Activation Of mTOR Signaling Pathway Research Articles

Failure of metabolic checkpoint control during late-stage granulopoiesis drives neutropenia in reticular dysgenesis

AbstractCellular metabolism is highly dynamic during hematopoiesis, yet the regulatory networks that maintain metabolic homeostasis during differentiation are incompletely understood. Herein, we have studied the grave immunodeficiency syndrome reticular dysgenesis caused by loss of mitochondrial adenylate kinase 2 (AK2) function. By coupling single-cell transcriptomics in samples from patients with reticular dysgenesis with a CRISPR model of this disorder in primary human hematopoietic stem cells, we found that the consequences of AK2 deficiency for the hematopoietic system are contingent on the effective engagement of metabolic checkpoints. In hematopoietic stem and progenitor cells, including early granulocyte precursors, AK2 deficiency reduced mechanistic target of rapamycin (mTOR) signaling and anabolic pathway activation. This conserved nutrient homeostasis and maintained cell survival and proliferation. In contrast, during late-stage granulopoiesis, metabolic checkpoints were ineffective, leading to a paradoxical upregulation of mTOR activity and energy-consuming anabolic pathways such as ribonucleoprotein synthesis in AK2-deficient cells. This caused nucleotide imbalance, including highly elevated adenosine monophosphate and inosine monophosphate levels, the depletion of essential substrates such as NAD+ and aspartate, and ultimately resulted in proliferation arrest and demise of the granulocyte lineage. Our findings suggest that even severe metabolic defects can be tolerated with the help of metabolic checkpoints but that the failure of such checkpoints in differentiated cells results in a catastrophic loss of homeostasis.

MTOR activity paces human blastocyst stage developmental progression.

Many mammals can temporally uncouple conception from parturition by pacing down their development around the blastocyst stage. In mice, this dormant state is achieved by decreasing the activity of the growth-regulating mTOR signaling pathway. It is unknown whether this ability is conserved in mammals in general and in humans in particular. Here, we show that decreasing the activity of the mTOR signaling pathway induces human pluripotent stem cells (hPSCs) and blastoids to enter a dormant state with limited proliferation, developmental progression, and capacity to attach to endometrial cells. These invitro assays show that, similar to other species, the ability to enter dormancy is active in human cells around the blastocyst stage and is reversible at both functional and molecular levels. The pacing of human blastocyst development has potential implications for reproductive therapies.

MARCH1 negatively regulates TBK1-mTOR signaling pathway by ubiquitinating TBK1

BackgroundTBK1 positively regulates the growth factor-mediated mTOR signaling pathway by phosphorylating mTOR. However, it remains unclear how the TBK1-mTOR signaling pathway is regulated. Considering that STING not only interacts with TBK1 but also with MARCH1, we speculated that MARCH1 might regulate the mTOR signaling pathway by targeting TBK1. The aim of this study was to determine whether MARCH1 regulates the mTOR signaling pathway by targeting TBK1.MethodsThe co-immunoprecipitation (Co-IP) assay was used to verify the interaction between MARCH1 with STING or TBK1. The ubiquitination of STING or TBK1 was analyzed using denatured co-immunoprecipitation. The level of proteins detected in the co-immunoprecipitation or denatured co-immunoprecipitation samples were determined by Western blotting. Stable knocked-down cells were constructed by infecting lentivirus bearing the related shRNA sequences. Scratch wound healing and clonogenic cell survival assays were used to detect the migration and proliferation of breast cancer cells.ResultsWe showed that MARCH1 played an important role in growth factor-induced the TBK1- mTOR signaling pathway. MARCH1 overexpression attenuated the growth factor-induced activation of mTOR signaling pathway, whereas its deficiency resulted in the opposite effect. Mechanistically, MARCH1 interacted with and promoted the K63-linked ubiquitination of TBK1. This ubiquitination of TBK1 then attenuated its interaction with mTOR, thereby inhibiting the growth factor-induced mTOR signaling pathway. Importantly, faster proliferation induced by MARCH1 deficiency was weakened by mTOR, STING, or TBK1 inhibition.ConclusionMARCH1 suppressed growth factors mediated the mTOR signaling pathway by targeting the STING-TBK1-mTOR axis.

Muscle-derived Stem Cell Exosomes Enhance Autophagy through the Regulation of the mTOR Signaling Pathway to Attenuate Glucolipotoxicity-induced Pancreatic Β-cell Injury

Background: Diabetes mellitus (DM) refers to a series of metabolic disorders, including elevated blood glucose level diseases due to insufficient insulin secretion or insulin resistance. Objective: To investigate the effect and protective mechanism of muscle-derived stem cell exosomes (MDSC-Exo) on glucolipotoxicity-induced pancreatic β cell injury. Methods: Primary rat muscle-derived stem cells (MDSCs) were isolated and cultured. After the completion of the third-generation culture for MDSCs, MDSC-Exo was isolated. Then, the morphology and diameter of exosomes were observed by means of electron microscopy and nanoparticle tracking analysis (NTA) instrument. The expression of exosome-related proteins CD63, TSG101 and Calnexin was detected by western blot. After stimulation of rat insulinoma cell line INS- 1 with high glucose/palmitic acid (HG/PA) and/or MDSC-Exo, cell viability and apoptosis were measured through MTT and flow cytometry (FCT), respectively. Biochemical reagents were utilized for the examination of the levels of superoxide dismutase (SOD) and malondialdehyde (MDA); enzyme-linked immunosorbent assay (ELISA) for the levels of cellular insulin secretion, and the western blot for the expression level of LC3, p62, AKT, p-AKT, mTOR and p-mTOR. Results: MDSC-Exo was successfully isolated and identified, and it was found that MDSC-Exo could reduce HG/PA-induced apoptosis as well as MDA levels in INS-1 cells. Also, MDSC-Exo could significantly increase cell viability, insulin secretion ability within 24 hours and SOD level. Besides, MDSC-Exo was able to significantly increase the LC3-II/I ratio, decrease the expression level of p62, and promote autophagy in the cells. Aside from what has been mentioned, MDSC- Exo showed a significant reduction effect on p-Akt and p-mTOR level as well as p-Akt/Akt and p-mTOR/mTOR ratios. Conclusion: MDSC-Exo can alleviate oxidative stress and enhance autophagy by inhibiting Akt/ mTOR signaling pathway activation. Then, the inhibition of apoptosis and the promotion of insulin secretion can be achieved to alleviate glucolipotoxicity-induced pancreatic β cell injury.

P170 SLC7A5 promotes the progression of Ulcerative Colitis by regulating amino acid transport and intestinal epithelial autophagy

Abstract Background IBD has become a common chronic intestinal disease in China. Considering the important role of amino acid metabolism in inflammatory diseases, inhibition amino acid metabolism of inflammatory sites may be effective in alleviating UC (ulcerative colitis). SLC7A5 is involved in the progression of UC, but the mechanism of its functional regulation still needs to be investigated. In this study, we aimed to clarify the link among SLC7A5 expression in UC, mTOR pathway activation and immunoregulation. Methods We previously discovered through high-throughput screening that SLC7A5, as a cellular amino acid transport carrier, is significantly elevated in the colonic tissues of UC patients; in vivo experiments have demonstrated for the first time that JPH203, a specific inhibitor of SLC7A5, alleviates DSS-induced intestinal inflammation and inhibits mTOR pathway activation to promote intestinal autophagy. This study utilized model animals, transcriptomics, amino acid-targeted metabolomics assays, and macro-genome sequencing to elucidate the mechanism of SLC7A5-regulated amino acids in mTOR pathway activation and intestinal autophagy dysfunction. Results SLC7A5 expression is increased in the colon of UC patients and DSS-induced mice lesions. SLC7A5 inhibitor (JPH203) restrained the inflammatory responses induced by DSS. SLC7A5 deletion or inhibition dampens the release of IL-1β, IL-18, and IL-23 and the production of ROS in the LPS-induced FHC and RAW264.7 cells. Moreover, upregulating SLC7A5 expression induces mTOR signaling pathway activation in FHC cells. Deletion or inhibition of SLC7A5 efficiently blocks the SLC7A5-dependent amino acid transport, inhibits the mTOR activation, and results in the activation of autophagy. Conclusion Targeting SLC7A5-mediated amino acid uptake is a potentially useful immunosuppressive strategy to regulate colonic inflammation through mTOR pathway and autophagy. This study is expected to reveal the intrinsic factors of metabolic disorders promoting the UC progression from the perspective of amino acid metabolism, and to lay a new theoretical and experimental foundation for potential UC treatment.

CASZ1 upregulates PI3K-AKT-mTOR signaling and promotes T-cell acute lymphoblastic leukemia.

CASZ1 is a conserved transcription factor involved in neural development, blood vessel assembly and heart morphogenesis. CASZ1 has been implicated in cancer, either suppressing or promoting tumor development depending on the tissue. However, the impact of CASZ1 on hematological tumors remains unknown. Here, we show that the T-cell oncogenic transcription factor TAL1 is a direct positive regulator of CASZ1, that T-cell acute lymphoblastic leukemia (T-ALL) samples at diagnosis overexpress CASZ1b isoform, and that CASZ1b expression in patient samples correlates with PI3K-AKT-mTOR signaling pathway activation. In agreement, overexpression of CASZ1b in both Ba/F3 and T-ALL cells leads to the activation of PI3K signaling pathway, which is required for CASZ1b-mediated transformation of Ba/F3 cells in vitro and malignant expansion in vivo. We further demonstrate that CASZ1b cooperates with activated NOTCH1 to promote T-ALL development in zebrafish, and that CASZ1b protects human T-ALL cells from serum deprivation and treatment with chemotherapeutic drugs. Taken together, our studies indicate that CASZ1b is a TAL1-regulated gene that promotes T-ALL development and resistance to chemotherapy.

FRI141 Dietary Salt Regulates mTORC1 Signaling In Humans And Mice With Hypertension

Abstract Disclosure: H. Aljaibeji: None. D.L. Brooks: None. A.E. Garza: None. P. Gerges: None. M. Heydarpour: None. P. Luminita: None. J.S. Williams: None. G. Williams: None. J.R. Romero: None. Raptor and the mammalian target of rapamycin (mTOR) form a complex (mTORC1) that has been proposed to play a role in aldosterone (ALDO) production. Pharmacological inhibition of mTORC1 reduces blood pressure (BP) and ALDO levels. However, the effects of environmental factors such as dietary salt on mTORC1 in humans are unclear. We studied the impact of one-week liberal Na+ (LIB, 200 mmol/day) and restricted (RES, 10 mmol/day) Na+ diets on Raptor and mTOR expression in human peripheral blood mononuclear cells (PBMC) from hypertensive subjects. Raptor and mTOR expression were higher on PBMCs from subjects on a LIB than on RES Na+ diet (n=13; P=0.036 and P=0.0008, respectively). We also studied Caucasian subjects (n=792) from the Hypertensive Pathotype (HyperPATH) cohort using the Multi-Ethnic Genotyping Array on the Illumina platform. Candidate gene association analyses showed that the single nucleotide polymorphic (SNP) Raptor gene variant, rs99001846, was associated with BP and ALDO levels. Multivariate linear regression analyses performed on hypertensive and normotensive participants showed that in hypertensives, rs99001846 risk allele carriers (AA/GA), as compared to non-risk carriers (GG), were associated with increased systolic BP levels on bothLIB and RES Na+ diets (P=0.02, P=0.01 respectively). In addition, risk allele carriers, as compared to non-risk carriers, showed increased ALDO and reduced Na+ excretion on a LIB Na+ diet (P=0.009, P=0.025, respectively). We also studied a mouse model of low nitric oxide and high AngII-mediated hypertension and observed increased cardiac Raptor and mTOR expression (n=4, P=0.0008 and P=0.03 respectively). We then performed in silico bioinformatic analyses of rat zona glomerulosa cells following 2 hr treatment with 100 nM angiotensin II (AngII) using the Gene Expression Omnibus (GEO). AngII treatment led to the activation of mTOR signaling pathways as shown by increased expression of insulin receptor substrate type 2 (P=0.00001) and Low-density lipoprotein-related receptor 5 (P=0.03). Also, AngII reduced the expression of Eukaryotic Translation Initiation Factor 4E Binding Protein 1 by 43% (P= 0.001) and increased expression of CAP-Gly Domain Containing Linker Protein 1 by 70% (P= 0.00003) - important signaling molecules that are downstream of mTOR activation. Our results suggest that in hypertension, Na+ intake and Raptor gene variants modify mTORC1. Thus, mTOR/Raptor expression are a potential biomarker of ALDO-mediated cardiovascular injury. Presentation: Friday, June 16, 2023

Metabolic reprogramming of renal epithelial cells contributes to lithium-induced nephrogenic diabetes insipidus

Lithium, mainstay treatment for bipolar disorder, frequently causes nephrogenic diabetes insipidus (NDI) and renal injury. However, the detailed mechanism remains unclear. Here we used the analysis of metabolomics and transcriptomics and metabolic intervention in a lithium-induced NDI model. Mice were treated with lithium chloride (40 mmol/kg chow) and rotenone (ROT, 100 ppm) in diet for 28 days. Transmission electron microscopy showed extensive mitochondrial structural abnormalities in whole nephron. ROT treatment markedly ameliorated lithium-induced NDI and mitochondrial structural abnormalities. Moreover, ROT attenuated the decrease of mitochondrial membrane potential in line with the upregulation of mitochondrial genes in kidney. Metabolomics and transcriptomics data demonstrated that lithium activated galactose metabolism, glycolysis, and amino sugar and nucleotide sugar metabolism. All these events were indicative of metabolic reprogramming in kidney cells. Importantly, ROT ameliorated metabolic reprogramming in NDI model. Based on transcriptomics analysis, we also found the activation of MAPK, mTOR and PI3K-Akt signaling pathways and impaired focal adhesion, ECM-receptor interaction and actin cytoskeleton in Li-NDI model were inhibited or attenuated by ROT treatment. Meanwhile, ROT administration inhibited the increase of Reactive Oxygen Species (ROS) in NDI kidneys along with enhanced SOD2 expression. Finally, we observed that ROT partially restored the reduced AQP2 and enhanced urinary sodium excretion along with the blockade of increased PGE2 output. Taken together, the current study demonstrates that mitochondrial abnormalities and metabolic reprogramming play a key role in lithium-induced NDI, as well as the dysregulated signaling pathways, thereby serving as a novel therapeutic target.

Advances in the mTOR signaling pathway and its inhibitor rapamycin in epilepsy.

Epilepsy is one of the most common and serious brain syndromes and has adverse consequences on a patient's neurobiological, cognitive, psychological, and social wellbeing, thereby threatening their quality of life. Some patients with epilepsy experiencepoor treatment effects due to the unclear pathophysiological mechanisms of the syndrome. Dysregulation of the mammalian target of the rapamycin (mTOR) pathway is thought to play an important role in the onset and progression of some epilepsies. This review summarizes the role of the mTOR signaling pathway in the pathogenesis of epilepsy and the prospects for the use of mTOR inhibitors. The mTOR pathway functions as a vital mediator in epilepsy development through diverse mechanisms, indicating that the it has great potential as an effective target for epilepsy therapy. The excessive activation of mTOR signaling pathway leads to structural changes in neurons, inhibits autophagy, exacerbates neuron damage, affects mossy fiber sprouting, enhances neuronal excitability, increases neuroinflammation, and is closely associated with tau upregulation in epilepsy. A growing number of studies have demonstrated that mTOR inhibitors exhibit significant antiepileptic effects in both clinical applications and animal models. Specifically, rapamycin, a specific inhibitor of TOR, reduces the intensity and frequency of seizures. Clinical studies in patients with tuberous sclerosis complex have shown that rapamycin has the function of reducing seizures and improving this disease. Everolimus, a chemically modified derivative of rapamycin, has been approved as an added treatment to other antiepileptic medicines. Further explorations are needed to evaluate the therapeutic efficacy and application value of mTOR inhibitors in epilepsy. Targeting the mTOR signaling pathway provides a promising prospect for the treatment of epilepsy.

25KDa branched polyethylenimine increases interferon-γ production in natural killer cells via improving translation efficiency

BackgroundEx vivo cultivation is a promising strategy for increasing the number of NK cells and enhancing their antitumor activity prior to clinical application. Recent studies show that stimulation with 25KDa branched polyethylenimine (25KbPEI) generates NK cells with enhanced antitumor activity. To better understand how 25KbPEI primes NK cells, we explored the mechanism underlying increase in production of IFN-γ.MethodsChemical priming was performed on NK-92MI cells by incubating them with 5 μg/ml of 25KbPEI. The production of IFN-γ was evaluated by RT-qPCR, ELISA, and Flow cytometry. By evaluating the effect of pharmacological inhibition of ERK/mTOR-eIF4E signaling pathways on IFN-γ translation, the function of these signaling pathways in IFN-γ translation was examined. To comprehend the level of 25KbPEI activity on immune-related components in NK cells, RNA sequencing and proteomics analyses were conducted.Results25KbPEI enhances the production of IFN-γ by NK cells without transcriptional activation. Activation of ERK and mTOR signaling pathways was found to be associated with 25KbPEI-mediated calcium influx in NK cells. The activation of ERK/mTOR signaling was linked to the phosphorylation of 4E-BP1, which resulted in the activation of translation initiation complex and subsequent IFN-γ translation. Analysis of RNA sequencing and proteomics data revealed that the activity of 25KbPEI to improve translation efficiency in NK cells could be extended to additional immune-related molecules.ConclusionsThis study provides substantial insight into the process by which 25KbPEI primes NK cells. Our data demonstrated that the 25KbPEI mediated activation of ERK/mTOR signaling and subsequent stimulation of eIF4E is the primary mechanism by which the chemical stimulates translation of IFN-γ in NK cells.BTHQ_5BYqzK7H1pGe9W3hFVideo abstract

Abstract 3129: Single-cell RNA sequencing of early-stage to refractory metastatic ER+ breast cancers reveal phenotypic states underlying resistance to multiple lines of therapy

Abstract Breast cancer is a leading cause of global cancer-related mortality. About 75% of breast tumors are estrogen receptor-positive (ER+). Nearly 30-50% of ER+ breast cancers eventually become refractory to multiple lines of therapy and progress to an uncurable metastatic stage despite positive 5-year results on primary endocrine therapy. To uncover the malignant phenotypes underlying the development of therapeutic resistance, we performed single-cell RNA sequencing (scRNA-seq) of 219,532 cells from 80 ER+ breast cancer patients. This included 28 solid tumors and 52 metastatic fluid samples (pleural effusions and ascites). We grouped samples in three groups to study the evolution of biological phenotypes based on the number of lines of therapy administered to a patient: 1. Early (non-metastatic), 2. Advanced (metastatic with less than 3 lines of therapy), and Refractory (metastatic with 3 or more lines of therapy). We used Pareto task inference analysis to model evolutionary task trade-offs across malignant cell populations from the early, advanced, and refractory tumors. Our model revealed 5 unique phenotypic states or “archetypes” that existed across ER+ cancer cells. A multitask learning model based on group lasso revealed the specialized biological tasks associated with each archetype, including 1. Metabolism and self-renewal, 2. EGF/STAT3 signaling, 3. Estrogen signaling, 4. Apoptosis, and 5. MYC activation with rapid proliferation. We found the estrogen signaling archetype was progressively lost in advanced and refractory tumors (P=7.2x10-110), while the MYC archetype was progressively gained in the advanced and refractory tumors (P=2.45x10-9). The EGF/STAT3 archetype was unique to refractory tumors (P= 1.42x10-37). We further investigated the evolution of resistant traits by modeling the pathway phenotypes in malignant cells against the number of lines of therapy received by the patients. Patients refractive to multiple lines of therapy progressively gained mTOR signaling pathway activity concurrent with loss of estrogen-signaling. Multiple pathways that crosstalk with the mTOR pathway were activated in the tumors receiving multiple lines of therapy, including IGFR, and EGF/ERBB signaling. In comparison to early and advanced tumors, refractory tumors also gained JNK/p38 signaling (P=0.006) and activated glycolysis (P=5x10-6). This study reveals how metastatic ER+ breast cancer cells acquire capabilities for resistance to multiple lines of therapy, thus serving as a unique resource for understanding refractory ER+ breast cancer biology. Citation Format: Aritro Nath, Patrick Cosgrove, Jason Griffiths, Jeffrey Chang, Adam Cohen, Andrea Bild. Single-cell RNA sequencing of early-stage to refractory metastatic ER+ breast cancers reveal phenotypic states underlying resistance to multiple lines of therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3129.

Abnormal mTOR Signaling Pathway Activity in Autism Spectrum Disorders: Prospects of Mechanism-Based Therapy

Abnormal mTOR Signaling Pathway Activity in Autism Spectrum Disorders: Prospects of Mechanism-Based Therapy

Abstract P1-13-15: Elevated TCEAL9 Expression Is Correlated With Trastuzumab-based Neoadjuvant Chemotherapy Resistance In HER2-positive Breast Cancer

Abstract Background: -Trastuzumab-based neoadjuvant chemotherapy has shown to have remarkable clinical benefits for HER2-positive breast cancer patients who had higher tumor burden. -Patients who achieved pathological complete response (pCR) are known to have better prognosis. -However, certain patients have little response or are not sensitive to trastuzumab-based treatment regimens. -Understanding the mechanism of trastuzumab resistance is crucial for the development of new therapeutic strategy. Objectives: To investigate the role of TCEAL9 in developing trastuzumab resistance in HER2-positive breast cancer Methods: A total of 83 patients who received paclitaxel, carboplatin and trastuzumab neoadjuvant chemotherapy in Fudan University Shanghai Cancer Center(FUSCC) from 2016 to 2018 were enrolled in this study. After completed neoadjuvant chemotherapy and surgery, gene expressions were compared between the pCR and non-pCR groups. Total RNA from formalin-fixed paraffin-embedded tissue sections was isolated and RNA-sequencing was performed. Gene sets from GEO dataset GSE52707 were used to analyze TCEAL9 expression in resistant and non-resistant cell lines. Gene expression levels were converted into log2 values and row-wised standardized. BT-474 and SK-BR-3 cell lines were transduced with each expression lentivirus, followed by selection with puromycin for stable expression. TCEAL9 mRNA and protein level evaluation was evaluated by qPCR and western blot. The influence of TCEAL9 expression on proliferation and sensitivity to HER2-targeted therapy was evaluated by CCK8. BT-474 and SK-BR-3 transfected cells were plated in 96-well plates with 4,000 cells per well. After 3 or 5 days of incubation with trastuzumab, pertuzumab or lapatinib, the viability of cells was measured using Cell Proliferation Assay. Comparisons between Kaplan-Meier curves were performed using the long-rank test. Results: TCEAL9 was elevated significantly (P< 0.05) in non-pCR patients in the FUSCC cohort and was associated with lapatinib resistance in GSE52707 from GEO datasets. Patients with elevated TCEAL9 expression had worse recurrence-free survival (RFS), distant metastasis-free survival (DMFS) and progression-free survival (PPS) (all P< 0.05)by using KM-plotter. Overexpression of TCEAL9 was associated with lapatinib(IC50= 5.56 vs 10.90nM) and trastuzumab + pertuzumab(IC50= 745 vs 635nM) resistance in BT-474 and SK-BR-3 respectively, but has no influence in proliferation. In this study, we found that TCEAL9 could induce HER2-positive breast cancer cells resistance to HER2-targeted therapy through the activation of mTOR signaling pathway. After EGFR stimulation, TCEAL9 has a higher mTOR phosphorylation level in BT-474 cells. TCEAL9 elevation also increased HER2 and mTOR phosphorylation after lapatinib treatment in SK-BR-3 cells. In addition, the elevation of TCEAL9 has a positive correlation with HER2 signaling pathways such as EGFR, PIK3R1, FOXO1 and AKT3 in TCGA datasets. Conclusions: TCEAL9 expression correlates with trastuzumab resistance and high TCEAL9 expression is associated with poor prognosis in HER2-positive breast cancer patients. Citation Format: Chih Wan Goh, Wei-Ru Chi, Liyi Zhang, Qi Zhang, Ming Chen, Min Xiong, Douwaner Liu, Hengyu Ren, Bingqiu Xiu, Jingyan Xue, Yayun Chi, Jiong Wu. Elevated TCEAL9 Expression Is Correlated With Trastuzumab-based Neoadjuvant Chemotherapy Resistance In HER2-positive Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-15.

Abnormal mTOR Signaling Pathway Activity in Autism Spectrum Disorders: Prospects of Mechanism-Based Therapy

Autism spectrum disorder (ASD) is a developmental disorder characterized by the early onset of communication, learning, and behavioral problems. The syndromic form of ASD is caused by monogenic mutations, in the case when it is not possible to find genetic or other known mechanisms, the term “idiopathic autism” is used. A significant part of both syndromic and idiopathic autism is associated with translational deregulation dependent on the mechanistic target of rapamycin (mTOR). In this review, we present both bioinformatic and experimental data that link the mTOR signaling pathway to maternal autoantibody-induced autism and childhood autoimmune neuropsychiatric disorders such as Sydenham’s chorea and pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS). The need for ASD subtyping and the possibility of mechanism-based therapy with inhibitors of the mTOR signaling pathway are also discussed.

Abnormal mTOR Signaling Pathway Activity in Autism Spectrum Disorders: Prospects of Mechanism-Based Therapy

Autism spectrum disorder (ASD) is a developmental disorder characterized by the early onset of problems with communication, learning, and behavior. The syndromic form of ASD is caused by monogenic mutations. When it is not possible to find genetic or other known mechanisms, the term "idiopathic autism" is used. A significant part of both syndromic and idiopathic autism is associated with translational deregulation dependent on the mechanistic target of rapamycin (mTOR). In this review, we present both bioinformatic and experimental data that link the mTOR signaling pathway to maternal autoantibody related autism and childhood autoimmune neuropsychiatric disorders such as Sydenham's chorea and pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS). The need for ASD subtyping and the prospects of mechanism-based therapy with inhibitors of the mTOR signaling pathway are also discussed.

Effects of L-arginine and arginine-arginine dipeptide on amino acids uptake and αS1-casein synthesis in bovine mammary epithelial cells.

Arginine (Arg), as an important functional amino acids (AA), is essential for milk protein synthesis in lactating ruminants. Arg shares transporters with cationic and neutral AA in mammary epithelial cells. Therefore, competitive inhibition might exist among these AA in uptake by mammary epithelial cells. In this study, cultured bovine mammary epithelial cells (BMEC) were used as the model to investigate whether the availability of L-Arg (0.7, 1.4, 2.8, 5.6, and 11.2 mM) affects the uptake of other AA and if this related to αS1-casein synthesis, and whether Arginine-Arginine (Arg-Arg) substituting part of free L-Arg can alleviate competitive inhibition among Arg and other AA, so as to promote αS1-casein synthesis. Our results showed that 2.8 mM L-Arg generated the greatest positive effects on αS1-casein synthesis and the activation of mammalian target of rapamycin (mTOR) signaling pathway (P < 0.01). With L-Arg supply increasing from 0.7 to 11.2 mM, the net-uptake of other AA (except Glu and Ala) decreased linearly and quadratically (Plinear < 0.01; Pquadratic < 0.01). Compared with 2.8 mM, the net-uptake of essential amino acids (EAA) and total amino acids (TAA) were lower at 11.2 mM L-Arg group, while greater at 1.4 mM L-Arg group (P < 0.01). Arg-Arg dipeptide replacing 10% free L-Arg increased αS1-casein synthesis (P < 0.05), net-uptake of EAA and TAA, as well as phosphorylation level of mTOR and p70 ribosomal protein S6 kinase (P70S6K) and mRNA expression of oligopeptide transporter 2 (PepT2; P < 0.01). These observations suggested that the increased αS1-casein synthesis by 10% Arg-Arg dipeptide might be related to the increase of AA availability and the activation of mTOR signaling pathway in BMEC.

Dietary sodium acetate improves high-fat diet utilization through promoting differential nutrients metabolism between liver and muscle in Nile tilapia (Oreochromis niloticus)

High-fat diet (HFD) often causes many negative effects and impairs fish growth. Short-chain fatty acids (SCFAs) such as acetates modify metabolic disorders and regulate body homeostasis. However, the effects of sodium acetate on alleviating HFD in fish is currently unknown. This study investigated the role of using dietary sodium acetate to alleviate adverse effects caused by HFD in fish. Three replicates (thirty fish each) of 4.8 ± 0.30 g Nile tilapia (Oreochromis niloticus) were fed with control diet (Con), high-fat diet (HFD) or HFD containing sodium acetate diet (HFD + NaAc) for eight weeks. After the feeding trial, Nile tilapia fed with HFD increased significantly tissue lipid deposition, reduced insulin sensitivity and suppressed glucose and lipid metabolism in both liver and muscle, accompanied with signs of metabolic disorders and liver damage. Moreover, HFD feeding inhibited muscle protein synthesis and impaired fish growth performance. However, the fish fed on HFD + NaAc improved significantly oxidative stress, inflammation, apoptosis and injury in liver compared to those fed on HFD. More importantly, dietary sodium acetate supplementation enhanced insulin sensitivity, promoted glucose catabolic utilization and protein synthesis in muscle through activation of insulin and mTOR signaling pathways, respectively, and markedly improved the growth performance. In contrast, dietary sodium acetate promoted hepatic pentose phosphate pathway, hepatic glycogen accumulation, and activated lipid catabolism to alleviate hepatic lipid deposition. Our study illustrates that sodium acetate alleviates differentially the adverse effects induced by feeding Nile tilapia with HFD in muscle and liver. SCFAs such as acetate may be used for improving HFD utilization in fish nutrition.

Castor1 overexpression regulates microglia M1/M2 polarization via inhibiting mTOR pathway.

Microglia are resident immune cells in the brain and are closely associated with central nervous system inflammation and neurodegenerative diseases. It is known that mammalian target of rapamycin (mTOR) pathway plays an important role in the polarization of microglia. Castor1 has been identified as the cytosolic arginine sensor for the mTOR complex 1 (mTORC1) pathway, but the role of Castor1 in microglial polarization is still unknown. The purpose of this study was to explore the regulatory effect of Castor1 on microglial polarization and the underlying mechanism. The results demonstrated that Castor1 expression was significantly decreased in lipopolysaccharides (LPS) and interferon (IFN)-γ treated microglia. Castor1 overexpression inhibited the microglia M1 polarization by reducing the expression of M1 related markers. However, the expression of M2-related genes was promoted when Castor1 was overexpressed in IL-4 treated microglia. Mechanistically, Castor1 overexpression inhibited the activation of mTOR signaling pathway. In addition, after treatment with the mTOR activator MHY1485, the inhibitory effect of Castor1 overexpression on M1 polarization was attenuated, indicating that the regulation effects of Castor1 on M1 polarization was dependent on its inhibition of mTOR pathway. We propose that Castor1-mTOR signaling pathway could be considered as a potential target for treatment and intervention of central nervous system-related diseases by regulating microglia polarization.

Rapamycin Inhibits Cytotoxic T Lymphocytes-Mediated Platelet Destruction in Immune Thrombocytopenia

Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized with increased risk of bleeding. CD8+ cytotoxic T lymphocytes (CTLs) of ITP patients express high level of lethal molecules, leading to the lysis and apoptosis of their own platelets, which is an important mechanism of platelet destruction in ITP patients. Accumulating evidence have highlighted that the activation of mTOR signaling pathway promotes the differentiation of CD8+ T cells to CTLs, which leads to the up-regulation of cell-mediated immunity. Several studies have showed that rapamycin, a mTOR inhibitor, was effective in the treatment of relapsed/refractory ITP. However, the endogenous mechanism remains to be explored. The activation of mTOR signaling pathway in CD8+ T cells was determined by flow cytometry. We found that the phosphorylation level of p-mTOR, p-Akt, p-4E-BP1 and p-P70S6K in ITP patients were significantly higher than that in healthy controls. Meanwhile, we found that the levels of perforin and granzyme B in CD8+ T cells in ITP patients were significantly higher than that in healthy controls. In the in vitro experiments, CTLs were isolated from PBMCs of ITP patients or healthy controls and cultured with different concentrations of rapamycin (0-1 uM) for 3 days, and we chose an optimal concentration of 20 nM. There was no significant difference in the expression levels of mTOR, Akt, 4E-BP1, and P70S6K after rapamycin treatment, but the phosphorylation level of p-mTOR, p-Akt, p-4E-BP1, p-P70S6K was significantly inhibited by rapamycin treatment. Likewise, the ratios of p-mTOR/mTOR, p-AKT/AKT, p-4E-BP1/4E-BP1, p-P70S6K/P70S6K were also significantly down-regulated. Flow cytometry analysis found that rapamycin significantly inhibited the expression of perforin and granzyme B (Figure 1) in CD8+ T cells of ITP patients. We further established the ITP murine model by transferring splenocytes of C57BL/6 CD61 knockout mice, immunized against platelets from wild-type syngeneic C57BL/6 mice, into severe combined immune deficient (SCID) mice. Rapamycin was injected intraperitoneally daily at a final concentration 2.0 mg/kg. Our data showed that the rapamycin group had significantly higher platelet counts compared with control group. In summary, our findings suggest that the activation of mTOR signaling pathway-related molecules of CD8+ T cells in patients with ITP are abnormal. These data shed new light on the molecular mechanism of rapamycin action by inhibiting CTLs cytotoxicity. This novel mechanism of rapamycin underlies the potential therapeutic strategy for ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

OR09-5 mTOR Signaling is Associated with Regulation of Mitochondrial Respiration in Thyroid Cancer

Abstract Background Thyroid cancers (TC) are divided into BRAF-like and RAS-like tumors based on their molecular signature. The mTOR (mammalian target of rapamycin) signaling pathway is commonly activated in RAS-like tumors. Oncogene-driven signaling pathways are involved in the regulation of intracellular metabolism, including glycolysis and oxidative phosphorylation (OXPHOS). There is inadequate information on the role of mTOR signaling in the regulation of TC metabolism. The purpose of this study was to determine the association between mTOR signaling and mitochondrial respiration in TC. Methods We conducted an in vitro study using 4 TC cell lines (2 BRAF-like, 2 RAS-like) characterized by variable activation of mTOR signaling pathway, as documented by immunoblotting to quantify a ratio of p-mTOR/mTOR. OXPHOS was measured through oxygen consumption rate (OCR) of TC cells by the Seahorse XF Cell Mito Stress Test Assay. Standard MTT assay was performed to evaluate cell proliferation. A specific mTOR inhibitor (rapamycin) as well as an mTOR activator (leucine) were used for functional studies. The association between OCR and p-mTOR/mTOR was calculated by Pearson correlation coefficient (r). The one-way Anova or Kruskal-Wallis test were used for between group comparisons, as appropriate. P-values of ≤ 0.05 were considered statistically significant. Results The baseline activation of mTOR signaling in TC cells correlated with baseline (r=0.75, p=0.04) and maximum OCR (r=0.91, p=0.01). In addition, the RAS-like cell lines with the highest activation of mTOR showed considerable response after inhibition of mTOR with rapamycin by inhibiting baseline OCR (FTC133: 6320 ± 1531 vs 3368 ± 651 pmol/min/mg, p=0.01; THJ29T: 4484 ± 514 vs 3067 ± 620 pmol/min/mg, p=0.047), while the TC cell lines with minimal baseline mTOR activation did not show change in mTOR phosphorylation status nor basal OCR after rapamycin treatment (TPC1: 1817 ± 201 vs 1974 ± 336 pmol/min/mg, p&amp;gt;0.99, BCPAP: 1051 ± 162 vs 1130 ± 228 pmol/min/mg, p&amp;gt;0.99). Treatment with rapamycin significantly reduced cell proliferation rates in all examined cells. On the other hand, treatment with the mTOR activator leucine increased both mTOR phosphorylation and basal OCR in TC cell lines characterized by minimal baseline mTOR activation (TPC1: 1778 ± 280 vs 2372 ± 249 pmol/min/mg, p=0.004, BCPAP: 1581 ± 603 vs 2064 ± 560 pmol/min/mg, p=0.04), but did not significantly increase OCR further in cell lines with high baseline mTOR activation and OXPHOS rate (FTC133: 4512 ± 683 vs 5355 ± 870 pmol/min/mg, p=0.27; THJ29T: 3881 ± 880 vs 4764 ± 1273 pmol/min/mg, p=0.14). Conclusion mTOR signaling is associated with the regulation of mitochondrial respiration as its inhibition decreases OXPHOS rate, while its activation results in increased mitochondrial respiration. The change in mitochondrial respiration might be one of the mechanisms of the growth inhibition caused by medications targeting mTOR signaling in TC. Presentation: Saturday, June 11, 2022 12:30 p.m. - 12:45 p.m.