The autosomal recessive white matter disorder LBSL (leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation) is caused by mutations in DARS2, coding for mtAspRS (mitochondrial aspartyl-tRNA synthetase). Generally, patients are compound heterozygous for mutations in DARS2. Many different mutations have been identified in patients, including several missense mutations. In the present study, we have examined the effects of missense mutations found in LBSL patients on the expression, enzyme activity, localization and dimerization of mtAspRS, which is important for understanding the cellular defect underlying the pathogenesis of the disease. Nine different missense mutations were analysed and were shown to have various effects on mtAspRS properties. Several mutations have a direct effect on the catalytic activity of the enzyme; others have an effect on protein expression or dimerization. Most mutations have a clear impact on at least one of the properties of mtAspRS studied, probably resulting in a small contribution of the missense variants to the mitochondrial aspartylation activity in the cell.

INTRODUCTION

LBSL (leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation) is an autosomal recessive white matter disorder [1]. Patients with LBSL suffer from slowly progressive pyramidal, cerebellar and dorsal column dysfunction. Signs of the disease typically start during childhood or adolescence, but can even start during adulthood [2,3]. The diagnosis is based on specific MRI (magnetic resonance imaging) abnormalities in the cerebral and cerebellar white matter and specific white matter tracts in the brain stem and spinal cord. The disease is caused by mutations in the gene DARS2 [4]. DARS2 codes for mtAspRS (mitochondrial aspartyl-tRNA synthetase), which is a homodimeric enzyme. This ubiquitous protein plays an essential role in mitochondrial protein translation. mtAspRS is responsible for attaching the amino acid aspartate to the correct tRNA in mitochondria. The charged tRNAs are used for the synthesis of the 13 proteins encoded by the mitochondrial genome.

Since the discovery of DARS2 mutations as the cause of LBSL, mutations in several other mitochondrial aminoacyl-tRNA synthetase genes (i.e. AARS2, EARS2, FARS2, HARS2, MARS2, RARS2, SARS2 and YARS2) have been demonstrated to lead to a variety of diseases with no apparent commonalities [512]. In some cases, the mutations affect the specific tRNA synthetase activities and sometimes mitochondrial respiratory chain activity is reduced, but this is not apparent in all cases. Although the central nervous system is affected in some of these diseases, only mutations in EARS2 lead to a leukoencephalopathy [12], similar to the situation for mutations in DARS2 and LBSL.

LBSL patients are typically compound heterozygous, although homozygous DARS2 mutations have been described recently in a Japanese family [13] and a German patient [14]. Relatives that are carriers of a DARS2 mutation are unaffected, indicating that mutations do not exert a strong dominant effect and DARS2 mutations probably result in a loss of function of the encoded protein mtAspRS.

A variety of different mutations have been found so far. Almost all LBSL patients have a mutation in a polypyrimidine tract at the 3′-end of intron 2, which is found on one allele. These mutations affect correct splicing of the third exon, which leads to a frameshift and a premature stop. These mutation are ‘leaky’, leading to a decreased, but not zero, expression of full-length mtAspRS [15]. The mutation on the other allele varies: other splice site mutations, deletions, nonsense and several different missense mutations have been found. A number of these missense mutations were shown to affect the catalytic activity [4]. A good understanding of the effects of the missense mutations might reveal a possible correlation between the mutations and the severity of the disease.

Many different missense mutations in DARS2 have been found in LBSL patients. In the present study, we examined the effect of a selection of these missense mutations that are spread over the gene (Figure 1 and Supplementary Figure S1 at http://www.biochemj.org/bj/450/bj4500345add.htm) on expression, enzyme activity, localization and dimerization of the mutant protein.

Position of missense mutations in mtAspRS

Figure 1
Position of missense mutations in mtAspRS

Two-dimensional representations of mtAspRS. Various domains and motifs, as described in [17], are indicated. Motifs 1, 2 and 3 form the catalytic platform. The positions of the mutations under examination in the present study are indicated on top. AC-binding domain, anticodon-binding domain.

Figure 1
Position of missense mutations in mtAspRS

Two-dimensional representations of mtAspRS. Various domains and motifs, as described in [17], are indicated. Motifs 1, 2 and 3 form the catalytic platform. The positions of the mutations under examination in the present study are indicated on top. AC-binding domain, anticodon-binding domain.

MATERIALS AND METHODS

Constructs

For expression in human cultured cells, constructs were made consisting of the DARS2 coding sequence followed by a 10-amino-acid linker sequence and a FLAG (DYKDDDDK) epitope tag or the Gluc (Gaussia luciferase) coding sequence at the C-terminus of DARS2, generating FLAG-tagged mtAspRS and Gluc-tagged mtAspRS respectively. The secretion signal sequence at the N-terminus (amino acids 1–17) of Gluc was excluded from the constructs (a gift from Dr R. Estévez, Department of Physiological Sciences, University of Barcelona, Barcelona, Spain). For bacterially expressed recombinant proteins, the pEX14-mtAspRS-His6 plasmid was used {this plasmid corresponds to the one encoding mtAspRS(I41) in [16]}. The plasmid contains base pairs corresponding to amino acids 41–645 of the coding sequence of DARS2 (excluding the theoretical mitochondrial targeting sequence), followed by a histidine tag. Mutations were introduced by site-directed mutagenesis using the QuikChange® kit (Stratagene). The primers used for mutagenesis can be found in Supplementary Table S1 at http://www.biochemj.org/bj/450/bj4500345add.htm. All mutations were confirmed by sequencing.

Aminoacylation assays

Expression of recombinant proteins was induced with 200 μM IPTG (isopropyl β-D-thiogalactopyranoside) for 20 h at 18°C in Escherichia coli BL21(DE3) rosetta2 strains (Novagen). Purification steps were conducted as described in [17]. Aminoacylation assays were performed at 37°C as described in [4].

Cell culture and transfections

HEK (human embryonic kidney)-293T cells were cultured in DMEM (Dulbecco's modified Eagle's medium) (Invitrogen) with 10% (v/v) FBS (fetal bovine serum) in 5% CO2 at 37°C. Cells were transfected with PEI (polyethyleneimine) (Polysciences) at 50% confluence, with constructs expressing FLAG- and Gluc-tagged wild-type and mutant mtAspRS. Cell lysates were obtained 44 h after transfection by harvesting in lysis buffer (20 mM Hepes/KOH, pH 7.4, 100 mM KCl, 10% glycerol, 1% Triton X-100 and 0.1 mM EDTA) with protease inhibitor cocktail (Roche Applied Sciences). After lysis, nuclei and cell debris were removed by centrifugation.

Dimerization assay

HEK-293T cells were transfected with a 1:1 ratio of mutant or wild-type FLAG- and Gluc-tagged mtAspRS constructs. Transfected cells were harvested and lysed after 44 h. Luminescence in the cell extracts was measured using a luminescence reader (GENios Plus, Tecan) using the Dual-Luciferase Reporter Assay kit (Promega). Immunoprecipitation was then carried out using a mouse monoclonal anti-FLAG antibody (Sigma). Precipitated proteins were dissolved in Passive Lysis Buffer (Promega). Luminescence of the co-immunoprecipitated mtAspRS–Gluc fusion proteins was measured. Dimerization efficiency was calculated as the amount of Gluc activity after immunoprecipitation of FLAG- and Gluc-tagged complexes in comparison with the total Gluc activity in the cell lysates before immunoprecipitation. Dimerization obtained with wild-type proteins was set as 100% for each experiment.

Immunocytochemistry

At 44 h after transfection, cells were stained with 200 nM MitoTracker Red CMXRos (Invitrogen) for 20 min. Cells were fixed in 4% (w/v) paraformaldehyde and blocked in 5% (v/v) FBS. Cells were incubated with mouse anti-FLAG antibodies (1:1000 dilution; Sigma) overnight at 4°C. After staining with secondary antibodies (Alexa Fluor® 488-tagged anti-mouse IgG; 1:1000 dilution; Invitrogen), coverslips were mounted on object glasses with Vectashield Mounting Medium for Fluorescence with DAPI (4′,6-diamidino-2-phenylindole) (Vector Laboratories) and photographed using a Leica DM6000B microscope.

SDS/PAGE and Western blotting

Cell lysates were run on SDS/PAGE (10% gels) and blotted on to PVDF membranes (Millipore). Membranes were blocked in PBS-T (PBS containing 0.1% Tween 20) and 5% (w/v) non-fat dried skimmed milk powder. Proteins of interest were detected by incubating the membranes with the appropriate antibodies in PBS-T containing 0.5% non-fat dried skimmed milk powder for either 2 h at room temperature (21°C) or overnight at 4°C. Mouse anti-FLAG (1:5000 dilution) and mouse anti-β-actin (1:100000 dilution; Abcam) antibodies were used. After washing with PBS-T, alkaline phosphatase-conjugated secondary antibodies (1:5000 dilution; Sigma) were added for 1 h and the immunoreactive bands were detected using ECF (enhanced chemifluorescence) substrate (GE Healthcare) on a FLA-5000 image reader (Fujifilm). Western blots were quantified using the AIDA Image Analyzer software package.

RESULTS

Expression

To study the basic properties of mtAspRS proteins with pathogenic missense mutations, we transfected HEK-293T cells with plasmids containing FLAG-tagged wild-type or mutant mtAspRS. Western blotting of cell lysates indicates strongly reduced protein levels for the C152F, Q184K and D560V mutant proteins (Figure 2). Similar results were obtained with mtAspRS mutants tagged with GFP (green fluorescent protein) or HA (haemagglutinin), indicating that the differences in expression levels are not due to the FLAG tag (results not shown). In experiments where cells were treated with cycloheximide to inhibit production of newly synthesized proteins, we observed that the three variants with low expression levels had reduced stability in comparison with the wild-type protein (Supplementary Figure S2 at http://www.biochemj.org/bj/450/bj4500345add.htm). Whereas the wild-type protein was nearly completely stable over the treatment period, the levels of the three mutant variants were reduced by approximately 50%. It should be noted that, due to the very low expression levels of C152F, Q184K and D560V, the results of the cycloheximide experiments were variable and difficult to quantify.

Expression of mutant mtAspRS

Figure 2
Expression of mutant mtAspRS

FLAG-tagged wild-type (WT) and mutant mtAspRS were expressed in HEK-293T cells. Expression of the variants was studied by SDS/PAGE and Western blotting.

Figure 2
Expression of mutant mtAspRS

FLAG-tagged wild-type (WT) and mutant mtAspRS were expressed in HEK-293T cells. Expression of the variants was studied by SDS/PAGE and Western blotting.

Enzyme activity

The enzyme activity of different mutant forms of mtAspRS has been measured previously [4]. Since the previous study, it has been found that recombinant mtAspRS with an additional seven amino acids at the N-terminus has substantially increased expression, stability, solubility and activity [16]. We have therefore expressed mature wild-type and several mutant mtAspRS proteins, starting at amino acid 41, in E. coli. Similarly to the experiments in cultured human cells, we noticed a strongly reduced expression of the recombinant protein with the D560V mutation in the E. coli expression system. Purified enzymes were used for in vitro aminoacylation assays to measure their ability to aminoacylate tRNAAsp. The results are shown in Table 1. R263Q, L626Q and, to a lesser degree, D560V significantly decrease the catalytic activity of mtAspRS. R58G, T136S, Q184K and L613F do not affect enzyme activity.

Table 1
Aminoacylation activity of recombinant proteins

Enzymatic activities were measured at 37°C in the presence of an excess of in vitro transcribed E. coli tRNAAsp under experimental conditions as described in [4]. Experiments were carried out at least three times.

Protein Enzyme activity (nmol/mg per min) Fold decrease in specific activity 
Wild-type 256.9 – 
R58G 324.1 – 
T136S 278.5 – 
Q184K 343.7 – 
R263Q 1.9 135 
D560V 42.6 
L613F 205.4 – 
L626Q 6.0 43 
Protein Enzyme activity (nmol/mg per min) Fold decrease in specific activity 
Wild-type 256.9 – 
R58G 324.1 – 
T136S 278.5 – 
Q184K 343.7 – 
R263Q 1.9 135 
D560V 42.6 
L613F 205.4 – 
L626Q 6.0 43 

Localization

MtAspRS is encoded by the nuclear DARS2 gene. To perform its function, the protein is, after synthesis in the cytosol, targeted into mitochondria with the help of a mitochondria-targeting sequence. We studied whether mutations affected the localization of mutant mtAspRS to mitochondria using the FLAG-tagged proteins transfected in HEK-293T cells. Mitochondria were stained with MitoTracker (Invitrogen), and mtAspRS was detected using immunocytochemistry with antibodies against the FLAG tag. The images are shown in Figure 3 and Supplementary Figure S3 at http://www.biochemj.org/bj/450/bj4500345add.htm. All mutant proteins showed a normal mitochondrial localization, indicating that these mutants do not affect proper targeting to mitochondria.

Localization of mutant mtAspRS to mitochondria

Figure 3
Localization of mutant mtAspRS to mitochondria

HEK-293T cells were transfected with wild-type (WT) and mutant constructs of FLAG-tagged mtAspRS. Mitochondria were stained with MitoTracker and mtAspRS was detected with anti-FLAG antibodies. A part of a transfected cell is shown to illustrate the similar punctute staining of mtAspRS and mitochondria. Supplementary Figure S3 at http://www.biochemj.org/bj/450/bj4500345add.htm shows colour images from similar experiments. The settings for the images were optimized for each variant to demonstrate the localization of the proteins.

Figure 3
Localization of mutant mtAspRS to mitochondria

HEK-293T cells were transfected with wild-type (WT) and mutant constructs of FLAG-tagged mtAspRS. Mitochondria were stained with MitoTracker and mtAspRS was detected with anti-FLAG antibodies. A part of a transfected cell is shown to illustrate the similar punctute staining of mtAspRS and mitochondria. Supplementary Figure S3 at http://www.biochemj.org/bj/450/bj4500345add.htm shows colour images from similar experiments. The settings for the images were optimized for each variant to demonstrate the localization of the proteins.

Dimerization

MtAspRS functions as a dimer [17] and several mutations (C152F, Q184K, R263Q and L626Q/V) are located close to the dimerization interface (Supplementary Figures S1 and S4 at http://www.biochemj.org/bj/450/bj4500345add.htm). We therefore studied whether mutations affect dimerization of mtAspRS. We analysed both the ability of mutant proteins to dimerize with wild-type mtAspRS as well as the ability to form homodimers (with the same mutant protein). We studied this by expressing FLAG-tagged mtAspRS together with Gluc-tagged mtAspRS in transfected HEK-293T cells, followed by co-immunoprecipitation using antibodies against the FLAG tag and measurement of the Gluc activity (Figure 4A). It is important to note that, with these fusion proteins, we observed relatively similar levels of luciferase activity in the lysates of the cells transfected with different mutants (see the representative experiment in Supplementary Figure S5 at http://www.biochemj.org/bj/450/bj4500345add.htm). Apparently, the expression levels of the Gluc-tagged proteins were less affected by the different mutations than previously seen for FLAG-, HA- or GFP-tagged variants. The combined result of five independent dimerization experiments are presented in Figures 4(B) and 4(C).

Dimerization is affected by missense mutations

Figure 4
Dimerization is affected by missense mutations

(A) Schematic overview of the experiment. Constructs of FLAG- and Gluc-tagged wild type and mutant mtAspRS were expressed in HEK-293T cells. The effects of mutations on dimerization were determined by immunoprecipitation of Gluc-fusion proteins that were bound to their FLAG-tagged counterparts. For simplicity, other possible dimers (containing two Gluc- or FLAG-tagged proteins) are omitted. Relative dimerization efficiencies were calculated as described in the Materials and methods section. The asterisk (*) indicates the mutant variant of mtAspRS. (B) Dimerization efficiency relative to wild-type (WT) protein. (C) Relative dimerization efficiency with mtAspRS harbouring the same mutation or another mutation (in the case of R58G+T136S and L613F+L626Q). Results in (B) and (C) are means±S.D. for five independent experiments.

Figure 4
Dimerization is affected by missense mutations

(A) Schematic overview of the experiment. Constructs of FLAG- and Gluc-tagged wild type and mutant mtAspRS were expressed in HEK-293T cells. The effects of mutations on dimerization were determined by immunoprecipitation of Gluc-fusion proteins that were bound to their FLAG-tagged counterparts. For simplicity, other possible dimers (containing two Gluc- or FLAG-tagged proteins) are omitted. Relative dimerization efficiencies were calculated as described in the Materials and methods section. The asterisk (*) indicates the mutant variant of mtAspRS. (B) Dimerization efficiency relative to wild-type (WT) protein. (C) Relative dimerization efficiency with mtAspRS harbouring the same mutation or another mutation (in the case of R58G+T136S and L613F+L626Q). Results in (B) and (C) are means±S.D. for five independent experiments.

The C152F, Q184K, R263Q and D560V mutations significantly decrease dimerization with the wild-type enzyme (Figure 4B). The same variants and R58G, T136S and L626Q decrease dimerization with the mutant enzyme (Figure 4C). Furthermore, we tested the two combinations of missense mutants that have been found in LBSL patients: R58G with T136S and L613F with L626Q (Supplementary Figure S6 at http://www.biochemj.org/bj/450/bj4500345add.htm). The ability to dimerize was reduced when R58G was combined with T136S, but to a similar degree as both mutations individually. Dimerization of L613F with L626Q was not impaired.

DISCUSSION

The aim of the present study was to examine the effects of missense mutations found in LBSL patients on the expression, enzyme activity, localization and dimerization of mtAspRS. The outcome of the different studies is summarized in Table 2.

Table 2
Effects of missense mutations in mtAspRS

+, similar to wild-type; −, reduced compared with wild-type; −−, strongly reduced compared with wild-type; ND, not determined.

Mutation    Dimerization 
DNA Protein Activity Expression Localization Wild-type Mutant Comment 
172C>G R58G − Compound heterozygous with T136S 
406A>T T136S − Compound heterozygous with R58G 
455G>T C152F ND − − − Compound heterozygous with intron 2 mutation 
550C>A Q184K − − −− Compound heterozygous with intron 2 mutation 
788G>A R263Q −− − − Compound heterozygous with intron 2 mutation 
1679A>T D560V − −− −− −− Compound heterozygous with intron 2 mutation 
1837C>T L613F Compound heterozygous with L626Q 
1877T>A L626Q −− − Compound heterozygous with L613F 
1876C>G L626V ND Compound heterozygous with intron 2 mutation 
Mutation    Dimerization 
DNA Protein Activity Expression Localization Wild-type Mutant Comment 
172C>G R58G − Compound heterozygous with T136S 
406A>T T136S − Compound heterozygous with R58G 
455G>T C152F ND − − − Compound heterozygous with intron 2 mutation 
550C>A Q184K − − −− Compound heterozygous with intron 2 mutation 
788G>A R263Q −− − − Compound heterozygous with intron 2 mutation 
1679A>T D560V − −− −− −− Compound heterozygous with intron 2 mutation 
1837C>T L613F Compound heterozygous with L626Q 
1877T>A L626Q −− − Compound heterozygous with L613F 
1876C>G L626V ND Compound heterozygous with intron 2 mutation 

Three mutations strongly reduced expression of mtAspRS and, in particular, the enzyme harbouring the D560V mutation was almost undetectable. These mutants also showed a decreased stability in the presence of cycloheximide, which could explain the decreased steady-state expression level.

We studied the enzyme activity of several mutant mtAspRS forms in aminoacylation assays, including some variants that were analysed previously [4]. It should be noted that the activity of the wild-type enzyme was approximately 10-fold higher than measured previously. The difference is most likely to be due to the presence of seven additional N-terminal amino acids in the recombinant proteins, demonstrated to increase the stability of the enzyme [16]. Again, we found strongly reduced activity of R263Q, which affects an amino acid located in the catalytic core of the protein, close to the highly conserved catalytic residue Arg266. L626Q also reduces catalytic activity, whereas D560V leads to a moderately reduced activity. In contrast with the previous study [4], we did not find a reduction in the activity of Q184K. The reconsideration of the N-terminus of the recombinant protein [16] might account for this.

It has been shown that the pathogenic mutation S45G, which affects a residue located near the cleavage site, alters correct import of the mutant protein into mitochondria [18]. None of the mutations tested in the present study affect residues located in the mitochondrial targeting sequence or near the cleavage site. As expected, all of the mutant proteins co-localize with a mitochondrial marker.

mtAspRS functions as a homodimer and some of the mutations studied are of residues on or near the predicted dimerization interface. We therefore studied whether the mutations affect dimerization. Most patients are compound heterozygous; almost all patients have a mutation in intron 2 of DARS2. This mutation is leaky, meaning that some wild-type full-length mtAspRS can still be formed from this allele. So, in cells of patients with one missense mutation, three possible dimers can be formed, i.e. wild-type–wild-type, wild-type–mutant and mutant–mutant. It is therefore interesting to see what the effect of missense mutations is on dimerization with the wild-type enzyme, as well as the effect on dimerization with the same mutant enzyme. Some of the missense mutations retain the ability to form dimers with the wild-type protein. If the missense variant can exert a dominant-negative effect on the wild-type part of the dimer, this will lead to a dysfunctional dimer. The only mtAspRS activity in a cell will then be provided by dimers that consist of two wild-type proteins, but the level of such dimers will be low because of the aberrant splicing of intron 2/exon 3. Furthermore, dimerization between mutant proteins is affected in several cases. In general, we see that mutants that have a decreased ability to form dimers also have a decreased expression. A possible explanation is that monomers are less stable, leading to a decreased amount of protein in the cells. Pathogenic mutations in other aminoacyl-tRNA synthetases have also been shown to affect dimerization. A study on mutations in glycyl-tRNA synthetase that cause Charcot–Marie–Tooth disease showed that many of these mutations either increase or decrease dimer formation [19]. We did not identify any mutations that increase the dimerization of mtAspRS.

There are only a few LBSL patients that are compound heterozygous for two missense mutations. R58G and T136S occur together in two patients from one family, and the L613F and L626Q mutations are present in another patient. For these mutations, we tested whether they affect dimerization with each other. Formation of R58G–R58G and T136S–T136S homodimers or R58G–T136S heterodimers is strongly decreased in all three cases. Produced as recombinant proteins in E. coli, homodimers of either mutant has normal aminoacylation activity and none of the other aspects that we studied was affected. Both amino acids reside in the anticodon-binding domain, although they are not predicted to interact directly with the anticodon of the tRNA. It is therefore still not completely understood how these mutations lead to LBSL.

An alternative effect of one of these two mutations could be a potential effect on the mRNA level. Prediction software on RNA splicing [20] suggests alternative folding of the pre-mRNA containing the 406A>T (corresponding to p.T136S) mutation. Owing to this alternative folding, the start of exon 5 would be in a closed conformation, making it less accessible for the spliceosome. Sequencing of cDNA from the patient's lymphoblasts carrying this mutation, however, did not reveal alternative splice products. We cannot fully exclude that the effect of 406A>T on splicing would be more pronounced in other cell types, which is the case for the common splice-site mutation in LBSL patients [15]. Yet another hypothesis is that the mutations affect the partial unfolding and refolding that these proteins undergo during the import into mitochondria. Such an effect has been shown for single nucleotide polymorphisms in mitochondrial phenylalanyl-tRNA synthetase and in mitochondrial leucyl-tRNA synthetase [21].

In the case of L613F and L626Q, it is interesting to note that L626Q decreases homodimerization, but does not decrease dimerization with the L613F mutant protein. In the patient's cells where both variants are present, a relatively large part of the dimers are expected to be dimers consisting of L613F and L626Q. Such dimers may have reduced catalytic activity because of the low activity of the L626Q mutant. Although L613F affects a highly conserved residue, we could not find any effect of this mutation on the expression, activity, localization and dimerization of mtAspRS with this single amino acid change. Altogether, it becomes evident that most missense mutations have a clear impact on at least one of the properties of mtAspRS studied. Mutations either decrease the catalytic activity (directly or through reduced dimerization) or substantially decrease the expression of the protein. The eventual outcome is a reduced aspartylation activity of proteins with missense mutations. Mitochondrial protein synthesis therefore probably relies on the reduced amount of wild-type protein that is expressed from the other allele with a typical splice site mutation in intron 2.

There does not seem to be an evident genotype–phenotype correlation for LBSL and the data from the present study did not reveal a correlation between the manner in which mtAspRS is affected by missense mutations and disease severity. For example, the disease course in patients with mtAspRS-R263Q or -D560V is relatively mild, despite the substantial effects on the catalytic activity or expression level respectively. It should be noted that only a selection of the LBSL mutations was studied, and the number of patients with the same mutation is very small, so determining whether differences between the effects of different missense mutations exist will be challenging.

Our data indicate that it will be difficult to develop a common therapy that aims at restoring the activity of mtAspRS with missense mutations. Probably, several different treatment options must be explored in order to counteract the variety of different effects that the amino acid changes exert on mtAspRS. Efforts to find a therapeutic intervention that would increase correct splicing of exon 3 are more likely to be successful, with the major advantage that almost all LBSL patients would benefit from such a treatment.

Abbreviations

     
  • FBS

    fetal bovine serum

  •  
  • GFP

    green fluorescent protein

  •  
  • Gluc

    Gaussia luciferase

  •  
  • HA

    haemagglutinin

  •  
  • HEK

    human embryonic kidney

  •  
  • LBSL

    leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation

  •  
  • mtAspRS

    mitochondrial aspartyl-tRNA synthetase

AUTHOR CONTRIBUTION

Laura van Berge was involved in the design of the study, performed the experiments and wrote the paper. Josta Kevenaar, Emiel Polder and Agnès Gaudry were involved in performing several experiments. Marie Sissler participated in the experimental design and data analysis and contributed to the critical revision of the paper. Catherine Florentz contributed to the interpretation of the experimental results and critical revision of the paper. Marjo van der Knaap contributed to conceptualization of the study and critical revision of the paper. Gert Scheper designed and supervised the study and contributed to writing of the paper.

FUNDING

This study was supported by the Prinses Beatrix Fonds [grant number WAR07/31], the Centre National de la Recherche Scientifique, Agence Nationale de la Recherche [grant number ANR-09-BLAN-0091-01/03], and partially supported by the French National Program Investissements d’Avenir (Labex MitoCross).

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Supplementary data