Fluvoxamine restores TFEB-mediated autophagy through Sigma-1R-controlled POM121 expression

doi:10.21203/rs.3.rs-2994070/v1

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Fluvoxamine restores TFEB-mediated autophagy through Sigma-1R-controlled POM121 expression

https://doi.org/10.21203/rs.3.rs-2994070/v1

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Expansion of the GGGGCC-RNA repeat is a known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two conditions that currently have no cure. Recent studies have shown that activation of the Sigma-1 receptor plays an important role in providing neuroprotection, particularly in ALS and Alzheimer’s disease. However, the mechanisms underlying Sigma-1R activation and its effect on (G₄C₂)n-RNA-induced cell death remain unclear. In this study, we demonstrated that fluvoxamine is a Sigma-1R agonist that can increase chaperone activity and rescue Pom121 intensity in (G₄C₂)₃₁-RNA-expressing NSC34 cells, thereby stabilizing the protein expression of Pom121. Interestingly, fluvoxamine treatment increased Pom121 protein expression without affecting transcription. In C9orf72-ALS, the TFEB autophagy factor translocation decreased owing to nucleocytoplasmic transport defects. Our results showed that pretreatment of NSC34 cells with fluvoxamine promoted TFEB translocation into the nucleus and increased LC3-II expression compared to the overexpression of (G₄C₂)₃₁-RNA alone. Collectively, fluvoxamine may act as a promising repurposed medicine for patients with C9orf72-ALS, as it stabilizes the nucleoporin Pom121 and promotes the translocation of TFEB in (G₄C₂)₃₁-RNA-expressing NSC34 cells.

C9orf72-Amyotrophic lateral sclerosis

Sigma-1 Receptor

Nucleoporin Pom121

Nucleocytoplasmic transport

Fluvoxamine

C9orf72 amyotrophic lateral sclerosis (ALS) is a familial ALS first identified in 2011 [1–3]. C9orf72-ALS is caused by a GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) and the accumulation of five dipeptide repeat proteins (poly-PR, GR, PA, GP, and GA), resulting in motor or cognitive dysfunction [4, 5]. The deficits in the nucleocytoplasmic transport led to C9orf72-ALS progression through inhibiting Ras-related GTPase (RAN) and transcription factor (for example, TFEB) translocation [3, 6, 7]. Therefore, there is an urgent need to develop therapeutic agents that promote nucleo-cytoplasmic transport.

Autophagy dysregulation is recently indicated to cause the progression of ALS, especially C9orf72-ALS [7–9]. One mechanism involves the accumulation of the transcriptional regulator of autophagy/lysosomal function, TFEB, in the cytosol, owing to impaired nucleocytoplasmic transport function [7, 9]. Nucleoporin POM121 reportedly acts as a nuclear pore regulator that facilitates Importin b1 and translocation of TFEB into the nucleus [7]. Because of the importance of POM121 in autophagy, further investigation into therapeutic approaches targeting POM121 in G4C2 RNA-induced NSC34 cellular models is required.

Sigma-1 receptor (Sig-1R) is a ligand-induced chaperone protein that has been demonstrated to exist in various cellular locations, including the mitochondria-endoplasmic reticulum (ER), nuclear envelope, and nuclear pore complex [6, 7, 10, 11]. Previous studies have demonstrated that Sig-1R agonists exert neuroprotective effects in various neurodegenerative diseases, such as ALS, Huntington’s disease, Parkinson's disease, and Alzheimer’s disease [7, 12–14]. Fluvoxamine, a selective serotonin reuptake inhibitor, is also a high affinity ligand of Sig-1R (Ki = 17.0 nM) [15, 16]. Fluvoxamine has been shown to exhibit a neuroprotective effect that prevents neuronal cell death resulting from ER stress [15] and decreases β-amyloid production by inhibiting γ-secretase activity [17]. However, the effects of fluvoxamine on autophagy through nucleocytoplasmic transport in G₄C₂ RNA-induced ALS require further investigation.

The present study showed that fluvoxamine functions as a Sig-1R agonist, as evidenced by its ability to decrease the association between BiP and Sig-1R and increase chaperone activity in a citrate synthase (CS) aggregation assay. Furthermore, treatment with fluvoxamine increased and stabilized the expression of nucleoporin Pom121. Subsequently, we observed that fluvoxamine treatment promoted the translocation of the autophagy transcription factor, TFEB, into the nucleus and increased LC3-II expression. These findings suggest that fluvoxamine can activate Sig-1R, increase the expression of the nucleoporin Pom121, and enhance autophagy function in C9orf72-ALS.

Cell culture and transfection

The motor neuron-like cell line NSC34 was purchased from CELLutions Biosystems Inc. (CLU140). The cells were maintained in Dulbecco's modified Eagle’s medium (DMEM; GIBCO, 11,965–092) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (GIBCO, 15,140–122). When the cell density reached 90% of a 10-cm dish, the cells were sub-cultured and seeded for gene transfection. Dishes (10 cm) and Poly-Jet reagent (SignaGen Laboratories, SL100688) were used, and the ratio of reagents to plasmids was 2:1. The mixture was incubated in serum-free DMEM (0.5 mL) for 20 min at 23℃ before being added to a 10-cm culture dish and incubated with the cells for the designated time.

Cell lysis and protein extraction

Cell pellets were incubated with ice-cold IP buffer (50 mM NaCl, 0.5% Nonidet P-40, 10 mM Tris-HCl, pH 8.0, and 1× protease inhibitor) for 30 min for lysis. The lysate was then centrifuged at 13,000 rpm for 10 min to extract proteins. The concentration of the extracted protein was measured using a Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific Inc.).

Immunoprecipitation (IP) assay

The extracted protein was supplemented with the required amount of IP buffer and incubated with the IgG antibody or the designated primary antibody at 4°C overnight. The mixture was then washed with IP buffer and incubated with sample buffer at 95°C for 10 min. Finally, western blot analysis was performed.

Nuclear-cytoplasmic fraction

A subcellular Protein Fractionation Kit for Cultured Cells (Thermo Fisher Scientific, 78840) was used to extract subcellular protein fractions from the harvested cells. All the reagents were mixed with protease inhibitors. Cytoplasmic extraction buffer was initially added to cell pellets, and the mixture was incubated at 4°C. The resulting mixture was centrifuged to obtain the supernatant (cytosolic extraction). Next, membrane extraction buffer was added to the pellets before vortexing and being incubated at 4°C. After obtaining the supernatant from the centrifugation of the complex, nuclear extraction buffer was added to the pellets, followed by centrifugation to obtain nuclear extraction.

Cycloheximide (CHX) chasing assay

The old medium was discarded from the dish containing the cells and fresh medium and CHX (100 µg/mL) was added. The cells were then incubated at 37°C for an appropriate amount of time. Subsequently, the cells were collected and lysed to extract proteins for western blot analysis [7].

Western blot analysis

Equal amounts of extracted protein were mixed with sample buffer before denatured at 95°C for 10 min. The proteins were then separated using sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk in tris-buffered saline containing 0.1% Tween-20 (v/v; TBST) for 1 h. Subsequently, the blocked membranes were incubated with the solution of various primary antibodies diluted with TBST and incubated overnight at 4°C. The membranes were washed thrice with TBST for 10 min and incubated with a solution of secondary antibodies diluted with TBST for 1 h. After washing thrice with TBST, the results were captured using an Azure 400 system.

Chaperone activity assay

CS (1.1 mM; Sigma-Aldrich, C-3260) was incubated in 100 µL of 50 mM HEPES-NaOH buffer (pH 7.5) with GST or GST-SIGMAR1/Sigma-1 receptor (1 mM) at 45°C in the presence or absence of fluvoxamine. Next, we monitored the scattered light at 320 nm for 70–90 min using a SpectraMax M2 (Molecular Devices, San Jose, CA, USA) detector [7].

Immunostaining assay

Cells were plated on poly L-lysine-coated coverslips and fixed with 4% paraformaldehyde. After permeabilization with 0.1% Triton X-100 in PB, cells were washed with phosphate-buffered saline (PBS) and blocked with 10% serum diluted in PBS for 1 h. The cells were incubated with various primary antibodies overnight to detect the target proteins. The coverslips were washed with PBS and TBST before incubation with secondary antibodies in PBS for 1 h. After further washing with TBST and PBS, the cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) solution to dye the chromatin, and images were captured using confocal microscopy.

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay

RNA was extracted using an RNA extraction kit (RNeasy Mini Kit; Qiagen, Hilden, Germany). Experimental cells were lysed with lysis buffer, and the complex was supplemented with 70% ethanol. After purification, the RNA was collected and reverse transcribed into DNA using an RT-qPCR kit [18]. The resulting cDNA was mixed with designed primers and enzymes in CFX96 Touch Real-Time PCR Detection System (BioRad), then we determined the CT values and calculated the relative expression of the target genes [19].

Statistical analysis

The data were collected from at least three independent experiments and analyzed using Prism software (version 9.4.0). The results are presented as the means ± standard error of the mean (SEM). Statistical significance was determined using unpaired Student’s t-test, one-way analysis of variance (ANOVA), or two-way ANOVA, along with a proper post-hoc test. For comparisons between nonlinear regression curves (Fig. 1D–F), the second-order polynomial (quadratic) model was first used for curve fitting. Next, the “extra sum-of-squares F-test” was used to test if the best-fit curve of a group is the same as the global (shared) fitting curve. Statistical significance was set at p-<0.05.

Fluvoxamine acted as an agonist to enhance the chaperone ability of Sig-1R

First, NSC34 cells were transfected with Sig-1R to evaluate the association between BiP and Sig-1R. These results indicated that the interaction between Sig-1R and BiP was enhanced by the overexpression of Sig-1R-EYFP, consistent with previous reports implying that Sig-1R dissociates from BiP when activated (Fig. 1a) [20]. We performed IP to determine the effect of Fluvoxamine on the BiP/Sig-1R interaction and found a significant decrease in the association of Sig-1R with BiP in NSC34 cells transfected with Sig-1R-YFP and pretreated with 10 µg/mL fluvoxamine for 1 h (Fig. 1b). CS, a thermosensitive enzyme, is commonly used to assess chaperone ability [21]. We investigated the chaperone function by the analysis of CS together with GST-Sig-1R and Fluvoxamine at 45 ℃ for 90 min (Fig. 1c). Data represented that GST-Sig-1R alone did not prevent CS aggregation at 70 min (Fig. 1d–e). However, pretreatment with 10 µg/mL Fluvoxamine and Sig-1R significantly reduced CS aggregation by facilitating the chaperone ability of Sig-1R, compared to treatment with 10 g/mL fluvoxamine alone (Fig. 1f–g).

Nucleoporin Pom121 protein expression was increased by fluvoxamine in NSC34 cells

Previous studies have shown that Pom121 is not regulated by EGFP-(G₄C₂)₃₁ and that Sigma-1R can chaperone Pom121, which participates in the nucleocytoplasmic transport of transcription factors [7, 22]. In Fig. 1, we confirmed fluvoxamine as a Sigma-1R agonist. Therefore, to investigate whether fluvoxamine plays a role in C9orf72-ALS and affects the nuclear pore complex (NPC), we pretreated NSC34 cells with 10 µg/mL fluvoxamine and overexpressed (G₄C₂)₃₁ RNA in NSC34 cells, followed by immunostaining and RT-qPCR. Z-stacks of images of fluorescently stained NSC34 cells transfected with EGFP-(G₄C₂)₃₁ are shown in the aerial view in Fig. 2a. Tracking the signal intensities in the 5th stack suggested that Pom121 (red) expression was upregulated in NSC34 cells treated with fluvoxamine (arrows shown in Fig. 2b). As shown in Fig. 2b, the total immunofluorescence intensity of Pom121 was upregulated in NSC34 cells transfected with EGFP-(G₄C₂)₃₁ compared to the control group (Fig. 2c, **P < 0.01). Additionally, the RT-qPCR results indicated no difference between NSC34 cells treated with EGFP-(G₄C₂)₃₁ plus fluvoxamine and EGFP-(G₄C₂)₃₁ alone (Fig. 2D, N = 3).

Pom121 was stabilized through supplementation with fluvoxamine in NSC34 cells

Based on the aforementioned results, we examined the connection between Sigma-1R, Pom121, and fluvoxamine. IP data revealed that Sigma-1R interacted with Pom121 in NSC34 cells overexpressing Sigma-1-EYFP, with or without treatment with 10 µg/mL fluvoxamine (Fig. 3a–b). As shown in Fig. 2d, Pom121 expression was not altered by EGFP-(G₄C₂)₃₁ at the transcriptional level. We used 100 µg/mL CHX, a protein translation inhibitor, to assess Pom121 stability. Western blotting demonstrated that fluvoxamine stabilized Pom121 in NSC34 cells transfected with EGFP-(G₄C₂)₃₁ (Fig. 3c–d, control group vs. fluvoxamine group at 6 h, **P < 0.01, N = 3). Pom121 protein expression was notably increased in the fluvoxamine group at 0 h (Fig. 3e, ^**P < 0.01, N = 3). Based on these findings, fluvoxamine enhanced Pom121 expression by reversing its degradation in NSC34 cells.

Treatment of Fluvoxamine in in NSC34 cells promoted autophagy through the translocation of TFEB and Importin b1

Autophagy defects are linked to importin b1 during nuclear import and are involved in the pathogenesis of C9orf72-ALS [7]. Previous studies have showed that Importin b1 together with Pom121 participates in the nuclear translocation of TFEB, a transcription factor regulating the expression of autophagy and lysosomal biogenesis-related genes. G₄C₂ repeat RNA alters the nuclear-to-cytoplasmic (N/C) ratio of TFEB [7]. In the present study, we investigated the role of fluvoxamine in the nucleocytoplasmic transport of TFEB and expression of LC3-II, which marks the formation of autophagosomes in NSC34 cells overexpressing G₄C₂ repeat RNA [7]. The results of the subcellular fraction assays suggested that fluvoxamine reversed the N/C ratio of TFEB in NSC34 cells (Fig. 4a-b, *P < 0.05, N = 4). Concomitantly, the N/C ratio of Importin b1 increased (Fig. 4c, *P < 0.05, N = 4). In addition, the western blotting data demonstrated EGFP-(G₄C₂)₃₁ decreased the expression of LC3-II, and fluvoxamine significantly restored the expression of LC3-II through stimulation of EGFP-(G₄C₂)₃₁ (Fig. 4d–e, **P < 0.01, N = 5). Collectively, these findings revealed the role of fluvoxamine in regulating autophagy in motor neurons.

In summary, this study indicated that fluvoxamine acts as an agonist to dissociate Sigma-1R from BiP and enhances the chaperone activity of Sigma-1R. Moreover, fluvoxamine upregulated the expression Pom121 by inhibiting protein degradation without influencing the interaction between Pom121 and Sigma-1R. Shuttling of TFEB and Importin b1 into the nucleus was promoted by fluvoxamine in NSC34 cells. Similarly, LC3-II expression was upregulated through the stimulation of G₄C₂ repeat RNA along with fluvoxamine compared to the control group. Overall, the current study suggests that fluvoxamine promotes the translocation of TFEB into the nucleus, and may serve as a therapeutic candidate for C9orf72-ALS (Fig. 5).

Several studies have indicated a noteworthy correlation between autophagy defects and the pathological progression of C9orf72-ALS in motoneurons [23–25]. Additionally, numerous experiments have demonstrated differentiating nuclear import under the stimulation of G₄C₂ repeat RNA, a toxic product from mutation of C9orf72 gene [7, 26]. To the best of our knowledge, this is the first study to demonstrate that fluvoxamine dissociated Sigma-1R from BiP and boosts chaperone activity. Furthermore, fluvoxamine increased the expression of Pom121, a crucial nucleoporin for nucleocytoplasmic shuttling, not by regulating its interaction with Sigma-1R but by enhancing the chaperone activity of Sigma-1R. Importantly, it influences the translocation of TFEB, a critical transcription factor for autophagy, from the cytoplasm to the nucleus, resulting in increased expression of LC3-II.

Autophagy dysregulation in motoneurons affected by C9orf72 mutation led to neuronal death [7, 9, 24]. Our previous study showed that pridopidine, a Sigma-1R agonist, stabilizes nucleoporin Pom121 protein expression and subsequently increases the translocation of TFEB into the nucleus to promote autophagy in G₄C₂ repeat RNA-expressing NSC34 cells, resulting in reduced neuronal death [7]. Similarly, fluvoxamine, also Sigma-1R agonist, stabilized Pom121 expression and promoted autophagy (Fig. 3 and Fig. 4). Repurposing existing drugs is important for the treatment of various diseases, including neurodegenerative diseases. Fluvoxamine is a selective serotonin reuptake inhibitor that is widely used to treat depression [15]. Fluvoxamine treatment increased the transcription and protein expression of Sigma-1R in N2a cells [15]. These results suggest that fluvoxamine may increase Pom121 expression by increasing Sigma-1R levels (Fig. 3e), thereby promoting nucleocytoplasmic transport.

Notably, the nuclear pore complex is composed of approximately 30 nucleoporins, with Pom121 playing a critical role in regulating nucleocytoplasmic transport [7, 27]. Moreover, Pom121 can control the activity of other nucleoporins such as GP210, NDC1, Nup133, Nup107, Nup50, TPR, and Nup98 [27]. In the C9orf72-iPSN model, decreased Pom121 expression was observed, leading to disruption of nucleocytoplasmic transport [27]. Therefore, our study aimed to elucidate strategies for stabilizing or increasing Pom121 expression. Based on our previous study, Sigma-1R agonists may serve as potential therapeutic options for the treatment of C9orf72-ALS [7].

Because of the complex mechanism, TFEB translocates into nucleus through the nuclear import cycle in which its nuclear localization signal (NLS) domain is conjugated with the Importin a/b complex in the cytoplasm [7]. This complex then interacts with nucleoporins containing hydrophobic phenylalanine-glycine domain (FG domain), allowing TFEB to shuttle into nucleoplasm [7, 28, 29]. A previous study revealed that the reduction in Pom121 correlated with decreased intensity of FG-domain-containing Nups (FG-nups) [30]. In the present study, we showed that Pom121 was stabilized by fluvoxamine and that nucleocytoplasmic transport was facilitated, as shown in Fig. 3c and Fig. 4a. This may indicate that Pom121 upregulation induced by Sigma-1R repairs the import of the cargo complex into the nucleus by restoring the number of FG-nups in the central channel of the NPC. In future studies, we will further analyze FG-Nups in NSC34 cells transfected with Pom121 to elucidate the detailed mechanism. Once inside the nucleus, Ran-GTP interacts with and releases Importin from TFEB, subsequently interacts with FG-nups, and moves into the cytoplasm, where hydrolysis of Ran-GTP with the assistance of GTPase-accelerating protein (GAP) takes place [31]. Ran-GDP then translocates to the nucleoplasm [31]. In addition, Sigma-1R binds to FG-Nups and maintains their stability. It was also exhibited that Sigma-1R stabilizes RanGAP1, which is crucial for the recycling of Ran protein and the continuation of nucleocytoplasmic transport [6].

Regarding other toxic products in C9orf72-ALS, dipeptide repeats (DPRs) are caused by mutations in C9orf72 gene and contribute to interference in normal neuronal metabolism, such as the death of neurons and defects in nucleocytoplasmic shuttling [32, 33]. It has been suggested that autophagy is responsible for the clearance of DPR; however, G₄C₂ repeat RNA changes the process by retaining TFEB outside the nuclei [24]. This implied an irreversible accumulation of DPRs. Collectively, we hypothesize that fluvoxamine may activate Sigma-1R, chaperoning the Pom121 protein to reverse the impaired nuclear import and export. This consequently induces autophagy activation and potentially protects motor neurons from DPR toxicity.

In conclusion, fluvoxamine exerts its innate function to activate Sigma-1R and rescues the impaired distribution of TFEB in the cytoplasm and nucleus by modulating the expression of Pom121. These factors, in turn, contribute to repaired autophagy in (G₄C₂) RNA repeat-induced C9orf72-ALS. The neuroprotective ability of fluvoxamine may mediate the progression of numerous diseases, such as ALS, Parkinson’s disease, and Huntington's disease. In this study, we validated the critical relationship between Sigma-1R and Pom121, as well as their ability to affect nucleocytoplasmic transport (Fig. 5). Moreover, we determined that fluvoxamine may act as a potential repurposed drug to prevent motoneurons from insulting C9orf72-ALS through its selective role as a Sigma-1R agonist.

ALS

amyotrophic lateral sclerosis

FTD

frontotemporal dementia

G4C2

GGGGCC

NSC34

mouse motor neuron-like hybrid cell line

HRE

hexanucleotide repeat expansion

RAN

Ras-related GTPase

TFEB

Transcription Factor EB

Sig-1R

Sigma-1 receptor

citrate synthase

NPC

nuclear pore complex

CHX

cycloheximide

PVDF

polyvinylidene difluoride

SDS-PAGE

SDS-polyacrylamide gel electrophoresis

FG domain

Phenylalanine-Glycine domain

DPRs

dipeptide repeats

Ethics approval and consent to participate

This research article does not involve any studies conducted on humans or animals.

Consent for publication

Not applicable.

Availability of data and materials

According to all data generated during this study are included in this article.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded by the National Science and Technology Council, Taiwan, grant number: MOST 111-2628-B-039-006-MY3.

Acknowledgements

We thank the Neuroscience and Brain Disease Center, China Medical University, Taiwan for the equipment support. This report was supported in part by the Intramural Research Program of the National Institute on Drug Abuse, NIH, DHHS.

Authors’ contributions

C-Y Lin, H-E Wu, and S-M Wang performed the experiments, organized all the figures and analyzed data. C-Y Lin, S-M Wang, Eddie F-J Weng, and H-E Wu wrote the manuscript. S-M Wang and T-P Su performed the investigation, methodology, project administration, writing review, and editing.

Data availability

The data that support this study are available from the corresponding authors upon reasonable request.

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Fluvoxamine restores TFEB-mediated autophagy through Sigma-1R-controlled POM121 expression

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Abstract

Figures

Introduction

Materials and methods

Cell lysis and protein extraction

Immunoprecipitation (IP) assay

Nuclear-cytoplasmic fraction

Cycloheximide (CHX) chasing assay

Western blot analysis

Chaperone activity assay

Immunostaining assay

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay

Statistical analysis

Results

Nucleoporin Pom121 protein expression was increased by fluvoxamine in NSC34 cells

Pom121 was stabilized through supplementation with fluvoxamine in NSC34 cells

Discussion

Abbreviations

Declarations

References

Supplementary Files

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