Phosphorylation Of Ribosomal Protein S6 Research Articles

Effect and mechanism of Zuogui Pills on neural function recovery in ischemic stroke mice based on OPN/IGF-1/mTOR

To explore the effect and mechanism of Zuogui Pills in promoting neural tissue recovery and functional recovery in mice with ischemic stroke. Male C57BL/6J mice were randomly divided into a sham group, a model group, and low-, medium, and high-dose Zuogui Pills groups(3.5, 7, and 14 g·kg~(-1)), with 15 mice in each group. The ischemic stroke model was established using photochemical embolization. Stiker remove and irregular ladder walking behavioral tests were conducted before modeling and on days 7, 14, 21, and 28 after medication. Triphenyl tetrazolium chloride(TTC) staining was performed on day 3 after modeling, and T2-weighted imaging(T2WI) and diffusion-weighted imaging(DWI) were performed on day 28 after medication to evaluate the extent of brain injury. Hematoxylin-eosin(HE) staining was performed to observe the histology of the cerebral cortex. Axonal marker proteins myelin basic protein(MBP), growth-associated protein 43(GAP43), mammalian target of rapamycin(mTOR), and its downstream phosphorylated s6 ribosomal protein(p-S6), as well as mechanism-related proteins osteopontin(OPN) and insulin-like growth factor 1(IGF-1), were detected using immunofluorescence and Western blot. Zuogui Pills had a certain restorative effect on the neural function impairment caused by ischemic stroke in mice. TTC staining showed white infarct foci in the sensory-motor cortex area, and T2WI imaging revealed cystic necrosis in the sensory-motor cortex area. The Zuogui Pills groups showed less brain tissue damage, fewer scars, and more capillaries. The number of neuronal axons in those groups was higher than that in the model group, and neuronal activity was stronger. The expression of GAP43, OPN, IGF-1, and mTOR proteins in the Zuogui Pills groups was higher than that in the model group. In summary, Zuogui Pills can promote the recovery of neural function and axonal growth in mice with ischemic stroke, and its mechanism may be related to the activation of the OPN/IGF-1/mTOR signaling pathway.

The effect of Dunaliella tertiolecta supplementation on diet-induced obesity in UCP1-deficient mice.

We previously demonstrated that dietary supplementation with Dunaliella tertiolecta (DT) increases uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) and improves diet-induced obesity (DIO) in C57BL/6J mice at thermoneutrality (30 °C). Here, we investigated whether DT improves DIO in a thermoneutral UCP1-deficient (KO) animal. KO mice were fed a high-fat diet supplemented with DT for 12 weeks. Compared to control group without DT, body weight was significantly reduced in DT group with no difference in food intake. Dunaliella tertiolecta-supplemented mice exhibited lower adiposity and well-maintained multilocular morphology in BAT, in which a significant increase in gene expression of PR domain containing 16 was detected in DT group compared to control group. Moreover, increase in UCP2 level and/or decrease in ribosomal protein S6 phosphorylation were detected in adipose tissues of DT group relative to control group. These results suggest that DT supplementation improves DIO by stimulating UCP1-independent energy dissipation at thermoneutrality.

Differential targeting of lysophosphatidic acid LPA1, LPA2, and LPA3 receptor signalling by tricyclic and tetracyclic antidepressants.

We previously reported that in different cell types antidepressant drugs activate lysophosphatidic acid (LPA) LPA1 receptor to induce proliferative and prosurvival responses. Here, we further characterize this unique action of antidepressants by examining their effects on two additional LPA receptor family members, LPA2 and LPA3. Human LPA1-3 receptors were stably expressed in HEK-293 cells (HEK-LPA1, -LPA2 and –LPA3 cells) and their functional activity was determined by Western blot and immunofluorescence. LPA effectively stimulated the phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in HEK-LPA1, -LPA2, and -LPA3 cells. The tricyclic antidepressants amitriptyline, clomipramine, imipramine and desipramine increased phospho-ERK1/2 levels in HEK-LPA1 and –LPA3 cells but were relatively poor agonists in LPA2-expressing cells. The tetracyclic antidepressants mianserin and mirtazapine were active at all three LPA receptors. When combined with LPA, both amitriptyline and mianserin potentiated Gi/o-mediated phosphorylation of ERK1/2 induced by LPA in HEK-LPA1, -LPA2 and -LPA3 cells, CHO–K1 fibroblasts and HT22 hippocampal neuroblasts. This potentiation was associated with enhanced phosphorylation of CREB and S6 ribosomal protein, two molecular targets of activated ERK1/2. The antidepressants also potentiated LPA-induced Gq/11-mediated phosphorylation of AMP-activated protein kinase in HEK-LPA1 and –LPA3 cells. Conversely, amitriptyline and mianserin were found to inhibit LPA-induced Rho activation in HEK-LPA1 and LPA2 cells. These results indicate that tricyclic and tetracyclic antidepressants can act on LPA1, LPA2 and LPA3 receptor subtypes and exert differential effects on LPA signalling through these receptors.

Extracellular Glutamine Promotes Intestinal Porcine Epithelial Cell Proliferation via Arf1-mTORC1 Pathway Independently of Rag GTPases.

Glutamine (Gln) is the major energy source of intestinal porcine epithelial cells (IPEC-J2 cells) and plays a critical role in the nutritional physiological function of the intestine. However, the underlying mechanism requires further investigation. Here, the Gln-sensing pathway in IPEC-J2 cells was investigated. The results showed that Gln increased the cell proliferation. Subsequently, an analysis of the phosphorylated proteome revealed that Gln markedly upregulated ribosomal protein S6 (RPS6) phosphorylation at serine 235/236, suggesting that Gln activated the mTORC1 pathway. mTOR inhibition revealed that Gln promotes cell proliferation through the mTORC1 pathway. Similarly, blocking ADP-ribosylation factor 1 (Arf1) activity indicated that Gln-induced mTORC1 activation promoted cell proliferation in an Arf1-dependent manner. Additionally, the RagA/B pathway did not participate in Gln-induced mTORC1 activation. Collectively, these findings suggest that Gln-induced mTORC1 activation promotes IPEC-J2 cell proliferation via Arf1, not Rag GTPases. These results broaden our understanding of functional-cell-sensing amino acids, particularly Gln, that are regulated by mTORC1.

Human iPSC-derived endothelial cells promote CNS remyelination via BDNF and mTORC1 pathway.

Damage of myelin is a component of many diseases in the central nervous system (CNS). The activation and maturation of the quiescent oligodendrocyte progenitor cells (OPCs) are the crucial cellular processes for CNS remyelination, which is influenced by neuroinflammation in the lesion microenvironment. Endothelial cells derived from human induced pluripotent stem cells (hiPSC-ECs) have shown promise in restoring function in various preclinical animal models. Here we ask whether and whether transplantation of hiPSC-ECs could benefit remyelination in a mouse model of CNS demyelination. Our results show that in vitro, hiPSC-ECs increase OPC proliferation, migration and differentiation via secreted soluble factors including brain-derived neurotrophic factor (BDNF). hiPSC-ECs also promote the survival of oligodendrocyte lineage cells in vitro and in vivo. Transplantation of hiPSC-ECs into a toxin-induced demyelination lesion in mouse corpus callosum (CC) leads to increased density of oligodendrocyte lineage cells and level of myelin in demyelinated area, correlated with a decreased neuroinflammation and an increased proportion of pro-regenerative M2 phenotype in microglia/macrophages. The hiPSC-EC-exposed oligodendrocyte lineage cells showed significant increase in the level of phosphorylated S6 ribosomal protein (pS6) both in vitro and in vivo, indicating an involvement of mTORC1 pathway. These results suggest that hiPSC-ECs may benefit myelin protection and regeneration which providing a potential source of cell therapy for a wide range of diseases and injuries associated with myelin damage.

Neuron-derived extracellular vesicles contain synaptic proteins, promote spine formation, activate TrkB-mediated signalling and preserve neuronal complexity.

Extracellular vesicles (EVs) play an important role in intercellular communication as carriers of signalling molecules such as bioactive miRNAs, proteins and lipids. EVs are key players in the functioning of the central nervous system (CNS) by influencing synaptic events and modulating recipient neurons. However, the specific role of neuron-to-neuron communication via EVs is still not well understood.Here, we provide evidence that primary neurons uptake neuron-derived EVs in the soma, dendrites, and even in the dendritic spines, and carry synaptic proteins. Neuron-derived EVs increased spine density and promoted the phosphorylation of Akt and ribosomal protein S6 (RPS6), via TrkB-signalling, without impairing the neuronal network activity. Strikingly, EVs exerted a trophic effect on challenged nutrient-deprived neurons. Altogether, our results place EVs in the spotlight for synaptic plasticity modulation as well as a possible therapeutic tool to fight neurodegeneration.

Randomized, double-blind, placebo-controlled trial of rapamycin in amyotrophic lateral sclerosis

In preclinical studies rapamycin was found to target neuroinflammation, by expanding regulatory T cells, and affecting autophagy, two pillars of amyotrophic lateral sclerosis (ALS) pathogenesis. Herein we report a multicenter, randomized, double-blind trial, in 63 ALS patients who were randomly assigned in a 1:1:1 ratio to receive rapamycin 2 mg/m2/day,1 mg/m2/day or placebo (EUDRACT 2016-002399-28; NCT03359538). The primary outcome, the number of patients exhibiting an increase >30% in regulatory T cells from baseline to treatment end, was not attained. Secondary outcomes were changes from baseline of T, B, NK cell subpopulations, inflammasome mRNA expression and activation status, S6-ribosomal protein phosphorylation, neurofilaments; clinical outcome measures of disease progression; survival; safety and quality of life. Of the secondary outcomes, rapamycin decreased mRNA relative expression of the pro-inflammatory cytokine IL-18, reduced plasmatic IL-18 protein, and increased the percentage of classical monocytes and memory switched B cells, although no corrections were applied for multiple tests. In conclusion, we show that rapamycin treatment is well tolerated and provides reassuring safety findings in ALS patients, but further trials are necessary to understand the biological and clinical effects of this drug in ALS.

Astaxanthin enhances autophagy, amyloid beta clearance and exerts anti-inflammatory effects in in vitro models of Alzheimer’s disease-related blood brain barrier dysfunction and inflammation

Defective degradation and clearance of amyloid-β as well as inflammation per se are crucial players in the pathology of Alzheimer’s disease (AD). A defective transport across the blood–brain barrier is causative for amyloid-β (Aβ) accumulation in the brain, provoking amyloid plaque formation. Using primary porcine brain capillary endothelial cells and murine organotypic hippocampal slice cultures as in vitro models of AD, we investigated the effects of the antioxidant astaxanthin (ASX) on Aβ clearance and neuroinflammation. We report that ASX enhanced the clearance of misfolded proteins in primary porcine brain capillary endothelial cells by inducing autophagy and altered the Aβ processing pathway. We observed a reduction in the expression levels of intracellular and secreted amyloid precursor protein/Aβ accompanied by an increase in ABC transporters ABCA1, ABCG1 as well as low density lipoprotein receptor-related protein 1 mRNA levels. Furthermore, ASX treatment increased autophagic flux as evidenced by increased lipidation of LC3B-II as well as reduced protein expression of phosphorylated S6 ribosomal protein and mTOR. In LPS-stimulated brain slices, ASX exerted anti-inflammatory effects by reducing the secretion of inflammatory cytokines while shifting microglia polarization from M1 to M2 phenotype. Our data suggest ASX as potential therapeutic compound ameliorating AD-related blood brain barrier impairment and inflammation.

Quantitative proteomics and phosphoproteomics of PP2A-PPP2R5D variants reveal deregulation of RPS6 phosphorylation via converging signaling cascades

Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for ∼40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420Kcell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients.

Intermittent fasting reduces mouse body fat while maintaining muscle mass by regulating protein synthesis and autophagy

ObjectivesThe aim of this study is to investigate the effect of intermittent fasting (IF) on the regulation of skeletal muscle protein metabolism in response to nutrient supplementation during fasting. MethodsTwelve-week-old male C57BL/6J mice were assigned to two groups: ad libitum and IF, with the latter having access to food for only 3 h/d. After 6 wk of experimental periods, an oral glucose tolerance test was performed. One week later, phosphate-buffered saline or a glucose and branched-chain amino acid mixture was administered orally, and blood and tissues were collected 30 min later. ResultsThe oral glucose tolerance test results revealed that the IF group had better insulin sensitivity. They also had lower body and fat weights while maintaining the same level of skeletal muscle mass as the ad libitum group. The phosphorylation of ribosomal protein S6 in the skeletal muscle, a marker for the activation of protein translation, was greater in the IF group after glucose and branched-chain amino acid mixture administration. Microtubule-associated protein light chain 3-II–to–light chain 3-I ratio, a marker for autophagosome formation, in skeletal muscle during fasting was significantly lower in the IF group than that in the ad libitum group. ConclusionsOur findings suggest that adaptation to IF regulates protein synthesis and breakdown, leading to the maintenance of skeletal muscle mass while reducing body fat.

Silibinin Alleviates Lipopolysaccharide Induced Inflammation in Porcine Mammary Epithelial Cells via mTOR/NF-κB Signaling Pathway.

Inflammatory responses reduce milk production in lactating sow. Silymarin (Silibinin is main component) reduces the inflammatory reaction and increases milk yield in lactating sow in the previous study. In the present study, silibinin may be a previously unrecognized nutrients in inflammatory resolution in porcine mammary epithelial cells (PMECs) is hypothesized. PMECs are treated with or without lipopolysaccharide (LPS) in the absence or presence of silibinin to test cell viability, cell cycle, cell apoptosis, cellular inflammatory factors, and signaling protein phosphorylation and expression. Silibinin promotes the proliferation of PMEC independent of the estrogen pathway. In LPS-induced damage of PMECs, silibinin protects cell proliferation, as well as reduced cell apoptosis. Silibinin reverses the LPS-induced increase in tumor necrosis factor-alpha (TNF-α) expression compared with control. In addition, silibinin accentuates the LPS-induced decrease in the key proteins phosphorylated-ribosomal protein S6 (p-S6) and phosphorylated-mammalian target of rapamycin (p-mTOR) of the mammalian target of rapamycin (mTOR) signaling pathway. Furthermore, silibinin reverses the increase in phosphorylated-nuclear factor-kappa B p65 (p-NF-κB p65), phosphorylated-Ikappab-alpha (p-IκB-α), and phosphorylated-Mitogen-activated protein kinase p38 (p-MAPK p38) expression in LPS-induced damage in PMECs. This study highlights silibinin-mTOR/NF-κB axis plays an important role in the control of inflammation in PMECs, and suggests that silibinin may be an effective dietary strategy to alleviate the inflammatory response in lactating sow.

Glucose metabolic upregulation via phosphorylation of S6 ribosomal protein affects tumor progression in distal cholangiocarcinoma

BackgroundThe prognosis of distal cholangiocarcinoma (dCCA) remains poor; thus, the identification of new therapeutic targets is warranted. Phosphorylated S6 ribosomal protein indicates a mammalian target of rapamycin complex 1 (mTORC1) activity, and mTORC1 plays a central role in controlling cell growth and regulating glucose metabolism. We aimed to clarify the effect of S6 phosphorylation on tumor progression and the glucose metabolic pathway in dCCA.MethodsThirty-nine patients with dCCA who underwent curative resection were enrolled in this study. S6 phosphorylation and the expression of GLUT1 were evaluated by immunohistochemistry, and their relationship with clinical factors was investigated. The effect of S6 phosphorylation on glucose metabolism with PF-04691502 treatment, an inhibitor of S6 phosphorylation, was examined in cancer cell lines by Western blotting and metabolomics analysis. Cell proliferation assays were performed with PF-04691502.ResultsS6 phosphorylation and the expression of GLUT1 were significantly higher in patients with an advanced pathological stage. Significant correlations between GLUT1 expression, S6 phosphorylation, and SUV-max of FDG-PET were shown. In addition, cell lines with high S6 phosphorylation levels showed high GLUT1 levels, and the inhibition of S6 phosphorylation reduced the expression of GLUT1 on Western blotting. Metabolic analysis revealed that inhibition of S6 phosphorylation suppressed pathways of glycolysis and the TCA cycle in cell lines, and then, cell proliferation was effectively reduced by PF-04691502.ConclusionUpregulation of glucose metabolism via phosphorylation of S6 ribosomal protein appeared to play a role in tumor progression in dCCA. mTORC1 may be a therapeutic target for dCCA.

The regeneration-responsive element careg monitors activation of Müller glia after MNU-induced damage of photoreceptors in the zebrafish retina.

In contrast to mammals, zebrafish can regenerate their damaged photoreceptors. This capacity depends on the intrinsic plasticity of Müller glia (MG). Here, we identified that the transgenic reporter careg, a marker of regenerating fin and heart, also participates in retina restoration in zebrafish. After methylnitrosourea (MNU) treatment, the retina became deteriorated and contained damaged cell types including rods, UV-sensitive cones and the outer plexiform layer. This phenotype was associated with the induction of careg expression in a subset of MG until the reconstruction of the photoreceptor synaptic layer. Single-cell RNA sequencing (scRNAseq) analysis of regenerating retinas revealed a population of immature rods, defined by high expression of rhodopsin and the ciliogenesis gene meig1, but low expression of phototransduction genes. Furthermore, cones displayed deregulation of metabolic and visual perception genes in response to retina injury. Comparison between careg:EGFP expressing and non-expressing MG demonstrated that these two subpopulations are characterized by distinct molecular signatures, suggesting their heterogenous responsiveness to the regenerative program. Dynamics of ribosomal protein S6 phosphorylation showed that TOR signaling became progressively switched from MG to progenitors. Inhibition of TOR with rapamycin reduced the cell cycle activity, but neither affected careg:EGFP expression in MG, nor prevented restoration of the retina structure. This indicates that MG reprogramming, and progenitor cell proliferation might be regulated by distinct mechanisms. In conclusion, the careg reporter detects activated MG, and provides a common marker of regeneration-competent cells in diverse zebrafish organs, including the retina.

Abstract LB288: Biomarker analysis from AMPECT correlating response to nab-sirolimus with TSC1 and TSC2 inactivating alterations

Abstract nab-Sirolimus is an mTOR inhibitor (mTORi) approved in the US for the treatment of adult patients with locally advanced, unresectable, or metastatic malignant perivascular epithelioid cell tumor (PEComa) based on clinical efficacy and safety data from the phase 2, multicenter, open-label AMPECT trial (NCT02494570). TSC1 and TSC2 are tumor suppressor genes and key upstream regulators of mTOR complex 1 (mTORC1); inactivating alterations (loss-of-function mutations or deletions) in these genes lead to mTORC1 hyperactivation, which may contribute to tumor formation. Phosphorylation of S6 ribosomal protein (pS6) is a reliable surrogate for mTORC1 activity. Here we present the results of an exploratory biomarker analysis performed on samples from patients enrolled in the AMPECT study. Targeted exome next-generation sequencing (NGS) using a 500-gene OncoPanel test (Center for Advanced Molecular Diagnostics, Brigham and Women’s Hospital, Boston, MA) assessed mutations, copy number changes, and translocation events. pS6 was assessed by immunohistochemistry (IHC). Twenty-five patients had tissue sufficient for NGS, 56% (14/25) had either TSC1 (N=5, 20%) or TSC2 (N=9, 36%) mutations: TSC1, 1 frameshift, 2 splice site, 1 missense, and 1 nonsense mutation; TSC2, 1 nonsense, 7 frameshift, and 1 homozygous deletion. In this dataset, TSC1 and TSC2 inactivating alterations were mutually exclusive. Patients with TSC1 or TSC2 inactivating alterations achieved a clinically meaningful benefit: 64.3% (9/14) objective overall responses (including complete and partial responses) and median (95% CI) duration of response (DOR) of 45.7 (5.6, not reached [NR]) months, progression-free survival (PFS) of 41.2 (5.5, NR) months, and overall survival (OS) of NR (31.6, NR) months. Mutations in TP53, RB1, and ATRX were also common (48%, 24%, and 20%, respectively). Of tissue samples evaluable for IHC (N=25), responses occurred in 58.8% (10 of 17) of patients with pS6 positive tumors versus 0% (0 of 8) with pS6 negative tumors; absent pS6 staining was negatively associated with response to nab-sirolimus (P=0.008, Fisher’s exact). Median DOR for pS6 positive responders was 39.7 (5.6, NR) months. In pS6 positive vs pS6 negative patients, the median PFS was 24.4 (2.8, 53.1) vs 5.5 (2.8, NR) months, and OS was 53.1 (18.0, NR) vs 37.0 (16.6, NR) months. A variety of pathogenic inactivating alterations were observed in TSC1 and TSC2 genes, though TSC2 mutations were most commonly frameshift mutations; no recurring mutations were observed. A tumor-agnostic study (PRECISION 1: NCT05103358) is now recruiting patients with pathogenic inactivating TSC1 or TSC2 alterations to further examine these biomarker findings. Citation Format: Lee D. Cranmer, Andrew J. Wagner, Vinod Ravi, Richard F. Riedel, Kristen N. Ganjoo, Brian A. Van Tine, Rashmi Chugh, Erlinda M. Gordon, David J. Kwiatkowski, Jason L. Hornick, Heng Du, Li Ding, Anita N. Schmid, Willis H. Navarro, Loretta M. Itri, Mark A. Dickson. Biomarker analysis from AMPECT correlating response to nab-sirolimus with TSC1 and TSC2 inactivating alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB288.

(100) Phosphorylated S6 Ribosomal Protein as an Additional Marker of Antibody-Mediated Rejection in Lung Allografts: A Multicentre Study

(100) Phosphorylated S6 Ribosomal Protein as an Additional Marker of Antibody-Mediated Rejection in Lung Allografts: A Multicentre Study

Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line

BackgroundAutophagy is an important part of pathogenesis of IBD. Thiopurines such as azathioprine (AZA) are approved drugs for clinical practices in IBD patients. Besides, as an escape strategy, Toxoplasma gondii can use the mTORC1 complex to inactivate autophagy.MethodsIn this study, we investigated whether T. gondii tachyzoites may modulate autophagy and interfere the effects of azathioprine in IBD treatment. PMA-activated human monocyte cell line (THP-1) was infected with fresh T. gondii RH tachyzoites. After 5 h of infection, the cells were treated with AZA for 6 h. The expression of atg5, atg7, atg12, lc3b, and β-actin (BACT) genes was evaluated using quantitative real-time PCR. To analyze the phosphorylation of ribosomal protein S6 (rpS6), western blot using specific primary antibodies was performed.ResultsThe results of real-time PCR revealed that AZA, T. gondii tachyzoites, and a combination of AZA and T. gondii tachyzoites upregulated atg5 gene for 4.297-fold (P-value = 0.014), 2.49-fold (P-value = 0.006), and 4.76-fold (P-value = 0.001), respectively. The atg7 gene showed significant upregulation (2.272-fold; P-value = 0.014) and (1.51-fold; P-value = 0.020) in AZA and AZA / T. gondii, respectively. The expression of atg12 gene was significantly downregulated in AZA and T. gondii tachyzoites for (8.85-fold; P-value = 0.004) and (2.005-fold; P-value = 0.038), respectively, but upregulated in T. gondii/AZA (1.52-fold; P-value = 0.037). In addition, the lc3b gene was only significantly changed in AZA / T. gondii (3.028-fold; P-value = 0.001). Western blot analysis showed that T. gondii tachyzoites significantly phosphorylated rpS6, and tachyzoites did not interfere the effects of AZA to phosphorylate the rpS6.ConclusionTaken together, although AZA and T. gondii similarly affects the expression levels of atg5, atg7, and atg12, but T. gondii does not seem to modulate the effects of AZA via mTORC functions.

The Role of mTOR and Injury in Developing Salispheres.

Salispheres are the representative primitive cells of salivary glands grown in vitro in a nonadherent system. In this study, we used the ligation model for salisphere isolation after seven days of obstruction of the main excretory duct of the submandibular gland. The mammalian target of rapamycin (mTOR) is a critical signalling pathway involved in many cellular functions and is suggested to play a role in atrophy. We determined the role of mTOR and injury in the formation and development of salispheres. Morphological assessments and Western blot analysis illustrated how mTOR inhibition by rapamycin impaired the assembly of salispheres and how indirect stimulation of mTOR by lithium chloride (LiCl) assisted in the expansion of the salispheres. The use of rapamycin highlighted the necessity of mTOR for the development of salispheres as it affected the morphology and inhibited the phosphorylation of the eukaryotic translation initiation factor 4E-binding protein (4e-bp1). mTOR activity also appeared to be a crucial regulator for growing salispheres, even from the ligated gland. However, atrophy induced by ductal ligation resulted in a morphological alteration. The phosphorylation of 4e-bp1 and S6 ribosomal protein in cultured salispheres from ligated glands suggests that mTOR was not responsible for the morphological modification, but other unexplored factors were involved. This exploratory study indicates that active mTOR is essential for growing healthy salispheres. In addition, mTOR stimulation by LiCl could effectively play a role in the expansion of salispheres. The impact of atrophy on salispheres suggests a complex mechanism behind the morphological alteration, which requires further investigation.

Ultra-Low Level Somatic Mutations and Structural Variations in Focal Cortical Dysplasia Type II.

Brain somatic mutations in mTOR pathway genes are a major genetic etiology of focal cortical dysplasia type II (FCDII). Despite a greater ability to detect low-level somatic mutations in the brain by deep sequencing and analytics, about 40% of cases remain genetically unexplained. We included 2 independent cohorts consisting of 21 patients with mutation-negative FCDII without apparent mutations on conventional deep sequencing of bulk brain. To find ultra-low level somatic variants or structural variants, we isolated cells exhibiting phosphorylation of the S6 ribosomal protein (p-S6) in frozen brain tissues using fluorescence-activated cell sorting (FACS). We then performed deep whole-genome sequencing (WGS; >90×) in p-S6+ cells in a cohort of 11 patients with mutation-negative. Then, we simplified the method to whole-genome amplification and target gene sequencing of p-S6+ cells in independent cohort of 10 patients with mutation-negative followed by low-read depth WGS (10×). We found that 28.6% (6 of 21) of mutation-negative FCDII carries ultra-low level somatic mutations (less than 0.2% of variant allele frequency [VAF]) in mTOR pathway genes. Our method showed ~34 times increase of the average mutational burden in FACS mediated enrichment of p-S6+ cells (average VAF=5.84%) than in bulky brain tissues (average VAF=0.17%). We found that 19% (4 of 21) carried germline structural variations in GATOR1 complex undetectable in whole exome or targeted gene sequencing. Our method facilitates the detection of ultra-low level somatic mutations, in specifically p-S6+ cells, and germline structural variations and increases the genetic diagnostic rate up to ~80% for the entire FCDII cohort. ANN NEUROL 2023;93:1082-1093.

IL-21R signal reprogramming cooperates with CD40 and BCR signals to select and differentiate germinal center B cells.

Both B cell receptor (BCR) and CD40 signaling are rewired in germinal center (GC) B cells (GCBCs) to synergistically induce c-MYC and phosphorylated S6 ribosomal protein (p-S6), markers of positive selection. How interleukin-21 (IL-21), a key T follicular helper (TFH)-derived cytokine, affects GCBCs is unclear. Like BCR and CD40 signals, IL-21 receptor (IL-21R) plus CD40 signals also synergize to induce c-MYC and p-S6 in GCBCs. However, IL-21R plus CD40 stimulation differentially affects GCBC fate compared with BCR plus CD40 ligation-engaging unique molecular mechanisms-as revealed by bulk RNA sequencing (RNA-seq), single-cell RNA-seq, and flow cytometry of GCBCs in vitro and in vivo. Whereas both signal pairs induced BLIMP1 in some GCBCs, only the IL-21R/CD40 combination induced IRF4hi/CD138+ cells, indicative of plasma cell differentiation, along with CCR6+/CD38+ memory B cell precursors. These findings reveal a second positive selection pathway in GCBCs, document rewired IL-21R signaling in GCBCs, and link specific TFH- and Ag-derived signals to GCBC differentiation.

Cell-type-specific translational control of spatial working memory by the cap-binding protein 4EHP

The consolidation of learned information into long-lasting memories requires the strengthening of synaptic connections through de novo protein synthesis. Translation initiation factors play a cardinal role in gating the production of new proteins thereby regulating memory formation. Both positive and negative regulators of translation play a critical role in learning and memory consolidation. The eukaryotic initiation factor 4E (eIF4E) homologous protein (4EHP, encoded by the gene Eif4e2) is a pivotal negative regulator of translation but its role in learning and memory is unknown. To address this gap in knowledge, we generated excitatory (glutamatergic: CaMKIIα-positive) and inhibitory (GABAergic: GAD65-positive) conditional knockout mice for 4EHP, which were analyzed in various behavioral memory tasks. Knockout of 4EHP in Camk2a-expressing neurons (4EHP-cKOexc) did not impact long-term memory in either contextual fear conditioning or Morris water maze tasks. Similarly, long-term contextual fear memory was not altered in Gad2-directed 4EHP knockout mice (4EHP-cKOinh). However, when subjected to a short-term T-maze working memory task, both mouse models exhibited impaired cognition. We therefore tested the hypothesis that de novo protein synthesis plays a direct role in working memory. We discovered that phosphorylation of ribosomal protein S6, a measure of mTORC1 activity, is dramatically reduced in the CA1 hippocampus of 4EHP-cKOexc mice. Consistently, genetic reduction of mTORC1 activity in either excitatory or inhibitory neurons was sufficient to impair working memory. Taken together, these findings indicate that translational control by 4EHP and mTORC1 in both excitatory and inhibitory neurons are necessary for working memory.