WO2018104172A1

WO2018104172A1 - Compounds for treating barth syndrome

Info

Publication number: WO2018104172A1
Application number: PCT/EP2017/081199
Authority: WO
Inventors: Déborah TRIBOUILLARD-TANVIER; Jean-Paul LASSERRE; Jean-Paul Di Rago
Original assignee: Centre National de la Recherche Scientifique CNRS; Association Francaise Contre les Myopathies; Universite de Bordeaux
Current assignee: Centre National de la Recherche Scientifique CNRS; Association Francaise Contre les Myopathies; Universite de Bordeaux
Priority date: 2016-12-06
Filing date: 2017-12-01
Publication date: 2018-06-14
Anticipated expiration: 2019-06-06

Abstract

The present invention relates to compounds and method for treating and/or preventing Barth syndrome as well as methods for identifying such compounds. In particular, the invention concerns compounds that inhibit the cytosolic protein synthesis for its use in the prevention and/or treatment of a disorder or disease associated to mitochondrial dysfunction such as Barth syndrome, wherein the mitochondrial dysfunction is associated with a deficiency in mature cardiolipin level in mitochondria membrane.

Description

COMPOUNDS FOR TREATING BARTH SYNDROME

The present invention relates to compounds and method for treating and/or preventing Barth syndrome as well as methods for identifying such compounds. Barth Syndrome (BTHS) is an X-linked recessive disorder that is characterized by cardiac and skeletal myopathies, growth retardation, hypocholesterolemia, increased urine levels of 3-methylglutaconic acid, increased susceptibility to bacterial infections due to cyclic neutropenia, and abnormal mitochondria and respiratory chain dysfunction. Consequences of Barth syndrome include cardiomyopathy, neutropenia, skeletal myopathy, and growth delay.

Mitochondria of BTHS patients have an abnormal ultrastructure and are characterized by pleiotropic respiratory defects in muscle, fibroblasts, and iPSCs- derived cardiomyocytes. The Barth syndrome is very often fatal in childhood due to the cardiac failure or sepsis and there is still no effective treatment. Moreover, a reduced level of mature cardiolipin (CL), and an accumulation of monolysocardiolipin (MLCL), an intermediate in the CL remodeling pathway has also been shown.

Cardiolipin (CL) is a lipid mostly found in the inner mitochondrial membrane (IMM) of eukaryotic cells. CL has been shown to play a role in numerous processes involving the mitochondria as oxidative phosphorylation and stabilization of supercomplexes, fusion, fission, protein import, iron-sulfur biogenesis, mitophagy, apoptosis and transport of metabolites for energy metabolism. CL plays a critical role in maintaining respiratory chain enzyme function, membrane stability, and mitochondrial dynamics. CL is synthesized exclusively in the mitochondria inner membrane as immature CL and is further remodeled into mature CL. Functional mature CL corresponds to cardiolipin with unsaturated fatty acyl chains, particularly four unsaturated fatty acyl chains, particularly C18:2 chains. The resulting combination of deficiency in mature CL and accumulation of monolysocardiolipin (MLCL) is believed to cause mitochondrial dysfunction and, consequentially, cardioskeletal myopathy, neutropenia, and growth delay. Detecting an increased monolysocardiolipin (MLCL) /mature cardiolipin (CL) ratio in blood, tissue or fibroblasts is a preferred diagnosis. The tafazzin protein, an acyl transferase located in the mitochondria, which is encoded by the nuclear TAZ gene, is located in the distal region of chromosome Xq28.12 and is crucial for the final remodelling and maturation of CL. Mutations in the TAZ gene have been shown to be responsible for Barth Syndrome.

Barth Syndrome is considered as a rare disease with less than 500 patients diagnosed in the world. It is very likely that this disease is highly under-diagnosed, affecting about 1 out of 300 000 children. Thanks to early diagnosis, which allows management of the various symptoms, the prognosis is strongly improved. However, in the past, children only rarely reached the age of three and often died of heart failure or infection. There is currently no definitive treatment specific to Barth syndrome and the treatment is essentially symptomatic and multidisciplinary.

Heart failure can be for instance treated by conventional treatments or by heart transplantation. The risk of bacterial sepsis in case of intermittent neutropenia can be controlled by prophylactic antibiotic therapy or intermittent administration of granulocytes growth factor (G - CSF). A treatment that could improve all components of the disease is expected.

Currently, to the applicants knowledge, one single product is in the clinical development phase, which consists in a small molecule developed by Lypro Bioscience, and is intended to replace deficient cardiolipin in mitochondria.

EP2012800 discloses compositions comprising ketoconazole 2S, 4R enantiomer, said pharmaceutical compositions being used to increase the synthesis of cardiolipin for the treatment of Barth syndrome, diabetic myopathy and/or cardiomyopathy associated with aging, as well as mitochondrial diseases and other ailments.

WO2014134554 concerns methods of prevention or treatment of Barth syndrome in a mammal using a cationic peptide aromatic (D-Arg-2'6'-Dmt-Lys-Phe-NH2), so as to increase the expression of TAZ in patients in need thereof. It is thus an object of the present invention to provide means and methods for the prevention and/or treatment of Barth syndrome.

The present invention concerns, in a first embodiment, a compound that inhibits the cytosolic protein synthesis for its use in the prevention and/or treatment of a disorder or disease associated to mitochondrial dysfunction, wherein the mitochondrial dysfunction is associated with a deficiency in mature cardiolipin level in mitochondria membrane.

According to the invention, the expression "disorder or disease associated to mitochondrial dysfunction" refers to disorders or diseases resulting from failures or dysfunction of the mitochondria, leading to less and less energy being generated within the cell. If this process is repeated throughout the body, whole systems begin to fail, and the life of the person in whom this is happening is severely compromised. The disease primarily affects children, but adult onset is becoming more and more common. Mitochondrial diseases or mitochondrial disorders may be the result of either inherited or spontaneous mutations in mtDNA or nDNA (nuclear DNA) which lead to altered functions of the proteins or RNA molecules that normally reside or play a role in mitochondria.

One can refer to the expression "disorder or disease associated to mitochondrial dysfunction" or to "mitochondrial disease" or "mitochondrial disorder".

Mitochondrial diseases appear to cause the most damage to cells of the brain, heart, liver, skeletal muscles, kidneys, eyes and endocrine and respiratory systems.

Depending on which cells are affected, symptoms may include loss of motor control, muscle weakness and pain, gastro-intestinal disorders and swallowing difficulties, growth delay, cardiac disease, liver disease, diabetes, respiratory complications, seizures, visual/hearing problems, lactic acidosis, developmental delays and susceptibility to infection.

The expression "prevention and/or treatment" as used in the present description refers to situations where one can prevent or slow down the occurrence of the disorder or of the disease or prevent or slow down the development of the disorder or of the disease or even can treat, i.e. revert, the disorder or the disease. The expression "prevention and/or treatment" may also include alleviation of at least one of the symptoms of the disorder or disease.

The expression "mature cardiolipin" refers to cardiolipin with unsaturated fatty acyl chains, particularly four unsaturated fatty acyl chains, particularly diunsaturated fatty acyl chains, more particularly C18:2 chain. Mature cardiolipin thus particularly corresponds to linoleoyl-cardiolipin (TLCL), i.e. cardiolipin in which the majority, particularly all four, phosphatidyl acyl groups are specified as linoleoyi.

The expression "deficiency in mature cardiolipin level" means that the ratio monolysocardiolipin (MLCL) /cardiolipin (CL) in the cell, particularly in white blood cell for a mammal, is comprised between 5 and 15

The method of direct analysis of cardiolipin and its derived lyso-compounds in intact blood white cells can represent a tool useful for the diagnosis, screening and clinical monitoring of mitochondrial diseases and disorders, such as Barth syndrome. Such a ratio can be used to identify deficiency in mature cardiolipin level and such a measurement can be made according to any appropriate method described in the literature such as in J Lipid Res. 2015 Sep;56(9):1787-94 or The FASEB Journal vol. 29 no. 1 Supplement 885.27 for example.

In a second embodiment, the invention concerns a compound for its use according the first embodiment, characterized in that the deficiency in mature cardiolipin level in mitochondria membrane is associated with a TAZ gene mutation.

Inherited mutations in the tafazzin {TAZ) gene are reported to be associated with Barth syndrome. Studies have revealed that TAZ gene encodes a phospholipid transacylase that is localized in mitochondria and functions in cardiolipin acyl chain remodeling and maturation. While cardiac tissue usually comprises > 90 % tetra- linoleoyl cardiolipin (CL), ie mature cardiolipin, subjects with Barth syndrome show characteristic differences in cardiolipin acyl chain composition, including decreased levels of mature cardiolipin.

The disorders or diseases associated to mitochondrial dysfunction that can be treated with the compound of the invention are thus preferably characterized by a mutation of TAZ gene. In such an embodiment, as a consequence, the protein encoded by mutated TAZ gene, a phospholipid transacylase, is missing and/or nonfunctional or mislocalized in cells.

By the term "missing" it is meant that the protein is not synthetized/translated or prone to degradation and thus degraded by the cellular machinery prior to performing its function.

By the term "non-functional" it is meant that the protein exists but is not able to perform its function, for example because of misfolding, unfolding, editing. A nonfunctional protein can also be degraded by the proteolysis machinery of the cell In a third embodiment, the invention concerns a compound for its use according to the second embodiment, characterized in that the mutation corresponds to an under- expression or extinction of the TAZ gene.

Such underexpression or extinction of the TAZ gene leads to a protein, a phospholipid transacylase, which is non-functional or missing. In a fourth and preferred embodiment, the invention concerns a compound for its use according to any preceding embodiments, characterized in that the disorder or disease is Barth syndrome.

In a fifth embodiment, the invention relates to a compound for its use according to any preceding embodiment, characterized in that the disorder or disease comprises one or more of the following pathologies : cardiomyopathy, skeletal muscle abnormalities, neutropenia, growth delay, weak muscle tone and bacterial infections.

The expression "cardiomyopathy" is to be understood as a weak heart muscle usually associated with enlargement of the heart (usually dilated with variable myocardial hypertrophy, sometimes with left ventricular noncompaction and/or endocardial fibroelastosis).

The expression "neutropenia" means a reduction in neutrophils, a type of white blood cells that is most important for fighting bacterial infections. Neutropenia may predispose an individual to mouth ulcers, fevers and bacterial infections such as bacterial pneumonia and skin abscesses.

The expression "underdeveloped skeletal musculature and muscle weakness" means that all muscles, including the heart, suffer from a cellular deficiency which limits their ability to produce energy. Muscle weakness and increased exertional fatigue are characteristic findings in Barth syndrome.

The expression "Growth delay" means that during childhood most affected individuals are below-average in height and/or weight.

In a preferred embodiment, the invention concerns a compound for its use in the treatment of Barth syndrome, wherein such compound inhibits the cytosolic protein synthesis.

In a preferred embodiment, the invention concerns a compound for its use in the treatment of Barth syndrome, wherein the compound inhibiting the cytosolic protein synthesis is selected in the group consisting of cycloheximide, emetin, and anisomycin ; or any pharmaceutically acceptable salt or prodrug thereof.

In the present invention, the term "prodrug" relates to a compound that, after administration, is metabolized (i.e., converted within the body) into a compound that inhibits cytosolic protein synthesis.

In the present invention, the term "pharmaceutically acceptable salt" relates to any pharmaceutically acceptable salt that can be prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. The pharmaceutical salts include any salt suitable to be administered to humans or animals. Examples of non-toxic pharmaceutically acceptable salts may include hydrochloride, carboxylate sodium, carboxylate disodium, sodium, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, citric acid, maleic acid, and succinic acid salts.

In a sixth embodiment, the invention deals with a compound for its use according to any preceding embodiment, characterized in that the compound is selected in the group consisting of cycloheximide, emetin, and anisomycin ; or any pharmaceutically acceptable salt or prodrug thereof. In one embodiment, the compound for its use according to the invention is selected in the group of compounds inhibiting cytosolic protein synthesis, i.e. cytosolic translation inhibitors. Suitable compounds may act at different levels of protein synthesis, and can inhibit any stage chosen among initiation, translocation, elongation and termination. As exposed above, some particular compounds are cycloheximide, emetin, and anisomycin.

Other antibiotics compounds capable of inhibiting cytosolic protein synthesis can be selected as suitable compounds according to the invention and one can cite various antibiotic inhibitors of 40S ribosome and/or 60S ribosome such as tubulosine, tylophorine, abrine, ricine or sarcine alpha for example.

However, the skilled person may easily find alternative compounds to the one listed above, that fulfill the requirement of inhibiting cytosolic protein synthesis and being usable in the treatment or prevention of disorders or diseases associated to mitochondrial dysfunction, in particular disorders or diseases characterized by a mutation of TAZ gene, and even more particularly Barth syndrome.

The functionality of inhibition of cytosolic protein synthesis is a routine measurement that can easily be put into practice for screening and identifying compounds according to the invention. The present description as well as abundant prior art describe usual techniques available for evaluation of protein homeostasis, thus for measuring cytosolic protein synthesis, including measuring the inhibition of cytosolic protein synthesis.

It is well described herein, including in the experimental part of the present description, that it is indeed the inhibition of cytosolic protein synthesis that corresponds to the essential feature of the compound of the invention. Indeed, it is proven that the selected exemplified compounds herein tested do have the ability to partially inhibit cytosolic protein synthesis and to reinstate the mitochondrial functions. However, other compounds having this property of inhibiting cytosolic protein synthesis can clearly, plausibly and reasonably be considered as compounds according to the invention. The expression "inhibits cytosolic protein synthesis" or "inhibition of cytosolic protein synthesis" is to be understood that the compound of the invention is able to affect protein homeostasis in the cytosol to such extent that less proteins are produced in the cytosol of the cell as a result of effect at various stage of protein synthesis, be it inhibition of the transcription or inhibition of the translation, at any stage, since interaction of a large number of factors that catalyze the assembly of ribosomes, mRNA templates, and aminoacylated tRNAs may play a role in protein synthesis.

The present description shows that the functional activity defined as the present invention is the essential feature of the invention whatever the structure of the compound which can be selected in a wide variety of chemical, , particularly among antibiotics that are inhibitors of eukaryotic cytosolic protein synthesis. Compounds with that functionality of inhibiting protein synthesis are part of the art and that functionality has clear technical meaning. Furthermore, this functionality can be measured/verified by tests and procedures well known or described in the present description and in any case without undue burden.

Various compounds capable of inhibiting cytosolic protein synthesis can be tested and used according to the invention. Cytosolic protein synthesis inhibitors and methods to evaluate those are described in RNA. 2004 Mar; 10(3): 528-543 (Eukaryotic protein synthesis inhibitors identified by comparison of cytotoxicity profiles), which one skilled in the art can use for further identification of alternative suitable compounds, particularly antibiotics, beside the specific ones described and disclosed herein.

In a further and seventh embodiment, the invention concerns a method, preferably an in vitro method for selecting a compound capable of inhibiting the cytosolic protein synthesis and restoring respiratory function of the mitochondria comprising the steps of :

- i) Providing an organism wherein an homolog of human TAZ gene is mutated and mature cardiolipin level in mitochondria membrane is deficient,

- ii) Contacting a compound to be screened with the organism of step i), - iii) Measuring the inhibition of cytosolic protein synthesis within the organism of step i), induced by the compound, - iv) Selecting the compound when the cytosolic protein synthesis is inhibited in comparison with an identical organism not contacted with the compound.

As described above, the deficiency of mature cardiolipin level corresponds to a ratio monolysocardiolipin (MLCL) /cardiolipin (CL) in the cell, comprised between 5 and 15.

In an eighth embodiment, the method, preferably the in vitro method, of the invention according to the seventh embodiment is characterized in that the cytosolic protein synthesis inhibition is partial.

In a ninth embodiment, the method, preferably the in vitro method, of the invention according to the eighth embodiment is characterized in that the cytosolic protein synthesis inhibition is comprised between 0.5 and 10%, particularly 0.5 and 5%, more particularly 0.5 and 2%. By "inhibition" it is understood that the protein synthesis is decreased by 0.5 to 10%, particularly by 0.5 to 5%, more particularly by 0.5 to 2%

It is noticeable that it is not necessary that the compound completely or almost completely inhibits the cytosolic protein synthesis (which would be lethal) and a slight inhibition appears to be sufficient for a compound to qualify as restoring respiratory function of the mitochondria.

In a tenth embodiment, the present invention also provides a method, preferably an in vitro, method according to previous seventh to ninth embodiments, characterized in that the cytosolic protein synthesis inhibition rescues the respiratory growth of TAZ mutated organism, wherein the TAZ mutated organism is a TAZ1 mutated yeast. In a eleventh embodiment, the present invention also provides a method, preferably an in vitro method, according to previous seventh to ninth embodiments, characterized in that partial cytosolic protein synthesis inhibition allows maintenance of the mitochondrial genome in the TAZ mutated organism.

In a twelfth embodiment, the present invention also provides a method, preferably an in vitro method, according to previous seventh to ninth embodiments, characterized in that partial cytosolic protein synthesis inhibition rescues the ATP production and mitochondrial membrane potential in the TAZ mutated organism.

In a thirteenth embodiment, the present invention also provides a method, preferably an in vitro method, according to seventh to ninth embodiments, characterized in that partial cytosolic protein synthesis inhibition rescues the OXPHOS assembly in the TAZ mutated organism.

In a further fourteenth embodiment, the invention provides a method, preferably an in vitro method, according to any one of seventh to thirteenth embodiments, characterized in that the organism wherein the TAZ gene is mutated is a TAZ1 mutated yeast and the mutation consists in a deletion, a substitution or an insertion which leads to a null allele. A null allele is herein understood as a mutant copy of a gene at a given locus that completely lacks the gene's normal function. This can be the result of the complete absence of the gene product (protein, RNA) at the molecular level, or the expression of a non-functional gene product. The step of contacting the compound with the organism can be achieved by any technology known to the skilled person provided that the compound to be tested enters the inside of the cell(s) of the organism in order it can exert its effect.

In a further aspect, the invention also relates to the use of an organism deficient in mature cardiolipin for the screening and selection of compounds capable of inhibiting the cytosolic protein synthesis and restoring respiratory mitochondrial function.

In one embodiment, the present invention also provides a method for treating Barth Syndrome in a mammalian subject, the method comprising: administering to the subject a therapeutically effective amount of a compound that partially inhibits the cytosolic protein synthesis and induces at least one effect selected in the group consisting of : rescue of the ATP production and mitochondrial membrane potential ; maintenance of the mitochondrial genome ; rescue of the OXPHOS assembly; rescue the respiratory rate and decrease the reactive oxygen species (ROS) production.

Legend of the Figures Figure 1. Cycloheximide (CHX) and anisomycin (ANI) suppress the respiratory growth defect of the tazl mutant strain. A. tazl mutant strain was spread onto respiratory ethanol-based media. Small sterile filters were placed on the agar surface and increasing concentrations of CHX and ANI were added to the filters at the indicated quantities (in nmoles). The plates were then incubated at 36°C and scanned after 5 days. B. Growth curve in liquid respiratory medium w/o drugs. The indicated cultures were inoculated with precultures in respiratory media at restrictive temperature diluted to 0.5OD/ml_ in 50ml_ of complete synthetic medium CSM + 0.5% galactose + 2% ethanol in presence of the indicated concentration of DMSO (vehicule, negative controle) or cycloheximide. Cultures were diluted every day in fresh media and drugs were added in the same time. Optical densities were taken at different time over a week. C. Serial dilutions of the indicated strains were spotted onto ethanol and glucose media. The plates were incubated at the indicated temperature and scanned after three (glucose) or six (ethanol) days. D. Growth curve in liquid respiratory medium. The indicated cultures were inoculated with precultures in respiratory media at restrictive temperature diluted to 0.5OD/ml_ in 50ml_ of complete synthetic medium CSM + 0.5% galactose + 2% ethanol. Optical densities were taken at different time over a week. E. Left panel. Cell growth on fermentable solid media. The indicated strains were streaked on solid fermentable media and plates were scanned after 1 week incubation at permissive temperature (28°C). Right panel. The percentage of petite colony in glucose media at 28°C was calculated in the indicated strains.

Figure 2. Mitochondrial membrane potential. Energization of the mitochondrial inner membrane. Variations in ΔΨ were monitored by the fluorescence quenching of rhodamine 123 as described in the material and methods section. 75 g of osmotically-protected mitochondria of the indicated strains were used in these experiments. (Mito: mitochondria; EtOH: ethanol; KCN: potassium cyanide; ADP: adenosine diphosphate; ATP: adenosine triphosphate; CCCP: carbonylcyanide-m- chlorophenyl hydrazone; Oligo: oligomycin). Fluorescence traces are representative of four experimental trials. Figure 3. Oxidative phosphorylation in tazIA mitochondria. A. Respiration rates. Mitochondria were extracted from indicated yeast strains cultured in CSM + 0.5% galactose + 2% ethanol at restrictive temperature (36°C) between 2 OD and 3 OD. 75 g of mitochondria were used and respiration rates were measured with a Clark- type electrode. In the left panel respiration rates were measured by adding NADH as a substrate and in the right panel respiration rates were measured by adding Ascorbate (Asc) and 1 .4 mM N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) as a substrate. n=4. B. Mitochondrial ATP production. ATP production of 75 g of mitochondria was quantified at different times by luciferase assay as described in the material and methods section, n = 4.

Figure 4. OXPHOS assembly/stability/activity. A. ATPase activity. Mitochondrial proteins were extracted with 2% digitonin, separated by BN-PAGE and incubated in solution to reveal the ATPase activity. Vi and V₂, monomeric and dimeric species of ATP synthase (complex V), respectively B. BN-PAGE and in-gel activities analysis. Mitochondrial proteins were extracted with digitonin (10g/g), separated by BN-PAGE, and assayed for in-gel complex IV activity. C. SDS-PAGE and BN-PAGE analysis. Left panel. 100 g of total mitochondrial proteins were separated in SDS-PAGE, transferred onto a nitrocellulose membrane and probed with the indicated antibodies. Right panel. Mitochondrial proteins were extracted with digitonin (4g/g), separated by BN-PAGE, and transferred to membranes for western blottings with the Cox2 and Cytb antibodies. D. SDS-PAGE analysis. 50 g of total proteins extracts were separated in SDS-PAGE, transferred onto a nitrocellulose membrane and probed with the Hsp60, Ccp1 and Ade13 antibodies. Data were standardized to the loading control Ade13. Data were shown as relative to the WT. Data are representive of at least 3 experiments. E. ROS production. Cells were grown at 36°C to the exponential phase in CSM + 0.5% galactose + 2% ethanol and the measures were done at the indicated time point as described under Experimental procedures. Data represent the average of three independent experiments. Wild-type is represented in black bars, tazIA mutant in open bars, tazIA/ reil in grey bars and tazIA/ rpl6bA and tazIA treated with 10nM CHX in striped bars. Figure 5. CHX improves cell proliferation and viability of human Tafazzin- deficient cells.

A. Growth curves in 200 μΙ_ wells inoculated with 7 500 cells. After reaching a plateau, the cells die and detach from their support. B. Relative slopes of proliferation curves shown in panel A.

C. xCELLigence recording of ShTazl proliferation in absence or presence of CHX at a concentration of 50 pM. CHX was added (Black arrow) after a 24 hour adhesion step. The cultures were inoculated with 5.103 cells in 200 μΙ_ wells. The optimal Cell Index values are indicated along the curves as well as the time (in hours) taken to reach the inflexion point. The horizontal red bar stands for the 48 hours lag phase induced by CHX. The shown curves are representative of four independent experiments with 4 wells for each growth condition (16 wells in total for establishing mean values).

EXAMPLE

In this study a yeast model for the Barth Syndrome that presents a proteostatic stress can be rescued by a subtle metabolic adaptation as a very little decrease of cytosolic protein synthesis. In addition, this study demonstrates that decreasing cytosolic protein synthesis is also beneficial to tafazzin-deficient human cells.This study allowed identifying metabolic suppression that could be used for mitochondrial diseases.

Materials and Methods

Yeast strains and growth media. The sources and genotypes of the strains used in this study are listed in Table 1 . Atazl mutant strain (wild-type, WT, MATa ade2-1 ura3-1 his3-1 1 , 15 trp1 -1 Ieu2-3,1 12 can1 -100, taz1::TRP1) was used for the construction of the double mutant strains Ataz1/Arpl6b and Ataz1/Arei1. Fermentable YPAD media contain 1 % (w/v) yeast extract, 2% (w/v) bacto peptone, 60 mg/L adenine and 2% (v/v) glucose; Respiratory rich media YPAEthanol contain 1 % (w/v) yeast extract, 2% (w/v) bacto peptone, 60 mg/L adenine and 2% (v/v) ethanol and non-fermentable complete synthetic media CSM/gal/ethanol contain 0.17% (w/v) yeast nitrogen base without aminoacids and ammoniumsulfate, 0.5% (w/v) ammonium sulfate, 0,5% (w/v) galactose, 2% ethanol and 0.8% (w/v) of a mixture of aminoacids and bases from Formedium. Ethanol was added after the sterilization. The solid media contained 2% (w/v) agar.

Construction of the strains. Ataz1/Arpl6b and Ataz1/Arei1 strains were constructed as follows. The entire open reading frame of RPL6B or REI1 was replaced by the KanMX marker using a PCR-mediated one-step gene replacement strategy. Concerning the Ataz1/Arpl6b strain, the KanMX cassette was amplified from pFA6a- Kan: Rpl6b-del-F (CTT TCT TGA ACT TGG AAG AGA AGC AAA TAT ATT CAA CGA A egg ate ccc ggg tta att aa) (SEQ ID Ν ) and Rpl6b-del-R (CTA TTT TAA ATC ATT TAT AAT TTT TTC AGT TCA AT gaa ttc gag etc gtt taa ac) (SEQ ID N°2), consisting of 40-nucleotide and 35-nucleotide sequences identical to the RPL6B flanking regions at the 5' ends (capital letters) and 20 nucleotides of sequence specific for the amplification of the KanMX marker gene at the 3' ends (small letters) of each primer. Concerning the Ataz1/Arei1 strain, the KanMX cassette was amplified from pFA6a-Kan: Rei1 -del-F (CAT TAG AAG TCA AGA AGA GAG CAT ATC AGT AAC AAT ACG egg ate ccc ggg tta attaa) (SEQ ID N°3) and Rei1 -del-R (GCG ACA AAA TAC TAA AAA AAG TAG TGC AAA AAG AA gaa ttc gag etc gtt taa ac) (SEQ ID N°4), consisting of 39-nucleotide and 35-nucleotide sequences identical to the REI1 flanking regions at the 5' ends (capital letters) and 20 nucleotides of sequence specific for the amplification of the KanMX marker gene at the 3' ends (small letters) of each primer. The PCR product was purified and transformed into Atazl strain, and transformants were selected on agar plates added with geneticin (G418 purchased from Sigma). The presence of a KanMX module instead of RPL6B or REI1 was verified by PCR analysis using primers specific for the flanking regions of RPL6B: Rpl6b-Fbis (CTG CGC TTC CGT TCA GCA TC) (SEQ ID N°5), and Rpl6b-Rbis (CGA TGA CCT GAT CTT GAA CCC) (SEQ ID N°6) or RE 11: Rei1 -Fbis (GTG GTG TAG CTA TTT GTA CAT G) (SEQ ID N°7), and Rei1 -Rbis (CAA CAT CTT CAG TCT TCA GCA GC) (SEQ ID N°8).

Yeast-based drug assay. 125 OD of exponentially growing cell cultures was spread homogeneously with sterile glass beads on a square Petri dish (12 cm x 12cm) containing YPAEthanol solid medium. Sterile filters were placed on the agar surface. Cycloheximide (purchased from Sigma) and Anisomycin (purchased from Sigma) were applied at the indicated quantities to each filter in addition to the top left filter where DMSO, the vehicle, was added as a negative control. Plates were then incubated at 36°C for several days and scanned.

Spot Assays. Indicated strains were grown overnight in YPAD at 29°C. Cells in the exponential phase (OD₆oo 0.2-0.4) were initially diluted to an OD₆oo of 0.05 and series of 5- fold dilutions were performed in YPA liquid media. Spots of 10 μΙ from each dilution series were then plated onto YPAD or YPAEthanol media and cultured at 28°C or 36°C for 4 days. All spot assays were repeated at least three times and a representative experiment is shown.

Growth curves. For liquid culture of yeast cells, YPAD overnight cultures at 29°C of the appropriate strains were diluted in YPAEthanol supplemented either with the compounds of interest dissolved in DMSO at a 1000X concentration or with the equivalent quantity of DMSO as a negative control and grown at 36°C. For growth curves, aliquots were removed at the appropriate times and their OD₆oo determined.

Estimation of the percentage of petites. YPAD overnight cultures at 29°C of the appropriate strains were spread onto YPAD solid media and incubated at 29°C. Strains were spread in order to have no more than 50 colonies per plate. Red colonies (rho+) and white colonies (rho- petite colonies) were counted. 10 plates per strains were counted and the experience has been made three times.

Bioenergetic experiments. The mitochondria were prepared by the enzymatic method as described. The protein amounts were determined by the Lowry method in the presence of 5% SDS. Oxygen consumption rates were measured on 75 g of fresh mitochondria using a Clarke electrode in the respiration buffer (0.65 M mannitol, 0.36 mM ethylene glycol tetraacetic acid, 5 mM Tris-phosphate, 10 mM Tris-maleate, pH 6.8) after consecutively adding 4 mM NADH (complex I respiration) or Ascorbate (Asc) and 1 .4 mM N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) (complex IV respiration) as a substrate, 150 μΜ ADP (state 3) and 4 μΜ carbonyl cyanide m-chlorophenylhydrazone (CCCP) (uncoupled respiration), as described. Variations in transmembrane potential (ΔΨ) were evaluated in the same respiration buffer as in by monitoring the quenching of rhodamine 123 fluorescence (0.5 μΜ) using a A_exc of 485 nm and a A_em of 533 nm under constant stirring using a FLX Spectrofluorimeter (SAFAS, Monaco). For ATP synthesis rate measurements, 75 g of fresh mitochondria were placed in a 2-ml thermostatically controlled chamber at 28°C in respiration buffer. The rates of ATP synthesis were determined in the presence of 4 mM NADH and 1 mM ADP; aliquots were withdrawn from the oxygraph cuvette every 15 seconds and the reaction was stopped with 3.5% (w/v) perchloric acid, 12.5 mM EDTA. The samples were then neutralized to pH 6.5 by addition of KOH, 0.3 M MOPS. Synthetized ATP was quantified using a luciferin/luciferase assay (ATPLite kit from Perkin Elmer) on a LKB bioluminometer. The specific ATPase activity was measured at pH 8.4 by using a previously described procedure . The participation of the FiF₀-ATP synthase in ATP production or hydrolysis was assessed by addition of oligomycin (2 mg/ml of protein).

BN-PAGE & SDS-PAGE. Blue native BN-PAGE and clear native CN-PAGE experiments were carried out as described previously in the art. Briefly, mitochondrial extracts solubilized with digitonin to an indicated protein ratio were separated in a native 3-12% acrylamide continuous gradient gel. After electrophoresis, the gel was either stained with coomassie blue or incubated in a solution of 8 mM ATP pH7, 14 mM MgCI₂, 0,2% lead nitrate, 35 mM Tris, 270 mM glycine pH 8.4, to detect the ATPase activity; a solution of Tris 5mM pH 7,4, Nitroblue tetrazolium (1 mg/ml), sodium succinate 20 mM, phenazine methosulfate 0,2 mM to detect the complex II activity; a solution of Tris 5 mM pH 7,4, diaminobenzidine 0,5 mg/ml, cytochrome C 0,05 mM to detect the complex IV activity or transferred to poly(vinylidene difluoride) membranes and analyzed by Western blotting. Western blot analyses were performed as previously described in the art. Polyclonal antibodies raised against yeast ATP synthase were used at a dilution of 1 :50,000 for subunit a; 1 :10,000 for subunits Y and a-Atp6 (kind gift from J. Velours); 1 :10,000 for Aac2; 1 :5000 for cytochrome b (kind gift from T. Langer); 1 :10000 for cytochrome c (kind gift from S. Manon); 1 :5000 for succinate deshydrogenase SDH2; 1 :5000 for tafazzin TAZ1 (kind gift from S. Claypool); 1 :1000 for Hsp60 (kind gift from Martin van der Laan) and 1 :5000 for ADE13. Monoclonal antibodies against yeast porin and yeast cytochrome c oxidase COX2 (from Molecular Probes) were used at a dilution of 1 :5000. Nitrocellulose membranes were incubated with peroxidase-labeled antibodies at a 1 :2500 dilution (Promega), and the blot visualization was conducted with an electrochemiluminescence reagent. Quantification was performed with the ImageJ software.

Lipidomic analysis. Lipids from isolated mitochondria were extracted with 2 ml of chloroform/methanol (2:1 , v/v). After centrifugation, the organic phase was isolated and the remaining lipids were further extracted twice by the addition of 2 ml of chloroform to the aqueous phase. After the phase separation, the organic phases were pooled and evaporated to dryness. The lipids were then suspended in chloroform/methanol (2:1 , v/v). Volumes equivalent to 50 g of acyl chains were spotted on silica plates four times for each strain. Polar lipids were separated by one dimensional TLC using chloroform/methanol/1 -propanol/methyl acetate/0.25% KCI (10:4:10:10:3.6, by vol.) as a solvent. Thin layer chromatography of phospholipids. The lipids were then located by immersing the plates in a solution of 0.001 % (w/v) primuline in PBS, followed by visualization under UV light. The silica gel zones corresponding to the various lipids (PE, CL, PI and PC) were then scraped from the plates and added to 1 ml of methanol/2.5% H2SO4 containing 5 μg of heptadecanoic acid methyl ester as a standard. After maintaining the lipids in the mixture at 80°C for 1 h, 1 .5 ml of water and 400 μΙ of hexane were added. After centrifugation, hexane phase containing FAMES (fatty acid methyl esters) was isolated. Separation of FAMES was performed as described in the literature (Biochem J, 2005. 387(Pt 3): p. 617-26).

Electron microscopy. Freezing and Freeze Substitution for Ultrastructural Studies. The yeast pellets were placed on the surface of a copper EM grid (400 mesh) coated with formvar. Each grid was very quickly submersed in liquid propane precooled and held at -180°C using liquid nitrogen. Loops were transferred in a precooled solution of 4% osmium tetroxide in dry acetone in a 1 .8 ml polypropylene vial at -82°C for 72 hr (substitution), warmed gradually to room temperature, and followed by three washes in dry acetone. The samples were infiltrated progressively with araldite (epoxy resin [Fluka]). Ultrathin sections were contrasted with lead citrate. Specimens were observed with a Hitachi 7650 (80 kV) electron microscope (Electronic Imaging Pole of Bordeaux Imaging Center). The number of mitochondria was averaged from 50 observed cells in which the nucleus and the vacuole were seen. ROS analysis by flow cytometry. 0.4 OD were taken from the indicated cultures, and the cells were pelleted in a microcentrifuge. After removal of the media, the cell pellets were resuspended in 1 ml of phosphate-buffered saline (PBS) containing 50 μΜ dihydroethidium (DHE; Molecular Probes) and incubated at room temperature for 5 min. Flow cytometry was carried out on a Becton-Dickinson Accuri C6 model flow cytometer. The DHE fluorescence indicated was the direct output of the FL2A (red fluorescence-detecting) channel without compensation. A total of 100,000 cells were analyzed for each curve. As a positive control, 5ul of H2O2 were added in the PBS suspension containing 5O μΜ DHE. xCELLigence real time cellular proliferation measurements

Experiments were carried out using the xCELLigence RTCA DP instrument (ACEA Biosciences, Ozyme, France) placed in a humidified incubator at 37°C and 5% CO2. Cell proliferation and cytotoxicity experiments were performed using 16-well plates (E-plate, Ozyme, Montigny le Bretonneux, France). The microelectrodes attached at the bottom of the wells allowed for impedance-based detection of the attachment, spreading and proliferation of the cells. Initially, 180 μί of cell-free growth medium (10% FBS) was added to the wells. After leaving the devices at room temperature for 30 min, the background impedance for each well was measured. Cells were harvested from exponential phase cultures by a standardized detachment procedure using 0.05% Trypsin-EDTA (Invitrogen). Flow cytometry was used to count the cells and test their viability (FSC versus propidium iodide staining). 5000 or 7500 cells in 20 μΙ were added in each well. After leaving the plates at room temperature for 30 min to allow early cell attachment, in accordance with the manufacturer's guidelines, they were locked in the RTCA DP device in the incubator and the impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index value (CI).

Water was added to the space surrounding the wells of the E-plate to avoid interference from evaporation. For proliferation assays, the cells were incubated during 120h for toxicity in growth medium (10% FBS) and CI was monitored every 15 min during the whole duration of the experiment. Four replicates of each condition were used in each test. After an initial assessment of the concentration of cycloheximide which would be non-toxic to the ShWT1 cells, we used a test of proliferation in presence and absence of low doses of cycloheximide (1 pM to 100 pM) either prior to cell seeding or after an initial adhesion phase and at an early proliferative step (at 24-26 hours). All experiments were conducted over 240 hours. All plots were normalized to the Cell Index.

Table 1 Genotypes and sources of yeast strains

Strain Nuclear genotype mtDNA Source

K699 W303-1B MATa ade2-1 ura3-1 his3-11, 15

trp1-1 Ieu2-3, 112 can1-100

Atazl ::TRP1 W303-1B MATa ade2-1 ura3-1 his3-11, 15 (submitted) trp1-1 leu2-3, 112 can1-100 Ataz1::TRP1

Ataz1 ::TRP1/ W303-1B MATa ade2-1 ura3-1 his3-11, 15 This study trp1-1 Ieu2-3, 112 can1-100 Ataz1::TRP1

Arei1 ::KanMX

Arei1::KanMX

Atazl ::TRP1/ W303-1B MATa ade2-1 ura3-1 his3-11, 15 This study trp1-1 Ieu2-3, 112 can1-100 Ataz1::TRP1

Arpl6b::KanMX

Results

Partial inhibition of cytosolic protein synthesis by chemical or genetic suppressors partially rescues the respiratory growth of the tazIA yeast mutant strain

Based on recent studies showing that a reduced cytosolic protein synthesis suppresses membrane depolarization and mitochondrial defects in mutant of the mitochondrial ADP/ATP translocase one wondered if a modulation of the cytosolic protein synthesis could rescue the tazIA mutant that also presents a deficiency in the mitochondrial inner membrane potential. To this end, the tazIA strain was spread on solid respiratory ethanol-based media and subsequently exposed to small filters (similar to the ones used for antibiograms) spotted with the compounds. It clearly demonstrates that cycloheximide (CHX) and anisomycin (ANI), two inhibitors of the cytosolic protein synthesis, are able to rescue the respiratory growth of the tazIA strain. Indeed, a halo of enhanced growth around filters was seen (Fig. 1A). The advantage of this method is that, in one simple experiment, it allows compounds to be tested across a large range of concentrations due to diffusion of the drugs in the growth medium. Both drugs, CHX and ANI were toxic at high concentration as seen by the halo of no growth near the filter but were very active at subtoxic concentrations. CHX is active at concentrations at least 10 fold lower than active concentrations of ANI (Fig. 1A). In addition both drugs partially suppressed, in a dose-dependent manner, the respiratory growth defects of the tazl mutant strains (Fig. 1A). Experiments were pursued with the most active of the drugs, CHX and confirmed its activity in liquid respiratory growth media. An optimal concentration of 10 nM of CHX was found that allowed to partially rescue the respiratory growth in the liquid media compared to the WT isogenic strain and the tazl mutant strain treated to DMSO (vehicle, negative control) or 3 nM CHX (Fig. 1 B). A genetic confirmation that inhibition of cytosolic protein synthesis rescued the respiratory growth of the tazIA mutant strain was done. To do this, the deletion of either the gene REI1 which is involved in ribosomal biogenesis or RPL6B which encodes a component of the 60S ribosomal subunit was made. Interestingly, the deletion of these genes in the tazIA mutant strain partially restored the respiratory growth compared to the WT isogenic strain (Fig. 1 C, 1 D) confirming that the activity of CHX and ANI went through the inhibition of the cytosolic protein synthesis. It worth to note that the modulation is very subtle. Indeed, other studies showed that the deletion of RPL6B or RE 11 inhibited only 20% and 30% of the cytosolic protein synthesis respectively. Moreover they showed that 100 ng/ml (355 nM) of CHX inhibits 20% of the cytosolic protein synthesis while it is herein demonstrated that 10 nM (30 fold less) was enough to partially rescue the respiratory growth of the tazIA mutant. Thus these results suggested that a very subtle modulation of the cytosolic protein synthesis is enough to cure the mutant strain tazIA. Nevertheless, in liquid culture, it is demonstrated that, contrary to a treatment of 10 nM of CHX, the deletion of REI1 and RPL6B in the tazIA mutant strain completely restored the respiratory growth of the tazIA mutant suggesting that the complete respiratory growth rescue go through an inhibition of at least 20% of the cytosolic protein synthesis. Very interestingly, the growth of the tazIA mutant showed a slower fermentable growth on glucose media at 28°C than the isogenic WT which correlated well with the slower fermentable growth in liquid respiratory media at 36°C (as long as galactose was present, the tazIA mutant grew by fermentation, then, the tazIA mutant stopped growing due to its respiratory deficiency that prevents it to utilize ethanol) (Fig. 1 C, 1 D). These results suggested the presence of a proteostatic stress in the tazIA mutant.

This proteostatic stress could explain the high mitochondrial instability in glucose at 28°C in the tazIA mutant compared to the WT (Fig. 1 E). The inhibition of cytosolic protein synthesis by deleting REI1 or RPL6B allowed to decrease the mitochondrial instability close to the level of the isogenic WT (Fig . 1 E). These results suggest that the decrease of the cytosolic protein synthesis allows to the tazl mutant to recover a mitochondrial membrane potential permitting the import of certain mitochondrial proteins that were accumulated in the cytosol and creating a proteostatic stress.

Partial inhibition of cytosolic protein synthesis rescues the mitochondrial membrane potential of the tazIA yeast mutant strain

In order to confirm this hypothesis, a complete bioenergetic study was achieved on the mutant strains grown in liquid respiratory ethanol-based media in which a growth defect can be observed. Because of the high mitochondrial genome instability of the mutant strain tazIA the percentage of petite colony was determined in cells that were using for all analysis and found significantly low in these particular conditions of culture (around 10%).

The mitochondrial electrical potential (ΔΨ) was analysed across the inner membrane via fluorescence quenching rhodamine 123. Adding ADP to WT mitochondria energized with ethanol induced a fluorescence variation that progressively disappeared due to the phosphorylating activity of the F₀Fi-ATP synthase (Figure 2A). The membrane potential increased when most of the ADP had been transformed into ATP. As expected, further additions of KCN and CCCP to these mitochondria led to a collapse of the membrane potential. In the mitochondria isolated from the tazIA strain, even though ethanol was able to establish an electrical membrane potential, ADP poorly induced a decrease in ΔΨ (Figure 2A). A much longer time was needed to recover the ethanol-induced ΔΨ in tazIA mitochondria, which reflects their compromised ability to synthesize ATP from ADP and Pi. Mitochondria isolated from the taz1A/rei1A and the taz1A/rpl6bA strains exhibited a behavior comparable to the one observed for the WT control (Fig. 2A) indicating that partial inhibition of cytosolic protein synthesis restored the mitochondrial ΔΨ. To investigate the intrinsic functionality of the F₀Fi-ATP synthase, the ability of ATP to energize the IMM through Frcatalyzed hydrolysis of ATP coupled to proton ejection out of the matrix through the F₀ (figure 2B) was tested. In the tazIA, taz1A/rei1A and the taz1A/rpl6bA strains, no difference with wild-type mitochondria was observed, indicating that the loss of Taz1 p did not seem to have major effects on the F₀Fi-ATPase activity.

Partial inhibition of cytosolic protein synthesis rescues the respiratory rate and the ATP production of the tazIA yeast mutant strain

The oxygen consumption activity of the indicated strains. As shown previously, using either NADH or Λ/,Λ/,Λ/',Λ/'-tetramethyl-p-phenylenediamine (TMPD) / Ascorbate) in the presence of CCCP as an electron donor, the oxygen consumption was reduced by approximately 50% in tazIA mitochondria with respect to the WT (Fig. 3A). However, it was observed that REI1 or RPL6B mutations in the tazIA mutant strain allowed to restore an oxygen consumption as high as the isogenic WT (Fig. 3A). This result was consistent with the respiratory growth obtained on solid and liquid media at 36°C.

Consequently, it has been found that the tazIA mutant strain deleted of REI1 or RPL6B was able to produce ATP at a level as high as the isogenic WT (Fig. 3B).

These results suggested that a subtle modulation in the cytosolic compartment can restore the oxidative phosphorylation functioning. Partial inhibition of cytosolic protein synthesis rescues the OXPHOS assembly of the tazIA yeast mutant strain

As previously showed, the rate of mitochondrial ATP hydrolysis was not significantly diminished in the tazIA mutant with respect to the WT and the deletion of REI1 or RPL6B in the tazIA mutant strain did not change this activity (Fig. 4A). However, the lower level in the assembly of the complexes II, III and IV seen in the tazIA mutant was increased in the tazIA mutant deleted of REI1 and RPL6B (Fig. 4B and Fig. 4C). Moreover, the steady-state level of several mitochondrial proteins, Cytc, Cox2, and Sdh2, involved in the OXPHOS that were also decreased in the tazl mutant was at the same level as the WT level in the tazl mutant deleted of REI1 and RPL6B (Fig. 4C).

Very interestingly, the level of Hsp60, a mitochondria-specific molecular chaperone that is the marker of the mitochondrial unfolded protein response (UPRmt), and Ccp1 , the cytochrome c peroxidase that modulate the antioxidant defense are overexpressed in the tazIA mutant in presence of CHX and in the double mutants taz1/rei1 and taz1/rpl6b (Fig. 4D).

Altogether these results confirmed that a modulation of the cytosolic protein synthesis can ameliorate a mutant that present a deficiency in the import pathway of mitochondrial proteins. This modulation of the cytosolic protein synthesis tends to initiate a response to the proteotoxic stress in mitochondria and to the presence of Reactive Oxygen Species (ROS) accumulation.

Partial inhibition of cytosolic protein synthesis decreases the level of ROS and increases the number of mitochondria in the tazIA yeast mutant strain

The effect of the modulation of the cytosolic protein synthesis on the production of ROS of the tazIA mutant strain was tested to confirm hypothesis. At 48 hours when the respiratory defect is well installed in the tazIA mutant compared to the WT (Fig. 1 D), the level of ROS in the tazIA mutant is 3 fold higher than the WT isogenic strain (Fig. 4E). Interestingly, the deletion of REI1 or RPL6B and even the addition of 10 nM of CHX in the tazl mutant strain allowed to decrease the level of ROS at a level comparable to the WT isogenic strain (Fig. 4E). Altogether, these results suggested that a metabolic modification in the cytosolic compartment can be beneficial to restore a mitochondrial functioning in the context of a mitochondrial proteostatic stress by inducing the UPRmt that includes the generation of mitochondrial proteostasis machinery such as mitochondrial molecular chaperones and proteases as well as antioxidant genes to limit mitochondrial damages.

Decreasing cytosolic protein synthesis is also beneficial to tafazzin-deficient human cells.

The inventors investigated whether partially decreasing cytosolic translation could also benefit human cells lacking Tafazzin. To this end, the previously described HeLa cells were used, in which the TAZ gene was knocked down by RNA interference (shTazl ) and two control cell lines, shWT1 and shTazI R in which the expression of TAZ1 was not inhibited (Gonzalvez F, Schug ZT, Houtkooper RH, MacKenzie ED, Brooks DG, Wanders RJ, Petit PX, Vaz FM, Gottlieb E (2008) Cardiolipin provides an essential activating platform for caspase-8 on mitochondria. The Journal of cell biology 183: 681 -96). As reported, ShTazl poorly accumulates Tafazzin, is defective in CL maturation, presents a destabilization of the supercomplexes, produces abnormally enlarged cells and has a higher content in mitochondria compared to shWT1 and shTazl R, as do cells from Barth syndrome patients.

Herein the inventors show that ShTazl cells proliferate four times slower and die more rapidly, in comparison to ShWT1 and ShTazRI cells (Fig.6 A, B).

The inventors decided to test the capacity of CHX at counteracting the detrimental effects of a lack in CL maturation in human cells. A large beneficial effect was observed at a 50 pM concentration of CHX. In the experiment shown in Fig. 6C, the drug was added 24-25 hours after inoculating 200 μΙ of media with 5000 cells. CHX induced a 48h lag phase, after which the cells grew for a long-lasting period of 170 hours, before dying and detaching from their support. In the absence of CHX, death was observed much more rapidly, after 72 hours of continuous growth.

These observations indicate that a partial decrease in cytosolic translation is, as in tazIA yeast, beneficial to human cells defective in CL remodeling.

Discussion The present description clearly chemically (by using CHX and ANI) and genetically (by deleting REI1 or RPL6B) shows that decreasing the eukaryotic cytosolic protein synthesis rescues the respiratory functions of the tazl mutant. REI1 and RPL6B genes are involved in the ribosomal biogenesis and in the 60S ribosomal subunit respectively, and their absence induces a decrease of about 20-30% of the eukaryotic cytosolic translation ; while a decrease of less than 1 % is observed with 10nM CHX. Thus a very subtle modulation of the cytosolic protein synthesis is enough to cure the mutant strain tazl.

The present description also shows that decreasing cytosolic protein synthesis is beneficial to human cells that are defective in CL remodeling.

It is thus demonstrated that a treatment with cytosolic protein synthesis inhibitors or deletion of gene coding for protein involved in the biogenesis of ribosomes helps the tazl mutant to recover a normal mitochondrial function. This is the first report that shows that the yeast mutant tazIA can be cured by a metabolic bypass without rescuing the primary defect of the mutant cell but in modulating the eukaryotic cytosolic protein synthesis. It also demonstrated that this treatment is also beneficial to human cells wherein the TAZ gene is knocked down.

Claims

1 . Compound that inhibits the cytosolic protein synthesis for its use in the prevention and/or treatment of a disorder or disease associated to mitochondrial dysfunction wherein the mitochondrial dysfunction is associated with a deficiency in mature cardiolipin level in mitochondria membrane.

2. Compound for its use according to claim 1 , characterized in that the deficiency in mature cardiolipin level in mitochondria membrane is associated with a TAZ gene mutation.

3. Compound for its use according to claim 2, characterized in that the mutation corresponds to an underexpression or extinction of the TAZ gene.

4. Compound for its use according to any preceding claim, characterized in that the disorder or disease is Barth syndrome.

5. Compound for its use according to any preceding claim, characterized in that the disorder or disease comprises one or more of the following pathologies: cardiomyopathy, skeletal muscle abnormalities, neutropenia, growth delay, weak muscle tone and bacterial infections.

6. Compound for its use according to any preceding claims, characterized in that the compound is selected in the group consisting of cycloheximide, emetin and anisomycin or any pharmaceutically acceptable salt or prodrug thereof.

7. An in vitro method for selecting a compound capable of inhibiting the cytosolic protein synthesis and restoring respiratory function of the mitochondria comprising the steps of :

- i) Providing an organism wherein an homolog of human TAZ gene is mutated and mature cardiolipin level is deficient, - ii) Contacting a compound to be screened with the organism of step i),

- iii) Measuring the inhibition of cytosolic protein synthesis within the organism, induced by the compound, - iv) Selecting the compound when the cytosolic protein synthesis is inhibited in comparison with an identical organism not contacted with the compound.

8. Method according to claim 7, characterized in that the cytosolic protein synthesis inhibition is partial, preferably the cytosolic protein synthesis inhibition is comprised between 0.5 and 10 %.

9. Method according to claim 7 or 8, characterized in that the cytosolic protein synthesis inhibition rescues of the respiratory growth TAZ mutated organism, wherein the TAZ mutated organism is a TAZ1 mutated yeast.

10. Method according to claim 7 or 8, characterized in that the cytosolic protein synthesis inhibition allows maintenance of the mitochondrial genome in the TAZ mutated organism.

1 1 . Method according to claim 7 or 8, characterized in that the cytosolic protein synthesis inhibition rescues the ATP production and mitochondrial membrane potential in the TAZ mutated organism.

12. Method according to claim 7 or 8, characterized in that the cytosolic protein synthesis inhibition rescues the OXPHOS assembly in the TAZ mutated organism.

13. Method according to any one of claims 7 to 12, characterized in that the organism wherein the TAZ gene is mutated is a TAZ1 mutated yeast and the mutation consists in a deletion, an insertion or a substitution which leads to a null allele.

14. Use of an organism deficient in cardiolipin for the screening and selection of compounds capable of inhibiting the cytosolic protein synthesis and/or restoring respiratory function of the mitochondria.

15. A method for reducing the risk of Barth Syndrome in a mammalian subject, the method comprising: administering to the subject a therapeutically effective amount of a compound that partially inhibits the cytosolic protein synthesis and induces at least one effect selected in the group consisting of rescue of the ATP production and mitochondrial membrane potential, maintenance of the mitochondrial genome, rescue of the OXPHOS assembly rescue of the respiratory rate; and decrease of the reactive oxygen species (ROS) production.