Peptide SS-31 upregulates frataxin expression and improves the quality of mitochondria: implications in the treatment of Friedreich ataxia

Friedreich ataxia is a progressive neurodegenerative disease caused by the expansion of GAA trinucleotide repeats within the first intron of the FXN gene, which encodes frataxin. The pathophysiology of the disease is thought to be derived from the decrease of Fe-S cluster biogenesis due to frataxin deficiency. There is currently no effective treatment for the disease. In our study, we demonstrated that treatment with the mitochondrion-targeted peptide SS-31 reduced frataxin deficiency-induced oxidative stress in lymphoblasts and fibroblasts derived from patients. Interestingly, SS-31 treatment translationally upregulated the protein level of frataxin in a dose-dependent manner. Furthermore, SS-31 treatment increased the enzymatic activities of the iron-sulphur enzymes, including aconitase and complex II and III of the respiratory chain. Further evaluation of the quality of mitochondria showed that mitochondrial membrane potential, ATP content, NAD+/NADH, and the morphology of mitochondria all improved. Our results suggest that SS-31 might potentially be a new drug for the early treatment of Friedreich ataxia.


SS-31 treatment upregulates FXN expression in FRDA patient-derived cells.
Efforts to find a drug capable of increasing the ability to cope with oxidative stress have long been made in the treatment of FRDA. Based on the characteristics of SS-31, a mitochondrion-targeted antioxidant, we chose it as a potential drug. Very strikingly, we found that SS-31 treatment increased the expression of FXN (Fig. 1). This prompted us to evaluate the value of the peptide. First, we optimised the condition for inducing the expression of FXN and found that the protein level of FXN increased in a dose dependent manner in both lymphoblasts and fibroblasts derived from patients ( Fig. 1a and Fig. S1). Relatively, FXN expression was much more robust in lymphoblasts than in fibroblasts derived from healthy controls (unpublished data) and the response to SS-31 treatment was stronger in lymphoblasts than in fibroblasts derived from patients ( Fig. 1a and Fig. S1). Thus, lymphocytes (GM15850) from FRDA patient are more straightforward for further study. The optimal concentration of SS-31 was determined to be within the range of 20 to 50 nM (Fig. 1a). A time course of the treatment was assessed, and the highest level was reached within 24 h (Fig. 1b). Finally, we chose 50 nM and 8 and 24 h as optimal conditions for later experiments. A one-way ANOVA was carried out, # p < 0.05 compared with Ctr; *p < 0.05 compared with the untreated cells (t = 0). GAPDH was used as an internal control.
Interestingly, our data showed that SS-31 treatment had no effect on FXN expression in healthy cells (Fig. S2), but significantly increased the protein level of FXN in cells derived from FRDA patients to reach 75% of the level of healthy control-derived cells (designated as "healthy control" later on) (Fig. 1d). To see if the effect of the upregulation stemmed from its antioxidant function, we used two common antioxidants lipoic acid (LA) and ascorbic acid (AA) as controls to check if FXN expression could be upregulated by them. Neither LA nor AA treatment had any effect on the protein level of FXN in patient-derived cells (Fig. S3). Thus, the effect of SS-31 on the upregulation of FXN is a novel function, and it is rational to further characterise the effect of SS-31-induced upregulation of FXN on iron metabolism and mitochondrial function. SS-31 treatment improves the quality of mitochondria in FRDA patient-derived cells. FRDA is a mitochondrial disease. Mitochondria derived from FRDA patient cells exhibit structural and functional impairment. Mitochondrial membrane potential (MMP) is considered as an indicator of mitochondrial integrity and bioenergetic function. To investigate the effects of SS-31 on mitochondria in cells derived from FRDA patients, we detected MMP with the lipophilic dye JC-1 by measuring a potential-dependent shift in fluorescence from green to red, which reflected its aggregation in mitochondria 29 . The increased ratio of red versus green fluorescence in patient-derived cells after SS-31 treatment indicated more polarised mitochondria (Fig. 2a). Intracellular ATP level is another pivotal measure of mitochondrial quality. We found that SS-31 treatment significantly raised ATP levels in patient-derived cells (Fig. 2b), indicating increased oxidative phosphorylation. The ratio of NADH/ NAD + in patient-derived lymphoblasts was also measured and was found to be significantly reduced to levels comparable to the healthy control cells (Fig. 2c). These results indicate much improvement of mitochondrial quality in patient-derived lymphoblasts post SS-31 treatment. Furthermore, we quantified the copy number of mitochondrial DNA and found that SS-31 treatment mildly increased the copy number of mitochondria in patient-derived lymphoblasts (Fig. 2d). Electron microscopic data substantiated these results, revealing structural improvements from abnormal cristae in patient-derived cells to regular invagination of the inner membrane after SS-31 treatment (Fig. 2e). Taken together, SS-31 improved mitochondrial quality significantly and increased mitochondria quantity mildly in patient-derived cells.

SS-31 treatment enhances the activities of mitochondrial iron-sulphur cluster-containing enzymes.
A deficiency in iron-sulphur cluster biogenesis is considered as the primary outcome of FXN deficiency. To check whether the increased FXN protein level was sufficient to improve iron-sulphur cluster biogenesis, we measured the activities of a number of iron-sulphur cluster-containing enzymes. First, in-gel activity assays showed that the mitochondrial aconitase activity (m-aco) in FRDA patient-derived cells increased Values represent mean ± SEM (n = 3, each duplicates). A one-way ANOVA was carried out, # p < 0.05, compared with Ctr. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the untreated GM15850 cells.
significantly after SS-31 treatment. The cytosolic aconitase activity (c-aco) increased as well, but mildly (Fig. 3a). However, the protein levels of the corresponding enzymes did not change (Fig. 3a, ACO2 and IRP1). Complex I, II, and III of the respiratory chain are the most abundant iron-sulphur cluster-containing enzymes in mitochondria. The activities of these enzymes in FRDA patient-derived cells are all reduced due to insufficient biogenesis of iron-sulphur clusters. No improvement in complex I was observed, while SS-31 treatment improved the activities of complex II and complex III (Fig. 3b). Xanthine oxidoreductase (XOD), an enzyme that generates ROS, needs iron-sulphur cluster as a prosthetic group. We found that the activity of XOD showed no changes after SS-31 treatment (Fig. 3c). These results suggest that SS-31-induced FXN upregulation facilitates iron-sulphur cluster biogenesis in the mitochondria to specifically promote the activities of several iron-sulphur cluster-containing enzymes.

SS-31 treatment modulates the iron metabolism of cells derived from FRDA patients.
To study the effects of SS-31 on the iron metabolism in FRDA patient-derived cells, we evaluated the expression of iron related proteins and measured the levels of cytosolic and mitochondrial labile iron pool (LIP). The expression level of ferritin in patient-derived cells increased to a level comparable to healthy control 24 h post SS-31 treatment, indicating that iron uptake was enhanced. In agreement with the result, both protein levels of IRP2 and TfR1 after SS-31 treatment increased, although IRP2 protein levels were already higher in patient-derived cells than in healthy control 21 (Fig. 4a). Then we used the fluorescent probe Calcein-AM to evaluate cytosolic LIP and RPA (rhodamine B-[(1,10-phenanthrolin-5-yl) aminocarbonyl] benzylester) for mitochondrial LIP. Cytosolic LIP level remained constant, in contrast to the increased level of ferritin ( Fig. 4a and b). However, mitochondrial LIP level was significantly elevated 24 h post SS-31 treatment (Fig. 4b, right panel), indicating an increase in bioavailable iron in mitochondria. These results demonstrate that SS-31 treatment modulates cellular iron metabolism.

SS-31 treatment decreases ROS level and enhances the antioxidative ability of FRDA patient-derived cells.
An increasing amount of data from different organisms support the notion that frataxin-deficiency results in oxidative stress, which is at least one of the pathological mechanisms of FRDA 30 . Reduction of oxidative stress by preventing ROS generation and/or enhancing the ROS scavenging ability may be a promising target for therapy 31 . To estimate the effect of SS-31 on the level of oxidative stress in patient-derived cells, we used the ROS probe DCFH-DA to analyse cellular ROS production by flow cytometry. The result showed that ROS levels were higher in the patient-derived lymphoblasts than in healthy control cells (Fig. 5a, left panel). SS-31 treatment drastically decreased ROS levels in patient-derived lymphoblasts (Fig. 5a, right panel). To reveal if the reduction in ROS levels stemmed from the ROS scavenging ability, we detected the protein levels and enzymatic activities of superoxide dismutase (SOD) and catalase, which are important cellular scavengers of superoxide and hydrogen peroxide (H 2 O 2 ), respectively. SS-31 treatment increased the expression and activity of catalase (Fig. 5b, left panel and c). Although SS-31 treatment did not change the protein level of SOD1 and SOD2 in patient-derived cells, total SOD activities significantly increased after incubation for 8 h with SS-31 (Fig. 5b, right panel and c). We then examined if patient-derived cells became more resistant to oxidative stress challenge after SS-31 treatment. Cell survival was estimated using a CCK8 viability assay after exposure to H 2 O 2 . Intriguingly, patient-derived lymphoblasts showed significantly more resistance to H 2 O 2 -mediated cellular toxicity after SS-31 treatment than before treatment (Fig. 5d). Altogether, these data suggest that SS-31 treatment diminishes ROS levels and enhances anti-oxidative ability of cells derived from FRDA patients.

SS-31 translationally upregulates FXN expression.
To investigate how SS-31 increases the protein level of FXN, we measured both the mRNA and protein levels of FXN. Treatment with SS-31 for 8 h resulted in no increase in the mRNA level of FXN (Fig. 6a), but a marked increase in FXN protein levels ( Fig. 1), suggesting posttranscriptional regulation of FXN expression. To confirm this, we treated the cells with SS-31 and simultaneously with actinomycin D to block gene transcription. Protein levels of FXN were then determined with immunoblotting. The result showed that the protein level of FXN was still elevated after 8 h treatment (Fig. 6b), further supporting translational or post-transcriptional upregulation. To further confirm that the increased FXN was indeed newly synthesised FXN, we used the SILAC (Stable isotope labelling with amino acids in cell culture) technology to distinguish newly synthesised FXN after SS-31 treatment from the FXN synthesised before SS-31 treatment. Figure 6c clearly showed that translation was markedly enhanced by SS-31 treatment. Combined with our previous study in which we showed that the half-life of FXN is about 48 h 21 , the current results demonstrate that SS-31 treatment translationally upregulates FXN expression, pointing to its potential in the treatment of FRDA.

Discussion
Currently no approved therapy is available for FRDA, and only limited treatment for the management of symptoms is available to patients 1,32 . Since the lack of mitochondrial FXN is the root cause, increasing the amount of FXN will be the most effective therapeutic approach 22 . The target cell types in FRDA, cardiocytes and neurons, are difficult to study in vitro. In contrast, lymphocytes from FRDA patients are more straightforward to study due to the very low level of FXN protein compared with healthy control cells. This lymphoblast model has been widely used in the FRDA field [33][34][35] . In this study, we demonstrate that SS-31 not only acts as an effective antioxidant, but also, most interestingly, rescues FXN deficiency in FRDA patient-derived cells. SS-31-induced upregulation of FXN occurs translationally and in a dose-dependent manner. Promisingly, enzymatic activities of most of the iron-sulphur cluster-containing enzymes tested increased and the quality of mitochondria was greatly improved after SS-31 treatment. In addition, SS-31 as an antioxidant not only reduced ROS accumulation, but also boosted the cell's ability to defend against oxidative stress. Therefore, the beneficial effect of SS-31 on the lymphoblasts derived from FRDA patients could result from these two mechanisms as described and modelled in Fig. 7.  Many studies showed that SS-31 is effective in cellular and animal models for a few diseases that are associated with bioenergetic failure 36,37 . Due to the superior properties of SS-31, more than 20 clinical trials (registered as elamipretide or Bendavia) have been being conducted in phase 1 or 2 (https://clinicaltrials.gov/), covering a number of mitochondrial disorders. So far, the safety and tolerability of SS-31 were well confirmed 38 . The efficacy of SS-31 revealed in the current study raises it as a candidate for a therapeutic strategy of FRDA treatment.
A deficit in iron-sulphur cluster biogenesis is considered the primary effect of FXN deficiency, a notion supported by a number of studies showing that the activities of the iron-sulphur cluster-dependent aconitase and mitochondrial respiratory chain complexes I, II and III drastically reduce in tissues of FRDA patients 8,39,40 . Here, SS-31 treatment induced upregulation of FXN and elevated the activities of iron-sulphur enzymes, such as mitochondrial aconitase and complex II and III, but not XOD and complex I. XOD is an enzyme that needs both iron-sulphur cluster and molybdenum cofactor as prosthetic groups to be functional, to generate cytosol-derived ROS. The lack of increased enzymatic activity of XOD after SS-31 treatment suggests iron-sulphur cluster alone is not enough to promote the activity of XOD. Complex I activity was not recovered, possibly because much more iron sulphur clusters are needed for complex I than other enzymes. Complex I is also the biggest complex with 46 subunits, which may take more time to be expressed and finely assembled than other complexes. Therefore, longer administration of SS-31 may be needed in clinical trials to reach full-scale beneficial effects.
Early histological and biochemical studies into the pathophysiology in patient samples revealed that dysregulation of iron metabolism is a key feature of the disease, mainly characterised by mitochondrial iron accumulation and cytoplasmic iron deficiency. Consistent with our previous study 21 , we found that IRP2 is upregulated and ferritin is downregulated in FRDA patient derived cells. After SS-31 treatment, the protein level of FXN increased along with ferritin upregulation, indicating more iron uptake into cytosol for ferritin to consume. Therefore, ferritin can be stabilised. Meanwhile, absorbed iron may bind the iron responsive element to activate the translation of ferritin 41 . Unexpectedly, IRP2 expression was still elevated, indicating iron-demand signalling. To track the absorbed iron after SS-31 treatment, we measured cytosolic and mitochondrial LIP levels with Calcein-AM and RPA, respectively. We found an increase in mitochondrial LIP level, but no changes in cytosolic LIP level after SS-31 treatment. Notably, no significant alteration in the concentration of mitochondrial LIP iron could be observed in cultured patient-derived cells, which had also been observed previously 42 . The results suggest that newly taken-up iron is supplied to ferritin and/or directly goes into mitochondria as bioavailable iron for iron-sulphur cluster biogenesis.
Oxidative damage and mitochondrial dysfunction are the key determinants of cellular damage in FRDA patient cells 43 . FXN deficient cells are much more sensitive to oxidative stress than wild type cells 9 . It has been shown that cells with FXN deficiency also have an impaired ability to recruit antioxidant to defend against endogenous oxidative stress 9,44 . Several therapies are aimed at reducing the consequences of FXN deficiency, including antioxidants to reduce cellular oxidative damage, such as idebenone, MitoQ, CoQ10, and vitamin E. However, clinical trials neither support nor refute an effect of antioxidants (idebenone, or a combination of coenzyme Q10 and vitamin E) so far on the neurological status of people with FRDA 45 . Many studies have shown that SS-31 can scavenge various ROS in animal models of neurodegenerative diseases 46,47 . In our cell model, SS-31 drastically decreased ROS level in patient-derived cells (Fig. 5a). Furthermore, the cells after SS-31 treatment exhibited significantly more resistance to H 2 O 2 -mediated cytotoxicity than the cells before treatment by increasing the enzymatic activities of SOD and catalase (Fig. 5b), two important ROS scavengers. Therefore, we conclude that SS-31 treatment reduces the ROS generation and enhances the competence to scavenge the ROS.
To judge the therapeutic strategy for a mitochondrial disease, any cellular improvement after drug treatment needs to be checked through mitochondrial morphology and function. SS-31 can improve the mitochondrial function in a number of cellular and preclinical models 48,49 . Our results demonstrate that SS-31 is very effective in restoring the MMP, increasing the ratio of NAD + /NADH, and promoting ATP production in FRDA patient-derived cells. Even mtDNA copy number mildly increased after SS-31 treatment, suggesting restored function. Though it was reported that SS-31reduced fission and increased fusion activity in mutant Htt neurons 50 , we did not find any clear difference when counting mitochondrial numbers (not shown). Remarkably, mitochondrial morphology changed structurally from disorganised into regular, finely arrayed cristae. However, SS-31 had no effect on mitochondrial morphology, mitochondrial proteome, respiration, ATP synthesis or ROS production in young healthy animals 26,51,52 . All the melioration in patient-derived cells suggests that SS-31 improves mitochondrial quality.
In summary, SS-31 treatment improves the morphology and function of mitochondria in the FRDA patient-derived cells by upregulating the expression of FXN at the translational level and reducing oxidative stress. In addition, SS-31 treatment significantly enhances the ability of patient-derived cells to withstand challenges from exogenous oxidative stress. The mechanism of the translational upregulation in FXN expression by mitochondrion-targeted SS-31 needs to be further addressed. However, improvement in the quality of mitochondria in FRDA patient-derived cells by SS-31 treatment appears promising. It is reasonable to suggest that SS-31 might potentially be a new drug for the early treatment of FRDA.
Measurement of the enzymatic activities of aconitase and complex I, II, and III. In-gel aconitase activity assays were performed as described previously 53 . The NADH dehydrogenase activity of isolated mitochondria was measured using the complex I enzyme activity microplate assay kit (Abcam). The procedure was adapted from the previous report 54 . The complex II assay was performed with the Succinate-coenzyme Q reductase activity assay kit (Comin Biotechnology Co., Suzhou, China) following the manufacturer's instructions. The activity was calculated by measuring the reduction of the absorbance of 2,6-dichloroindole at 605 nm. The Mitochondrial complex III activity was measured, based on the reduction of cytochrome c through the activity of complex III following the manufacturer's instructions (Biovision Inc., Milpitas, CA, USA).

Determination of mitochondrial membrane potential (MMP) level and ATP content. MMP
level was determined using a 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) mitochondrial membrane potential detection kit (Solarbio Science & Technology Co. Ltd.). The fluorescent intensity (excitation 490 nm, emission 520 nm) was determined using a fluorescence microplate reader. ATP levels were measured in cells derived from healthy control and FRDA patients by ATP content assay kit (Beyotime Biotech). The bioluminescence assay is based on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin. Luciferase catalyses the formation of light from ATP and luciferin, which was measured using a luminometer. After an hour, the reagent (Cell Counting Kit-8, Dojindo, Kumamoto, Japan) was added (10 μL/well) and cells were incubated for additional 4 h. Results were expressed as the percentage of reduction of absorbance at 450 nm by calibration with the absorbance of the control (untreated) cells.

Measurement of cellular ROS production and SOD
Quantitative real-time PCR. Total genomic DNA was extracted from the cells using Genomic DNA Mini Preparation Kit with Spin Column (Beyotime Biotech.) and eluted in sterile distilled H 2 O. Total RNA was extracted using TRizol (Bioteke, Beijing, China), and converted to cDNA with Transcriptor First Strand cDNA Synthesis Kit (Roche). Total genomic DNA or cDNA was subjected to qPCR analysis with SYBR Green Master Mix (Thermo Fisher Scientific) in Applied Biosystems 7300. Results were considered positive when the fluorescent signal above the baseline was detected and the cycle number was recorded. The relative copy number of a gene was determined by calibration with the internal control (GAPDH for RNA, β-actin for DNA). Primers for the genes are listed as follows (gene: forward primer; reserve primer): FXN: 5′-CCTTGCAGACAAGCCATACA-3′; 5′-CCACTGGATGGAGAAGATAG-3′; GAPDH: 5′-TGCA CCAC CAACTGCT TAGC-3′ ; 5′ -GGCATGGACTGTGGTCATGAG-3′ ; Cy tB: 5′ -TATCCGC CATCCCATACATT-3′; 5′-GGTGATTCCTAGGGGGTTGT-3′; β-actin: 5′-AGAAAATCTGGCACCACACC-3′; 5′-AACGGCAGAAGAGAGAACCA-3′ Stable isotope labelling with amino acids in cell culture (SILAC). SILAC is a simple and straightforward approach for in vivo incorporation of a label into proteins for mass spectrometry (MS)-based quantitative proteomics. Cells were cultured in conventional RPMI 1640 medium containing 12 C 14 N-Lysine and 12 C 14 N-Arginine ("light") overnight. The cells were then transferred into RMPI 1640 medium containing 13 C 6 15 N 2 -Lysine and 13 C 6 15 N 4 -Arginine ("heavy") (Sigma) with or without 50 nM of SS-31. After 24 h, the cells were collected and lysed with lysis buffer. Total FXN was enriched by immunoprecipitation with FXN antibody and assayed by LC-MS/MS using machine Q Exactive (Thermo Fisher Scientific). Newly synthesised FXN containing the "heavy" isotopes is distinguishable from the "old" FXN containing the "light" isotopes.