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Changes in the endoplasmic reticulum‑mitochondria communication in dermal fibroblasts from early‑stage bipolar disorder patients: Skin‑brain axis as a new route to understand the pathophysiology of mental illness?

  • Authors:
    • Ana Catarina Pereira
    • Ana Patrícia Marques
    • Rosa Resende
    • Laura Serrano‑cuñarro
    • Margarida Caldeira
    • Tânia Fernandes
    • Mariana Batista
    • António Macedo
    • Joana Barbosa De Melo
    • Nuno Madeira
    • Cláudia Cavadas
    • Maria Teresa Cruz
    • Cláudia Fragão Pereira
  • View Affiliations / Copyright

    Affiliations: Center for Neuroscience and Cell Biology (CNC‑UC), Center for Innovative Biomedicine and Biotechnology (CiBB), University of Coimbra, 3004‑504 Coimbra, Portugal, Clinical and Academic Center of Coimbra (CACC), 3004‑561 Coimbra, Portugal, Faculty of Medicine, University of Coimbra, 3000‑370 Coimbra, Portugal
    Copyright: © Pereira et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 213
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    Published online on: September 30, 2025
       https://doi.org/10.3892/ijmm.2025.5654
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Abstract

Compromised cellular resilience in bipolar disorder (BD) has been associated with structural brain changes and cognitive deficits caused by perturbation of redox status, endoplasmic reticulum (ER) stress and innate immunity. These crucial cellular events are regulated by the ER‑mitochondria close contacts at mitochondria‑associated membranes (MAM) through Ca2+ transfer and lipids exchange between these organelles. The present study aimed to investigate the structural and functional alterations in MAM during BD early stages using patient‑ and control‑derived cellular models, namely dermal fibroblasts. Morphological alterations in close ER‑mitochondria contacts at MAM occur in BD cells and correlate with functional changes, as shown by lipid droplets accumulation. The MAM dysfunction in BD cells parallels changes in Ca2+ homeostasis, namely inhibition of store‑operated Ca2+ entry (SOCE), ER Ca2+ depletion and attenuation of ER‑mitochondria Ca2+ transfer, as well as enhanced ER and oxidative stress and NOD‑like receptor family pyrin domain‑containing 3 (NLRP3) inflammasome activation leading to sterile inflammation. The absence of inflammasome activation upon lipopolysaccharide exposure supports the compromised ability of BD cells (fibroblasts as well as monocytes) to deal with stressful conditions. In conclusion, MAM disruption is highlighted as a potential pathophysiological mechanism driving impaired cellular resilience in BD. Skin fibroblasts are a particularly attractive cellular model for studying mental illnesses, such as BD, due to the shared developmental origin of epidermal and neural tissues. The ectodermal origins of the skin‑brain axis have been proposed as a novel route for understanding brain development, neurodevelopmental conditions and behavior modulation. 
View Figures

Figure 1

ER-mitochondria contacts in primary
fibroblasts derived from patients with BD and healthy controls.
Colocalization of Mitotracker and ER-Tracker fluorescent probes was
evaluated by live cell imaging. (A) Representative confocal
microscopy images of Mitotracker (green) and ER-Tracker (red)
immunoreactivity and nuclei labelling with Hoechst 33342 (blue;
scale bar, 50 μm). (B) The mitochondrial fraction
(Mitotracker) that colocalizes with the ER (ER-Tracker) was
measured with the Mander's coefficient of colocalization using the
ImageJ software. Colocalization of the IP3R (ER marker) and the
VDAC (mitochondrial marker) was evaluated by immunocytochemistry.
(C) Representative confocal microscopy images of VDAC (green) and
IP3R (red) immunoreactivity and nuclei labelling with Hoechst 33342
(blue; scale bar, 50 μm). (D) The mitochondrial fraction
(VDAC) that colocalizes with the ER (IP3R) was measured with the
Mander's coefficient of colocalization (ImageJ software). Data
represent the means ± standard error of the mean of results
obtained by the analysis of five images from five participants per
group. The number of IP3R/VDAC dots per cell was determined by the
PLA. (E) Representative confocal microscopy images of PLA signal
IP3R/VDAC dots (red) and nuclei labelling with Hoechst 33342 (blue;
scale bar, 50 μm). (F) PLA fluorescent quantification was
performed using the Particle Analysis function of ImageJ software.
Data represent the means ± standard error of the mean of results
obtained by the analysis of five images from five controls and four
patients. The mitochondrial network was analyzed by
immunocytochemistry. (G) Representative confocal microscopy images
of TOM20 immunoreactivity (green) and Hoechst 33342-stained nuclei
(blue; scale bar, 50 μm). (H) TOM20 staining was analyzed
using the ImageJ software. Data represent the means ± standard
error of the mean of results obtained by the analysis of five
images collected from four participants per group. (I) The
mitochondrial area was quantified in the confocal images of
Mitotracker (Fig. 1A) using a
macro designed in the ImageJ software. Statistical significance of
differences between experimental groups was determined by unpaired
Student's t-test. The ER-mitochondria contacts were evaluated by
transmission electron microscopy. (J) Representative images of the
ER-mitochondria contacts (arrow; scale bar, 200 nm). (K)
ER-mitochondria distance was measured using the ImageJ software.
(L) Distribution of the ER-mitochondria contacts: close contacts
(≤20 nm; MAM) and large contacts (>20 nm). Data represent the
means ± standard error of the mean of results obtained by the
analysis of at least five images collected from three participants
per group. Statistical significance between groups was determined
using the unpaired Student's t-test *P<0.05,
**P<0.01; and the comparison of close (≤20 nm) and
large contacts (>20 nm) between the two groups was obtained
using the two-way ANOVA test, followed by the Sidak's post hoc
test: *P<0.05. ER, endoplasmic reticulum; BD, bipolar
disorder; IP3R, inositol trisphosphate receptor; VDAC,
voltage-dependent anion channel; PLA, proximity ligation assay;
TOM20, translocase of outer mitochondrial membrane 20; MAM,
mitochondria-associated membranes.

Figure 2

Levels of components of
ER-mitochondria contacts (Mfn2, VDAC, VAPB, PTPIP51 and Sigma-1R)
and ER stress-induced UPR markers (GRP78, ATF4 and ATF6) in primary
fibroblasts derived from patients with BD and healthy controls.
Protein levels of (A and B) Mfn2, (C and D) VDAC, (C, E and F)
ATF6, (G and H) VAPB, (G and I) GRP78, (G and J) ATF4, (K and L)
PTPIP51 and (K and M) Sigma-1R, were quantified by western blotting
in total cellular extracts obtained from primary fibroblasts
derived from patients with BD and healthy controls. β-actin was
used to control protein loading and to normalize the levels of the
proteins of interest. Data represent the means ± standard error of
the mean of results obtained in samples from five healthy controls
and four patients with BD. Statistical differences between
fibroblasts from patients with BD and healthy controls were
analyzed using the unpaired Student's t-test:
*P<0.05, **P<0.01. (A) The Mfn2
antibody was incubated in the first western blotting. VDAC and ATF6
antibodies were both incubated in the second western blotting. (C)
The ATF6 antibody, which recognizes both full-length (75 kDa) and
cleaved protein (50 kDa), was incubated on the VDAC western
blotting membrane, after a stripping procedure. (G) The VAPB, GRP78
and ATF4 antibodies were labelled on the third western blotting.
Upon incubation with the VAPB antibody, the western blotting
membrane was then incubated with ATF4 and GRP78, after a stripping.
(K) PTPIP51 and Sigma-1R antibodies were both incubated in the
fourth western blotting. The Sigma-1R antibody was incubated on the
PTPIP51 western blotting membrane, after a stripping. ER,
endoplasmic reticulum; UPR, unfolded protein response; BD, bipolar
disorder; MAM, mitochondria-associated membranes; Mfn2, Mitofusin
2; VDAC, voltage-dependent anion channel; VAPB, vesicle-associated
membrane protein associated protein B; PTPIP51, protein tyrosine
phosphatase-interacting protein-51; GRP78, glucose-regulated
protein 78; ATF4. activating transcription factor 4;
ATF6-activating transcription factor 6.

Figure 3

Lipid droplets accumulation and
Ca2+ dynamics in primary fibroblasts derived from
patients with BD and healthy controls. The number of lipid droplets
per cell was determined using the LipidTOX fluorescent probe. (A)
Representative confocal microscopy images of lipid droplets spots
(red) and nuclei labelling with Hoechst 33342 (blue; scale bar, 50
μm). (B) Quantification of LipidTOX fluorescent was
performed using the Spot function of Imaris Microscopy Image
Analysis Software. Data represent the means ± standard error of the
mean of results obtained by the analysis of at least five images
collected from four participants per group. IP3R-mediated ER
Ca2+ efflux and its influx into mitochondria were
measured by SCCI with the fluorescent probes (C) Fluo-4 and (D)
Rhod-2, respectively, after treatment with histamine (100
μM). (E and F) The SCCI results were baseline-corrected and
normalized to calculate the normalized fluorescent calcium signals.
Data were expressed as ΔF/F0, where ΔF=F-F0. F represents the
highest post-stimulus value and F0 the mean baseline level. Data
represent the means ± standard error of the mean of results
obtained from 15-30 cells from each of the four participants per
group. (G) SOCE was evaluated by measuring Ca2+
oscillations with Fluo-4 after thapsigargin (1 μM,
TG)-induced ER Ca2+ depletion in Ca2+-free
medium and after addition of 2 mM CaCl2 to the
extracellular medium. (H and I) The SCCI results were
baseline-corrected and normalized to calculate the normalized
fluorescent calcium signals. Data were expressed as ΔF/F0, where
ΔF=F-F0. F represents the highest post-stimulus value and F0 the
mean baseline level. Data represent the means ± standard error of
the mean of results obtained from 15-30 cells from each of the four
participants per group. (J and K) MCU and (J and L) STIM1 protein
levels were evaluated in total cellular extracts obtained from
control and BD fibroblasts through western blotting. β-actin was
used to control protein loading and to normalize the levels of the
proteins of interest. Data represent the means ± standard error of
the mean of results obtained in samples from five healthy controls
and four patients with BD. The STIM1 antibody was incubated on the
Sigma-1R western blotting membrane, after a stripping. Statistical
difference between groups was evaluated with unpaired Student's
t-test (B, D, H, I and L) or with Mann-Whitney non-parametric test
(F and K): *P<0.05; **P<0.01;
***P<0.001; ****P<0.0001. BD, bipolar
disorder; IP3R, inositol trisphosphate receptor; ER, endoplasmic
reticulum; SCCI, single cell calcium imaging; STIM1, stromal
interaction molecule 1; MCU, mitochondrial Ca2+
uniporter protein.

Figure 4

Redox state in primary fibroblasts
derived from patients with BD and healthy controls. The
accumulation of ROS in control and BD fibroblasts was assessed
using the CellROX fluorescent probe. (A) Representative microscopy
images of CellROX immunoreactivity (green; scale bar, 100
μm). (B) Quantification of the CellROX green staining using
ImageJ software. Data represent the means ± standard error of the
mean of results obtained in cells from three participants per
group. At least five images from each participant were analyzed.
(C) The levels of mitochondrial ROS were measured by using the
MitoPY fluorescent probe. Data represent the means ± standard error
of the mean of results obtained in cells from five healthy controls
and four patients with BD. (D) The activity of SOD2 mitochondrial
antioxidant enzyme was analyzed in primary fibroblasts derived from
patients with BD and healthy controls by using a commercial kit.
Data represent the means ± standard error of the mean of results
obtained in total lysates obtained from cells isolated from four
participants per group. The statistical significance of differences
between control and BD fibroblasts was determined using the
unpaired Student's t-test: *P<0.05;
**P<0.01. BD, bipolar disorder; ROS, reactive oxygen
species; SOD2, superoxide dismutase 2.

Figure 5

NLRP3 inflammasome activity and
inflammatory status in primary fibroblasts and monocytes derived
from patients with BD and healthy controls. Basal mRNA levels of
(A) NLRP3 and pro-inflammatory cytokines (B) pro-IL-1β, (C) TNFα,
(D) IL-6 and (E) IL-8 were evaluated by reverse
transcription-quantitative PCR in fibroblasts from patients with BD
and healthy controls. (F) Baseline levels of caspase-1 activity in
control and BD fibroblasts. (G) Caspase-1 activity was analyzed in
total extracts from control and BD fibroblasts treated in the
absence or presence of 5 μg/ml LPS for 32 h. (H) Fold
increase of caspase-1 activity was calculated by normalizing the
levels of caspase-1 activity in control and BD fibroblasts treated
with LPS to the baseline values. Data represent the means ±
standard error of the mean of results obtained in cells from 3-5
participants per group. (I) IL-1β levels in supernatants from
control and BD fibroblasts incubated in the presence or absence of
5 μg/ml LPS for 32 h, were quantified using an ELISA kit an
expressed as pg/ml. (J) Fold increase of IL-1β secretion was
calculated by the difference between LPS-treated and basal values.
Data represent the means ± standard error of the mean of results
obtained in cells from three controls and four patients with BD.
(K) IL-1β levels in supernatants from primary monocytes derived
from healthy controls and patients with BD incubated in the
presence or absence of 1 μg/ml LPS for 32 h, were quantified
by ELISA. Results were normalized to the basal levels and represent
the means ± standard error of the mean of results obtained in cells
from four participants per group. (L) IL-1β secretion in monocytes
was also calculated as the fold increase observed upon LPS
treatment. Data represent means ± standard error of the mean of
results obtained in cells from four participants per group.
Statistical significance of differences between groups was
determined by the unpaired Student's t-test (A-F, H, J and L):
*P>0.05; **P>0.01; significance between
both basal and LPS-treated cells within each experimental group and
between the two groups were obtained using the two-way ANOVA test,
followed by the Tukey's post hoc test (G, I and K):
*P>0.05; **P>0.01. NLRP3, NOD-like
receptor family pyrin domain-containing 3; BD, bipolar disorder;
LPS, lipopolysaccharide.

Figure 6

The MAM hypothesis for BD.
Fibroblasts derived from patients diagnosed with BD exhibit
increased ER-mitochondria apposition and markedly higher percentage
of close ER-mitochondria contacts sites (MAM) in comparison with
healthy controls, in particular those <20 nm. This structural
alteration in the ER-mitochondria juxtaposition directly affects
the main functions of MAM, which are the inter-organelle transfer
of Ca2+ and lipids. As a consequence, ER-mitochondria
lipids transfer is enhanced increasing the formation of lipid
droplets while Ca2+ transfer is inhibited.
Concomitantly, BD fibroblasts exhibit lower SOCE and higher
oxidative stress levels due to the inhibition of SOD2 mitochondrial
antioxidant enzyme and accumulation of ROS in the cytosol and in
the mitochondria. In addition to these alterations in
Ca2+ and redox homeostasis as well as in lipid
metabolism, BD-specific changes in MAM are also associated with
induction of ER stress and sterile inflammation. A basal
pro-inflammatory status in BD is supported by the upregulation of
pro-inflammatory cytokines, which seems to be implicated in the
diminished susceptibility of both fibroblasts and monocytes derived
from patients with BD to inflammatory stimuli, as shown by the
lower activation of the NLRP3 inflammasome in response to LPS. PM,
plasma membrane; MAM, mitochondria-associated membranes; BD,
bipolar disorder; MCU, mitochondrial calcium uniport; Cyt C,
cytochrome c; TCA cycle, tricarboxylic acid cycle; ADP,
adenosine diphosphate; ATP, adenosine triphosphate; O2,
superoxide anion; O2, oxygen;
H2O2, hydrogen peroxide; SOD2, superoxide
dismutase 2; ROS, reactive oxygen species; VDAC, voltage-dependent
anion channels; Mfn2, mitofusin 2; IP3R, inositol trisphosphate
receptor; Sig-1R, sigma-1 receptor; ACAT1, Acetyl-CoA
acetyltransferase 1; CE, cholesterol ester; SOCE, store-operated
calcium entry; STIM1, stromal interaction molecule 1; UPR, unfolded
protein response; ER, endoplasmic reticulum; NLRP3, NOD-like
receptor family pyrin domain-containing 3; BD, bipolar disorder;
LPS, lipopolysaccharide.
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Copy and paste a formatted citation
Spandidos Publications style
Pereira AC, Marques AP, Resende R, Serrano‑cuñarro L, Caldeira M, Fernandes T, Batista M, Macedo A, De Melo JB, Madeira N, Madeira N, et al: Changes in the endoplasmic reticulum‑mitochondria communication in dermal fibroblasts from early‑stage bipolar disorder patients: Skin‑brain axis as a new route to understand the pathophysiology of mental illness?. Int J Mol Med 56: 213, 2025.
APA
Pereira, A.C., Marques, A.P., Resende, R., Serrano‑cuñarro, L., Caldeira, M., Fernandes, T. ... Pereira, C. (2025). Changes in the endoplasmic reticulum‑mitochondria communication in dermal fibroblasts from early‑stage bipolar disorder patients: Skin‑brain axis as a new route to understand the pathophysiology of mental illness?. International Journal of Molecular Medicine, 56, 213. https://doi.org/10.3892/ijmm.2025.5654
MLA
Pereira, A. C., Marques, A. P., Resende, R., Serrano‑cuñarro, L., Caldeira, M., Fernandes, T., Batista, M., Macedo, A., De Melo, J. B., Madeira, N., Cavadas, C., Cruz, M., Pereira, C."Changes in the endoplasmic reticulum‑mitochondria communication in dermal fibroblasts from early‑stage bipolar disorder patients: Skin‑brain axis as a new route to understand the pathophysiology of mental illness?". International Journal of Molecular Medicine 56.6 (2025): 213.
Chicago
Pereira, A. C., Marques, A. P., Resende, R., Serrano‑cuñarro, L., Caldeira, M., Fernandes, T., Batista, M., Macedo, A., De Melo, J. B., Madeira, N., Cavadas, C., Cruz, M., Pereira, C."Changes in the endoplasmic reticulum‑mitochondria communication in dermal fibroblasts from early‑stage bipolar disorder patients: Skin‑brain axis as a new route to understand the pathophysiology of mental illness?". International Journal of Molecular Medicine 56, no. 6 (2025): 213. https://doi.org/10.3892/ijmm.2025.5654
Copy and paste a formatted citation
x
Spandidos Publications style
Pereira AC, Marques AP, Resende R, Serrano‑cuñarro L, Caldeira M, Fernandes T, Batista M, Macedo A, De Melo JB, Madeira N, Madeira N, et al: Changes in the endoplasmic reticulum‑mitochondria communication in dermal fibroblasts from early‑stage bipolar disorder patients: Skin‑brain axis as a new route to understand the pathophysiology of mental illness?. Int J Mol Med 56: 213, 2025.
APA
Pereira, A.C., Marques, A.P., Resende, R., Serrano‑cuñarro, L., Caldeira, M., Fernandes, T. ... Pereira, C. (2025). Changes in the endoplasmic reticulum‑mitochondria communication in dermal fibroblasts from early‑stage bipolar disorder patients: Skin‑brain axis as a new route to understand the pathophysiology of mental illness?. International Journal of Molecular Medicine, 56, 213. https://doi.org/10.3892/ijmm.2025.5654
MLA
Pereira, A. C., Marques, A. P., Resende, R., Serrano‑cuñarro, L., Caldeira, M., Fernandes, T., Batista, M., Macedo, A., De Melo, J. B., Madeira, N., Cavadas, C., Cruz, M., Pereira, C."Changes in the endoplasmic reticulum‑mitochondria communication in dermal fibroblasts from early‑stage bipolar disorder patients: Skin‑brain axis as a new route to understand the pathophysiology of mental illness?". International Journal of Molecular Medicine 56.6 (2025): 213.
Chicago
Pereira, A. C., Marques, A. P., Resende, R., Serrano‑cuñarro, L., Caldeira, M., Fernandes, T., Batista, M., Macedo, A., De Melo, J. B., Madeira, N., Cavadas, C., Cruz, M., Pereira, C."Changes in the endoplasmic reticulum‑mitochondria communication in dermal fibroblasts from early‑stage bipolar disorder patients: Skin‑brain axis as a new route to understand the pathophysiology of mental illness?". International Journal of Molecular Medicine 56, no. 6 (2025): 213. https://doi.org/10.3892/ijmm.2025.5654
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