tRNA biogenesis and specific aminoacyl-tRNA synthetases regulate senescence stability under the control of mTOR.

Oncogenes or chemotherapy treatments trigger the induction of suppressive pathways such as apoptosis or senescence. Senescence was initially defined as a definitive arrest of cell proliferation but recent results have shown that this mechanism is also associated with cancer progression and chemotherapy resistance. Senescence is therefore much more heterogeneous than initially thought. How this response varies is not really understood, it has been proposed that its outcome relies on the secretome of senescent cells and on the maintenance of their epigenetic marks. Using experimental models of senescence escape, we now described that the stability of this proliferative arrest relies on specific tRNAs and aminoacyl-tRNA synthetases. Following chemotherapy treatment, the DNA binding of the type III RNA polymerase was reduced to prevent tRNA transcription and induce a complete cell cycle arrest. By contrast, during senescence escape, specific tRNAs such as tRNA-Leu-CAA and tRNA-Tyr-GTA were up-regulated. Reducing tRNA transcription appears necessary to control the strength of senescence since RNA pol III inhibition through BRF1 depletion maintained senescence and blocked the generation of escaping cells. mTOR inhibition also prevented chemotherapy-induced senescence escape in association with a reduction of tRNA-Leu-CAA and tRNA-Tyr-GTA expression. Further confirming the role of the tRNA-Leu-CAA and tRNA-Tyr-GTA, results showed that their corresponding tRNA ligases, LARS and YARS, were necessary for senescence escape. This effect was specific since the CARS ligase had no effect on persistence. By contrast, the down-regulation of LARS and YARS reduced the emergence of persistent cells and this was associated with the modulation of E2F1 target genes expression. Overall, these findings highlight a new regulation of tRNA biology during senescence and suggest that specific tRNAs and ligases contribute to the strength and heterogeneity of this tumor suppressive pathway.

Fatty acyl availability modulates cardiolipin composition and alters mitochondrial function in HeLa cells.

The molecular assembly of cells depends not only on the balance between anabolism and catabolism but to a large degree on the building blocks available in the environment. For cultured mammalian cells, this is largely determined by the composition of the applied growth medium. Here, we study the impact of lipids in the medium on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum-free and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins strongly depends on the lipid environment in cultured cells and favors the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This raises the question on a link between membrane composition and respiratory control. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. This underlines the importance of considering these factors when using and establishing cell culture models in biomedical research. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium.

Resveratrol induces a mitochondrial complex I-dependent increase in NADH oxidation responsible for sirtuin activation in liver cells.

Resveratrol (RSV) has been shown to be involved in the regulation of energetic metabolism, generating increasing interest in therapeutic use. SIRT1 has been described as the main target of RSV. However, recent reports have challenged the hypothesis of its direct activation by RSV, and the signaling pathways remain elusive. Here, the effects of RSV on mitochondrial metabolism are detailed both in vivo and in vitro using murine and cellular models and isolated enzymes. We demonstrate that low RSV doses (1-5 µM) directly stimulate NADH dehydrogenases and, more specifically, mitochondrial complex I activity (EC50 ∼1 µM). In HepG2 cells, this complex I activation increases the mitochondrial NAD(+)/NADH ratio. This higher NAD(+) level initiates a SIRT3-dependent increase in the mitochondrial substrate supply pathways (i.e. the tricarboxylic acid cycle and fatty acid oxidation). This effect is also seen in liver mitochondria of RSV-fed animals (50 mg/kg/day). We conclude that the increase in NADH oxidation by complex I is a crucial event for SIRT3 activation by RSV. Our results open up new perspectives in the understanding of the RSV signaling pathway and highlight the critical importance of RSV doses used for future clinical trials.

Anterior gradient protein 2 is a marker of tumor aggressiveness in breast cancer and favors chemotherapy­induced senescence escape.

Among the different chemotherapies available, genotoxic drugs are widely used. In response to these drugs, particularly doxorubicin, tumor cells can enter into senescence. Chemotherapy­induced senescence (CIS) is a complex response. Long described as a definitive arrest of cell proliferation, the present authors and various groups have shown that this state may not be complete and could allow certain cells to reproliferate. The mechanism could be due to the activation of new signaling pathways. In the laboratory, the proteins involved in these pathways and triggering cell proliferation were studied. The present study determined a new role for anterior gradient protein 2 (AGR2) in vivo in patients and in vitro in a senescence escape model. AGR2's implication in breast cancer patients and proliferation of senescent cells was assessed based on a SWATH­MS proteomic study of patients' samples and RNA interference technology on cell lines. First, AGR2 was identified and it was found that its concentration is higher in the serum of patients with breast cancer and that this high concentration is associated with metastasis occurrence. An inverse correlation between intratumoral AGR2 expression and the senescence marker p16 was also observed. This observation led to the study of the role of AGR2 in the CIS escape model. In this model, it was found that AGR2 is overexpressed in cells during senescence escape and that its loss considerably reduces this phenomenon. Furthermore, it was shown that the extracellular form of AGR2 stimulated the reproliferation of senescent cells. The power of proteomic analysis based on the SWATH­MS approach allowed the present study to highlight the mammalian target of rapamycin (mTOR)/AKT signaling pathway in the senescence escape mechanism mediated by AGR2. Analysis of the two signaling pathways revealed that AGR2 modulated RICTOR and AKT phosphorylation. All these results showed that AGR2 expression in sera and tumors of breast cancer patients is a marker of tumor progression and metastasis occurrence. They also showed that its overexpression regulates CIS escape via activation of the mTOR/AKT signaling pathway.

Mitochondrial Activity Is Upregulated in Nonlesional Atopic Dermatitis and Amenable to Therapeutic Intervention.

Previous work has shown increased expression of genes related to oxidative stress in nonlesional atopic dermatitis (ADNL) skin. Although mitochondria are key regulators of ROS production, their function in AD has never been investigated. Energy metabolism and the oxidative stress response were studied in keratinocytes (KCs) from patients with ADNL or healthy controls. Moreover, ADNL human epidermal equivalents were treated with tigecycline or MitoQ. We found that pyruvate and glucose were used as energy substrates by ADNL KCs. Increased mitochondrial oxidation of (very) long-chain fatty acids, associated with enhanced complexes I and II activities, was observed in ADNL KCs. Metabolomic analysis revealed increased tricarboxylic acid cycle turnover. Increased aerobic metabolism generated oxidative stress in ADNL KCs. ADNL human epidermal equivalents displayed increased mitochondrial function and an enhanced oxidative stress response compared with controls. Treatment of ADNL human epidermal equivalents with tigecycline or MitoQ largely corrected the AD profile, including high p-65 NF-κB, abnormal lamellar bodies, and cellular damage. Furthermore, we found that glycolysis supports but does not supersede mitochondrial metabolism in ADNL KCs. Thus, aerobic metabolism predominates in ADNL but leads to oxidative stress. Therefore, mitochondria could be a reservoir of potential therapeutic targets in atopic dermatitis.

Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis.

Alterations of the lipid profile of the stratum corneum have an important role in the pathogenesis of atopic dermatitis (AD) because they contribute to epidermal barrier impairment. However, they have not previously been envisioned as a cellular response to altered metabolic requirements in AD epidermis. In this study, we report that the lipid composition in the epidermis of flaky tail, that is, ft/ft mice mimics that of human lesional AD (ADL) epidermis, both showing a shift toward shorter lipid species. The amounts of C24 and C26 free fatty acids and C24 and C26 ceramides-oxidized exclusively in peroxisomes-were reduced in the epidermis of ft/ft mice despite increased lipid synthesis, similar to that seen in human ADL edpidermis. Increased ACOX1 protein and activity in granular keratinocytes of ft/ft epidermis, altered lipid profile in human epidermal equivalents overexpressing ACOX1, and increased ACOX1 immunostaining in skin biopsies from patients with ADL suggest that peroxisomal ß-oxidation significantly contributes to lipid signature in ADL epidermis. Moreover, we show that increased anaerobic glycolysis in ft/ft mouse epidermis is essential for keratinocyte proliferation and adenosine triphosphate synthesis but does not contribute to local inflammation. Thus, this work evidenced a metabolic shift toward enhanced peroxisomal ß-oxidation and anaerobic glycolysis in ADL epidermis.

3D-Organotypic Cultures to Unravel Molecular and Cellular Abnormalities in Atopic Dermatitis and Ichthyosis Vulgaris.

Atopic dermatitis (AD) is characterized by dry and itchy skin evolving into disseminated skin lesions. AD is believed to result from a primary acquired or a genetically-induced epidermal barrier defect leading to immune hyper-responsiveness. Filaggrin (FLG) is a protein found in the cornified envelope of fully differentiated keratinocytes, referred to as corneocytes. Although FLG null mutations are strongly associated with AD, they are not sufficient to induce the disease. Moreover, most patients with ichthyosis vulgaris (IV), a monogenetic skin disease characterized by FLG homozygous, heterozygous, or compound heterozygous null mutations, display non-inflamed dry and scaly skin. Thus, all causes of epidermal barrier impairment in AD have not yet been identified, including those leading to the Th2-predominant inflammation observed in AD. Three dimensional organotypic cultures have emerged as valuable tools in skin research, replacing animal experimentation in many cases and precluding the need for repeated patient biopsies. Here, we review the results on IV and AD obtained with epidermal or skin equivalents and consider these findings in the context of human in vivo data. Further research utilizing complex models including immune cells and cutaneous innervation will enable finer dissection of the pathogenesis of AD and deepen our knowledge of epidermal biology.

Warburg-like effect is a hallmark of complex I assembly defects.

Due to its pivotal role in NADH oxidation and ATP synthesis, mitochondrial complex I (CI) emerged as a crucial regulator of cellular metabolism. A functional CI relies on the sequential assembly of nuclear- and mtDNA-encoded subunits; however, whether CI assembly status is involved in the metabolic adaptations in CI deficiency still remains largely unknown. Here, we investigated the relationship between CI functions, its structure and the cellular metabolism in 29 patient fibroblasts representative of most CI mitochondrial diseases. Our results show that, contrary to the generally accepted view, a complex I deficiency does not necessarily lead to a glycolytic switch, i.e. the so-called Warburg effect, but that this particular metabolic adaptation is a feature of CI assembly defect. By contrast, a CI functional defect without disassembly induces a higher catabolism to sustain the oxidative metabolism. Mechanistically, we demonstrate that reactive oxygen species overproduction by CI assembly intermediates and subsequent AMPK-dependent Pyruvate Dehydrogenase inactivation are key players of this metabolic reprogramming. Thus, this study provides a two-way-model of metabolic responses to CI deficiencies that are central not only in defining therapeutic strategies for mitochondrial diseases, but also in all pathophysiological conditions involving a CI deficiency.

Resveratrol Directly Binds to Mitochondrial Complex I and Increases Oxidative Stress in Brain Mitochondria of Aged Mice.

Resveratrol is often described as a promising therapeutic molecule for numerous diseases, especially in metabolic and neurodegenerative disorders. While the mechanism of action is still debated, an increasing literature reports that resveratrol regulates the mitochondrial respiratory chain function. In a recent study we have identified mitochondrial complex I as a direct target of this molecule. Nevertheless, the mechanisms and consequences of such an interaction still require further investigation. In this study, we identified in silico by docking study a binding site for resveratrol at the nucleotide pocket of complex I. In vitro, using solubilized complex I, we demonstrated a competition between NAD+ and resveratrol. At low doses (<5µM), resveratrol stimulated complex I activity, whereas at high dose (50 µM) it rather decreased it. In vivo, in brain mitochondria from resveratrol treated young mice, we showed that complex I activity was increased, whereas the respiration rate was not improved. Moreover, in old mice with low antioxidant defenses, we demonstrated that complex I activation by resveratrol led to oxidative stress. These results bring new insights into the mechanism of action of resveratrol on mitochondria and highlight the importance of the balance between pro- and antioxidant effects of resveratrol depending on its dose and age. These parameters should be taken into account when clinical trials using resveratrol or analogues have to be designed.

Assembly defects induce oxidative stress in inherited mitochondrial complex I deficiency.

Complex I (CI) deficiency is the most common respiratory chain defect representing more than 30% of mitochondrial diseases. CI is an L-shaped multi-subunit complex with a peripheral arm protruding into the mitochondrial matrix and a membrane arm. CI sequentially assembled into main assembly intermediates: the P (pumping), Q (Quinone) and N (NADH dehydrogenase) modules. In this study, we analyzed 11 fibroblast cell lines derived from patients with inherited CI deficiency resulting from mutations in the nuclear or mitochondrial DNA and impacting these different modules. In patient cells carrying a mutation located in the matrix arm of CI, blue native-polyacrylamide gel electrophoresis (BN-PAGE) revealed a significant reduction of fully assembled CI enzyme and an accumulation of intermediates of the N module. In these cell lines with an assembly defect, NADH dehydrogenase activity was partly functional, even though CI was not fully assembled. We further demonstrated that this functional N module was responsible for ROS production through the reduced flavin mononucleotide. Due to the assembly defect, the FMN site was not re-oxidized leading to a significant oxidative stress in cell lines with an assembly defect. These findings not only highlight the relationship between CI assembly and oxidative stress, but also show the suitability of BN-PAGE analysis in evaluating the consequences of CI dysfunction. Moreover, these data suggest that the use of antioxidants may be particularly relevant for patients displaying a CI assembly defect.