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Environmental factors are important regulators of cell growth and proliferation. Mechanistic target of rapamycin (mTOR) is a central kinase that maintains cellular homeostasis in response to a variety of extracellular and intracellular inputs. Dysregulation of mTOR signaling is associated with many diseases, including diabetes and cancer. Calcium ion (Ca[sup.2+]) is important as a second messenger in various biological processes, and its intracellular concentration is tightly regulated. Although the involvement of Ca[sup.2+] mobilization in mTOR signaling has been reported, the detailed molecular mechanisms by which mTOR signaling is regulated are not fully understood. The link between Ca[sup.2+] homeostasis and mTOR activation in pathological hypertrophy has heightened the importance in understanding Ca[sup.2+]-regulated mTOR signaling as a key mechanism of mTOR regulation. In this review, we introduce recent findings on the molecular mechanisms of regulation of mTOR signaling by Ca[sup.2+]-binding proteins, particularly calmodulin (CaM).

Safe and efficacious systemic delivery of messenger RNA (mRNA) to specific organs and cells in vivo remains the major challenge in the development of mRNA-based therapeutics. Targeting of systemically administered lipid nanoparticles (LNPs) coformulated with mRNA has largely been confined to the liver and spleen. Using a library screening approach, we identified that N-series LNPs (containing an amide bond in the tail) are capable of selectively delivering mRNA to the mouse lung, in contrast to our previous discovery that O-series LNPs (containing an ester bond in the tail) that tend to deliver mRNA to the liver. We analyzed the protein corona on the liver- and lung-targeted LNPs using liquid chromatography–mass spectrometry and identified a group of unique plasma proteins specifically absorbed onto the surface that may contribute to the targetability of these LNPs. Different pulmonary cell types can also be targeted by simply tuning the headgroup structure of N-series LNPs. Importantly, we demonstrate here the success of LNP-based RNA therapy in a preclinical model of lymphangioleiomyomatosis (LAM), a destructive lung disease caused by loss-of-function mutations in the Tsc2 gene. Our lung-targeting LNP exhibited highly efficient delivery of the mouse tuberous sclerosis complex 2 (Tsc2) mRNA for the restoration of TSC2 tumor suppressor in tumor and achieved remarkable therapeutic effect in reducing tumor burden. This research establishes mRNA LNPs as a promising therapeutic intervention for the treatment of LAM.

The autosomal dominant disorder tuberous sclerosis complex (TSC) is characterized by an array of manifestations both within and outside of the central nervous system (CNS), including hamartomas and other malformations. TSC is caused by mutations in the TSC1 or TSC2 gene resulting in activation of the mechanistic target of rapamycin (mTOR) signaling pathway. Study of TSC has shed light on the critical role of the mTOR pathway in neurodevelopment. This update reviews the genetic basis of TSC, its cardinal phenotypic CNS features, and recent developments in the field of TSC and other mTOR-altered disorders.

Despite advances in molecular characterization, glioblastoma (GBM) remains the most common and lethal brain tumour with high mortality rates in both paediatric and adult patients. The signal transducer and activator of transcription 3 (STAT3) is an important oncogenic driver of GBM. Although STAT3 reportedly plays a role in autophagy of some cells, its role in cancer cell autophagy remains unclear. In this study, we found Serine‐727 and Tyrosine‐705 phosphorylation of STAT3 was constitutive in GBM cell lines. Tyrosine phosphorylation of STAT3 in GBM cells suppresses autophagy, whereas knockout (KO) of STAT3 increases ULK1 gene expression, increases TSC2‐AMPKα‐ULK1 signalling, and increases lysosomal Cathepsin D processing, leading to the stimulation of autophagy. Rescue of STAT3‐KO cells by the enforced expression of wild‐type (WT) STAT3 reverses these pathways and inhibits autophagy. Conversely, expression of Y705F‐ and S727A‐STAT3 phosphorylation deficient mutants in STAT3‐KO cells did not suppress autophagy. Inhibition of ULK1 activity (by treatment with MRT68921) or its expression (by siRNA knockdown) in STAT3‐KO cells inhibits autophagy and sensitizes cells to apoptosis. Taken together, our findings suggest that serine and tyrosine phosphorylation of STAT3 play critical roles in STAT3‐dependent autophagy in GBM, and thus are potential targets to treat GBM.

[This corrects the article DOI: 10.1371/journal.pone.0226400.].

Background Disease severity is tremendously variable in tuberous sclerosis complex (TSC). In contrast with the detailed guidelines available for TSC diagnosis and management, clinical practice lacks adequate tools to evaluate the prognosis, especially in the case of in utero diagnosis. In addition, the correlation between genotypes and phenotypes remains a challenge, in part due to the large number of mutations linked to TSC. In this report, we describe a case of severe TSC diagnosed in utero and associated with a specific mutation in the gene tuberous sclerosis complex 2 ( TSC2 ). Case presentation A mother was referred for a thorough investigation following the observation by ultrasound of cardiac abnormalities in her fetus. The mother was healthy and reported frequent, intense and long-lasting hiccups/spasms in the fetus. The fetus of gestational age 33 weeks and 4 days was found to have multiple cardiac tumors with cardiac ultrasound. Brain magnetic resonance imaging (MRI) performed in utero revealed the presence of sub-ependymal nodules and of abnormal signals disseminated in the white matter, in the cerebral cortex and in the cerebellum. Following diagnosis of definite TSC, pregnancy interruption was chosen by the parents. Genetic testing of the fetus exposed a duplication in exon 41 of TSC2 (c.5169dupA), which was absent in the parents. The autopsy ascertained the high severity of brain damage characterized by an extensive disorganisation of white and grey matter in most cerebral lobes. Conclusions This case presentation is the first to depict the association between a de novo TSC2 c.5169dupA and multi-organ manifestation together with indications of a particularly high disease severity. This report can help physicians to perform early clinical diagnosis of TSC and to evaluate the prognosis.

Background: Adult T-cell leukemia/lymphoma (ATLL) is a hematologic malignancy associated with human T-cell lymphotropic virus type 1 (HTLV-1) infection. Gene expression changes play a role in its pathogenesis. Objectives:  In this study, we examined the gene expression profiles of alpha-actinin-4 (ACTN4), Tuberous Sclerosis Complex 2 (TSC2), C-X-C chemokine receptor type 4 (CXCR4), and Activating Transcription Factor 1 (ATF1) in Iranian ATLL patients for the first time, contrasting the findings with those from healthy controls. Methods: This case-control study (2023 - 2024) included 20 male Iranian participants (10 ATLL patients and 10 healthy controls). From each participant, 6 ml of whole blood was collected. Samples from eligible participants were screened for HTLV-1 infection using enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). RNA was extracted and complementary DNA (cDNA) synthesis was performed. The expression of ACTN4, TSC2, CXCR4, ATF1, and viral HBZ genes was measured by Real-time PCR, using RPLP0 as the reference gene. Results: Expression of TSC2 and ACTN4 genes was significantly decreased in ATLL patients compared to healthy controls (TSC2: mean ± SD: 0.00003 ± 0.00004 vs. 0.00006 ± 0.00004, P < 0.05; ACTN4: 0.0129 ± 0.024 vs. 0.0207 ± 0.009, P < 0.05); CXCR4 and ATF1 expression levels were slightly increased but not significantly different between groups (CXCR4: 0.147 ± 0.154 vs. 0.139 ± 0.09, P > 0.05; ATF1: 0.0028 ± 0.0029 vs. 0.0015 ± 0.0009, P > 0.05). Conclusions: Dysregulation of target genes in ATLL patients suggests HTLV-1 involvement in disease progression by modulating host cellular pathways. Although CXCR4 and ATF1 showed non-significant upward trends, the observed downregulation of TSC2 and ACTN4 warrants further investigation in a larger sample size to clarify their potential roles in ATLL pathogenesis.

Purpose To perform comprehensive genotyping of TSC1 and TSC2 in a cohort of 94 infants with tuberous sclerosis complex (TSC) and correlate with clinical manifestations. Methods Infants were enrolled at age <4 months, and subject to intensive clinical monitoring including electroencephalography (EEG), brain magnetic resonance imaging (MRI), and neuropsychological assessment. Targeted massively parallel sequencing (MPS), genome sequencing, and multiplex ligation-dependent probe amplification (MLPA) were used for variant detection in TSC1 / TSC2 . Results Pathogenic variants in TSC1 or TSC2 were identified in 93 of 94 (99%) subjects, with 23 in TSC1 and 70 in TSC2 . Nine (10%) subjects had mosaicism. Eight of 24 clinical features assessed at age 2 years were significantly less frequent in those with TSC1 versus TSC2 variants including cortical tubers, hypomelanotic macules, facial angiofibroma, renal cysts, drug-resistant epilepsy, developmental delay, subependymal giant cell astrocytoma, and median seizure-free survival. Additionally, quantitative brain MRI analysis showed a marked difference in tuber and subependymal nodule/giant cell astrocytoma volume for TSC1 versus TSC2 . Conclusion TSC2 pathogenic variants are associated with a more severe clinical phenotype than mosaic TSC2 or TSC1 variants in TSC infants. Early assessment of gene variant status and mosaicism might have benefit for clinical management in infants and young children with TSC.

Background Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that is associated with neurological symptoms, including autism spectrum disorder. Tuberous sclerosis complex is caused by pathogenic germline mutations of either the TSC1 or TSC2 gene, but somatic mutations were identified in both genes, and the combined effects of TSC1 and TSC2 mutations have been unknown. Methods The present study investigated social behaviors by the social interaction test and three-chambered sociability tests, effects of rapamycin treatment, and gene expression profiles with a gene expression microarray in Tsc1 and Tsc2 double heterozygous mutant ( TscD +/− ) mice. Results TscD +/− mice exhibited impairments in social behaviors, and the severity of impairments was similar to Tsc2 +/− mice rather than Tsc1 +/− mice. Impairments in social behaviors were rescued by rapamycin treatment in all mutant mice. Gene expression profiles in the brain were greatly altered in TscD +/− mice more than in Tsc1 +/− and Tsc2 +/− mice. The gene expression changes compared with wild type (WT) mice were similar between TscD +/− and Tsc2 +/− mice, and the overlapping genes whose expression was altered in mutant mice compared with WT mice were enriched in the neoplasm- and inflammation-related canonical pathways. The “signal transducer and activator of transcription 3, interferon regulatory factor 1, interferon regulatory factor 4, interleukin-2R α chain, and interferon-γ” signaling pathway, which is initiated from signal transducer and activator of transcription 4 and PDZ and LIM domain protein 2, was associated with impairments in social behaviors in all mutant mice. Limitations It is unclear whether the signaling pathway also plays a critical role in autism spectrum disorders not caused by Tsc1 and Tsc2 mutations. Conclusions These findings suggest that TSC1 and TSC2 double mutations cause autistic behaviors similarly to TSC2 mutations, although significant changes in gene expression were attributable to the double mutations. These findings contribute to the knowledge of genotype–phenotype correlations in TSC and suggest that mutations in both the TSC1 and TSC2 genes act in concert to cause neurological symptoms, including autism spectrum disorder.

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