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Abstract

Background: Mitochondrial encephalomyopathies are severe, relentlessly progressive conditions and there are very few effective therapies available to date. We have previously suggested that in two rare forms of reversible mitochondrial disease (reversible infantile respiratory chain deficiency and reversible infantile hepatopathy) supplementation with L-cysteine can improve mitochondrial protein synthesis, since cysteine is required for the 2-thiomodification of mitochondrial tRNAs.

Objectives: We studied whether supplementation with L-cysteine or N-acetyl-cysteine (NAC) results in any improvement of the mitochondrial function in vitro in fibroblasts of patients with different genetic forms of abnormal mitochondrial translation.

Methods: We studied in vitro in fibroblasts of patients carrying the common m.3243A>G and m.8344A>G mutations or autosomal recessive mutations in genes affecting mitochondrial translation, whether L-cysteine or N-acetyl-cysteine supplementation have an effect on mitochondrial respiratory chain function.

Results: Here we show that supplementation with L-cysteine, but not with N-acetyl-cysteine partially rescues the mitochondrial translation defect in vitro in fibroblasts of patients carrying the m.3243A>G and m.8344A>G mutations. In contrast, N-acetyl-cysteine had a beneficial effect on mitochondrial translation in TRMU and MTO1 deficient fibroblasts.

Conclusions: Our results suggest that L-cysteine or N-acetyl-cysteine supplementation may be a potential treatment for selected subgroups of patients with mitochondrial translation deficiencies. Further studies are needed to explore the full potential of cysteine supplementation as a treatment for patients with mitochondrial disease.

Keywords

Cysteine supplementation mitochondrial translation mt-tRNA modification mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS)myoclonic epilepsy ragged red fibres (MERRF)

INTRODUCTION

Most mitochondrial diseases are disabling, progressive or fatal, affecting the brain, liver, skeletal muscle, heart and other organs [1, 2]. Currently there are no effective cures and treatment is at best symptomatic [3]. Although defective oxidative phosphorylation is the common final pathway, it is unknown why different mitochondrial DNA (mtDNA) or nuclear mutations result in largely heterogeneous clinical presentations [1]. The diagnosis in patients with multiple respiratory chain complex defects due to abnormal mitochondrial translation, which contribute to ∼30% of all mitochondrial disease, is particularly difficult because of the large number of nuclear genes involved in intra-mitochondrial protein synthesis [46]. Many of these genes have not yet been linked to human disease.

Mitochondrial protein synthesis requires about 150 different proteins, involved in the translation of the 13 mitochondrial-encoded proteins [4, 5]. Mitochondrial tRNAs, ribosomal proteins, ribosomal assembly proteins, aminoacyl-tRNA synthetases, tRNA modifying enzymes, initiation, elongation and termination factors are needed for an optimal translation, and mutations in these genes are common causes of mitochondrial disease. More than half of the pathogenic mtDNA mutations occur in tRNA genes and associated with common forms of mitochondrial diseases such as mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes (MELAS) and myoclonic epilepsy ragged red fibres (MERRF) and the heterogeneous clinical manifestations usually reflect variable heteroplasmy [7, 8, 7, 8]. Homoplasmic tRNA mutations with variable penetrance and clinical presentations suggest the role of genetic, epigenetic and/or environmental modifiers in mitochondrial translation [9]. The disorders caused by nuclear defects of mitochondrial protein synthesis are childhood-onset, severe, often fatal diseases frequently affecting the brain, skeletal muscle, heart, liver and peripheral nerves [5, 6]. Whole exome sequencing (WES) rapidly changed the diagnostic pathway by identifying the primary genetic defect, thus making invasive and complex biochemical testing unnecessary [4]. Interestingly, similar to mt-tRNA mutations in mtDNA-related disease, the majority of patients with nuclear genes causing a combined defect of the respiratory chain carry mutations in mitochondrial tRNA synthetases and tRNA modifying genes, suggesting that these genes are common causes of mitochondrial disease. Therefore, understanding the mitochondrial protein synthesis apparatus and the expression of mitochondrial proteins is of utmost importance in human health and disease, and an attractive target for the development of personalized therapies.

While most mitochondrial diseases are progressive conditions, two unique mitochondrial conditions termed reversible infantile respiratory chain deficiency (RIRCD, homoplasmic mt-tRNA^Glu mutation) and reversible infantile hepatopathy (TRMU mutations) stand out by showing complete spontaneous recovery [6, 10]. TRMU is an enzyme responsible for the thiouridylation ofmt-tRNA^Glu, mt-tRNA^Gln and mt-tRNA^Lys [11]. Posttranscriptional modifications of tRNAs offer rigidity to the U34 wobble base. We postulated that the mutation in mt-tRNA^Glu that causes RIRCD changes the tRNA structure and thus indirectly affects tRNA thiouridylation. Furthermore, cysteine (the substrate for TRMU) is an essential amino acid in infants until the developmental activation of the cysthationase enzyme, and infants rely on dietary cysteine intake in the first few months of life [10, 12]. The combination of low cysteine and the presence of the tRNA^Glu/TRMU mutations in infants lead to low thiouridylation of mt-tRNAs, thereby interfering with mitochondrial protein translation and contributing to disease manifestation (myopathy/hepatopathy) in newborns [13]. In return, increasing thiouridylation levels of mt-tRNA^Glu by increasing substrate (cysteine) availability for TRMU may improve mitochondrial translation and result in clinical recovery [13]. Indeed, addition of L-cysteine to culture of muscle cells carrying the homoplasmic m.14674T>C in the mt-tRNA^Glu or autosomal recessive mutations in the nuclear encoded TRMU gene partially rescued the reduced respiratory chain complex activities [13]. MTO1 is another mt-tRNA modifying enzyme, catalyzing the 5-carboxymethylaminomethylation of the wobble uridine base (U34) on mt-tRNA^Lys, mt-tRNA^Glu and mt-tRNA^Gln [14, 15]. Furthermore, altered post-transcriptional modifications at the wobble positions of mitochondrial tRNAs for leucine and lysine has been related to MELAS and MERRF, respectively [16]. The underlying mutations, m.3243A>G and m.8344A>G, cause a mitochondrial respiratory chain defect due to impaired mitochondrial protein synthesis. Based on these data we tested whether supplementation of growth media with L-cysteine or N-acetyl-cysteine (NAC) can reverse the defect in fibroblasts from patients carrying the m.3243A>G and m.8344A>G mtDNA mutations, or autosomal recessive mutations in the MTO1 and TRMU genes, and also in two further cell lines with nuclear mitochondrial disease of different pathomechanisms (ELAC2 and COX10). ELAC2 encodes an endonuclease, responsible for the removal of the 3-prime extensions from tRNA precursors, which is an essential step in tRNA biogenesis and mutations of this gene were associated with early onset cardioencephalomyopathy [17]. COX10 encodes a cytochrome c oxidase (COX) assembly protein involved in the mitochondrial heme biosynthetic pathway, by catalyzing the farnesylation of a vinyl group, resulting in the conversion of protoheme (heme B) to heme O. The COX10 protein is required for the expression of functional COX [18] and mutations in this gene result in multisystem mitochondrial disorders [18, 19, 18, 19].

Here we studied whether supplementation with L-cysteine or N-acetyl-cysteine (NAC) results in any improvement of the mitochondrial function in vitro in fibroblasts of patients with different genetic forms of abnormal mitochondrial translation.

MATERIALS AND METHODS

Primary cell cultures

Primary fibroblast cultures of patients and controls were obtained from the Newcastle Biobank. All studies were carried out with informed consent of the patients or their parents and were approved by institutional ethics review boards. The patients carried pathogenic mutations in mitochondrial tRNA genes (m.3243A>G MELAS, m.8344A>G MERRF), in nuclear genes affecting mitochondrial protein synthesis (MTO1, ELAC2, TRMU) or respiratory chain complex assembly (COX10) (Table 1). Primary fibroblasts were studied from patients carrying mutations in COX10 and ELAC2 and immortalised fibroblasts from the patients carrying mutations in the MTO1 and TRMU genes. Fibroblasts were grown in high glucose Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% foetal bovine serum. Supplementation with L-cysteine has been done in several concentrations: 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 2 mM, 4 mM, 5 mM and 8 mM. The higher concentration inhibited cell growth and resulted in precipitation in the medium. The low concentrations <2 mM had no effect in Seahorse analysis therefore we used 4 mM concentrations for supplementation. To identify the best dose for N-acetyl-cysteine (NAC) (Sigma-Aldrich), we performed supplementation first in 5 different concentrations [1, 2, 3, 4 and 5 mM], and 4 mM had the highest increase in oxygen consumption. Therefore, in this study we used supplementation with 4 mM of L-cysteine or NAC for 9 to 14 days. The supplementation medium was changed every 48 hours. L-cysteine and NAC solutions (50 mM stock solutions) were prepared fresh every time.

Oxygen consumption rate

Oxygen consumption rate (OCR) in fibroblasts was measured with a XF96 Extracellular Flux Analyser (Seahorse Bioscience Billerica), as described previously [20]. Each cell line was seeded in 12 wells of a XF96-well cell culture microplate (Seahorse Bioscience) at a density 30×10³ cells/well in 80 μL of DMEM and incubated for at 37°C in 5% CO₂ atmosphere. After replacing the growth medium with 180 μL of bicarbonate-free DMEM pre-warmed at 37°C cells were pre-incubated for 30 min before starting the assay. Oxygen consumption rate (OCR), leaking respiration (LR), maximal capacity respiration (MCR) and not electron transport chain respiration (NMR) were determined by adding 1 μM oligomycin (Sigma-Aldrich) (LR), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, Sigma-Aldrich, Dorset UK) (MCR): 2 injections of 0.5 μM and 1 μM respectively) and 1 uM Rotenone/Antimycin (Sigma-Aldrich) (NMR). For immortalised fibroblast lines 1.5 μM oligomycin, 1.25 μM and 2 μM FCCP and 1 μM Rotenone/Antimycin were used. The data were corrected by the NMR and expressed as pmol of oxygen/min/mg of protein. Protein quantitation was determined by the Bradford method.

BN-PAGE

Levels of the holoenzymes of the electron transport chain were determined with blue native polyacrylamide gel electrophoresis as has been described before [21]. Cells were lysed with digitonin (4 mg/mL, Sigma-Aldrich) 2.5 mg/mL final concentration in the cells. Afterwards, the cells were incubated for 10 minutes on ice and centrifuged at 10000 g at 4°C for 10 minutes. The obtained cell pellet was resuspended in Blue native (BN) sample buffer [0.5 ml 3X gel buffer (1.5 M aminocaproic acid (Sigma-Aldrich), 150 mM Bis-tris (Sigma-Aldrich), pH 7.0), 0.5 ml 2 M aminocaproic acid, and 4 ml 500 mM EDTA] and lauryl malthoside (Sigma-Aldrich) was added to a final concentration of 1%. The samples were vortexed, incubated on ice for 15 minutes and centrifuged at 20000 g at 4°C for 20 minutes. The supernatant was kept and the protein was quantified by the Bradford assay. NativePAGEtrademark Novextrademark 312% Bis-Tris Protein Gels, NativePAGEtrademark Running Buffer and NativePAGEtrademark Cathode Buffer Additive (Thermo Fisher Scientific) were utilised for the pre-cast gel and running buffer. Coomassie Brilliant Blue G-250 (SBG) (Bio-Rad) as a 5% solution in 0.75 mM aminocaproic acid was used as loading buffer to 20 μg of protein. The following commercial antibodies were used for the detection of the different mitochondrial complexes: Anti-NDUFA9 (ab14713) for Complex I, Anti-SDHA for Complex II (ab14715), Anti-Ubiquinol-Cytochrome C Reductase Core Protein I for Complex III (ab110252), Anti-COX4 + COX4L2 for Complex IV and Anti-ATP5A antibody for Complex V (ab14748). The antibodies were added sequentially. First the membranes were incubated with antibodies for CI, then CIV and finally CII, CIII and CV, respectively. The detection method was ECL.

DNA purification and quantification of mitochondrial point mutations

DNeasy Blood and Tissue Kit (Qiagen) was used for purification of the DNA. Heteroplasmy levels of MERRF and MELAS cell lines before and after the supplementation were quantified by pyrosequencing technology as it was described before (White et al., 2005). The assay method was designed using the PyroMarktrademark Q24 software (Qiagen, Manchester, UK) and the primers used are described in Supplementary Table 1. Amplicons were generated in 25 μL reaction volume with 1x MyTaq Reaction Buffer (Bioline), 0.4 μM of forward and reverse primer (IDT), 1U of MyTaqtrademark HS DNA Polymerase (Bioline), 50 ng/ μL DNA and autoclaved PCR-grade deionised water up to 25 μL. Afterwards, 10 μL of PCR product were mixed with 2 μL sepharose beads (GE Life sciences), 40 μL binding buffer (Qiagen) and 28 μL autoclaved PCR-grade deionised water. The samples were transferred to a BioShake thermoshaker (Quantifoil Instruments GmbH) and agitated for 10 minutes at 2000 rpm. The beads were captured on the filter probes and were processed through 70% ethanol for 15 seconds, denaturation buffer for 15 seconds and then wash buffer for 30 seconds. Following, the beads were released into a PyroMark Q24 sequencing plate containing 0.3 μM of sequencing primers diluted in 25 μL of annealing buffer. The sequencing plate was then heated for 2 minutes at 80°C on a digital dry water bath hot block (Benchmark Scientific) and allowed to cool to room temperature. The sequencing cartridge was then loaded with enzyme mixture, substrate mixture and dNTPs and placed in the dispensing unit in PyroMark Q24. Once the assay was finished, the data was analysed using the PyroMarktrademark Q24 software.

Quantification of mitochondrial DNA copy number

Quantification of mtDNA copy number was performed using CFX96 Touchtrademark Real-Time PCR Detection System (Bio-Rad) in triplicate by duplex TaqMan qPCR amplification of the mitochondrial gene MTND1 and the nuclear encoded gene B2M as described previously (Grady et al., 2014). The primers used for template generation of standard curves and the qPCR reaction are described in Supplementary Table 2. The copies per μL of each template were standardised to 1×10¹⁰ and a serial dilution in 1Log₁₀ dilution steps, was amplified along with a DNA negative control on each qPCR plate. This was performed in 20 μL reactions in a 96-well plate (Bio-Rad, Hertfordshire, UK), sealed using microplate ‘B’ plate sealers, using the iTaqtrademark Universal Probes Supermix (Bio-Rad). Each reaction contained: 1×iTaqtrademark Universal Probes Supermix (Bio-Rad), 0.3 μM forward and reverse primers, 0.2 μM ND1-HEX and β2M-FAM probes andPCR-grade autoclaved sterile deionised water (to make up to 20 μL reaction). The cycling conditions consisted of initial denaturation at 95°C for 3 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, with annealing and extension at 62.5°C for 1 minute. The relative mtDNA copy number per cell was calculated using the ΔCt data by the following equation: ΔCt = Ct MT-ND1 – Ct β2M. Relative mtDNA copy number per cell = 2(2^-ΔCt).

Statistical analysis

Data are presented as±standard deviation using the ANOVA test on Sigma plot version 11 and paired t-test. A P-value of≤0.05 was considered significant.

RESULTS

L-cysteine supplementation in MELAS and MERRF resulted in slight increase in oxidative phosphorylation by Seahorse analysis

In order to identify the most effective dose, several concentrations between 0.258 mM L-cysteine (or NAC) were studied in both control and patient cell lines for 914 days. Concentrations higher than 5 mM inhibited cell growth and caused precipitation, whereas concentrations lower than 2 mM had no effect on oxygen consumption rates. Further experiments were therefore conducted with 4 mML-cysteine (or NAC) added to the cell culture medium. The untreated cells were maintained in culture in parallel and the media was changed every 2 days, in both treated and untreated cell lines.

Two cell lines carrying the most common MELAS and MERRF mutations, m.3243A>G and m.8344A>G (Table 1), were supplemented with 4 mM L-cysteine and/or NAC during 9 days. The two cell lines carrying the m.8344A>G mutation (MERRF1 and MERRF2) (Table 1) showed increased levels of basal respiration accompanied with increased levels of maximal respiration after 9 days of supplementation (Fig. 1A). Interestingly, MERRF2 showed slightly greater increase in terms of basal respiration compared to MERRF1, although none of the changes were statistically significant. We measured the heteroplasmy levels of the m.3243A>G (MELAS) and m.8344A>G (MERRF) mutations before and after supplementation to exclude that the changes in oxygen consumption were caused by a shift in the level of heteroplasmy, but detected no significant change. Heteroplasmy levels were 83% and 95% for MERRF1 and MERRF2, respectively, and remained the same after supplementation. We noted that the greatest increase in levels of oxygen consumption was detected in the cell line with the higher mutation rate (MERRF2). One of two primary fibroblast cell lines (MELAS2), carrying the m.3243A>G mutation, demonstrated increased levels of basal and maximal respiration after L-cysteine supplementation, while no change was observed in the basal respiration, and slight decrease in maximal respiration in the other cell line (MELAS1) (Fig. 1B). Heteroplasmy levels of the MELAS cell lines were different, with the levels of mutated mtDNA being lower in MELAS1 (54 %) compared to the MELAS2 cell line (88%). The level of heteroplasmy influenced the severity of the mitochondrial defect. Oxygen consumption was more compromised in the cell line with higher heteroplasmy level (Fig. 3B), whereas the MELAS1 cell line with lower heteroplasmy level showed only slightly decreased levels of basal respiration compared to the control. Interestingly, similar to MERRF, the effect of L-cysteine was more pronounced in the cell line with a larger defect and higher heteroplasmylevel.

L-cysteine supplementation in MELAS and MERRF resulted in a slight increase of mitochondrial respiratory chain complex levels on BN-PAGE

Subsequently, we also studied whether L-cysteine supplementation affects the steady state of mitochondrial proteins by BN-PAGE (Fig. 2A-2B). Originally all complexes except for complex II were low in both MERRF and MELAS cells confirming the severe defect of mitochondrial protein synthesis. Mitochondrial protein complexes in general showed a tendency to improve after supplementation, however the very weak or non-detectable bands for complexes I and IV in MERRF cells did not enable us to conclude the effect of L-cysteine after supplementation. In both MERRF cell lines we detected increased levels of complexes III and V after supplementation with L-cysteine (Fig. 2A). In cells carrying the m.3243A>G mutation, the more affected MELAS2 cell line showed an increase in all 4 complexes containing mtDNA-encoded subunits after L-cysteine supplementation, especially for complexes III and V, which both showed a more prominent increase. In the less deficient MELAS1 cell line, complexes I, III and V increased, and complex IV showed a very mild decrease (Fig. 2B). Here, we noted that complex IV was not significantly compromised in this cell line before supplementation.

Although none of the alterations in the single complexes were statistically significant, almost all complexes/mitochondrial proteins showed some level of improvement after supplementation with L-cysteine, suggesting a beneficial effect in the translation of mtDNA encoded proteins. Taken together, our data support that L-cysteine improves the mitochondrial function in cells carrying the m.8344A>G and m.3243A>G mutations. Furthermore, it seems that the improvement is more prominent in cells with higher levels of heteroplasmy and a more obvious defect of mitochondrial translation. Some bands representing potentially abnormal subassembly intermediates in MELAS and MERRF cells became slightly weaker after supplementation with L-cysteine, but this was not quantifiable due to variable involvement of multiple bands. The multiple bands detected in Fig. 2 represent subcomplexes of complex V, as they appear on the BN-PAGE with complex V antibodies.

L-cysteine supplementation in patient fibroblasts with nuclear mutations caused no significant improvement of mitochondrial oxidative phosphorylation

To study whether the positive effect of L-cysteine is specific for MELAS and MERRF, we also analysed 3 fibroblast lines from patients carrying various nuclear defects of mitochondrial protein synthesis (MTO1, ELAC2, TRMU), and one cell line carrying a homozygous pathogenic mutation in COX10, a nuclear-encoded COX assembly gene (Table 1). As shown in Fig. 3A-3B, the oxygen consumption rate and basal respiration prior to supplementation in all four patient cell lines is not significantly lower, or higher than in controls, implying that the mutated fibroblasts do not have a very severe mitochondrial dysfunction, or that compensatory mechanisms enable a normal respiration. Supplementation with L-cysteine caused a slight to moderate decrease of basal respiration in all cell lines (Fig. 3B). Remarkably, the basal and maximal respiration in TRMU cells were significantly higher and fell back to similar levels as the control cell line after L-cysteine supplementation, raising the possibility that less active compensatory upregulation was needed after supplementation. Interestingly, the maximal respiration level in the MTO1 mutant cell line increased after L-cysteine treatment, which was statistically significant (p < 0.05), and the proton leak, which represents the flow of electrons across the inner mitochondrial membrane that produce heat and not ATP, decreased in all four patient fibroblast lines (but not in the controls). The increase in maximal respiration was statistically significant in cells with recessive mutations in COX10 (p < 0.05) and in ELAC2 (p < 0.05) (Fig. 3B).

BN-PAGE detected no improvement of respiratory chain complex levels in cells with nuclear mutations

Prior to supplementation, a significant reduction of complex I was observed in MTO1 deficient fibroblasts only (Fig. 4A), and complex IV was decreased in cells with the homozygous COX10 mutation (Fig. 4B). Other complexes were not significantly different compared to the control (Fig. 4A-4B). Supplementation with L-cysteine resulted in a slight, but not significant increase in complexes I, III and V in cells with MTO1 mutations, and an even milder increase of the same three complexes occurred in deficiency of ELAC2 (Fig. 4A-4B). Although a uniform decrease of complex IV was present in all patient cells and controls; this may be due to the sequestration of complex IV into supercomplex formation and subsequent disappearance of the monomeric form. Complexes I, II and IV have been shown to form large supercomplexes of different composition [22], and these supercomplexes might not have been well separated by the type of BN-PAGE gels used in this study. These data are correlated to the decreased basal respiration levels (Fig. 3A-3B).

N-acetyl-cysteine (NAC) resulted in a slightly higher mitochondrial oxidative phosphorylation only in fibroblasts with mutations in MTO1 and TRMU

We performed supplementation with 4 mM NAC in the same cell lines for 9 to 14 days as done before with L-cysteine. Supplementation of MERRF and MELAS cell lines did not result in the same effect as observed previously with L-cysteine (Fig. 5A-5B). Levels of heteroplasmy remained stable during the experiments in both MERRF and MELAS cells.

Surprisingly, however, supplementation with NAC improved mitochondrial function of both MTO1 and TRMU deficient cells as demonstrated by the elevated levels of basal and maximal respiration (Fig. 6A), although the results are not statistically significant. Differences in basal respiration for both MTO1 and TRMU deficient cells were statistically significant (p < 0.05 and p < 0.01, respectively). The difference in the maximal respiration was not significant for TRMU, but highly significant for MTO1 (p < 0.001). ELAC2 and COX10 deficient cell lines showed a significantly decreased basal and maximal respiration after treatment with NAC.

MtDNA copy numbers showed no major alteration after supplementation with L-cysteine or NAC

We analysed mtDNA copy numbers in all cell lines before and after L-cysteine and NAC supplementation, but no significant changes were observed. A slight increase was noted after L-cysteine treatment in MERRF, COX10 and MTO1 cells, no change was observed in ELAC2, and a decrease in TRMU (Fig. 7). MtDNA copy numbers tended to decrease after treatment with NAC in most cells except for COX10 and ELAC2 cells (Fig. 7).

DISCUSSION

Recent developments in genetic technology (next generation sequencing panels, whole exome sequencing, whole genome sequencing) have enabled the majority of patients with mitochondrial diseases to receive a confirmed molecular diagnosis [4]. However, effective therapies remain limited and they are only available for a few patients with very specific defects of the biosynthesis or transport of cofactors, vitamins and coenzyme Q₁₀ [3]. It is therefore of utmost importance to identify potentially beneficial substrates, which can be further investigated in clinical trials, although the clinical and genetic variability of mitochondrial diseases pose significant challenges to assemble large collectives with homogeneous patient groups. Recent developments in national and international registries, biomarkers and outcome measures development have improved trial readiness for patients with mitochondrial disease. One approach to search for a beneficial drug is high throughput screening with hundreds or thousands of substrates in cell lines or animal models (Drosophila, C. elegans, etc) [23]. Others (including us) have defined single pathways or molecular mechanisms, which can be specifically targeted. In this paper, we used a targeted approach and studied whether cysteine supplementation (L-cysteine or NAC) could result in benefit for patients suffering from abnormal mitochondrial protein synthesis caused by mutations in mt-tRNA genes or in nuclear mutations affecting mitochondrial translation.

L-cysteine is an amino acid that is needed for optimal cellular function and based on our previous results, it was effective in improving mitochondrial translation in myoblasts of patients with reversible infantile mitochondrial diseases in vitro.N-acetyl-cysteine (NAC) has been used to treat a variety of medical conditions due to its antioxidant effect [24]. NAC has been tested as a potential treatment in several neurological and psychiatric conditions and it seemed an excellent compound with a good safety profile [25]. A benefit of supplementation with N-acetyl-cysteine (NAC), a precursor of sulfide-buffering glutathione, was suggested in ethylmalonic encephalopathy [26]. Similarly, a whey-based cysteine donor resulted in significantly reduced oxidative stress in mitochondrial myopathies [26] and lower levels of reduced cysteine and thiols were detected in plasma of children with mitochondrial diseases [27]. NAC is a precursor of intracellular cysteine andglutathione, and it has an antioxidant effect by direct interaction with radicals and by stimulating cytosolic enzymes in glutathione metabolism [28]. It has been shown to attenuate oxidative stress, increase cell survival, reverse mitochondrial depolarisation and increase the activities of complexes I, IV and V in a variety of models and cell lines [28]. Similarly to our data, NAC showed encouraging results in a recent paper in fibroblasts carrying pathogenic mutations in the TRMU and TSFM genes [28].

Although NAC was originally administered in acetaminophen overdose and as a mucolytic, it has been used with variable effect in numerous clinical trials in a large variety of neurological and psychiatric diseases with distinct pathomechanisms including oxidative stress, apoptosis, mitochondrial dysfunction, neuroinflammation, glutamate and dopamine dysregulation [25]. Favourable results have been shown in autism, Alzheimer‘s disease, drug induced neuropathy and in some psychiatric diseases, whilst the evidence of its effectiveness for other disorders was less clear. Supplementation with NAC reverses abnormalities in diseased cells and in mouse models of Huntington‘s disease [29] where major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine has been shown to contribute to the pathomechanism [29]. Recently, it has been shown that orally administered NAC is able to reach the brain efficiently and restore neuronal glutathione levels in Parkinson‘s disease [30]. These findings support the feasibility of NAC as a potential disease-modifying factor in neurological disease and it has been clearly proven to be safe.

Cysteine plays a pivotal role in a number of important cellular processes, such as protein synthesis, oxidative stress response, iron-sulfur (Fe-S) cluster biogenesis and regulatory and structural changes in proteins. The transport of cysteine is important in regulating cellular cysteine biosynthesis as well as modulating the availability of sulfur for mitochondrial metabolism [31]. Experimental evidence suggests that cysteine can penetrate the lipid bilayer of the cell and can enter mitochondria through specific cellular cysteine uptake mechanisms [31]. Exogenously administered ³⁵S-cysteine accumulates in the mitochondrial fraction and is taken up into isolated mitochondria for intra-mitochondrial protein synthesis [31].

All tRNA molecules require a wide variety of posttranscriptional modifications which stabilize tRNA structure, enable efficient interaction with the ribosome, and fine-tune protein translation [32, 33, 32, 33]. After the update of the RNA modifications pathways database in 2008, 119 different posttranscriptional modifications in RNA were revealed [34, 35]. The bulk of these modifications were present in mt-tRNAs. The uridine at position 34 (U34), which is the 5’ position (wobble position) of the anticodon in the tRNAs Lys, Glu and Gln, is modified at carbons 2 and 5. Carbon 2 is modified exclusively through thiolation (s²), whereas carbon 5 can be methyl modified in various ways along different species. In the mammalian tRNAs there is evidence that the U34 mitochondrial tRNA taurinomethyl (τm⁵) modification contribute to the mitochondrial translation defect in association with the m.3243A>G mutation. The xm⁵s² modification offers rigidity to the U34 wobble base of tRNAs Lys, Glu and Gln [11]. Abnormal modification of the U34 has been also shown to contribute to the disease mechanism in patients with autosomal recessive mutations in the genes TRMU and MTO1. Although MTO1 does not require cysteine, it acts at the U34 wobble base of mt-tRNAs Lys, Glu and Gln and its mutations may indirectly alter thiolation. It has been shown that the steady-state level of mt-tRNAs was decreased in these diseases [36] resulting in a severe defect of mitochondrial translation [14]. The absence of post-transcriptional modifications at the wobble positions of mitochondrial tRNA^Leu and tRNA^Lys has been correlated to some common mt-tRNA mutations [16]. The m.3243A>G and m.3271T>C mutations in tRNA^Leu result in the lack of taurine modification at the wobble position, leading to reduced translation of UUG, but not of UUA codons [37]. The rationale to use cysteine in case of the m.3243A>G mutation is based on the fact, that cysteine is oxidised by cysteine dioxygenase to cysteine sulfonate, which is further metabolized to taurine and may affect taurinomethylation of mt-tRNA^Leu [38]. Furthermore, the pathogenic MERRF mutation m.8344A>G causes lack of both s2 and τm5 modifications in tRNA^Lys [39] and as a result none of the codons for lysine are decoded [39] (Fig. 8).

Based on these previous studies, we tested whether supplementation with cysteine can alter mt-tRNA modifications linked to U34 and may have an impact on mitochondrial translation. Here we show that supplementation with L-cysteine, but not with N-acetyl-cysteine partially rescues the mitochondrial translation defect in vitro in fibroblasts of patients carrying the m.3243A>G and m.8344A>G mutations. Although none of the alterations in the single complexes were statistically significant, almost all complexes showed some improvement after supplementation with L-cysteine, suggesting a beneficial effect in the translation of mtDNA encoded proteins. Furthermore, it seems that the improvement is more prominent in cells with higher levels of heteroplasmy and a more obvious defect of mitochondrial translation. In contrast, NAC, which is predicted to have a positive effect in various neurodegenerative diseases, did not improve mitochondrial translation in mt-tRNA mutations. However, NAC significantly improved mitochondrial oxidative phosphorylation in cells carrying mutations in MTO1 and TRMU. The observed variability after supplementation could be caused by differences in glutathione (GSH) homeostasis directly affecting the steady-state level of cellular GSH that cannot be accurately measured due to its extreme instability. Furthermore, we used high glucose levels in the medium, which might have been masking the mitochondrial defect and therefore the margin of change caused by L-cysteine and NAC was quite small [40].

In summary, our results show that selected groups of mitochondrial translation defects caused by altered modification of the wobble base U34 may be rescued at least partially by either L-cysteine or NAC supplementation (Fig. 8). Since these compounds are not toxic and pharmacokinetic studies confirmed that they reach the target tissues, our data provide a rationale to use cysteine or NAC in animal models and clinical trials in patients. These further studies are needed to explore the in vivo effects of cysteine supplementation and will clarify whether this can be a future therapy option for patients with abnormal mitochondrial translation.

CONFLICT OF INTEREST

The authors report no conflict of interest.

Footnotes

ACKNOWLEDGMENTS

RH is a Wellcome Trust Investigator (109915/Z/15/Z), who receives support from the Medical Research Council (UK) (MR/N025431/1), the European Research Council (309548), the Wellcome Trust Pathfinder Scheme (201064/Z/16/Z), the Newton Fund (UK/Turkey, MR/N027302/1) and the Mitochondrial European Educational Training (MEET) ITN MARIE CURIE PEOPLE (317433). PYWM is supported by a Clinician Scientist Fellowship Award (G1002570) from the Medical Research Council (UK), and also receives funding from Fight for Sight (UK), the UK National Institute of Health Research (NIHR) as part of the Rare Diseases Translational Research Collaboration, and the NIHR Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. We are grateful to the Medical Research Council (MRC) Centre for Neuromuscular Diseases Biobank Newcastle and for the EuroBioBank for supporting this project and for providing primary human cells.

Appendix

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Cysteine Supplementation May be Beneficial in a Subgroup of Mitochondrial Translation Deficiencies

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Primary cell cultures

Oxygen consumption rate

BN-PAGE

DNA purification and quantification of mitochondrial point mutations

Quantification of mitochondrial DNA copy number

Statistical analysis

RESULTS

L-cysteine supplementation in MELAS and MERRF resulted in slight increase in oxidative phosphorylation by Seahorse analysis

L-cysteine supplementation in MELAS and MERRF resulted in a slight increase of mitochondrial respiratory chain complex levels on BN-PAGE

L-cysteine supplementation in patient fibroblasts with nuclear mutations caused no significant improvement of mitochondrial oxidative phosphorylation

BN-PAGE detected no improvement of respiratory chain complex levels in cells with nuclear mutations

N-acetyl-cysteine (NAC) resulted in a slightly higher mitochondrial oxidative phosphorylation only in fibroblasts with mutations in MTO1 and TRMU

MtDNA copy numbers showed no major alteration after supplementation with L-cysteine or NAC

DISCUSSION

CONFLICT OF INTEREST

Footnotes

ACKNOWLEDGMENTS

Appendix

References