In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

IntroductionFibromyalgia is a chronic pain syndrome with unknown etiology. Recent studies have shown some evidence demonstrating that oxidative stress may have a role in the pathophysiology of fibromyalgia. However, it is still not clear whether oxidative stress is the cause or the effect of the abnormalities documented in fibromyalgia. Furthermore, the role of mitochondria in the redox imbalance reported in fibromyalgia also is controversial. We undertook this study to investigate the role of mitochondrial dysfunction, oxidative stress, and mitophagy in fibromyalgia.MethodsWe studied 20 patients (2 male, 18 female patients) from the database of the Sevillian Fibromyalgia Association and 10 healthy controls. We evaluated mitochondrial function in blood mononuclear cells from fibromyalgia patients measuring, coenzyme Q10 levels with high-performance liquid chromatography (HPLC), and mitochondrial membrane potential with flow cytometry. Oxidative stress was determined by measuring mitochondrial superoxide production with MitoSOX™ and lipid peroxidation in blood mononuclear cells and plasma from fibromyalgia patients. Autophagy activation was evaluated by quantifying the fluorescence intensity of LysoTracker™ Red staining of blood mononuclear cells. Mitophagy was confirmed by measuring citrate synthase activity and electron microscopy examination of blood mononuclear cells.ResultsWe found reduced levels of coenzyme Q10, decreased mitochondrial membrane potential, increased levels of mitochondrial superoxide in blood mononuclear cells, and increased levels of lipid peroxidation in both blood mononuclear cells and plasma from fibromyalgia patients. Mitochondrial dysfunction was also associated with increased expression of autophagic genes and the elimination of dysfunctional mitochondria with mitophagy.ConclusionsThese findings may support the role of oxidative stress and mitophagy in the pathophysiology of fibromyalgia.

Mitochondria play a central role in apoptosis through release of cytochrome c and activation of caspases. In the present study, we showed that, in Jurkat human T cells, camptothecininduced apoptosis is preceded by (i) an increase in cytochrome c and subunit IV of cytochrome c oxidase (COX IV) levels in mitochondria; and (ii) an elevation of the mitochondrial membrane potential (DC m ). These events are followed by cytochrome c release into the cytosol, cytochrome c and COX IV depletion from mitochondria, externalization of phosphatidylserine (PS), disruption of DC m , caspase activation, poly(ADP-ribose)polymerase cleavage and DNA fragmentation. The pan-caspase inhibitor z-VAD.fmk blocked camptothecin-induced PS externalization, disruption of DC m and DNA fragmentation, suggesting that these events are mediated by caspase activation. In contrast, z-VAD did not prevent cytochrome c release, despite preventing cytochrome c and COX IV depletion from mitochondria. Together, these data suggest that mitochondrial cytochrome c and COX IV enrichment are early events preceding the onset of apoptosis and that cytochrome c release is upstream of caspase activation and loss of DC m . Furthermore, prevention by z-VAD of cytochrome c and COX IV depletion in mitochondria suggests the possibility that a caspase-like activity in mitochondria is involved in the proteolytic depletion of respiratory chain proteins. Activation of this activity may play an important role in drug-induced apoptosis.

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(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.

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.

No abstract

No abstract