Inhibition of mammalian mtDNA transcription acts paradoxically to reverse diet-induced hepatosteatosis and obesity.

The oxidative phosphorylation system1 in mammalian mitochondria plays a key role in transducing energy from ingested nutrients2. Mitochondrial metabolism is dynamic and can be reprogrammed to support both catabolic and anabolic reactions, depending on physiological demands or disease states. Rewiring of mitochondrial metabolism is intricately linked to metabolic diseases and promotes tumour growth3-5. Here, we demonstrate that oral treatment with an inhibitor of mitochondrial transcription (IMT)6 shifts whole-animal metabolism towards fatty acid oxidation, which, in turn, leads to rapid normalization of body weight, reversal of hepatosteatosis and restoration of normal glucose tolerance in male mice on a high-fat diet. Paradoxically, the IMT treatment causes a severe reduction of oxidative phosphorylation capacity concomitant with marked upregulation of fatty acid oxidation in the liver, as determined by proteomics and metabolomics analyses. The IMT treatment leads to a marked reduction of complex I, the main dehydrogenase feeding electrons into the ubiquinone (Q) pool, whereas the levels of electron transfer flavoprotein dehydrogenase and other dehydrogenases connected to the Q pool are increased. This rewiring of metabolism caused by reduced mtDNA expression in the liver provides a principle for drug treatment of obesity and obesity-related pathology.

A role for BCL2L13 and autophagy in germline purifying selection of mtDNA.

Mammalian mitochondrial DNA (mtDNA) is inherited uniparentally through the female germline without undergoing recombination. This poses a major problem as deleterious mtDNA mutations must be eliminated to avoid a mutational meltdown over generations. At least two mechanisms that can decrease the mutation load during maternal transmission are operational: a stochastic bottleneck for mtDNA transmission from mother to child, and a directed purifying selection against transmission of deleterious mtDNA mutations. However, the molecular mechanisms controlling these processes remain unknown. In this study, we systematically tested whether decreased autophagy contributes to purifying selection by crossing the C5024T mouse model harbouring a single pathogenic heteroplasmic mutation in the tRNAAla gene of the mtDNA with different autophagy-deficient mouse models, including knockouts of Parkin, Bcl2l13, Ulk1, and Ulk2. Our study reveals a statistically robust effect of knockout of Bcl2l13 on the selection process, and weaker evidence for the effect of Ulk1 and potentially Ulk2, while no statistically significant impact is seen for knockout of Parkin. This points at distinctive roles of these players in germline purifying selection. Overall, our approach provides a framework for investigating the roles of other important factors involved in the enigmatic process of purifying selection and guides further investigations for the role of BCL2L13 in the elimination of non-synonymous mutations in protein-coding genes.

Metabolic resistance to the inhibition of mitochondrial transcription revealed by CRISPR-Cas9 screen.

Cancer cells depend on mitochondria to sustain their increased metabolic need and mitochondria therefore constitute possible targets for cancer treatment. We recently developed small-molecule inhibitors of mitochondrial transcription (IMTs) that selectively impair mitochondrial gene expression. IMTs have potent antitumor properties in vitro and in vivo, without affecting normal tissues. Because therapy-induced resistance is a major constraint to successful cancer therapy, we investigated mechanisms conferring resistance to IMTs. We employed a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats)-(CRISP-associated protein 9) whole-genome screen to determine pathways conferring resistance to acute IMT1 treatment. Loss of genes belonging to von Hippel-Lindau (VHL) and mammalian target of rapamycin complex 1 (mTORC1) pathways caused resistance to acute IMT1 treatment and the relevance of these pathways was confirmed by chemical modulation. We also generated cells resistant to chronic IMT treatment to understand responses to persistent mitochondrial gene expression impairment. We report that IMT1-acquired resistance occurs through a compensatory increase of mitochondrial DNA (mtDNA) expression and cellular metabolites. We found that mitochondrial transcription factor A (TFAM) downregulation and inhibition of mitochondrial translation impaired survival of resistant cells. The identified susceptibility and resistance mechanisms to IMTs may be relevant for different types of mitochondria-targeted therapies.

The one-carbon pool controls mitochondrial energy metabolism via complex I and iron-sulfur clusters.

Induction of the one-carbon cycle is an early hallmark of mitochondrial dysfunction and cancer metabolism. Vital intermediary steps are localized to mitochondria, but it remains unclear how one-carbon availability connects to mitochondrial function. Here, we show that the one-carbon metabolite and methyl group donor S-adenosylmethionine (SAM) is pivotal for energy metabolism. A gradual decline in mitochondrial SAM (mitoSAM) causes hierarchical defects in fly and mouse, comprising loss of mitoSAM-dependent metabolites and impaired assembly of the oxidative phosphorylation system. Complex I stability and iron-sulfur cluster biosynthesis are directly controlled by mitoSAM levels, while other protein targets are predominantly methylated outside of the organelle before import. The mitoSAM pool follows its cytosolic production, establishing mitochondria as responsive receivers of one-carbon units. Thus, we demonstrate that cellular methylation potential is required for energy metabolism, with direct relevance for pathophysiology, aging, and cancer.

Mitochondrial DNA copy number in human disease: the more the better?

Most of the genetic information has been lost or transferred to the nucleus during the evolution of mitochondria. Nevertheless, mitochondria have retained their own genome that is essential for oxidative phosphorylation (OXPHOS). In mammals, a gene-dense circular mitochondrial DNA (mtDNA) of about 16.5 kb encodes 13 proteins, which constitute only 1% of the mitochondrial proteome. Mammalian mtDNA is present in thousands of copies per cell and mutations often affect only a fraction of them. Most pathogenic human mtDNA mutations are recessive and only cause OXPHOS defects if present above a certain critical threshold. However, emerging evidence strongly suggests that the proportion of mutated mtDNA copies is not the only determinant of disease but that also the absolute copy number matters. In this review, we critically discuss current knowledge of the role of mtDNA copy number regulation in various types of human diseases, including mitochondrial disorders, neurodegenerative disorders and cancer, and during ageing. We also provide an overview of new exciting therapeutic strategies to directly manipulate mtDNA to restore OXPHOS in mitochondrial diseases.

Defects of mitochondrial RNA turnover lead to the accumulation of double-stranded RNA in vivo.

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.

Superoxide Dismutases SOD1 and SOD2 Rescue the Toxic Effect of Dopamine-Derived Products in Human SH-SY5Y Neuroblastoma Cells.

The preferential loss of dopaminergic neurons in the substantia nigra pars compacta is one of the pathological hallmarks characterizing Parkinson's disease. Although the pathogenesis of this disorder is not fully understood, oxidative stress plays a central role in the onset and/or progression of Parkinson's disease and dopamine itself has been suggested to participate in the preferential neuronal degeneration through the induction of oxidative conditions. In fact, the accumulation of dopamine into the cytosol can lead to the formation of reactive oxygen species as well as highly reactive dopamine-quinones. In the present work, we first analyzed the cellular damage induced by the addition of dopamine (DA) in the culture medium of SH-SY5Y cells, discriminating whether the harmful effects were related to the generation of reactive oxygen species or to the toxicity associated to dopamine-derived quinones. Then, we tested and demonstrated the capability of the antioxidant enzymes SOD1 and SOD2 to protect cells from the noxious effects induced by DA treatment. Our results support further exploration of superoxide dismutating molecules as a therapeutic strategy against Parkinson's disease.

The Clinical Use of the Neutrophil to Lymphocyte Ratio (NLR) in Urothelial Cancer: A Systematic Review.

The neutrophil to lymphocyte ratio (NLR) is an inflammatory index that has been considered as a potential prognostic factor in human cancer. The aim of this study was to evaluate the available evidence regarding the NLR as a prognostic value in patients affected by urothelial cancer. This literature review, including papers on NLR in urothelial cancers, was done on PubMed/Medline and Cochrane libraries in November 2015. The selection of the articles followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses process. Twenty-three of 99 articles fulfilled all the inclusion criteria, including data on 6240 patients affected by urothelial cancers. Overall, cancer-specific, and recurrence-free survival were evaluated as the main oncological outcomes. There was significant heterogeneity among studies, and the majority of studies were of poor quality. Overall, NLR was considered as a prognostic marker in 87.5%, 80%, and 60% of the studies on upper tract urothelial cancer, urothelial bladder cancer, and metastatic and advanced disease, respectively. The NLR cut-off value ranged between 2 and 5. A high NLR was associated with worse overall, cancer-specific, and recurrence-free survival. NLR is a widely available, easy-to-collect, costless, prognostic marker in urothelial cancers. Its clinical use still remains under investigation, especially for the need for cut-off values, particularly in different subsets of patients.

Superoxide Dismutase (SOD)-mimetic M40403 Is Protective in Cell and Fly Models of Paraquat Toxicity: IMPLICATIONS FOR PARKINSON DISEASE.

Parkinson disease is a debilitating and incurable neurodegenerative disorder affecting ∼1-2% of people over 65 years of age. Oxidative damage is considered to play a central role in the progression of Parkinson disease and strong evidence links chronic exposure to the pesticide paraquat with the incidence of the disease, most probably through the generation of oxidative damage. In this work, we demonstrated in human SH-SY5Y neuroblastoma cells the beneficial role of superoxide dismutase (SOD) enzymes against paraquat-induced toxicity, as well as the therapeutic potential of the SOD-mimetic compound M40403. Having verified the beneficial effects of superoxide dismutation in cells, we then evaluated the effects using Drosophila melanogaster as an in vivo model. Besides protecting against the oxidative damage induced by paraquat treatment, our data demonstrated that in Drosophila M40403 was able to compensate for the loss of endogenous SOD enzymes, acting both at a cytosolic and mitochondrial level. Because previous clinical trials have indicated that the M40403 molecule is well tolerated in humans, this study may have important implication for the treatment of Parkinson disease.

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation.

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field.