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  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
• • G—PHYSICS
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  • G01N2333/395—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Saccharomyces

Definitions

• the present invention falls within the general field of biomedicine and in particular refers to a method for screening and / or evaluation of the efficacy of a treatment for mitochondrial diseases and MELAS syndrome.
• Mitochondrial diseases cover a broad spectrum of neurodegenerative, chronic and progressive disorders, with phenotypic manifestations and varying degrees of affection, as a consequence of alterations in mitochondrial oxidative metabolism [Zeviani M, Carelli V. Mitochondrial disorders. Curr Opin Neurol 2007; 20: 564-571].
• the pathogenesis of these disorders has its origin in a chronic state of energy insufficiency, due to the inability of the affected mitochondria to generate enough ATP through the OXPHOS system (oxidative phosphorylation).
• OXPHOS system oxidative phosphorylation
• a conversion of pyruvate to lactate occurs, which systemically manifests itself as a chronic lactic acidosis.
• mitochondrial cytopathies have a multisystemic pattern, with tissues with a strong energy demand such as the brain and muscle, the organs that are most frequently affected.
• the 37 mitochondrial DNA (mtDNA) genes are essential for oxidative phosphorylation. Of these, 13 encode subunits of respiratory chain complexes: seven subunits of complex I, one subunit of complex III, three subunits of complex IV and two subunits of complex V. Mutations of these genes cause various mitochondrial alterations and generally present maternal inheritance In addition, 22 tRNA and 2 ribosomal RNAs (rRNA) are required for mitochondrial protein synthesis. In the last decade, clinical researchers have also been interested in mitochondrial alterations with Mendelian inheritance.
• nDNA Nuclear DNA
• nDNA nuclear DNA
• genes necessary for oxidative phosphorylation including 72 polypeptide subunits, as well as all the factors required for the correct assembly of the respiratory chain and the machinery necessary for integrity, replication, repair and expression of mtDNA.
• mutations in the factors required for the translation of proteins in the mitochondria, the importation of proteins, and the fusion / fission of mitochondria also cause mitochondrial abnormalities [Debray FG, Lambert M, Mitchell GA. Disorders of mitochondrial function. Curr Opin Pediatr 2008; 20: 471-482].
• Mitochondrial diseases are clinically heterogeneous due to the uneven distribution of mutations in the different tissues, the degree of heteroplasmia of the affected tissues, the mitotic segregation and the variability of penetrance and threshold effect of the different mutations.
• the prevalence of mitochondrial diseases is approximately 1: 5000 among the population worldwide.
• MELAS syndrome owes its name to the English acronym of Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (mitochondrial encephalomyopathy, lactic acidosis and episodes similar to strokes). It was first described by Pavlakis et al., In 1984. Patients present with clinical manifestations that include the triad of symptoms that give name to the disease. Stroke mainly affects the parieto-occipital region of the brain which leads to defects in the visual field. Seizures are common in these patients associated with stroke episodes or as an isolated phenomenon.
• MELAS syndrome is a polygenic disorder, associated with at least 29 specific point mutations in mtDNA.
• the most common mutation related to this syndrome is the transition from an adenine to a guanine in the position 3243 of the mitochondrial genome (A3243G), in the gene that codes for tRNA (UUR), with a prevalence of 0.06% of the general population [Sproule DM, Kaufmann P. Mitochondrial encephalopathy, lactic acidosis, and strokelike episodes: basic concepts, clinical phenotype, and therapeutic management of MELAS syndrome. Annals of the New York Academy of Sciences 2008; 1 142: 133-158].
• the A3243G mutation makes it difficult to modify the base U of balancing, hindering the translation of the UUA and UUG codons, resulting in an altered incorporation of the amino acids to the proteins synthesized in the mitochondria [Kirino Y, Yasukawa T, Ohta S, Akira S, Ishihara K, Watanabe K et al. Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease. Proceedings of the National Academy of Sciences of the United States of America 2004; 101: 15070-15075].
• Other proposed factors that also influence the altered synthesis of mitochondrial proteins are: disorders in the processing of mRNAs, incorrect aminoacylation kinetics of tRNALeu (UUR) or incorrect conjugation of amino acids to tRNALeu (UUR).
• mitochondria are not capable of producing sufficient amounts of ATP. This leads to a chronic state of energy deficiency, due to an imbalance between energy requirements and available energy. Finally, this energy imbalance causes cellular and tissue damage.
• the A3243G mutation causes a higher glycolytic rate, increased lactate production, reduced glucose oxidation, an altered response to NADH, low ⁇ ⁇ , decreased ATP production, increased ROS and intracellular calcium homeostasis altered decrease in insulin secretion, premature aging and a deregulation of amino acid metabolism and urea synthesis.
• mtDNA mutations cause cell damage and the compensatory mechanisms that the cell activates to survive.
• MELAS fibroblasts showed reduced respiratory enzymatic activities, CoQ deficiency and mitochondrial depolarization.
• Mitochondrial dysfunction was associated with an increase in ROS production, activation of the mitochondrial permeability transition (MPT) and the elimination of mitochondria altered by mitophagy.
• MPT mitochondrial permeability transition
• Saccharomyces cerevisiae yeast is a useful tool, since specific mutations can be introduced into your mtDNA by biobalistics, such as base substitution in mitochondrial tRNA (mt tRNA) genes equivalent to those that originate human neurodegenerative diseases. This is possible because mitochondrial yeast and human tRNAs are similar in sequence and structure, except for the presence of a longer loop in yeasts than in humans.
• mt tRNA mitochondrial tRNA
• the advantages of the use of S. cerevisiae as a model organism are diverse: high growth rate, economic maintenance, classification as GRAS microorganism (generally recognized as safe), fully sequenced genome, suitable for the expression of heterologous proteins, contains a multitude of selective markers including auxotrophic and resistance markers.
• Yeasts are also particularly useful for the study of human mitochondrial diseases thanks to their ability to survive in a medium with a fermentable carbon source, even though their respiratory chain is not functional.
• concentration of glucose When the concentration of glucose is reduced, the mutant yeasts deficient in respiration grow slowly, giving rise to small colonies (petite).
• These petite mutants have abnormalities in mtDNA in the form of multiple rearrangements (petites rho-) or loss of mtDNA (petites rho 5 ).
• the The causative mutation of 80% of MELAS syndrome cases is the transition from an adenine to a guanine at position 3243 of the mitochondrial genome (m.3243A> G), in the gene encoding the tRNA (UUR).
• This mutation is found in a highly conserved region between the human mitochondrial genome and the yeast genome ( Figure 1).
• the A14 nucleotide of the tRNA participates in a canonical tertiary interaction with the nucleotidouridine in position 8 ( Figure 2), which stabilizes the secondary structure of the tRNA and determines its functionality. Therefore, the A14G mutation in yeast results in a conformational rearrangement of the D arm of the tRNA and a decrease in the efficiency of aminoacylation [Montanari A, Besagni C, De Luca C, Morea V, Olive R, Tramontane A et al. Yeast as a model of human mitochondrial tRNA base substitutions: investigation of the molecular basis of respiratory defects. RNA (New York, NY 2008; 14: 275-283].
• the A3243G mutation like other mutations related to MELAS syndrome in humans, has been shown to prevent the modification of uridine with a taurine residue (5-taurinomethyl uridine, xm 5 U) in the staggered position of the anticodon, and The lack of this modification has been proposed as responsible for the pathological effect. It seems that the enzyme responsible for carrying out this modification recognizes the tertiary structure of the complete tRNA, which is affected as a result of this mutation.
• the alteration of mitochondrial tRNA (UUR) shows a reduced translation of UUG, while there is no decrease in the translation of UUA ( Figure 3).
• the main molecular cause of MELAS syndrome is the poor translation of the UUG codon as a result of the defect in the modification of taurine in the staggering position of the anticodon, which translates into a reduction in the activity of the Complex I, which is one of the characteristic symptoms that has been found in patients.
• the A14G mutation in tRNA (Leu) (UUR) equivalent to that produced in humans, causes severe respiratory deficiencies with a high production of mtDNA-deficient mutants (rho 5 ). The percentage of rho 5 colonies is a good indicator of the severity of the respiratory phenotype.
• yeast strains carrying the A14G mutation can grow in fermentation medium (with glucose or galactose as a carbon source), but rapidly lose mtDNA, indicating that they have a serious defect in the synthesis of mitochondrial proteins. Instead, these yeasts are unable to grow in between respiratory (with glycerol as a carbon source).
• the use of yeasts for the study of mitochondrial diseases due to alterations in the mitochondrial tRNA presents as a limitation that they are homoplasmic unlike human cells, which are heteroplastic. Therefore, the yeast models of these pathologies do not allow the threshold effect to be evaluated. In spite of this, they constitute a very useful tool for the simplification of a complex system.
• Yeasts are not exclusively useful for understanding the effects of mutations of mitochondrial diseases, they can also be used for mass screening of drugs capable of reversing the defects in the respiration-dependent growth characteristic of mitochondrial mutant yeasts. This is a fast and sensitive trial that allows the screening of thousands of drugs in a robotic and economical way. However, and despite the fact that dozens of yeast models of mitochondrial diseases are available, there are few studies of their usefulness for mass drug screening.
• Drugs that modify the function of the respiratory chain or prevent oxidative stress CoQ, idebenone, succinate, vitamin C, vitamin K3, riboflavin-B2, thiamine-B1, cytochrome c, creatine monohydrate, copper, uridine.
• the present invention relates to a method for the identification and evaluation of the efficacy of drugs for the treatment of diseases that occur with mitochondrial dysfunction and / or MELAS syndrome characterized in that it comprises the following steps: a) drug screening in a model of Saccharomices cerevisiae mutant A14G, by exposing said yeasts to at least one drug and determining whether said drug produces cellular growth of the A14G mutant yeasts.
• the cell growth of the A14G mutant yeasts of step a) can be performed by any prior art method known to a person skilled in the art.
• the cell growth of the mutant yeasts is determined by optical density.
• step b) evaluation of the efficacy of the drug that produces cellular growth of the A14G mutant yeasts of step a) in cellular models derived from patients with MELAS syndrome and to determine if the drug is effective by means of the ability of said drug to restore pathophysiological alterations said cellular models,
• the cellular models derived from patients with MELAS syndrome in step b) of the method of the present invention are fibroblasts derived from patients with MELAS syndrome and / or in MELAS transmitochondrial cybrids.
• the pathophysiological alterations restored in the cellular models of step b) of the method of the present invention are an increase in cell proliferation, an increase in ATP levels, a decrease in ROS, a decrease in mitophageal activity, an increase in mitochondrial protein expression and / or increased mitochondrial activity.
• mitochondrial dysfunction refers to mitochondrial and other adult diseases such as diabetes, Parkinson's disease, arteriosclerosis, cerebrovascular disease, Alzheimer's disease, and cancer in which mitochondrial dysfunction plays a determining role in the course of the disease.
• the present invention relates to a drug identified by the method of the present invention for the treatment of diseases that occur with mitochondrial dysfunction and / or MELAS syndrome. Description of the figures
• Figure 1 shows the structure of the Leu tRNA (UUR) of yeasts (A) and humans (B), showing the position of the A14G and A3243G mutation.
• UUR Leu tRNA
• Figure 2 shows the three-dimensional structure of tRNA (A) and the base pairing of A14 and U8 (B).
• Figure 3 shows the chemical structure of 5-Taurinomethyl uridine (xm 5 U) (A) and taurine (B).
• C Punctual mutation of the tRNA Leu (UUR) , which prevents the modification of a uridine to taurine in the staggering position of the anticodon, resulting in an abnormal pattern of codon recognition.
• Figure 4 shows the results obtained with the method described in the present invention: A) CoQ, B) riboflavin, C) carnitine, D) creatinine, E) vitamin E, F) Lithium, G) menadione, H) lipoic acid, I) thiamine, J) uridine, K) vitamin C, L) resveratrol.
• FIG. 5 Immunofluorescence images that show that treatment with CoQ and riboflavin dramatically decreases the mitophagy present in MELAS fibroblasts (panel A). The mitophagy is quantified as the number of discrete points where the mitochondrial cytochrome c marker and the autophagic marker LC3 panel B and C are placed.
• FIG. 6 A: wild yeasts (WT) grown in YPD medium, B: mutant yeasts (MUT) A14G in YPD medium, C: WT yeasts in YPG medium, D: A14G MUT yeasts in YPG medium.
• Yeasts were grown in complete medium containing 2% glucose (YPD) or 3% glycerol (YPG). The media were solidified with 1 , 5% agar. For the experiments, the yeasts were collected from colonies on YPG plates and transferred to 5ml of YPG medium to obtain an optical density of 0.5.
• each well was replicated 4 times in YPG. The average of the 4 replicates was normalized. The control wells were also placed on each plate.
• the drugs tested at different concentrations were: Creatine, Thiamine, Vitamin E, Vitamin C (ascorbic acid), Menadione, Lipoic acid, L-Arginine, Carnitine, Riboflavin, Resveratrol, Lithium, Uridine and CoQ.
• Example 2 Evaluation of selected drugs in fibroblast models derived from MELAS patients and in MELAS transmitochondrial cybrids.
• the active ingredients selected in the initial screening in yeasts were subsequently evaluated for their ability to improve pathophysiological alterations in fibroblasts and MELAS cybrids.
• the optimal concentration of the two drugs (CoQ and riboflavin) was determined, by means of the yeast growth test, the fibroblasts and cybrids were treated for two weeks to determine their performance on the pathophysiological alterations previously detected.
• the proliferation of the cells was determined by microscopic counting with Neubauer chamber.
• the enzymatic activities of the respiratory chain and oxygen consumption in the cultured cells were first determined. Specifically, the activities of NADH dehydrogenase (Complex I), succinate dehydrogenase (complex II), NADH-cytochrome c reductase sensitive to Rotenone (complex l + lll), succinate-cytochrome c reductase (complex ll + lll) and cytochrome c oxidase (complex IV).
• citrate synthase an enzyme of mitochondrial hue
• Cytochemical stains for cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) completed the biochemical studies.
• the respiratory capacity of the cells was analyzed by polarographic techniques that measure the rate of oxygen consumption in intact fibroblasts with a Clark electrode (Yellow Spring).
• the levels of lactic acid production in the culture medium were measured as a measure of the degree of mitochondrial dysfunction and compensation for the glycolytic pathway and the intracellular ATP levels as a guideline measure in the degree of bioenergetic alteration.
• Oxygen consumption was determined using a Clark electrode (Yellow Springs Instruments Co, Yellow Springs, OH).
• Lactic acid levels in the culture medium were measured using the Boehringer-Mannhein L-lactic acid commercial kit.
• ATP levels were determined using the commercial ATP bioluminescence assay kit HS II (Roche Applied Science) according to the manufacturer's instructions. Samples were measured using a luminometer equipped with an injector (Lumat LB 9505, Berthold).
• the synthesis of mitochondrial proteins in cultures of control fibroblasts and MELAS was essentially evaluated by measuring the incorporation of [ 35 S] -methionine in the presence of emetine (an inhibitor of cytoplasmic protein synthesis). Radioactively labeled proteins corresponding to mitochondrial synthesis were precipitated with trichloroacetic acid (10%) and analyzed by a liquid scintillation counter.
• ATP is synthesized from ADP and phosphate by mitochondrial oxidative phosphorylation.
• the three redox centers of the mitochondrial respiratory chain pump hydrogenions from the mitochondrial matrix to the intermembrane space, developing a protonmotrial force or mitochondrial membrane potential of the order of - 180 mV.
• This protonmotrial force allows the synthesis of ATP by means of ATPsintetase.
• the alterations of the mitochondrial membrane potential reflect functional alterations of the mitochondria.
• the mitochondrial membrane potential was measured using JC-1 fluorochrome.
• the 2'-7 'dichlorofluorescein diacetate (DCFDA) probe is preferably oxidized by H 2 0 2 generating a green fluorescence. Fluorescence was measured in a flow cytometer.
• isolated mitochondria were resuspended in PBS and lipoperoxides were measured using an LPO-560 kit (OxisResearch, Portland, OR).
• the mitophagy was characterized by the following techniques: 1) Autophagosome observation by electron microscopy; 2) Increased lysosomal activity: beta-galactosidase assay; 3) Colocalization of mitochondrial markers such as cytochrome c with lysosomal markers such as cathepsin or Lysotracker (invitrogen); 4) Mitochondrial localization of autophagy markers such as LC3, ATG12; 5) Expression of genes (ATG genes) and proteins that participate in the mitophagy by Western Blotting and real-time PCR. Autophageal flow integrity was measured by incubating the MELAS fibroblasts and cybrids in the presence of bafilomycin and subsequently determining the increase in LC3-II by Western blotting and real-time PCR. Autophageal flow integrity was measured by incubating the MELAS fibroblasts and cybrids in the presence of bafilomycin and subsequently determining the increase in LC3-II
• Mitochondrial biogenesis was determined by mitochondrial mass (citrate synthase activity) and expression levels of mitochondrial biogenesis factors as a compensatory mechanism before eliminating dysfunctional mitochondria.
• the expression and activation of factors related to mitochondrial biogenesis such as mitochondrial transcription factor Tfam, nuclear respiratory factor NRF-1 and NFR-2, and activated peroxisome proliferation receptor (PGC-1-alpha) were studied.
• the number of mitochondrial DNA copies was determined by real-time PCR.
• apoptosis was studied by treating cells with camptothecin (a topoisomerase I inhibitor) and serum withdrawal or treatment with staurosporine (protein kinase C inhibitor). Apoptosis was assessed by flow cytometry and immunofluorescence. Cells with the activation of caspases, cytochrome c release, cell condensation and fragmentation and other characteristic parameters of apoptotic cells were detected in fibroblast cultures.
• camptothecin a topoisomerase I inhibitor
• staurosporine protein kinase C inhibitor

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