Alpha-lipoic acid supplementation corrects pathological alterations in cellular models of pantothenate kinase-associated neurodegeneration with residual PANK2 expression levels.

BACKGROUND: Neurodegeneration with brain iron accumulation (NBIA) disorders are a group of neurodegenerative diseases that have in common the accumulation of iron in the basal nuclei of the brain which are essential components of the extrapyramidal system. Frequent symptoms are progressive spasticity, dystonia, muscle rigidity, neuropsychiatric symptoms, and retinal degeneration or optic nerve atrophy. One of the most prevalent subtypes of NBIA is Pantothenate kinase-associated neurodegeneration (PKAN). It is caused by pathogenic variants in the gene of pantothenate kinase 2 (PANK2) which encodes the enzyme responsible for the first reaction on the coenzyme A (CoA) biosynthesis pathway. Thus, deficient PANK2 activity induces CoA deficiency as well as low expression levels of 4'-phosphopantetheinyl proteins which are essential for mitochondrial metabolism. METHODS: This study is aimed at evaluating the role of alpha-lipoic acid (α-LA) in reversing the pathological alterations in fibroblasts and induced neurons derived from PKAN patients. Iron accumulation, lipid peroxidation, transcript and protein expression levels of PANK2, mitochondrial ACP (mtACP), 4''-phosphopantetheinyl and lipoylated proteins, as well as pyruvate dehydrogenase (PDH) and Complex I activity were examined. RESULTS: Treatment with α-LA was able to correct all pathological alterations in responsive mutant fibroblasts with residual PANK2 enzyme expression. However, α-LA had no effect on mutant fibroblasts with truncated/incomplete protein expression. The positive effect of α-LA in particular pathogenic variants was also confirmed in induced neurons derived from mutant fibroblasts. CONCLUSIONS: Our results suggest that α-LA treatment can increase the expression levels of PANK2 and reverse the mutant phenotype in PANK2 responsive pathogenic variants. The existence of residual enzyme expression in some affected individuals raises the possibility of treatment using high dose of α-LA.

Pantothenate and L-Carnitine Supplementation Improves Pathological Alterations in Cellular Models of KAT6A Syndrome.

Mutations in several genes involved in the epigenetic regulation of gene expression have been considered risk alterations to different intellectual disability (ID) syndromes associated with features of autism spectrum disorder (ASD). Among them are the pathogenic variants of the lysine-acetyltransferase 6A (KAT6A) gene, which causes KAT6A syndrome. The KAT6A enzyme participates in a wide range of critical cellular functions, such as chromatin remodeling, gene expression, protein synthesis, cell metabolism, and replication. In this manuscript, we examined the pathophysiological alterations in fibroblasts derived from three patients harboring KAT6A mutations. We addressed survival in a stress medium, histone acetylation, protein expression patterns, and transcriptome analysis, as well as cell bioenergetics. In addition, we evaluated the therapeutic effectiveness of epigenetic modulators and mitochondrial boosting agents, such as pantothenate and L-carnitine, in correcting the mutant phenotype. Pantothenate and L-carnitine treatment increased histone acetylation and partially corrected protein and transcriptomic expression patterns in mutant KAT6A cells. Furthermore, the cell bioenergetics of mutant cells was significantly improved. Our results suggest that pantothenate and L-carnitine can significantly improve the mutant phenotype in cellular models of KAT6A syndrome.

Therapeutic approach with commercial supplements for pantothenate kinase-associated neurodegeneration with residual PANK2 expression levels.

BACKGROUND: Neurodegeneration with brain iron accumulation (NBIA) is a group of rare neurogenetic disorders frequently associated with iron accumulation in the basal nuclei of the brain characterized by progressive spasticity, dystonia, muscle rigidity, neuropsychiatric symptoms, and retinal degeneration or optic nerve atrophy. Pantothenate kinase-associated neurodegeneration (PKAN) is one of the most widespread NBIA subtypes. It is caused by mutations in the gene of pantothenate kinase 2 (PANK2) that result in dysfunction in PANK2 enzyme activity, with consequent deficiency of coenzyme A (CoA) biosynthesis, as well as low levels of essential metabolic intermediates such as 4'-phosphopantetheine, a necessary cofactor for essential cytosolic and mitochondrial proteins. METHODS: In this manuscript, we examined the therapeutic effectiveness of pantothenate, panthetine, antioxidants (vitamin E and omega 3) and mitochondrial function boosting supplements (L-carnitine and thiamine) in mutant PANK2 cells with residual expression levels. RESULTS: Commercial supplements, pantothenate, pantethine, vitamin E, omega 3, carnitine and thiamine were able to eliminate iron accumulation, increase PANK2, mtACP, and NFS1 expression levels and improve pathological alterations in mutant cells with residual PANK2 expression levels. CONCLUSION: Our results suggest that several commercial compounds are indeed able to significantly correct the mutant phenotype in cellular models of PKAN. These compounds alone or in combinations are of common use in clinical practice and may be useful for the treatment of PKAN patients with residual enzyme expression levels.

Vitamin E prevents lipid peroxidation and iron accumulation in PLA2G6-Associated Neurodegeneration.

BACKGROUND: PLA2G6-Associated Neurodegeneration (PLAN) is a rare neurodegenerative disease with autosomal recessive inheritance, which belongs to the NBIA (Neurodegeneration with Brain Iron Accumulation) group. Although the pathogenesis of the disease remains largely unclear, lipid peroxidation seems to play a central role in the pathogenesis. Currently, there is no cure for the disease. OBJECTIVE: In this work, we examined the presence of lipid peroxidation, iron accumulation and mitochondrial dysfunction in two cellular models of PLAN, patients-derived fibroblasts and induced neurons, and assessed the effects of α-tocopherol (vitamin E) in correcting the pathophysiological alterations in PLAN cell cultures. METHODS: Pathophysiological alterations were examined in fibroblasts and induced neurons generated by direct reprograming. Iron and lipofuscin accumulation were assessed using light and electron microscopy, as well as biochemical analysis techniques. Reactive Oxygen species production, lipid peroxidation and mitochondrial dysfunction were measured using specific fluorescent probes analysed by fluorescence microscopy and flow cytometry. RESULTS: PLAN fibroblasts and induced neurons clearly showed increased lipid peroxidation, iron accumulation and altered mitochondrial membrane potential. All these pathological features were reverted with vitamin E treatment. CONCLUSIONS: PLAN fibroblasts and induced neurons reproduce the main pathological alterations of the disease and provide useful tools for disease modelling. The main pathological alterations were corrected by Vitamin E supplementation in both models, suggesting that blocking lipid peroxidation progression is a critical therapeutic target.

Coenzyme Q10 Analogues: Benefits and Challenges for Therapeutics.

Coenzyme Q10 (CoQ10 or ubiquinone) is a mobile proton and electron carrier of the mitochondrial respiratory chain with antioxidant properties widely used as an antiaging health supplement and to relieve the symptoms of many pathological conditions associated with mitochondrial dysfunction. Even though the hegemony of CoQ10 in the context of antioxidant-based treatments is undeniable, the future primacy of this quinone is hindered by the promising features of its numerous analogues. Despite the unimpeachable performance of CoQ10 therapies, problems associated with their administration and intraorganismal delivery has led clinicians and scientists to search for alternative derivative molecules. Over the past few years, a wide variety of CoQ10 analogues with improved properties have been developed. These analogues conserve the antioxidant features of CoQ10 but present upgraded characteristics such as water solubility or enhanced mitochondrial accumulation. Moreover, recent studies have proven that some of these analogues might even outperform CoQ10 in the treatment of certain specific diseases. The aim of this review is to provide detailed information about these Coenzyme Q10 analogues, as well as their functionality and medical applications.

Parkin-mediated mitophagy and autophagy flux disruption in cellular models of MERRF syndrome.

Mitochondrial diseases are considered rare genetic disorders characterized by defects in oxidative phosphorylation (OXPHOS). They can be provoked by mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). MERRF (Myoclonic Epilepsy with Ragged-Red Fibers) syndrome is one of the most frequent mitochondrial diseases, principally caused by the m.8344A>G mutation in mtDNA, which affects the translation of all mtDNA-encoded proteins and therefore impairs mitochondrial function. In the present work, we evaluated autophagy and mitophagy flux in transmitochondrial cybrids and fibroblasts derived from a MERRF patient, reporting that Parkin-mediated mitophagy is increased in MERRF cell cultures. Our results suggest that supplementation with coenzyme Q10 (CoQ), a component of the electron transport chain (ETC) and lipid antioxidant, prevents Parkin translocation to the mitochondria. In addition, CoQ acts as an enhancer of autophagy and mitophagy flux, which partially improves cell pathophysiology. The significance of Parkin-mediated mitophagy in cell survival was evaluated by silencing the expression of Parkin in MERRF cybrids. Our results show that mitophagy acts as a cell survival mechanism in mutant cells. To confirm these results in one of the main affected cell types in MERRF syndrome, mutant induced neurons (iNs) were generated by direct reprogramming of patients-derived skin fibroblasts. The treatment of MERRF iNs with Guttaquinon CoQ10 (GuttaQ), a water-soluble derivative of CoQ, revealed a significant improvement in cell bioenergetics. These results indicate that iNs, along with fibroblasts and cybrids, can be utilized as reliable cellular models to shed light on disease pathomechanisms as well as for drug screening.

Atherosclerosis and Coenzyme Q10.

Atherosclerosis is the most common cause of cardiac deaths worldwide. Classically, atherosclerosis has been explained as a simple arterial lipid deposition with concomitant loss of vascular elasticity. Eventually, this condition can lead to consequent blood flow reduction through the affected vessel. However, numerous studies have demonstrated that more factors than lipid accumulation are involved in arterial damage at the cellular level, such as inflammation, autophagy impairment, mitochondrial dysfunction, and/or free-radical overproduction. In order to consider the correction of all of these pathological changes, new approaches in atherosclerosis treatment are necessary. Ubiquinone or coenzyme Q10 is a multifunctional molecule that could theoretically revert most of the cellular alterations found in atherosclerosis, such as cholesterol biosynthesis dysregulation, impaired autophagy flux and mitochondrial dysfunction thanks to its redox and signaling properties. In this review, we will show the latest advances in the knowledge of the relationships between coenzyme Q10 and atherosclerosis. In addition, as atherosclerosis phenotype is closely related to aging, it is reasonable to believe that coenzyme Q10 supplementation could be beneficial for both conditions.

Precision medicine in pantothenate kinase-associated neurodegeneration.

Neurodegeneration with brain iron accumulation is a broad term that describes a heterogeneous group of progressive and invalidating neurologic disorders in which iron deposits in certain brain areas, mainly the basal ganglia. The predominant clinical symptoms include spasticity, progressive dystonia, Parkinson's disease-like symptoms, neuropsychiatric alterations, and retinal degeneration. Among the neurodegeneration with brain iron accumulation disorders, the most frequent subtype is pantothenate kinase-associated neurodegeneration (PKAN) caused by defects in the gene encoding the enzyme pantothenate kinase 2 (PANK2) which catalyzed the first reaction of the coenzyme A biosynthesis pathway. Currently there is no effective treatment to prevent the inexorable course of these disorders. The aim of this review is to open up a discussion on the utility of using cellular models derived from patients as a valuable tool for the development of precision medicine in PKAN. Recently, we have described that dermal fibroblasts obtained from PKAN patients can manifest the main pathological changes of the disease such as intracellular iron accumulation accompanied by large amounts of lipofuscin granules, mitochondrial dysfunction and a pronounced increase of markers of oxidative stress. In addition, PKAN fibroblasts showed a morphological senescence-like phenotype. Interestingly, pantothenate supplementation, the substrate of the PANK2 enzyme, corrected all pathophysiological alterations in responder PKAN fibroblasts with low/residual PANK2 enzyme expression. However, pantothenate treatment had no favourable effect on PKAN fibroblasts harbouring mutations associated with the expression of a truncated/incomplete protein. The correction of pathological alterations by pantothenate in individual mutations was also verified in induced neurons obtained by direct reprograming of PKAN fibroblasts. Our observations indicate that pantothenate supplementation can increase/stabilize the expression levels of PANK2 in specific mutations. Fibroblasts and induced neurons derived from patients can provide a useful tool for recognizing PKAN patients who can respond to pantothenate treatment. The presence of low but significant PANK2 expression which can be increased in particular mutations gives valuable information which can support the treatment with high dose of pantothenate. The evaluation of personalized treatments in vitro of fibroblasts and neuronal cells derived from PKAN patients with a wide range of pharmacological options currently available, and monitoring its effect on the pathophysiological changes, can help for a better therapeutic strategy. In addition, these cell models will be also useful for testing the efficacy of new therapeutic options developed in the future.

Pantothenate Rescues Iron Accumulation in Pantothenate Kinase-Associated Neurodegeneration Depending on the Type of Mutation.

Neurodegeneration with brain iron accumulation (NBIA) is a group of inherited neurologic disorders in which iron accumulates in the basal ganglia resulting in progressive dystonia, spasticity, parkinsonism, neuropsychiatric abnormalities, and optic atrophy or retinal degeneration. The most prevalent form of NBIA is pantothenate kinase-associated neurodegeneration (PKAN) associated with mutations in the gene of pantothenate kinase 2 (PANK2), which is essential for coenzyme A (CoA) synthesis. There is no cure for NBIA nor is there a standard course of treatment. In the current work, we describe that fibroblasts derived from patients harbouring PANK2 mutations can reproduce many of the cellular pathological alterations found in the disease, such as intracellular iron and lipofuscin accumulation, increased oxidative stress, and mitochondrial dysfunction. Furthermore, mutant fibroblasts showed a characteristic senescent morphology. Treatment with pantothenate, the PANK2 enzyme substrate, was able to correct all pathological alterations in responder mutant fibroblasts with residual PANK2 enzyme expression. However, pantothenate had no effect on mutant fibroblasts with truncated/incomplete protein expression. The positive effect of pantothenate in particular mutations was also confirmed in induced neurons obtained by direct reprograming of mutant fibroblasts. Our results suggest that pantothenate treatment can stabilize the expression levels of PANK2 in selected mutations. These results encourage us to propose our screening model as a quick and easy way to detect pantothenate-responder patients with PANK2 mutations. The existence of residual enzyme expression in some affected individuals raises the possibility of treatment using high dose of pantothenate.

Intracellular cholesterol accumulation and coenzyme Q10 deficiency in Familial Hypercholesterolemia.

Familial Hypercholesterolemia (FH) is an autosomal co-dominant genetic disorder characterized by elevated low-density lipoprotein (LDL) cholesterol levels and increased risk for premature cardiovascular disease. Here, we examined FH pathophysiology in skin fibroblasts derived from FH patients harboring heterozygous mutations in the LDL-receptor. Fibroblasts from FH patients showed a reduced LDL-uptake associated with increased intracellular cholesterol levels and coenzyme Q10 (CoQ10) deficiency, suggesting dysregulation of the mevalonate pathway. Secondary CoQ10 deficiency was associated with mitochondrial depolarization and mitophagy activation in FH fibroblasts. Persistent mitophagy altered autophagy flux and induced inflammasome activation accompanied by increased production of cytokines by mutant cells. All the pathological alterations in FH fibroblasts were also reproduced in a human endothelial cell line by LDL-receptor gene silencing. Both increased intracellular cholesterol and mitochondrial dysfunction in FH fibroblasts were partially restored by CoQ10 supplementation. Dysregulated mevalonate pathway in FH, including increased expression of cholesterogenic enzymes and decreased expression of CoQ10 biosynthetic enzymes, was also corrected by CoQ10 treatment. Reduced CoQ10 content and mitochondrial dysfunction may play an important role in the pathophysiology of early atherosclerosis in FH. The diagnosis of CoQ10 deficiency and mitochondrial impairment in FH patients may also be important to establish early treatment with CoQ10.

Coenzyme Q10 partially restores pathological alterations in a macrophage model of Gaucher disease.

BACKGROUND: Gaucher disease (GD) is caused by mutations in the GBA1 gene which encodes lysosomal ß-glucocerebrosidase (GCase). In GD, partial or complete loss of GCase activity causes the accumulation of the glycolipids glucosylceramide (GlcCer) and glucosylsphingosine in the lysosomes of macrophages. In this manuscript, we investigated the effects of glycolipids accumulation on lysosomal and mitochondrial function, inflammasome activation and efferocytosis capacity in a THP-1 macrophage model of Gaucher disease. In addition, the beneficial effects of coenzyme Q10 (CoQ) supplementation on cellular alterations were evaluated. Chemically-induced Gaucher macrophages were developed by differentiateing THP-1 monocytes to macrophages by treatment with phorbol 12-myristate 13-acetate (PMA) and then inhibiting intracellular GCase with conduritol B-epoxide (CBE), a specific irreversible inhibitor of GCase activity, and supplementing the medium with exogenous GlcCer. This cell model accumulated up to 16-fold more GlcCer compared with control THP-1 cells. RESULTS: Chemically-induced Gaucher macrophages showed impaired autophagy flux associated with mitochondrial dysfunction and increased oxidative stress, inflammasome activation and impaired efferocytosis. All abnormalities were partially restored by supplementation with CoQ. CONCLUSION: These data suggest that targeting mitochondria function and oxidative stress by CoQ can ameliorate the pathological phenotype of Gaucher cells. Chemically-induced Gaucher macrophages provide cellular models that can be used to investigate disease pathogenesis and explore new therapeutics for GD.

AMPK Regulation of Cell Growth, Apoptosis, Autophagy, and Bioenergetics.

In eukaryotic cells, AMP-activated protein kinase (AMPK) generally promotes catabolic pathways that produce ATP and at the same time inhibits anabolic pathways involved in different processes that consume ATP. As an energy sensor, AMPK is involved in the main cellular functions implicated in cell fate, such as cell growth and autophagy.Recently, AMPK has been connected with apoptosis regulation, although the molecular mechanism by which AMPK induces and/or inhibits cell death is not clear.This chapter reviews the essential role of AMPK in signaling pathways that respond to cellular stress and damage, highlighting the complex and reciprocal regulation between AMPK and their targets and effectors. The therapeutic implications of the role of AMPK in different pathologies such as diabetes, cancer, or mitochondrial dysfunctions are still controversial, and it is necessary to further investigate the molecular mechanisms underlying AMPK activation.

Pharmacological Chaperones and Coenzyme Q10 Treatment Improves Mutant ß-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 ß-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 ß-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.

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.

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. EXPERIMENTAL APPROACH: We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. KEY RESULTS: According to our results, supplementation with riboflavin or coenzyme Q(10) effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. CONCLUSIONS AND IMPLICATIONS: Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment.

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 RNA(Lysine), 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 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 CoQ10 deficiency, mitochondrial dysfunction, and increased mitophagy activity. All these abnormalities in patient-derived fibroblasts and cybrids were partially restored by CoQ10 supplementation, indicating that these cell culture models may be suitable for screening and validation of novel drug candidates for MERRF disease.

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 Q(10) (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.

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 (beta-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.

Missense mutation of the COQ2 gene causes defects of bioenergetics and de novo pyrimidine synthesis.

Coenzyme Q(10) (CoQ(10)) deficiency has been associated with an increasing number of clinical phenotypes that respond to CoQ(10) supplementation. In two siblings with encephalomyopathy, nephropathy and severe CoQ(10) deficiency, a homozygous mutation was identified in the CoQ(10) biosynthesis gene COQ2, encoding polyprenyl-pHB transferase. To confirm the pathogenicity of this mutation, we have demonstrated that human wild-type, but not mutant COQ2, functionally complements COQ2 defective yeast. In addition, an equivalent mutation introduced in the yeast COQ2 gene also decreases both CoQ(6) concentration and growth in respiratory-chain dependent medium. Polyprenyl-pHB transferase activity was 33-45% of controls in COQ2 mutant fibroblasts. CoQ-dependent mitochondrial complexes activities were restored in deficient fibroblasts by CoQ(10) supplementation, and growth rate was restored in these cells by either CoQ(10) or uridine supplementation. This work is the first direct demonstration of the pathogenicity of a COQ2 mutation involved in human disease, and establishes yeast as a useful model to study human CoQ(10) deficiency. Moreover, we demonstrate that CoQ(10) deficiency in addition to the bioenergetics defect also impairs de novo pyrimidine synthesis, which may contribute to the pathogenesis of the disease.

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.