Autophagy initiation triggers p150Glued-AP-2ß interaction on the lysosomes and facilitates their transport.

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2ß and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued-AP-2ß complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued-AP-2ß interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued-AP-2ß interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued-AP-2ß interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued-AP-2ß interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.

Monocytes prevent apoptosis of iPSCs and promote differentiation of kidney organoids.

BACKGROUND: Induced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin. METHODS: Differentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy. RESULTS: We observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles. CONCLUSION: Our data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

HMGA1 sensitizes esophageal squamous cell carcinoma to mTOR inhibitors through the ETS1-FKBP12 axis.

Esophageal carcinoma is amongst the prevalent malignancies worldwide, characterized by unclear molecular classifications and varying clinical outcomes. The PI3K/AKT/mTOR signaling, one of the frequently perturbed dysregulated pathways in human malignancies, has instigated the development of various inhibitory agents targeting this pathway, but many ESCC patients exhibit intrinsic or adaptive resistance to these inhibitors. Here, we aim to explore the reasons for the insensitivity of ESCC patients to mTOR inhibitors. We assessed the sensitivity to rapamycin in various ESCC cell lines by determining their respective IC50 values and found that cells with a low level of HMGA1 were more tolerant to rapamycin. Subsequent experiments have supported this finding. Through a transcriptome sequencing, we identified a crucial downstream effector of HMGA1, FKBP12, and found that FKBP12 was necessary for HMGA1-induced cell sensitivity to rapamycin. HMGA1 interacted with ETS1, and facilitated the transcription of FKBP12. Finally, we validated this regulatory axis in in vivo experiments, where HMGA1 deficiency in transplanted tumors rendered them resistance to rapamycin. Therefore, we speculate that mTOR inhibitor therapy for individuals exhibiting a reduced level of HMGA1 or FKBP12 may not work. Conversely, individuals exhibiting an elevated level of HMGA1 or FKBP12 are more suitable candidates for mTOR inhibitor treatment.

Uncharacterized yeast gene YBR238C, an effector of TORC1 signaling in a mitochondrial feedback loop, accelerates cellular aging via HAP4- and RMD9-dependent mechanisms.

Uncovering the regulators of cellular aging will unravel the complexity of aging biology and identify potential therapeutic interventions to delay the onset and progress of chronic, aging-related diseases. In this work, we systematically compared genesets involved in regulating the lifespan of Saccharomyces cerevisiae (a powerful model organism to study the cellular aging of humans) and those with expression changes under rapamycin treatment. Among the functionally uncharacterized genes in the overlap set, YBR238C stood out as the only one downregulated by rapamycin and with an increased chronological and replicative lifespan upon deletion. We show that YBR238C and its paralog RMD9 oppositely affect mitochondria and aging. YBR238C deletion increases the cellular lifespan by enhancing mitochondrial function. Its overexpression accelerates cellular aging via mitochondrial dysfunction. We find that the phenotypic effect of YBR238C is largely explained by HAP4- and RMD9-dependent mechanisms. Furthermore, we find that genetic- or chemical-based induction of mitochondrial dysfunction increases TORC1 (Target of Rapamycin Complex 1) activity that, subsequently, accelerates cellular aging. Notably, TORC1 inhibition by rapamycin (or deletion of YBR238C) improves the shortened lifespan under these mitochondrial dysfunction conditions in yeast and human cells. The growth of mutant cells (a proxy of TORC1 activity) with enhanced mitochondrial function is sensitive to rapamycin whereas the growth of defective mitochondrial mutants is largely resistant to rapamycin compared to wild type. Our findings demonstrate a feedback loop between TORC1 and mitochondria (the TORC1-MItochondria-TORC1 (TOMITO) signaling process) that regulates cellular aging processes. Hereby, YBR238C is an effector of TORC1 modulating mitochondrial function.

Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans.

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.

Ultrathin-strut versus thin-strut stent healing and outcomes in preclinical and clinical subjects.

BACKGROUND: Compared with thin-strut durable-polymer drug-eluting stents (DP-DES), ultrathin-strut biodegradable-polymer sirolimus-eluting stents (BP-SES) improve stent-related clinical outcomes in patients undergoing percutaneous coronary intervention (PCI). Reduced stent strut thickness is hypothesised to underlie these benefits, but this conjecture remains unproven. AIMS: We aimed to assess the impact of strut thickness on stent healing and clinical outcomes between ultrathin-strut and thin-strut BP-SES. METHODS: First, we performed a preclinical study of 8 rabbits implanted with non-overlapping thin-strut (diameter/thickness 3.5 mm/80 µm) and ultrathin-strut (diameter/thickness 3.0 mm/60 µm) BP-SES in the infrarenal aorta. On day 7, the rabbits underwent intravascular near-infrared fluorescence optical coherence tomography (NIRF-OCT) molecular-structural imaging of fibrin deposition and stent tissue coverage, followed by histopathological analysis. Second, we conducted an individual data pooled analysis of patients enrolled in the BIOSCIENCE and BIOSTEMI randomised PCI trials treated with ultrathin-strut (n=282) or thin-strut (n=222) BP-SES. The primary endpoint was target lesion failure (TLF) at 1-year follow-up, with a landmark analysis at 30 days. RESULTS: NIRF-OCT image analyses revealed that ultrathin-strut and thin-strut BP-SES exhibited similar stent fibrin deposition (p=0.49) and percentage of uncovered stent struts (p=0.63). Histopathological assessments corroÂborated these findings. In 504 pooled randomised trial patients, TLF rates were similar for those treated with ultrathin-strut or thin-strut BP-SES at 30-day (2.5% vs 1.8%; p=0.62) and 1-year follow-up (4.3% vs 4.7%; p=0.88). CONCLUSIONS: Ultrathin-strut and thin-strut BP-SES demonstrate similar early arterial healing profiles and 30-day and 1-year clinical outcomes.

A role of ROS-dependent defects in mitochondrial dynamic and autophagy in carbon black nanoparticle-mediated myocardial cell damage.

Carbon black nanoparticles (CBNPs) are widely distributed in the environment and are increasingly recognized as a contributor in the development of cardiovascular disease. A variety of cardiac injuries and diseases result from structural and functional damage to cardiomyocytes. This study explored the mechanisms of CBNPs-mediated myocardial toxicity. CBNPs were given to mice through intra-tracheal instillation and it was demonstrated that the particles can be taken up into the cardiac tissue. Exposure to CBNPs induced cardiomyocyte inflammation and apoptosis. In combination with in vitro experiments, we showed that CBNPs increased the ROS and induced mitochondria fragmentation. Functionally, CBNPs-exposed cardiomyocyte exhibited depolarization of the mitochondrial membrane potential, release of cytochrome c, and activation of pro-apoptotic BAX, thereby initiating programmed cell death. On the other hand, CBNPs impaired autophagy, leading to the inadequate removal of dysfunctional mitochondria. The excess accumulation of damaged mitochondria further stimulated NF-κB activation and triggered the NLRP3 inflammasome pathway. Both the antioxidant N-acetylcysteine and the autophagy activator rapamycin were effective to attenuate the damage of CBNPs on cardiomyocytes. Taken together, this study elucidated the potential mechanism underlying CBNPs-induced myocardial injury and provided a scientific reference for the evaluation and prevention of the CBNPs-related heart risk.

Anti-aging effects of medicinal plants and their rapid screening using the nematode Caenorhabditis elegans.

BACKGROUND: Aging is the primary risk factor of most chronic diseases in humans, including cardiovascular diseases, osteoporosis and neurodegenerative diseases, which extensively damage the quality of life for elderly individuals. Aging is a multifaceted process with numerous factors affecting it. Efficient model organisms are essential for the research and development of anti-aging agents, particularly when investigating pharmacological mechanisms are needed. PURPOSE: This review discusses the application of Caenorhabditis elegans for studying aging and its related signaling pathways, and presents an overview of studies exploring the mechanism and screening of anti-aging agents in C. elegans. Additionally, the review summarizes related clinical trials of anti-aging agents to inspire the development of new medications. METHOD: Literature was searched, analyzed, and collected using PubMed, Web of Science, and Science Direct. The search terms used were "anti-aging", "medicinal plants", "synthetic compounds", "C. elegans", "signal pathway", etc. Several combinations of these keywords were used. Studies conducted in C. elegans or humans were included. Articles were excluded, if they were on studies conducted in silico or in vitro or could not offer effective data. RESULTS: Four compounds mainly derived through synthesis (metformin, rapamycin, nicotinamide mononucleotide, alpha-ketoglutarate) and four active ingredients chiefly obtained from plants (resveratrol, quercetin, Astragalus polysaccharide, ginsenosides) are introduced emphatically. These compounds and active ingredients exhibit potential anti-aging effects in preclinical and clinical studies. The screening of these anti-aging agents and the investigation of their pharmacological mechanisms can benefit from the use of C. elegans. CONCLUSION: Medicinal plants provide valuable resource for the treatment of diseases. A wide source of raw materials for the particular plant medicinal compounds having anti-aging effects meet diverse pharmaceutical requirements, such as immunomodulatory, anti-inflammation and alleviating oxidative stress. C. elegans possesses advantages in scientific research including short life cycle, small size, easy maintenance, genetic tractability and conserved biological processes related to aging. C. elegans can be used for the efficient and rapid evaluation of compounds with the potential to slow down aging.

Rescue of impaired blood-brain barrier in tuberous sclerosis complex patient derived neurovascular unit.

BACKGROUND: Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene and dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood-brain barrier (BBB). METHODS: We generated TSC disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies. RESULTS: Using microphysiological systems, we demonstrate that a BBB generated from TSC2 heterozygous mutant cells shows increased permeability. This can be rescued by wild type astrocytes or by treatment with rapamycin, an mTOR kinase inhibitor. CONCLUSION: Our results demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of cell lineages contributing to TSC pathogenesis and informs future therapeutics.

Pharmacological inhibition of mTORC1 reduces neural death and damage volume after MCAO by modulating microglial reactivity.

Ischemic stroke is a sudden and acute disease characterized by neuronal death, increment of reactive gliosis (reactive microglia and astrocytes), and a severe inflammatory process. Neuroinflammation is an early event after cerebral ischemia, with microglia playing a leading role. Reactive microglia involve functional and morphological changes that drive a wide variety of phenotypes. In this context, deciphering the molecular mechanisms underlying such reactive microglial is essential to devise strategies to protect neurons and maintain certain brain functions affected by early neuroinflammation after ischemia. Here, we studied the role of mammalian target of rapamycin (mTOR) activity in the microglial response using a murine model of cerebral ischemia in the acute phase. We also determined the therapeutic relevance of the pharmacological administration of rapamycin, a mTOR inhibitor, before and after ischemic injury. Our data show that rapamycin, administered before or after brain ischemia induction, reduced the volume of brain damage and neuronal loss by attenuating the microglial response. Therefore, our findings indicate that the pharmacological inhibition of mTORC1 in the acute phase of ischemia may provide an alternative strategy to reduce neuronal damage through attenuation of the associated neuroinflammation.

Activation of M2 macrophage autophagy by rapamycin increases the radiosensitivity of colorectal cancer xenografts.

BACKGROUND: Tumor-associated macrophages (TAMs) are intimately involved in cancer radiochemotherapy resistance. However, the mechanism by which macrophages affect radiosensitivity through autophagy remains unclear. The purpose of our study was to investigate how activating autophagy in type-II macrophages (M2) by using rapamycin (RAP) would affect the radiosensitivity of colorectal cancer (CRC) xenografts. MATERIALS AND METHODS: A nude mouse CRC model was established by injecting LoVo CRC cells. After tumor formation, supernatant from M2 cells (autophagy-unactivated), autophagy-activated M2 cells, or autophagy-downregulated M2 cells was injected peritumorally. All tumor-bearing mice were irradiated with 8-Gy X-rays twice, and the radiosensitivity of CRC xenografts was analyzed in each group. RESULTS: The mass, volume, and microvessel density (MVD) of tumors in the autophagy-unactivated M2 group significantly increased; however, supernatant from M2 cells that were autophagy-activated by rapamycin significantly decreased tumor weight, volume, and MVD compared with negative control. Combining bafilomycin A1 (BAF-A1) with RAP treatment restored the ability of the M2 supernatant to increase tumor mass, volume, and MVD. Immunohistochemical and Western blot results showed that compared with the negative control group, supernatant from M2 cells that were not activated by autophagy downregulated the expression of Livin and Survivin in tumor tissues; activation of M2 autophagy further downregulated the protein levels. CONCLUSIONS: Therefore, autophagy-activated M2 supernatant can downregulate the expression of the antiapoptotic genes Livin and Survivin in CRC xenografts, improving the radiosensitivity of CRC by inducing apoptosis in combination with radiotherapy and inhibiting the growth of transplanted tumors.

Sulfated Polysaccharide-Based Nanocarrier Drives Microenvironment-Mediated Cerebral Neurovascular Remodeling for Ischemic Stroke Treatment.

Stroke is a leading cause of global mortality and severe disability. However, current strategies used for treating ischemic stroke lack specific targeting capabilities, exhibit poor immune escape ability, and have limited drug release control. Herein, we developed an ROS-responsive nanocarrier for targeted delivery of the neuroprotective agent rapamycin (RAPA) to mitigate ischemic brain damage. The nanocarrier consisted of a sulfated chitosan (SCS) polymer core modified with a ROS-responsive boronic ester enveloped by a red blood cell membrane shell incorporating a stroke homing peptide. When encountering high levels of intracellular ROS in ischemic brain tissues, the release of SCS combined with RAPA from nanoparticle disintegration facilitates effective microglia polarization and, in turn, maintains blood-brain barrier integrity, reduces cerebral infarction, and promotes cerebral neurovascular remodeling in a mouse stroke model involving transient middle cerebral artery occlusion (tMCAO). This work offers a promising strategy to treat ischemic stroke therapy.

Impact of glucocorticoids and rapamycin on autophagy in Candida glabrata-infected macrophages from BALB/c mice.

Objective: In the defense against microorganisms like Candida albicans, macrophages recruit LC3(Microtubule-associated protein 1A/1B-light chain 3) to the periplasm, engaging in the elimination process through the formation of a single-membrane phagosome known as LC3-associated phagocytosis (LAP). Building on this, we propose the hypothesis that glucocorticoids may hinder macrophage phagocytosis of Candida glabrata by suppressing LAP, and rapamycin could potentially reverse this inhibitory effect. Methods: RAW264.7 cells were employed for investigating the immune response to Candida glabrata infection. Various reagents, including dexamethasone, rapamycin, and specific antibodies, were utilized in experimental setups. Assays, such as fluorescence microscopy, flow cytometry, ELISA (Enzyme-Linked Immunosorbent Assay), Western blot, and confocal microscopy, were conducted to assess phagocytosis, cytokine levels, protein expression, viability, and autophagy dynamics. Results: Glucocorticoids significantly inhibited macrophage autophagy, impairing the cells' ability to combat Candida glabrata. Conversely, rapamycin exhibited a dual role, initially inhibiting and subsequently promoting phagocytosis of Candida glabrata by macrophages. Glucocorticoids hinder macrophage autophagy in Candida glabrata infection by suppressing the MTOR pathway(mammalian target of rapamycin pathway), while the activation of MTOR pathway by Candida glabrata diminishes over time. Conclusion: Our study elucidates the intricate interplay between glucocorticoids, rapamycin, and macrophage autophagy during Candida glabrata infection. Understanding the implications of these interactions not only sheds light on the host immune response dynamics but also unveils potential therapeutic avenues for managing fungal infections.

Inhibition of p70 Ribosomal S6 Kinase (S6K1) Reduces Cortical Blood Flow in a Rat Model of Autism-Tuberous Sclerosis.

The manifestations of tuberous sclerosis complex (TSC) in humans include epilepsy, autism spectrum disorders (ASD) and intellectual disability. Previous studies suggested the linkage of TSC to altered cerebral blood flow and metabolic dysfunction. We previously reported a significant elevation in cerebral blood flow in an animal model of TSC and autism of young Eker rats. Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin could restore normal oxygen consumption and cerebral blood flow. In this study, we investigated whether inhibiting a component of the mTOR signaling pathway, p70 ribosomal S6 kinase (S6K1), would yield comparable effects. Control Long Evans and Eker rats were divided into vehicle and PF-4708671 (S6K1 inhibitor, 75 mg/kg for 1 h) treated groups. Cerebral regional blood flow (14C-iodoantipyrine) was determined in isoflurane anesthetized rats. We found significantly increased basal cortical (+ 32%) and hippocampal (+ 15%) blood flow in the Eker rats. PF-4708671 significantly lowered regional blood flow in the cortex and hippocampus of the Eker rats. PF-4708671 did not significantly lower blood flow in these regions in the control Long Evans rats. Phosphorylation of S6-Ser240/244 and Akt-Ser473 was moderately decreased in Eker rats but only the latter reached statistical significance upon PF-4708671 treatment. Our findings suggest that moderate inhibition of S6K1 with PF-4708671 helps to restore normal cortical blood flow in Eker rats and that this information might have therapeutic potential in tuberous sclerosis complex and autism.

Acute physical exercise prevents memory amnesia caused by protein synthesis inhibition in rats' hippocampus.

The benefits of physical exercise (PE) on memory consolidation have been well-documented in both healthy and memory-impaired animals. However, the underlying mechanisms through which PE exerts these effects are still unclear. In this study, we aimed to investigate the role of hippocampal protein synthesis in memory modulation by acute PE in rats. After novel object recognition (NOR) training, rats were subjected to a 30-min moderate-intensity acute PE on the treadmill, while control animals did not undergo any procedures. Using anisomycin (ANI) and rapamycin (RAPA), compounds that inhibit protein synthesis through different mechanisms, we manipulated protein synthesis in the CA1 region of the hippocampus to examine its contribution to memory consolidation. Memory was assessed on days 1, 7, and 14 post-training. Our results showed that inhibiting protein synthesis by ANI or RAPA impaired NOR memory consolidation in control animals. However, acute PE prevented this impairment without affecting memory persistence. We also evaluated brain-derived neurotrophic factor (BDNF) levels after acute PE at 0.5h, 2h, and 12h afterward and found no differences in levels compared to animals that did not engage in acute PE or were only habituated to the treadmill. Therefore, our findings suggest that acute PE could serve as a non-pharmacological intervention to enhance memory consolidation and prevent memory loss in conditions associated with hippocampal protein synthesis inhibition. This mechanism appears not to depend on BDNF synthesis in the early hours after exercise.

Rapamycin improves the survival of epilepsy model cells by blocking phosphorylation of mTOR base on computer simulations and cellular experiments.

PURPOSE: Epilepsy is a chronic brain dysfunction characterized by recurrent epileptic seizures. Rapamycin is a naturally occurring macrolide from Streptomyces hygroscopicus, and rapamycin may provide a protective effect on the nervous system by affecting mTOR. Therefore, we investigated the pharmacologic mechanism of rapamycin treating epilepsy through bioinformatics analysis, cellular experiments and supercomputer simulation. METHODS: Bioinformatics analysis was used to analyze targets of rapamycin treating epilepsy. We established epilepsy cell model by HT22 cells. RT-qPCR, WB and IF were used to verify the effects of rapamycin on mTOR at gene level and protein level. Computer simulations were used to model and evaluate the stability of rapamycin binding to mTOR protein. RESULTS: Bioinformatics indicated mTOR played an essential role in signaling pathways of cell growth and cell metabolism. Cellular experiments showed that rapamycin could promote cell survival, and rapamycin did not have an effect on mRNA expression of mTOR. However, rapamycin was able to significantly inhibit the phosphorylation of mTOR at protein level. Computer simulations indicated that rapamycin was involved in the treatment of epilepsy through regulating phosphorylation of mTOR at protein level. CONCLUSION: We found that rapamycin was capable of promoting the survival of epilepsy cells by inhibiting the phosphorylation of mTOR at protein level, and rapamycin did not have an effect on mRNA expression of mTOR. In addition to the traditional study that rapamycin affects mTORC1 complex by acting on FKBP12, this study found rapamycin could also directly block the phosphorylation of mTOR, therefore affecting the assembly of mTORC1 complex and mTOR signaling pathway.

Infectious bronchitis virus (IBV) triggers autophagy to enhance viral replication by activating the VPS34 complex.

Autophagy plays an important role in the lifecycle of viruses. However, there is currently a lack of systematic research on the relationship between Infectious Bronchitis Virus (IBV) and autophagy. This study aims to investigate the impact of IBV on autophagy and the role of autophagy in viral replication. We observed that IBV infection increased the expression of microtubule-associated protein 1 light chain 3, a marker of autophagy, decreased the expression of sequestosome 1, and led to elevated intracellular LC3 puncta levels. These findings suggest that IBV infection activates the autophagic process in cells. To investigate the impact of autophagy on the replication of IBV, we utilized rapamycin as an autophagy activator and 3-methyladenine as an autophagy inhibitor. Our results indicate that IBV promotes viral replication by inducing autophagy. Further investigation revealed that IBV induces autophagosome formation by inhibiting the mTOR-ULK1 pathway and activating the activity of vacuolar protein sorting 34 (VPS34), autophagy-related gene 14, and the Beclin-1 complex. VPS34 plays a crucial role in this process, as inhibiting VPS34 protein activity enhances cell proliferation after IBV infection. Additionally, inhibiting VPS34 significantly improves the survival rate of IBV-infected chicks, suppresses IBV replication in the kidney, and alleviates tracheal, lung, and kidney damage caused by IBV infection. In summary, IBV infection can induce autophagy by modulating the mTOR/ULK1 signaling pathway and activating the VPS34 complex, while autophagy serves to promote virus replication.

Acquired NF2 mutation confers resistance to TRK inhibition in an ex vivo LMNA::NTRK1-rearranged soft-tissue sarcoma cell model.

Genomic rearrangements of the neurotrophic receptor tyrosine kinase genes (NTRK1, NTRK2, and NTRK3) are the most common mechanism of oncogenic activation for this family of receptors, resulting in sustained cancer cell proliferation. Several targeted therapies have been approved for tumours harbouring NTRK fusions and a new generation of TRK inhibitors has already been developed due to acquired resistance. We established a patient-derived LMNA::NTRK1-rearranged soft-tissue sarcoma cell model ex vivo with an acquired resistance to targeted TRK inhibition. Molecular profiling of the resistant clones revealed an acquired NF2 loss of function mutation that was absent in the parental cell model. Parental cells showed continuous sensitivity to TRK-targeted treatment, whereas the resistant clones were insensitive. Furthermore, resistant clones showed upregulation of the MAPK and mTOR/AKT pathways in the gene expression based on RNA sequencing data and increased sensitivity to MEK and mTOR inhibitor therapy. Drug synergy was seen using trametinib and rapamycin in combination with entrectinib. Medium-throughput drug screening further identified small compounds as potential drug candidates to overcome resistance as monotherapy or in combination with entrectinib. In summary, we developed a comprehensive model of drug resistance in an LMNA::NTRK1-rearranged soft-tissue sarcoma and have broadened the understanding of acquired drug resistance to targeted TRK therapy. Furthermore, we identified drug combinations and small compounds to overcome acquired drug resistance and potentially guide patient care in a functional precision oncology setting. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

In Vitro Profiling of Commonly Used Post-transplant Immunosuppressants Reveals Distinct Impact on Antiviral T-cell Immunity Towards CMV.

Infectious complications, including widespread human cytomegalovirus (CMV) disease, frequently occur after hematopoietic stem cell and solid organ transplantation due to immunosuppressive treatment causing impairment of T-cell immunity. Therefore, in-depth analysis of the impact of immunosuppressants on antiviral T cells is needed. We analyzed the impact of mTOR inhibitors sirolimus (SIR/S) and everolimus (EVR/E), calcineurin inhibitor tacrolimus (TAC/T), purine synthesis inhibitor mycophenolic acid (MPA/M), glucocorticoid prednisolone (PRE/P) and common double (T+S/E/M/P) and triple (T+S/E/M+P) combinations on antiviral T-cell functionality. T-cell activation and effector molecule production upon antigenic stimulation was impaired in presence of T+P and triple combinations. SIR, EVR and MPA exclusively inhibited T-cell proliferation, TAC inhibited activation and cytokine production and PRE inhibited various aspects of T-cell functionality including cytotoxicity. This was reflected in an in vitro infection model, where elimination of CMV-infected human fibroblasts by CMV-specific T cells was reduced in presence of PRE and all triple combinations. CMV-specific memory T cells were inhibited by TAC and PRE, which was also reflected with double (T+P) and triple combinations. EBV- and SARS-CoV-2-specific T cells were similarly affected. These results highlight the need to optimize immune monitoring to identify patients who may benefit from individually tailored immunosuppression.

Effect and Mechanism of Rapamycin on Cognitive Deficits in Animal Models of Alzheimer's Disease: A Systematic Review and Meta-analysis of Preclinical Studies.

Background: Alzheimer's disease (AD), the most common form of dementia, remains long-term and challenging to diagnose. Furthermore, there is currently no medication to completely cure AD patients. Rapamycin has been clinically demonstrated to postpone the aging process in mice and improve learning and memory abilities in animal models of AD. Therefore, rapamycin has the potential to be significant in the discovery and development of drugs for AD patients. Objective: The main objective of this systematic review and meta-analysis was to investigate the effects and mechanisms of rapamycin on animal models of AD by examining behavioral indicators and pathological features. Methods: Six databases were searched and 4,277 articles were retrieved. In conclusion, 13 studies were included according to predefined criteria. Three authors independently judged the selected literature and methodological quality. Use of subgroup analyses to explore potential mechanistic effects of rapamycin interventions: animal models of AD, specific types of transgenic animal models, dosage, and periodicity of administration. Results: The results of Morris Water Maze (MWM) behavioral test showed that escape latency was shortened by 15.60 seconds with rapamycin therapy, indicating that learning ability was enhanced in AD mice; and the number of traversed platforms was increased by 1.53 times, indicating that the improved memory ability significantly corrected the memory deficits. CONCLUSIONS: Rapamycin therapy reduced age-related plaque deposition by decreasing AßPP production and down-regulating ß-secretase and γ-secretase activities, furthermore increased amyloid-ß clearance by promoting autophagy, as well as reduced tau hyperphosphorylation by up-regulating insulin-degrading enzyme levels.