Juan Garrido-Maraver

Secondary coenzyme Q10 deficiency triggers mitochondria degradation by mitophagy in MELAS fibroblasts

Mitochondrial encephalomyopathy, lactic acidosis, and stroke‐like episodes (MELAS) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mtDNA. Here, we report on how this mutation affects mitochondrial function in primary fibroblast cultures established from 2 patients with MELAS who harbored the A3243G mutation. Both mitochondrial respiratory chain enzyme activities and coenzyme Q10 (CoQ) levels were significantly decreased in MELAS fibroblasts. A similar decrease in mitochondrial membrane potential was found in intact MELAS fibroblasts. Mitochondrial dysfunction was associated with increased oxidative stress and the activation of mitochondrial permeability transition (MPT), which triggered the degradation of impaired mitochondria. Furthermore, we found defective autophagosome elimination in MELAS fibroblasts. Electron and fluorescence microscopy studies confirmed a massive degradation of mitochondria and accumulation of autophagosomes, suggesting mitophagy activation and deficient autophagic flux. Transmitochondrial cybrids harboring the A3243G mutation also showed CoQ deficiency and increased autophagy activity. All these abnormalities were partially restored by CoQ supplementation. Autophagy in MELAS fibroblasts was also abolished by treatment with antioxidants or cyclosporine, suggesting that both reactive oxygen species and MPT participate in this process. Furthermore, prevention of autophagy in MELAS fibroblasts resulted in apoptotic cell death, suggesting a protective role of autophagy in MELAS fibroblasts.—Cotán, D., Cordero, M. D., Garrido‐Maraver, J., Oropesa‐Ávila, M., Rodríguez‐Hernández, A., Gómez Izquierdo, L., De la Mata, M., De Miguel, M., Bautista Lorite, J., Rivas Infante, E., Jackson, S., Navas, P., Sánchez‐Alcázar, J. A. Secondary coenzyme Q10 deficiency triggers mitochondria degradation by mitophagy in MELAS fibroblasts. FASEB J. 25, 2669–2687 (2011). www.fasebj.org

Pharmacological Chaperones and Coenzyme Q10 Treatment Improves Mutant β-Glucocerebrosidase Activity and Mitochondrial Function in Neuronopathic Forms of Gaucher Disease

Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes lysosomal β-glucocerebrosidase. Homozygosity for the L444P mutation in GBA1 is associated with high risk of neurological manifestations which are not improved by enzyme replacement therapy. Alternatively, pharmacological chaperones (PCs) capable of restoring the correct folding and trafficking of the mutant enzyme represent promising alternative therapies.Here, we report on how the L444P mutation affects mitochondrial function in primary fibroblast derived from GD patients. Mitochondrial dysfunction was associated with reduced mitochondrial membrane potential, increased reactive oxygen species (ROS), mitophagy activation and impaired autophagic flux.Both abnormalities, mitochondrial dysfunction and deficient β-glucocerebrosidase activity, were partially restored by supplementation with coenzyme Q10 (CoQ) or a L-idonojirimycin derivative, N-[N’-(4-adamantan-1-ylcarboxamidobutyl)thiocarbamoyl]-1,6-anhydro-L-idonojirimycin (NAdBT-AIJ), and more markedly by the combination of both treatments. These data suggest that targeting both mitochondria function by CoQ and protein misfolding by PCs can be promising therapies in neurological forms of GD.

Coenzyme Q10 Therapy

For a number of years, coenzyme Q10 (CoQ10) was known for its key role in mitochondrial bioenergetics; later studies demonstrated its presence in other subcellular fractions and in blood plasma, and extensively investigated its antioxidant role. These 2 functions constitute the basis for supporting the clinical use of CoQ10. Also, at the inner mitochondrial membrane level, CoQ10 is recognized as an obligatory cofactor for the function of uncoupling proteins and a modulator of the mitochondrial transition pore. Furthermore, recent data indicate that CoQ10 affects the expression of genes involved in human cell signaling, metabolism and transport, and some of the effects of CoQ10 supplementation may be due to this property. CoQ10 deficiencies are due to autosomal recessive mutations, mitochondrial diseases, aging-related oxidative stress and carcinogenesis processes, and also statin treatment. Many neurodegenerative disorders, diabetes, cancer, and muscular and cardiovascular diseases have been associated with low CoQ10 levels as well as different ataxias and encephalomyopathies. CoQ10 treatment does not cause serious adverse effects in humans and new formulations have been developed that increase CoQ10 absorption and tissue distribution. Oral administration of CoQ10 is a frequent antioxidant strategy in many diseases that may provide a significant symptomatic benefit.

Clinical applications of coenzyme Q10.

Coenzyme Q10 (CoQ10) or ubiquinone was known for its key role in mitochondrial bioenergetics as electron and proton carrier; later studies demonstrated its presence in other cellular membranes and in blood plasma, and extensively investigated its antioxidant role. These two functions constitute the basis for supporting the clinical indication of CoQ10. Furthermore, recent data indicate that CoQ10 affects expression of genes involved in human cell signalling, metabolism and transport and some of the effects of CoQ10 supplementation may be due to this property. CoQ10 deficiencies are due to autosomal recessive mutations, mitochondrial diseases, ageing-related oxidative stress and carcinogenesis processes, and also a secondary effect of statin treatment. Many neurodegenerative disorders, diabetes, cancer, fibromyalgia, muscular and cardiovascular diseases have been associated with low CoQ10 levels. CoQ10 treatment does not cause serious adverse effects in humans and new formulations have been developed that increase CoQ10 absorption and tissue distribution. Oral CoQ10 treatment is a frequent mitochondrial energizer and antioxidant strategy in many diseases that may provide a significant symptomatic benefit.

Recovery of MERRF Fibroblasts and Cybrids Pathophysiology by Coenzyme Q10

Mitochondrial DNA mutations are an important cause of human disease for which there is no effective treatment. Myoclonic epilepsy with ragged-red fibers (MERRF) is a mitochondrial disease usually caused by point mutations in transfer RNA genes encoded by mitochondrial DNA. The most common mutation associated with MERRF syndrome, m.8344A > G in the gene MT-TK, which encodes transfer RNALysine, affects the translation of all mitochondrial DNA encoded proteins. This impairs the assembly of the electron transport chain complexes leading to decreased mitochondrial respiratory function. Here we report on how this mutation affects mitochondrial function in primary fibroblast cultures established from patients harboring the A8344G mutation. Coenzyme Q10 (CoQ) levels, as well as mitochondrial respiratory chain activity, and mitochondrial protein expression levels were significantly decreased in MERRF fibroblasts. Mitotracker staining and imaging analysis of individual mitochondria indicated the presence of small, rounded, depolarized mitochondria in MERRF fibroblasts. Mitochondrial dysfunction was associated with increased oxidative stress and increased degradation of impaired mitochondria by mitophagy. Transmitochondrial cybrids harboring the A8344G mutation also showed CoQ deficiency, mitochondrial dysfunction, and increased mitophagy activity. All these abnormalities in patient-derived fibroblasts and cybrids were partially restored by CoQ supplementation, indicating that these cell culture models may be suitable for screening and validation of novel drug candidates for MERRF disease.

Oral treatment with amitriptyline induces coenzyme Q deficiency and oxidative stress in psychiatric patients

Amitriptyline is a commonly prescribed tricyclic antidepressant, which has been shown to impair mitochondrial function and increase oxidative stress in a variety of in vitro assays. Coenzyme Q(10) (CoQ(10)), an essential component of the mitochondrial respiratory chain and a potent antioxidant, has been proposed as a mitochondrial dysfunction marker. In order to evaluate the putative mitochondrial toxicity of amitriptyline, we have analyzed CoQ(10) and ATP levels, oxidative damage and mitochondrial mass in peripheral blood cells from control healthy volunteers and psychiatric patients with depressive episodes treated or non-treated with amitriptyline. In patients not following amitriptyline treatment, CoQ(10) and ATP levels and mitochondrial mass were reduced when compared to normal individuals while lipid peroxidation was clearly increased. All these alterations were aggravated in patients following oral amitriptyline therapy. These results suggest that mitochondrial dysfunction could be involved in the pathophysiology of depression and may be worsened by amitriptyline treatment. CoQ(10) supplementation is postulated to counteract the adverse effects of amitriptyline treatment in psychiatric patients.

Acute oxidant damage promoted on cancer cells by amitriptyline in comparison with some common chemotherapeutic drugs

Oxidative therapy is a relatively new anticancer strategy based on the induction of high levels of oxidative stress, achieved by increasing intracellular reactive oxygen species (ROS) and/or by depleting the protective antioxidant machinery of tumor cells. We focused our investigations on the antitumoral potential of amitriptyline in three human tumor cell lines: H460 (lung cancer), HeLa (cervical cancer), and HepG2 (hepatoma); comparing the cytotoxic effect of amitriptyline with three commonly used chemotherapeutic drugs: camptothecin, doxorubicin, and methotrexate. We evaluated apoptosis, ROS production, mitochondrial mass and activity, and antioxidant defenses of tumor cells. Our results show that amitriptyline produces the highest cellular damage, inducing high levels of ROS followed by irreversible serious mitochondrial damage. Interestingly, an unexpected decrease in antioxidant machinery was observed only for amitriptyline. In conclusion, based on the capacity of generating ROS and inhibiting antioxidants in tumor cells, amitriptyline emerges as a promising new drug to be tested for anticancer therapy.

Screening of effective pharmacological treatments for MELAS syndrome using yeasts, fibroblasts and cybrid models of the disease.

BACKGROUND AND PURPOSE MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke‐like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT‐TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors.

Metformin and caloric restriction induce an AMPK-dependent restoration of mitochondrial dysfunction in fibroblasts from Fibromyalgia patients

Impaired AMPK is associated with a wide spectrum of clinical and pathological conditions, ranging from obesity, altered responses to exercise or metabolic syndrome, to inflammation, disturbed mitochondrial biogenesis and defective response to energy stress. Fibromyalgia (FM) is a world-wide diffused musculoskeletal chronic pain condition that affects up to 5% of the general population and comprises all the above mentioned pathophysiological states. Here, we tested the involvement of AMPK activation in fibroblasts derived from FM patients. AMPK was not phosphorylated in fibroblasts from FM patients and was associated with decreased mitochondrial biogenesis, reduced oxygen consumption, decreased antioxidant enzymes expression levels and mitochondrial dysfunction. However, mtDNA sequencing analysis did not show any important alterations which could justify the mitochondrial defects. AMPK activation in FM fibroblast was impaired in response to moderate oxidative stress. In contrast, AMPK activation by metformin or incubation with serum from caloric restricted mice improved the response to moderate oxidative stress and mitochondrial metabolism in FM fibroblasts. These results suggest that AMPK plays an essential role in FM pathophysiology and could represent the basis for a valuable new therapeutic target/strategy. Furthermore, both metformin and caloric restriction could be an interesting therapeutic approach in FM.

Amitriptyline induces mitophagy that precedes apoptosis in human HepG2 cells

Systemic treatments for hepatocellular carcinoma (HCC) have been largely unsuccessful. This study investigated the antitumoral activity of Amitriptyline, a tricyclic antidepressant, in hepatoma cells. Amitriptyline-induced toxicity involved early mitophagy activation that subsequently switched to apoptosis. Amitriptyline induced mitochondria dysfunction and oxidative stress in HepG2 cells. Amitriptyline specifically inhibited mitochondrial complex III activity that is associated with decreased mitochondrial membrane potential (∆Ψm) and increased reactive oxygen species (ROS) production. Transmission electron microscopy (TEM) studies revealed structurally abnormal mitochondria that were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitophagy activation, fluorescence microscopy analysis showed mitochondrial Parkin recruitment and colocalization of mitochondria with autophagosome protein markers. Pharmacological or genetic inhibition of autophagy exacerbated the deleterious effects of Amitriptyline on hepatoma cells and led to increased apoptosis. These results suggest that mitophagy acts as an initial adaptive mechanism of cell survival. However persistent mitochondrial damage induced extensive and lethal mitophagy, autophagy stress and autophagolysome permeabilization leading eventually to cell death by apoptosis. Amitriptyline also induced cell death in hepatoma cells lines with mutated p53 and non-sense p53 mutation. Our results support the hypothesis that Amitriptyline-induced mitochondrial dysfunction can be a useful therapeutic strategy for HCC treatment, especially in tumors showing p53 mutations and/or resistant to genotoxic treatments.