Activated Fibroblast Growth Factor Receptor 1 Mitigated Poly-PR–Induced Oxidative Stress and Protein Translational Impairment

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neuron cell death. A GGGGCC hexanucleotide repeat expansion (HRE) within the chromosome 9 open reading frame 72 (C9orf72) gene is a major causative factor in ALS. This abnormal HRE triggers five types of dipeptide repeat protein (DPR), each composed of two alternating amino acid expressions. Among the DPRs, arginine-rich Poly-PR localizes predominantly to the nucleus, exerting particularly strong toxicity on motor and cortical neurons. Several mechanisms have been proposed for poly-PR–induced neurotoxicity. In this study, poly-PR–expressing NSC34 motor neuron-like cells showed an increase in oxidative stress. Fibroblast growth factor receptor 1 (FGFR1) is known to promote neurogenesis and inhibit apoptosis in neurons. However, its neuroprotective effects against DPR-induced toxicity have not been previously reported. Here, we demonstrated that FGFR1 activation reduced oxidative stress by upregulating nuclear factor erythroid 2-related factor 2 (NRF2) expression. Furthermore, we propose that the increase in NRF2 through FGFR1 activation may result from the alleviation of protein translation impairment. Overall, these findings suggest that FGFR1 activation provides neuroprotection against poly-PR toxicity and may represent a potential therapeutic strategy for ALS.

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neuron cell death. Symptoms include muscle weakness, atrophy, and paralysis, eventually leading to death due to respiratory failure from respiratory muscle paralysis.^1,2) Although 90–95% of ALS cases are sporadic, 5–10% are familial.³⁾ Among the genetic mutations associated with ALS and frontotemporal dementia (FTD), a hexanucleotide repeat expansion (HRE) consisting of six bases (GGGGCC) within the intron of chromosome 9 open reading frame 72 (C9orf72) is the most common.^4,5) In unaffected individuals, the number of repeats typically ranges from 2 to 23. In contrast, patients with ALS harboring C9orf72 mutations (C9-ALS) exhibit a substantially larger range, spanning several hundred to thousands of repeats.

This abnormal HRE triggers dipeptide repeat protein (DPR) expression through repeat-associated non-AUG (RAN) translation of the expanded repeat RNA.^6–8) DPRs are translated from both the sense (GGGGCC) and antisense (CCCCGG) strands of abnormal HRE transcripts, producing five types: Glycine (G)–Alanine (A) DPR (poly-GA), Glycine (G)–Arginine (R) DPR (poly-GR), Glycine (G)–Proline (P) DPR (poly-GP), Proline (P)–Alanine (A) DPR (poly-PA), and Proline (P)–Arginine (R) DPR (poly-PR).⁹⁾ Among DPRs, poly-PR preferentially accumulates in nucleolar aggregates and demonstrates potent neurotoxicity, even with a lower number of repeats compared to other DPRs.⁹⁾ Several mechanisms have been proposed for poly-PR–induced neurotoxicity, including oxidative stress, protein translational impairment, RNA splicing abnormalities, nucleocytoplasmic trafficking dysfunction, aberrant liquid–liquid phase separation, and nucleolar stress.^10–17)

Oxidative stress is a well-known factor that damages motor neurons in C9-ALS.^18–20) Reducing oxidative stress is an important approach to treating ALS. Indeed, edaravone, an antioxidant, and a powerful radical scavenger has been approved for the treatment of ALS.^21,22) On the other hand, the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), a key regulator of antioxidant activity, is known to respond to oxidative stress and exert protective effects on neurons. However, poly-PR has been shown to impair NRF2 functions, contributing to neuronal vulnerability.²³⁾

Fibroblast growth factor receptors (FGFRs) constitute a family of receptor tyrosine kinases comprising four subtypes (FGFR1–FGFR4). They play a role in a diverse range of biological processes, such as morphogenesis and tissue repair.^24–26) Activated FGFR1, which shows high-level expressions in the central nervous system, has been known to exert neuroprotective effects in mouse models of Alzheimer’s disease and Parkinson’s Disease.^27,28) Patients with ALS exhibiting sustained expression of FGF2, a FGFR1 ligand, reportedly experience slower disease progression, and circulating levels of FGF2 have been associated with a lower ALS risk.^29,30) In addition, FGFR1 activation is expected to suppress oxidative stress via up-regulation of NRF2 expression.^31,32) However, the role of FGFR1 in ALS remains poorly understood, and its relationship to poly-PR toxicity has yet to be investigated. This study aims to evaluate the effects of FGFR1 activation on oxidative stress in NSC34 motor neuron-like cells expressing poly-PR.

MATERIALS AND METHODS

Plasmids, Cell Culture, and Transfection

Expression plasmids pEGFP-1 (Clontech Laboratories Inc., CA, U.S.A.) or pmCherry-N1 (Clontech Laboratories Inc.) harboring PR50 were prepared as described in a previous report.⁹⁾ pBRPB CAG-mCherry-IP was a gift from Prof. Thomas Tuschl (Addgene plasmid # 106333; http://n2t.net/addgene:106333; RRID: Addgene_106333).³³⁾ NSC34-mCherry cells were established by co-transfected with pBRPB CAG-mCherry-IP and the piggyBac transposase into NSC34 cells (Mouse Neuroblastoma × Spinal Cord-34, CELLutions Biosystems, Canada) using Lipofectamine 3000, following the manufacturer’s protocol (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.).

NSC34 cells (Mouse Neuroblastoma × Spinal Cord-34, CELLutions Biosystems, Canada) and NSC34-mCherry cells were maintained in Dulbecco’s modified Eagle medium (DMEM; Wako Pure Chemical Corporation, Osaka, Japan) containing 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific Inc.) in a humidified atmosphere of 5% CO₂ at 37 °C. Cells were passaged every 3–4 d via trypsinization. Transient plasmid expression in NSC34 cells was achieved using Lipofectamine 2000, following the manufacturer’s protocol (Thermo Fisher Scientific Inc.). After 48 h of plasmid transfection, NSC34 cells, and NSC34-mCherry cells were differentiated for 48 h in DMEM/Ham’s F-12 (Nacalai Tesque, Inc., Kyoto, Japan), containing non-essential amino acid, supplemented with 1.5% FBS with reference to the previous studies and used for several experiments.^34,35)

Cell Viability

NSC34-mCherry cells were seeded at 5.0 × 10⁴ cells/mL in 24-well plates with DMEM containing 10% FBS. After 48 h of plasmid transfection, cells were differentiated for 48 h in DMEM/Ham’s F-12 supplemented with 1.5% FBS and varying concentrations of FGF2 (10 ng/mL; REPROCELL Inc., Yokohama, Japan) with or without 100 nM PD173074 (ChemScene LLC, NJ, U.S.A.), pretreated for 30 min. Cell viability was assessed by quantifying the number of mCherry-positive cells. Briefly, 48 h after FGF2 treatment, fluorescence microscopy images were captured 9 images/well using an all-in-one fluorescence microscope (EVOS™ FL AUTO 2, Invitrogen, Waltham, MA, U.S.A.). The number of mCherry-positive cells was analyzed by Cellste 6 (Invitrogen).

Oxidative Stress

To measure oxidative stress, CellRO™ Green (Thermo Fisher Scientific Inc.) and MitoSOX^® Red (Thermo Fisher Scientific Inc.) were used, following the manufacturer’s instructions. At 48 h after FGF2 treatment, cells were treated with 5 μM CellROX™ Green and 5 μM MitoSOX^® Red for 30 min at 37°C. Nuclear staining was performed with Hoechst 33342 (Thermo Fisher Scientific Inc.). Fluorescence microscopy images were captured using a confocal fluorescence microscope (LSM700, Carl Zeiss). ImageJ was used to measure fluorescence intensity, which was normalized to the number of Hoechst-stained cells.

Immunoblotting

At 48 h after FGF2 treatment, cells were lysed with RIPA buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 1% sodium dodecyl sulfate (SDS), 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1% phosphatase inhibitor, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] and centrifuged at 15000 × g and 4°C for 15 min. Supernatant protein samples were then collected. Protein concentrations were quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific Inc.), with bovine serum albumin used as the standard. Lysates were mixed with sample buffer containing 10% 2-mercaptoethanol and subjected to 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was performed under a constant voltage of 200 V at room temperature for 50 min. The separated proteins were transferred from polyacrylamide gel to a polyvinylidene difluoride (PVDF) membrane in a transfer buffer (0.3% Tris, 1.44% glycine, and 20% methanol) under a constant voltage of 100 V at 4°C for 90 min. The membranes were incubated with 5% skim milk and Blocking One (Nacalai Tesque, Inc.) for 60 min, followed by primary antibody incubation overnight with the following antibodies: mouse antibodies against β-actin (1 : 2000 dilution; Sigma-Aldrich, St. Louis, MO, U.S.A.) and rabbit antibodies against NRF2 (1 : 1000 dilution; Cell Signaling Technology), and heme oxygenase (HO)-1 (1 : 1000 dilution; Enzo Life Sciences, Farmingdale, NY, U.S.A.). After primary antibody incubation, the membranes were incubated with a secondary antibody: goat anti-rabbit antibody conjugated with HRP (1 : 2500 dilution; Sigma-Aldrich) or goat anti-mouse HRP antibody conjugated with HRP (1 : 2500 dilution; Sigma-Aldrich). Chemiluminescence was developed from HRP antibodies using ECL Prime (GE Healthcare, Buckinghamshire, U.K.) and detected using a Fusion system (Vilber-Lourmat, Collégien, France). Band density was then measured using ImageJ.

RNA Preparation and Reverse Transcriptase-Quantitative PCR (RT-qPCR)

Reverse transcription was performed using the ReverTra Ace qPCR RT Master Mix, following the manufacturer’s instructions (TOYOBO, Osaka, Japan). RT-qPCR was performed using SYBR Green on a StepOne Real-Time PCR System, according to the manufacturer’s protocol (Life Technologies, Carlsbad, CA, U.S.A.). Gene-specific primer set sequences are listed in Table 1. mRNA expression levels were normalized to Gapdh mRNA expression.

Table 1. Primer Pairs Used for RT-qPCR

mRNA	Forward	Reverse
Nfe2l2	5'-ctttagtcaccgacagaaggac-3'	5'-aggcatcttgtttgggaatgtg-3'
Hmox1	5'-aagccgagaatgctgagttca-3'	5'-gccgtgtagatatggtacaagga-3'
Gapdh	5'-cctcgtcccgtagacaaaatg-3'	5'-tctccactttgccactgcaa-3'

RT-qPCR, RT-quantitative PCR; Nfe2l2, nuclear factor erythroid 2-related factor 2; Hmox1, heme oxygenase 1; Gapdh, glyceraldehyde-3-phosphate dehydrogenase.

Protein Synthesis Assay

The Click-iT^® HPG Alexa Fluor^® Protein Synthesis Assay Kit (Invitrogen) was used for the protein synthesis assay, following the manufacturer’s instructions. Click-iT^® HPG (l-homopropargylglycine) is an amino acid analog of methionine. Similar to 35S-methionine, Click-iT^® HPG is incorporated into proteins during active protein synthesis. Measuring the incorporation of Click-iT into proteins indicates newly synthesized protein levels. Detection of the incorporated amino acid utilizes a chemoselective ligation or click reaction between an azide and alkyne, where the alkyne-modified protein is detected with Alexa Fluor^® 594 azide. At 48 h after FGF2 treatment, cells were treated with 50 μM Click-iT^® HPG working solution for 30 min. Following reagent removal, cells were fixed with 4% paraformaldehyde and permeabilized using 0.1% Triton X-100 in phosphate-buffered saline (PBS). The Click-iT^® reaction cocktail was then added and incubated in the dark at room temperature for 30 min. Nuclear staining was achieved with Hoechst 33342 (Thermo Fisher Scientific Inc.). Fluorescence microscopy images were captured using a confocal fluorescence microscope (LSM700, Carl Zeiss, Oberkochen, Germany). ImageJ was employed to measure fluorescence intensity, which was normalized to the number of Hoechst-stained cells.

Statistical Analysis

Data are presented as means ± standard errors of means (S.E.Ms.). Significance was determined using ANOVA, and post-hoc comparisons were performed using the Tukey–Kramer test (RStudio, Abacus). p < 0.05 was considered statistically significant.

RESULTS

FGFR1 Activation Exerts Neuroprotective Effects against Poly-PR Toxicity

In this study, we transfected GFP or mCherry-fused poly-PR plasmid vectors into NSC34 cells or NSC34-mCherry. Poly-PR plasmid vector contained 50 repeats of proline (P) and arginine (R) (PR50) exhibiting substantial toxicity and designed to express PR50-green fluorescent protein (GFP) (hereafter referred to as PR50) or PR50-mCherry through ATG-dependent translation, as demonstrated in prior research⁹⁾ (Fig. 1A).

Fig. 1. FGF2 Prevents Poly-PR–Induced Neurotoxicity

At 48 h post-transfection of each vector into NSC34 cells or NSC34-mCherry cells, cells were treated with 10 ng/mL FGF2 for 48 h, with or without 100 nM PD173074 pretreated 30 min prior. (A) Schematic of the PR50-GFP and PR50-mCherry plasmid vector. (B) Representative fluorescence microscopy images captured using a confocal fluorescence microscope. Scale bar: 10 μm. (C) Representative fluorescence microscopy images captured using an all-in-one fluorescence microscope. Scale bar: 250 μm. (D) Cell viability assessed through the quantifying the number of mCherry-positive cells. Data are presented as means ± S.E.Ms. from six independent experiments. ^###p < 0.005 vs. GFP; *** p < 0.005 vs. PR50 (–); ^†††p < 0.005 vs. PR50 FGF2. Scale bar: 250 μm.

NSC34 cells transfected with PR50 showed substantial nuclear accumulation of poly-PR aggregates (Fig. 1B). In this study, NSC34-mCherry cells, stably expressed mCherry by a piggyBac transposon system, were used for measuring cell viability (Figs. 1C and 1D). A significant decrease in cell viability was observed in PR50–expressing NSC34-mCherry cells, and FGF2 treatment significantly increased cell viability. In addition, pretreatment with PD173074, a selective FGFR1 inhibitor, abolished the FGF2-induced increase in cell viability (Figs. 1C and 1D). These findings indicate that FGFR1 activation by FGF2 treatment exerts a neuroprotective effect against poly-PR toxicity.

FGFR1 Activation Suppresses Oxidative Stress

We also investigated the effect of FGFR1 activation on oxidative stress by poly-PR. In this study, CellROX Green was used to measure overall oxidative stress in cells. In PR50-mCherry–expressing NSC34 cells, an increase in oxidative stress was observed. FGF2 treatment effectively prevented the poly-PR–induced increase in oxidative stress (Figs. 2A and 2B). Additionally, we investigated using MitoSox Red, a selective live cell probe for detecting superoxide–the predominant ROS in the mitochondria. FGF2 treatment also prevented an increase of superoxide in the mitochondria in PR50–expressing NSC34 cells (Figs. 2C and 2D). These findings indicate that FGFR1 activation suppresses poly-PR–induced oxidative stress.

Fig. 2. FGFR1 Activation Are Suppressed Oxidative Stress

At 48 h post-transfection of each vector into NSC34 cells, cells were treated with 10 ng/mL FGF2 for 48 h, with or without 100 nM PD173074 pretreated 30 min prior. (A, C) Representative fluorescence microscopy images of CellROX Green and MitoSOX Red captured using a confocal fluorescence microscope. Scale bar: 10 μm. (B, D) Quantified intensity normalized to the number of Hoechst-stained cells. Data are expressed as means ± S.E.Ms. from six independent experiments. ^##p < 0.01, ^###p < 0.005 vs. GFP; * p < 0.05, *** p < 0.005 vs. PR50 (–); ^†††p < 0.005 vs. PR50 FGF2. Scale bar: 10 μm.

FGFR1 Activation Increases NRF2 Expression and Improves Poly-PR–Induced Protein Translational Impairment

Poly-PR expression has been reported to impair the activation of NRF2, a transcription factor responsible for regulating cellular redox balance and protective antioxidant responses. In addition, protein translation impairment, one of the major dysfunctions caused by poly-PR, is involved as a factor in this impairment of Nrf2.²³⁾ Therefore, improving protein translation may ameliorate this dysfunction. In this study, using a protein synthesis assay, we observed reduced protein synthesis capacity in PR50-expressing NSC34 cells. However, FGFR1 activation via FGF2 treatment enhanced protein synthesis (Figs. 3A and 3B). These results indicate that FGFR1 activation improves protein translational function in PR50-expressing NSC34 cells.

Fig. 3. FGFR1 Activation Mitigates Protein Translational Impairment

At 48 h post-transfection of each vector into NSC34 cells, cells were treated with 10 ng/mL FGF2 for 48 h, with or without 100 nM PD173074 pretreated 30 min prior. (A) Representative fluorescence microscopy images of Click-iT HPG Alexa Fluor 594. Scale bar: 10 μm. (B) Quantified Click-iT HPG Alexa Fluor 594 intensity, normalized to the number of Hoechst-stained cells. Data are presented as means ± S.E.Ms. from six independent experiments. ^###p < 0.005 vs. GFP; *** p < 0.005 vs. PR50 (–); ^†††p <0.005 vs. PR50 FGF2. Scale bar: 10 μm.

In addition, FGF2 treatment increased the expression of NRF2 as well as its downstream target HO-1, a Nrf2-regulated antioxidant enzyme (Figs. 4A–4D). Moreover, we measured mRNA expression levels of Nfe2l2 and Hmox1, the genes encoding Nrf2 and HO-1, to examine the changes in gene levels of Nrf2 and HO-1. RT-qPCR analysis also showed that FGF2 treatment increased the mRNA expression levels of Nfe2l2 and Hmox1 in PR50-expressing NSC34 cells (Figs. 4E and 4F). These findings suggest that the FGFR1 signaling pathway may play a crucial role in upregulating NRF2 expression and exerting neuroprotective effects through promoting antioxidant effects.

Fig. 4. FGFR1 Activation Reduces Oxidative Stress via Increased NRF2 and HO-1 Expression

At 48 h post-transfection of each vector into NSC34 cells, cells were treated with 10 ng/mL FGF2 for 48 h, with or without 100 nM PD173074 pretreated 30 min prior. (A, C) Lysates were analyzed via immunoblotting with antibodies for NRF2, HO-1, and β-actin. (B, D) Relative levels normalized to β-actin expression were quantified. (E, F) At 24 h post-treatment with FGF2, mRNA expression levels of Nfe2l2 and Hmox1 were analyzed using SYBR Green RT-qPCR, with expression normalized to Gapdh mRNA levels. Data are expressed as means ± S.E.Ms. from six independent experiments. * p < 0.05, *** p < 0.005 vs. PR50 (–); ^††p < 0.01, ^†††p < 0.005 vs. PR50 FGF2.

DISCUSSION

This study highlights the neuroprotective effects of FGFR1 activation in mitigating against poly-PR toxicity, a hallmark of ALS driven by abnormal HREs in the C9orf72 gene, a major causative factor of the disease. Poly-PR, characterized by its nuclear accumulation and pronounced cytotoxicity,^6–20) exhibits similar toxic properties in PR50-expressing NSC34 cells. Our findings reveal that FGFR1 activation through FGF2 treatment significantly ameliorates the poly-PR–induced decrease in cell viability and increase in cytotoxicity.

One mechanism underlying poly-PR toxicity is an increase in oxidative stress. Our results suggest that FGFR1 activation suppresses oxidative stress by promoting NRF2 expression, thereby contributing to neuroprotection. Edaravone, an approved medication for ALS, is known not only to function as an antioxidant and a potent radical scavenger but also to induce NRF2 activation and enhance antioxidant defenses.²²⁾ The role of NRF2 is thus considered critical in the therapeutic approach to ALS. However, poly-PR expression has been reported to impair NRF2 activation. This impairment may be ameliorated through improving protein translational impairment caused by poly-PR.²³⁾ In this study, we demonstrated that FGFR1 activation ameliorates protein translational impairment. Therefore, the increase in NRF2 through FGFR1 activation may be associated with the restoration of protein translation.

In summary, FGFR1 activation exhibited neuroprotective effects in PR50-expressing NSC34 cells, likely by suppressing oxidative stress via NRF2 upregulation. Additionally, we propose that the increase in NRF2 through FGFR1 activation may result from the alleviation of protein translation impairment.

Poly-PR–induced neurotoxicity is also linked to its interactions with ribosomal proteins and translation initiation factors, and sequestration of these proteins may lead to translational impairment.^11,12) FGFR1 activation is known to promote the translation of various proteins.³⁶⁾ Therefore, the increase in protein synthesis with FGF2 treatment observed in this study may result from enhanced translation through FGFR1 activation. Many proteins whose translation is enhanced by FGFR1 activation include translation initiation factors and ribosomal proteins.³⁶⁾ Thus, FGFR1 activation may have alleviated the poly-PR–induced translational impairment and restored the function of ribosomal proteins and translation initiation factors. However, further studies are needed as the specific role of FGFR1 in protein synthesis was not fully investigated in this study.

Although this study suggests that improved protein translation contributes to NRF2 upregulation, it is possible that FGFR1 activation also enhances NRF2 activation through other mechanisms. FGFR1 activation may increase NRF2 expression by activating downstream signaling pathways, such as phosphatidylinositol 3-kinase (PI3K)–protein kinase B (AKT) and MEK–extracellular signal-regulated kinase (ERK).^32,37) Additionally, FGFR1 activation has been reported to enhance NRF2 stability by interacting with KEAP1, a key regulator of NRF2 degradation, and inhibiting its function.³¹⁾ Previous studies have shown that treatment with dimethyl fumarate, which inhibits KEAP1-mediated degradation of NRF2, does not affect NRF2 expression levels in poly-PR-expressing cells.²³⁾ FGFR1 activation may employ multiple mechanisms to increase NRF2 expression, thereby improving poly-PR–induced impairment in NRF2 activation and suppressing oxidative stress.

The therapeutic effect of edaravone, an antioxidant, is limited, as it only prolongs patient survival for a few months.³⁸⁾ This limitation underscores the challenge of inhibiting the onset and progression of ALS solely by targeting oxidative stress. While this study primarily focused on oxidative stress and protein translation, FGFR1 activation is known to regulate various signaling pathways.^24–26) For example, FGFR1 activation has been shown to inhibit the accumulation of p53, a protein implicated in poly-PR-induced neurotoxicity.^39–41) Furthermore, FGFR1 may serve as a promising therapeutic target, particularly in consideration of glial cells, which play a critical role in ALS pathology. FGFR1 activation has been reported to suppress glial cell activation, reduce inflammation, and mitigate excitotoxicity by promoting glutamate clearance.^42–45) Although the mechanisms underlying FGFR1 signaling are diverse and require further investigation, our findings suggest that FGFR1 activation holds significant potential as a therapeutic target for ALS.

Acknowledgments

This work was supported by Grants from the Takeda Science Foundation, the Japan Society for the Promotion of Science (JSPS), a Grant from the Research Fellow of JSPS (Grant No. JP22KJ2579).

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

Data will be made available at a reasonable request from the corresponding author.

Supplementary Materials

This article contains supplementary materials.

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