AIMTOR, a BRET biosensor for live imaging, reveals subcellular mTOR signaling and dysfunctions.

BACKGROUND: mTOR signaling is an essential nutrient and energetic sensing pathway. Here we describe AIMTOR, a sensitive genetically encoded BRET (Bioluminescent Resonance Energy Transfer) biosensor to study mTOR activity in living cells. RESULTS: As a proof of principle, we show in both cell lines and primary cell cultures that AIMTOR BRET intensities are modified by mTOR activity changes induced by specific inhibitors and activators of mTORC1 including amino acids and insulin. We further engineered several versions of AIMTOR enabling subcellular-specific assessment of mTOR activities. We then used AIMTOR to decipher mTOR signaling in physio-pathological conditions. First, we show that mTORC1 activity increases during muscle cell differentiation and in response to leucine stimulation in different subcellular compartments such as the cytosol and at the surface of the lysosome, the nucleus, and near the mitochondria. Second, in hippocampal neurons, we found that the enhancement of neuronal activity increases mTOR signaling. AIMTOR further reveals mTOR-signaling dysfunctions in neurons from mouse models of autism spectrum disorder. CONCLUSIONS: Altogether, our results demonstrate that AIMTOR is a sensitive and specific tool to investigate mTOR-signaling dynamics in living cells and phenotype mTORopathies.

Regulation of mitochondrial activity controls the duration of skeletal muscle regeneration in response to injury.

Thyroid hormone is a major regulator of skeletal muscle development and repair, and also a key regulator of mitochondrial activity. We have previously identified a 43 kDa truncated form of the nuclear T3 receptor TRα1 (p43) which stimulates mitochondrial activity and regulates skeletal muscle features. However, its role in skeletal muscle regeneration remains to be addressed. To this end, we performed acute muscle injury induced by cardiotoxin in mouse tibialis in two mouse models where p43 is overexpressed in or depleted from skeletal muscle. The measurement of muscle fiber size distribution at different time point (up to 70 days) upon injury lead us to unravel requirement of the p43 signaling pathway for satellite cells dependent muscle regeneration; strongly delayed in the absence of p43; whereas the overexpression of the receptor enhances of the regeneration process. In addition, we found that satellite cells derived from p43-Tg mice display higher proliferation rates when cultured in vitro when compared to control myoblasts, whereas p43-/- satellites shows reduced proliferation capacity. These finding strongly support that p43 plays an important role in vivo by controling the duration of skeletal muscle regeneration after acute injury, possibly through the regulation of mitochondrial activity and myoblasts proliferation.

Glucocorticoid-dependent REDD1 expression reduces muscle metabolism to enable adaptation under energetic stress.

BACKGROUND: Skeletal muscle atrophy is a common feature of numerous chronic pathologies and is correlated with patient mortality. The REDD1 protein is currently recognized as a negative regulator of muscle mass through inhibition of the Akt/mTORC1 signaling pathway. REDD1 expression is notably induced following glucocorticoid secretion, which is a component of energy stress responses. RESULTS: Unexpectedly, we show here that REDD1 instead limits muscle loss during energetic stresses such as hypoxia and fasting by reducing glycogen depletion and AMPK activation. Indeed, we demonstrate that REDD1 is required to decrease O2 and ATP consumption in skeletal muscle via reduction of the extent of mitochondrial-associated endoplasmic reticulum membranes (MAMs), a central hub connecting energy production by mitochondria and anabolic processes. In fact, REDD1 inhibits ATP-demanding processes such as glycogen storage and protein synthesis through disruption of the Akt/Hexokinase II and PRAS40/mTORC1 signaling pathways in MAMs. Our results uncover a new REDD1-dependent mechanism coupling mitochondrial respiration and anabolic processes during hypoxia, fasting, and exercise. CONCLUSIONS: Therefore, REDD1 is a crucial negative regulator of energy expenditure that is necessary for muscle adaptation during energetic stresses. This present study could shed new light on the role of REDD1 in several pathologies associated with energetic metabolism alteration, such as cancer, diabetes, and Parkinson's disease.

Evaluation of the ROS Inhibiting Activity and Mitochondrial Targeting of Phenolic Compounds in Fibroblast Cells Model System and Enhancement of Efficiency by Natural Deep Eutectic Solvent (NADES) Formulation.

PURPOSE: Many phenolics have already been tested for their antioxidant activities using in vitro methods. However, such assays do not consider the complexity of real cellular systems, and most of the phenolics characterized with such assays shows disappointing results when evaluated in cells. Accordingly, there is a need to develop effective screening methods. METHODS: Antioxidants were first evaluated by CAT assay and then, evaluated for their ability (i) to reduce the level of ROS using fluorescent probe, (ii) to cross fibroblast cell membranes using confocal microscopy, and (iii) to target mitochondria. Antioxidants were also formulated in NADES. RESULTS: Correlation was obtained when comparing CAT results with short term inhibition (2 h) in the fibroblast cells. On the contrary, it was difficult to anticipate ROS inhibiting efficiency at long term (24 h) from both the CAT assay and the short term inhibition measurements. Indeed, some molecules displayed activity rapidly but lost it over time. In contrast, other molecules were better for long term. The comparable efficiency at long term of Bis-Ethylhexyl Hydroxydimethoxy Benzylmalonate (Bis-EHBm) and decyl rosmarinate, prompted us to further investigate the potential mitochondrial targeting of the former. Using mitochondrial probes, our results confirmed its mitochondrial location. Finally, the formulation of antioxidants in NADES could greatly improve their activity. CONCLUSIONS: Combinations of fast acting and slow acting molecules could be promising strategies to identify a performant antioxidant system. Bis-EHBm behaves as decyl rosmarinate with a confirmed mitochondrial location. Finally, the formulation of antioxidants in NADES could greatly improve their activity for ROS inhibition.

Muscle Regeneration with Intermuscular Adipose Tissue (IMAT) Accumulation Is Modulated by Mechanical Constraints.

Sports trauma are able to induce muscle injury with fibrosis and accumulation of intermuscular adipose tissue (IMAT), which affect muscle function. This study was designed to investigate whether hypoactivity would influence IMAT accumulation in regenerating mouse skeletal muscle using the glycerol model of muscle regeneration. The animals were immediately hindlimb unloaded for 21 days after glycerol injection into the tibialis anterior (TA) muscle. Muscle fiber and adipocyte cross-sectional area (CSA) and IMAT accumulation were determined by histomorphometric analysis. Adipogenesis during regenerative processes was examined using RT-qPCR and Western blot quantification. Twenty-one days of hindlimb unloading resulted in decreases of 38% and 50.6% in the muscle weight/body weight ratio and CSA, respectively, in soleus muscle. Glycerol injection into TA induced IMAT accumulation, reaching 3% of control normal-loading muscle area. This IMAT accumulation was largely inhibited in unloading conditions (0.09%) and concomitant with a marked reduction in perilipin and FABP4 protein content, two key markers of mature adipocytes. Induction of PPARγ and C/EBPα mRNA, two markers of adipogenesis, was also decreased. Furthermore, the protein expression of PDGFRα, a cell surface marker of fibro/adipogenic progenitors, was much lower in regenerating TA from the unloaded group. Exposure of regenerating muscle to hypoactivity severely reduces IMAT development and accumulation. These results provide new insight into the mechanisms regulating IMAT development in skeletal muscle and highlight the importance of taking into account the level of mechanical constraint imposed on skeletal muscle during the regeneration processes.

Formation of vacuolar tannin deposits in the chlorophyllous organs of Tracheophyta: from shuttles to accretions.

Most Tracheophyta synthesize-condensed tannins (also called proanthocyanidins), polymers of catechins, which appear in the vacuole as uniformly stained deposits-termed tannin accretions-lining the inner face of the tonoplast. A large body of evidence argues that tannins are formed in recently described thylakoid-derived organelles, the tannosomes, which are packed in membrane-bound shuttles (Brillouet et al. 2013); it has been suggested that shuttles agglomerate into tannin accretions. The aim of the study was to describe the ontogenesis of tannin accretions in members of the Tracheophyta. For this purpose, fresh specimens of young tissues from diverse Tracheophyta were cut, gently lacerated in paraformaldehyde, and examined using light, epifluorescence, confocal, and transmission electron microscopy. Fresh samples were also incubated with gelatin-Oregon Green, a fluorescent marker of condensed tannins. Our observations showed that vacuolar accretions (1 → 40 µm), that constitute the typical form of tannin storage in tannin-producing Tracheophyta, are formed by agglomeration (not fusion) of shuttles containing various proportions of chlorophylls and tannins.

In vivo intracellular pH measurements in tobacco and Arabidopsis reveal an unexpected pH gradient in the endomembrane system.

The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH -1.5) and Arabidopsis thaliana root cells (ΔpH -2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H(+) ATPase-dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.

Boosting antioxidants by lipophilization: a strategy to increase cell uptake and target mitochondria.

PURPOSE: To explore the possibility to boost phenolic antioxidants through their structural modification by lipophilization and check the influence of such covalent modification on cellular uptake and mitochondria targeting. METHODS: Rosmarinic acid was lipophilized by various aliphatic chain lengths (butyl, octyl, decyl, dodecyl, hexadecyl, and octadecyl) to give rosmarinate alkyl esters which were then evaluated for their ability (i) to reduce the level of reactive oxygen species (ROS) using 2',7'-dichlorodihydrofluorescein diacetate probe, (ii) to cross fibroblast cell membranes using confocal microscopy, and (iii) to target mitochondria using MitoTracker® Red CMXRos. RESULTS: Increasing the chain length led to an improvement of the antioxidant activity until a threshold is reached for medium chain (10 carbon atoms) and beyond which lengthening resulted in a decrease of activity. This nonlinear phenomenon-also known as the cut-off effect-is discussed here in connection to the previously similar results observed in emulsified, liposomal, and cellular systems. Moreover, butyl, octyl, and decyl rosmarinates passed through the membranes in less than 15 min, whereas longer esters did not cross membranes and formed extracellular aggregates. Besides cell uptake, alkyl chain length also determined the subcellular localization of esters: mitochondria for medium chains esters, cytosol for short chains and extracellular media for longer chains. CONCLUSION: The localization of antioxidants within mitochondria, the major site and target of ROS, conferred an advantage to medium chain rosmarinates compared to both short and long chains. In conjunction with changes in cellular uptake, this result may explain the observed decrease of antioxidant activity when lengthening the lipid chain of esters. This brings a proof-of-concept that grafting medium chain allows the design of mitochondriotropic antioxidants.

Myostatin deficiency is associated with an increase in number of total axons and motor axons innervating mouse tibialis anterior muscle.

INTRODUCTION: Myostatin (Mstn) is a secreted protein that acts as a negative regulator of skeletal muscle mass. However, a critical evaluation of neuromuscular aspects of hypertrophied muscles induced by Mstn deficiency has not been done. METHODS: We compared the tibialis anterior muscle-nerve interrelationships in wild-type and Mstn-null mice of both genders by immunohistochemical analyses, which allowed us to count the number of total axons and motor axons and estimate the size of motor units and the innervation ratio of the tibialis anterior muscle (TAm). RESULTS: There was an increase in the number of total axons and motor axons, and higher values in both the motor unit size and the innervation ratio of Mstn-null TAm compared with those of wild-type TAm. CONCLUSIONS: We found that myostatin is involved either directly in the control of neuromuscular interrelationships or indirectly through its effect on muscle size.

Myostatin inactivation increases myotube size through regulation of translational initiation machinery.

Myostatin deficiency leads in skeletal muscle overgrowth but the precise molecular mechanisms underlying this hypertrophy are not well understood. In this study, to gain insight into the role of endogenous myostatin in the translational regulation, we used an in vitro model of cultured satellite cells derived from myostatin knock-out mice. Our results show that myostatin knock-out myotubes are larger than control myotubes and that this phenotype is associated with an increased activation of the Akt/mTOR signaling pathway, a known regulator of muscle hypertrophy. These results demonstrate that hypertrophy due to myostatin deficiency is preserved in vitro and suggest that myostatin deletion results in an increased protein synthesis. Accordingly, the rates of global RNA content, polysome formation and protein synthesis are all increased in myostatin-deficient myotubes while they are counteracted by the addition of recombinant myostatin. We furthermore demonstrated that genetic deletion of myostatin stimulates cap-dependent translation by positively regulating assembly of the translation preinitiation complex. Together the data indicate that myostatin controls muscle hypertrophy in part by regulating protein synthesis initiation rates, that is, translational efficiency.