José A. Sánchez-Alcázar

Coenzyme Q deficiency triggers mitochondria degradation by mitophagy

Coenzyme Q10 (CoQ) is a small lipophilic molecule critical for the transport of electrons from complexes I and II to complex III in the mitochondrial respiratory chain. CoQ deficiency is a rare human genetic condition that has been associated with a variety of clinical phenotypes. With the aim of elucidating how CoQ deficiency affects an organism, we have investigated the pathophysiologic processes present within fibroblasts derived from 4 patients with CoQ deficiency. Assays of cultured fibroblasts revealed decreased activities of complex II+III, complex III, and complex IV, reduced expression of mitochondrial proteins involved in oxidative phosphorylation, decreased mitochondrial membrane potential, increased production of reactive oxygen species (ROS), activation of mitochondrial permeability transition (MPT), and reduced growth rates. These abnormalities were partially restored by CoQ supplementation. Moreover, we demonstrate that CoQ deficient fibroblasts exhibited increased levels of lysosomal markers (β-galactosidase, cathepsin, LC3, and Lyso Tracker), and enhanced expression of autophagic genes at both transcriptional and translational levels, indicating the presence of autophagy. Electron microscopy studies confirmed a massive degradation of the altered mitochondria by mitophagy. Autophagy in CoQ deficient fibroblasts was abolished by antioxidants or cyclosporin treatments suggesting that both ROS and MPT participate in this process. Furthermore, prevention of autophagy in CoQ deficient fibroblasts by 3-methyl adenine or wortmannin, as well as the induction of CoQ deficiency in cells lacking autophagy (by means of genetic knockout of the Atg5 gene in mouse embryonic fibroblasts) resulted in apoptotic cell death, suggesting a protective role of autophagy in CoQ deficiency.

NLRP3 inflammasome is activated in mononuclear blood cells from patients with major depressive disorder

INTRODUCTION Major depressive disorder (MDD) is a very prevalent disease which pathogenic mechanism remains elusive. There are some hypotheses and pilot studies suggesting that cytokines may play an important role in MDD. In this respect, we have investigated the role of NLRP3 inflammasome complex in the maturation of caspase-1 and the processing of its substrates, IL-1β and IL-18, in blood cells from MDD patients. METHODS Forty MDD patients were selected for this study, twenty without treatments and twenty treated with amitriptyline, a common tricyclic antidepressant. Blood samples from twenty healthy volunteers were included in the study. The inflammasome activation was studied by Western blot and real-time PCR of NLRP3 and caspase 1 and serum levels of IL-1β and 18. RESULTS We observed increased gene expression of NLRP3 and caspase-1 in blood cells, and increased serum levels of IL-1β and IL-18 in non-treated patients. IL-1β and IL-18 correlated with Beck Depression Inventory (BDI) scores of MDD patients. Interestingly, amitriptyline treatment reduced NLRP3 and caspase-1 gene expression, and IL-1β and IL-18 serum levels. As it is well established that oxidative stress is associated with NLRP3 inflammasome activation, we next studied mitochondrial ROS and lipid peroxidation (LPO) levels in MDD patients. Increased levels of mitochondrial ROS and LPO were observed in MDD patients, however oxidative damage was higher in MDD patients treated with amitriptyline. CONCLUSIONS These findings provide new insight into the pathogenesis of MDD and the effects of amitriptyline treatment on NLRP3 inflammasome activation and IL-1β and IL-18 serum levels.

Cerebellar ataxia with coenzyme Q10 deficiency: diagnosis and follow-up after coenzyme Q10 supplementation.

UNLABELLED Our aim was to report a new case with cerebellar ataxia associated with coenzyme Q10 (CoQ) deficiency, the biochemical findings caused by this deficiency and the response to CoQ supplementation. PATIENT A 12-year-old girl presenting ataxia and cerebellar atrophy. BIOCHEMICAL STUDIES: Coenzyme Q10 in muscle was analysed by HPLC with electrochemical detection and mitochondrial respiratory chain (MRC) enzyme activities by spectrophotometric methods. CoQ biosynthesis in fibroblasts was assayed by studying the incorporation of radiolabeled 4-hydroxy[U 14C] benzoic acid by HPLC with radiometric detection. RESULTS Mitochondrial respiratory chain enzyme analysis showed a decrease in complex I + III and complex II + III activities. CoQ concentration in muscle was decreased (56 nmol/g of protein: reference values: 157-488 nmol/g protein). A reduced incorporation of radiolabeled 4-hydroxy[U- 14C] benzoic acid was observed in the patient (19% of incorporation respect to the median control values). After 16 months of CoQ supplementation, the patient is now able to walk unaided and cerebellar signs have disappeared. CONCLUSIONS Cerebellar ataxia associated with CoQ deficiency in our case might be allocated in the transprenylation pathway or in the metabolic steps after condensation of 4-hydroxybenzoate and the prenyl side chain of CoQ. Clinical improvement after CoQ supplementation was remarkable, supporting the importance of an early diagnosis of this kind of disorders.

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

Cyclooxygenase (COX) inhibitors induce apoptosis in non-small cell lung cancer through cyclooxygenase independent pathways.

Cyclooxygenase (COX) inhibitors are chemopreventive in many tumours but the role of COX inhibition in their effects is contentious. Here we determined if COX inhibitors influenced apoptosis in two non-small cell lung cancer cells one which over expresses COX-2 (MOR-P) and one which expresses neither isoform (H-460). NS398, a selective COX inhibitor, and indomethacin, a non-selective COX inhibitor, were cytotoxic in both cell lines, independently of their COX-2 expression. Furthermore, the cytotoxic concentrations were far greater than the concentrations required to inhibit COX. As indomethacin was more effective we used it in mechanistic studies. Indomethacin induced apoptotic cell death assessed as cytochrome c and apoptotic inducing factor (AIF) release, caspase activation, PARP, lamin B and gelsolin cleavage, chromatin condensation and nuclear fragmentation. The pan-caspase inhibitor, z-VAD, attenuated cell death, and blocked caspase activation, PARP cleavage and nuclear fragmentation without preventing cytochrome c release, suggesting that cytochrome c release is upstream of caspase activation. These observations suggest that COX inhibitors induce apoptosis in non-small lung cancer cells through cytochrome c and AIF release, and subsequent caspase activation, independently of COX-2 expression and prostaglandin production.

Tumor necrosis factor-alpha increases ATP content in metabolically inhibited L929 cells preceding cell death.

The effects of tumor necrosis factor-α (TNF) on ATP levels were studied in metabolically inhibited L929 cells. Treatment of these cells with TNF in the presence of actinomycin D or cycloheximide induces cyclic changes in the intracellular ATP content preceding cell death. After 3 h of incubation, the intracellular ATP content increased by 48 ± 6% (p < 0.001), but at 4 h, it decreased to the control level. Two hours later, it increased again by 23 ± 5% over the control level (p < 0.001). Coinciding with cell death, ATP content decreased progressively until almost complete depletion. These changes in ATP content were associated with parallel alterations in the respiratory coupling and with increased generation of reactive oxygen species. The mechanism by which TNF/actinomycin D or TNF/cycloheximide increased cellular ATP seemed to be dependent on the mitochondrial ATP synthesis and related to the cytotoxic effect of TNF, since blockade of mitochondrial electron transport prevented the increase in cellular ATP, the formation of reactive oxygen species, and the apoptotic cell death caused by TNF. We suggest that the TNF/actinomycin D- or TNF/cycloheximide-induced changes in intracellular ATP levels may be involved in the cytotoxic effect of TNF in metabolically inhibited L929 cells.

Analysis of Coenzyme Q10 in muscle and fibroblasts for the diagnosis of CoQ10 deficiency syndromes

OBJECTIVES To study CoQ(10) concentrations in muscle and fibroblast from 6 patients with a CoQ(10) deficiency syndrome. DESIGN AND METHODS CoQ(10) was quantified by HPLC with electrochemical detection. RESULTS Four out of the 6 cases showed muscle CoQ(10) deficiency plus a reduction of mitochondrial respiratory chain enzyme activities. All cases showed decreased CoQ(10) values in fibroblasts when compared with controls. CONCLUSIONS Biochemical study of CoQ(10) in both muscle and fibroblasts seems advisable to demonstrate the deficiency in all patients.

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.

Oxidative Stress Correlates with Headache Symptoms in Fibromyalgia: Coenzyme Q10 Effect on Clinical Improvement

Background Fibromyalgia (FM) is a chronic pain syndrome with unknown etiology and a wide spectrum of symptoms such as allodynia, debilitating fatigue, joint stiffness and migraine. Recent studies have shown some evidences demonstrating that oxidative stress is associated to clinical symptoms in FM of fibromyalgia. We examined oxidative stress and bioenergetic status in blood mononuclear cells (BMCs) and its association to headache symptoms in FM patients. The effects of oral coenzyme Q10 (CoQ10) supplementation on biochemical markers and clinical improvement were also evaluated. Methods We studied 20 FM patients and 15 healthy controls. Clinical parameters were evaluated using the Fibromyalgia Impact Questionnaire (FIQ), visual analogues scales (VAS), and the Headache Impact Test (HIT-6). Oxidative stress was determined by measuring CoQ10, catalase and lipid peroxidation (LPO) levels in BMCs. Bioenergetic status was assessed by measuring ATP levels in BMCs. Results We found decreased CoQ10, catalase and ATP levels in BMCs from FM patients as compared to normal control (P<0.05 and P<0.001, respectively) We also found increased level of LPO in BMCs from FM patients as compared to normal control (P<0.001). Significant negative correlations between CoQ10 or catalase levels in BMCs and headache parameters were observed (r = −0.59, P<0.05; r = −0.68, P<0.05, respectively). Furthermore, LPO levels showed a significant positive correlation with HIT-6 (r = 0.33, P<0.05). Oral CoQ10 supplementation restored biochemical parameters and induced a significant improvement in clinical and headache symptoms (P<0.001). Discussion The results of this study suggest a role for mitochondrial dysfunction and oxidative stress in the headache symptoms associated with FM. CoQ10 supplementation should be examined in a larger placebo controlled trial as a possible treatment in FM.

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.