SIGMA-1 RECEPTOR ACTIVATION BY FLUVOXAMINE RESTORES TFEB- MEDIATED AUTOPHAGY VIA NUCLEOPORIN POM121 IN A NSC34 C9ALS/FTD MODEL

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 Wang, S.-M., Wu, H.-E., Yasui, Y., Geva, M., Hayden, M., Maurice, T., Cozzolino, M., and Su,T.-P. (2023). Nucleoporin POM121 signals TFEB-mediated autophagy via activation of SIGMAR1/sigma-1 receptor chaperone by pridopidine. Autophagy, 19(1), pp. 126-151. Cunningham,K.M., Maulding K., Ruan K., Senturk M., Grima J.C., Sung H., Zuo Z., Song H., Gao J., Dubey S., Rothstein J.D., Zhang K., Bellen H.J., and Lloyd T.E. (2020). TFEB/Mitf links impaired nuclear import to autophagolysosomal dysfunction in C9-ALS. Elife (online), Article e59419, doi:10.7554/eLife.59419. Lee P.-T., Lievens J.-C., Wang S.-M., Chuang J.-Y., Khalil B., Wu H.-E., Chang W.-C., Maurice T., and Su T.-P. (2020). Sigma-1 receptor chaperones rescue nucleocytoplasmic transport deficit seen in cellular and Drosophila ALS/FTD models. Nat Commun, 11(1):5580, doi: 10.1038/s41467-020-19396-3. Estevez-Silva H.M., Cuesto G., Romero N., Brito-Armas J.M., Acevedo-Arozena A., Acebes A., and Marcellino D.J. (2022). Pridopidine promotes synaptogenesis and reduces spatial memory deficits in the Alzheimer’ s Disease APP/PS1 mouse model. Neurotherapeutics, 19(5), pp. 1566-1587. Omi T., Tanimukai H., Kanayama D., Sakagami Y., Tagami S., Okochi M., Morihara T., Sato M., Yanagida K., Kitasyoji A., Hara H., Imaizumi K., Maurice T., Chevallier N., Marchal S., Takeda M,m Kudo T. (2014). Fluvoxamine alleviates ER stress via induction of sigma-1 receptor. Cell Death Dis, 5(7), Article e1332, doi:10.1038/cddis.2014.301. Prasanth M.I., Malar D.S., tencomnao T., and Brimson J.M. (2021). The emerging role of the sigma-1 receptor in autophagy: hand-in-hand targets for the treatment of Alzheimer’ s. Expert Opin Ther Targets, 25(5), pp. 401-414.