mTOR Reactivation Research Articles

Stress-induced perturbations in intracellular amino acids reprogram mRNA translation in osmoadaptation independently of the ISR.

SUMMARYThe integrated stress response (ISR) plays a pivotal role in adaptation of translation machinery to cellular stress. Here, we demonstrate an ISR-independent osmoadaptation mechanism involving reprogramming of translation via coordinated but independent actions of mTOR and plasma membrane amino acid transporter SNAT2. This biphasic response entails reduced global protein synthesis and mTOR signaling followed by translation of SNAT2. Induction of SNAT2 leads to accumulation of amino acids and reactivation of mTOR and global protein synthesis, paralleled by partial reversal of the early-phase, stress-induced translatome. We propose SNAT2 functions as a molecular switch between inhibition of protein synthesis and establishment of an osmoadaptive translation program involving the formation of cytoplasmic condensates of SNAT2-regulated RNA-binding proteins DDX3X and FUS. In summary, we define key roles of SNAT2 in osmotolerance.

Abstract 1120: Tipifarnib potentiates the antitumor effects of PI3Ka blockade in HNSCC via convergent inhibition of mTOR activity

Abstract The PI3K-AKT-mTOR signaling cascade is the most frequently activated pathway in the head and neck squamous cell carcinomas (HNSCC). PIK3CA (encoding PI3K’s α catalytic subunit), is activated by gain-of-function mutation or amplification in approximately 30% of HNSCCs, making PI3Kα an attractive therapeutic candidate. While the PI3Kα inhibitor alpelisib has shown some promise in HNSCC in a phase I setting, its single agent efficacy will likely be limited by feedback reactivation of PI3K or compensatory parallel pathways, necessitating the development of rational combination strategies. Here, we utilize cell line and patient-derived xenograft (PDX) models to evaluate the therapeutic potential of the farnesyltransferase inhibitor (FTI) tipifarnib, in combination with alpelisib in the PIK3CA-dysregulated subset of HNSCC. Because FTIs potentially block hyperactivated growth factor signaling at multiple nodes, including HRAS and RHEB, we examined tipifarnib’s impact on growth of PIK3CA-altered HNSCC models in vitro and in vivo. In cell lines harboring PIK3CA mutation or amplification, tipifarnib reduced proliferation of both monolayer and spheroid cultures and when combined with alpelisib, induced cytotoxicity. Consistently, in PIK3CA mutant/amplified PDX models, the tipifarnib-alpelisib doublet led to deeper antitumor responses compared to alpelisib monotherapy. Simultaneous administration was superior to split intermittent dosing, hinting at cooperativity between the mechanistic targets of tipifarnib and alpelisib. To interrogate the mechanistic underpinnings of this synergy, we exposed HNSCC cell lines to tipifarnib, alpelisib, or the combination, and assessed their effect on RAS/PI3K pathway activity. In PIK3CA dysregulated lines, single agent tipifarnib or alpelisib reduced phosphorylation of p90 RSK, and mTOR substrates, particularly S6 kinase and ribosomal protein S6. Combination treatment effects were more robust and induced rapid apoptosis. In cells exposed to alpelisib alone, marked rebound of RSK and mTOR substrate phosphorylation occurred after 24 hours, correlating with restored AKT activity. In contrast, although AKT activity rebounded in cells treated with the combination, RSK phosphorylation and markers of mTOR activity (including 4EBP1) remained suppressed. Thus, tipifarnib appears to blunt both MAPK and mTOR reactivation following PI3K inhibition. This dual effect implies that the efficacy of tipifarnib in this context may stem from simultaneous defarnesylation of multiple targets, likely HRAS and RHEB, which converge upon mTOR and synergize with alpelisib to durably block tumor growth. We contend that combination of alpelisib and tipifarnib holds therapeutic potential for treatment of recurrent/metastatic HNSCCs harboring dysregulated PIK3CA, which will be evaluated in the recently initiated KURRENT clinical trial (NCT04997902). Citation Format: Shivani Malik, Alison Smith, Stacia Chan, Asako McCloskey, Hetika Vora, Quinn Reilly, Francis Burrows. Tipifarnib potentiates the antitumor effects of PI3Ka blockade in HNSCC via convergent inhibition of mTOR activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1120.

Loss of LAT1 sex-dependently delays recovery after caerulein-induced acute pancreatitis.

BACKGROUNDThe expression of amino acid transporters is known to vary during acute pancreatitis (AP) except for LAT1 (slc7a5), the expression of which remains stable. LAT1 supports cell growth by importing leucine and thereby stimulates mammalian target of rapamycin (mTOR) activity, a phenomenon often observed in cancer cells. The mechanisms by which LAT1 influences physiological and pathophysiological processes and affects disease progression in the pancreas are not yet known.AIMTo evaluate the role of LAT1 in the development of and recovery from AP.METHODSAP was induced with caerulein (cae) injections in female and male mice expressing LAT1 or after its knockout (LAT1 Cre/LoxP). The development of the initial AP injury and its recovery were followed for seven days after cae injections by daily measuring body weight, assessing microscopical tissue architecture, mRNA and protein expression, protein synthesis, and enzyme activity levels, as well as by testing the recruitment of immune cells by FACS and ELISA.RESULTSThe initial injury, evaluated by measurements of plasma amylase, lipase, and trypsin activity, as well as the gene expression of dedifferentiation markers, did not differ between the groups. However, early metabolic adaptations that support regeneration at later stages were blunted in LAT1 knockout mice. Especially in females, we observed less mTOR reactivation and dysfunctional autophagy. The later regeneration phase was clearly delayed in female LAT1 knockout mice, which did not regain normal expression of the pancreas-specific differentiation markers recombining binding protein suppressor of hairless-like protein (rbpjl) and basic helix-loop-helix family member A15 (mist1). Amylase mRNA and protein levels remained lower, and, strikingly, female LAT1 knockout mice presented signs of fibrosis lasting until day seven. In contrast, pancreas morphology had returned to normal in wild-type littermates.CONCLUSIONLAT1 supports the regeneration of acinar cells after AP. Female mice lacking LAT1 exhibited more pronounced alterations than male mice, indicating a sexual dimorphism of amino acid metabolism.

Antitumor Activity of Tipifarnib and PI3K Pathway Inhibitor in HRAS-associated HNSCC

Purpose/Objective(s) HRAS-MAPK and PI3K-AKT-mTOR are important oncogenic pathways in squamous cell carcinomas (SCCs) including those of the head and neck (HNSCC). Although HRAS mutations occur at a rate of ∼5% in HNSCCs, HRAS overexpression is present in up to 30% of HNSCC tumors, raising the possibility that some HRAS wild-type (WT) HNSCCs may also display a degree of HRAS dependence. PI3Kα (the catalytic subunit of PI3K), another prominent driver in HNSCC, is activated by gain-of-function mutations or PIK3CA gene amplification in about 30% of HNSCC patients. Multiple reports indicate that the HRAS and PI3K pathways cooperate and crosstalk to drive tumor progression and resistance to targeted therapies in SCCs. In this study, we explored whether combined inhibition of HRAS (via tipifarnib) and PI3K signaling (via PI3Ka inhibitor alpelisib) would have greater anti-tumor activity in cell line and patient-derived xenograft (PDX) models of HRAS-associated SCCs relative to monotherapy approaches. Materials/Methods HNSCC PDX combination studies were performed in Balb/c nu/nu or SCID mice. PDX were allowed to establish to 250-300mm3 after which the animals were randomized and treated orally with vehicle or tipifarnib (60-80 mg/kg BID) alone or in combination with alpelisib (40mg/kg QD) for 25-30 days. Results In a panel of HNSCC cell lines harboring HRAS and/or PIK3CA alterations (mutation, overexpression or amplification), tipifarnib reduced cell growth and, in combination with PI3K-a inhibitor alpelisib, induced cytotoxicity. Consistent with in vitro findings, regressions with the tipifarnib-PI3Ka inhibitor doublet were observed in tumors bearing HRAS mutation or overexpression, PIK3CA mutation or gene amplification, as well as tumors harboring alterations in both genetic drivers, suggesting that concomitant inhibition of HRAS/PI3Ka may have surprisingly broad and potent anti-tumor activity in HNSCC. In dose-scheduling experiments in PDX models, simultaneous blockade of both targets was superior to split intermittent dosing of the two drugs, underlining the extent of pathway cooperativity in these models. Mechanistically, the tipifarnib/alpelisib combination inhibited mTOR activity more effectively than single-agent treatment. Tipifarnib blocked the compensatory mTOR reactivation induced by alpelisib alone, resulting in durable inhibition of mTOR signaling and cell growth. Conclusion Tipifarnib muted mTOR reactivation induced by alpelisib explaining cytotoxic cellular response to the combination in HRAS/PIk3CA dysregulated HNSCC models. We contend that combination of tipifarnib with a PI3Ka inhibitor holds therapeutic potential for the treatment of R/M HNSCCs harboring alterations in HRAS and/or PIK3CA which will be evaluated in the recently initiated KURRENT clinical trial (NCT04997902). HRAS-MAPK and PI3K-AKT-mTOR are important oncogenic pathways in squamous cell carcinomas (SCCs) including those of the head and neck (HNSCC). Although HRAS mutations occur at a rate of ∼5% in HNSCCs, HRAS overexpression is present in up to 30% of HNSCC tumors, raising the possibility that some HRAS wild-type (WT) HNSCCs may also display a degree of HRAS dependence. PI3Kα (the catalytic subunit of PI3K), another prominent driver in HNSCC, is activated by gain-of-function mutations or PIK3CA gene amplification in about 30% of HNSCC patients. Multiple reports indicate that the HRAS and PI3K pathways cooperate and crosstalk to drive tumor progression and resistance to targeted therapies in SCCs. In this study, we explored whether combined inhibition of HRAS (via tipifarnib) and PI3K signaling (via PI3Ka inhibitor alpelisib) would have greater anti-tumor activity in cell line and patient-derived xenograft (PDX) models of HRAS-associated SCCs relative to monotherapy approaches. HNSCC PDX combination studies were performed in Balb/c nu/nu or SCID mice. PDX were allowed to establish to 250-300mm3 after which the animals were randomized and treated orally with vehicle or tipifarnib (60-80 mg/kg BID) alone or in combination with alpelisib (40mg/kg QD) for 25-30 days. In a panel of HNSCC cell lines harboring HRAS and/or PIK3CA alterations (mutation, overexpression or amplification), tipifarnib reduced cell growth and, in combination with PI3K-a inhibitor alpelisib, induced cytotoxicity. Consistent with in vitro findings, regressions with the tipifarnib-PI3Ka inhibitor doublet were observed in tumors bearing HRAS mutation or overexpression, PIK3CA mutation or gene amplification, as well as tumors harboring alterations in both genetic drivers, suggesting that concomitant inhibition of HRAS/PI3Ka may have surprisingly broad and potent anti-tumor activity in HNSCC. In dose-scheduling experiments in PDX models, simultaneous blockade of both targets was superior to split intermittent dosing of the two drugs, underlining the extent of pathway cooperativity in these models. Mechanistically, the tipifarnib/alpelisib combination inhibited mTOR activity more effectively than single-agent treatment. Tipifarnib blocked the compensatory mTOR reactivation induced by alpelisib alone, resulting in durable inhibition of mTOR signaling and cell growth. Tipifarnib muted mTOR reactivation induced by alpelisib explaining cytotoxic cellular response to the combination in HRAS/PIk3CA dysregulated HNSCC models. We contend that combination of tipifarnib with a PI3Ka inhibitor holds therapeutic potential for the treatment of R/M HNSCCs harboring alterations in HRAS and/or PIK3CA which will be evaluated in the recently initiated KURRENT clinical trial (NCT04997902).

Disruption of morphogenic and growth pathways in lysosomal storage diseases.

The lysosome achieved a new protagonism that highlights its multiple cellular functions, such as in the catabolism of complex substrates, nutrient sensing, and signaling pathways implicated in cell metabolism and growth. Lysosomal storage diseases (LSDs) cause lysosomal accumulation of substrates and deficiency in trafficking of macromolecules. The substrate accumulation can impact one or several pathways which contribute to cell damage. Autophagy impairment and immune response are widely studied, but less attention is paid to morphogenic and growth pathways and its impact on the pathophysiology of LSDs. Hedgehog pathway is affected with abnormal expression and changes in distribution of protein levels, and a reduced number and length of primary cilia. Moreover, growth pathways are identified with delay in reactivation of mTOR that deregulate termination of autophagy and reformation of lysosomes. Insulin resistance caused by changes in lipids rafts has been described in different LSDs. While the genetic and biochemical bases of deficient proteins in LSDs are well understood, the secondary molecular mechanisms that disrupt wider biological processes associated with LSDs are only now becoming clearer. Therefore, we explored how specific signaling pathways can be related to specific LSDs, showing that a system medicine approach could be a valuable tool for the better understanding of LSD pathogenesis. This article is categorized under: Metabolic Diseases > Molecular and Cellular Physiology.

Indoximod opposes the immunosuppressive effects mediated by IDO and TDO via modulation of AhR function and activation of mTORC1.

Indoximod has shaped our understanding of the biology of IDO1 in the control of immune responses, though its mechanism of action has been poorly understood. Previous studies demonstrated that indoximod creates a tryptophan (Trp) sufficiency signal that reactivates mTOR in the context of low Trp concentrations, thus opposing the effects caused by IDO1. Here we extend the understanding of indoximod’s mechanism of action by showing that it has pleiotropic effects on immune regulation. Indoximod can have a direct effect on T cells, increasing their proliferation as a result of mTOR reactivation. Further, indoximod modulates the differentiation of CD4+ T cells via the aryl hydrocarbon receptor (AhR), which controls transcription of several genes in response to different ligands including kynurenine (Kyn). Indoximod increases the transcription of RORC while inhibiting transcription of FOXP3, thus favoring differentiation to IL-17-producing helper T cells and inhibiting the differentiation of regulatory T cells. These indoximod-driven effects on CD8+ and CD4+ T cells were independent from the activity of IDO/TDO and from the presence of exogenous Kyn, though they do oppose the effects of Kyn produced by these Trp catabolizing enzymes. Indoximod can also downregulate expression of IDO protein in vivo in murine lymph node dendritic cells and in vitro in human monocyte-derived dendritic cells via a mechanism that involves signaling through the AhR. Together, these data improve the understanding of how indoximod influences the effects of IDO, beyond and distinct from direct enzymatic inhibition of the enzyme.

TNFAIP8L2/TIPE2 impairs autolysosome reformation via modulating the RAC1-MTORC1 axis

ABSTRACT Macroautophagy/autophagy is an evolutionarily conserved process that involves the selective degradation of cytoplasmic components within lysosomes in response to starvation. Autophagy is an ancient defense mechanism that has been closely integrated with the immune system and has multiple effects on innate and adaptive immunity. The pro-inflammatory and anti-inflammatory cytokines can activate and inhibit autophagy, respectively. TNFAIP8L2/TIPE2 (tumor necrosis factor, alpha-induced protein 8-like 2) is a newly identified immune negative regulator of innate and adaptive immunity that plays an important role in immune homeostasis. However, whether and how TNFAIP8L2 controls autophagy is still unknown. Murine TNFAIP8L2 can directly bind to and block the RAC1 GTPase activity to regulate innate immunity. RAC1 can also bind to MTOR and regulate MTORC1 cellular localization and activity. Here, we find that TNFAIP8L2 can compete with MTOR for binding to the GTP-bound state of RAC1 and negatively regulate MTORC1 activity. Interestingly, TNFAIP8L2 overexpression fails to induce autophagy flux by the suppression of the MTOR activity under glutamine and serum starvation. Instead, TNFAIP8L2 appears to impair autophagic lysosome reformation (ALR) during prolonged starvation. Finally, we demonstrate that TNFAIP8L2 overexpression leads to a defect in MTOR reactivation and disrupts autophagy flux, thereby leading to cell death. Furthermore, TNFAIP8L2 deficiency can exacerbate the inflammatory response and lung injury by controlling the MTOR activity in an LPS-induced mouse endotoxemia model. Our study reveals a novel role of TNFAIP8L2 in autophagy by regulating the RAC1-MTORC1 axis that supports its potential as a target for therapeutic treatment.Abbreviations: ALR: autophagic lysosome reformation; BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; Co-IP: Co-Immunoprecipitation; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase complex 1; RAPA: rapamycin; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; Starv: Starvation; TNFAIP8L2/TIPE2: tumor necrosis factor-alpha-induced protein-8 like-2.

Phosphorylated mTORC1 represses autophagic-related mRNA translation in neurons exposed to ischemia-reperfusion injury.

The sequential reactivation of mechanistic target of rapamycin (mTOR) inhibited autophagic flux in neurons exposed to oxygen-glucose deprivation/reperfusion (OGD/R), which was characterized by reduction of autophagosome formation and restriction of autolysosome degradation. However, its detailed molecular mechanism was still unknown. In this study, we further explore the existing form of mTOR and its suppression on the transcriptional levels of related mRNA from neurons exposed to ischemia-reperfusion injury. The OGD/R or middle cerebral artery occlusion/reperfusion (MCAO/R)-treated neurons was used to simulate ischemia/reperfusion injury . Autophagy flux was monitored by means of microtubule-associated protein 1 light chain 3 (LC3) and p62. The reactivation of mTOR was determined by phosphorylation of ribosomal protein S6 kinase 1 (S6K1). Then the inhibitors of mTOR were used to confirm its existence form. Finally, the mRNA transcription levels were analyzed to observe the negative regulation of mTOR. The sequential phosphorylation of mTOR contributed to the neuronal autophagy flux blocking. mTOR was re-phosphorylated and existed as mTOR complex 1 (mTORC1), which was supported by phosphorylation of S6K1 at Thr 389 in neurons. In addition, the phosphorylation of S6K1 was decreased roughly by applying mTORC1 inhibitors, rapamycin and torin 1. However, the administration of mTORC1/2 inhibitor PP242 could recover the phosphorylation of S6K1, which suggested that mTORC2 was involved in the regulation of mTORC1 activity. In paralleling with reactivation of mTORC1, related mRNA transcription was repressed in neurons under ischemia-reperfusion exposure in vivo and in vitro. The mRNA expression levels of LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b were decreased in neurons after reperfusion, comparing with ischemia-treated neurons. The reactivated mTORC1 could suppress the transcription levels of related mRNA, such as LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b. The research will expand the horizons that mTOR would negatively regulate autophagy at transcription and post-translation levels in neurons suffering ischemia-reperfusion injury.

Adaptive Resistance to EGFR-Targeted Therapy by Calcium Signaling in NSCLC Cells.

Targeted therapies against oncogenic receptor tyrosine kinases (RTK) show promising results in the clinic. Unfortunately, despite the initial positive response, most patients develop therapeutic resistance. Most research has focused on acquired resistance occurring after an extensive time of treatment; however, the question remains as to how cells can survive an initial treatment, as early resistance to apoptosis will enable cells to develop any growth-stimulating mechanism. Here, the non-small cell lung cancer (NSCLC) PC9 cell line was used to systematically profile, by mass spectrometry, changes in the proteome, kinome, and phosphoproteome during early treatment with the EGFR inhibitor afatinib. Regardless of the response, initial drug-sensitive cells rapidly adapt to targeted therapy, and within days, cells regained the capacity to proliferate, despite persisting target inhibition. These data reveal a rapid reactivation of mTOR and MAPK signaling pathways after initial inhibition and an increase in abundance and activity of cytoskeleton and calcium signaling-related proteins. Pharmacologic inhibition of reactivated pathways resulted in increased afatinib efficacy. However more strikingly, cells that were restricted from accessing extracellular calcium were extremely sensitive to afatinib treatment. These findings were validated using three additional inhibitors tested in four different NSCLC cell lines, and the data clearly indicated a role for Ca2+ signaling during the development of adaptive resistance. From a therapeutic point of view, the increased inhibitor efficacy could limit or even prevent further resistance development.Implications: Combined targeting of calcium signaling and RTKs may limit drug resistance and improve treatment efficacy. Mol Cancer Res; 16(11); 1773-84. ©2018 AACR.

Deep analysis of acquired resistance to FGFR1 inhibitor identifies MET and AKT activation and an expansion of AKT1 mutant cells.

The development of acquired resistance (AR) to tyrosine kinase inhibitors (TKIs) of FGFR1 activation is currently not well understood. To gain a deeper insight into this matter in lung cancer, we used the FGFR1-amplified DMS114 cell line and generated multiple clones with AR to an FGFR1-TKI. We molecularly scrutinized the resistant cells, using whole-exome sequencing, RNA sequencing and global DNA methylation analysis. Our results show a de novo activation of AKT and ERK, and a reactivation of mTOR. Furthermore, the resistant cells exhibited strong upregulation and activation of MET, indicating crosstalk between the FGFR1 and MET axes. The resistant cells also underwent a global decrease in promoter hypermethylation of the CpG islands. Finally, we observed clonal expansion of a pre-existing change in AKT1, leading to S266L substitution, within the kinase domain of AKT. Our results demonstrate that AR to FGFR1-TKI involves deep molecular changes that promote the activation of MET and AKT, coupled with common gene expression and DNA methylation profiles. The expansion of a substitution at AKT1 was the only shared genetic change, and this may have contributed to the AR.

CPKCγ-Modulated Sequential Reactivation of mTOR Inhibited Autophagic Flux in Neurons Exposed to Oxygen Glucose Deprivation/Reperfusion.

We have reported that conventional protein kinase Cγ (cPKCγ)-modulated neuron-specific autophagy improved the neurological outcome of mice following ischemic stroke through the Akt-mechanistic target of rapamycin (mTOR) pathway. However, its detailed molecular mechanism remains unclear. In this study, primary cortical neurons from postnatal one-day-old C57BL/6J cPKCγ wild-type (cPKCγ+/+) and knockout (cPKCγ−/−) mice suffering oxygen glucose deprivation/reperfusion (OGD/R) were used to simulate ischemia/reperfusion injury in vitro. A block of autophagic flux was observed in cPKCγ+/+ neurons under OGD/R exposure, characterized by accumulation of p62. Immunofluorescent results showed a decrease in colocalization between LC3 and Atg14 or Stx17 in cPKCγ+/+ neurons when compared with cPKCγ−/− neurons after OGD/R. However, the colocalization between LC3 and Lamp2 was barely decreased, indicating the presence of autolysosomes. The larger lysotracker-positive structures were also significantly increased. These results suggest that cPKCγ-induced inhibition of autophagy occurred at the stages of autophagosome formation, Stx17 anchoring, and the degradation of autolysosomes in particular. In addition, cPKCγ-modulated phosphorylation of mTOR at Ser 2481 was dependent on the site of Ser 2448, which may have blocked autophagic flux. cPKCγ-modulated sequential reactivation of mTOR inhibited autophagic flux in neurons exposed to OGD/R, which may provide endogenous interventional strategies for stroke, especially ischemia/reperfusion injury.

Loss of CLN7 results in depletion of soluble lysosomal proteins and impaired mTOR reactivation.

Defects in the MFSD8 gene encoding the lysosomal membrane protein CLN7 lead to CLN7 disease, a neurodegenerative lysosomal storage disorder belonging to the group of neuronal ceroid lipofuscinoses. Here, we have performed a SILAC-based quantitative analysis of the lysosomal proteome using Cln7-deficient mouse embryonic fibroblasts (MEFs) from a Cln7 knockout (ko) mouse model. From 3335 different proteins identified, we detected 56 soluble lysosomal proteins and 29 highly abundant lysosomal membrane proteins. Quantification revealed that the amounts of 12 different soluble lysosomal proteins were significantly reduced in Cln7 ko MEFs compared with wild-type controls. One of the most significantly depleted lysosomal proteins was Cln5 protein that underlies another distinct neuronal ceroid lipofuscinosis disorder. Expression analyses showed that the mRNA expression, biosynthesis, intracellular sorting and proteolytic processing of Cln5 were not affected, whereas the depletion of mature Cln5 protein was due to increased proteolytic degradation by cysteine proteases in Cln7 ko lysosomes. Considering the similar phenotypes of CLN5 and CLN7 patients, our data suggest that depletion of CLN5 may play an important part in the pathogenesis of CLN7 disease. In addition, we found a defect in the ability of Cln7 ko MEFs to adapt to starvation conditions as shown by impaired mammalian target of rapamycin complex 1 reactivation, reduced autolysosome tubulation and increased perinuclear accumulation of autolysosomes compared with controls. In summary, depletion of multiple soluble lysosomal proteins suggest a critical role of CLN7 for lysosomal function, which may contribute to the pathogenesis and progression of CLN7 disease.

MiR-199a-3p is involved in estrogen-mediated autophagy through the IGF-1/mTOR pathway in osteocyte-like MLO-Y4 cells.

To date, evidence indicates that estrogen partially modulates cellular processes through microRNAs. Autophagy is a catabolic process that is regulated by multiple factors and is associated with skeletal diseases. However, whether estrogen regulates osteocyte autophagy via microRNAs is largely unknown. In this study, we observed the up-regulation of microRNA-199a-3p, a post-transcriptional regulatory factor, in osteocytic areas in ovariectomized (OVX) mice. The mature forms of miR-199a-3p and pri-miR-199a were produced in response to estrogen signaling in osteocyte-like MLO-Y4 cells. Western blotting, autophagic flux detection, mRFP-GFP-LC3 fluorescence, and electron microscopy confirmed that miR-199a-3p induced autophagy in MLO-Y4 cells, although cellular apoptosis was not affected. Additionally, we documented the ability of estrogen to mediate osteocyte autophagy. Based on our in vivo data, estrogen deficiency induced autophagy in osteocytes. Treatment of starved MLO-Y4 cells with 17β-estradiol suppressed the excess autophagy induced by starvation via activation of mammalian target of rapamycin (mTOR)-related signaling cascades, while administration of rapamycin reversed the effects of 17β-estradiol. Meanwhile, miR-199a-3p overexpression reversed 17β-estradiol-mediated regulation of autophagy in MLO-Y4 cells. According to mechanistic studies, miR-199a-3p inhibited the mTOR pathway by directly binding to the 3'-untranslated regions of insulin growth factor-1 (IGF-1) and mTOR. However, overexpression of miR-199a-3p inhibited IGF-1 phosphorylation and mTOR-related pathways. Knockdown of mTOR and IGF-1 abolished estrogen signaling and restored LC3-II expression through mTOR re-activation, respectively. Thus, miR-199a-3p appears to be involved in the estrogen regulatory networks that mediate bone cell autophagy, potentially by targeting IGF-1 and mTOR.

Modulation of mTOR signaling as a strategy for the treatment of Pompe disease

Mechanistic target of rapamycin (mTOR) coordinates biosynthetic and catabolic processes in response to multiple extracellular and intracellular signals including growth factors and nutrients. This serine/threonine kinase has long been known as a critical regulator of muscle mass. The recent finding that the decision regarding its activation/inactivation takes place at the lysosome undeniably brings mTOR into the field of lysosomal storage diseases. In this study, we have examined the involvement of the mTOR pathway in the pathophysiology of a severe muscle wasting condition, Pompe disease, caused by excessive accumulation of lysosomal glycogen. Here, we report the dysregulation of mTOR signaling in the diseased muscle cells, and we focus on potential sites for therapeutic intervention. Reactivation of mTOR in the whole muscle of Pompe mice by TSC knockdown resulted in the reversal of atrophy and a striking removal of autophagic buildup. Of particular interest, we found that the aberrant mTOR signaling can be reversed by arginine. This finding can be translated into the clinic and may become a paradigm for targeted therapy in lysosomal, metabolic, and neuromuscular diseases.

GADD34 Keeps the mTOR Pathway Inactivated in Endoplasmic Reticulum Stress Related Autophagy.

The balance of protein synthesis and proteolysis (i.e. proteostasis) is maintained by a complex regulatory network in which mTOR (mechanistic target of rapamycin serine/threonine kinase) pathway and unfolded protein response are prominent positive and negative actors. The interplay between the two systems has been revealed; however the mechanistic details of this crosstalk are largely unknown. The aim of the present study was to investigate the elements of crosstalk during endoplasmic reticulum stress and to verify the key role of GADD34 in the connection with the mTOR pathway. Here, we demonstrate that a transient activation of autophagy is present in endoplasmic reticulum stress provoked by thapsigargin or tunicamycin, which is turned into apoptotic cell death. The transient phase can be characterized by the elevation of the autophagic marker LC3II/I, by mTOR inactivation, AMP-activated protein kinase activation and increased GADD34 level. The switch from autophagy to apoptosis is accompanied with the appearance of apoptotic markers, mTOR reactivation, AMP-activated protein kinase inactivation and a decrease in GADD34. Inhibition of autophagy by 3-methyladenine shortens the transient phase, while inhibition of mTOR by rapamycin or resveratrol prolongs it. Inhibition of GADD34 by guanabenz or transfection of the cells with siGADD34 results in down-regulation of autophagy-dependent survival and a quick activation of mTOR, followed by apoptotic cell death. The negative effect of GADD34 inhibition is diminished when guanabenz or siGADD34 treatment is combined with rapamycin or resveratrol addition. These data confirm that GADD34 constitutes a mechanistic link between endoplasmic reticulum stress and mTOR inactivation, therefore promotes cell survival during endoplasmic reticulum stress.

Autophagic lysosomal reformation depends on mTOR reactivation in H2O2-induced autophagy

Autophagic lysosomal reformation, a key cellular process for maintaining lysosome homeostasis in elevated autophagy, so far has only been reported for cells under certain forms of starvation. For this reason, it is controversial that whether this phenomenon is starvation-specific and its importance in lysosomal regeneration at the late stage of autophagy is often challenged. Here we show that exogenous hydrogen peroxide (H2O2) induced lysosome depletion and recovery characteristic of autophagic lysosomal reformation, and we confirmed the occurrence of autophagic lysosomal reformation after H2O2 treatment by demonstrating Rab7 dissociation from autolysosomes, recruitment of Phosphatidylinositol 4-phosphate (PI4P) and clathrin to the surface of autolysosomes, and the existence of tubular “pro-lysosome” structures extending from autolysosomes. Similar to starvation, H2O2 caused an initial deactivation and a subsequent reactivation for mTOR, and mTOR reactivation was essential for ALR. Our results provided a first non-starvation example of autophagic lysosomal reformation and provide evidence for its importance for some autophagic processes other than that of starvation.

MTOR Silencing in Parkinson's Disease both in vitro and in vivo

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates processes including mRNA translation, proliferation, and survival. As a central element signaling cell growth and enhancing protein translation, (mTOR), when inhibited, induces autophagy. Moreover, as a critical feedback mechanism, reactivation of mTOR terminates autophagy and initiates lysosome reformation

ASAP1和ARF1通过调节mTOR重激活调控自噬性溶酶体再生

Autophagy is an evolutionarily conserved lysosome-based degradation process. Stimuli such as nutrients starvation induce formation of autophagosome, which then fuse with lysosome to form autolysosome. After degradation of engulfed cellular content, lysosomes are recycled from autolysosomes through an evolutionarily conserved process named autophagic lysosome reformation (ALR), which is triggered by reactivation of mTOR during prolonged starvation. Our previous study identified ASAP1, the GAP protein for ARF1, is a regulator of ALR. In this study, we report that ARF1 is translocated to autolysosome during ALR. Knocking down ASAP1, or overexpression GTP binding form of ARF1, can block the mTOR reactivation and ALR. Thus, ARF1 and ASAP1 regulate ALR through regulating mTOR reactivation.

The general amino acid control pathway regulates mTOR and autophagy during serum/glutamine starvation

Organisms have evolved elaborate mechanisms to adjust intracellular nutrient levels in response to fluctuating availability of exogenous nutrients. During starvation, cells can enhance amino acid uptake and synthesis through the general amino acid control (GAAC) pathway, whereas nonessential cellular contents are recycled by autophagy. How these two pathways are coordinated in response to starvation is currently unknown. Here we show that the GAAC pathway couples exogenous amino acid availability with autophagy. Starvation caused deactivation of mTOR, which then activated autophagy. In parallel, serum/glutamine starvation activated the GAAC pathway, which up-regulated amino acid transporters, leading to increased amino acid uptake. This elevated the intracellular amino acid level, which in turn reactivated mTOR and suppressed autophagy. Knockdown of activating transcription factor 4, the major transcription factor in the GAAC pathway, or of SLC7A5, a leucine transporter, caused impaired mTOR reactivation and much higher levels of autophagy. Thus, the GAAC pathway modulates autophagy by regulating amino acid uptake and mTOR reactivation during serum/glutamine starvation.

AB0229 Senescence associated β-galactosisdase expression in systemic sclerosis skin and fibroblasts

BackgroundSystemic sclerosis (SSc) is connective tissue disease characterized by fibrosis and vascular changes in the skin and internal organs with autoimmune background. However, the mechanism is still unknown. Recently, it...