Estudio preclínico para el tratamiento de la encefalopatía mitocondrial asociada a la deficiencia en coenzima q

Resumen

Summary Mitochondrion is one of the most fascinating organelles of the cell. It is formed by a lipid bilayer and it is present in hundreds to thousands of copies per cell. It acts as the powerplant of the cell and, therefore, it is necessary for the generation of the energy needed for the activity of the cell. Furthermore, it is a critical organelle for many metabolic processes. Because of it, a defect in mitochondria, mainly produced by mutations of genes encoding mitochondrial proteins, could triggers some of the so-called mitochondrial diseases (MD). MD are a group of rare neurometabolic disorders that are extremely complex and refer to a heterogeneous group of genetic disorders resulting from abnormal oxidative phosphorylation, including the mitochondrial electron transfer chain (mtETC) and leading to defective cellular energy production in the form of adenosine triphosphate (ATP). MD affect 12.5 per 100,000 (1/8,000) persons, and the management of these patients remains difficult since, in most cases, there are no interventions that provide a realistic prospect of cure, and the therapeutic options for these disorders are mostly limited to palliative care and remain woefully inadequate. The treatment of MD is, in most cases, still limited to a palliative care. An affection considered as MD is primary CoQ deficiency, which can be associated to different clinical presentations. In many cases, even the treatments with the best results still fail in improving the mitochondrial bioenergetics and/or phenotypic features on mouse models, as weight or strength gaining, and consequently, the quality of life or survival are not improved. So, it is of vital importance to find therapeutic options that approach to a real cure for these patients. The conventional treatment is based in the exogenous administration of high doses of CoQ10. This treatment, however, has limited effects in a high percentage of patients due to different factors: 1) the low absorption and bioavailability of the exogenous CoQ10, together with its low capacity to cross the Blood Brain Barrier (BBB); 2) the lack of effect over the accumulation of intermediate metabolites in the synthesis of CoQ (importantly, some of these metabolites, e.g. Demethoxyubiquinone, may contribute to the disease phenotype by inhibiting the transfer of electrons in the mtETC); and 3) the lack of effect over the Complex Q and the endogenous biosynthesis of CoQ. For that reason, CoQ10 therapy does not induce any change in the levels of CoQ9. In the Coq9R239X mouse model with fatal mitochondrial encephalopathy due to CoQ deficiency, we have tested the therapeutic potential of β‐resorcylic acid (β‐RA), a structural analog of the CoQ precursor 4‐hydroxybenzoic acid (4-HB) and the anti‐inflammatory salicylic acid. β‐RA noticeably rescued the phenotypic, morphological, and histopathological signs of the encephalopathy, leading to a significant increase in the survival. Those effects were due to thedecrease of the levels of DMQ9 and the increase of mitochondrial bioenergetics in peripheral tissues. However, neither CoQ biosynthesis nor mitochondrial function changed in the brain after the therapy, suggesting that some endocrine interactions may induce the reduction of the astrogliosis, spongiosis, and the secondary down‐regulation of astrocytes‐related neuroinflammatory genes. Because the therapeutic outcomes of β‐RA administration were superior to those after CoQ10 supplementation, its use in the clinic should be considered in CoQ deficiencies. Somehow, to apply the β‐RA treatment into the clinic, the safety and dose–response studies included in a clinical trial should be first developed. b-RA, as other hydroxybenzoic acids derivatives, are natural phenolic compounds that are being used for medical purposes. Some of these compounds (e.g. salicylic acid), however, have reported toxicity in particular conditions but the mechanisms of this effect remain obscure. In this study, we used biased and unbiased approaches to evaluate the effect of a high dose of b-RA, approximately 1g/kg body weight/day in mice, previously used for the preclinical treatment of CoQ deficiency, in wild-type animals, and to test the therapeutic potential of a lower dose of b-RA, three time less than the high dose, in both wild-type and our mouse model Coq9R239X of mitochondrial encephalopathy caused by CoQ deficiency. We show that the high dose of b-RA, previously used for the preclinical treatment of CoQ deficiency, induces hepatic, renal and cerebral toxicity in wild-type animals, with a disruption in transcriptomic profiles and mitochondrial proteomes. Those toxic effects are dependent of the metabolic use of b-RA in the CoQ biosynthetic pathway. Thus, the low dose of b-RA, dramatically decreases the toxicity in wild-type animals, preserving the therapeutic capability in Coq9R239X mice by modulating CoQ metabolism. Collectively, our results highlight novel toxic mechanisms of HBAs and provide a safe translational perspective for the use the b-RA in the treatment of CoQ deficiencies. Besides, CoQ is involved in many other metabolic processes, receiving electrons from enzymes of other metabolic pathways. CoQ is cofactor of the dihydroorotate dehydrogenase (DHOH), which is involved in the synthesis de novo of pyrimidines; the electron transfer flavoprotein (ETF), which receives electrons from the fatty acids b-oxidation; the mitochondrial glycerol 3- phosphate dehydrogenase (G3PDH), which connects glycolysis, OXPHOS and fatty acids metabolism; the choline dehydrogenase (CHDH), which is involved in the glycine metabolism; the proline dehydrogenase (PDH), which is related with proline and arginine metabolism; and the sulfide:quinone oxidoreductase (SQOR), catalytic enzyme of the sulfide oxidation metabolic pathway. So, it is possible that a defect in CoQ could affect any of these pathways and influence in the pathology. In fact, our group, together with the group of Dr. Catarina Quinzii in New York, have recently published two independent studies that show that CoQ deficiency severely decreases the levels of SQOR. Thus, the decrease in SQOR leads to an impairment of H2S oxidation, leading to accumulation of H2S and depletion on the glutathione system. Consequently, the modulation of sulfur amino acids availability in the diet, as sources of H2S could provide therapeutic benefits. Thus, we treated Coq9R239X mice with N-acetylcysteine (NAC), which is a sulfur amino acid and it can stimulate glutathione biosynthesis, or a diet with sulfur amino acids restriction (SAAR). However, we did not find any changes in the glutathione system or sulfide metabolism enzymes after the treatments. Consequently, the survival or phenotype of the mutant mice were not improved. Globally, these data probe the inefficiency of the modulation of sulfur amino acids availability in rescuing the pathology in Coq9R239X mice and confirm that sulfide metabolism influences the levels of glutathione, independently of the sulfur amino acids availability. Resumen