPL11, RPL23)
and proteins such as ARF bind to the E3 ubiquitin li-
gase MDM2 and inhibit it[22]. Some of the important
nucleolar proteins are used to mark nucleolar com-
ponents– fibrillar centers, dense fibrillar component,
and granular component. These markers include the
nucleophosmin protein (NPM/B23), a member of the
nuclear chaperone family. This is a multifunctional
protein involved in various biological processes, but
the main function of NPM/B23 is ribosome biogenesis.
NPM/B23 is localized in the granular component of
the nucleolus [18]. Another common nucleolar mark-
er is fibrillarin, which is specifically localized in the
fibrillar region of the nucleolus, covering both the
dense fibrillar component and the fibrillar centers.
It has a highly conserved methyltransferase domain
and is a ribosomal RNA methyltransferase. Fibril-
larin is a key regulator in the early stages of ribo-
some biogenesis. It is localized in the nucleolus at
the boundary between the fibrillar centers and the
dense fibrillar component, where rRNA transcription
occurs [16]. When nucleolar stress is induced, NPM/
B23 is redistributed from the nucleoli into the nu-
cleoplasm [11], but we observed this effect only in
some individual cells. Based on our data, incubation
of astrocytes with Cu
2+
for 24 h leads to increase in
the size of both the nucleolus itself and the nucleolar
areas of NPM/B23 localization. The observed increase
in the nucleolus in astrocytes is likely not due to the
enhanced function but rather to the disruption of ri-
bosome biogenesis and accumulation of ribonucleop-
rotein components, because it was previously shown,
that formation of pre-ribosomal particles is disrupt-
ed in various cell lines under the action of Cu
2+
,
and 47S and 45S rRNA accumulate due to improper
distribution of nucleophosmin and fibrillarin [23].
It should be noted that in addition to the increase in
the size of the nucleolus and of the NPM/B23 clus-
ters, distribution of NPM/B23 relative to fibrillarin
also changed. In our experiments, in the control cells,
fibrillarin is detected in the nucleoli as distinct clus-
ters, while in the Cu
2+
-treated cells, fibrillarin clusters
lose their clear outlines. It should be noted that such
changes for this protein in the nucleoli were previ-
ously shown in the HeLa cells under the action of
hydrogen peroxide [24]. Migration of the nucleolar
components stained with acridine orange into the
nucleoplasm was observed during longer 48-h incu-
bation of astrocytes with Cu
2+
. Thus, increase in size
(expansion) and following fragmentation of the nucle-
olus are classical morphological signs of the nucleo-
lar stress. Studying of various cell lines shows that
the action of stress factors that suppress ribosome
biogenesis leads to nucleolar hypertrophy (increase)
followed by fragmentation or formation of nucleo-
lar “caps”, in which nucleolar components segregate.
This is visualized using the NPM/B23 immunofluores-
cence analysis [12]. Disruptions in the distribution of
fibrillarin within the nucleolus were shown in dif-
ferent cell lines under the action of mercury, but the
authors of the study associate this more with apopto-
sis rather than disruption of rRNA transcription [25].
Additionally, increase in the nucleolar size under the
action of copper was noted in the experiments with
the HEK293T cell line [23].
Inhibition of rRNA synthesis and processing is
a key functional disruption in the nucleolar stress.
A consequence of this disruption should be decrease
in the overall level of protein synthesis. It should be
noted that copper is required for functioning of more
than thirty proteins, including superoxide dismutase,
ceruloplasmin, lysyl oxidase, cytochrome-C oxidase,
tyrosinase, and dopamine-β-hydroxylase. However,
excess of Cu
2+
causes a proteotoxic effect through
direct binding to proteins, displacing other metals,
causing their misfolding, aggregation, and inhibiting
their functions [26]. For example in hepatocytes, cop-
per exhibits toxicity through the ER stress [27]. There-
fore, we tested how Cu
2+
affects the state of the ER
and the content of the GRP78 protein in the cultured
astrocytes. GRP78 is a luminal ER chaperone and one
of the markers of ER stress. When misfolded proteins
accumulate in the lumen, the protein detaches from
the transmembrane ER stress sensors and activates a
signaling cascade, leading, among other things, to the
increased expression of GRP78 itself. Increase in the
GRP78 content in the lumen enhances the mechanism
of protein folding and assembly [28-30]. It should be
noted that ER stress not only activates GRP78 to con-
trol protein quality in endoplasmic reticulum but also
promotes its movement into the other cellular com-
partments, where it significantly expands its func-
tional repertoire [31]. Although after 24-h incubation
of astrocytes with Cu
2+
, we did not observe the char-
acteristic increase in the GRP78 content in the cells
typical of ER stress, however, longer incubation of as-
trocytes with Cu
2+
caused pronounced accumulation
of GRP78 in the astrocytes, which was accompanied
by an even greater increase in the nucleolar size.
Although some studies have shown increase in
the nucleolar size during nucleolar stress, Lu et al.
consider that the main changes during the nucleo-
lar stress include reduction in the nucleolar size and
volume, suppression of rRNA synthesis by RNA poly-
merase I, segregation of the nucleolar components,
translocation of nucleophosmin from the nucleolus
into the nucleoplasm [32]. Probably, this conclusion
summarizes the terminal stage of nucleolar stress
EFFECT OF Cu
2+
ON ASTROCYTE NUCLEOLI 1467
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
development caused by harsh exposures to such
agents as actinomycin D, hydrogen peroxide, high
temperature (42°C), and ultraviolet radiation [11].
However, even with nucleolar stress stimulation
by ultraviolet radiation, transient expression of the
NPM/B23 mRNA and temporary increase in the NPM/
B23 protein content in the nucleoli have been ob-
served [22]. Based on our studies, the effect of Cu
2+
on astrocytes at a mildly toxic concentration is ac-
companied by: (i) increase in the nucleolar size with
subsequent migration of the nucleolar components
into the nucleoplasm; (ii) increase in the zone of lo-
calization of the nucleolar protein NPM/B23; (iii) dis-
ruption of the nucleolar ultrastructure. It should be
noted that under acute stress, not only the “canoni-
cal” p53-dependent pathway with phosphorylation of
TIF-IA – a protein that plays a key role in the initi-
ation of ribosomal RNA transcription – but also an
alternative mechanism – proteasomal degradation of
TIF-IA, which leads to a paradoxical increase in the
nucleolus and activation of NF-κB, may be possible.
This mechanism leads to the increase in the nucleolar
size and inhibition of rDNA transcription[33]. Ingen-
eral, nucleolar stress appears as a cellular response to
disruptions in ribosome biogenesis, characterized by
the changes in the structure and function of the nu-
cleolus, activation of signaling pathways, and aimed
at maintaining cellular homeostasis. Therefore, un-
der the toxic effect of Cu
2+
, we observe intracellular
changes in astrocytes that could reflect the develop-
ment of nucleolar stress. Additionally, morphological
changes in the nucleolus under the action of Cu
2+
begin earlier than accumulation of the GRP78 pro-
tein, which is a luminal ER chaperone and one of
the markers of ER stress. Apparently, nucleolus could
be considered as a universal stress sensor that inte-
grates various exposures and triggers a unified re-
sponse.
Abbreviations
Cu
2+
copper ions
DFC dense fibrillar component
ER endoplasmic reticulum
FC fibrillar center
GC granular component
GRP78 endoplasmic reticulum stress marker
protein
NPM/B23 nucleophosmin
PBS phosphate-buffered saline
Contributions
E. V. Stelmashook and N. K. Isaev: concept and su-
pervision of the work. E. E. Genrikhs, E. V. Stelma-
shook, N. K. Isaev, O. P. Alexandrova, M. R. Kapkaeva,
S. A. Golyshev, and A. E. Lapieva: conducting exper-
iments, data processing, and analysis. E. A. Smirno-
va, N. K. Isaev, and E. V. Stelmashook: discussion of
the research results. N. K. Isaev: writing of the text.
E. E. Genrikhs, E. V. Stelmashook, and E. A. Smirnova:
editing of the article text.
Funding
This work was carried out under the planned theme
of the Federal State Budgetary Scientific Institution
“Russian Center of Neurology and Neurosciences.
Ethics approval and consent to participate
All applicable international, national, and/or insti-
tutional principles of care and use of animals were
followed. All experimental procedures with animals
were conducted in compliance with bioethical stan-
dards for working with laboratory animals in accor-
dance with the Recommendation of the Collegium of
the Eurasian Economic Commission dated November
14, 2023, no. 33 “On the Guidelines for Working with
Laboratory (Experimental) Animals in the Conduct of
Preclinical (Nonclinical) Studies”, as well as guided by
the “Rules for Working with Laboratory Rodents and
Rabbits” (GOST 33216-2014). All methods of isolation
and culturing comply with ethical standards approved
by the legal acts of the Russian Federation, principles
of the Basel Declaration, and position on the ethics of
using animals in research. The experimental protocols
were approved by the Ethics Committee of the Re-
search Center of Neurology (Protocol no. 5-5/22 dated
June 1, 2022).
Conflict of interest
The authors of this work declare that they have no
conflicts of interest.
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