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Regulation of nuclear transport of the transcriptional factor REST improves axon regeneration in peripheral nerves

  • Authors:
    • Takamaru Suzuki
    • Kiyohito Naito
    • Daisuke Kubota
    • Yuji Ueno
    • Takako Negishi‑Koga
    • Yasuhiro Yamamoto
    • So Kawakita
    • Norizumi Imazu
    • Kenjiro Kawamura
    • Nobutaka Hattori
    • Muneaki Ishijima
  • View Affiliations / Copyright

    Affiliations: Department of Medicine for Orthopaedics and Motor Organ, Juntendo University Graduate School of Medicine, Tokyo 113‑8421, Japan, Department of Neurology, Graduate School of Medical Sciences, University of Yamanashi, Chuo‑shi, Yamanashi 409‑3898, Japan, Department of Orthopaedics, Juntendo University Faculty of Medicine, Tokyo 113‑8421, Japan, Department of Neurology, Juntendo University Faculty of Medicine, Tokyo 113‑8421, Japan
    Copyright: © Suzuki et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 338
    |
    Published online on: October 1, 2025
       https://doi.org/10.3892/mmr.2025.13703
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Abstract

The expression of repressor element 1 silencing transcription factor (REST) in peripheral nerves increases with age, which leads to a decline in axon regeneration. However, the detailed mechanisms behind the decline in axon regeneration with age remain to be elucidated. The present study investigated the mechanism of nuclear transport of REST using hydrogen (H2), which has neuroprotective effects. First, aged mice as an animal model and REST‑overexpressed (REST‑OE) cells generated from the NIH‑3T3 fibroblast cell line as a cellular model were treated with H2 to examine REST expression and growth‑associated protein 43 (GAP43) as an axon regeneration marker. Subsequently, to examine differences in the localization of REST expression, in vitro cell lines were fractionated into cytoplasmic and nuclear fractions for immunofluorescence staining and western blotting, and REST expression was quantified. Furthermore, to investigate the mechanisms of nuclear transport, REST nuclear transport proteins (REST‑interacting LIM domain protein, Huntingtin and dynactin subunit 1/p150Glued) and the autophagy‑related protein LC3 were semi‑quantified by western blotting. REST expression was decreased, and GAP43 expression was increased following H2 administration in animal models and REST‑OE cells. REST intracellular localization analysis revealed that REST expression was significantly increased in the cytoplasm and significantly decreased in the nucleus in the REST‑OE + H2 group compared with the REST‑OE group. Furthermore, the present findings revealed that the addition of H2 resulted in a significant decrease in several REST nuclear transport proteins, subsequently suppressing the nuclear translocation of REST. These findings suggest that regulation of the nuclear transport of REST by H2 improves the decline in axon regeneration.
View Figures

Figure 1

REST and GAP43 expression in SN of
the young, aged and aged + H2 groups. The aged group was
compared with the young group, and the aged + H2 group
was compared with the aged group. The graphs show the
quantification of relative protein and mRNA abundance. Mean ± SD,
n=6 mice per group. *P<0.05, **P<0.01, ***P<0.001 and
****P<0.0001 (one-way ANOVA). (A) qPCR analysis of Rest
expression. (B) qPCR analysis of Gap43 expression. (C)
Western blot analysis of REST and GAP43 expression. (D)
Histochemical assessment of REST expression by immunofluorescence
staining. Scale bar, 100 µm. (E) Histochemical assessment of GAP43
expression by immunofluorescence staining. Scale bar, 100 µm.
GAP43, growth-associated protein 43; H2, hydrogen; qPCR,
quantitative PCR; REST, repressor element 1 silencing transcription
factor; SN, sciatic nerves.

Figure 2

REST and GAP43 expression in the
control, REST-OE and REST-OE + H2 groups. The REST-OE
group was compared with the control group, and the REST-OE +
H2 group was compared with the REST-OE group. The graphs
show the quantification of relative protein and mRNA abundance.
Mean ± SD, n=3 per group. *P<0.05, **P<0.01 and
****P<0.0001 (one-way ANOVA). (A) qPCR analysis of Rest
expression. (B) qPCR analysis of Gap43 expression. (C)
Western blot analysis of REST and GAP43 expression. GAP43,
growth-associated protein 43; H2, hydrogen; qPCR,
quantitative PCR; REST, repressor element 1 silencing transcription
factor; REST-OE, REST-overexpressed.

Figure 3

REST expression in the cytoplasm and
nucleus of the control, REST-OE and REST-OE + H2 groups.
The REST-OE group was compared with the control group, and the
REST-OE + H2 group was compared with the REST-OE group.
Mean ± SD, n=30 cells for (A-C), n=3 per group for (D-F).
*P<0.05, **P<0.01 and ****P<0.0001 (one-way ANOVA). (A)
Immunofluorescence staining of REST expression. REST is labeled in
green. Scale bar, 50.0 µm. (B) Histochemical assessment of REST
expression in the cytoplasm by immunofluorescence staining. (C)
Histochemical assessment of REST expression in the nucleus by
immunofluorescence staining. (D) Western blot analysis of REST
expression in the cytoplasm. (E) Western blot analysis of REST
expression in the nucleus. (F) Ratio of REST expression in the
nucleus to that in the cytoplasm. H2, hydrogen; REST,
repressor element 1 silencing transcription factor; REST-OE,
REST-overexpressed.

Figure 4

RILP, Huntingtin,
DCTN1/p150Glued and LC3 expression in the control,
REST-OE and REST-OE + H2 groups evaluated by western
blotting. The REST-OE group was compared with the control group,
and the REST-OE + H2 group was compared with the REST-OE
group. The graphs show the semi-quantification of relative protein
expression. Mean ± SD, n=3 per group. **P<0.01, ***P<0.001
and ****P<0.0001 (one-way ANOVA). (A) RILP expression. (B)
Huntingtin expression. (C) DCTN1/p150Glued expression.
(D) LC3 expression. DCTN1, dynactin subunit 1; H2,
hydrogen; N.S., no significant difference; REST, repressor element
1 silencing transcription factor; REST-OE, REST-overexpressed;
RILP, REST-interacting LIM domain protein.

Figure 5

RILP, Huntingtin,
DCTN1/p150Glued and LC3 expression in the cytoplasm of
the control, REST-OE and REST-OE + H2 groups evaluated
by western blotting. The REST-OE group was compared with the
control group, and the REST-OE + H2 group was compared
with the REST-OE group. The graphs show the semi-quantification of
relative protein expression. Mean ± SD, n=3 per group. *P<0.05,
**P<0.01 and ****P<0.0001 (one-way ANOVA). (A) RILP
expression. (B) Huntingtin expression. (C)
DCTN1/p150Glued expression. (D) LC3 expression. DCTN1,
dynactin subunit 1; H2, hydrogen; N.S., no significant
difference; REST, repressor element 1 silencing transcription
factor; REST-OE, REST-overexpressed; RILP, REST-interacting LIM
domain protein.

Figure 6

RILP, Huntingtin,
DCTN1/p150Glued and LC3 expression in the nucleus of the
control, REST-OE and REST-OE + H2 groups evaluated by
western blotting. The REST-OE group was compared with the control
group, and the REST-OE + H2 group was compared with the
REST-OE group. The graphs show the semi-quantification of relative
protein expression. Mean ± SD, n=3 per group. The data were
analyzed using one-way ANOVA. (A) RILP expression. (B) Huntingtin
expression. (C) DCTN1/p150Glued expression. (D) LC3
expression. DCTN1, dynactin subunit 1; H2, hydrogen;
N.S., no significant difference; REST, repressor element 1
silencing transcription factor; REST-OE, REST-overexpressed; RILP,
REST-interacting LIM domain protein.
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Copy and paste a formatted citation
Spandidos Publications style
Suzuki T, Naito K, Kubota D, Ueno Y, Negishi‑Koga T, Yamamoto Y, Kawakita S, Imazu N, Kawamura K, Hattori N, Hattori N, et al: Regulation of nuclear transport of the transcriptional factor REST improves axon regeneration in peripheral nerves. Mol Med Rep 32: 338, 2025.
APA
Suzuki, T., Naito, K., Kubota, D., Ueno, Y., Negishi‑Koga, T., Yamamoto, Y. ... Ishijima, M. (2025). Regulation of nuclear transport of the transcriptional factor REST improves axon regeneration in peripheral nerves. Molecular Medicine Reports, 32, 338. https://doi.org/10.3892/mmr.2025.13703
MLA
Suzuki, T., Naito, K., Kubota, D., Ueno, Y., Negishi‑Koga, T., Yamamoto, Y., Kawakita, S., Imazu, N., Kawamura, K., Hattori, N., Ishijima, M."Regulation of nuclear transport of the transcriptional factor REST improves axon regeneration in peripheral nerves". Molecular Medicine Reports 32.6 (2025): 338.
Chicago
Suzuki, T., Naito, K., Kubota, D., Ueno, Y., Negishi‑Koga, T., Yamamoto, Y., Kawakita, S., Imazu, N., Kawamura, K., Hattori, N., Ishijima, M."Regulation of nuclear transport of the transcriptional factor REST improves axon regeneration in peripheral nerves". Molecular Medicine Reports 32, no. 6 (2025): 338. https://doi.org/10.3892/mmr.2025.13703
Copy and paste a formatted citation
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Spandidos Publications style
Suzuki T, Naito K, Kubota D, Ueno Y, Negishi‑Koga T, Yamamoto Y, Kawakita S, Imazu N, Kawamura K, Hattori N, Hattori N, et al: Regulation of nuclear transport of the transcriptional factor REST improves axon regeneration in peripheral nerves. Mol Med Rep 32: 338, 2025.
APA
Suzuki, T., Naito, K., Kubota, D., Ueno, Y., Negishi‑Koga, T., Yamamoto, Y. ... Ishijima, M. (2025). Regulation of nuclear transport of the transcriptional factor REST improves axon regeneration in peripheral nerves. Molecular Medicine Reports, 32, 338. https://doi.org/10.3892/mmr.2025.13703
MLA
Suzuki, T., Naito, K., Kubota, D., Ueno, Y., Negishi‑Koga, T., Yamamoto, Y., Kawakita, S., Imazu, N., Kawamura, K., Hattori, N., Ishijima, M."Regulation of nuclear transport of the transcriptional factor REST improves axon regeneration in peripheral nerves". Molecular Medicine Reports 32.6 (2025): 338.
Chicago
Suzuki, T., Naito, K., Kubota, D., Ueno, Y., Negishi‑Koga, T., Yamamoto, Y., Kawakita, S., Imazu, N., Kawamura, K., Hattori, N., Ishijima, M."Regulation of nuclear transport of the transcriptional factor REST improves axon regeneration in peripheral nerves". Molecular Medicine Reports 32, no. 6 (2025): 338. https://doi.org/10.3892/mmr.2025.13703
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