TIA-1 promotes FUNDC1-mediated mitophagy to protect against stress-induced cellular senescence

TIA-1 promotes FUNDC1-mediated mitophagy to protect against stress-induced cellular senescence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article TIA-1 promotes FUNDC1-mediated mitophagy to protect against stress-induced cellular senescence Eun Kyung Lee, Seongho Cha, Myeongwoo Jung, Hyosun Tak, Seungyeon Ryu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7588642/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Mitochondrial dysfunction, characterized by reduced mitophagy, excessive mitochondrial elongation, and elevated reactive oxygen species (ROS) production, is a hallmark of cellular senescence. However, the molecular mechanisms linking impairment of redox balance to mitophagy suppression during senescence remain poorly understood. In this study, we identified TIA-1, an RNA-binding protein, as a positive regulator of FUNDC1 expression, a key receptor for ubiquitin-independent mitophagy. Sodium butyrate (NaBu) and UV-B irradiation triggered oxidative stress-associated senescence in HaCaT cells, leading to reduced TIA-1 expression, decreased FUNDC1 levels, impaired mitophagy flux, excessive mitochondrial elongation, and upregulation of senescence markers. Conversely, ectopic expression of TIA-1 restored FUNDC1 levels, enhanced mitophagy activity, improved mitochondrial function, and reduced the expression of senescence markers. Ribonucleoprotein immunoprecipitation assays confirmed that TIA-1 directly interacts with FUNDC1 mRNA to promote its expression. Together, these findings establish TIA-1 as a pivotal regulator of mitochondrial homeostasis during cellular stress, acting through FUNDC1 to sustain mitophagy and limit senescence. Targeting TIA-1 may offer new strategies to mitigate mitochondrial dysfunction and redox balance in aging and age-related diseases. Biological sciences/Cell biology/Senescence Biological sciences/Molecular biology/RNA metabolism/RNA quality control Biological sciences/Cell biology/Organelles/Mitochondria Redox Reactive oxygen species Mitophagy Senescence FUNDC1 Mitochondria Mitochondrial dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Mitochondrial homeostasis plays a central role in maintaining cellular function by regulating energy production, metabolic processes and various signal pathways 1 , 2 . Dysregulation of mitochondrial homeostasis leads to mitochondrial dysfunction, which has been implicated in several pathological conditions, including neurodegenerative diseases, cancer and metabolic disorders 2 , 3 . Cellular senescence, a state of irreversible cell cycle arrest, is characterized by mitochondrial dysfunction 4 . Features of this dysfunction include impaired mitochondrial dynamics, pronounced mitochondrial elongation, increased production of reactive oxygen species (ROS) and accumulation of damaged mitochondria, all of which contribute to the progression of age-related diseases and tissue degeneration 5 , 6 . In senescent cells, mitochondria exhibit abnormal elongation and dysfunction 7 , 8 ; however, the precise molecular mechanisms underlying these changes remain unclear. Our previous studies have shown that reducing TIA-1 in senescent cells induces mitochondrial elongation 9 . Building on these findings, we extended our investigation to investigate how the loss of TIA-1 contributes to mitochondrial elongation during cellular senescence. Mitophagy, the selective clearance of damaged mitochondria via autophagy, is essential for maintaining mitochondrial quality and overall cellular homeostasis 10 , 11 , 12 . While mitophagy plays a critical role in preserving mitochondrial integrity, its activity is notably reduced during cellular senescence 10 , 13 . This decline contributes to the accumulation of dysfunctional mitochondria, exacerbating mitochondrial elongation and ROS production. Despite its recognized importance, the molecular mechanisms underlying the reduction of mitophagy during senescence remain poorly understood, representing a critical gap in our knowledge. TIA-1, an RNA binding protein, is another key player in mitochondrial homeostasis 14 , 15 . It is known to regulate RNA metabolism, including alternative splicing, stability, and translation, and is involved in the formation of stress granules under various stress conditions 16 . TIA-1 has been shown to play important role in the regulation of mitochondrial homeostasis 14 , 17 , 18 . Mutations in TIA-1 have been associated with diseases such as amyotrophic lateral sclerosis (ALS) and dementia 19 , highlighting its potential clinical importance. However, the precise role of TIA-1 in mitophagy and its regulatory mechanisms during cellular senescence remain largely unexplored. In this study, we investigated the molecular mechanisms by which TIA-1 regulates mitochondrial elongation and mitophagy during stress-induced cellular senescence. Our results showed that TIA-1 regulates the expression of FUNDC1, a mitochondria-specific receptor of mitophagy 20 . Furthermore, we showed that the reduction of TIA-1 during cellular senescence leads to decreased FUNDC1 expression, which in turn impairs mitophagy. These findings provide new insights into how TIA-1 downregulation contributes to mitochondrial dysfunction in senescent cells. By providing new insights into the molecular regulation of mitophagy and mitochondrial homeostasis, our findings have the potential to advance our understanding of cellular senescence and its contribution to age-related and mitochondrial-associated diseases. In addition, this work highlights TIA-1 as potential therapeutic target for alleviating mitochondrial dysfunction in pathological conditions. Materials and methods Cell culture and transfection HaCaT (human skin keratinocytes), HEK293T (human embryonic kidney cells), and SH-SY5Y (human neuroblastoma) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Cytiva, Wilmington DE, USA) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37°C. The transfection of siRNA against TIA-1 (Genolution Pharmaceuticals, Inc., Seoul, South Korea), HA-tagged TIA-1 plasmid 9 , and their appropriate controls was carried out using Lipofectamine™ 2000 (Invitrogen™, Waltham, MA, USA), according to the manufacturer’s instruction. To establish the stress-induced senescence model, HaCaT cells were incubated with media containing 1 mM sodium butyrate (NaBu) (Sigma-Aldrich, Burlington, MA, USA) or exposed to UV-B (75 mJ/cm²) using UV Transilluminator (Core Bio System, Seoul, Korea) during the indicated time. RNA analysis Total RNA was extracted from whole cells using RNAiso Plus (Takara Bio, Inc., Shiga, Japan) and cDNA was synthesized by reverse transcription (RT) with the ReverTra® Ace qPCR RT kit (Toyobo Co., Ltd, Osaka, Japan). Quantitative PCR (qPCR) was conducted with the SensiFAST™ SYBR Hi-ROX kit (Meridian Bioscience, Inc., Cincinnati, OH, USA) and gene-specific primer sets listed in Supplementary Table S1 using the StepOnePlus™ Real-Time PCR System (Applied Biosystems™, Waltham, MA, USA). Relative levels of mRNAs were calculated using the ΔΔ CT method, comparing control and each experimental group. GAPDH mRNA was used as an internal reference gene for the normalization. Western blot (WB) analysis Whole cell lysates were prepared using the RIPA buffer containing 1× protease inhibitor cocktail (Roche, Basel, Switzerland). The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated overnight at 4°C with primary antibodies against TIA-1, p21, p16, GAPDH, p-IRF3, ISG15, GFP, Lamin B, TOM40, FUNDC1, or β-actin (Supplementary Table S2). Following incubation with primary antibodies, the membranes were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Chemiluminescence was detected by applying the Clarity Western ECL Substrate (Bio-Rad, Inc., Hercules, CA, USA) to the membranes and luminescent signals were acquired using the ChemiDoc Imaging Systems (Bio-Rad, Inc.). Analysis of mitochondrial morphology Cells were incubated with 100 nM MitoTracker Red CMXRos (Invitrogen™) in serum-free medium for 30 min at 37℃. Mitochondrial morphology was observed by tracing fluorescent signals using the Observer Z1 inverted microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using the Mitochondrial Analyzer plugin for ImageJ/Fiji software ( https://github.com/AhsenChaudhry/Mitochondria-Analyzer ). Median valued in control group were used as intermediated group. 21 The elongated group refers to cells with increased mitochondrial length compared to the control. Mitochondrial elongation was determined by measuring the ratio of cells have elongated forms of mitochondria to total cells. For transmission electron microscopy (TEM) analysis, cells were fixed with 1% osmium tetroxide and embedded in Epon 812. Ultrathin sections were analyzed with a transmission electron microscope (JEM 1010, Tokyo, Japan). Perimeter of mitochondria in TEM image of each experimental group was analyzed using ImageJ software 9 , 14 . mt-Keima assay The modified mt-Keima reporter plasmid was generated by cloning the fragment corresponding to the COX8/COX8/hmKeima-Red sequence from pCHAC-mt-mKeima (Addgene #72342) into the pcDNA 3.1. Following transfection with the pcDNA 3.1-mt-Keima plasmid (phmt-Keima), cells were incubated with 20 µM FCCP and 5 µM oligomycin (Sigma-Aldrich) for 6 hours, then the mt-Keima signal was observed using the Observer Z1 inverted microscope (Carl Zeiss) to detect acidic (586 nm excitation, "red") and pH-neutral (480 nm excitation, "green") signals. Mitophagy events were determined by calculating the ratio of red (mitophagy) to green (mitochondria) fluorescence signals from microscopic images using ImageJ software. Immunostaining and Fluorescence Microscopy Cells were labeled with 100 nM LysoTracker Red DND-99 (MedChemExpress, Monmouth Junction, NJ, USA) in complete medium for 30 minutes at 37°C, followed by fixation with 4% formaldehyde at room temperature. After permeabilization with Triton X-100 solution, cells were sequentially incubated with blocking solution and primary antibody against NDUFV2, a subunit of mitochondrial complex I located in the inner membrane, followed by incubation with Alexa Fluor® 488-conjugated secondary antibodies (Supplementary Table S2). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen). Fluorescence images were acquired using an ObserverZ1 inverted fluorescence microscope (Carl Zeiss, Germany) with consistent imaging parameters across all conditions. The number of lysosomes–mitochondria overlay events was manually quantified by counting discrete yellow puncta, indicative of colocalization, within individual cells as described in 22 . At least 200 cells selected randomly per condition were analyzed. Analysis of mitochondrial function Mitochondrial membrane potential (Δψm) and ATP generation were measured using the JC-1 assay kit (Abcam, Cambridge, UK) and the Mitochondrial ToxGlo™ system (Promega, Madison, WI, USA), respectively, according to the manufacturers’ protocols. JC-1 fluorescence was detected at 535 nm (excitation) and 590 nm (emission), while ToxGlo™ signals were recorded at 485 nm/530 nm using a Synergy H1 microplate reader (BioTek, Santa Clara, CA, USA). Mitochondrial ROS levels were assessed by incubating cells with 2.5 µM MitoSOX™ Red (Invitrogen) in HBSS for 15 mins. Fluorescence images were acquired using the Observer Z1 inverted fluorescence microscope (Carl Zeiss), and signal quantification was performed using ImageJ software. Analysis of cytosolic mitochondrial DNA release Cytosolic mtDNA was analyzed as described in 23, 24 . In brief, the cytosolic fraction was isolated by differential centrifugation using a lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 18 µg/ml digitonin, and 1× protease inhibitor cocktail. Genomic DNA was extracted from the cytosolic fraction using the AccuPrep® Genomic DNA Extraction Kit (Bioneer, Daejeon, South Korea) according to the manufacturer’s instructions. Cytosolic mitochondrial DNA (mtDNA) levels were quantified by qPCR using gene-specific primers for MT-ND1 and MT-D-loop , as listed in Supplementary Table S1 . qPCR was performed on the StepOnePlus™ Real-Time PCR System (Applied Biosystems™,). To normalize for total mtDNA content, cytosolic mtDNA levels were calibrated against mtDNA levels measured in whole-cell lysates. The nuclear gene KCNJ10 was used as the reference gene for normalization of total mtDNA quantification. Senescence-associated β-galactosidase assay Cells were fixed in 4% formaldehyde (FA) and incubated in a staining solution containing 40 mM citric acid/sodium phosphate (pH 6.0), 150 mM sodium chloride, 2 mM magnesium chloride, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/mL X-gal (BEAMS Biotechnology, Seongnam, South Korea) at 37°C for 16 hours in the dark 9 . After fixation in 20% glycerol, β-galactosidase levels were observed using an IX70w microscope (Olympus Corp., Tokyo, Japan). The number of β-gal-positive cells was counted from at least 150 cells per experiment, across three independent experiments. Ribonucleoprotein immunoprecipitation (RNP IP) RNP complexes were immunoprecipitated from cell lysates using Protein A beads (Invitrogen™) coated with anti-TIA-1 or normal rabbit IgG antibody (Santa Cruz Biotechnology, Inc.). The immunoprecipitated RNP complexes were then sequentially treated with DNase I and proteinase K. RNAs isolated from the complexes were analyzed by RT-qPCR using gene-specific primer sets listed in Supplementary Table S1 . Biotin pulldown assay T7 promoter-incorporated DNA fragments corresponding to the 3′UTR of FUNDC1 mRNA (NM_173794.4) were amplified by PCR using specific primers (Supplementary Table S1 ). Biotinylated RNA probes were synthesized by in vitro transcription using the MaxiScript T7 kit (Ambion, Waltham, MA, USA) and biotin-CTP (Enzo Life Sciences, Farmingdale, NY, USA). Cell lysates were incubated with purified biotinylated RNA probes for 30 min at room temperature. Proteins bound to biotinylated RNA probes were isolated using streptavidin magnetic beads (Invitrogen™), and further analyzed by WB using TIA-1 antibody 9 . EGFP reporter analysis The EGFP reporter containing the 3′UTR of FUNDC1 mRNA (pEGFP-FUNDC1 3U) was generated by inserting 3′UTR fragment of FUNDC1 mRNA (NM_173794.4, 496–1054 nt, 559 bp) into pEGFP-C1 plasmid (BD Bioscience, Franklin Lakes, NJ, USA). After transfection with siRNA (siCtrl or siTIA-1) or plasmid (pCtrl or pTIA-1), the cells were sequentially transfected with reporter plasmids (control C1 or EGFP-FUNDC1 3U) for 24 hours. The relative expression of the EGFP reporter was assessed by WB analysis. Statistical analysis Data are presented as the mean ± SEM from three independent experiments. Statistical significance was assessed using the Student’s t-test (n.s., not significant with p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001). Results Stress-induced senescence is associated with mitochondrial hyperfusion and reduced mitophagy in HaCaT cells To establish a model of stress-induced senescence in human keratinocyte HaCaT cells, we treated cells with sodium butyrate (NaBu) or UV-B irradiation. Both stressors significantly increase senescence-associated β-galactosidase (SA β-gal) activity, as demonstrated by enhanced SA β-gal staining in treated cells (Fig. 1 A). Western blot analysis reveals that NaBu and UV-B exposure upregulate the expression of canonical senescence markers p16 and p21, while reducing Lamin B levels (Fig. 1 B). Notably, both treatments also lead to a marked decrease in the expression of TIA-1 (Fig. 1 B), as observed in our previous study 9 . Consistently, RT-qPCR analysis confirms that TIA-1 mRNA levels are significantly reduced under both stress conditions (Fig. 1 C). Next, we examined mitochondrial morphology using MitoTracker staining. Both NaBu and UV-B treatment significantly increase the proportion of cells exhibiting elongated mitochondria, indicating stress-induced mitochondrial hyperfusion (Fig. 1 D). Transmission electron microscopy (TEM) analysis further showed that NaBu treatment increased mitochondrial perimeter compared to controls (Fig. 1 E). The enhanced mitochondrial elongation in senescent cells could result from the altered expression of proteins that regulate mitochondrial dynamics 6 , 23 ; however, it could also be due to a decrease in mitophagy activity, a form of selective autophagy leading to mitochondrial degradation 25 , 26 . To investigate the alteration in mitophagy in cellular senescence, we assessed mitophagy activity in these stress conditions using the mt-Keima reporter assay 27 , 28 . HaCaT cells transfected with mt-Keima were treated with NaBu or UV-B and then challenged with FCCP to induce mitophagic flux. Both NaBu and UV-B treatment significantly reduced the FCCP-induced mt-Keima red-to-green fluorescence ratio, indicating impaired mitophagy activity under stress (Figs. 2 A and B). Together, these findings demonstrate that NaBu and UV-B-induced stress promote cellular senescence in HaCaT cells, which is associated with decreased TIA-1 expression, mitochondrial hyperfusion, and reduced mitophagic clearance. TIA-1 knockdown exacerbates stress-induced senescence and suppresses mitophagy To investigate the role of TIA-1 in regulating mitophagy under stress conditions, we first assessed mt-Keima reporter activity after TIA-1 knockdown. TIA-1 downregulation significantly reduced FCCP-induced mt-Keima red fluorescence signal in HaCaT, HEK293T, and SH-SY5Y cells (Supplementary Fig. S1 ), indicating that TIA-1 downregulation broadly impairs mitophagic flux. Next, we examined how TIA-1 knockdown influence stress-induced senescence and mitophagy in the NaBu treatment model. In HaCaT cells, TIA-1 knockdown led to a greater increase in SA β-gal-positive staining (Fig. 3 A) and an elevation of senescence marker p21 and p16 (Fig. 3 B) after NaBu treatment compared to control. Mitophagy activity, measured by the mt-Keima assay, was even more decreased by TIA-1 knockdown in NaBu-treated cells, as reflected by a further reduced red-to green fluorescence ratio (Fig. 3 C). Similar enhancement of NaBu-induced mitophagy suppression was observed in HEK293T cells following TIA-1 downregulation (Supplementary Fig. S2). These findings suggest that loss of TIA-1 level exacerbates stress-induced senescence and further impairs mitophagic clearance in HaCaT cells. TIA-1 overexpression mitigates stress-induced senescence and restores mitophagy activity To determine whether TIA-1 overexpression can alleviate stress-induced senescence, we investigated senescence markers in HaCaT cells exposed to either NaBu or UV-B irradiation. SA β-gal staining revelated that ectopic expression of TIA-1 significantly reduced the number of SA β-gal-positive cells under both NaBu (Fig. 4 A) and UV-B (Fig. 4 B) treatment conditions. These results indicate that elevated TIA-1 level decreases the acquisition of SA β-gal expression in response to different stressors. In addition, TIA-1 overexpression markedly attenuated the stress-induced increase in p21 and p16 levels in both NaBu and UV-B treated cells (Figs. 4 C and 4 D), further supporting its role in regulating cellular senescence. Finally, we examined whether TIA-1 overexpression could restore mitophagy activity suppressed by stress. Using the mt-Keima reporter assay, we observed that both treatments reduced FCCP-induced mitophagic flux, as indicated by a decreased red-to-green fluorescence ratio. Importantly, ectopic expression of TIA-1 significantly rescued mitophagy activity under both NaBu and UV-B (Figs. 4 D and 4 F) treatment conditions. Together, these results demonstrate that TIA-1 overexpression consistently attenuates stress-induced senescence and restored mitophagy activity in response to senescence-inducing stimuli, underscoring its critical protective role in maintaining mitochondrial quality control under stress conditions. TIA-1 overexpression alleviates NaBu-induced mitochondrial dysfunction and limits mtDNA-mediated inflammatory signaling To further investigate how TIA-1 overexpression modulates cellular responses to stress, we examined its effects on mitochondrial function in NaBu-treated HaCaT cells. NaBu exposure significantly reduced mitochondrial membrane potential, as determined by decreased JC-1 staining intensity. Notably, ectopic expression of TIA-1 significantly preserved mitochondrial membrane potential under NaBu treatment (Fig. 5 A). Mitochondrial ATP production assays showed similar results: while NaBu treatment decreased ATP levels, TIA-1 overexpression rescued this decline (Fig. 5 B). We also assessed mitochondrial reactive oxygen species (ROS) production using MitoSOX staining. NaBu treatment markedly increased ROS levels in HaCaT cells, but TIA-1 overexpression significantly attenuated this stress-induced ROS accumulation (Fig. 5 C). Together, these data indicate that TIA-1 overexpression can mitigate NaBu-induced mitochondrial dysfunction. Because mitophagy is closely linked to the control of cytosolic mitochondrial DNA (mtDNA) release 29 , 30 , we next investigated whether stress-induced senescence or TIA-1 knockdown altered mtDNA release. Fractionation and qPCR analysis revealed that both NaBu treatment and TIA-1 knockdown increased cytosolic mtDNA (MT-ND1 and D-loop) levels in HaCaT cells without changing total mtDNA copy number (Supplementary Fig. S3). Importantly, we found that TIA-1 overexpression significantly reduced the relative amount of mtDNA detected in the cytosolic fraction after NaBu treatment, again without altering total mtDNA copy number (Fig. 5 D). These findings suggest that TIA-1 overexpression suppresses stress-induced mtDNA release into the cytosol. Given that cytosolic mtDNA release can activate the cGAS–STING pathway and induce pro-inflammatory gene expression 31 , we examined downstream signaling events. Western blot analysis showed that NaBu treatment increased phosphorylation of IRF3 and the level of free ISG15 protein, while TIA-1 overexpression partially reduced these stress-induced increases (Fig. 5 E). Consistently, NaBu treatment upregulated mRNA levels of IL6 , IL8 , IL1β , and IFNγ , whereas TIA-1 overexpression modestly attenuated this transcriptional activation (Fig. 5 F). Together, these results demonstrate that TIA-1 overexpression not only protects against NaBu-induced mitochondrial dysfunction but also limits mtDNA-mediated activation of the cGAS–STING inflammatory pathway in senescent HaCaT cells. TIA-1 directly associates with the FUNDC1 mRNA to promote its expression TIA-1 is an RNA-binding protein that binds to its target mRNAs and can affect their expression by regulating RNA metabolism 16 . To understand the molecular mechanism by which TIA-1 regulates mitophagy, we sought to identify the downstream molecules of TIA-1. By analyzing the TIA-1 crosslinking immunoprecipitation sequencing (CLIP-seq) dataset 32 and POSTAR3 database 33 , we identified a set of putative TIA-1-bound mRNAs encoding selective autophagy receptors (SARs), which are essential mediators for mitophagy 11 , 12 (Fig. 6 A). To confirm whether TIA-1 physically associates with these SAR mRNAs, we performed ribonucleoprotein (RNP) immunoprecipitation using TIA-1 antibody, followed by RT-qPCR. Several SAR mRNAs, including NIX , BNIP3 , FUNDC1 , and BCL2L13 , were significantly enriched in TIA-1-containing RNP complexes compared to control IgG immunoprecipitation, indicating direct binding (Fig. 6 B). Among these candidates, FUNDC1 mRNA showed statistically significant and consistent enrichment. FUNDC1 (a FUN14 domain containing 1) is a known SAR involved in ubiquitin-independent mitophagy 34 and contains a ~ 500 nt 3'UTR in human (NM_173794.4) (Fig. 6 C). To test direct interaction between TIA-1 and FUNDC1 mRNA, we performed in vitro binding assays with biotin-labeled transcripts corresponding to the 3'UTR of FUNDC1 mRNA. TIA-1 bound to the 3'UTR of FUNDC1 mRNA (Fig. 6 C), supporting a direct interaction. To investigate whether TIA-1 modulates FUNDC1 expression via its 3'UTR, we used an EGFP reporter construct containing the FUNDC1 3′UTR. TIA-1 knockdown significantly reduced reporter expression, whereas TIA-1 overexpression increased it (Fig. 6 D). These results indicate that TIA-1 binds to 3'UTR of FUNDC1 mRNA and promotes its expression. Consistent with this observation, modulation of TIA-1 expression in HaCaT cells altered endogenous FUNDC1 protein levels: TIA-1 knockdown reduced FUNDC1 protein, while overexpression increased it (Figs. 6 E and 6 F). Importantly, these changes occurred without significant alterations in total FUNDC1 mRNA levels, suggesting that TIA-1 primarily regulates FUNDC1 at the level of translation. Ectopic expression of TIA-1 restores FUNDC1 expression and promotes mitophagy under stress conditions To further validate the mechanistic link between TIA-1 and mitophagy regulation under stress, we investigated whether TIA-1 overexpression could restore FUNDC1 expression in HaCaT cells in response to NaBu or UV-B treatment. Western blot analysis revealed that both stress conditions reduced FUNDC1 protein levels, whereas ectopic expression of TIA-1 significantly rescued FUNDC1 expression under both NaBu (Fig. 7 A) and UV-B (Fig. 7 B) conditions. We next examined whether changes in FUNDC1 levels driven by TIA-1 overexpression were associated with alterations in mitophagy. Immunofluorescence analysis using FUNDC1 antibody and Lysotracker to label lysosomes showed that NaBu treatment increased co-localized FUNDC1 and lysosomal signals, indicating stress-induced mitophagy activation. Importantly, TIA-1 overexpression further enhanced the overlap of FUNDC1 and lysosomal signals under NaBu stress (Fig. 7 C). These results suggest that TIA-1 overexpression promotes FUNDC1-mediated mitophagy by enhancing mitochondrial–lysosomal fusion in stressed cells. Based on these findings, we propose a mechanistic model (Fig. 7 D) in which stress-induced downregulation of TIA-1 leads to decreased FUNDC1 expression, suppression of mitophagy, mitochondrial hyperfusion, and ultimately mitochondrial dysfunction. Conversely, ectopic expression of TIA-1 restores FUNDC1 levels, enhances mitophagy activity, and alleviates stress-induced mitochondrial dysfunction and cellular senescence. Discussion Tight regulation of mitochondrial homeostasis is essential for the maintenance of normal cellular functions 2 , 10 . Mitochondrial homeostasis is controlled at multiple levels, including biogenesis, dynamics and mitophagy 10 . Dysregulation of mitochondrial homeostasis leads to mitochondrial dysfunction, which contributes to the pathogenesis of several human diseases 2 , 5 , 35 . Mitochondrial dysfunction, as indicated by imbalanced mitochondrial dynamics, decreased membrane potential, and elevated ROS production, has been identified as a significant contributor to cellular senescence 6 , 23 . In this study, we demonstrated that mitophagy is reduced in stress-induced senescence model, accompanied by decreased expression of TIA-1. We found that TIA-1 knockdown decreased mitophagy, while its ectopic expression enhanced mitophagy activity and mitigated senescence marker expression under NaBu or UV-B treatment. These results suggest that TIA-1 may serve as a novel regulator of mitophagy in the context of cellular senescence. Several studies have shown that senescent cells have highly elongated mitochondria 1 , 4 . Enhanced elongation of mitochondria has been linked to alterations in mitochondrial dynamics, specifically the downregulation of fission factors such as DRP1, FIS1, and MFF, and the upregulation of fusion factors such as MFN1/2 and OPA1. Changes in the expression levels of these proteins have been consistently observed in senescent cells 7 , 8 , 9 . Furthermore, the reduction in mitophagy activity, which is crucial for the removal of damaged mitochondria, may contribute to the excessive elongation of mitochondria in senescent cells 13 , 26 , 36 . Our previous research documented reduced MFF expression in senescent HaCaT cells 9 , and in this study, we further identified a reduction in FUNDC1 expression. Importantly, we have shown that TIA-1 plays a key role in regulating the expression of MFF and FUNDC1 in senescent cells. These findings underscore the central role of TIA-1 in maintaining mitochondrial homeostasis and highlight its potential as a therapeutic target for diseases caused by mitochondrial dysfunction, including cancer and age-related diseases. Despite its critical role in regulating mitochondrial homeostasis and cellular senescence, much remains unknown about the molecular functions of TIA-1 and the mechanisms governing its expression. As an RNA-binding protein, TIA-1 modulates RNA metabolism processes such as alternative splicing and translation 16 . In response to various stress stimuli, it regulates the expression of its target mRNAs by forming stress granules or interacting with other proteins 37 . Mutations in the TIA-1 gene have been associated with several human diseases such as amyotrophic lateral sclerosis (ALS) and myopathy, highlighting its clinical significance 19 , 38 . While certain factors, including microRNA-27a and microRNA-30-5p , have been identified as regulators of TIA-1 expression by our group 9 , 14 , the precise mechanisms controlling TIA-1 expression and activity remain largely unclear. Notably, how TIA-1 expression is diminished during cellular senescence is a question that requires further investigation. Our findings demonstrated that TIA-1 expression is reduced following NaBu treatment (Fig. 1 ). Exploring how HDAC inhibitor butyrate mediates this downregulation could provide valuable insights into the regulatory mechanisms underlying TIA-1 expression. FUNDC1 is a mitochondrial receptor that directly interacts with LC3 and mediates a PINK1/Parkin-independent pathway of mitophagy in response to various stimuli 20 , 39 . Dysregulation of mitophagy due to alterations in FUNDC1 expression has been implicated in several pathological conditions, including cancer, fibrosis and cardiovascular disease 34 , 40 , 41 , 42 . In this study, we observed the downregulation of FUNDC1 and a corresponding reduction in mitophagy in the NaBu-induced senescence model. In addition to its role in regulating mitophagy, FUNDC1 plays a critical role in maintaining mitochondrial homeostasis by contributing to the formation of mitochondrial-endoplasmic reticulum (ER) contact sites (MERCs), mitochondrial biogenesis and mitochondrial dynamics 43 , 44 . Therefore, a detailed understanding of FUNDC1 expression and its regulatory mechanisms is important for advancing our knowledge of FUNDC1-mediated pathology. While our study has identified TIA-1 as a novel regulator of FUNDC1 expression, further investigations are required to identify additional regulatory factors and to elucidate the pathways governing FUNDC1 function across multiple levels including transcription, post-transcription, post-translation, or protein-protein interaction. These efforts will contribute to a more comprehensive understanding of mitochondrial homeostasis mediated by FUNDC1. In conclusion, our findings provide new insights into the regulatory roles of TIA-1 and FUNDC1 in mitochondrial homeostasis and cellular senescence. By identifying TIA-1 as a novel regulator of FUNDC1 expression and highlighting its impact on mitophagy, this study underscores the importance of these factors in maintaining mitochondrial structure and functionality. These results not only advance our understanding of the molecular mechanisms underlying mitochondrial dysregulation in the stress-induced senescence model but also lay the groundwork for future investigations into potential therapeutic strategies targeting mitochondrial dysfunction in cellular senescence and age-related and pathological conditions. Declarations Conflict of interest Authors have no conflicts of interest to declare. Funding This work was supported by the Basic Science Research Programs through the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (RS-2021-NR058095 and RS-2024-00405790) and the GRRC program of Gyeonggi province (GRRCAjou2023-B01). Author contribution EKL conceived and supervised the study. DC, JK, SMJ acquired funding for the study. SC, HT, WK and EKL conceptualized and designed the experiment. SC, MJ, SR, and SH conducted experiments. SC analyzed the data and created the figures. SC and EKL wrote manuscript. SC and WK revised the manuscript. All authors have read and approved the manuscript. Acknowledgement The authors appreciate Professor DH Cho and DS Jo, PhD at Kyungpook National University and Professor JH Kim for the technical advice and sharing materials for the mt-Keima assay and cmtDNA analysis, respectively. Availability of Data and Materials The data analyzed during this study are included in this published article and the supplemental data files. References Chen W, Zhao H, Li Y. Mitochondrial dynamics in health and disease: mechanisms and potential targets. Signal Transduct Target Ther 2023, 8(1): 333. Zong Y, Li H, Liao P, Chen L, Pan Y, Zheng Y, et al. Mitochondrial dysfunction: mechanisms and advances in therapy. Signal Transduct Target Ther 2024, 9(1): 124. Suomalainen A, Nunnari J. Mitochondria at the crossroads of health and disease. Cell 2024, 187(11): 2601–2627. Martini H, Passos JF. Cellular senescence: all roads lead to mitochondria. Febs J 2023, 290(5): 1186–1202. Amorim JA, Coppotelli G, Rolo AP, Palmeira CM, Ross JM, Sinclair DA. Mitochondrial and metabolic dysfunction in ageing and age-related diseases. Nat Rev Endocrinol 2022, 18(4): 243–258. Miwa S, Kashyap S, Chini E, von Zglinicki T. Mitochondrial dysfunction in cell senescence and aging. J Clin Invest 2022, 132(13). Yoon YS, Yoon DS, Lim IK, Yoon SH, Chung HY, Rojo M, et al. Formation of elongated giant mitochondria in DFO-induced cellular senescence: involvement of enhanced fusion process through modulation of Fis1. J Cell Physiol 2006, 209(2): 468–480. Mai S, Klinkenberg M, Auburger G, Bereiter-Hahn J, Jendrach M. Decreased expression of Drp1 and Fis1 mediates mitochondrial elongation in senescent cells and enhances resistance to oxidative stress through PINK1. J Cell Sci 2010, 123(Pt 6): 917–926. Tak H, Cha S, Hong Y, Jung M, Ryu S, Han S, et al. The miR-30-5p/TIA-1 axis directs cellular senescence by regulating mitochondrial dynamics. Cell Death Dis 2024, 15(6): 404. Ma K, Chen G, Li W, Kepp O, Zhu Y, Chen Q. Mitophagy, Mitochondrial Homeostasis, and Cell Fate. Front Cell Dev Biol 2020, 8: 467. Picca A, Faitg J, Auwerx J, Ferrucci L, D'Amico D. Mitophagy in human health, ageing and disease. Nat Metab 2023, 5(12): 2047–2061. Onishi M, Yamano K, Sato M, Matsuda N, Okamoto K. Molecular mechanisms and physiological functions of mitophagy. Embo J 2021, 40(3). Korolchuk VI, Miwa S, Carroll B, von Zglinicki T. Mitochondria in Cell Senescence: Is Mitophagy the Weakest Link? Ebiomedicine 2017, 21: 7–13. Tak H, Eun JW, Kim J, Park SJ, Kim C, Ji E, et al. T-cell-restricted intracellular antigen 1 facilitates mitochondrial fragmentation by enhancing the expression of mitochondrial fission factor. Cell Death Differ 2017, 24(1): 49–58. Sanchez-Jimenez C, Izquierdo JM. T-cell intracellular antigen (TIA)-proteins deficiency in murine embryonic fibroblasts alters cell cycle progression and induces autophagy. PLoS One 2013, 8(9): e75127. Fernandez-Gomez A, Izquierdo JM. The Multifunctional Faces of T-Cell Intracellular Antigen 1 in Health and Disease. Int J Mol Sci 2022, 23(3). Carrascoso I, Alcalde J, Sanchez-Jimenez C, Gonzalez-Sanchez P, Izquierdo JM. T-Cell Intracellular Antigens and Hu Antigen R Antagonistically Modulate Mitochondrial Activity and Dynamics by Regulating Optic Atrophy 1 Gene Expression. Mol Cell Biol 2017, 37(17). Carrascoso I, Velasco BR, Izquierdo JM. Deficiency of T-Cell Intracellular Antigen 1 in Murine Embryonic Fibroblasts Is Associated with Changes in Mitochondrial Morphology and Respiration. Int J Mol Sci 2021, 22(23). Mackenzie IR, Nicholson AM, Sarkar M, Messing J, Purice MD, Pottier C, et al. TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics. Neuron 2017, 95(4): 808–816 e809. Chen M, Chen Z, Wang Y, Tan Z, Zhu C, Li Y, et al. Mitophagy receptor FUNDC1 regulates mitochondrial dynamics and mitophagy. Autophagy 2016, 12(4): 689–702. Chaudhry A, Shi R, Luciani DS. A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells. Am J Physiol-Endoc M 2020, 318(2): E87–E101. Liu B, Li A, Qin Y, Chen L, Gao M, Gong G. Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome. J Vis Exp 2022(189). Victorelli S, Salmonowicz H, Chapman J, Martini H, Vizioli MG, Riley JS, et al. Apoptotic stress causes mtDNA release during senescence and drives the SASP. Nature 2023, 622(7983): 627–636. Bryant JD, Lei Y, VanPortfliet JJ, Winters AD, West AP. Assessing Mitochondrial DNA Release into the Cytosol and Subsequent Activation of Innate Immune-related Pathways in Mammalian Cells. Curr Protoc 2022, 2(2): e372. Zhang KH, Li QL, Zhang YX, Nuerlan G, Li YY, Mao J, et al. Targeting Mitophagy as a Potential Therapeutic Approach for Age-Related Bone Diseases. Adv Ther-Germany 2024, 7(7). Kelly G, Kataura T, Panek J, Ma GL, Salmonowicz H, Davis A, et al. Suppressed basal mitophagy drives cellular aging phenotypes that can be reversed by a p62-targeting small molecule. Dev Cell 2024, 59(15). Vargas JNS, Wang CX, Bunker E, Hao L, Maric D, Schiavo G, et al. Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy. Molecular Cell 2019, 74(2): 347–+. Puri R, Cheng XT, Lin MY, Huang N, Sheng ZH. Mul1 restrains Parkin-mediated mitophagy in mature neurons by maintaining ER-mitochondrial contacts. Nat Commun 2019, 10. Zhong W, Rao Z, Xu J, Sun Y, Hu H, Wang P, et al. Defective mitophagy in aged macrophages promotes mitochondrial DNA cytosolic leakage to activate STING signaling during liver sterile inflammation. Aging Cell 2022, 21(6): e13622. Jimenez-Loygorri JI, Villarejo-Zori B, Viedma-Poyatos A, Zapata-Munoz J, Benitez-Fernandez R, Frutos-Lison MD, et al. Mitophagy curtails cytosolic mtDNA-dependent activation of cGAS/STING inflammation during aging. Nat Commun 2024, 15(1): 830. Kim J, Kim HS, Chung JH. Molecular mechanisms of mitochondrial DNA release and activation of the cGAS-STING pathway. Exp Mol Med 2023, 55(3): 510–519. Meyer C, Garzia A, Mazzola M, Gerstberger S, Molina H, Tuschl T. The TIA1 RNA-Binding Protein Family Regulates EIF2AK2-Mediated Stress Response and Cell Cycle Progression. Mol Cell 2018, 69(4): 622–635 e626. Zhao WH, Zhang S, Zhu YM, Xi XC, Bao PF, Ma ZY, et al. POSTAR3: an updated platform for exploring post-transcriptional regulation coordinated by RNA-binding proteins. Nucleic Acids Res 2022, 50(D1): D287–D294. Bai XZ, Zhang Z, Li X, Yang YJ, Ding SZ. FUNDC1: An Emerging Mitochondrial and MAMs Protein for Mitochondrial Quality Control in Heart Diseases. International Journal of Molecular Sciences 2023, 24(11). Gong G, Song M, Csordas G, Kelly DP, Matkovich SJ, Dorn GW, 2nd. Parkin-mediated mitophagy directs perinatal cardiac metabolic maturation in mice. Science 2015, 350(6265): aad2459. Rambold AS, Kostelecky B, Elia N, Lippincott-Schwartz J. Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation. P Natl Acad Sci USA 2011, 108(25): 10190–10195. Rayman JB, Kandel ER. TIA-1 Is a Functional Prion-Like Protein. Csh Perspect Biol 2017, 9(5). Hackman P, Sarparanta J, Lehtinen S, Vihola A, Evilä A, Jonson PH, et al. Welander distal myopathy is caused by a mutation in the RNA-binding protein TIA1. Ann Neurol 2013, 73(4): 500–509. Liu L, Feng D, Chen G, Chen M, Zheng Q, Song P, et al. Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells. Nat Cell Biol 2012, 14(2): 177–185. Hou HL, Er PC, Cheng JJ, Chen XL, Ding XF, Wang YW, et al. High expression of FUNDC1 predicts poor prognostic outcomes and is a promising target to improve chemoradiotherapy effects in patients with cervical cancer. Cancer Med-Us 2017, 6(8): 1871–1881. Yuan QC, Sun N, Zheng JY, Wang YX, Yan XL, Mai WQ, et al. Prognostic and Immunological Role of FUN14 Domain Containing 1 in Pan-Cancer: Friend or Foe? Front Oncol 2020, 9. Ma LJ, Li KF, Wei WX, Zhou JH, Li Z, Zhang T, et al. Exercise protects aged mice against coronary endothelial senescence via FUNDC1-dependent mitophagy. Redox Biol 2023, 62. Wu WX, Lin CX, Wu K, Jiang L, Wang XJ, Li W, et al. FUNDC1 regulates mitochondrial dynamics at the ER-mitochondrial contact site under hypoxic conditions. Embo J 2016, 35(13): 1368–1384. Liu L, Li YJ, Wang JN, Zhang D, Wu H, Li WH, et al. Mitophagy receptor FUNDC1 is regulated by PGC-1α/NRF1 to fine tune mitochondrial homeostasis. Embo Rep 2021, 22(3). 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1","display":"","copyAsset":false,"role":"figure","size":507859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial hyperfusion is induced by stress in HaCaT cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaCaT cells were treated with 1 mM sodium butyrate (NaBu) or exposed to UV-B irradiation (75 mJ/cm²), followed by incubation for 72 hours. (A) SA β-gal activity was assessed by X-gal staining (pH 6.0) and the number of SA β-gal-positive cells were quantified. (B and C) TIA-1 expression was analyzed by Western blotting (WB) and RT-qPCR. β-actin is a loading control for WB and \u003cem\u003eGAPDH\u003c/em\u003emRNA was used for RT-qPCR normalization. (D and E) Mitochondrial morphology was analyzed using MitoTracker staining (D) and transmission electron microscopy (E). The number of cells with elongated mitochondria and the mitochondrial perimeter were determined using ImageJ software. Representative images are shown and data represent mean ± SEM from three independent experiments. Scale bars: 20 μm (A and D), 0.5 μm (E). *, p \u0026lt; 0.05; ***, p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7588642/v1/56af28ca4d7d50cb82b18ed2.png"},{"id":97367684,"identity":"6e13530a-45ab-48c4-9c11-802c3467e3cd","added_by":"auto","created_at":"2025-12-03 16:20:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":261399,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitophagy is reduced in stress-induced senescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter transfection with phmt-Keima, HaCaT cells were incubated with NaBu (A) or exposed to UV-B irradiation (75 mJ/cm²) (B) for 72 hours. Mitophagy activity was assessed by monitoring the red fluorescence signals of the hmt-Keima reporter followingtreatment with FCCP (20 μM) and oligomycin (5 μM) for 6 hours. The fluorescence ratio (red/green) was determined using ImageJ software, as described in the Materials and Methods. Representative images are shown and data represent mean ± SEM from three independent experiments. Scale bars: 20 μm. ***, p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7588642/v1/83576baf9f5559668ccd2f55.png"},{"id":97263488,"identity":"4033de52-8296-4763-becd-14166883c099","added_by":"auto","created_at":"2025-12-02 14:21:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":366236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTIA-1 knockdown promotes cellular senescence and reduces mitophagy in response to NaBu treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing TIA-1 knockdown, HaCaT cells were treated with 1 mM NaBu for 48 hours. (A) SA β-gal activity was assessed by X-gal staining (pH 6.0) and the number of SA β-gal-positive cells were quantified. (B) Protein expression was analyzed by WB. β-actin was used as the loading control. (C) Mitophagy activity was assessed by monitoring the red fluorescence signals of the hmtKeima reporter following treatment with FCCP (20 μM), and the fluorescence ratio (red/green) was determined using ImageJ software. Representative images are shown and data represent mean ± SEM from three independent experiments. Scale bar, 20 μm. **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7588642/v1/d9eed675ad26f6d2da80da6e.png"},{"id":97367067,"identity":"163b14f0-e853-4834-af11-c7d64717d9a1","added_by":"auto","created_at":"2025-12-03 16:16:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":322208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTIA-1 overexpression attenuates mitophagy reduction during stress-induced senescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing ectopic expression of pTIA-1, HaCaT cells were treated with 1 mM NaBu or exposed to UV-B (75 mJ/cm²) and incubated for 48 hours. (A and B) SA β-gal activity was assessed by X-gal staining (pH 6.0) and the number of SA β-gal-positive cells were quantified in NaBu (A) and UV-B (B) treated groups.(C and D) Protein expression was analyzed by WB in NaBu (C) and UV-B (D) treated groups. β-actin was used as the loading control. (E and F) Mitophagy activity was assessed by monitoring the red fluorescence signals of the hmtKeima reporter followingtreatment with FCCP (20 μM) and the fluorescence ratio (red/green) was determined in NaBu (E) and UV-B (F) treated groups using ImageJ software. Representative images are shown and data represent mean ± SEM from three independent experiments. Scale bar: 20 μm. **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7588642/v1/17ed699e8e42b63ce8f5c349.png"},{"id":97263487,"identity":"198ae194-fee6-4c9f-8c46-166672352de5","added_by":"auto","created_at":"2025-12-02 14:21:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":208989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTIA-1 overexpression attenuates mitochondrial dysfunction induced by NaBu treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing ectopic expression of pTIA-1, HaCaT cells were treated with 1 mM NaBu for 48 hours. (A–C) Mitochondrial function was evaluated by measuring membrane potential (Δψm, JC-1 staining) (A), ATP production (Mitochondrial ToxGlo™) (B), and mitochondrial superoxide levels (MitoSOX™) (C). (D) Cytosolic release of mitochondrial DNA (mtDNA) was evaluated by measuring the levels of mtDNA (ND1 and D-loop) in the cytosolic fraction using qPCR. TOM40 and GAPDH served as markers for the mitochondrial and cytosolic faction, respectively, and β-actin was used as a loading control. The relative level of mtDNA in the cytosolic fraction was determined by qPCR following normalization to the total mtDNA copy number in whole-cell lysate. Total mtDNA copy number was determined by qPCR using genomic DNA from whole-cell lysates, with normalization to KCNJ10 as the reference gene. (E) Levels of IRF3 phosphorylation and free ISG15 were analyzed by WB. β-actin was used as the loading control. (F) \u003cem\u003eIL6, IL8, IL1β\u003c/em\u003e, and \u003cem\u003eIFNγ\u003c/em\u003emRNA levels were analyzed by RT-qPCR.\u003cem\u003e GAPDH\u003c/em\u003e mRNA was used for normalization. Representative images are shown and data represent mean ± SEM from three independent experiments. n.s., not significant (p \u0026gt; 0.05); *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7588642/v1/fed4a62a7fef5c349f42b772.png"},{"id":97367562,"identity":"30116291-fc56-4c35-9179-e20180593874","added_by":"auto","created_at":"2025-12-03 16:19:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":212059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTIA-1 promotes FUNDC1 expression by associating with the 3′UTR of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFUNDC1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mRNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram illustrating the identification of putative target mRNAs of TIA-1. \u003cem\u003eIn silico \u003c/em\u003eanalysis using TIA-1 CLIP-seq data combined with GO analysis for mitophagy related genes identified several selective autophagy receptors (SARs) genes. (B) The interaction between TIA-1 and SAR gene mRNAs was analyzed by RNP immunoprecipitation (RNP IP) followed by RT-qPCR with anti-TIA-1 and normal IgG antibodies. \u003cem\u003eGAPDH\u003c/em\u003e mRNA was used as the reference gene for normalization. (C) Biotinylated transcripts corresponding to the 3′UTR of \u003cem\u003eFUNDC1\u003c/em\u003e mRNA (NM_173794.4, denoted as FUNDC1 3U) or control GAPDH\u003cem\u003e \u003c/em\u003e3U were incubated with HaCaT cell lysates, and their association with TIA-1 was determined by biotin pulldown assay. (D) A schematic representation of EGFP reporter constructs containing the 3′UTR of \u003cem\u003eFUNDC1\u003c/em\u003emRNA (upper). Reporter expression was analyzed by WB after TIA-1 knockdown or overexpression (bottom). (E and F) FUNDC1 expression levels were analyzed using WB and RT-qPCR following TIA-1 knockdown or overexpression. β-actin served as the loading control for WB, and \u003cem\u003eGAPDH\u003c/em\u003e mRNA was used as the reference gene for normalization of RT-qPCR data. Representative images are shown and data represent mean ± SEM from three independent experiments. n.s., not significant (p \u0026gt; 0.05); *, p \u0026lt; 0.05; ***, p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7588642/v1/ef6635a6893147160eb52c87.png"},{"id":97263491,"identity":"4da8ee26-17bb-4764-9994-ffd925d3cccf","added_by":"auto","created_at":"2025-12-02 14:21:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":365405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTIA-1 promotes mitophagy and prevents mitochondrial hyperfusion by upregulating FUNDC1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A and B) HaCaT cells were transfected with pTIA-1 and further incubated with 1 mM NaBu (A) or exposed to UV-B irradiation (75 mJ/cm²) (B). Protein levels were determined by WB, and β-actin was used as the loading control. (C) HaCaT cells were transfected with pTIA-1 and further incubated with 1 mM NaBu. Intracellular distribution of FUNDC1 (green) and lysosomes (red) was examined by immunofluorescence microscopy. Nuclei were counterstained with DAPI (blue). Representative images are shown and data represent mean ± SEM from three independent experiments. Scale bars: 20 μm. (D) Schematic model depicting the proposed mechanism: stress-induced downregulation of TIA-1 reduces FUNDC1 expression, leading to impaired mitophagy and mitochondrial hyperfusion, thereby promoting cellular senescence. Ectopic expression of TIA-1 restores FUNDC1 levels, enhances mitophagy, and alleviates mitochondrial dysfunction and senescence.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7588642/v1/eea3635bc5d2dc0b6b0061bd.png"},{"id":97664542,"identity":"9c35a94d-5735-4a9a-893f-c07e84687532","added_by":"auto","created_at":"2025-12-08 09:09:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3126047,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7588642/v1/2d8006fb-899a-4948-ae1d-0f5b05be9c0b.pdf"},{"id":97368465,"identity":"33d65f46-7eec-4383-b530-7cea8519c036","added_by":"auto","created_at":"2025-12-03 16:22:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":867073,"visible":true,"origin":"","legend":"Supplementary materials","description":"","filename":"ChaetalSupplementarymaterialsEMM.docx","url":"https://assets-eu.researchsquare.com/files/rs-7588642/v1/c5d19e061d28ad4ec59b0311.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"TIA-1 promotes FUNDC1-mediated mitophagy to protect against stress-induced cellular senescence","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMitochondrial homeostasis plays a central role in maintaining cellular function by regulating energy production, metabolic processes and various signal pathways \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Dysregulation of mitochondrial homeostasis leads to mitochondrial dysfunction, which has been implicated in several pathological conditions, including neurodegenerative diseases, cancer and metabolic disorders \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Cellular senescence, a state of irreversible cell cycle arrest, is characterized by mitochondrial dysfunction \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Features of this dysfunction include impaired mitochondrial dynamics, pronounced mitochondrial elongation, increased production of reactive oxygen species (ROS) and accumulation of damaged mitochondria, all of which contribute to the progression of age-related diseases and tissue degeneration \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In senescent cells, mitochondria exhibit abnormal elongation and dysfunction \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e; however, the precise molecular mechanisms underlying these changes remain unclear. Our previous studies have shown that reducing TIA-1 in senescent cells induces mitochondrial elongation \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Building on these findings, we extended our investigation to investigate how the loss of TIA-1 contributes to mitochondrial elongation during cellular senescence.\u003c/p\u003e\u003cp\u003eMitophagy, the selective clearance of damaged mitochondria via autophagy, is essential for maintaining mitochondrial quality and overall cellular homeostasis \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. While mitophagy plays a critical role in preserving mitochondrial integrity, its activity is notably reduced during cellular senescence \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This decline contributes to the accumulation of dysfunctional mitochondria, exacerbating mitochondrial elongation and ROS production. Despite its recognized importance, the molecular mechanisms underlying the reduction of mitophagy during senescence remain poorly understood, representing a critical gap in our knowledge.\u003c/p\u003e\u003cp\u003eTIA-1, an RNA binding protein, is another key player in mitochondrial homeostasis \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. It is known to regulate RNA metabolism, including alternative splicing, stability, and translation, and is involved in the formation of stress granules under various stress conditions \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. TIA-1 has been shown to play important role in the regulation of mitochondrial homeostasis \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Mutations in TIA-1 have been associated with diseases such as amyotrophic lateral sclerosis (ALS) and dementia \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, highlighting its potential clinical importance. However, the precise role of TIA-1 in mitophagy and its regulatory mechanisms during cellular senescence remain largely unexplored.\u003c/p\u003e\u003cp\u003eIn this study, we investigated the molecular mechanisms by which TIA-1 regulates mitochondrial elongation and mitophagy during stress-induced cellular senescence. Our results showed that TIA-1 regulates the expression of FUNDC1, a mitochondria-specific receptor of mitophagy \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Furthermore, we showed that the reduction of TIA-1 during cellular senescence leads to decreased FUNDC1 expression, which in turn impairs mitophagy. These findings provide new insights into how TIA-1 downregulation contributes to mitochondrial dysfunction in senescent cells. By providing new insights into the molecular regulation of mitophagy and mitochondrial homeostasis, our findings have the potential to advance our understanding of cellular senescence and its contribution to age-related and mitochondrial-associated diseases. In addition, this work highlights TIA-1 as potential therapeutic target for alleviating mitochondrial dysfunction in pathological conditions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eCell culture and transfection\u003c/p\u003e\u003cp\u003eHaCaT (human skin keratinocytes), HEK293T (human embryonic kidney cells), and SH-SY5Y (human neuroblastoma) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Cytiva, Wilmington DE, USA) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37\u0026deg;C. The transfection of siRNA against TIA-1 (Genolution Pharmaceuticals, Inc., Seoul, South Korea), HA-tagged TIA-1 plasmid \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and their appropriate controls was carried out using Lipofectamine\u0026trade; 2000 (Invitrogen\u0026trade;, Waltham, MA, USA), according to the manufacturer\u0026rsquo;s instruction.\u003c/p\u003e\u003cp\u003eTo establish the stress-induced senescence model, HaCaT cells were incubated with media containing 1 mM sodium butyrate (NaBu) (Sigma-Aldrich, Burlington, MA, USA) or exposed to UV-B (75 mJ/cm\u0026sup2;) using UV Transilluminator (Core Bio System, Seoul, Korea) during the indicated time.\u003c/p\u003e\u003cp\u003eRNA analysis\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from whole cells using RNAiso Plus (Takara Bio, Inc., Shiga, Japan) and cDNA was synthesized by reverse transcription (RT) with the ReverTra\u0026reg; Ace qPCR RT kit (Toyobo Co., Ltd, Osaka, Japan). Quantitative PCR (qPCR) was conducted with the SensiFAST\u0026trade; SYBR Hi-ROX kit (Meridian Bioscience, Inc., Cincinnati, OH, USA) and gene-specific primer sets listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e using the StepOnePlus\u0026trade; Real-Time PCR System (Applied Biosystems\u0026trade;, Waltham, MA, USA). Relative levels of mRNAs were calculated using the \u003csup\u003e\u003cem\u003eΔΔ\u003c/em\u003e\u003c/sup\u003eCT method, comparing control and each experimental group. \u003cem\u003eGAPDH\u003c/em\u003e mRNA was used as an internal reference gene for the normalization.\u003c/p\u003e\u003cp\u003eWestern blot (WB) analysis\u003c/p\u003e\u003cp\u003eWhole cell lysates were prepared using the RIPA buffer containing 1\u0026times; protease inhibitor cocktail (Roche, Basel, Switzerland). The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated overnight at 4\u0026deg;C with primary antibodies against TIA-1, p21, p16, GAPDH, p-IRF3, ISG15, GFP, Lamin B, TOM40, FUNDC1, or β-actin (Supplementary Table S2). Following incubation with primary antibodies, the membranes were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Chemiluminescence was detected by applying the Clarity Western ECL Substrate (Bio-Rad, Inc., Hercules, CA, USA) to the membranes and luminescent signals were acquired using the ChemiDoc Imaging Systems (Bio-Rad, Inc.).\u003c/p\u003e\u003cp\u003eAnalysis of mitochondrial morphology\u003c/p\u003e\u003cp\u003eCells were incubated with 100 nM MitoTracker Red CMXRos (Invitrogen\u0026trade;) in serum-free medium for 30 min at 37℃. Mitochondrial morphology was observed by tracing fluorescent signals using the Observer Z1 inverted microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using the Mitochondrial Analyzer plugin for ImageJ/Fiji software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/AhsenChaudhry/Mitochondria-Analyzer\u003c/span\u003e\u003cspan address=\"https://github.com/AhsenChaudhry/Mitochondria-Analyzer\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Median valued in control group were used as intermediated group. \u003csup\u003e21\u003c/sup\u003e The elongated group refers to cells with increased mitochondrial length compared to the control. Mitochondrial elongation was determined by measuring the ratio of cells have elongated forms of mitochondria to total cells.\u003c/p\u003e\u003cp\u003eFor transmission electron microscopy (TEM) analysis, cells were fixed with 1% osmium tetroxide and embedded in Epon 812. Ultrathin sections were analyzed with a transmission electron microscope (JEM 1010, Tokyo, Japan). Perimeter of mitochondria in TEM image of each experimental group was analyzed using ImageJ software \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003emt-Keima assay\u003c/p\u003e\u003cp\u003eThe modified mt-Keima reporter plasmid was generated by cloning the fragment corresponding to the COX8/COX8/hmKeima-Red sequence from pCHAC-mt-mKeima (Addgene #72342) into the pcDNA 3.1. Following transfection with the pcDNA 3.1-mt-Keima plasmid (phmt-Keima), cells were incubated with 20 \u0026micro;M FCCP and 5 \u0026micro;M oligomycin (Sigma-Aldrich) for 6 hours, then the mt-Keima signal was observed using the Observer Z1 inverted microscope (Carl Zeiss) to detect acidic (586 nm excitation, \"red\") and pH-neutral (480 nm excitation, \"green\") signals. Mitophagy events were determined by calculating the ratio of red (mitophagy) to green (mitochondria) fluorescence signals from microscopic images using ImageJ software.\u003c/p\u003e\u003cp\u003eImmunostaining and Fluorescence Microscopy\u003c/p\u003e\u003cp\u003eCells were labeled with 100 nM LysoTracker Red DND-99 (MedChemExpress, Monmouth Junction, NJ, USA) in complete medium for 30 minutes at 37\u0026deg;C, followed by fixation with 4% formaldehyde at room temperature. After permeabilization with Triton X-100 solution, cells were sequentially incubated with blocking solution and primary antibody against NDUFV2, a subunit of mitochondrial complex I located in the inner membrane, followed by incubation with Alexa Fluor\u0026reg; 488-conjugated secondary antibodies (Supplementary Table S2). Nuclei were stained with DAPI (4\u0026prime;,6-diamidino-2-phenylindole; Invitrogen). Fluorescence images were acquired using an ObserverZ1 inverted fluorescence microscope (Carl Zeiss, Germany) with consistent imaging parameters across all conditions.\u003c/p\u003e\u003cp\u003eThe number of lysosomes\u0026ndash;mitochondria overlay events was manually quantified by counting discrete yellow puncta, indicative of colocalization, within individual cells as described in \u003csup\u003e22\u003c/sup\u003e. At least 200 cells selected randomly per condition were analyzed.\u003c/p\u003e\u003cp\u003eAnalysis of mitochondrial function\u003c/p\u003e\u003cp\u003eMitochondrial membrane potential (Δψm) and ATP generation were measured using the JC-1 assay kit (Abcam, Cambridge, UK) and the Mitochondrial ToxGlo\u0026trade; system (Promega, Madison, WI, USA), respectively, according to the manufacturers\u0026rsquo; protocols. JC-1 fluorescence was detected at 535 nm (excitation) and 590 nm (emission), while ToxGlo\u0026trade; signals were recorded at 485 nm/530 nm using a Synergy H1 microplate reader (BioTek, Santa Clara, CA, USA).\u003c/p\u003e\u003cp\u003eMitochondrial ROS levels were assessed by incubating cells with 2.5 \u0026micro;M MitoSOX\u0026trade; Red (Invitrogen) in HBSS for 15 mins. Fluorescence images were acquired using the Observer Z1 inverted fluorescence microscope (Carl Zeiss), and signal quantification was performed using ImageJ software.\u003c/p\u003e\u003cp\u003eAnalysis of cytosolic mitochondrial DNA release\u003c/p\u003e\u003cp\u003eCytosolic mtDNA was analyzed as described in \u003csup\u003e23, 24\u003c/sup\u003e. In brief, the cytosolic fraction was isolated by differential centrifugation using a lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 18 \u0026micro;g/ml digitonin, and 1\u0026times; protease inhibitor cocktail. Genomic DNA was extracted from the cytosolic fraction using the AccuPrep\u0026reg; Genomic DNA Extraction Kit (Bioneer, Daejeon, South Korea) according to the manufacturer\u0026rsquo;s instructions. Cytosolic mitochondrial DNA (mtDNA) levels were quantified by qPCR using gene-specific primers for \u003cem\u003eMT-ND1\u003c/em\u003e and \u003cem\u003eMT-D-loop\u003c/em\u003e, as listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. qPCR was performed on the StepOnePlus\u0026trade; Real-Time PCR System (Applied Biosystems\u0026trade;,). To normalize for total mtDNA content, cytosolic mtDNA levels were calibrated against mtDNA levels measured in whole-cell lysates. The nuclear gene \u003cem\u003eKCNJ10\u003c/em\u003e was used as the reference gene for normalization of total mtDNA quantification.\u003c/p\u003e\u003cp\u003eSenescence-associated β-galactosidase assay\u003c/p\u003e\u003cp\u003eCells were fixed in 4% formaldehyde (FA) and incubated in a staining solution containing 40 mM citric acid/sodium phosphate (pH 6.0), 150 mM sodium chloride, 2 mM magnesium chloride, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/mL X-gal (BEAMS Biotechnology, Seongnam, South Korea) at 37\u0026deg;C for 16 hours in the dark\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. After fixation in 20% glycerol, β-galactosidase levels were observed using an IX70w microscope (Olympus Corp., Tokyo, Japan). The number of β-gal-positive cells was counted from at least 150 cells per experiment, across three independent experiments.\u003c/p\u003e\u003cp\u003eRibonucleoprotein immunoprecipitation (RNP IP)\u003c/p\u003e\u003cp\u003eRNP complexes were immunoprecipitated from cell lysates using Protein A beads (Invitrogen\u0026trade;) coated with anti-TIA-1 or normal rabbit IgG antibody (Santa Cruz Biotechnology, Inc.). The immunoprecipitated RNP complexes were then sequentially treated with DNase I and proteinase K. RNAs isolated from the complexes were analyzed by RT-qPCR using gene-specific primer sets listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eBiotin pulldown assay\u003c/p\u003e\u003cp\u003eT7 promoter-incorporated DNA fragments corresponding to the 3\u0026prime;UTR of \u003cem\u003eFUNDC1\u003c/em\u003e mRNA (NM_173794.4) were amplified by PCR using specific primers (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Biotinylated RNA probes were synthesized by \u003cem\u003ein vitro\u003c/em\u003e transcription using the MaxiScript T7 kit (Ambion, Waltham, MA, USA) and biotin-CTP (Enzo Life Sciences, Farmingdale, NY, USA). Cell lysates were incubated with purified biotinylated RNA probes for 30 min at room temperature. Proteins bound to biotinylated RNA probes were isolated using streptavidin magnetic beads (Invitrogen\u0026trade;), and further analyzed by WB using TIA-1 antibody \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEGFP reporter analysis\u003c/p\u003e\u003cp\u003eThe EGFP reporter containing the 3\u0026prime;UTR of \u003cem\u003eFUNDC1\u003c/em\u003e mRNA (pEGFP-FUNDC1 3U) was generated by inserting 3\u0026prime;UTR fragment of \u003cem\u003eFUNDC1\u003c/em\u003e mRNA (NM_173794.4, 496\u0026ndash;1054 nt, 559 bp) into pEGFP-C1 plasmid (BD Bioscience, Franklin Lakes, NJ, USA). After transfection with siRNA (siCtrl or siTIA-1) or plasmid (pCtrl or pTIA-1), the cells were sequentially transfected with reporter plasmids (control C1 or EGFP-FUNDC1 3U) for 24 hours. The relative expression of the EGFP reporter was assessed by WB analysis.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from three independent experiments. Statistical significance was assessed using the Student\u0026rsquo;s t-test (n.s., not significant with p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStress-induced senescence is associated with mitochondrial hyperfusion and reduced mitophagy in HaCaT cells\u003c/h2\u003e\u003cp\u003eTo establish a model of stress-induced senescence in human keratinocyte HaCaT cells, we treated cells with sodium butyrate (NaBu) or UV-B irradiation. Both stressors significantly increase senescence-associated β-galactosidase (SA β-gal) activity, as demonstrated by enhanced SA β-gal staining in treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Western blot analysis reveals that NaBu and UV-B exposure upregulate the expression of canonical senescence markers p16 and p21, while reducing Lamin B levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Notably, both treatments also lead to a marked decrease in the expression of TIA-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), as observed in our previous study \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Consistently, RT-qPCR analysis confirms that TIA-1 mRNA levels are significantly reduced under both stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Next, we examined mitochondrial morphology using MitoTracker staining. Both NaBu and UV-B treatment significantly increase the proportion of cells exhibiting elongated mitochondria, indicating stress-induced mitochondrial hyperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Transmission electron microscopy (TEM) analysis further showed that NaBu treatment increased mitochondrial perimeter compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe enhanced mitochondrial elongation in senescent cells could result from the altered expression of proteins that regulate mitochondrial dynamics \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e; however, it could also be due to a decrease in mitophagy activity, a form of selective autophagy leading to mitochondrial degradation \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. To investigate the alteration in mitophagy in cellular senescence, we assessed mitophagy activity in these stress conditions using the mt-Keima reporter assay \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. HaCaT cells transfected with mt-Keima were treated with NaBu or UV-B and then challenged with FCCP to induce mitophagic flux. Both NaBu and UV-B treatment significantly reduced the FCCP-induced mt-Keima red-to-green fluorescence ratio, indicating impaired mitophagy activity under stress (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). Together, these findings demonstrate that NaBu and UV-B-induced stress promote cellular senescence in HaCaT cells, which is associated with decreased TIA-1 expression, mitochondrial hyperfusion, and reduced mitophagic clearance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTIA-1 knockdown exacerbates stress-induced senescence and suppresses mitophagy\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of TIA-1 in regulating mitophagy under stress conditions, we first assessed mt-Keima reporter activity after TIA-1 knockdown. TIA-1 downregulation significantly reduced FCCP-induced mt-Keima red fluorescence signal in HaCaT, HEK293T, and SH-SY5Y cells (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), indicating that TIA-1 downregulation broadly impairs mitophagic flux. Next, we examined how TIA-1 knockdown influence stress-induced senescence and mitophagy in the NaBu treatment model. In HaCaT cells, TIA-1 knockdown led to a greater increase in SA β-gal-positive staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and an elevation of senescence marker p21 and p16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) after NaBu treatment compared to control. Mitophagy activity, measured by the mt-Keima assay, was even more decreased by TIA-1 knockdown in NaBu-treated cells, as reflected by a further reduced red-to green fluorescence ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Similar enhancement of NaBu-induced mitophagy suppression was observed in HEK293T cells following TIA-1 downregulation (Supplementary Fig. S2). These findings suggest that loss of TIA-1 level exacerbates stress-induced senescence and further impairs mitophagic clearance in HaCaT cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eTIA-1 overexpression mitigates stress-induced senescence and restores mitophagy activity\u003c/h3\u003e\n\u003cp\u003eTo determine whether TIA-1 overexpression can alleviate stress-induced senescence, we investigated senescence markers in HaCaT cells exposed to either NaBu or UV-B irradiation. SA β-gal staining revelated that ectopic expression of TIA-1 significantly reduced the number of SA β-gal-positive cells under both NaBu (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and UV-B (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) treatment conditions. These results indicate that elevated TIA-1 level decreases the acquisition of SA β-gal expression in response to different stressors. In addition, TIA-1 overexpression markedly attenuated the stress-induced increase in p21 and p16 levels in both NaBu and UV-B treated cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), further supporting its role in regulating cellular senescence. Finally, we examined whether TIA-1 overexpression could restore mitophagy activity suppressed by stress. Using the mt-Keima reporter assay, we observed that both treatments reduced FCCP-induced mitophagic flux, as indicated by a decreased red-to-green fluorescence ratio. Importantly, ectopic expression of TIA-1 significantly rescued mitophagy activity under both NaBu and UV-B (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) treatment conditions. Together, these results demonstrate that TIA-1 overexpression consistently attenuates stress-induced senescence and restored mitophagy activity in response to senescence-inducing stimuli, underscoring its critical protective role in maintaining mitochondrial quality control under stress conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eTIA-1 overexpression alleviates NaBu-induced mitochondrial dysfunction and limits mtDNA-mediated inflammatory signaling\u003c/h2\u003e\u003cp\u003eTo further investigate how TIA-1 overexpression modulates cellular responses to stress, we examined its effects on mitochondrial function in NaBu-treated HaCaT cells. NaBu exposure significantly reduced mitochondrial membrane potential, as determined by decreased JC-1 staining intensity. Notably, ectopic expression of TIA-1 significantly preserved mitochondrial membrane potential under NaBu treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Mitochondrial ATP production assays showed similar results: while NaBu treatment decreased ATP levels, TIA-1 overexpression rescued this decline (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We also assessed mitochondrial reactive oxygen species (ROS) production using MitoSOX staining. NaBu treatment markedly increased ROS levels in HaCaT cells, but TIA-1 overexpression significantly attenuated this stress-induced ROS accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Together, these data indicate that TIA-1 overexpression can mitigate NaBu-induced mitochondrial dysfunction.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBecause mitophagy is closely linked to the control of cytosolic mitochondrial DNA (mtDNA) release \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, we next investigated whether stress-induced senescence or TIA-1 knockdown altered mtDNA release. Fractionation and qPCR analysis revealed that both NaBu treatment and TIA-1 knockdown increased cytosolic mtDNA (MT-ND1 and D-loop) levels in HaCaT cells without changing total mtDNA copy number (Supplementary Fig. S3). Importantly, we found that TIA-1 overexpression significantly reduced the relative amount of mtDNA detected in the cytosolic fraction after NaBu treatment, again without altering total mtDNA copy number (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These findings suggest that TIA-1 overexpression suppresses stress-induced mtDNA release into the cytosol.\u003c/p\u003e\u003cp\u003eGiven that cytosolic mtDNA release can activate the cGAS\u0026ndash;STING pathway and induce pro-inflammatory gene expression \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, we examined downstream signaling events. Western blot analysis showed that NaBu treatment increased phosphorylation of IRF3 and the level of free ISG15 protein, while TIA-1 overexpression partially reduced these stress-induced increases (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Consistently, NaBu treatment upregulated mRNA levels of \u003cem\u003eIL6\u003c/em\u003e, \u003cem\u003eIL8\u003c/em\u003e, \u003cem\u003eIL1β\u003c/em\u003e, and \u003cem\u003eIFNγ\u003c/em\u003e, whereas TIA-1 overexpression modestly attenuated this transcriptional activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Together, these results demonstrate that TIA-1 overexpression not only protects against NaBu-induced mitochondrial dysfunction but also limits mtDNA-mediated activation of the cGAS\u0026ndash;STING inflammatory pathway in senescent HaCaT cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTIA-1 directly associates with the\u003c/b\u003e \u003cb\u003eFUNDC1\u003c/b\u003e \u003cb\u003emRNA to promote its expression\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTIA-1 is an RNA-binding protein that binds to its target mRNAs and can affect their expression by regulating RNA metabolism \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. To understand the molecular mechanism by which TIA-1 regulates mitophagy, we sought to identify the downstream molecules of TIA-1. By analyzing the TIA-1 crosslinking immunoprecipitation sequencing (CLIP-seq) dataset \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and POSTAR3 database \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, we identified a set of putative TIA-1-bound mRNAs encoding selective autophagy receptors (SARs), which are essential mediators for mitophagy \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To confirm whether TIA-1 physically associates with these SAR mRNAs, we performed ribonucleoprotein (RNP) immunoprecipitation using TIA-1 antibody, followed by RT-qPCR. Several SAR mRNAs, including \u003cem\u003eNIX\u003c/em\u003e, \u003cem\u003eBNIP3\u003c/em\u003e, \u003cem\u003eFUNDC1\u003c/em\u003e, and \u003cem\u003eBCL2L13\u003c/em\u003e, were significantly enriched in TIA-1-containing RNP complexes compared to control IgG immunoprecipitation, indicating direct binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Among these candidates, \u003cem\u003eFUNDC1\u003c/em\u003e mRNA showed statistically significant and consistent enrichment. FUNDC1 (a FUN14 domain containing 1) is a known SAR involved in ubiquitin-independent mitophagy \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and contains a\u0026thinsp;~\u0026thinsp;500 nt 3'UTR in human (NM_173794.4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). To test direct interaction between TIA-1 and \u003cem\u003eFUNDC1\u003c/em\u003e mRNA, we performed \u003cem\u003ein vitro\u003c/em\u003e binding assays with biotin-labeled transcripts corresponding to the 3'UTR of \u003cem\u003eFUNDC1\u003c/em\u003e mRNA. TIA-1 bound to the 3'UTR of \u003cem\u003eFUNDC1\u003c/em\u003e mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), supporting a direct interaction. To investigate whether TIA-1 modulates FUNDC1 expression via its 3'UTR, we used an EGFP reporter construct containing the \u003cem\u003eFUNDC1\u003c/em\u003e 3\u0026prime;UTR. TIA-1 knockdown significantly reduced reporter expression, whereas TIA-1 overexpression increased it (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These results indicate that TIA-1 binds to 3'UTR of \u003cem\u003eFUNDC1\u003c/em\u003e mRNA and promotes its expression. Consistent with this observation, modulation of TIA-1 expression in HaCaT cells altered endogenous FUNDC1 protein levels: TIA-1 knockdown reduced FUNDC1 protein, while overexpression increased it (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Importantly, these changes occurred without significant alterations in total \u003cem\u003eFUNDC1\u003c/em\u003e mRNA levels, suggesting that TIA-1 primarily regulates FUNDC1 at the level of translation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEctopic expression of TIA-1 restores FUNDC1 expression and promotes mitophagy under stress conditions\u003c/h3\u003e\n\u003cp\u003eTo further validate the mechanistic link between TIA-1 and mitophagy regulation under stress, we investigated whether TIA-1 overexpression could restore FUNDC1 expression in HaCaT cells in response to NaBu or UV-B treatment. Western blot analysis revealed that both stress conditions reduced FUNDC1 protein levels, whereas ectopic expression of TIA-1 significantly rescued FUNDC1 expression under both NaBu (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) and UV-B (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) conditions. We next examined whether changes in FUNDC1 levels driven by TIA-1 overexpression were associated with alterations in mitophagy. Immunofluorescence analysis using FUNDC1 antibody and Lysotracker to label lysosomes showed that NaBu treatment increased co-localized FUNDC1 and lysosomal signals, indicating stress-induced mitophagy activation. Importantly, TIA-1 overexpression further enhanced the overlap of FUNDC1 and lysosomal signals under NaBu stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These results suggest that TIA-1 overexpression promotes FUNDC1-mediated mitophagy by enhancing mitochondrial\u0026ndash;lysosomal fusion in stressed cells. Based on these findings, we propose a mechanistic model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) in which stress-induced downregulation of TIA-1 leads to decreased FUNDC1 expression, suppression of mitophagy, mitochondrial hyperfusion, and ultimately mitochondrial dysfunction. Conversely, ectopic expression of TIA-1 restores FUNDC1 levels, enhances mitophagy activity, and alleviates stress-induced mitochondrial dysfunction and cellular senescence.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTight regulation of mitochondrial homeostasis is essential for the maintenance of normal cellular functions \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Mitochondrial homeostasis is controlled at multiple levels, including biogenesis, dynamics and mitophagy \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Dysregulation of mitochondrial homeostasis leads to mitochondrial dysfunction, which contributes to the pathogenesis of several human diseases \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Mitochondrial dysfunction, as indicated by imbalanced mitochondrial dynamics, decreased membrane potential, and elevated ROS production, has been identified as a significant contributor to cellular senescence \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In this study, we demonstrated that mitophagy is reduced in stress-induced senescence model, accompanied by decreased expression of TIA-1. We found that TIA-1 knockdown decreased mitophagy, while its ectopic expression enhanced mitophagy activity and mitigated senescence marker expression under NaBu or UV-B treatment. These results suggest that TIA-1 may serve as a novel regulator of mitophagy in the context of cellular senescence.\u003c/p\u003e\u003cp\u003eSeveral studies have shown that senescent cells have highly elongated mitochondria \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Enhanced elongation of mitochondria has been linked to alterations in mitochondrial dynamics, specifically the downregulation of fission factors such as DRP1, FIS1, and MFF, and the upregulation of fusion factors such as MFN1/2 and OPA1. Changes in the expression levels of these proteins have been consistently observed in senescent cells \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Furthermore, the reduction in mitophagy activity, which is crucial for the removal of damaged mitochondria, may contribute to the excessive elongation of mitochondria in senescent cells \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Our previous research documented reduced MFF expression in senescent HaCaT cells \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and in this study, we further identified a reduction in FUNDC1 expression. Importantly, we have shown that TIA-1 plays a key role in regulating the expression of MFF and FUNDC1 in senescent cells. These findings underscore the central role of TIA-1 in maintaining mitochondrial homeostasis and highlight its potential as a therapeutic target for diseases caused by mitochondrial dysfunction, including cancer and age-related diseases.\u003c/p\u003e\u003cp\u003eDespite its critical role in regulating mitochondrial homeostasis and cellular senescence, much remains unknown about the molecular functions of TIA-1 and the mechanisms governing its expression. As an RNA-binding protein, TIA-1 modulates RNA metabolism processes such as alternative splicing and translation \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In response to various stress stimuli, it regulates the expression of its target mRNAs by forming stress granules or interacting with other proteins \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Mutations in the \u003cem\u003eTIA-1\u003c/em\u003e gene have been associated with several human diseases such as amyotrophic lateral sclerosis (ALS) and myopathy, highlighting its clinical significance \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. While certain factors, including \u003cem\u003emicroRNA-27a\u003c/em\u003e and \u003cem\u003emicroRNA-30-5p\u003c/em\u003e, have been identified as regulators of TIA-1 expression by our group \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, the precise mechanisms controlling TIA-1 expression and activity remain largely unclear. Notably, how TIA-1 expression is diminished during cellular senescence is a question that requires further investigation. Our findings demonstrated that TIA-1 expression is reduced following NaBu treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Exploring how HDAC inhibitor butyrate mediates this downregulation could provide valuable insights into the regulatory mechanisms underlying TIA-1 expression.\u003c/p\u003e\u003cp\u003eFUNDC1 is a mitochondrial receptor that directly interacts with LC3 and mediates a PINK1/Parkin-independent pathway of mitophagy in response to various stimuli \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Dysregulation of mitophagy due to alterations in FUNDC1 expression has been implicated in several pathological conditions, including cancer, fibrosis and cardiovascular disease \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In this study, we observed the downregulation of FUNDC1 and a corresponding reduction in mitophagy in the NaBu-induced senescence model. In addition to its role in regulating mitophagy, FUNDC1 plays a critical role in maintaining mitochondrial homeostasis by contributing to the formation of mitochondrial-endoplasmic reticulum (ER) contact sites (MERCs), mitochondrial biogenesis and mitochondrial dynamics \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Therefore, a detailed understanding of FUNDC1 expression and its regulatory mechanisms is important for advancing our knowledge of FUNDC1-mediated pathology. While our study has identified TIA-1 as a novel regulator of FUNDC1 expression, further investigations are required to identify additional regulatory factors and to elucidate the pathways governing FUNDC1 function across multiple levels including transcription, post-transcription, post-translation, or protein-protein interaction. These efforts will contribute to a more comprehensive understanding of mitochondrial homeostasis mediated by FUNDC1.\u003c/p\u003e\u003cp\u003eIn conclusion, our findings provide new insights into the regulatory roles of TIA-1 and FUNDC1 in mitochondrial homeostasis and cellular senescence. By identifying TIA-1 as a novel regulator of FUNDC1 expression and highlighting its impact on mitophagy, this study underscores the importance of these factors in maintaining mitochondrial structure and functionality. These results not only advance our understanding of the molecular mechanisms underlying mitochondrial dysregulation in the stress-induced senescence model but also lay the groundwork for future investigations into potential therapeutic strategies targeting mitochondrial dysfunction in cellular senescence and age-related and pathological conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eAuthors have no conflicts of interest to declare.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Basic Science Research Programs through the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (RS-2021-NR058095 and RS-2024-00405790) and the GRRC program of Gyeonggi province (GRRCAjou2023-B01).\u003c/p\u003e\u003ch2\u003eAuthor contribution\u003c/h2\u003e\u003cp\u003eEKL conceived and supervised the study. DC, JK, SMJ acquired funding for the study. SC, HT, WK and EKL conceptualized and designed the experiment. SC, MJ, SR, and SH conducted experiments. SC analyzed the data and created the figures. SC and EKL wrote manuscript. SC and WK revised the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors appreciate Professor DH Cho and DS Jo, PhD at Kyungpook National University and Professor JH Kim for the technical advice and sharing materials for the mt-Keima assay and cmtDNA analysis, respectively.\u003c/p\u003e\u003ch2\u003eAvailability of Data and Materials\u003c/h2\u003e\u003cp\u003eThe data analyzed during this study are included in this published article and the supplemental data files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen W, Zhao H, Li Y. Mitochondrial dynamics in health and disease: mechanisms and potential targets. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e 2023, 8(1): 333.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZong Y, Li H, Liao P, Chen L, Pan Y, Zheng Y, \u003cem\u003eet al.\u003c/em\u003e Mitochondrial dysfunction: mechanisms and advances in therapy. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e 2024, 9(1): 124.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSuomalainen A, Nunnari J. Mitochondria at the crossroads of health and disease. \u003cem\u003eCell\u003c/em\u003e 2024, 187(11): 2601\u0026ndash;2627.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartini H, Passos JF. Cellular senescence: all roads lead to mitochondria. \u003cem\u003eFebs J\u003c/em\u003e 2023, 290(5): 1186\u0026ndash;1202.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAmorim JA, Coppotelli G, Rolo AP, Palmeira CM, Ross JM, Sinclair DA. Mitochondrial and metabolic dysfunction in ageing and age-related diseases. \u003cem\u003eNat Rev Endocrinol\u003c/em\u003e 2022, 18(4): 243\u0026ndash;258.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiwa S, Kashyap S, Chini E, von Zglinicki T. Mitochondrial dysfunction in cell senescence and aging. \u003cem\u003eJ Clin Invest\u003c/em\u003e 2022, 132(13).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoon YS, Yoon DS, Lim IK, Yoon SH, Chung HY, Rojo M, \u003cem\u003eet al.\u003c/em\u003e Formation of elongated giant mitochondria in DFO-induced cellular senescence: involvement of enhanced fusion process through modulation of Fis1. \u003cem\u003eJ Cell Physiol\u003c/em\u003e 2006, 209(2): 468\u0026ndash;480.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMai S, Klinkenberg M, Auburger G, Bereiter-Hahn J, Jendrach M. Decreased expression of Drp1 and Fis1 mediates mitochondrial elongation in senescent cells and enhances resistance to oxidative stress through PINK1. \u003cem\u003eJ Cell Sci\u003c/em\u003e 2010, 123(Pt 6): 917\u0026ndash;926.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTak H, Cha S, Hong Y, Jung M, Ryu S, Han S, \u003cem\u003eet al.\u003c/em\u003e The miR-30-5p/TIA-1 axis directs cellular senescence by regulating mitochondrial dynamics. \u003cem\u003eCell Death Dis\u003c/em\u003e 2024, 15(6): 404.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa K, Chen G, Li W, Kepp O, Zhu Y, Chen Q. Mitophagy, Mitochondrial Homeostasis, and Cell Fate. \u003cem\u003eFront Cell Dev Biol\u003c/em\u003e 2020, 8: 467.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePicca A, Faitg J, Auwerx J, Ferrucci L, D'Amico D. Mitophagy in human health, ageing and disease. \u003cem\u003eNat Metab\u003c/em\u003e 2023, 5(12): 2047\u0026ndash;2061.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOnishi M, Yamano K, Sato M, Matsuda N, Okamoto K. Molecular mechanisms and physiological functions of mitophagy. \u003cem\u003eEmbo J\u003c/em\u003e 2021, 40(3).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKorolchuk VI, Miwa S, Carroll B, von Zglinicki T. Mitochondria in Cell Senescence: Is Mitophagy the Weakest Link? \u003cem\u003eEbiomedicine\u003c/em\u003e 2017, 21: 7\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTak H, Eun JW, Kim J, Park SJ, Kim C, Ji E, \u003cem\u003eet al.\u003c/em\u003e T-cell-restricted intracellular antigen 1 facilitates mitochondrial fragmentation by enhancing the expression of mitochondrial fission factor. \u003cem\u003eCell Death Differ\u003c/em\u003e 2017, 24(1): 49\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanchez-Jimenez C, Izquierdo JM. T-cell intracellular antigen (TIA)-proteins deficiency in murine embryonic fibroblasts alters cell cycle progression and induces autophagy. \u003cem\u003ePLoS One\u003c/em\u003e 2013, 8(9): e75127.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFernandez-Gomez A, Izquierdo JM. The Multifunctional Faces of T-Cell Intracellular Antigen 1 in Health and Disease. \u003cem\u003eInt J Mol Sci\u003c/em\u003e 2022, 23(3).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarrascoso I, Alcalde J, Sanchez-Jimenez C, Gonzalez-Sanchez P, Izquierdo JM. T-Cell Intracellular Antigens and Hu Antigen R Antagonistically Modulate Mitochondrial Activity and Dynamics by Regulating Optic Atrophy 1 Gene Expression. \u003cem\u003eMol Cell Biol\u003c/em\u003e 2017, 37(17).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarrascoso I, Velasco BR, Izquierdo JM. Deficiency of T-Cell Intracellular Antigen 1 in Murine Embryonic Fibroblasts Is Associated with Changes in Mitochondrial Morphology and Respiration. \u003cem\u003eInt J Mol Sci\u003c/em\u003e 2021, 22(23).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMackenzie IR, Nicholson AM, Sarkar M, Messing J, Purice MD, Pottier C, \u003cem\u003eet al.\u003c/em\u003e TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics. \u003cem\u003eNeuron\u003c/em\u003e 2017, 95(4): 808\u0026ndash;816 e809.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen M, Chen Z, Wang Y, Tan Z, Zhu C, Li Y, \u003cem\u003eet al.\u003c/em\u003e Mitophagy receptor FUNDC1 regulates mitochondrial dynamics and mitophagy. \u003cem\u003eAutophagy\u003c/em\u003e 2016, 12(4): 689\u0026ndash;702.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChaudhry A, Shi R, Luciani DS. A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells. \u003cem\u003eAm J Physiol-Endoc M\u003c/em\u003e 2020, 318(2): E87\u0026ndash;E101.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu B, Li A, Qin Y, Chen L, Gao M, Gong G. Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome. \u003cem\u003eJ Vis Exp\u003c/em\u003e 2022(189).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVictorelli S, Salmonowicz H, Chapman J, Martini H, Vizioli MG, Riley JS, \u003cem\u003eet al.\u003c/em\u003e Apoptotic stress causes mtDNA release during senescence and drives the SASP. \u003cem\u003eNature\u003c/em\u003e 2023, 622(7983): 627\u0026ndash;636.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBryant JD, Lei Y, VanPortfliet JJ, Winters AD, West AP. Assessing Mitochondrial DNA Release into the Cytosol and Subsequent Activation of Innate Immune-related Pathways in Mammalian Cells. \u003cem\u003eCurr Protoc\u003c/em\u003e 2022, 2(2): e372.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang KH, Li QL, Zhang YX, Nuerlan G, Li YY, Mao J, \u003cem\u003eet al.\u003c/em\u003e Targeting Mitophagy as a Potential Therapeutic Approach for Age-Related Bone Diseases. \u003cem\u003eAdv Ther-Germany\u003c/em\u003e 2024, 7(7).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKelly G, Kataura T, Panek J, Ma GL, Salmonowicz H, Davis A, \u003cem\u003eet al.\u003c/em\u003e Suppressed basal mitophagy drives cellular aging phenotypes that can be reversed by a p62-targeting small molecule. \u003cem\u003eDev Cell\u003c/em\u003e 2024, 59(15).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVargas JNS, Wang CX, Bunker E, Hao L, Maric D, Schiavo G, \u003cem\u003eet al.\u003c/em\u003e Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy. \u003cem\u003eMolecular Cell\u003c/em\u003e 2019, 74(2): 347\u0026ndash;+.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePuri R, Cheng XT, Lin MY, Huang N, Sheng ZH. Mul1 restrains Parkin-mediated mitophagy in mature neurons by maintaining ER-mitochondrial contacts. \u003cem\u003eNat Commun\u003c/em\u003e 2019, 10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhong W, Rao Z, Xu J, Sun Y, Hu H, Wang P, \u003cem\u003eet al.\u003c/em\u003e Defective mitophagy in aged macrophages promotes mitochondrial DNA cytosolic leakage to activate STING signaling during liver sterile inflammation. \u003cem\u003eAging Cell\u003c/em\u003e 2022, 21(6): e13622.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJimenez-Loygorri JI, Villarejo-Zori B, Viedma-Poyatos A, Zapata-Munoz J, Benitez-Fernandez R, Frutos-Lison MD, \u003cem\u003eet al.\u003c/em\u003e Mitophagy curtails cytosolic mtDNA-dependent activation of cGAS/STING inflammation during aging. \u003cem\u003eNat Commun\u003c/em\u003e 2024, 15(1): 830.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim J, Kim HS, Chung JH. Molecular mechanisms of mitochondrial DNA release and activation of the cGAS-STING pathway. \u003cem\u003eExp Mol Med\u003c/em\u003e 2023, 55(3): 510\u0026ndash;519.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeyer C, Garzia A, Mazzola M, Gerstberger S, Molina H, Tuschl T. The TIA1 RNA-Binding Protein Family Regulates EIF2AK2-Mediated Stress Response and Cell Cycle Progression. \u003cem\u003eMol Cell\u003c/em\u003e 2018, 69(4): 622\u0026ndash;635 e626.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao WH, Zhang S, Zhu YM, Xi XC, Bao PF, Ma ZY, \u003cem\u003eet al.\u003c/em\u003e POSTAR3: an updated platform for exploring post-transcriptional regulation coordinated by RNA-binding proteins. \u003cem\u003eNucleic Acids Res\u003c/em\u003e 2022, 50(D1): D287\u0026ndash;D294.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBai XZ, Zhang Z, Li X, Yang YJ, Ding SZ. FUNDC1: An Emerging Mitochondrial and MAMs Protein for Mitochondrial Quality Control in Heart Diseases. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e 2023, 24(11).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGong G, Song M, Csordas G, Kelly DP, Matkovich SJ, Dorn GW, 2nd. Parkin-mediated mitophagy directs perinatal cardiac metabolic maturation in mice. \u003cem\u003eScience\u003c/em\u003e 2015, 350(6265): aad2459.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRambold AS, Kostelecky B, Elia N, Lippincott-Schwartz J. Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation. \u003cem\u003eP Natl Acad Sci USA\u003c/em\u003e 2011, 108(25): 10190\u0026ndash;10195.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRayman JB, Kandel ER. TIA-1 Is a Functional Prion-Like Protein. \u003cem\u003eCsh Perspect Biol\u003c/em\u003e 2017, 9(5).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHackman P, Sarparanta J, Lehtinen S, Vihola A, Evil\u0026auml; A, Jonson PH, \u003cem\u003eet al.\u003c/em\u003e Welander distal myopathy is caused by a mutation in the RNA-binding protein TIA1. \u003cem\u003eAnn Neurol\u003c/em\u003e 2013, 73(4): 500\u0026ndash;509.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu L, Feng D, Chen G, Chen M, Zheng Q, Song P, \u003cem\u003eet al.\u003c/em\u003e Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells. \u003cem\u003eNat Cell Biol\u003c/em\u003e 2012, 14(2): 177\u0026ndash;185.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHou HL, Er PC, Cheng JJ, Chen XL, Ding XF, Wang YW, \u003cem\u003eet al.\u003c/em\u003e High expression of FUNDC1 predicts poor prognostic outcomes and is a promising target to improve chemoradiotherapy effects in patients with cervical cancer. \u003cem\u003eCancer Med-Us\u003c/em\u003e 2017, 6(8): 1871\u0026ndash;1881.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYuan QC, Sun N, Zheng JY, Wang YX, Yan XL, Mai WQ, \u003cem\u003eet al.\u003c/em\u003e Prognostic and Immunological Role of FUN14 Domain Containing 1 in Pan-Cancer: Friend or Foe? \u003cem\u003eFront Oncol\u003c/em\u003e 2020, 9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa LJ, Li KF, Wei WX, Zhou JH, Li Z, Zhang T, \u003cem\u003eet al.\u003c/em\u003e Exercise protects aged mice against coronary endothelial senescence via FUNDC1-dependent mitophagy. \u003cem\u003eRedox Biol\u003c/em\u003e 2023, 62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu WX, Lin CX, Wu K, Jiang L, Wang XJ, Li W, \u003cem\u003eet al.\u003c/em\u003e FUNDC1 regulates mitochondrial dynamics at the ER-mitochondrial contact site under hypoxic conditions. \u003cem\u003eEmbo J\u003c/em\u003e 2016, 35(13): 1368\u0026ndash;1384.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu L, Li YJ, Wang JN, Zhang D, Wu H, Li WH, \u003cem\u003eet al.\u003c/em\u003e Mitophagy receptor FUNDC1 is regulated by PGC-1α/NRF1 to fine tune mitochondrial homeostasis. \u003cem\u003eEmbo Rep\u003c/em\u003e 2021, 22(3).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Redox, Reactive oxygen species, Mitophagy, Senescence, FUNDC1, Mitochondria, Mitochondrial dysfunction","lastPublishedDoi":"10.21203/rs.3.rs-7588642/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7588642/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMitochondrial dysfunction, characterized by reduced mitophagy, excessive mitochondrial elongation, and elevated reactive oxygen species (ROS) production, is a hallmark of cellular senescence. However, the molecular mechanisms linking impairment of redox balance to mitophagy suppression during senescence remain poorly understood. In this study, we identified TIA-1, an RNA-binding protein, as a positive regulator of FUNDC1 expression, a key receptor for ubiquitin-independent mitophagy. Sodium butyrate (NaBu) and UV-B irradiation triggered oxidative stress-associated senescence in HaCaT cells, leading to reduced TIA-1 expression, decreased FUNDC1 levels, impaired mitophagy flux, excessive mitochondrial elongation, and upregulation of senescence markers. Conversely, ectopic expression of TIA-1 restored FUNDC1 levels, enhanced mitophagy activity, improved mitochondrial function, and reduced the expression of senescence markers. Ribonucleoprotein immunoprecipitation assays confirmed that TIA-1 directly interacts with \u003cem\u003eFUNDC1\u003c/em\u003e mRNA to promote its expression. Together, these findings establish TIA-1 as a pivotal regulator of mitochondrial homeostasis during cellular stress, acting through FUNDC1 to sustain mitophagy and limit senescence. Targeting TIA-1 may offer new strategies to mitigate mitochondrial dysfunction and redox balance in aging and age-related diseases.\u003c/p\u003e","manuscriptTitle":"TIA-1 promotes FUNDC1-mediated mitophagy to protect against stress-induced cellular senescence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 14:21:31","doi":"10.21203/rs.3.rs-7588642/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-12-17T05:52:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-12-16T10:21:23+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-12-12T05:02:48+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-12-08T09:34:51+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-12-01T08:35:33+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-12-01T06:07:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-15T00:41:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2025-09-14T23:51:51+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-09-14T23:42:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-11T06:57:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e7429083-ff56-4b96-8419-22a3c7d972f8","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58857273,"name":"Biological sciences/Cell biology/Senescence"},{"id":58857274,"name":"Biological sciences/Molecular biology/RNA metabolism/RNA quality control"},{"id":58857275,"name":"Biological sciences/Cell biology/Organelles/Mitochondria"}],"tags":[],"updatedAt":"2026-03-26T07:28:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-02 14:21:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7588642","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7588642","identity":"rs-7588642","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}