Temporal regulation of transgene expression controlled by amino acid availability in human T cells.

T cell therapy strategies, from allogeneic stem cell transplantation toward genetically-modified T cells infusion, develop powerful anti-tumor effects but are often accompanied by side effects and their efficacy remains sometimes to be improved. It therefore appears important to provide a flexible and easily reversible gene expression regulation system to control T cells activity. We developed a gene expression regulation technology that exploits the physiological GCN2-ATF4 pathway's ability to induce gene expression in T cells in response to one essential amino acid deficiency. We first demonstrated the functionality of NUTRIREG in human T cells by transient expression of reporter genes. We then validated that NUTRIREG can be used in human T cells to transiently express a therapeutic gene such as IL-10. Overall, our results represent a solid basis for the promising use of NUTRIREG to regulate transgene expression in human T cells in a reversible way, and more generally for numerous preventive or curative therapeutic possibilities in cellular immunotherapy strategies.

Role of liver AMPK and GCN2 kinases in the control of postprandial protein metabolism in response to mid-term high or low protein intake in mice.

PURPOSE: Protein synthesis and proteolysis are known to be controlled through mammalian target of rapamycin, AMP-activated kinase (AMPK) and general control non-derepressible 2 (GCN2) pathways, depending on the nutritional condition. This study aimed at investigating the contribution of liver AMPK and GCN2 on the adaptation to high variations in protein intake. METHODS: To evaluate the answer of protein pathways to high- or low-protein diet, male wild-type mice and genetically modified mice from C57BL/6 background with liver-specific AMPK- or GCN2-knockout were fed from day 25 diets differing in their protein level as energy: LP (5%), NP (14%) and HP (54%). Two hours after a 1 g test meal, protein synthesis rate was measured after a 13C valine flooding dose. The gene expression of key enzymes involved in proteolysis and GNC2 signaling pathway were quantified. RESULTS: The HP diet but not the LP diet was associated with a decrease in fractional synthesis rate by 29% in the liver compared to NP diet. The expression of mRNA encoding ubiquitin and Cathepsin D was not sensitive to the protein content. The deletion of AMPK or GCN2 in the liver did not affect nor protein synthesis rates and neither proteolysis markers in the liver or in the muscle, whatever the protein intake. In the postprandial state, protein level alters protein synthesis in the liver but not in the muscle. CONCLUSIONS: Taken together, these results suggest that liver AMPK and GCN2 are not involved in this adaptation to high- and low-protein diet observed in the postprandial period.

Activation of the eIF2α-ATF4 Pathway by Chronic Paracetamol Treatment Is Prevented by Dietary Supplementation with Cysteine.

Chronic treatment with acetaminophen (APAP) induces cysteine (Cys) and glutathione (GSH) deficiency which leads to adverse metabolic effects including muscle atrophy. Mammalian cells respond to essential amino acid deprivation through the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). Phosphorylated eIF2α leads to the recruitment of activating transcription factor 4 (ATF4) to specific CCAAT/enhancer-binding protein-ATF response element (CARE) located in the promoters of target genes. Our purpose was to study the activation of the eIF2α-ATF4 pathway in response to APAP-induced Cys deficiency, as well as the potential contribution of the eIF2α kinase GCN2 and the effect of dietary supplementation with Cys. Our results showed that chronic treatment with APAP activated both GCN2 and PERK eIF2α kinases and downstream target genes in the liver. Activation of the eIF2α-ATF4 pathway in skeletal muscle was accompanied by muscle atrophy even in the absence of GCN2. The dietary supplementation with cysteine reversed APAP-induced decreases in plasma-free Cys, liver GSH, muscle mass, and muscle GSH. Our new findings demonstrate that dietary Cys supplementation also reversed the APAP-induced activation of GCN2 and PERK and downstream ATF4-target genes in the liver.

When idiopathic male infertility is rooted in maternal malnutrition during the perinatal period in mice†.

Infertility represents a growing burden worldwide, with one in seven couples presenting difficulties conceiving. Among these, 10-15% of the men have idiopathic infertility that does not correlate with any defect in the classical sperm parameters measured. In the present study, we used a mouse model to investigate the effects of maternal undernutrition on fertility in male progeny. Our results indicate that mothers fed on a low-protein diet during gestation and lactation produce male offspring with normal sperm morphology, concentration, and motility but exhibiting an overall decrease of fertility when they reach adulthood. Particularly, in contrast to control, sperm from these offspring show a remarkable lower capacity to fertilize oocytes when copulation occurs early in the estrus cycle relative to ovulation, due to an altered sperm capacitation. Our data demonstrate for the first time that maternal nutritional stress can have long-term consequences on the reproductive health of male progeny by affecting sperm physiology, especially capacitation, with no observable impact on spermatogenesis and classical quantitative and qualitative sperm parameters. Moreover, our experimental model could be of major interest to study, explain, and ultimately treat certain categories of infertilities.

Induction of ATF4-Regulated Atrogenes Is Uncoupled from Muscle Atrophy during Disuse in Halofuginone-Treated Mice and in Hibernating Brown Bears.

Activating transcription factor 4 (ATF4) is involved in muscle atrophy through the overexpression of some atrogenes. However, it also controls the transcription of genes involved in muscle homeostasis maintenance. Here, we explored the effect of ATF4 activation by the pharmacological molecule halofuginone during hindlimb suspension (HS)-induced muscle atrophy. Firstly, we reported that periodic activation of ATF4-regulated atrogenes (Gadd45a, Cdkn1a, and Eif4ebp1) by halofuginone was not associated with muscle atrophy in healthy mice. Secondly, halofuginone-treated mice even showed reduced atrophy during HS, although the induction of the ATF4 pathway was identical to that in untreated HS mice. We further showed that halofuginone inhibited transforming growth factor-ß (TGF-ß) signalling, while promoting bone morphogenetic protein (BMP) signalling in healthy mice and slightly preserved protein synthesis during HS. Finally, ATF4-regulated atrogenes were also induced in the atrophy-resistant muscles of hibernating brown bears, in which we previously also reported concurrent TGF-ß inhibition and BMP activation. Overall, we show that ATF4-induced atrogenes can be uncoupled from muscle atrophy. In addition, our data also indicate that halofuginone can control the TGF-ß/BMP balance towards muscle mass maintenance. Whether halofuginone-induced BMP signalling can counteract the effect of ATF4-induced atrogenes needs to be further investigated and may open a new avenue to fight muscle atrophy. Finally, our study opens the way for further studies to identify well-tolerated chemical compounds in humans that are able to fine-tune the TGF-ß/BMP balance and could be used to preserve muscle mass during catabolic situations.

Cyclosporin A but not FK506 activates the integrated stress response in human cells.

Cyclosporin A (CsA) and tacrolimus (FK506) are valuable immunosuppressants for a range of clinical settings, including (but not limited to) organ transplantation and the treatment of autoimmune diseases. They function by inhibiting the activity of the Ca2+/calmodulin-dependent phosphatase calcineurin toward nuclear factor of activated T-cells (NF-AT) in T-lymphocytes. However, use of CsA is associated with more serious side effects and worse clinical outcomes than FK506. Here we show that CsA, but not FK506, causes activation of the integrated stress response (ISR), an event which is normally an acute reaction to various types of intracellular insults, such as nutrient deficiency or endoplasmic reticulum stress. These effects of CsA involve at least two of the stress-activated protein kinases (GCN2 and PERK) that act on the translational machinery to slow down protein synthesis via phosphorylation of the eukaryotic initiation factor (eIF) 2α and thereby induce the ISR. These actions of CsA likely contribute to the adverse effects associated with its clinical application.

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.

Liver GCN2 controls hepatic FGF21 secretion and modulates whole body postprandial oxidation profile under a low-protein diet.

General control nonderepressible 2 (GCN2) is a kinase that detects amino acid deficiency and is involved in the control of protein synthesis and energy metabolism. However, the role of hepatic GCN2 in the metabolic adaptations in response to the modulation of dietary protein has been seldom studied. Wild-type (WT) and liver GCN2-deficient (KO) mice were fed either a normo-protein diet, a low-protein diet, or a high-protein diet for 3 wk. During this period, body weight, food intake, and metabolic parameters were followed. In mice fed normo- and high-protein diets, GCN2 pathway in the liver is not activated in WT mice, leading to a similar metabolic profile with the one of KO mice. On the contrary, a low-protein diet activates GCN2 in WT mice, inducing FGF21 secretion. In turn, FGF21 maintains a high level of lipid oxidation, leading to a different postprandial oxidation profile compared with KO mice. Hepatic GCN2 controls FGF21 secretion under a low-protein diet and modulates a whole body postprandial oxidation profile.

Decreased ATF4 expression as a mechanism of acquired resistance to long-term amino acid limitation in cancer cells.

The uncontrolled growth of tumor can lead to the formation of area deprived in nutrients. Due to their high genetic instability, tumor cells can adapt and develop resistance to this pro-apoptotic environment. Among the resistance mechanisms, those involved in the resistance to long-term amino acid restriction are not elucidated. A long-term amino acid restriction is particularly deleterious since nine of them cannot be synthetized by the cells. In order to determine how cancer cells face a long-term amino acid deprivation, we developed a cell model selected for its capacity to resist a long-term amino acid limitation. We exerted a selection pressure on mouse embryonic fibroblast to isolate clones able to survive with low amino acid concentration. The study of several clones revealed an alteration of the eiF2α/ATF4 pathway. Compared to the parental cells, the clones exhibited a decreased expression of the transcription factor ATF4 and its target genes. Likewise, the knock-down of ATF4 in parental cells renders them resistant to amino acid deprivation. Moreover, this association between a low level of ATF4 protein and the resistance to amino acid deprivation was also observed in the cancer cell line BxPC-3. This resistance was abolished when ATF4 was overexpressed. Therefore, decreasing ATF4 expression may be one important mechanism for cancer cells to survive under prolonged amino acid deprivation.

GCN2 contributes to mTORC1 inhibition by leucine deprivation through an ATF4 independent mechanism.

It is well known that the GCN2 and mTORC1 signaling pathways are regulated by amino acids and share common functions, in particular the control of translation. The regulation of GCN2 activity by amino acid availability relies on the capacity of GCN2 to sense the increased levels of uncharged tRNAs upon amino acid scarcity. In contrast, despite recent progress in the understanding of the regulation of mTORC1 by amino acids, key aspects of this process remain unsolved. In particular, while leucine is well known to be a potent regulator of mTORC1, the mechanisms by which this amino acid is sensed and control mTORC1 activity are not well defined. Our data establish that GCN2 is involved in the inhibition of mTORC1 upon leucine or arginine deprivation. However, the activation of GCN2 alone is not sufficient to inhibit mTORC1 activity, indicating that leucine and arginine exert regulation via additional mechanisms. While the mechanism by which GCN2 contributes to the initial step of mTORC1 inhibition involves the phosphorylation of eIF2α, we show that it is independent of the downstream transcription factor ATF4. These data point to a novel role for GCN2 and phosphorylation of eIF2α in the control of mTORC1 by certain amino acids.

Regulating the expression of therapeutic transgenes by controlled intake of dietary essential amino acids.

Widespread application of gene therapy will depend on the development of simple methods to regulate the expression of therapeutic genes. Here we harness an endogenous signaling pathway to regulate therapeutic gene expression through diet. The GCN2-eIF2α signaling pathway is specifically activated by deficiencies in any essential amino acid (EAA); EAA deficiency leads to rapid expression of genes regulated by ATF4-binding cis elements. We found that therapeutic genes under the control of optimized amino acid response elements (AAREs) had low basal expression and high induced expression. We applied our system to regulate the expression of TNFSF10 (TRAIL) in the context of glioma therapy and found that intermittent activation of this gene by EEA-deficient meals retained its therapeutic efficacy while abrogating its toxic effects on normal tissue. The GCN2-eIF2α pathway is expressed in many tissues, including the brain, and is highly specific to EAA deficiency. Our system may be particularly well suited for intermittent regulation of therapeutic transgenes over short or long time periods.

Method for collecting mouse milk without exogenous oxytocin stimulation.

It has been reported that breast-feeding more than 6 months strongly decreases the risk of allergy, diabetes, obesity, and hypertension in humans. In order to understand the mechanisms responsible for this benefit, it is important to evaluate precisely the composition of maternal milk, especially in response to environmental cues. Mouse models offer a unique opportunity to study the impact of maternal milk composition on the development and health of offspring. Oxytocin injection of the dam is usually used to stimulate milk ejection; however, exogenous oxytocin might have deleterious effects under some experimental conditions by modifying milk content as well as the physiology and behavior of the dam. Taking advantage of the natural stimulation of the mammary gland that occurs after the reunion of a dam that has been separated from her pups, we developed a new procedure to collect mouse milk without the injection of oxytocin. This method is easy to use, low-cost ,and non-invasive. Moreover, it provides a sufficient amount of milk for use in a wide range of biological analyses.

In vivo imaging of the spatiotemporal activity of the eIF2α-ATF4 signaling pathway: Insights into stress and related disorders.

The eIF2α-ATF4 pathway is involved in cellular adaptation to stress and is dysregulated in numerous diseases. Activation of this pathway leads to phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and the recruitment of the transcription factor ATF4 (activating transcription factor 4) to specific CCAAT/enhancer binding protein (C/EBP)-ATF response elements (CAREs) located in the promoters of target genes. To monitor the spatiotemporal modulation of this pathway in living animals, we generated a novel CARE-driven luciferase mouse model (CARE-LUC). These transgenic mice enable the investigation of the eIF2α-ATF4 pathway activity in the whole organism and at the tissue and cellular levels by combining imaging, luciferase assays, and immunochemistry. Using this mouse line, we showed the tissue-specific activation pattern of this pathway in response to amino acid deficiency or endoplasmic reticulum stress and the hepatic induction of this pathway in a stress-related pathology model of liver fibrosis. The CARE-LUC mouse model represents an innovative tool to investigate the eIF2α-ATF4 axis and to develop drugs targeting this important pathway in the remediation of related pathologies.

[Adaptation to the availability of essential amino-acids: role of GCN2/eIF2α/ATF4 pathway]. / Mécanismes d'adaptation à la disponibilité en acides aminés indispensables: rôles de la voie GCN2/eIF2α/ATF4.

In mammals, metabolic adaptations are required to overcome nutritional deprivation in amino-acids/proteins as well as episodes of malnutrition. GCN2 protein kinase, which phosphorylates the α subunit of the translation initiation factor eIF2, is a sensor of amino-acid(s) deficiencies. On one hand, this review briefly describes the main features of amino-acid metabolism. On the other hand, it describes the role of GCN2 in regulating numerous physiological functions.

Perinatal protein malnutrition affects mitochondrial function in adult and results in a resistance to high fat diet-induced obesity.

Epidemiological findings indicate that transient environmental influences during perinatal life, especially nutrition, may have deleterious heritable health effects lasting for the entire life. Indeed, the fetal organism develops specific adaptations that permanently change its physiology/metabolism and that persist even in the absence of the stimulus that initiated them. This process is termed "nutritional programming". We previously demonstrated that mothers fed a Low-Protein-Diet (LPD) during gestation and lactation give birth to F1-LPD animals presenting metabolic consequences that are different from those observed when the nutritional stress is applied during gestation only. Compared to control mice, adult F1-LPD animals have a lower body weight and exhibit a higher food intake suggesting that maternal protein under-nutrition during gestation and lactation affects the energy metabolism of F1-LPD offspring. In this study, we investigated the origin of this apparent energy wasting process in F1-LPD and demonstrated that minimal energy expenditure is increased, due to both an increased mitochondrial function in skeletal muscle and an increased mitochondrial density in White Adipose Tissue. Importantly, F1-LPD mice are protected against high-fat-diet-induced obesity. Clearly, different paradigms of exposure to malnutrition may be associated with differences in energy expenditure, food intake, weight and different susceptibilities to various symptoms associated with metabolic syndrome. Taken together these results demonstrate that intra-uterine environment is a major contributor to the future of individuals and disturbance at a critical period of development may compromise their health. Consequently, understanding the molecular mechanisms may give access to useful knowledge regarding the onset of metabolic diseases.

Requirement for lysosomal localization of mTOR for its activation differs between leucine and other amino acids.

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and metabolism. It controls many cell functions by integrating nutrient availability and growth factor signals. Amino acids, and in particular leucine, are among the main positive regulators of mTORC1 signaling. The current model for the regulation of mTORC1 by amino acids involves the movement of mTOR to the lysosome mediated by the Rag-GTPases. Here, we have examined the control of mTORC1 signaling and mTOR localization by amino acids and leucine in serum-fed cells, because both serum growth factors (or, e.g., insulin) and amino acids are required for full activation of mTORC1 signaling. We demonstrate that mTORC1 activity does not closely correlate with the lysosomal localization of mTOR. In particular, leucine controls mTORC1 activity without any detectable modification of the lysosomal localization of mTOR, indicating that the signal(s) exerted by leucine is likely distinct from those exerted by other amino acids. In addition, knock-down of the Rag-GTPases attenuated the inhibitory effect of amino acid- or leucine-starvation on the phosphorylation of mTORC1 targets. Furthermore, data from cells where Rag expression has been knocked down revealed that leucine can promote mTORC1 signaling independently of the lysosomal localization of mTOR. Our data complement existing models for the regulation of mTORC1 by amino acids and provide new insights into this important topic.

Dual role for CHOP in the crosstalk between autophagy and apoptosis to determine cell fate in response to amino acid deprivation.

CHOP encodes a ubiquitous transcription factor that is one of the most important components in the network of stress-inducible transcription. In particular, this factor is known to mediate cell death in response to stress. The focus of this work is to study its pivotal role in the control of cell viability according to the duration of a stress like amino acid starvation. We show that during the first 6h of starvation, CHOP upregulates a number of autophagy genes but is not involved in the first steps of the autophagic process. By contrast, when the amino acid starvation is prolonged (16-48h), we demonstrated that CHOP has a dual role in both inducing apoptosis and limiting autophagy through the transcriptional control of specific target genes. Overall, this study reveals a novel regulatory role for CHOP in the crosstalk between autophagy and apoptosis in response to stress.

Impairing the production of ribosomal RNA activates mammalian target of rapamycin complex 1 signalling and downstream translation factors.

Ribosome biogenesis is a key process for maintaining protein synthetic capacity in dividing or growing cells, and requires coordinated production of ribosomal proteins and ribosomal RNA (rRNA), including the processing of the latter. Signalling through mammalian target of rapamycin complex 1 (mTORC1) activates all these processes. Here, we show that, in human cells, impaired rRNA processing, caused by expressing an interfering mutant of BOP1 or by knocking down components of the PeBoW complex elicits activation of mTORC1 signalling. This leads to enhanced phosphorylation of its substrates S6K1 and 4E-BP1, and stimulation of proteins involved in translation initiation and elongation. In particular, we observe both inactivation and downregulation of the eukaryotic elongation factor 2 kinase, which normally inhibits translation elongation. The latter effect involves decreased expression of the eEF2K mRNA. The mRNAs for ribosomal proteins, whose translation is positively regulated by mTORC1 signalling, also remain associated with ribosomes. Therefore, our data demonstrate that disrupting rRNA production activates mTORC1 signalling to enhance the efficiency of the translational machinery, likely to help compensate for impaired ribosome production.