Tristetraprolin inhibits mitochondrial function through suppression of α-Synuclein expression in cancer cells

Mitochondrial dynamics play critical roles in maintaining mitochondrial functions. Here, we report a novel mechanism for regulation of mitochondrial dynamics mediated by tristetraprolin (TTP), an AU-rich element (ARE)-binding protein. Overexpression of TTP resulted in elongated mitochondria, down-regulation of mitochondrial oxidative phosphorylation, reduced membrane potential, cytochrome c release, and increased apoptotic cell death in cancer cells. TTP overexpression inhibited the expression of α-Synuclein (α-Syn). TTP bound to the ARE within the mRNA 3′-untranslated regions (3′-UTRs) of α-Syn and enhanced the decay of α-Syn mRNA. Overexpression of α-Syn without the 3′-UTR restored TTP-induced defects in mitochondrial morphology, mitochondrial oxidative phosphorylation, membrane potential, and apoptotic cell death. Taken together, our data demonstrate that TTP acts as a regulator of mitochondrial dynamics through enhancing degradation of α-Syn mRNA in cancer cells. This finding will increase understanding of the molecular basis of mitochondrial dynamics.


INTRODUCTION
Mitochondria are involved in a variety of cellular functions [1]. Alterations in mitochondrial function are often associated with neurodegenerative disorders and cancer [2]. Depending on the cell type and physiological conditions, mitochondria can be present either as numerous morphologically small organelles, or they can form large interconnected networks [3][4][5]. Mitochondrial dynamics are determined by mitochondrial fusion and fission. Mitochondrial fusion and fission are mediated by three large GTPases and their interacting factors [6]. Fusion between mitochondrial outer membranes is mediated by the membrane-anchored dynamin family members mitofusin (Mfn)1 and Mfn2, whereas fusion between mitochondrial inner membranes is mediated by a single dynamin family member called optic atrophy 1 (OPA1) [6]. Fission is mediated by a cytosolic dynamin family member dynamin related protein 1 (Drp1). Mitochondrial fission protein 1 (Fis1) acts as receptors that recruit Drp1 to mitochondrial surface [7], often at sites where mitochondria make contact with the endoplasmic reticulum [8]. Drp1 induces mitochondrial fission by forming helical structures that wrap around mitochondria [9][10][11]. Impairments in mitochondrial fission and fusion cause breakdown of the mitochondrial network, loss of mitochondrial DNA (mtDNA), respiratory defects, and increases in reactive oxygen species (ROS), mitophagy, and apoptosis [12][13][14][15][16] and play a role in the pathogenesis of mitochondrial disease [17][18][19][20].
Post-transcriptional regulation of gene expression is mediated by AU-rich elements (AREs) located in the 3′-UTR of a variety of short-lived mRNAs such as cytokines and proto-oncogenes [40]. The destabilizing function of AREs is regulated by ARE-binding proteins [41]. One of the best-characterized ARE-binding proteins is tristetraprolin (TTP), which promotes degradation of ARE-containing transcripts [42][43][44]. TTP expression is significantly decreased in various cancers [45], which correlates with increased expression of proto-oncogenes and, as a result, may lead to abnormalities that contribute to cancer processes. Re-expression of TTP induces growth inhibitory effects [46][47][48][49].
In this study, we demonstrate for the first time that TTP expression alters mitochondrial morphology. Overexpression of TTP enhanced mitochondrial fusion in cancer cell lines. TTP did not increase the degradation of mRNAs from the three large GTPases involved in mitochondrial fission and fusion but did enhance mRNA degradation of a-Syn. Exogenous expression of a-Syn without the 3′-UTR recovered the mitochondrial morphology, suggesting that downregulation of a-Syn induces mitochondrial fusion. Down-regulation of a-Syn by TTP impaired mitochondrial functions, which decreased mitochondrial membrane potential, increased ROS production, induced apoptosis, and inhibited growth of cancer cells. Taken together, these findings suggest that TTP plays an important role as a regulator of mitochondrial dynamics through down-regulating expression of a-Syn in cancer cells.

TTP overexpression promotes an elongation of mitochondria
Previously, we reported that overexpression of TTP suppresses cellular proliferation [46,47,50] and induces a change in cell morphology from a mesenchymal shape to an epithelial shape [51]. Here we assessed whether TTP overexpression modifies mitochondrial morphology. To test this, SHSY5Y neuroblastoma cells and HeLa cervical carcinoma cells were transiently transfected with pcDNA6/V5-TTP (SHSY5Y/TTP and HeLa/TTP) or a control pcDNA6/V5 (SHSY5Y/pcDNA and HeLa/ pcDNA) vector. After confirming the overexpression of TTP by RT-PCR and western blot analysis ( Figure 1A), mitochondria in the cells were stained with Mitotracker. Confocal microscopic imaging of mitochondria showed that TTP overexpression promoted the elongation of the mitochondrial compared with control cells in both SHSY5Y and HeLa cells ( Figure 1B, 1C). To confirm this, mitochondrial morphology was observed using electron microscopy. Mitochondria of SHSY5Y cells overexpressing TTP demonstrated an elongated ultrastructure compared with control cells, and the average length of mitochondria in TTP-overexpressing cells was significantly increased over that of control cells ( Figure 1D, 1E). We next tested whether down-regulation of TTP increased fragmentation of mitochondria. We used siRNA against TTP to reduce the expression level of TTP in SHSY5Y and HeLa cells. Down-regulation of TTP ( Figure 1F) significantly increased fragmentation of mitochondria in both SHSY5Y and HeLa cells ( Figure  1G-1I). Our results suggest that TTP plays an important role in the regulation of mitochondrial morphology.

TTP does not decrease the expression of large GTPases involved in mitochondrial fusion and fission but does inhibit the expression of α-Syn
Mitochondrial morphology is regulated by mitochondrial dynamics, fusion, and fission [52]. Mfn1, Mfn2, and OPA1 have been identified for the mitochondrial fusion process, while Drp1, and Fis1 are thought to play critical roles in the fission process [53]. This prompted us to investigate whether TTP overexpression inhibits the expression of these mitochondrial fusion and fission proteins in SHSY5Y and HeLa cells. When we analyzed the expression levels of Mfn1, Mfn2, OPA1, Drp1, and Fis1 in SHSY5Y and HeLa cells by western blot, RT-PCR, and qRT-PCR, we unexpectedly found that overexpression of TTP did not decrease expression levels of these genes in either SHSY5Y or HeLa cells ( Figure 2A).
Previously, it was reported that α-Syn decreased mitochondrial fusion [36] and enhanced mitochondrial fragmentation [54]. Analysis of a-Syn 3′-UTR revealed the presence of several AREs within its 3′-UTR (see below). Thus, we tested whether TTP overexpression inhibited a-Syn expression. Interestingly, we found that the expression levels of a-Syn were decreased in TTPoverexpressing SHSY5Y/TTP and HeLa/TTP cells ( Figure 2B, 2C). To test whether down-regulation of TTP increased a-Syn expression, we used siRNA against TTP to reduce the expression level of TTP in SHSY5Y and HeLa cells. Down-regulation of TTP significantly increased the expression level of a-Syn ( Figure 2D, 2E). These results suggest TTP is functionally involved in regulation of a-Syn expression.  cells. We first determined whether mitochondrial morphology could be modulated by the expression level of a-Syn in SHSY5Y and HeLa cells. Consistent with previous reports [36,54], inhibition of a-Syn expression by siRNA ( Figure 3A) increased mitochondrial length ( Figure 3B, 3C and Supplementary Figure 1) and, on the contrary, overexpression of a-Syn decreased the length of mitochondria (Supplementary Figure 2). Next, we determined whether overexpression of a-Syn which did not contain a 3′-UTR restored the mitochondrial morphology in SHSY5Y/TTP and HeLa/TTP cells. To test this, SHSY5Y and HeLa cells were co-transfected with pcDNA6/V5-TTP and pcDNA/α-Syn. Overexpression of α-Syn ( Figure 3D) significantly inhibited the elongation of mitochondria induced by TTP overexpression ( Figure 3E-3G). These results indicate that TTP overexpression induced elongation of mitochondria through the down-regulation of a-Syn in SHSY5Y and HeLa cells.

TTP destabilizes a-Syn mRNA
The TTP protein decreases mRNA stability through binding to the AREs within the mRNA 3′-UTR [42,43,46,47,55]. Analysis of the 2529-bplong human a-Syn 3′-UTR revealed the presence of five pentameric AUUUA (ARE) motifs ( Figure 4A). To determine whether TTP-induced inhibition of a-Syn expression resulted from decrease in the stability of a-Syn mRNA, the half-life of a-Syn mRNA was measured by qRT-PCR in SHSY5Y cells transfected with pcDNA6/V5-TTP or with the pcDNA6/V5 control vector. In the control SHSY5Y/pcDNA cells, the half-life of a-Syn mRNA was >2 h after actinomycin D treatment. However, in TTP-overexpressing cells, the half-life was reduced to 1 h in SHSY5Y/TTP cells ( Figure 4B). These results indicated that overexpression of TTP contributed to a decrease in a-Syn levels through the destabilization of a-Syn mRNA. www.impactjournals.com/oncotarget To determine whether down-regulation of a-Syn expression by TTP was mediated through the a-Syn mRNA 3′-UTR, we used a luciferase reporter gene linked to the a-Syn 3′-UTR fragment containing all five AREs, Frag-ARE-1-5, in the plasmid psiCHECK2. When SHSY5Y cells were transfected to overexpress TTP, luciferase activity was inhibited ( Figure 4C). We next determined which AREs within the a-Syn 3′-UTR were important for TTP activity. We prepared a luciferase reporter gene linked to oligonucleotides containing each ARE within the a-Syn 3′-UTR (Oligo-ARE-1, Oligo-ARE-2, Oligo-ARE-3,4, and Oligo-ARE-5) in the plasmid psiCHECK2. In SHSY5Y cells overexpressing TTP, the luciferase activity of Oligo-ARE-2 was inhibited by 60% ( Figure 4D), while the other Oligo-AREs did not respond or only slightly responded. To determine the importance of the second ARE (ARE-2), we prepared luciferase reporter genes containing wild-type (Oligo-ARE-2W, containing wild-type ARE-2) and mutant (Oligo-ARE-2M, containing AUUUA sequences substituted with AGCA) oligonucleotides. While Oligo-ARE-2W responded to TTP (60% inhibition), Oligo-ARE-2M did not respond to TTP ( Figure 4E). These results suggested that the second ARE within the a-Syn mRNA 3′-UTR was involved in TTP inhibitory activity.

TTP binds to the second ARE within the a-Syn mRNA 3′-UTR
To determine whether TTP interacted with ARE-2 of the a-Syn 3′-UTR, SHSY5Y cells were co-transfected with pcDNA6/V5-TTP and psiCHECK2-Oligo-ARE-2W (Oligo-ARE-2W) or psiCHECK2-Oligo-ARE-2M (Oligo-ARE-2M). After immunoprecipitation with anti-V5 or control antibody (IgG), the presence of TTP was determined by western blot analysis using an anti-V5 antibody ( Figure 4F). Total RNA was extracted from the immunoprecipitates, and the presence of luciferase mRNA was analyzed by RT-PCR using PCR primers specific to the luciferase gene. The amplified PCR product was observed in immunoprecipitates from cells transfected with Oligo-ARE-2W and pcDNA6/V5-TTP ( Figure 4F). However, no PCR products were detected in samples from cells transfected with Oligo-ARE-2M or pcDNA6/V5-TTP ( Figure 4F). PCR product was not also detected in immunoprecipitates obtained using control antibody. These results demonstrated that TTP interacted specifically with the a-Syn ARE-2.
To confirm the interaction of TTP with ARE-2 of the a-Syn 3′-UTR, RNA EMSA was conducted using a biotinylated RNA probe containing wild-type or mutant ARE-2 of a-Syn. The RNA probes used for RNA EMSA were the same as those for the luciferase assay. Cytoplasmic extracts were prepared from SHSY5Y cells transfected with pcDNA6/V5-TTP to overexpress TTP and were incubated with a biotinylated RNA probe containing wild-type or mutant ARE-2 of a-Syn. When RNA EMSA was conducted using the wild-type ARE-2 probe of a-Syn, a dominant probe-protein complex was observed. However, the mutant ARE-2 of a-Syn prevented the formation of this complex. The complex was supershifted with the anti-V5 antibody ( Figure 4G). These results confirmed that a-Syn ARE-2 was essential for TTP binding. Taken together, these data strongly suggest that repression of a-Syn occurs through binding of TTP to ARE-2 of a-Syn 3′-UTR.

TTP overexpression decreases mitochondrial membrane potential and ATP production
Imbalances in mitochondrial fission/fusion have been linked to mitochondrial dysfunction such as a decrease in mitochondrial membrane potential and OXPHOS, loss of mitochondrial DNA (mtDNA), increase in ROS production, and release of cytochrome c [56][57][58][59][60][61][62][63], leading to defects in energy production and induction of apoptosis. To investigate whether elongation of mitochondria in TTP-overexpressing cells was associated with mitochondrial dysfunction, we first examined mitochondrial membrane potential using the dye tetramethylrhodamine methyl ester (TMRM). TTP overexpression caused an approximately 38% and 57% decrease in green fluorescence in SHSY5Y and HeLa cells, respectively, indicating a marked loss (E) Graphs represent ratio of maximum axis to minimum axis of mitochondria. Values are mean ± s.e.m. from three separate experiments (*p < 0.05; **p < 0.01). www.impactjournals.com/oncotarget of mitochondrial membrane potential ( Figure 5A). Inhibition of a-Syn by using a-Syn-siRNA decreased mitochondrial membrane potential in control SHSY5Y and HeLa cells ( Figure 5B), and overexpression of α-Syn by transfection with α-Syn without mRNA 3′-UTR reversed the decrease in mitochondrial membrane potential in TTP-overexpressing SHSY5Y and HeLa cells ( Figure 5A). Mitochondrial membrane potential (Δym) is critical for maintaining respiratory chain physiological function to generate ATP. Consistent with the decrease in mitochondrial membrane potential, total ATP production was decreased by TTP overexpression or inhibition of α-Syn in both SHSY5Y and HeLa cells ( Figure 5C, 5D). This phenomenon was reversed by co-transfection with α-Syn without mRNA 3′-UTR ( Figure 5C). Thus, these results suggest the elongation of mitochondria induced by TTP overexpression was accompanied by mitochondrial depolarization and loss of ATP production, which were reversed by overexpression of α-Syn.

TTP overexpression increases ROS generation and decreases mtDNA contents
Mitochondrial dysfunction is often associated with increased ROS production by mitochondria [64] and ER [65]. ROS levels were significantly increased in SHSY5Y and HeLa cells in which α-Syn level was downregulated by transient transfection with pcDNA6/V5-TTP ( Figure 5E). Similarly, inhibition of α-Syn by using siRNA increased ROS levels in both SHSY5Y and HeLa cells ( Figure 5F). Overexpression of α-Syn by transfection with α-Syn without the mRNA 3′-UTR significantly attenuated increased ROS levels induced by TTP overexpression in SHSY5Y and HeLa cells ( Figure 5E). mtDNA is located in close proximity to the respiratory chain, the main cellular source of ROS. Generation of ROS induces degradation of mtDNA [66]. Thus, we investigated whether ROS induced by TTP overexpression decreased mtDNA content. Overexpression of TTP or inhibition of a-Syn significantly decreased mtDNA content as quantified by immunofluorescence in both SHSY5Y and HeLa cells ( Figure 5G, 5H). Transfection of α-Syn without mRNA 3′-UTR reversed the decrease in mtDNA content in TTP-overexpressing SHSY5Y and HeLa cells ( Figure 5G).

TTP overexpression decreases oxygen consumption
We further characterized mitochondrial dysfunction induced by TTP overexpression by analyzing oxygen consumption rate (OCR). SHSY5Y and HeLa cells overexpressing TTP showed decreases in basal respiration rates compared to control cells ( Figure 5I, 5J). We also measured OCR after sequential addition of the ATP synthase inhibitor oligomycin, the uncoupler FCCP, the RC complex I inhibitor rotenone, and the RC complex III inhibitor antimycin A. TTP overexpression decreased ATP production (coupled respiration, the drop in the OCR after addition of oligomycin) in HeLa cells ( Figure 5I, 5J). However, TTP overexpression did not affect the proton leak (uncoupled respiration, the difference between oligomycinand rotenone and antimycin A-responsive OCRs) in HeLa cells ( Figure 5I, 5J). Transfection of α-Syn without mRNA 3′-UTR reversed the decrease in basal respiration and ATP production in TTP-overexpressing HeLa cells ( Figure 5I, 5J). We concluded from these results that TTP overexpression decreased respiration rate.

TTP overexpression promotes apoptosis in cancer cells
A reduction in mitochondrial membrane potential can induce caspase-dependent apoptosis by release of cytochrome c [67]. We thus determined whether a decrease in the mitochondrial membrane potential induced by TTP overexpression lead to cytochrome c release and apoptosis in HeLa cells. Release of cytochrome c was assessed by confocal microscopic observation and subcellular fractionation of control and TTP-overexpressing HeLa cells. TTP overexpression induced cytochrome c release into the cytosolic fraction ( Figure 6A, 6B) and increased caspase-3 activity ( Figure 6C). Consistently, inhibition of α-Syn using a-Syn-siRNA led to an increase in cytochrome c release ( Figure 6A) and caspase-3 activity ( Figure 6D). Overexpression of α-Syn without mRNA 3′-UTR inhibited cytochrome c release ( Figure 6A, 6B) and caspase-3 activity ( Figure 6C) in TTP-overexpressing cells. Next, apoptotic cells were detected by Annexin V staining. TTP overexpression or inhibition of α-Syn using a-Syn-siRNA increased apoptosis in both SHSY5Y and HeLa cells ( Figure 6E, 6F) and expression of α-Syn without mRNA 3′-UTR reversed apoptosis in TTP-overexpressing cells ( Figure 6E). Consistently, overexpression of TTP or inhibition of α-Syn using a-Syn-siRNA suppressed cell proliferation ( Figure 6G, 6H), and expression of α-Syn without mRNA 3′-UTR rescued cell proliferation in TTPoverexpressing SHSY5Y and HeLa cells ( Figure 6G). These results suggested that TTP overexpression induced cytochrome c release from mitochondria, activation of caspase-3, and apoptosis in cancer cells.

DISCUSSION
The many functions of mitochondria are intimately linked to their morphology, which is maintained by mitochondrial dynamics [68,69]. Here, we found that TTP regulated mitochondrial morphology and function: overexpression of TTP induced elongation of mitochondria and led to mitochondrial dysfunction and apoptosis. Mitochondrial dynamics and morphology are finely tuned by mitochondrial fusion and fission proteins Mfn1, Mfn2, OPA1, Drp1, and Fis1 [6,7]. However, we found that TTP did not decrease the expression levels of the well-known mitochondrial fusion and fission proteins Mfn1, Mfn2, Values are mean ± s.e.m. from three separate experiments (*p < 0.05; ***p < 0.001). www.impactjournals.com/oncotarget Drp1, and OPA1. Instead, TTP inhibited the expression of α-Syn, which lead to mitochondrial elongation and mitochondrial dysfunction. We here provide supporting evidence that 1) TTP binds to the 3′-UTR of α-Syn mRNA and enhances degradation of α-Syn mRNA, 2) transfection of a-Syn without mRNA 3′-UTR reverses mitochondrial morphology and 3) mitochondrial dysfunction is induced by TTP overexpression.
It is widely agreed that α-Syn expression levels are critical for the development of PD and that reducing α-Syn levels represents an attractive strategy to counteract the detrimental effects of the overexpression. Consistent with this hypothesis, overexpression of α-Syn results in severe neurodegeneration, suggesting a dose-dependent toxic effect [70,71]. While accumulation of α-Syn by the inhibition of autophagy results in increased toxicity of α-Syn, reducing α-Syn levels by activation of autophagy has shown therapeutic effects in PD models [72][73][74][75]. Overexpression of α-Syn leads to mitochondrial fragmentation and neuronal cell death [36,54], and reducing α-Syn levels by RNAi makes mitochondria more tubular in a neural cell line [36]. However, it is not likely that reduction of α-Syn levels always improves mitochondrial function and/or diseases. We here found that even though reducing α-Syn levels by TTP overexpression induces elongation of mitochondria, it also causes mitochondrial dysfunction including reduction in mitochondrial membrane potential and ATP generation, and cytochrome c release. Consistent with our results, it has been reported that robust reduction of α-Syn by siRNA increased neurotoxicity in rat models [76,77] suggesting that either reducing or increasing the α-Syn level beyond the normal physiological level can cause defects in mitochondrial dynamics and mitochondrial dysfunction. Taken together, these results imply that regulation of α-Syn expression to maintain normal physiological levels is important for optimal mitochondrial function.
Besides in the central nervous system, α-Syn is expressed in a variety of tumors [30][31][32] and α-Syn expression has been proposed to affect tumorigenesis [78,79]. These suggest the common pathogenic mechanisms between cancer and neurodegenerative diseases such as PD. Consistently, several studies have shown elevated risk for malignant melanoma in PD patients [80][81][82]. In this study, we found that TTP downregulates α-Syn expression. Previously it has been reported that TTP exerts inhibitory effects on the growth of cancer cells [46][47][48][49]. Combining our results and previous reports, it is possible to suggest that the inhibitory effect of TTP on the growth of cancer cells may be partly mediated by down-regulation of α-Syn expression.
Expression of α-Syn is modulated by posttranscriptional control mechanisms targeting the 3′-UTR of mRNAs. Several reports have suggested that α-Syn mRNA levels are post-transcriptionally decreased by endogenous microRNAs (miRNAs) targeting the 3′-UTR of α-Syn mRNA. In rodent primary neurons, α-Syn levels are downregulated by miR-7 and miR-153 [83]. In humans, miR-7, miR-34b and miR-34c target the 3′-UTR of α-Syn mRNA and reduce α-Syn mRNA levels, suggesting that downregulation of miR-34b and miR-34c in the brain, as well as a single-nucleotide polymorphism (SNP) in the 3′-UTR of α-Syn, can increase α-Syn expression, possibly contributing to PD pathogenesis [84,85]. Consistent with these studies, it has been reported that expression of these two miRNAs is down-regulated in human brains [86,87]. In this study, we found TTP as a factor responsible for post-transcriptional regulation of α-Syn expression. TTP is an RNA-binding protein that binds to AU-rich elements within the mRNA 3′-UTR and inhibits gene expression through enhancing mRNA degradation [42][43][44]. TTP binds to a -AUUUAmotif within the 3′-UTR of α-Syn and inhibits expression of α-Syn through enhancing mRNA degradation. Mutation in this AUUUA motif blocks binding of TTP to the motif and, subsequently, the inhibitory effects of TTP. Based on our results, we predict that dysregulation of TTP or SNPs in the TTP target AUUUA motif within the 3′-UTR of α-Syn mRNA may increase α-Syn levels and cause mitochondrial dysfunction.
In conclusion, our data suggest that TTP could reduce the expression of α-Syn and cause defects in mitochondrial morphology and function. We demonstrated that TTP enhances α-Syn mRNA decay through binding to an AUUUA motif within the α-Syn mRNA 3′-UTR, and disruption of this AUUUA motif diminished the repressive activity of TTP. Considering the involvement of α-Syn in the pathological processes of neurodegenerative diseases such as PD and Alzheimer's disease [26][27][28][29] our present findings raise the possibility that TTP dysregulation and sequence variations in its target sequence within α-Syn mRNA 3′-UTR might trigger neurodegenerative diseases as well as mitochondrial dysfunction.

Cell culture
Human cancer cell lines, SHSY5Y and HeLa were purchased from the Korean Cell Line Bank (KCLB-Seoul, Korea). SHSY5Y and HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM). All cell lines were supplemented with 10% FBS (heat-inactivated fetal bovine serum) (Welgene) and were maintained at 37°C in a humidified at atmosphere of 5% CO 2 .
For the luciferase assays, cells were co-transfected with various psiCHECK2-α-Syn 3′-UTR constructs and pcDNA6/V5-TTP using the TurboFect ™ in vitro transfection reagent (R0531, Thermo Scientific). Transfected cells were lysed with lysis buffer and mixed with luciferase assay reagent (017757, Promega), then the chemiluminescent signal was measured using a SpectraMax L Microplate reader (Molecular Devices). Firefly luciferase was normalized to Renilla luciferase in each sample. All luciferase assays reported here represent at least three independent experiments, each consisting of three wells per transfection.

Ribonucleoprotein immunoprecipitation (RNP) assay
RNP complexes were immunoprecipitated after reverse cross-linking between target RNA and proteins as described previously (47). Briefly, 1 × 10 7 SHSY5Y cells were co-transfected with 10 μg of pcDNA6/V5-TTP and psiCHECK2-α-Syn-Oligo-ARE-2W or psiCHECK2α-Syn-Oligo-ARE-2M. At 24 h after transfection, the cell suspension was incubated in 1% formaldehyde for 20 min at room temperature. The reaction was stopped with 0.25 M glycine (pH 7.0), and cells were www.impactjournals.com/oncotarget sonicated in modified radioimmune precipitation assay buffer containing protease inhibitors (Roche Applied Science). RNP complexes were immunoprecipitated using protein G-agarose beads preincubated with 1 μg of anti-V5 Tag antibody (GWB-7DC53A, Genway Biotech) or 1 μg of isotype control (Sigma). After crosslinking reversion at 70 o C for 45 min, RNA was isolated from the immunoprecipitates and treated with DNase I (Qiagen). cDNA was synthesized from the RNA, and the Renilla luciferase gene was amplified by PCR using Taq polymerase and Renilla luciferase-specific primers (Up, 5′-ACGTGCTGGACTCCTTCATC-3′; and Down, 5′-GACACTCTCAGCATGGACGA-3′). TTP proteins in the immunoprecipitated samples were detected by western blot analysis using anti-V5 Tag antibody.

Electrophoretic mobility shift assay (EMSA)
Biotinylated RNA probes for the wild type (α-Syn-ARE-2W, 5′-UAAAUCUACCUAAAGC AGCAUA UUUUAAAAAUUUAAAAGUAUUGGUAUUAAAUU AAGAAAUAGAG-3′) and mutant (α-Syn-ARE-2M, 5′-UAAAUCUACCUAAAGCAGCAUAUUUUAAAAA GCAAAAGUAUUGGUAUUAAAUUAAGAAAUAGA G-3′) constructs were generated by ST Pharm. Co., Ltd. (Korea). A mutant RNA probe in which two AUUUA pentamers were each substituted with AGCA was used as a negative control. Cytoplasmic extracts were prepared from SHSY5Y cells and TTP-transfected SHSY5Y cells using NE-PER nuclear and cytoplasmic extraction reagent (78833, Thermo Scientific). An electrophoretic mobility shift assay (EMSAs) was performed using the LightShift chemiluminescent EMSA kit (20158, Thermo Scientific) according to the manufacturer's instructions. Briefly, 20 fmol of biotinylated RNA was combined with 4 µg of cytoplasmic protein from cell extract in binding buffer. For the supershift EMSA, rabbit anti-human TTP polyclonal antibody (ab36558, Abcam) or control antibody (I-5381, Sigma) was added to the reaction mixture. After the addition of antibodies, reaction mixtures were incubated on ice. The reaction mixtures were resolved on 5% nondenaturing polyacrylamide gels in 0.5× Tris borate/ EDTA buffer. Gels were transferred to nylon membrane (Hybond TM -N) in 0.5× Tris borate/EDTA at 70 V for 40 min. Transferred RNAs were cross-linked to the membrane and detected using horseradish peroxidaseconjugated streptavidin (LightShift chemiluminescent EMSA kit, Thermo Scientific) according to the manufacturer's instructions.

Annexin V staining
Annexin V staining was conducted using an Annexin-V-FLUOS staining kit, according to the protocol supplied by the manufacturer (11858777001, Roche

Cell proliferation
Cells were transfected with a combination of pcDNA6/V5-TTP and pcDNA/α-Syn or with scramble or α-Syn-siRNA for 48 h. Cells were seeded in triplicate in 24-well culture plates at 5 × 10 4 cells/well and incubated for 24 h. Cells were harvested by treating with Trypsin-EDTA (25200-072, Invitrogen). Cells were then stained by Trypan-Blue Solution (0.4%) (T8154, Sigma) and counted using a hematocytometer. paraformaldehyde. Cells were permeabilized with 0.2% Triton X-100 for 10 min at room temperature and then incubated in blocking buffer (0.1% Triton X-100, 3% goat serum in PBS) for 40 min. Cells were incubated with an anti-DNA antibody (61014, PROGEN Biotechnik GmbH) or anti-cytochrome c antibody (556432, BD Pharmingen ™ ) for overnight followed by incubation with anti-mouse Alexa-Fluor-488-conjugated secondary antibodies (A-11001, Life Technologies) for 1 h. Cells were washed three times for 5 min each with 1% Triton X-100 in PBS. During the final wash step, cells were incubated with 10 μg/ml DAPI (D1306, Life Technologies) in PBS for 5 min and analyzed for mtDNA and cytochrome c by fluorescence microscopy using an Olympus FV1200-OSR microscope. Image analysis was performed using Image J software. The percentage of mtDNA stain remaining was calculated using the following formula: (cDNA v -nDNA v )/n, in which cDNA v was the total cellular DNA volume determined by staining using anti-DNA antibodies, and nDNA v was the total nuclear DNA stain volume determined using DAPI, where n denotes the number of cells.
Active caspase-3 was detected using the Image-iT LIVE Red Caspase Detection Kit (I35102, Life Technologies) according to the manufacturer's instructions. Briefly, cells were incubated with fluorescent inhibitor of caspases (FLICA) reagent for 60 min while protected from light. The solutions were then removed. After incubating with 1µM Hoechst for 2-10 min, confocal fluorescence images were obtained using an Olympus FV1200-OSR microscope.

Luminescent assays
Cellular ATP levels were measured using CellTiter-Glo ® Luminescent cell viability assay kit (G7570, Promega) according to the manufacturer's instructions. Briefly, SHSY5Y or HeLa cells were plated on 96-well white-walled plates with clear bottoms in 100 μl culture media then 100 μl of CellTiter-Glo ® reagent was added to each well. The contents were mixed for 2 min on an orbital shaker to induce cellular lysis followed by incubation at room temperature for 10 min to stabilize the signal, then luminescence was recorded immediately.
Cellular ROS levels were measured using the ROS-Glo ™ H 2 O 2 Assay Kit (G8820, Promega) according to the manufacturer's instructions. Briefly, SHSY5Y or HeLa cells were plated on 96-well white-walled plates with clear bottoms in 80 μl culture media. To record luminescence, 20 μl H 2 O 2 substrate solution was added to each well, and cells were incubated in a 37ºC CO 2 incubator for 2 hr. ROS-Glo ™ detection solution (100 μl) was added to each well, and cells were incubated for 20 min at room temperature.

Electron microscopy
Cells were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in a 0.1M phosphate buffer for overnight. After washing in a 0.1M phosphate buffer, cells were postfixed with 1% osmium tetroxide in the same buffer for 1 h. Then, the samples were dehydrated with a series of the graded ethyl alcohol. The samples were embedded in Epon 812 and then polymerization was performed at 60°C for 3 days. Ultrathin sections (60-70 nm) were obtained by ultramicrotome (Leica Ultracut UCT). Ultrathin sections collected on grids (200 mesh) were examined in the transmission electron microscope (TEM) (JEOL JEM-1010) operating at 60 kV and images in the TEM were recorded by the CCD camera (SC1000; Gatan).

Detection of cytosolic cytochrome c
SHSY5Y or HeLa cells were seeded in 100 mm diameter dishes (Thermo Scientific) and then transfected as indicated in the figure legends. After transfection, cells were harvested using a Qproteome Mitochondria isolation kit (37612, Qiagen) according to the manufacturer's instructions. Briefly, cells were lysed by adding Mitochondria Isolation Reagents A, B, and C, and the solution was centrifuged at 12,000 × g for 15 min to separate the cytosol and mitochondria fractions. Cytosol was used for western blot analysis. www.impactjournals.com/oncotarget

Statistical analysis
For statistical comparisons, p values were determined using Student's t-test or one-way ANOVA. A p value of < 0.05 was consider significant.