The present study was approved by the Animal Care Committee of Juntendo University, Tokyo, Japan (registration no. 1555; approval no. 2023202, date of approval: December 11, 2023).

Ten male C57BL/6J mice (Young group: 10-week-old mice, n=5; Aged group: 70-week-old mice, n=5) for immunofluorescence staining; 10 male C57BL/6J mice (Young group: 8-week-old mice, n=5; Aged group: 78-week-old mice, n=5) for qPCR and western blotting; and 6 male aged C57BL/6J mice for treatment of GP130 receptor agonist-1 (Ga1, Selleck, Tokyo, Japan) analysis (78-week-old, treated with vehicle, n=3; treated with Ga1, n=3) were purchased from JAPAN SLC, Inc. Mice were housed at five animals/cage in a sterile environment controlled at a temperature of 22± 2°C, humidity of 40–60%, and 12-h light and dark cycle, and were given water and CRF-1 gamma-ray-irradiated (15 kGy) (Oriental Yeast Co., Ltd.) ad libitum. Ten mice for immunofluorescence staining, and 10 mice for qPCR and western blotting were monitored only once when they were carried out, then they were sacrificed immediately. Six mice treated with vehicle and Ga1 were monitored 5 times, when they were carried out, before and after treatment, 1 h after treatment and 24 h after treatment. Humane endpoints were defined as a loss of 20% of body weight, difficulty breathing, coughing, wheezing, severe diarrhea, vomiting, flaccid or spastic paralysis, convulsions, coupled with body temperature significantly below normal. No mice were reached humane endpoints. All mice were anaesthetized using 5% isoflurane and sacrificed by cervical dislocation and used for each experiment. Death was confirmed when breathing and heart rate had stopped.

There are some reports that low estrogen affects peripheral neuropathy. Because estrogen decreases with age, males with less estrogen fluctuations and susceptibility to estrogen were used in this study.

Mouse embryonic fibroblast cell line NIH3T3 (Cell Line Service) was cultured in a humidified incubator with 5% CO₂ at 37°C. The culture medium was Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F-12) (Sigma-Aldrich) supplemented with 10% fetal bovine serum and penicillin (100 U/ml).

Using cultured cell lines, REST expression-regulated cells were constructed. The lentiviral vector used to overexpress REST in our study was constructed by VectorBuilder Inc. The vector ID is VB900006-3284rup. Meanwhile, the mock plasmid acted as the negative control. They were propagated in Escherichia coli DH5α. All plasmid DNA used for transfection was isolated using QIAGEN^® Plasmid Maxi kit from propagated Escherichia coli. To make REST-overexpressed (REST-OE) cells, cells were transfected with isolated REST plasmid using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's instructions. To make REST-low expressed (siREST) cells, cells were transfected with REST-targeting siRNA (Sigma-Aldrich, SASI_Mm01_00196017) and control siRNA (Sigma-Aldrich, SIC001-10NMOL) using Lipofectamine RNAimax (Thermo Fisher Scientific) according to the manufacturer's instructions. REST-targeting siRNA can be purchased by registration and logging in to website of Sigma-Aldrich at https://www.sigmaaldrich.com/JP/ja/login?redirect=%2FJP%2Fja, and entering ‘REST’ into the gene search box and choosing the mouse at https://www.sigmaaldrich.com/JP/ja/semi-configurators/sirna?activeLink=selectAssays. Control siRNA can be purchased at https://www.sigmaaldrich.com/JP/ja/product/sigma/sic001.

The harvested sciatic nerve (SN) and dorsal root ganglia (DRG) were fixed in 4% paraformaldehyde at room temperature for 72 h and paraffin blocks were prepared. Immunofluorescence staining was performed to assess the expression of REST and growth associated protein 43 (GAP43). Tissue sections were prepared by cutting the harvested SN and DRG at a thickness of 3 µm. Samples were deparaffinized and autoclaved at 121°C for 10 min for antigen retrieval. After treatment with True View^TM (SP-8400, Vector, CA, USA) to suppress autofluorescence, samples were blocked using 2% bovine serum albumin (A2153, Sigma Aldrich, MO, USA) in PBS containing 0.05% Tween 20 for 30 min. Samples were then reacted with antibodies against the target proteins at 4°C for 15 h. After washing with Tris-buffered saline with Tween 20 (TBST), a goat anti-mouse IgG antibody labeled with Alexa Fluor 488 (A11001, Thermo Fisher Scientific) was used as a secondary antibody, and a rabbit IgG monoclonal antibody as a negative control. The intensity of fluorescence in each section was quantified in the photon counting mode using a fluorescence imaging microscope (Leica, TCSSP5). The antibodies used in the present study were against REST, a transcription factor that regulates the expression of nerve-specific proteins, and GAP43, a neuronal protein known for its important role in axonal outgrowth. Primary antibodies were as follows: rabbit polyclonal anti-REST (1:100, 22242-1-AP, ProteinTech, IL, USA), and rabbit polyclonal anti-GAP43 (1:100, 16971-1-AP, ProteinTech).

In the photon counting mode, fluorescence intensity (gray value) was measured at 10 randomly selected sites from the perikaryon in a region of interest set in a fluorescence-emitting area, and mean fluorescence intensity was calculated. Fluorescence intensity measured using each antibody was compared between the Young and Aged groups.

For western blotting, protein was extracted by 1×radio immunoprecipitation assay buffer. Equal amounts of proteins from the SN and DRG in animal models, and REST expression-regulated cells were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a poly vinylidene di-fluoride (PVDF) membrane by Trans-Blot Turbo (BIORAD). Non-specific sites were blocked with PVDF Blocking Reagent (Toyobo Co., Ltd.) for 1 h at room temperature following which the membrane was washed with TBST three times, for 10 min each. The blot was then incubated overnight at 4°C with appropriate primary antibody in solution 1 (Toyobo Co., Ltd.) according to the supplier's specific instructions. Primary antibodies were as follows: rabbit polyclonal anti-REST (1:1,000, 22242-1-AP; ProteinTech), rabbit polyclonal anti-GAP43 (1:1,000, 16971-1-AP; ProteinTech), rabbit polyclonal anti-glycoprotein 130 (GP130, 1:1,000, #3732; Cell Signaling), anti-signal transducer and activator of transcription 3 (STAT3, 1:1,000, #9132; Cell Signaling), anti-phosphorylated STAT3 (pSTAT3, 1:1,000, #9131; Cell Signaling), and mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2,000, sc-32233; Santa Cruz). The blots were washed with TBST and incubated with appropriate secondary antibody for 2 h at room temperature. After washing, Amersham Imager 680 (GE Healthcare Life SciencesA) was applied, and blot images were captured using a gel documentation system. Relative optical density of protein bands was analyzed using gel software image lab 3.0. The membranes were stripped and reprobed with GAPDH as a loading control.

Total RNA was isolated from the SN, DRG in animal models and REST expression-regulated cells by using RNeasy Microkit (Qiagen) in accordance with the manufacturer's instructions. Complementary DNA was synthesized using PrimeScript™ RT reagent Kit (Takara). Next, qPCR was performed with SYBR Green real-time PCR assay (Thermo Fisher Scientific) according to the ΔΔCq method. The expression levels of targets were normalized to GAPDH. Used primers are listed in Table I.

Young mice (n=5) and aged mice (n=5) were sacrificed by cervical dislocation on the day that SN and DRG were harvested. The expression of REST and GAP43 in SN and DRG was compared between the young and aged mice by immunofluorescence staining, qPCR, and western blotting.

The expression of interleukin 6 (IL6), IL6 receptor, GP130, janus kinase 1 (JAK1), and STAT3, which are components of the JAK1/STAT3 pathway involved in regulating GAP43 expression (15), was evaluated by qPCR in REST-regulated cells. The expression of GP130 was evaluated by western blot additionally. As the expression of GAP43 is promoted when STAT3 is phosphorylated by JAK1, we evaluated STAT3 and pSTAT3 by western blotting (16).

Since expression of JAK1/STAT3 pathway was investigated and GP130 was found to be important, we treated REST-OE cells with Ga1 to investigate the effect on axonal regeneration markers. The stock solution of Ga1 was prepared by transferring 5 mg to dimethyl sulfoxide (DMSO) at a concentration of 10 mM. Further dilutions were made fresh in cell culture medium. The final concentration of DMSO was 0.1% and Ga1 was 10 µM. After 48 h culture, REST-OE cells were incubated with 10 µM Ga1 for an additional 30 min at 37°C. After being treated by Ga1, the expression of REST, GP130, and GAP43 in REST-OE was evaluated by western blotting and qPCR. DMSO was used as the vehicle control.

Six 78-week-old mice were used and divided into a DMSO group (n=3) and Ga1 group (n=3). DMSO was used as the vehicle control. The Ga1 group mice received an intraperitoneal injection of Ga1 dissolved by DMSO at a dose of 10 mg/kg. Since 10 mg/kg Ga1 was used to mice to evaluate the nervous system in the previous study reported by Alam et al (17), a dose of 10 mg/kg was administered to mice in this study. Only one dose was given during the entire experimental period. The mice were sacrificed at 24 h after treatment and SN was harvested to confirm the effect of Ga1 on the expression of axon regeneration marker GAP43. The expression of REST and GAP43 in SN was evaluated by western blotting and qPCR.

Data are presented as the mean ± standard deviation and were analyzed for significant differences using unpaired Student's t-test, significance was defined as P<0.05 (Prism 7; GraphPad Software).

To investigate the difference in the expression of REST and GAP43 in young (Young group) and aged (Aged group) mice, the expression of REST and GAP43 in the SN and DRG were quantified by immunofluorescence staining, western blotting, and qPCR. Immunofluorescence staining revealed a significant increase in the fluorescence intensity of REST in the Aged group compared to the Young group in the SN (Young group 132.3±14.5; Aged group 189.4±12.1, P=0.0003) (Fig. 1A) and DRG (Young group 115.9±25.5; Aged group 174.7±41.1, P=0.026) (Fig. 1B). Furthermore, a significant decrease in the fluorescence intensity of GAP43 in the Aged group compared to the Young group was found in the SN (Young group 192.3±16.3; Aged group 119.2±9.0, P<0.0001) (Fig. 1C) and DRG (Young group 168.5±16.3; Aged group 89.0±45.0, P=0.005) (Fig. 1D).

Western blotting revealed a significant increase in the expression of REST in the Aged group compared to Young group in the SN (1.29±0.04-fold, P=0.002) (Fig. 1E) and DRG (3.57±0.52-fold, P=0.009) (Fig. 1F). On the other hand, a significant decrease was observed in the expression of GAP43 in the Aged group compared to the Young group in the SN (0.68±0.07-fold, P=0.026) (Fig. 1E) and DRG (0.74±0.09-fold, P=0.012) (Fig. 1F).

qPCR revealed a significant increase in the expression of REST in the Aged group compared to the Young group in the SN (2.12±0.46-fold, P=0.006) (Fig. 1G) and DRG (1.75±0.29-fold, P=0.009) (Fig. 1H). A significant decrease was found in the expression of GAP43 in the Aged group compared to the Young group in the SN (0.75±0.21-fold, P=0.031) (Fig. 1I). No significant difference between the expression of GAP43 in the Aged group and the Young group was observed in the DRG (0.99±0.21-fold, P=0.935) (Fig. 1J). GAP43 is known to be strictly regulated in terms of its expression at the mRNA level and protein level, by regulating mRNA stability post-transcriptionally and by post-translational modification including phosphorylation and palmitoylation (18). Future study may clarify whether GAP43 is degraded by post-translational modification. In addition, GAP43 mRNA and protein may be regulated by different mechanisms that are tissue-dependent, such as in the SN and DRG.

To determine whether REST plays a role in GAP43 expression, REST plasmid and siRNA were used to construct REST-OE cells and siREST cells using NIH3T3. Western blotting and qPCR revealed a significant increase in the expression of REST in REST-OE compared to the Control (protein: 18.8±5.5-fold, P=0.038; mRNA: 662.6±53.6-fold, P<0.0001) (Fig. 2A and B) and a significant decrease in the expression of REST in siREST compared to the Control (protein: 0.63±0.25-fold, P=0.026; mRNA: 0.33±0.18-fold, P<0.0001) (Fig. 2C and D). Therefore, REST-OE cells and siREST cells were used for the following studies. Interestingly, qPCR revealed a significant decrease in the expression of GAP43 in REST-OE and a significant increase in the expression of GAP43 in siREST compared to the Control (REST-OE: 0.58±0.31-fold, P=0.016; siREST: 2.60±0.50-fold, P<0.0001) (Fig. 2E and F). This relationship between REST and GAP43 supports the findings in the animal models.

To determine the role of REST in GAP43 expression, the involvement of molecules in the JAK1/STAT3 pathway was investigated. The expression levels of IL-6, IL-6 receptor, GP130, JAK1, and STAT3 were investigated using qPCR. qPCR revealed a significant increase in IL-6 expression in REST-OE and siREST compared to the Control (REST-OE: 1.98±0.18-fold, P=0.017; siREST: 2.81±0.26-fold, P=0.010) (Fig. 3A and B). There was no significant difference in IL-6 receptor expression between REST-OE and the Control (0.96±0.02-fold, P=0.184) (Fig. 3C), or between siREST and Control (1.20±0.29-fold, P=0.442) (Fig. 3D). A significant decrease in GP130 expression was observed in REST-OE, whereas a significant increase was observed in siREST compared to the Control (REST-OE: 0.52±0.01-fold, P=0.004; siREST: 2.26±0.11-fold, P=0.004) (Fig. 3E and F). JAK1 expression significantly decreased in REST-OE and significantly increased in siREST compared to the Control (REST-OE: 0.68±0.08-fold, P=0.003; siREST: 1.66±0.20-fold, P=0.005) (Fig. 3G and H). No significant difference in STAT3 expression was observed between REST-OE and Control (1.14±0.02-fold, P=0.095) (Fig. 3I), whereas a significant increase was noted in siREST compared to the Control (1.51±0.04-fold, P=0.012) (Fig. 3J). Since GP130 was considered to be an important molecule, GP130 expression was also evaluated by western blotting. As a result, a significant decrease in GP130 expression was observed in REST-OE, whereas a significant increase was observed in siREST compared to the Control (REST-OE: 0.69±0.04-fold, P=0.003; siREST: 1.48±0.17-fold, P=0.013) (Fig. 3K and L).

The expression of GAP43 is promoted when STAT3 is phosphorylated by JAK1 (16). Therefore, the expression of STAT3 and pSTAT3 protein was evaluated by western blotting. Western blotting revealed no significant difference between the expression of STAT3 in REST-OE and Control, however a significant decrease in the expression of pSTAT3 in REST-OE compared to the Control (STAT3: 1.10 ± 0.15-fold, P=0.459, pSTAT3: 0.76±0.04-fold, P=0.015) (Fig. 4A). Moreover, a significant increase in the expression of STAT3 and pSTAT3 was observed in siREST compared to the Control (STAT3: 1.28±0.08-fold; P=0.043, pSTAT3: 1.33±0.07-fold, P=0.033) (Fig. 4B). Furthermore, the ratio of phosphorylation/total STAT3 protein was significantly decreased in REST-OE compared to the Control (0.69±0.09-fold; P=0.005) (Fig. 4C), whereas the ratio of that was significantly increased in siREST compared to the Control (1.21±0.07-fold; P=0.005) (Fig. 4D). These findings suggest that the expression of STAT3 does not change, but the activation of STAT3 is low in REST-OE.

In summary, despite similar changes of IL6 and IL6 receptor in REST-OE and siREST, the expression of GP130, JAK1, and phosphorylation of STAT3 were decreased in REST-OE, whereas the expression of GP130, JAK1, and phosphorylation of STAT3 were increased in siREST. These findings suggest that REST may regulate the expression of GAP43 by the JAK1/STAT3 pathway via the expression of GP130 (Fig. 5).

As REST possibly regulates the expression of GAP43 by the JAK1/STAT3 pathway via the expression of GP130, we predicted that regulation of GP130-related molecular expression may change the expression of GAP43 in REST-OE cells. To investigate this hypothesis, we used Ga1, a GP130 agonist. The expression of REST, GP130, and GAP43 was investigated by western blotting and qPCR. Western blotting and qPCR revealed no significant difference between the expression of REST in Ga1 and DMSO control (protein: 1.01±0.03-fold, P=0.54; mRNA: 1.05±0.33-fold, P=0.85) (Fig. 6A and B), no significant difference between the expression of GP130 in Ga1 and a DMSO control (protein: 0.95±0.07-fold, P=0.28; mRNA: 0.92±0.13-fold, P=0.34) (Fig. 6A and C). Interestingly, a significant increase was observed in the expression of GAP43 in Ga1 compared to a DMSO control (protein: 1.41±0.10-fold, P=0.018; mRNA: 1.66±0.26-fold, P=0.040) (Fig. 6A and D). Thus, Ga1 enhanced GAP43 expression without changes of REST and GP130 expression.

Ga1 enhanced GAP43 expression in REST-OE cells; therefore, we dosed Ga1 into aged mice that had high REST expression to investigate its influence in vivo. The expression of REST and GAP43 was investigated by western blotting and qPCR. Western blotting and qPCR revealed no significant difference between the expression of REST in Ga1 and a DMSO control (protein: 1.48±0.72-fold, P=0.54; mRNA: 1.07±0.19-fold, P=0.55) (Fig. 7A and B). Interestingly, a significant increase was observed in the expression of GAP43 in Ga1 compared to a DMSO control (protein: 2.25±0.55-fold, P=0.016, mRNA: 1.54±0.22-fold; P=0.013) (Fig. 7A and C). Our findings suggest that Ga1 enhanced GAP43 expression in vitro and in vivo.