SIGMA-1 RECEPTOR ACTIVATION BY FLUVOXAMINE RESTORES TFEB- MEDIATED AUTOPHAGY VIA NUCLEOPORIN POM121 IN A NSC34 C9ALS/FTD MODEL
Authors: Wu *Hsiang-En , Wang Shao-Ming , Lin Chun-Yu , Weng Fenf-Ju , Wu Hsuan-Cheng , Sands Leeodella , Su Tsung-Ping 
Published: February 2025

Abstract

Background
Dysregulated autophagy relates to neurodegenerative diseases, in particular the C9orf72- amyotrophic lateral sclerosis (ALS) (Wang et al. 2023). In C9orf72-ALS, (G4C2)-hexanucleotide repeats expansion (HRE) does so by disrupting the nucleocytoplasmic transport of TFEB required for autophagy (Wang et al. 2023; Cunningham et al. 2020).

Aims & Objectives
Recent studies have shown that the Sigma-1 receptor (Sig-1R), a multiple-functional chaperone protein, can provide neuroprotection, particularly in ALS and Alzheimer’ s disease (Lee et al. 2020; Estevez-Silva et al. 2022). However, the exact mechanism underlying Sig-1R on (G4C2)n-RNA- induced cell death remains unclear.

Method
The NSC34 cell line was overexpressed with Sig-1R-EYFP, EYFP, EGFP-(G4C2)31, or GFP-Tfeb for 24h. Fluvoxamine (10 & micro;g/mL) or vehicle was administered for 1h in transfected cells for immunoprecipitation or chaperone activity assay (citrate synthase with/without GST measured at 320nm wavelength every 5 min for 70 min). In other experiments, fluvoxamine (10 & micro;g/mL) was administered 1h before transfection and during transfection. In cycloheximide chasing assay, cycloheximide (100 & micro;g/mL) was added in fluvoxamine pretreated EGFP-(G4C2)31 NSC34 cells and cell lysate was collected at 0 or 6h later. To study how fluvoxamine modulates autophagy, fluvoxamine (10 & micro;g/mL) pretreated EGFP-(G4C2)31 overexpressed NSC34 cells were used for immunocytochemistry and western blotting.

Results
Here we found that fluvoxamine, a Sig-1R agonist which causes the dissociation of Sig-1R from BiP and inhibits the aggregation of citrate synthase, increased chaperoning nucleoporin POM121 in (G4C2)31-RNA-expression NSC34 cells, thereby stabilizing POM121 protein expression. Interestingly, fluvoxamine administration increased POM121 expression without affecting transcription. In C9orf72- ALS, the TFEB autophagy factor translocation decreases due to nucleocytoplasmic transport defects. HRE causes the dissociation of Sig-1R from POM121, resulting in a deficiency of TFEB and an autophagy marker LC3-II. Our results showed that pretreatment with fluvoxamine promoted TFEB translocation into the nucleus and increased LC3-II expression compared to the overexpression of (G4C2)31-RNA alone in NSC34 cells.

Discussion & Conclusions
Fluvoxamine activates by dissociating Sig-1R from BiP to boost chaperone activity (Omi et al. 2014; Prasanth et al. 2021) and increases the expression of POM121, a crucial nucleoporin for nucleocytoplasmic shuttling. Importantly, it affects the translocation of TFEB, a critical transcription factor for autophagy, from cytoplasm to the nucleus, resulting in increased LC3-II expression. Autophagy dysregulation in motoneurons affected by C9orf72 mutation leads to neuronal death. Fluvoxamine activates Sig-1R and hence stabilizes POM121 expression and facilitates autophagy in G4C2 repeat RNA-expression NSC34 cells, resulting in reduced neuronal death. Together, fluvoxamine, a selective serotonin reuptake inhibitor widely used to treat depression, could be a promising repurposed drug for treating C9orf72-ALS, as it stabilizes the nucleoporin POM121 and foster the translocation of TFEB to repair autophagy in (G4C2)31-RNA-expressing NSC34 cells. (Supported by the IRP, NIDA, NIH, and National Science and Technology Council, Taiwan).

References
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