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Hepatocellular carcinoma (HCC) is a common malignant tumor with high mortality. Human phenylalanine tRNA synthetase (PheRS) comprises two α catalytic subunits encoded by the FARSA gene and two β regulatory subunits encoded by the FARSB gene. FARSB is a potential oncogene, but no experimental data show the relationship between FARSB and HCC progression. We found that the high expression of FARSB in liver cancer is closely related to patients' low survival and poor prognosis. In liver cancer cells, the mRNA and protein expression levels of FARSB are increased and promote cell proliferation and migration. Mechanistically, FARSB activates the mTOR complex 1 (mTORC1) signaling pathway by binding to the component Raptor of the mTORC1 complex to play a role in promoting cancer. In addition, we found that FARSB can inhibit erastin-induced ferroptosis by regulating the mTOR signaling pathway, which may be another mechanism by which FARSB promotes HCC progression. In summary, FARSB promotes HCC progression and is associated with the poor prognosis of patients. FARSB is expected to be a biomarker for early screening and treatment of HCC. [ABSTRACT FROM AUTHOR]

Abstract Background mTORC1 (mechanistic target of rapamycin complex 1) is associated with lymphoma progression. Oncogenic RRAGC (Rag guanosine triphosphatase C) mutations identified in patients with follicular lymphoma facilitate the interaction between Raptor (regulatory protein associated with mTOR) and Rag GTPase. It promotes the activation of mTORC1 and accelerates lymphomagenesis. Cardamonin inhibits mTORC1 by decreasing the protein level of Raptor. In the present study, we investigated the inhibitory effect and possible mechanism of action of cardamonin in RRAGC-mutant lymphoma. This could provide a precise targeted therapy for lymphoma with RRAGC mutations. Methods Cell viability was measured using a cell counting kit-8 (CCK-8) assay. Protein expression and phosphorylation levels were determined using western blotting. The interactions of mTOR and Raptor with RagC were determined by co-immunoprecipitation. Cells overexpressing RagC wild-type (RagCWT) and RagC Thr90Asn (RagCT90N) were generated by lentiviral infection. Raptor knockdown was performed by lentivirus-mediated shRNA transduction. The in vivo anti-tumour effect of cardamonin was assessed in a xenograft model. Results Cardamonin disrupted mTOR complex interactions by decreasing Raptor protein levels. RagCT90N overexpression via lentiviral infection increased cell proliferation and mTORC1 activation. The viability and tumour growth rate of RagCT90N-mutant cells were more sensitive to cardamonin treatment than those of normal and RagCWT cells. Cardamonin also exhibited a stronger inhibitory effect on the phosphorylation of mTOR and p70 S6 kinase 1 in RagCT90N-mutant cells. Raptor knockdown abolishes the inhibitory effects of cardamonin on mTOR. An in vivo xenograft model demonstrated that the RagCT90N-mutant showed significantly higher sensitivity to cardamonin treatment. Conclusions Cardamonin exerts selective therapeutic effects on RagCT90N-mutant cells. Cardamonin can serve as a drug for individualised therapy for follicular lymphoma with RRAGC mutations.

The mTOR pathway is an essential regulator of cell growth and metabolism. Midbrain dopamine neurons are particularly sensitive to mTOR signaling status as activation or inhibition of mTOR alters their morphology and physiology. mTOR exists in two distinct multiprotein complexes termed mTORC1 and mTORC2. How each of these complexes affect dopamine neuron properties, and whether they have similar or distinct functions is unknown. Here, we investigated this in mice with dopamine neuron-specific deletion of Rptor or Rictor, which encode obligatory components of mTORC1 or mTORC2, respectively. We find that inhibition of mTORC1 strongly and broadly impacts dopamine neuron structure and function causing somatodendritic and axonal hypotrophy, increased intrinsic excitability, decreased dopamine production, and impaired dopamine release. In contrast, inhibition of mTORC2 has more subtle effects, with selective alterations to the output of ventral tegmental area dopamine neurons. Disruption of both mTOR complexes leads to pronounced deficits in dopamine release demonstrating the importance of balanced mTORC1 and mTORC2 signaling for dopaminergic function.

Background: mTORC1 (mechanistic target of rapamycin complex 1) is associated with lymphoma progression. Oncogenic RRAGC (Rag guanosine triphosphatase C) mutations identified in patients with follicular lymphoma facilitate the interaction between Raptor (regulatory protein associated with mTOR) and Rag GTPase. It promotes the activation of mTORC1 and accelerates lymphomagenesis. Cardamonin inhibits mTORC1 by decreasing the protein level of Raptor. In the present study, we investigated the inhibitory effect and possible mechanism of action of cardamonin in RRAGC-mutant lymphoma. This could provide a precise targeted therapy for lymphoma with RRAGC mutations. Methods: Cell viability was measured using a cell counting kit-8 (CCK-8) assay. Protein expression and phosphorylation levels were determined using western blotting. The interactions of mTOR and Raptor with RagC were determined by co-immunoprecipitation. Cells overexpressing RagC wild-type (RagCWT) and RagC Thr90Asn (RagCT90N) were generated by lentiviral infection. Raptor knockdown was performed by lentivirus-mediated shRNA transduction. The in vivo anti-tumour effect of cardamonin was assessed in a xenograft model. Results: Cardamonin disrupted mTOR complex interactions by decreasing Raptor protein levels. RagCT90N overexpression via lentiviral infection increased cell proliferation and mTORC1 activation. The viability and tumour growth rate of RagCT90N-mutant cells were more sensitive to cardamonin treatment than those of normal and RagCWT cells. Cardamonin also exhibited a stronger inhibitory effect on the phosphorylation of mTOR and p70 S6 kinase 1 in RagCT90N-mutant cells. Raptor knockdown abolishes the inhibitory effects of cardamonin on mTOR. An in vivo xenograft model demonstrated that the RagCT90N-mutant showed significantly higher sensitivity to cardamonin treatment. Conclusions: Cardamonin exerts selective therapeutic effects on RagCT90N-mutant cells. Cardamonin can serve as a drug for individualised therapy for follicular lymphoma with RRAGC mutations. [ABSTRACT FROM AUTHOR]

The mechanistic target of rapamycin (mTOR) is a kinase at the center of an evolutionarily conserved signaling pathway that orchestrates cell growth and metabolism. mTOR responds to an array of intra- and extracellular stimuli and in turn controls multiple cellular anabolic and catabolic processes. Aberrant mTOR activity is associated with numerous diseases, with particularly profound impact on the nervous system. mTOR is found in two protein complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2), which are governed by different upstream regulators and have distinct cellular actions. Mutations in genes encoding for mTOR regulators result in a collection of neurodevelopmental disorders known as mTORopathies. While these disorders can affect multiple organs, neuropsychiatric conditions such as epilepsy, intellectual disability, and autism spectrum disorder have a major impact on quality of life. The neuropsychiatric aspects of mTORopathies have been particularly challenging to treat in a clinical setting. Current therapeutic approaches center on rapamycin and its analogs, drugs that are administered systemically to inhibit mTOR activity. While these drugs show some clinical efficacy, adverse side effects, incomplete suppression of mTOR targets, and lack of specificity for mTORC1 or mTORC2 may limit their utility. An increased understanding of the neurobiology of mTOR and the underlying molecular, cellular, and circuit mechanisms of mTOR-related disorders will facilitate the development of improved therapeutics. Animal models of mTORopathies have helped unravel the consequences of mTOR pathway mutations in specific brain cell types and developmental stages, revealing an array of disease-related phenotypes. In this review, we discuss current progress and potential future directions for the therapeutic treatment of mTORopathies with a focus on findings from genetic mouse models.

Raptor, a key component of mTORC1, is required for recruiting substrates to mTORC1 and contributing to its subcellular localization. Raptor has a highly conserved N-terminus domain and seven WD40 repeats, which interact with mTOR and other mTORC1-related proteins. mTORC1 participates in various cellular events and mediates differentiation and metabolism. Directly or indirectly, many factors mediate the differentiation and function of lymphocytes that is essential for immunity. In this review, we summarize the role of Raptor in lymphocytes differentiation and function, whereby Raptor mediates the secretion of cytokines to induce early lymphocyte metabolism, development, proliferation and migration. Additionally, Raptor regulates the function of lymphocytes by regulating their steady-state maintenance and activation. [ABSTRACT FROM AUTHOR]

Objective: Obesity is a complex disorder and is linked to chronic diseases such as type 2 diabetes. Major intrinsically disordered NOTCH2-associated receptor2 (MINAR2) is an understudied protein with an unknown role in obesity and metabolism. The purpose of this study was to determine the impact of Minar2 on adipose tissues and obesity. Method: We generated Minar2 knockout (KO) mice and used various molecular, proteomic, biochemical, histopathology, and cell culture studies to determine the pathophysiological role of Minar2 in adipocytes. Results: We demonstrated that the inactivation of Minar2 results in increased body fat with hypertrophic adipocytes. Minar2 KO mice on a high-fat diet develop obesity and impaired glucose tolerance and metabolism. Mechanistically, Minar2 interacts with Raptor, a specific and essential component of mammalian TOR complex 1 (mTORC1) and inhibits mTOR activation. mTOR is hyperactivated in the adipocytes deficient for Minar2 and over-expression of Minar2 in HEK-293 cells inhibited mTOR activation and phosphorylation of mTORC1 substrates, including S6 kinase, and 4E-BP1. Conclusion: Our findings identified Minar2 as a novel physiological negative regulator of mTORC1 with a key role in obesity and metabolic disorders. Impaired expression or activation of MINAR2 could lead to obesity and obesity-associated diseases.

Concentric lung vascular wall thickening due to enhanced proliferation of pulmonary arterial smooth muscle cells is an important pathological cause for the elevated pulmonary vascular resistance reported in patients with pulmonary arterial hypertension. We identified a differential role of mammalian target of rapamycin (mTOR) complex 1 and complex 2, two functionally distinct mTOR complexes, in the development of pulmonary hypertension (PH). Inhibition of mTOR complex 1 attenuated the development of PH; however, inhibition of mTOR complex 2 caused spontaneous PH, potentially due to up-regulation of platelet-derived growth factor receptors in pulmonary arterial smooth muscle cells, and compromised the therapeutic effect of the mTOR inhibitors on PH. In addition, we describe a promising therapeutic strategy using combination treatment with the mTOR inhibitors and the platelet-derived growth factor receptor inhibitors on PH and right ventricular hypertrophy. The data from this study provide an important mechanism-based perspective for developing novel therapies for patients with pulmonary arterial hypertension and right heart failure.

Hepatocellular carcinoma (HCC) is a common malignant tumor with high mortality. Human phenylalanine tRNA synthetase (PheRS) comprises two α catalytic subunits encoded by the FARSA gene and two β regulatory subunits encoded by the FARSB gene. FARSB is a potential oncogene, but no experimental data show the relationship between FARSB and HCC progression. We found that the high expression of FARSB in liver cancer is closely related to patients’ low survival and poor prognosis. In liver cancer cells, the mRNA and protein expression levels of FARSB are increased and promote cell proliferation and migration. Mechanistically, FARSB activates the mTOR complex 1 (mTORC1) signaling pathway by binding to the component Raptor of the mTORC1 complex to play a role in promoting cancer. In addition, we found that FARSB can inhibit erastin-induced ferroptosis by regulating the mTOR signaling pathway, which may be another mechanism by which FARSB promotes HCC progression. In summary, FARSB promotes HCC progression and is associated with the poor prognosis of patients. FARSB is expected to be a biomarker for early screening and treatment of HCC.

The mTOR pathway is an essential regulator of cell growth and metabolism. Midbrain dopamine neurons are particularly sensitive to mTOR signaling status as activation or inhibition of mTOR alters their morphology and physiology. mTOR exists in two distinct multiprotein complexes termed mTORC1 and mTORC2. How each of these complexes affect dopamine neuron properties, and whether they have similar or distinct functions is unknown. Here, we investigated this in mice with dopamine neuron-specific deletion of Rptor or Rictor, which encode obligatory components of mTORC1 or mTORC2, respectively. We find that inhibition of mTORC1 strongly and broadly impacts dopamine neuron structure and function causing somatodendritic and axonal hypotrophy, increased intrinsic excitability, decreased dopamine production, and impaired dopamine release. In contrast, inhibition of mTORC2 has more subtle effects, with selective alterations to the output of ventral tegmental area dopamine neurons. Disruption of both mTOR complexes leads to pronounced deficits in dopamine release demonstrating the importance of balanced mTORC1 and mTORC2 signaling for dopaminergic function.

Background: The coordinated wiring of neurons, glia and endothelial cells into neurovascular units is critical for central nervous system development. This is best exemplified in the mammalian retina where interneurons, astrocytes and retinal ganglion cells sculpt their vascular environment to meet the metabolic demands of visual function. Identifying the molecular networks that underlie neurovascular unit formation is an important step towards a deeper understanding of nervous system development and function. Results: Here, we report that cell‐to‐cell mTORC1‐signaling is essential for neurovascular unit formation during mouse retinal development. Using a conditional knockout approach we demonstrate that reduced mTORC1 activity in asymmetrically positioned retinal ganglion cells induces a delay in postnatal vascular network formation in addition to the production of rudimentary and tortuous vessel networks in adult animals. The severity of this vascular phenotype is directly correlated to the degree of mTORC1 down regulation within the neighboring retinal ganglion cell population. Conclusions: This study establishes a cell nonautonomous role for mTORC1‐signaling during retinal development. These findings contribute to our current understanding of neurovascular unit formation and demonstrate how ganglion cells actively sculpt their local environment to ensure that the retina is perfused with an appropriate supply of oxygen and nutrients. Key Findings: Reduced mTORC1‐signaling in RPCs leads to asymmetric retinal ganglion cell mosaics.Asymmetric retinal ganglion cell mosaics induce delayed vascular network formation.Delayed vascular network formation leads to tortuous vessels with reduced complexity.Vascular phenotype severity is correlated with the degree of reduced mTORC1 activity. [ABSTRACT FROM AUTHOR]

Abstract Mammalian target of rapamycin complex 1 (mTORC1) is evolutionally conserved and frequently activated in various tumors, including colorectal cancer (CRC). It has been reported that the ribosome assembly factor Urb1 acts downstream of mTORC1/raptor signaling and contributes to digestive organ development in zebrafish. Previously, we highlighted that URB1 was overexpressed in CRC. Here, we assessed the mTORC1/regulatory associated protein with mTOR (RAPTOR)‐URB1 axis in CRC tumorigenesis. We found that RAPTOR was overexpressed in CRC tissues and cell lines, was a favorable predictor in patients with CRC, and positively correlated with URB1. Silencing of RAPTOR suppressed CRC cell proliferation and migration and induced cell cycle arrest and apoptosis in vitro and inhibited xenograft growth in vivo. Moreover, ectopic overexpression of RAPTOR exerted an inverse biological phenotype. Knockdown of RAPTOR quenched mTORC1 activity and reduced the expression of URB1 and cyclinA2 (CCNA2). In contrast, overexpression of RAPTOR activated mTORC1 and upregulated URB1 and CCNA2. Furthermore, URB1 and CCNA2 expression were also impeded by rapamycin, which is a specific inhibitor of mTORC1. Thus, RAPTOR promoted CRC proliferation, migration, and cell cycle progression by inducing mTORC1 signaling and transcriptional activation of both URB1 and CCNA2. Taken together, we concluded that RAPTOR has the potential to serve as a novel biomarker and therapeutic target for CRC.

The antiproliferative effect of cardamonin on mTORC1 is related with downregulation of Raptor. We investigated the mechanism that cardamonin decreases Raptor expression through caspase-mediated protein degradation. SKOV3 cells and HeLa cells were pretreated with caspase inhibitor z-VAD-fmk for 30 min and then exposed to different doses of cardamonin and cisplatin, respectively. We analyzed the gene expression of caspases based on TCGA and GTEx gene expression data in serous cystadenocarcinoma and normal tissues, monitored caspase activity by caspase colorimetric assay kit, detected expression of mTORC1-associated proteins and apoptosis-associated proteins by western blotting, and finally detected cell viability by methyl thiazolyl tetrazolium (MTT) assay. A different expression of caspases except caspase-1 was found between serous cystadenocarcinoma and normal tissues. Raptor was cleaved when caspases were activated by cisplatin and caspase-6/caspase-8 was activated by cardamonin in SKOV3 cells. We further used a monoclonal antibody recognizing the N-terminal part of Raptor to find that Raptor was cleaved into a smaller fragment of about 70 kDa by cardamonin and was rescued by z-VAD-fmk treatment. As a result of Raptor cleavage, mTORC1 activity was decreased and cell viability was inhibited, while cell apoptosis was induced in SKOV3 cells. Notably, similar results are only observed in HeLa cells with a high dose of cardamonin. We concluded that caspase-mediated cleavage of Raptor might be an important mechanism in that cardamonin regulated Raptor and mTORC1 activity. [ABSTRACT FROM AUTHOR]

• The kinase Dapk2 is quickly induced in activated T cells. • Dapk2 deficient T cells promote GC responses by increasing cognate T-B interactions. • mTORC1 pathway mediates Dapk2 function in T cell. Germinal center (GC) formation is a critical step during T-dependent humoral immune responses. We report Death Associated Protein Kinase 2, a serine/threonine kinase, is rapidly induced in T cells following activation and plays an inhibitory role in T cell-mediated help for the GC formation. Specifically, T cells deficient in Dapk2 have an increased ability to physically conjugate with antigen-presenting B cells and to promote GC formation. However, Dapk2 does not regulate T cell receptor signaling strength and does not influence cytokine-driven T-cell subset polarization. Instead, Dapk2 dampens mTORC1 activities by associating with Raptor. Silencing of Raptor rescues defects observed with the Dapk2 insufficiency. Our study thus identifies Dapk2 as a new kinase likely involved in negative regulation of contact-dependent help delivery to B cells and GC formation. [ABSTRACT FROM AUTHOR]

The mechanistic target of Rapamycin (mTOR) is essential for multiple cellular processes. The unique roles of mTOR complex 1 (mTORC1) or mTOR2 in regulating immune functions are emerging. NK cells are the major lymphocyte subset of innate immunity, and their development and effector functions require metabolic reprogramming. Recent studies demonstrate that in NK cells, conditionally disrupting the formation of mTORC1 or mTOR complex 2 (mTORC2) alters their development significantly. Transcriptomic profiling of NK cells at the single-cell level demonstrates that mTORC1 was critical for the early developmental progression, while mTORC2 regulated the terminal maturation. In this review, we summarize the essential roles of mTOR complexes in NK development and functions.

Summary: mTOR is a serine/threonine kinase and a master regulator of cell growth and proliferation. Raptor, a scaffolding protein that recruits substrates to mTOR complex 1 (mTORC1), is known to be phosphorylated during mitosis, but the significance of this phosphorylation remains largely unknown. Here we show that raptor expression and mTORC1 activity are dramatically reduced in cells arrested in mitosis. Expression of a non-phosphorylatable raptor mutant reactivates mTORC1 and significantly reduces cytotoxicity of the mitotic poison Taxol. This effect is mediated via degradation of PDCD4, a tumor suppressor protein that inhibits eIF4A activity and is negatively regulated by the mTORC1/S6K pathway. Moreover, pharmacological inhibition of eIF4A is able to enhance the effects of Taxol and restore sensitivity in Taxol-resistant cancer cells. These findings indicate that the mTORC1/S6K/PDCD4/eIF4A axis has a pivotal role in the death versus slippage decision during mitotic arrest and may be exploited clinically to treat tumors resistant to anti-mitotic agents.

The mammalian target of rapamycin (mTOR) is an evolutionarily highly conserved atypical serine/threonine protein kinase, which regulates cell growth, proliferation, apoptosis, autophagy, and metabolism. As a regulatory protein, Raptor is awfully important for the stability and function of mTOR complex 1 (mTORC1). However, the studies about how Raptor/mTORC1 participates in and regulates immune response in lower vertebrates are still limited. In this study, we investigated the regulation of immune response by the Raptor/mTORC1 signaling pathway in rockfish Sebastes schlegelii. Sebastes schlegelii Raptor (Ss-Raptor) is a highly conserved protein during the evolution, in both primary and tertiary structure. Ss-Raptor mRNA was widely distributed in various tissues of rockfish and has a relative higher expression in spleen and blood. After infected by Micrococcus luteus or Listonella anguillarum, mRNA expression of Ss-Raptor rapidly increased within 48 h. Once Raptor/mTORC1 signaling was blocked by rapamycin, expression of the pro-inflammatory cytokines IL-1β and IL-8 was severely impaired, suggesting potential regulatory role of Raptor/mTORC1 signaling in the innate immune response of rockfish. In addition, Raptor/mTORC1 pathway participated in lymphocyte activation of rockfish through promoting 4EBP1 and S6 phosphorylation. Inhibition of Raptor/mTORC1 signaling crippled the lymphocyte expansion during primary adaptive immune response, manifesting by the decrease of lymphoid organ weight and lymphocyte numbers. More importantly, inhibition of Raptor/mTORC1 signaling impaired the lymphocyte mediated cytotoxic response, and made the fish more vulnerable to the bacterial infection. Together, our results suggested that Raptor and its tightly regulated mTORC1 signaling acts as modulatory center to regulate both innate and lymphocyte-mediated adaptive immune response during bacterial infection. This research has shed new light on regulatory mechanism of teleost immune response, and provide helpful evidences to understand the evolution of immune system.

Mammalian target of rapamycin complex 1 (mTORC1) is evolutionally conserved and frequently activated in various tumors, including colorectal cancer (CRC). It has been reported that the ribosome assembly factor Urb1 acts downstream of mTORC1/raptor signaling and contributes to digestive organ development in zebrafish. Previously, we highlighted that URB1 was overexpressed in CRC. Here, we assessed the mTORC1/regulatory associated protein with mTOR (RAPTOR)‐URB1 axis in CRC tumorigenesis. We found that RAPTOR was overexpressed in CRC tissues and cell lines, was a favorable predictor in patients with CRC, and positively correlated with URB1. Silencing of RAPTOR suppressed CRC cell proliferation and migration and induced cell cycle arrest and apoptosis in vitro and inhibited xenograft growth in vivo. Moreover, ectopic overexpression of RAPTOR exerted an inverse biological phenotype. Knockdown of RAPTOR quenched mTORC1 activity and reduced the expression of URB1 and cyclinA2 (CCNA2). In contrast, overexpression of RAPTOR activated mTORC1 and upregulated URB1 and CCNA2. Furthermore, URB1 and CCNA2 expression were also impeded by rapamycin, which is a specific inhibitor of mTORC1. Thus, RAPTOR promoted CRC proliferation, migration, and cell cycle progression by inducing mTORC1 signaling and transcriptional activation of both URB1 and CCNA2. Taken together, we concluded that RAPTOR has the potential to serve as a novel biomarker and therapeutic target for CRC. [ABSTRACT FROM AUTHOR]

The mammalian target of rapamycin (mTOR) is an evolutionarily highly conserved atypical serine/threonine protein kinase, which regulates cell growth, proliferation, apoptosis, autophagy, and metabolism. As a regulatory protein, Raptor is awfully important for the stability and function of mTOR complex 1 (mTORC1). However, the studies about how Raptor/mTORC1 participates in and regulates immune response in lower vertebrates are still limited. In this study, we investigated the regulation of immune response by the Raptor/mTORC1 signaling pathway in rockfish Sebastes schlegelii. Sebastes schlegelii Raptor (Ss-Raptor) is a highly conserved protein during the evolution, in both primary and tertiary structure. Ss-Raptor mRNA was widely distributed in various tissues of rockfish and has a relative higher expression in spleen and blood. After infected by Micrococcus luteus or Listonella anguillarum , mRNA expression of Ss-Raptor rapidly increased within 48 h. Once Raptor/mTORC1 signaling was blocked by rapamycin, expression of the pro-inflammatory cytokines IL-1β and IL-8 was severely impaired, suggesting potential regulatory role of Raptor/mTORC1 signaling in the innate immune response of rockfish. In addition, Raptor/mTORC1 pathway participated in lymphocyte activation of rockfish through promoting 4EBP1 and S6 phosphorylation. Inhibition of Raptor/mTORC1 signaling crippled the lymphocyte expansion during primary adaptive immune response, manifesting by the decrease of lymphoid organ weight and lymphocyte numbers. More importantly, inhibition of Raptor/mTORC1 signaling impaired the lymphocyte mediated cytotoxic response, and made the fish more vulnerable to the bacterial infection. Together, our results suggested that Raptor and its tightly regulated mTORC1 signaling acts as modulatory center to regulate both innate and lymphocyte-mediated adaptive immune response during bacterial infection. This research has shed new light on regulatory mechanism of teleost immune response, and provide helpful evidences to understand the evolution of immune system. [ABSTRACT FROM AUTHOR]

Development of the vertebrate central nervous system involves the co-ordinated differentiation of progenitor cells and the establishment of functional neural networks. This neurogenic process is driven by both intracellular and extracellular cues that converge on the mammalian target of rapamycin complex 1 (mTORC1). Here we demonstrate that mTORC1-signalling mediates multi-faceted roles during central nervous system development using the mouse retina as a model system. Downregulation of mTORC1-signalling in retinal progenitor cells by conditional ablation of Rptor leads to proliferation deficits and an over-production of retinal ganglion cells during embryonic development. In contrast, reduced mTORC1-signalling in postnatal animals leads to temporal deviations in programmed cell death and the consequent production of asymmetric retinal ganglion cell mosaics and associated loss of axonal termination topographies in the dorsal lateral geniculate nucleus of adult mice. In combination these developmental defects induce visually mediated behavioural deficits. These collective observations demonstrate that mTORC1-signalling mediates critical roles during visual pathway development and function.

The mechanistic/mammalian target of rapamycin (mTOR) is widely implicated in the pathogenesis of various diseases, including cancer, obesity, and cardiovascular disease. Bone homeostasis is maintained by the actions of bone-resorbing osteoclasts and bone-forming osteoblasts. An imbalance in the sophisticated regulation of osteoclasts and osteoblasts leads to the pathogenesis as well as etiology of certain metabolic bone diseases, including osteoporosis and osteopetrosis. Here, we identified mTOR complex 1 (mTORC1) as a pivotal mediator in the regulation of bone resorption and bone homeostasis under pathological conditions through its expression in osteoclasts. The activity of mTORC1, which was indicated by the phosphorylation level of its downstream target p70S6 kinase, was reduced during osteoclast differentiation, in accordance with the upregulation of Hamartin (encoded by tuberous sclerosis complex 1 [Tsc1]), a negative regulator of mTORC1. Receptor activator of nuclear factor-κB ligand (RANKL)-dependent osteoclastogenesis was impaired in Tsc1-deficient bone marrow macrophages. By contrast, osteoclastogenesis was markedly enhanced by Raptor deficiency but was unaffected by Rictor deficiency. The deletion of Tsc1 in osteoclast lineage cells in mice prevented bone resorption and bone loss in a RANKL-induced mouse model of osteoporosis, although neither bone volume nor osteoclastic parameter was markedly altered in these knockout mice under physiological conditions. Therefore, these findings suggest that mTORC1 is a key potential target for the treatment of bone diseases.

The mechanistic/mammalian target of rapamycin (mTOR) is widely implicated in the pathogenesis of various diseases, including cancer, obesity, and cardiovascular disease. Bone homeostasis is maintained by the actions of bone-resorbing osteoclasts and bone-forming osteoblasts. An imbalance in the sophisticated regulation of osteoclasts and osteoblasts leads to the pathogenesis as well as etiology of certain metabolic bone diseases, including osteoporosis and osteopetrosis. Here, we identified mTOR complex 1 (mTORC1) as a pivotal mediator in the regulation of bone resorption and bone homeostasis under pathological conditions through its expression in osteoclasts. The activity of mTORC1, which was indicated by the phosphorylation level of its downstream target p70S6 kinase, was reduced during osteoclast differentiation, in accordance with the upregulation of Hamartin (encoded by tuberous sclerosis complex 1 [ Tsc1 ]), a negative regulator of mTORC1. Receptor activator of nuclear factor-κB ligand (RANKL)-dependent osteoclastogenesis was impaired in Tsc1 -deficient bone marrow macrophages. By contrast, osteoclastogenesis was markedly enhanced by Raptor deficiency but was unaffected by Rictor deficiency. The deletion of Tsc1 in osteoclast lineage cells in mice prevented bone resorption and bone loss in a RANKL-induced mouse model of osteoporosis, although neither bone volume nor osteoclastic parameter was markedly altered in these knockout mice under physiological conditions. Therefore, these findings suggest that mTORC1 is a key potential target for the treatment of bone diseases. [ABSTRACT FROM AUTHOR]

Mechanistic target of rapamycin (mTOR) resides as two complexes within skeletal muscle. mTOR complex 1 [mTORC1-regulatory associated protein of mTOR (Raptor) positive] regulates skeletal muscle growth, whereas mTORC2 [rapamycininsensitive companion of mTOR (Rictor) positive] regulates insulin sensitivity. To examine the regulation of these complexes in human skeletal muscle, we utilized immunohistochemical analysis to study the localization of mTOR complexes before and following proteincarbohydrate feeding (FED) and resistance exercise plus proteincarbohydrate feeding (EXFED) in a unilateral exercise model. In basal samples, mTOR and the lysosomal marker lysosomal associated membrane protein 2 (LAMP2) were highly colocalized and remained so throughout. In the FED and EXFED states, mTOR/LAMP2 complexes were redistributed to the cell periphery [wheat germ agglutinin (WGA)-positive staining] (time effect; P = 0.025), with 39% (FED) and 26% (EXFED) increases in mTOR/WGA association observed 1 h post-feeding/exercise. mTOR/WGA colocalization continued to increase in EXFED at 3 h (48% above baseline) whereas colocalization decreased in FED (21% above baseline). A significant effect of condition (P = 0.05) was noted suggesting mTOR/WGA colocalization was greater during EXFED. This pattern was replicated in Raptor/WGA association, where a significant difference between EXFED and FED was noted at 3 h post-exercise/feeding (P = 0.014). Rictor/WGA colocalization remained unaltered throughout the trial. Alterations in mTORC1 cellular location coincided with elevated S6K1 kinase activity, which rose to a greater extent in EXFED compared with FED at 1 h post-exercise/feeding (P < 0.001), and only remained elevated in EXFED at the 3 h time point (P = 0.037). Collectively these data suggest that mTORC1 redistribution within the cell is a fundamental response to resistance exercise and feeding, whereas mTORC2 is predominantly situated at the sarcolemma and does not alter localization. [ABSTRACT FROM AUTHOR]

How neuronal signaling affects brain myelination remains poorly understood. We show dysregulated neuronal RHEB-mTORC1-DLK1 axis impairs brain myelination. Neuronal Rheb cKO impairs oligodendrocyte differentiation/myelination, with activated neuronal expression of the imprinted gene Dlk1. Neuronal Dlk1 cKO ameliorates myelination deficit in neuronal Rheb cKO mice, indicating that activated neuronal Dlk1 expression contributes to impaired myelination caused by Rheb cKO. The effect of Rheb cKO on Dlk1 expression is mediated by mTORC1; neuronal mTor cKO and Raptor cKO and pharmacological inhibition of mTORC1 recapitulate elevated neuronal Dlk1 expression. We demonstrate that both a secreted form of DLK1 and a membrane-bound DLK1 inhibit the differentiation of cultured oligodendrocyte precursor cells into oligodendrocytes expressing myelin proteins. Finally, neuronal expression of Dlk1 in transgenic mice reduces the formation of mature oligodendrocytes and myelination. This study identifies Dlk1 as an inhibitor of oligodendrocyte myelination and a mechanism linking altered neuronal signaling with oligodendrocyte dysfunction. [Display omitted] • Neuronal mTORC1 is required for proper brain myelination • Disrupting RHEB-mTORC1 axis activates neuronal Dlk1 expression • DLK1 is an inhibitor of oligodendrocyte differentiation and myelination • Activated neuronal Dlk1 expression contributes to hypomyelination caused by Rheb cKO Huang et al. identify the genetically imprinted gene Dlk1 as an inhibitor to oligodendrocyte myelination in vitro and in vivo. Disrupting neuronal RHEB-mTORC1 axis activates DLK1 expression that contributes to impairment in oligodendrocyte differentiation and myelination in neuronal Rheb knockout mouse. [ABSTRACT FROM AUTHOR]

Obesity is a complex disorder and is linked to chronic diseases such as type 2 diabetes. Major intrinsically disordered NOTCH2-associated receptor2 (MINAR2) is an understudied protein with an unknown role in obesity and metabolism. The purpose of this study was to determine the impact of Minar2 on adipose tissues and obesity. We generated Minar2 knockout (KO) mice and used various molecular, proteomic, biochemical, histopathology, and cell culture studies to determine the pathophysiological role of Minar2 in adipocytes. We demonstrated that the inactivation of Minar2 results in increased body fat with hypertrophic adipocytes. Minar2 KO mice on a high-fat diet develop obesity and impaired glucose tolerance and metabolism. Mechanistically, Minar2 interacts with Raptor, a specific and essential component of mammalian TOR complex 1 (mTORC1) and inhibits mTOR activation. mTOR is hyperactivated in the adipocytes deficient for Minar2 and over-expression of Minar2 in HEK-293 cells inhibited mTOR activation and phosphorylation of mTORC1 substrates, including S6 kinase, and 4E-BP1. Our findings identified Minar2 as a novel physiological negative regulator of mTORC1 with a key role in obesity and metabolic disorders. Impaired expression or activation of MINAR2 could lead to obesity and obesity-associated diseases. [Display omitted] • Inactivation of Minar2 in mice predisposes mice to diet-induced obesity. • Minar2 KO mice on high-fat diet develop obesity and display impaired glucose metabolism. • Minar2 interacts with Raptor and regulates mTORC1 signaling. [ABSTRACT FROM AUTHOR]

The mammalian target of rapamycin complex 1 (mTORC1) is activated by extracellular factors that control bone accrual. However, the direct role of this complex in osteoblast biology remains to be determined. To investigate this question, we disrupted mTORC1 function in preosteoblasts by targeted deletion of Raptor (Rptor) in Osterix-expressing cells. Deletion of Rptor resulted in reduced limb length that was associated with smaller epiphyseal growth plates in the postnatal skeleton. Rptor deletion caused a marked reduction in pre- and postnatal bone accrual, which was evident in skeletal elements derived from both intramembranous and endochondrial ossification. The decrease in bone accrual, as well as the associated increase in skeletal fragility, was due to a reduction in osteoblast function. In vitro, osteoblasts derived from knockout mice display a reduced osteogenic potential, and an assessment of bone-developmental markers in Rptor knockout osteoblasts revealed a transcriptional profile consistent with an immature osteoblast phenotype suggesting that osteoblast differentiation was stalled early in osteogenesis. Metabolic labeling and an assessment of cell size of Rptor knockout osteoblasts revealed a significant decrease in protein synthesis, a major driver of cell growth. These findings demonstrate that mTORC1 plays an important role in skeletal development by regulating mRNA translation during preosteoblast differentiation. [ABSTRACT FROM AUTHOR]

Objective Normal adipose tissue growth and function is critical to maintaining metabolic homeostasis and its excess (e.g. obesity) or absence (e.g. lipodystrophy) is associated with severe metabolic disease. The goal of this study was to understand the mechanisms maintaining healthy adipose tissue growth and function. Methods Adipose tissue senses and responds to systemic changes in growth factor and nutrient availability; in cells mTORC1 regulates metabolism in response to growth factors and nutrients. Thus, mTORC1 is poised to be a critical intracellular regulator of adipocyte metabolism. Here, we investigate the role of mTORC1 in mature adipocytes by generating and characterizing mice in which the Adiponectin-Cre driver is used to delete floxed alleles of Raptor , which encodes an essential regulatory subunit of mTORC1. Results Raptor Adipoq-cre mice have normal white adipose tissue (WAT) mass for the first few weeks of life, but soon thereafter develop lipodystrophy associated with hepatomegaly, hepatic steatosis, and insulin intolerance. Raptor Adipoq-cre mice are also resistant to becoming obese when consuming a high fat diet (HFD). Resistance to obesity does not appear to be due to increased energy expenditure, but rather from failed adipose tissue expansion resulting in severe hepatomegaly associated with hyperphagia and defective dietary lipid absorption. Deleting Raptor in WAT also decreases C/EBPα expression and the expression of its downstream target adiponectin, providing one possible mechanism of mTORC1 function in WAT. Conclusions mTORC1 activity in mature adipocytes is essential for maintaining normal adipose tissue growth and its selective loss in mature adipocytes leads to a progressive lipodystrophy disorder and systemic metabolic disease that shares many of the hallmarks of human congenital generalized lipodystrophy. [ABSTRACT FROM AUTHOR]

Orexigenic agouti-related protein/neuropeptide Y (Agrp/NPY) neurons and an orexigenic pro-opiomelanocortin (POMC) neurons of the hypothalamus regulate feeding behavior and energy homeostasis. An understanding of the molecular signaling pathways that regulate Agrp/NPY and POMC function could lead to novel treatments for metabolic disorders. Target of Rapamycin Complex 1 (TORC1) is a nutrient-activated protein kinase and central controller of growth and metabolism. We therefore investigated the role of mammalian TORC1 (mTORC1) in Agrp neurons. We generated and characterized Agrp neuron-specific raptor knockout (Agrp- raptor KO) mice. Agrp- raptor KO mice displayed reduced, non-circadian expression of Agrp and NPY but normal feeding behavior and energy homeostasis on both normal and high fat diet. Thus, mTORC1 in Agrp neurons controls circadian expression of orexigenic neuropeptides but is dispensable for the regulation of feeding behavior and energy metabolism. [ABSTRACT FROM AUTHOR]

Much of the mammalian skeleton is derived from a cartilage template that undergoes rapid growth during embryogenesis, but the molecular mechanism of growth regulation is not well understood. Signaling by mammalian target of rapamycin complex 1 (mTORC1) is an evolutionarily conserved mechanism that controls cellular growth. Here we report that mTORC1 signaling is activated during limb cartilage development in the mouse embryo. Disruption of mTORC1 signaling through deletion of either mTOR or the associated protein Raptor greatly diminishes embryonic skeletal growth associated with severe delays in chondrocyte hypertrophy and bone formation. The growth reduction of cartilage is not due to changes in chondrocyte proliferation or survival, but is caused by a reduction in cell size and in the amount of cartilagematrix. Metabolic labeling reveals a notable deficit in the rate of protein synthesis in Raptor-deficient chondrocytes. Thus, mTORC1 signaling controls limb skeletal growth through stimulation of protein synthesis in chondrocytes. [ABSTRACT FROM AUTHOR]

Breast cancer is the second leading cause of cancer death in women. Targeted therapies are available for HER2-positive and endocrine-sensitive disease while chemotherapy remains the mainstay of treatment for triple-negative breast cancer. The efficacy of all targeted interventions is, however, limited by primary or secondary resistance. Preclinical data show that active PI3K/AKT/mTOR signaling contributes to therapy resistance in HER2-positive and hormone-receptor-positive breast cancer. In line with these preclinical observations, clinical trials such as BOLERO-2 demonstrated a benefit of additional inhibition of mTOR signaling in advanced estrogen-receptor-positive breast cancer patients refractory to prior aromatase-inhibitor therapy. Besides the mTOR, several other proteins involved in the PI3K-pathway serve as potential therapeutic targets, such as PI3K and AKT. In this review, we summarize the current available knowledge and experimental and clinical research results about targeting the PI3K-pathway in breast cancer and, thus, provide the rationale for PI3K- and AKT-inhibitor use in the clinic. [ABSTRACT FROM AUTHOR]

Background: mTOR, which can form mTOR Complex 1 (mTORC1) or mTOR Complex 2 (mTORC2) depending on its binding partners, is frequently deregulated in the pulmonary neoplastic conditions and interstitial lung diseases of the patients treated with rapalogs. In this study, we investigated the relationship between mTOR signaling and epithelial mesenchymal transition (EMT) by dissecting mTOR pathways. Methods: Components of mTOR signaling pathway were silenced by shRNA in a panel of non-small cell lung cancer cell lines and protein expression of epithelial and mesenchymal markers were evaluated by immunoblotting and immunocytochemistry. mRNA level of the E-cadherin repressor complexes were evaluated by qRT-PCR. Results: IGF-1 treatment decreased expression of the E-cadherin and rapamycin increased its expression, suggesting hyperactivation of mTOR signaling relates to the loss of E-cadherin. Genetic ablation of rapamycin-insensitive companion of mTOR (Rictor), a component of mTORC2, did not influence E-cadherin expression, whereas genetic ablation of regulatory-associated protein of mTOR (Raptor), a component of mTORC1, led to a decrease in E-cadherin expression at the mRNA level. Increased phosphorylation of AKT at Ser473 and GSK-3β at Ser9 were observed in the Raptor-silenced NSCLC cells. Of the E-cadherin repressor complexes tested, Snail, Zeb2, and Twist1 mRNAs were elevated in raptor-silenced A549 cells, and Zeb2 and Twist1 mRNAs were elevated in Raptor-silenced H2009 cells. These findings were recapitulated by treatment with the GSK-3β inhibitor, LiCl. Raptor knockdown A549 cells showed increased expression of N-cadherin and vimentin with mesenchymal phenotypic changes. Conclusions: In conclusion, selective inhibition of mTORC1 leads to hyperactivation of the AKT/GSK-3β pathway, inducing E-cadherin repressor complexes and EMT. These findings imply the existence of a feedback inhibition loop of mTORC1 onto mTORC2 that plays a role in the homeostasis of E-cadherin expression and EMT, requiring caution in the clinical use of rapalog and selective mTORC1 inhibitors. [ABSTRACT FROM AUTHOR]

Regulated associated protein of mTOR (Raptor) and rapamycin-insensitive companion of mTOR (rictor) are two proteins that delineate two different mTOR complexes, mTORC1 and mTORC2 respectively. Recent studies demonstrated the role of rictor in the development and function of b-cells. mTORC1 has long been known to impact β-cell function and development. However, most of the studies evaluating its role used either drug treatment (i.e. rapamycin) or modification of expression of proteins known to modulate its activity, and the direct role of raptor in insulin secretion is unclear. In this study, using siRNA, we investigated the role of raptor and rictor in insulin secretion and production in INS-1 cells and the possible cross talk between their respective complexes, mTORC1 and mTORC2. Reduced expression of raptor is associated with increased glucose-stimulated insulin secretion and intracellular insulin content. Downregulation of rictor expression leads to impaired insulin secretion without affecting insulin content and is able to correct the increased insulin secretion mediated by raptor siRNA. Using dominant-negative or constitutively active forms of Akt, we demonstrate that the effect of both raptor and rictor is mediated through alteration of Akt signaling. Our finding shed new light on the mechanism of control of insulin secretion and production by the mTOR, and they provide evidence for antagonistic effect of raptor and rictor on insulin secretion in response to glucose by modulating the activity of Akt, whereas only raptor is able to control insulin biosynthesis. [ABSTRACT FROM AUTHOR]