Sustained Activation Of mTORC1 Research Articles

TSC2 S1365A mutation potently regulates CD8+T cell function and differentiation improving adoptive cellular cancer therapy.

MTORC1 integrates signaling from the immune microenvironment to regulate T cell activation, differentiation, and function. TSC2 in the tuberous sclerosis complex tightly regulates mTORC1 activation. CD8+ T cells lacking TSC2 have constitutively enhanced mTORC1 activity and generate robust effector T cells; however sustained mTORC1 activation prevents generation of long-lived memory CD8+ T cells. Here we show manipulating TSC2 at Ser1365 potently regulates activated but not basal mTORC1 signaling in CD8+ T cells. Unlike non-stimulated TSC2 knockout cells, CD8+ T cells expressing a phospho-silencing mutant TSC2-S1365A (SA) retain normal basal mTORC1 activity. PKC and T-cell Receptor (TCR) stimulation induces TSC2 S1365 phosphorylation and preventing this with the SA mutation markedly increases mTORC1 activation and T-cell effector function. Consequently, SA CD8+ T cells display greater effector responses while retaining their capacity to become long-lived memory T cells. SA CD8+ T cells also display enhanced effector function under hypoxic and acidic conditions. In murine and human solid-tumor models, CD8+ SA T cells used as adoptive cell therapy display greater anti-tumor immunity than WT CD8+ T cells. These findings reveal an upstream mechanism to regulate mTORC1 activity in T cells. The TSC2-SA mutation enhances both T cell effector function and long-term persistence/memory formation, supporting an approach to engineer better CAR-T cells for treating cancer.

Hyperglucagonaemia in diabetes: altered amino acid metabolism triggers mTORC1 activation, which drives glucagon production.

Hyperglycaemia is associated with alpha cell dysfunction, leading to dysregulated glucagon secretion in type 1 and type 2 diabetes; however, the mechanisms involved are still elusive. The nutrient sensor mammalian target of rapamycin complex 1 (mTORC1) plays a major role in the maintenance of alpha cell mass and function. We studied the regulation of alpha cell mTORC1 by nutrients and its role in the development of hyperglucagonaemia in diabetes. Alpha cell mTORC1 activity was assessed by immunostaining for phosphorylation of its downstream target, the ribosomal protein S6, and glucagon, followed by confocal microscopy on pancreatic sections and flow cytometry on dispersed human and mouse islets and the alpha cell line, αTC1-6. Metabolomics and metabolic flux were studied by 13C glucose labelling in 2.8 or 16.7 mmol/l glucose followed by LC-MS analysis. To study the role of mTORC1 in mediating hyperglucagonaemia in diabetes, we generated an inducible alpha cell-specific Rptor knockout in the Akita mouse model of diabetes and tested the effects on glucose tolerance by IPGTT and on glucagon secretion. mTORC1 activity was increased in alpha cells from diabetic Akita mice in parallel to the development of hyperglycaemia and hyperglucagonaemia (two- to eightfold increase). Acute exposure of mouse and human islets to amino acids stimulated alpha cell mTORC1 (3.5-fold increase), whereas high glucose concentrations inhibited mTORC1 (1.4-fold decrease). The mTORC1 response to glucose was abolished in human and mouse diabetic alpha cells following prolonged islet exposure to high glucose levels, resulting in sustained activation of mTORC1, along with increased glucagon secretion. Metabolomics and metabolic flux analysis showed that exposure to high glucose levels enhanced glycolysis, glucose oxidation and the synthesis of glucose-derived amino acids. In addition, chronic exposure to high glucose levels increased the expression of Slc7a2 and Slc38a4, which encode amino acid transporters, as well as the levels of branched-chain amino acids and methionine cycle metabolites (~1.3-fold increase for both). Finally, conditional Rptor knockout in alpha cells from adult diabetic mice inhibited mTORC1, thereby inhibiting glucagon secretion (~sixfold decrease) and improving diabetes, despite persistent insulin deficiency. Alpha cell exposure to hyperglycaemia enhances amino acid synthesis and transport, resulting in sustained activation of mTORC1, thereby increasing glucagon secretion. mTORC1 therefore plays a major role in mediating alpha cell dysfunction in diabetes. All sequencing data are available from the Gene Expression Omnibus (GEO) repository (accession no. GSE154126; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE154126 ).

Age-Related Dysfunction in Proteostasis and Cellular Quality Control in the Development of Sarcopenia

Sarcopenia is a debilitating skeletal muscle disease that accelerates in the last decades of life and is characterized by marked deficits in muscle strength, mass, quality, and metabolic health. The multifactorial causes of sarcopenia have proven difficult to treat and involve a complex interplay between environmental factors and intrinsic age-associated changes. It is generally accepted that sarcopenia results in a progressive loss of skeletal muscle function that exceeds the loss of mass, indicating that while loss of muscle mass is important, loss of muscle quality is the primary defect with advanced age. Furthermore, preclinical models have suggested that aged skeletal muscle exhibits defects in cellular quality control such as the degradation of damaged mitochondria. Recent evidence suggests that a dysregulation of proteostasis, an important regulator of cellular quality control, is a significant contributor to the aging-associated declines in muscle quality, function, and mass. Although skeletal muscle mammalian target of rapamycin complex 1 (mTORC1) plays a critical role in cellular control, including skeletal muscle hypertrophy, paradoxically, sustained activation of mTORC1 recapitulates several characteristics of sarcopenia. Pharmaceutical inhibition of mTORC1 as well as caloric restriction significantly improves muscle quality in aged animals, however, the mechanisms controlling cellular proteostasis are not fully known. This information is important for developing effective therapeutic strategies that mitigate or prevent sarcopenia and associated disability. This review identifies recent and historical understanding of the molecular mechanisms of proteostasis driving age-associated muscle loss and suggests potential therapeutic interventions to slow or prevent sarcopenia.

Muscle 4EBP1 activation modifies the structure and function of the neuromuscular junction in mice

Dysregulation of mTOR complex 1 (mTORC1) activity drives neuromuscular junction (NMJ) structural instability during aging; however, downstream targets mediating this effect have not been elucidated. Here, we investigate the roles of two mTORC1 phosphorylation targets for mRNA translation, ribosome protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), in regulating NMJ structural instability induced by aging and sustained mTORC1 activation. While myofiber-specific deletion of S6k1 has no effect on NMJ structural integrity, 4EBP1 activation in murine muscle induces drastic morphological remodeling of the NMJ with enhancement of synaptic transmission. Mechanistically, structural modification of the NMJ is attributed to increased satellite cell activation and enhanced post-synaptic acetylcholine receptor (AChR) turnover upon 4EBP1 activation. Considering that loss of post-synaptic myonuclei and reduced NMJ turnover are features of aging, targeting 4EBP1 activation could induce NMJ renewal by expanding the pool of post-synaptic myonuclei as an alternative intervention to mitigate sarcopenia.

Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle

Preserving skeletal muscle function is essential to maintain life quality at high age. Calorie restriction (CR) potently extends health and lifespan, but is largely unachievable in humans, making “CR mimetics” of great interest. CR targets nutrient-sensing pathways centering on mTORC1. The mTORC1 inhibitor, rapamycin, is considered a potential CR mimetic and is proven to counteract age-related muscle loss. Therefore, we tested whether rapamycin acts via similar mechanisms as CR to slow muscle aging. Here we show that long-term CR and rapamycin unexpectedly display distinct gene expression profiles in geriatric mouse skeletal muscle, despite both benefiting aging muscles. Furthermore, CR improves muscle integrity in mice with nutrient-insensitive, sustained muscle mTORC1 activity and rapamycin provides additive benefits to CR in naturally aging mouse muscles. We conclude that rapamycin and CR exert distinct, compounding effects in aging skeletal muscle, thus opening the possibility of parallel interventions to counteract muscle aging.

Depdc5 deficiency exacerbates alcohol-induced hepatic steatosis via suppression of PPAR\u03b1 pathway

Alcohol-related liver disease (ALD), a condition caused by alcohol overconsumption, occurs in three stages of liver injury including steatosis, hepatitis, and cirrhosis. DEP domain-containing protein 5 (DEPDC5), a component of GAP activities towards Rags 1 (GATOR1) complex, is a repressor of amino acid-sensing branch of the mammalian target of rapamycin complex 1 (mTORC1) pathway. In the current study, we found that aberrant activation of mTORC1 was likely attributed to the reduction of DEPDC5 in the livers of ethanol-fed mice or ALD patients. To further define the in vivo role of DEPDC5 in ALD development, we generated Depdc5 hepatocyte-specific knockout mouse model (Depdc5-LKO) in which mTORC1 pathway was constitutively activated through loss of the inhibitory effect of GATOR1. Hepatic Depdc5 ablation leads to mild hepatomegaly and liver injury and protects against diet-induced liver steatosis. In contrast, ethanol-fed Depdc5-LKO mice developed severe hepatic steatosis and inflammation. Pharmacological intervention with Torin 1 suppressed mTORC1 activity and remarkably ameliorated ethanol-induced hepatic steatosis and inflammation in both control and Depdc5-LKO mice. The pathological effect of sustained mTORC1 activity in ALD may be attributed to the suppression of peroxisome proliferator activated receptor α (PPARα), the master regulator of fatty acid oxidation in hepatocytes, because fenofibrate (PPARα agonist) treatment reverses ethanol-induced liver steatosis and inflammation in Depdc5-LKO mice. These findings provide novel insights into the in vivo role of hepatic DEPDC5 in the development of ALD.

Activation of mTORC1 at late endosomes misdirects T cell fate decision in older individuals.

The nutrient-sensing mammalian target of rapamycin (mTOR) is integral to cell fate decisions after T cell activation. Sustained mTORC1 activity favors the generation of terminally differentiated effector T cells instead of follicular helper and memory T cells. This is particularly pertinent for T cell responses of older adults who have sustained mTORC1 activation despite dysfunctional lysosomes. Here, we show that lysosome-deficient T cells rely on late endosomes rather than lysosomes as an mTORC1 activation platform, where mTORC1 is activated by sensing cytosolic amino acids. T cells from older adults have an increased expression of the plasma membrane leucine transporter SLC7A5 to provide a cytosolic amino acid source. Hence, SLC7A5 and VPS39 deficiency (a member of the HOPS complex promoting early to late endosome conversion) substantially reduced mTORC1 activities in T cells from older but not young individuals. Late endosomal mTORC1 is independent of the negative-feedback loop involving mTORC1-induced inactivation of the transcription factor TFEB that controls expression of lysosomal genes. The resulting sustained mTORC1 activation impaired lysosome function and prevented lysosomal degradation of PD-1 in CD4+ T cells from older adults, thereby inhibiting their proliferative responses. VPS39 silencing of human T cells improved their expansion to pertussis and to SARS-CoV-2 peptides in vitro. Furthermore, adoptive transfer of CD4+ Vps39-deficient LCMV-specific SMARTA cells improved germinal center responses, CD8+ memory T cell generation, and recall responses to infection. Thus, curtailing late endosomal mTORC1 activity is a promising strategy to enhance T cell immunity.

Metabolic remodeling precedes mTORC1-mediated cardiac hypertrophy

RationaleThe nutrient sensing mechanistic target of rapamycin complex 1 (mTORC1) and its primary inhibitor, tuberin (TSC2), are cues for the development of cardiac hypertrophy. The phenotype of mTORC1 induced hypertrophy is unknown. ObjectiveTo examine the impact of sustained mTORC1 activation on metabolism, function, and structure of the adult heart. Methods and resultsWe developed a mouse model of inducible, cardiac-specific sustained mTORC1 activation (mTORC1iSA) through deletion of Tsc2. Prior to hypertrophy, rates of glucose uptake and oxidation, as well as protein and enzymatic activity of glucose 6-phosphate isomerase (GPI) were decreased, while intracellular levels of glucose 6-phosphate (G6P) were increased. Subsequently, hypertrophy developed. Transcript levels of the fetal gene program and pathways of exercise-induced hypertrophy increased, while hypertrophy did not progress to heart failure. We therefore examined the hearts of wild-type mice subjected to voluntary physical activity and observed early changes in GPI, followed by hypertrophy. Rapamycin prevented these changes in both models. ConclusionActivation of mTORC1 in the adult heart triggers the development of a non-specific form of hypertrophy which is preceded by changes in cardiac glucose metabolism.

Sustained mTORC1 activity during palbociclib-induced growth arrest triggers senescence in ER+ breast cancer cells

ABSTRACT Palbociclib, a selective CDK4/6 kinase inhibitor, is approved in combination with endocrine therapies for the treatment of advanced estrogen receptor positive (ER+) breast cancer. In pre-clinical cancer models, CDK4/6 inhibitors act primarily as cytostatic agents. In two commonly studied ER+ breast cancer cell lines (MCF7 and T47D), CDK4/6 inhibition drives G1-phase arrest and the acquisition of a senescent-like phenotype, both of which are reversible upon palbociclib withdrawal (incomplete senescence). Here we identify an ER+ breast cancer cell line, CAMA1, in which palbociclib treatment induces irreversible cell cycle arrest and senescence (complete senescence). In stark contrast to T47D and MCF7 cells, mTORC1 activity is not stably suppressed in CAMA1 cells during palbociclib treatment. Importantly, inhibition of mTORC1 signaling either by the mTORC1 inhibitor rapamycin or by knockdown of Raptor, a unique component of mTORC1, during palbociclib treatment of CAMA1 cells blocks the induction of complete senescence. These results indicate that sustained mTORC1 activity promotes complete senescence in ER+ breast cancer cells during CDK4/6 inhibitor-induced cell cycle arrest. Consistent with this mechanism, genetic depletion of TSC2, a negative regulator of mTORC1, in MCF7 cells resulted in sustained mTORC1 activity during palbociclib treatment and evoked a complete senescence response. These findings demonstrate that persistent mTORC1 signaling during palbociclib-induced G1 arrest is a potential liability for ER+ breast cancer cells, and suggest a strategy for novel drug combinations with palbociclib.

Oncogenic activation of PI3K-AKT-mTOR signaling suppresses ferroptosis via SREBP-mediated lipogenesis.

Ferroptosis, a form of regulated necrosis driven by iron-dependent peroxidation of phospholipids, is regulated by cellular metabolism, redox homeostasis, and various signaling pathways related to cancer. In this study, we found that activating mutation of phosphatidylinositol 3-kinase (PI3K) or loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) function, highly frequent events in human cancer, confers ferroptosis resistance in cancer cells, and that inhibition of the PI3K-AKT-mTOR signaling axis sensitizes cancer cells to ferroptosis induction. Mechanistically, this resistance requires sustained activation of mTORC1 and the mechanistic target of rapamycin (mTOR)C1-dependent induction of sterol regulatory element-binding protein 1 (SREBP1), a central transcription factor regulating lipid metabolism. Furthermore, stearoyl-CoA desaturase-1 (SCD1), a transcriptional target of SREBP1, mediates the ferroptosis-suppressing activity of SREBP1 by producing monounsaturated fatty acids. Genetic or pharmacologic ablation of SREBP1 or SCD1 sensitized ferroptosis in cancer cells with PI3K-AKT-mTOR pathway mutation. Conversely, ectopic expression of SREPB1 or SCD1 restored ferroptosis resistance in these cells, even when mTORC1 was inhibited. In xenograft mouse models for PI3K-mutated breast cancer and PTEN-defective prostate cancer, the combination of mTORC1 inhibition with ferroptosis induction resulted in near-complete tumor regression. In conclusion, hyperactive mutation of PI3K-AKT-mTOR signaling protects cancer cells from oxidative stress and ferroptotic death through SREBP1/SCD1-mediated lipogenesis, and combination of mTORC1 inhibition with ferroptosis induction shows therapeutic promise in preclinical models.

Adult-Onset Myopathy with Constitutive Activation of Akt following the Loss of hnRNP-U.

SummarySkeletal muscle has the remarkable ability to modulate its mass in response to changes in nutritional input, functional utilization, systemic disease, and age. This is achieved by the coordination of transcriptional and post-transcriptional networks and the signaling cascades balancing anabolic and catabolic processes with energy and nutrient availability. The extent to which alternative splicing regulates these signaling networks is uncertain. Here we investigate the role of the RNA-binding protein hnRNP-U on the expression and splicing of genes and the signaling processes regulating skeletal muscle hypertrophic growth. Muscle-specific Hnrnpu knockout (mKO) mice develop an adult-onset myopathy characterized by the selective atrophy of glycolytic muscle, the constitutive activation of Akt, increases in cellular and metabolic stress gene expression, and changes in the expression and splicing of metabolic and signal transduction genes. These findings link Hnrnpu with the balance between anabolic signaling, cellular and metabolic stress, and physiological growth.

ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation

Background:The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. Objective:The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. Methods:Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0–16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. Results:Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. Conclusions:The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.

Macropinocytosis drives T cell growth by sustaining the activation of mTORC1

Macropinocytosis is an evolutionarily-conserved, large-scale, fluid-phase form of endocytosis that has been ascribed different functions including antigen presentation in macrophages and dendritic cells, regulation of receptor density in neurons, and regulation of tumor growth under nutrient-limiting conditions. However, whether macropinocytosis regulates the expansion of non-transformed mammalian cells is unknown. Here we show that primary mouse and human T cells engage in macropinocytosis that increases in magnitude upon T cell activation to support T cell growth even under amino acid (AA) replete conditions. Mechanistically, macropinocytosis in T cells provides access of extracellular AA to an endolysosomal compartment to sustain activation of the mechanistic target of rapamycin complex 1 (mTORC1) that promotes T cell growth. Our results thus implicate a function of macropinocytosis in mammalian cell growth beyond Ras-transformed tumor cells via sustained mTORC1 activation.

Sarcopenia: Tilting the Balance of Protein Homeostasis.

Sarcopenia, defined as age-associated decline of muscle mass and function, is a risk factor for mortality and disability, and comorbid with several chronic diseases such as type II diabetes and cardiovascular diseases. Clinical trials showed that nutritional supplements had positive effects on muscle mass, but not on muscle function and strength, demonstrating our limited understanding of the molecular events involved in the ageing muscle. Protein homeostasis, the equilibrium between protein synthesis and degradation, is proposed as the major mechanism underlying the development of sarcopenia. As the key central regulator of protein homeostasis, the mammalian target of rapamycin (mTOR) is proposed to be essential for muscle hypertrophy. Paradoxically, sustained activation of mTOR complex 1 (mTORC1) is associated with a loss of sensitivity to extracellular signaling in the elderly. It is not understood why sustained mTORC1 activity, which should induce muscle hypertrophy, instead results in muscle atrophy. Here, recent findings on the implications of disrupting protein homeostasis on muscle physiology and sarcopenia development in the context of mTOR/protein kinase B (AKT) signaling are reviewed. Understanding the role of these molecular mechanisms during the ageing process will contribute towards the development of targeted therapies that will improve protein metabolism and reduce sarcopenia.

MTORC1 and PKB/Akt control the muscle response to denervation by regulating autophagy and HDAC4

Loss of innervation of skeletal muscle is a determinant event in several muscle diseases. Although several effectors have been identified, the pathways controlling the integrated muscle response to denervation remain largely unknown. Here, we demonstrate that PKB/Akt and mTORC1 play important roles in regulating muscle homeostasis and maintaining neuromuscular endplates after nerve injury. To allow dynamic changes in autophagy, mTORC1 activation must be tightly balanced following denervation. Acutely activating or inhibiting mTORC1 impairs autophagy regulation and alters homeostasis in denervated muscle. Importantly, PKB/Akt inhibition, conferred by sustained mTORC1 activation, abrogates denervation-induced synaptic remodeling and causes neuromuscular endplate degeneration. We establish that PKB/Akt activation promotes the nuclear import of HDAC4 and is thereby required for epigenetic changes and synaptic gene up-regulation upon denervation. Hence, our study unveils yet-unknown functions of PKB/Akt-mTORC1 signaling in the muscle response to nerve injury, with important implications for neuromuscular integrity in various pathological conditions.

Insulin Signaling and Insulin Resistance Facilitate Trained Immunity in Macrophages Through Metabolic and Epigenetic Changes.

Adaptation of the innate immune system has been recently acknowledged, explaining sustained changes of innate immune responses. Such adaptation is termed trained immunity. Trained immunity is initiated by extracellular signals that trigger a cascade of events affecting cell metabolism and mediating chromatin changes on genes that control innate immune responses. Factors demonstrated to facilitate trained immunity are pathogenic signals (fungi, bacteria, viruses) as well non-pathogenic signals such as insulin, cytokines, adipokines or hormones. These signals initiate intracellular signaling cascades that include AKT kinases and mTOR as well as histone methylases and demethylases, resulting in metabolic changes and histone modifications. In the context of insulin resistance, AKT signaling is affected resulting in sustained activation of mTORC1 and enhanced glycolysis. In macrophages elevated glycolysis readily impacts responses to pathogens (bacteria, fungi) or danger signals (TLR-driven signals of tissue damage), partly explaining insulin resistance-related pathologies. Thus, macrophages lacking insulin signaling exhibit reduced responses to pathogens and altered metabolism, suggesting that insulin resistance is a state of trained immunity. Evidence from Insulin Receptor as well as IGF1Receptor deficient macrophages support the contribution of insulin signaling in macrophage responses. In addition, clinical evidence highlights altered macrophage responses to pathogens or metabolic products in patients with systemic insulin resistance, being in concert with cell culture and animal model studies. Herein, we review the current knowledge that supports the impact of insulin signaling and other insulin resistance related signals as modulators of trained immunity.

Macropinocytosis of amino acids regulates T cell growth by promoting the sustained activation of mTORC1

Abstract Macropinocytosis, a non-selective form of bulk endocytosis, has been associated with diverse processes in mammalian cells, including antigen presentation and regulation of receptor density. In Ras-transformed tumor cells, macropinocytosis of protein regulates growth when amino acids are limiting in the extracellular space. A requirement of macropinocytosis for the growth of non-transformed cells has not been described previously. Here we show that primary mouse and human T cells engage in constitutive macropinocytosis that is enhanced approximately three-fold in response to CD3/CD28 antibody stimulation. We further show that macropinocytosis is essential for the growth of CD3/CD28 antibody-stimulated T cells during the G1 phase of the cell cycle, even under amino acid-replete conditions. Macropinocytosis in activated T cells provides a means of access of extracellular amino acids to an endolysosomal compartment necessary for sustained activation of the mechanistic target of rapamycin complex 1 (mTORC1), which in part explains its function in G1 growth. In addition, macropinocytosis promotes activated T cell growth by means other than activation of mTORC1, possibly by furnishing bulk substrates for anabolism and anaplerosis. Regulation of mammalian cell growth by macropinocytosis, therefore, is not limited to Ras-transformed tumor cells but may instead represent a more general means of promoting sustained mTORC1 activation in highly proliferative cells. These findings provide important new information upon the mechanisms by which T cells acquire nutrients for growth and suggest means by which T cell growth might be manipulated for therapeutic benefit.

Exosome Release Is Regulated by mTORC1.

Exosomes are small membrane‐bound vesicles released into extracellular spaces by many types of cells. These nanovesicles carry proteins, mRNA, and miRNA, and are involved in cell waste management and intercellular communication. In the present study, it is shown that exosome release, which leads to net loss of cellular membrane and protein content, is negatively regulated by mechanistic target of rapamycin complex 1 (mTORC1). It is found that in cells and animal models exosome release is inhibited by sustained activation of mTORC1, leading to intracellular accumulation of CD63‐positive exosome precursors. Inhibition of mTORC1 by rapamycin or nutrient and growth factor deprivation stimulates exosome release, which occurs concomitantly with autophagy. The drug‐stimulated release is blocked by siRNA‐mediated downregulation of small GTPase Rab27A. Analysis of the cargo content in exosomes released from rapamycin‐treated cells reveals that inhibition of mTORC1 does not significantly alter its majority protein and miRNA profiles. These observations demonstrate that exosome release, like autophagy, is negatively regulated by mTORC1 in response to changes in nutrient and growth factor conditions.

Remodeling mTORC1 Responsiveness to Amino Acids by the Herpes Simplex Virus UL46 and Us3 Gene Products Supports Replication during Nutrient Insufficiency.

By sensing fundamental parameters, including nutrient availability, activated mechanistic target of rapamycin complex 1 (mTORC1) suppresses catabolic outcomes and promotes anabolic processes needed for herpes simplex virus 1 (HSV-1) productive growth. While the virus-encoded Us3 Ser/Thr kinase is required to activate mTORC1, whether stress associated with amino acid insufficiency impacts mTORC1 activation in infected cells and virus reproduction was unknown. In contrast to uninfected cells, where amino acid withdrawal inhibits mTORC1 activation, we demonstrate that mTORC1 activity is sustained in HSV-1-infected cells during amino acid insufficiency. We show that in the absence of Us3, the insensitivity of mTORC1 to amino acid withdrawal in infected cells was dependent on the host kinase Akt and establish a role for the HSV-1 UL46 gene product, which stimulates phosphatidylinositol (PI) 3-kinase signaling. Significantly, virus reproduction during amino acid insufficiency was stimulated by the viral UL46 gene product. By synergizing with Us3, UL46 reprograms mTORC1 such that it is insensitive to amino acid withdrawal and supports sustained mTORC1 activation and virus reproduction during amino acid insufficiency. This identifies an unexpected function for UL46 in supporting virus reproduction during physiological stress and identifies a new class of virus-encoded mTORC1 regulators that selectively uncouple mTORC1 activation from amino acid sufficiency.IMPORTANCE Mechanistic target of rapamycin complex 1 (mTORC1) is a multisubunit cellular kinase that coordinates protein synthesis with changing amino acid levels. During amino acid insufficiency, mTORC1 is repressed in uninfected cells, dampening protein synthesis and potentially restricting virus reproduction. Here, we establish that HSV-1 alters the responsiveness of mTORC1 to metabolic stress resulting from amino acid insufficiency. Unlike in uninfected cells, mTORC1 remains activated in HSV-1-infected cells deprived of amino acids. Synergistic action of the HSV-1 UL46 gene product, which stimulates PI 3-kinase, and the Us3 kinase supports virus reproduction during amino acid withdrawal. These results define how HSV-1, a medically important human pathogen associated with a range of diseases, uncouples mTORC1 activation from amino acid availability. Furthermore, they help explain how the virus reproduces during physiological stress. Reproduction triggered by physiological stress is characteristic of herpesvirus infections, where lifelong latency is punctuated by episodic reactivation events.

Nociceptor Translational Profiling Reveals the Ragulator-Rag GTPase Complex as a Critical Generator of Neuropathic Pain.

Nociceptors, sensory neurons in the DRG that detect damaging or potentially damaging stimuli, are key drivers of neuropathic pain. Injury to these neurons causes activation of translation regulation signaling, including the mechanistic target of rapamycin complex 1 (mTORC1) and mitogen-activated protein kinase interacting kinase (MNK) eukaryotic initiation factor (eIF) 4E pathways. This is a mechanism driving changes in excitability of nociceptors that is critical for the generation of chronic pain states; however, the mRNAs that are translated to lead to this plasticity have not been elucidated. To address this gap in knowledge, we used translating ribosome affinity purification in male and female mice to comprehensively characterize mRNA translation in Scn10a-positive nociceptors in chemotherapy-induced neuropathic pain (CIPN) caused by paclitaxel treatment. This unbiased method creates a new resource for the field, confirms many findings in the CIPN literature and also find extensive evidence for new target mechanisms that may cause CIPN. We provide evidence that an underlying mechanism of CIPN is sustained mTORC1 activation driven by MNK1-eIF4E signaling. RagA, a GTPase controlling mTORC1 activity, is identified as a novel target of MNK1-eIF4E signaling. This demonstrates a novel translation regulation signaling circuit wherein MNK1-eIF4E activity drives mTORC1 via control of RagA translation. CIPN and RagA translation are strongly attenuated by genetic ablation of eIF4E phosphorylation, MNK1 elimination or treatment with the MNK inhibitor eFT508. We identify a novel translational circuit for the genesis of neuropathic pain caused by chemotherapy with important implications for therapeutics.SIGNIFICANCE STATEMENT Neuropathic pain affects up to 10% of the population, but its underlying mechanisms are incompletely understood, leading to poor treatment outcomes. We used translating ribosome affinity purification technology to create a comprehensive translational profile of DRG nociceptors in naive mice and at the peak of neuropathic pain induced by paclitaxel treatment. We reveal new insight into how mechanistic target of rapamycin complex 1 is activated in neuropathic pain pointing to a key role of MNK1-eIF4E-mediated translation of a complex of mRNAs that control mechanistic target of rapamycin complex 1 signaling at the surface of the lysosome. We validate this finding using genetic and pharmacological techniques. Our work strongly suggests that MNK1-eIF4E signaling drives CIPN and that a drug in human clinical trials, eFT508, may be a new therapeutic for neuropathic pain.