bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2023‒03‒19
fifty-one papers selected by
Viktor Korolchuk
Newcastle University

  1. J Cell Biol. 2023 Apr 03. pii: e202302118. [Epub ahead of print]222(4):
      When mitochondrial damage threatens to disrupt cell and tissue homeostasis, selective autophagy (mitophagy) provides an important route to neutralize dysfunctional organelles. Whilst we understand much about stress-induced mitophagy, steady-state and spatial mechanisms remain elusive. In this issue, Gok et al. (2023. J. Cell Biol. reveal an unexpected role for TMEM11 in mitophagy regulation.
  2. Autophagy. 2023 Mar 16.
      The critical intracellular pattern recognition receptor NLRP3 senses pathogenic organisms and endogenous danger signals via forming inflammasomes to orchestrate innate immune responses. Dysfunction of NLRP3 inflammasomes is implicated in several inflammatory disorders. Hence, it is important to uncover the mechanisms preventing sustained NLRP3 inflammasome activation. Recently, we revealed that ZDHHC12-mediated palmitoylation enhances NLRP3 degradation through the chaperone-mediated autophagy pathway, and identified gain-of-function variants of NLRP3 in autoinflammatory disease, which induce excessive NLRP3 inflammasome activation through decreased palmitoylation level and impaired chaperone-mediated autophagic degradation.
    Keywords:  NLRP3; chaperone-mediated autophagy; inflammasome; inflammation; palmitoylation
  3. Biochem Soc Trans. 2023 Mar 16. pii: BST20210038. [Epub ahead of print]
      The Rag GTPases are an evolutionarily conserved family that play a crucial role in amino acid sensing by the mammalian target of rapamycin complex 1 (mTORC1). mTORC1 is often referred to as the master regulator of cell growth. mTORC1 hyperactivation is observed in multiple diseases such as cancer, obesity, metabolic disorders, and neurodegeneration. The Rag GTPases sense amino acid levels and form heterodimers, where RagA or RagB binds to RagC or RagD, to recruit mTORC1 to the lysosome where it becomes activated. Here, we review amino acid signaling to mTORC1 through the Rag GTPases.
    Keywords:  Rag GTPases; amino acid sensing; mTORC1; signaling
  4. Vet Res. 2023 Mar 14. 54(1): 22
      Orf virus (ORFV) is the causative agent of contagious ecthyma, which is an important zoonotic pathogen with a widespread distribution affecting sheep, goats and humans. Our previous research showed that autophagy can be induced in host cells by ORFV infection. However, the exact mechanism of ORFV-induced autophagy remains unknown. In this study, we investigated the underlying mechanisms of autophagy induced by ORFV in OFTu cells and the impact of autophagy on ORFV replication. By using specific autophagy inhibitors and activators, Western blotting, immunofluorescence and transmission electron microscopy imaging, we confirmed that ORFV infection triggered intracellular autophagosome accumulation and the activation of autophagic flux. Moreover, ORFV-induced autophagic activity was found to rely on an increase in the phosphorylation of tuberous sclerosis complex 2 (TSC2) and a decrease in the phosphorylation of mammalian target of rapamycin (mTOR), which is mediated by the suppression of the PI3K/AKT/mTOR signalling pathway and activation of the ERK1/2/mTOR signalling pathway. Furthermore, we investigated the role of mTOR-mediated autophagy during ORFV replication using pharmacological agents and demonstrated that ORFV-induced autophagy correlated positively with viral replication. Taken together, our data reveal the pathways of ORFV-induced autophagy and the impact of autophagy on ORFV replication, providing new insights into ORFV pathogenesis.
    Keywords:  ERK1/2/mTOR; ORFV; PI3K/AKT/mTOR; autophagy; replication
  5. Am J Pathol. 2023 Mar 09. pii: S0002-9440(23)00084-6. [Epub ahead of print]
      SQSTM1/p62 (hereafter referred to as p62) is an autophagy receptor protein for selective autophagy primarily due to its direct interaction with the microtubule light chain 3 (LC3) protein that specifically localizes on autophagosome membranes. As a result, impaired autophagy leads to the accumulation of p62. p62 is also a common component of many human liver disease-related cellular inclusion bodies, such as Mallory-Denk Bodies (MDB), intracytoplasmic hyaline bodies (IHBs), α1 antitrypsin aggregates, as well as p62 bodies/condensates. p62 also acts as an intracellular signaling hub, and it involves multiple signaling pathways including nuclear factor erythroid 2-related factor 2 (NRF2), nuclear factor kappa B (NFκB), and the mechanistic target of rapamycin (mTOR), which are critical for oxidative stress, inflammation, cell survival, metabolism, and liver tumorigenesis. In this review, we will discuss the recent insights of p62 in protein quality control, including the role of p62 in the formation and degradation of p62 stress granules and protein aggregates as well as regulation of multiple signaling pathways in the pathogenesis of alcohol-associated liver disease (ALD).
    Keywords:  TFEB; autophagy; protein aggregates; proteostasis; stress granules
  6. Acta Physiol (Oxf). 2023 Mar 12. e13960
      The mammalian target of rapamycin (mTOR) signalling pathway is crucial in maintaining cell growth and metabolism. The mTOR protein kinase constitutes the catalytic subunit of two multimeric protein complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). As such, this pathway is indispensable for many organs, including the kidney. Since its discovery, mTOR has been associated with major renal disorders such as acute kidney injury, chronic kidney disease, and polycystic kidney disease. On top of that, emerging studies using pharmacological interventions and genetic disease models have unveiled mTOR role in renal tubular ion handling. Along the tubule, mTORC1 and mTORC2 subunits are ubiquitously expressed at mRNA level. Nevertheless, at the protein level, current studies suggest that a tubular segment-specific balance between mTORC1 and mTORC2 exist. In the proximal tubule, mTORC1 regulates nutrients transports through various transporters located in this segment. On the other hand, in the thick ascending limb of the loop of Henle, both complexes play a role in regulating NKCC2 expression and activity. Lastly, in the principal cells of the collecting duct, mTORC2 determines Na+ reabsorption and K+ excretion by regulating of SGK1 activation. Altogether, these studies establish the relevance of the mTOR signalling pathway in the pathophysiology of tubular solute transport. Despite extensive studies on the effectors of mTOR, the upstream activators of mTOR signalling remains elusive in most nephron segments. Further understanding of the role of growth factor signalling and nutrient sensing is essential to establish the exact role of mTOR in kidney physiology.
    Keywords:  Mammalian target of rapamycin; electrolyte transport; kidney physiology; tubular disorder
  7. Aging Brain. 2022 ;2 100056
      Alzheimer's disease (AD) is one of the most persistent and devastating neurodegenerative disorders of old age, and is characterized clinically by an insidious onset and a gradual, progressive deterioration of cognitive abilities, ranging from loss of memory to impairment of judgement and reasoning. Despite years of research, an effective cure is still not available. Autophagy is the cellular 'garbage' clearance system which plays fundamental roles in neurogenesis, neuronal development and activity, and brain health, including memory and learning. A selective sub-type of autophagy is mitophagy which recognizes and degrades damaged or superfluous mitochondria to maintain a healthy and necessary cellular mitochondrial pool. However, emerging evidence from animal models and human samples suggests an age-dependent reduction of autophagy and mitophagy, which are also compromised in AD. Upregulation of autophagy/mitophagy slows down memory loss and ameliorates clinical features in animal models of AD. In this review, we give an overview of autophagy and mitophagy and their link to the progression of AD. We also summarize approaches to upregulate autophagy/mitophagy. We hypothesize that age-dependent compromised autophagy/mitophagy is a cause of brain ageing and a risk factor for AD, while restoration of autophagy/mitophagy to more youthful levels could return the brain to health.
    Keywords:  Ageing; Alzheimer’s disease; Autophagy; Mitophagy
  8. bioRxiv. 2023 Feb 28. pii: 2023.02.27.530319. [Epub ahead of print]
      Autophagosomes fuse with lysosomes, forming autolysosomes that degrade engulfed cargo. To maintain lysosomal capacity, autolysosome reformation (ALR) must regenerate lysosomes from autolysosomes using a membrane tubule-based process. Maintaining lysosomal capacity is required to maintain proteostasis and cellular health, especially in neurons where lysosomal dysfunction has been repeatedly implicated in neurodegenerative disease. Cell biological studies have linked the DNA-J domain Hsc70 co-chaperone RME-8/DNAJC13 to endosomal coat protein regulation, while human genetics studies have linked RME-8/DNAJC13 to neurological disease, including Parkinsonism and Essential Tremor. We report new analysis of the requirements for the RME-8/DNAJC13 protein in neurons, focusing on C. elegans mechanosensory neurons in the intact animal, and in primary mouse cortical neurons in culture. We find that loss of RME-8/DNAJC13 in both systems results in accumulation of grossly elongated autolysosomal tubules. Further C. elegans analysis revealed a similar autolysosome tubule accumulation defect in mutants known to be required for ALR in mammals, including bec-1/beclin and vps-15/PIK3R4/p150 that regulate type-III PI3-kinase VPS-34, and dyn-1 /dynamin that severs ALR tubules. Clathrin is also an important ALR regulator implicated in autolysosome tubule formation and release. In C. elegans we found that loss of RME-8 causes severe depletion of clathrin from neuronal autolysosomes, a phenotype shared with bec-1 and vps-15 mutants. We conclude that RME-8/DNAJC13 plays a conserved but previously unrecognized role in autolysosome reformation, likely affecting ALR tubule initiation and/or severing. Additionally, in both systems, we found that loss of RME-8/DNAJC13 appeared to reduce autophagic flux, suggesting feedback regulation from ALR to autophagy. Our results connecting RME-8/DNAJC13 to ALR and autophagy provide a potential mechanism by which RME-8/DNAJC13 could influence neuronal health and the progression of neurodegenerative disease.
  9. Front Cell Dev Biol. 2023 ;11 1137870
      Autophagy, one of the arms of proteostasis, influences aging and age-related diseases. Recently, the discovery of additional roles of autophagy-related proteins in non-canonical degradation and secretion has revealed alternative fates of autophagic cargo. Some of these non-canonical pathways have been linked to neurodegenerative diseases and improving the understanding of this link is crucial for their potential targetability in aging and age-related diseases. This review discusses recent investigations of the involvement of non-canonical autophagy players and pathways in age-related diseases that are now beginning to be discovered. Unraveling these pathways and their relation to classical autophagy could unearth a fascinating new layer of proteostasis regulation during normal aging and in longevity.
    Keywords:  LANDO; LAP (LC3 associated phagocytosis); aging; neurodegenerative diseasaes; non-canonical autophagy; secretory autophagy
  10. Proc Natl Acad Sci U S A. 2023 Mar 21. 120(12): e2221712120
      Selective macroautophagy (hereafter referred to as autophagy) describes a process in which cytosolic material is engulfed in a double membrane organelle called an autophagosome. Autophagosomes are carriers responsible for delivering their content to a lytic compartment for destruction. The cargo can be of diverse origin, ranging from macromolecular complexes to protein aggregates, organelles, and even invading pathogens. Each cargo is unique in composition and size, presenting different challenges to autophagosome biogenesis. Among the largest cargoes targeted by the autophagy machinery are intracellular bacteria, which can, in the case of Salmonella, range from 2 to 5 μm in length and 0.5 to 1.5 μm in width. How phagophores form and expand on such a large cargo remains mechanistically unclear. Here, we used HeLa cells infected with an auxotrophic Salmonella to study the process of phagophore biogenesis using in situ correlative cryo-ET. We show that host cells generate multiple phagophores at the site of damaged Salmonella-containing vacuoles (SCVs). The observed double membrane structures range from disk-shaped to expanded cup-shaped phagophores, which have a thin intermembrane lumen with a dilating rim region and expand using the SCV, the outer membrane of Salmonella, or existing phagophores as templates. Phagophore rims establish different forms of contact with the endoplasmic reticulum (ER) via structurally distinct molecular entities for membrane formation and expansion. Early omegasomes correlated with the marker Double-FYVE domain-Containing Protein 1 (DFCP1) are observed in close association with the ER without apparent membrane continuity. Our study provides insights into the formation of phagophores around one of the largest selective cargoes.
    Keywords:  Salmonella; autophagosome; cryo-electron t​omo​gra​phy​; omegasome; xenophagy
  11. Trends Pharmacol Sci. 2023 Mar 15. pii: S0165-6147(23)00037-8. [Epub ahead of print]
      Autophagy is a cellular homeostasis mechanism that fuels the proliferation and survival of advanced cancers by degrading and recycling organelles and proteins. Preclinical studies have identified that within an established tumor, tumor cell autophagy and host cell autophagy conspire to support tumor growth. A growing body of evidence suggests that autophagy inhibition can augment the efficacy of chemotherapy, targeted therapy, or immunotherapy to enhance tumor shrinkage. First-generation autophagy inhibition trials in cancer using the lysosomal inhibitor hydroxychloroquine (HCQ) have produced mixed results but have guided the way for the development of more potent and specific autophagy inhibitors in clinical trials. In this review, we will discuss the role of autophagy in cancer, newly discovered molecular mechanisms of the autophagy pathway, the effects of autophagy modulation in cancer and host cells, and novel autophagy inhibitors that are entering clinical trials.
    Keywords:  autophagy; cancer; hydroxychloroquine; immunotherapy, chemotherapy; lysosome
  12. Nat Commun. 2023 Mar 13. 14(1): 1362
      Adipocytes robustly synthesize fatty acids (FA) from carbohydrate through the de novo lipogenesis (DNL) pathway, yet surprisingly DNL contributes little to their abundant triglyceride stored in lipid droplets. This conundrum raises the hypothesis that adipocyte DNL instead enables membrane expansions to occur in processes like autophagy, which requires an abundant supply of phospholipids. We report here that adipocyte Fasn deficiency in vitro and in vivo markedly impairs autophagy, evident by autophagosome accumulation and severely compromised degradation of the autophagic substrate p62. Our data indicate the impairment occurs at the level of autophagosome-lysosome fusion, and indeed, loss of Fasn decreases certain membrane phosphoinositides necessary for autophagosome and lysosome maturation and fusion. Autophagy dependence on FA produced by Fasn is not fully alleviated by exogenous FA in cultured adipocytes, and interestingly, imaging studies reveal that Fasn colocalizes with nascent autophagosomes. Together, our studies identify DNL as a critical source of FAs to fuel autophagosome and lysosome maturation and fusion in adipocytes.
  13. Biomater Res. 2023 Mar 13. 27(1): 20
      BACKGROUND: Macroautophagy is an essential cellular self-protection mechanism, and defective autophagy has been considered to contribute to a variety of diseases. During the process, cytoplasmic components are transported via autophagosomes to acidic lysosomes for metabolism and recycling, which represents application niches for lysosome-targeted fluorescent probes. Additionally, in view of the complexity of the autophagy pathway, it entails more stringent requirements for probes suitable for monitoring autophagy. Meanwhile, aggregation-induced emission (AIE) fluorescent probes have been impressively demonstrated in the biomedical field, which bring fascinating possibilities to the autophagy visualization.METHODS: We reported a generalizable de novo design of a novel pH-sensitive AIE probe ASMP-AP tailored to lysosome targeting for the interpretation of autophagy. Firstly, the theoretical calculation was carried out followed by the investigation of optical properties. Then, the performance of ASMP-AP in visualizing autophagy was corroborated by starvation or drugs treatments. Furthermore, the capability of ASMP-AP to monitor autophagy was demonstrated in ex vivo liver tissue and zebrafish in vivo.
    RESULTS: ASMP-AP displays a large stokes shift, great cell permeability and good biocompatibility. More importantly, ASMP-AP enables a good linear response to pH, which derives from the fact that its aggregation state can be manipulated by the acidity. It was successfully applied for imaging autophagy in living cells and was proved capable of monitoring mitophagy. Moreover, this novel molecular tool was validated by ex vivo visualization of activated autophagy in drug-induced liver injury model. Interestingly, it provided a meaningful pharmacological insight that the melanin inhibitor 1-phenyl-2-thiourea (PTU)-induced autophagy was clearly presented in wild-type zebrafish.
    CONCLUSIONS: ASMP-AP offers a simple yet effective tool for studying lysosome and autophagy. This is the first instance to visualize autophagy in zebrafish using a small-molecule probe with AIE characters, accurate lysosome targeting and simultaneous pH sensitivity. Ultimately, this novel fluorescent system has great potential for in vivo translation to fuel autophagy research.
    Keywords:  AIE fluorescent probe; Autophagy visualization; Biomaterials imaging; Lysosome-targeting
  14. BMC Vet Res. 2023 Mar 15. 19(1): 60
      BACKGROUND: Canine distemper virus (CDV) is one of the most contagious and lethal viruses known to the Canidae, with a very broad and expanding host range. Autophagy serves as a fundamental stabilizing response against pathogens, but some viruses have been able to evade or exploit it for their replication. However, the effect of autophagy mechanisms on CDV infection is still unclear.RESULTS: In the present study, autophagy was induced in CDV-infected Vero cells as demonstrated by elevated LC3-II levels and aggregation of green fluorescent protein (GFP)-LC3 spots. Furthermore, CDV promoted the complete autophagic process, which could be determined by the degradation of p62, co-localization of LC3 with lysosomes, GFP degradation, and accumulation of LC3-II and p62 due to the lysosomal protease inhibitor E64d. In addition, the use of Rapamycin to promote autophagy promoted CDV replication, and the inhibition of autophagy by Wortmannin, Chloroquine and siRNA-ATG5 inhibited CDV replication, revealing that CDV-induced autophagy facilitated virus replication. We also found that UV-inactivated CDV still induced autophagy, and that nucleocapsid (N) protein was able to induce complete autophagy in an mTOR-dependent manner.
    CONCLUSIONS: This study for the first time revealed that CDV N protein induced complete autophagy to facilitate viral replication.
    Keywords:  Autophagy; Canine distemper virus; Nucleocapsid protein; Replication
  15. Front Cell Neurosci. 2023 ;17 1140916
      Mitochondrial dysfunction is associated with ototoxicity, which is caused by external factors. Mitophagy plays a key role in maintaining mitochondrial homeostasis and function and is regulated by a series of key mitophagy regulatory proteins and signaling pathways. The results of ototoxicity models indicate the importance of this process in the etiology of ototoxicity. A number of recent investigations of the control of cell fate by mitophagy have enhanced our understanding of the mechanisms by which mitophagy regulates ototoxicity and other hearing-related diseases, providing opportunities for targeting mitochondria to treat ototoxicity.
    Keywords:  PINK1-Parkin; autophagy receptors; mitochondria; mitophagy; ototoxicity
  16. J Cell Biochem. 2023 Mar 16.
      The coordinated interaction between mitochondria and lysosomes, mainly manifested by mitophagy, mitochondria-derived vesicles, and direct physical contact, is essential for maintaining cellular life activities. The VPS39 subunit of the homotypic fusion and protein sorting complex could play a key role in the regulation of organelle dynamics, such as endolysosomal trafficking and mitochondria-vacuole/lysosome crosstalk, thus contributing to a variety of physiological functions. The abnormalities of VPS39 and related subunits have been reported to be involved in the pathological process of some diseases. Here, we analyze the potential mechanisms and the existing problems of VPS39 in regulating organelle dynamics, which, in turn, regulate physiological functions and disease pathogenesis, so as to provide new clues for facilitating the discovery of therapeutic targets for mitochondrial and lysosomal diseases.
    Keywords:  HOPS complex; VPS39; diseases; endolysosomal trafficking; mitochondria-lysosome crosstalk
  17. Nutr Hosp. 2023 Mar 16.
      Autophagy is a very active process that plays an important role in cell and organ differentiation and remodelling, being a crucial system to guarantee health. This physiological process is activated in starvation and inhibited in the presence of nutrients. This short review comments on the three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy, as well as different aspects that control autophagy and its relationship with health and degenerative diseases. As autophagy is highly dependent on functional autophagy (ATG) proteins integrating the phagophore, the role of some key ATG genes and epigenes are briefly commented on. The manuscript deepens discussing some central aspects of type-2 diabetes mellitus and their relationship with the cell cleaning process and mitochondria homeostasis maintenance, as well as the mechanisms through which antidiabetic drugs affect autophagy. Well-designed studies are needed to elucidate whether autophagy plays a casual or causal role in T2DM.
  18. J Equine Vet Sci. 2023 Mar 09. pii: S0737-0806(23)00072-2. [Epub ahead of print] 104281
      Skeletal muscle is a highly dynamic organ that is essential for locomotion as well as endocrine regulation in all populations of horses. However, despite the importance of adequate muscle development and maintenance, the mechanisms underlying protein anabolism in horses on different diets, exercise programs, and at different life stages remain obscure. Mechanistic target of rapamycin (mTOR) is a key component of the protein synthesis pathway and is regulated by biological factors such as insulin and amino acid availability. Providing a diet ample in vital amino acids, such as leucine and glutamine, is essential in activating sensory pathways that recruit mTOR to the lysosome and assist in the translation of important downstream targets. When the diet is well balanced, mitochondrial biogenesis and protein synthesis are activated in response to increased exercise bouts in the performing athlete. It is important to note that the mTOR kinase pathways are multi-faceted and very complex, with several binding partners and targets that lead to specific functions in protein turnover of the cell, and ultimately, the capacity to maintain or grow muscle mass. Further, these pathways are likely altered across the lifespan, with an emphasis of growth in young horses while decreases in musculature with aged horses appears to be attributable to degradation or other regulators of protein synthesis rather than alterations in the mTOR pathway. Previous work has begun to pinpoint ways in which the mTOR pathway is influenced by diet, exercise, and age; however, future research is warranted to quantify the functional outcomes related to changes in mTOR. Promisingly, this could provide direction on appropriate management techniques to support skeletal muscle growth and maximize athletic potential in differing equine populations.
    Keywords:  anabolic; diet; disease; equine; exercise; skeletal muscle
  19. Eur J Cell Biol. 2023 Mar 09. pii: S0171-9335(23)00020-1. [Epub ahead of print]102(2): 151305
      The neuronal ceroid lipofuscinoses (NCLs), collectively referred to as Batten disease, are a group of fatal neurodegenerative disorders that primarily affect children. The etiology of Batten disease is linked to mutations in 13 genes that encode distinct CLN proteins, whose functions have yet to be fully elucidated. The social amoeba Dictyostelium discoideum has been adopted as an efficient and powerful model system for studying the diverse cellular roles of CLN proteins. The genome of D. discoideum encodes several homologs of human CLN proteins, and a growing body of literature supports the conserved roles and networking of CLN proteins in D. discoideum and humans. In humans, CLN proteins have diverse cellular roles related to autophagy, signal transduction, lipid homeostasis, lysosomal ion homeostasis, and intracellular trafficking. Recent work also indicates that CLN proteins play an important role in protein secretion. Remarkably, many of these findings have found parallels in studies with D. discoideum. Accordingly, this review will highlight the translatable value of novel work with D. discoideum in the field of NCL research and propose further avenues of research using this biomedical model organism for studying the NCLs.
    Keywords:  Autophagy; Batten disease; Lysosomal storage diseases; Model system; Neuronal ceroid lipofuscinosis; Secretion
  20. PLoS One. 2023 ;18(3): e0280650
      Tay-Sachs disease is a lethal lysosomal storage disorder caused by mutations in the HexA gene encoding the α subunit of the lysosomal β-hexosaminidase enzyme (HEXA). Abnormal GM2 ganglioside accumulation causes progressive deterioration in the central nervous system in Tay-Sachs patients. Hexa-/- mouse model failed to display abnormal phenotype. Recently, our group generated Hexa-/-Neu3-/- mouse showed severe neuropathological indications similar to Tay-Sachs patients. Despite excessive GM2 ganglioside accumulation in the brain and visceral organs, the regulation of autophagy has not been clarified yet in the Tay-Sachs disease mouse model. Therefore, we investigated distinct steps of autophagic flux using markers including LC3 and p62 in four different brain regions from the Hexa-/-Neu3-/- mice model of Tay-Sachs disease. Our data revealed accumulated autophagosomes and autophagolysosomes indicating impairment in autophagic flux in the brain. We suggest that autophagy might be a new therapeutic target for the treatment of devastating Tay-Sachs disease.
  21. J Cell Physiol. 2023 Mar 16.
      Circular dorsal ruffles (CDRs) are rounded membrane ruffles induced by growth factors to function as precursors of the large-scale endocytosis called macropinocytosis. In addition to their role in cellular uptake, recent research using cell line systems has shown that CDRs/macropinocytosis regulate the canonical AKT-mTORC1 growth factor signaling pathway. However, as CDRs have not been observed in tissues, their physiological relevance has remained unclear. Here, utilizing ultrahigh-resolution scanning electron microscopy, we first report that CDRs are expressed in glomerular podocytes ex vivo and in vivo, and we visually captured the transformation process to macropinocytosis. Moreover, through biochemical and imaging analyses, we show that AKT phosphorylation localized to CDRs upstream of mTORC1 activation in podocyte cell lines and isolated glomeruli. These results demonstrate the physiological role of CDRs as signal platforms for the AKT-mTORC1 pathway in glomerular podocytes at the tissue level. As mTORC1 plays critical roles in podocyte metabolism, and aberrant activation of mTORC1 triggers podocytopathies, our results strongly suggest that targeting CDR formation could represent a potential therapeutic approach for these diseases.
    Keywords:  AKT; circular dorsal ruffle; mTORC2; macropinocytosis; podocyte
  22. Sci China Life Sci. 2023 Mar 09.
      Multiple viral infections in insect vectors with synergistic effects are common in nature, but the underlying mechanism remains elusive. Here, we find that rice gall dwarf reovirus (RGDV) facilitates the transmission of rice stripe mosaic rhabdovirus (RSMV) by co-infected leafhopper vectors. RSMV nucleoprotein (N) alone activates complete anti-viral autophagy, while RGDV nonstructural protein Pns11 alone induces pro-viral incomplete autophagy. In co-infected vectors, RSMV exploits Pns11-induced autophagosomes to assemble enveloped virions via N-Pns11-ATG5 interaction. Furthermore, RSMV could effectively propagate in Sf9 cells. Expression of Pns11 in Sf9 cells or leafhopper vectors causes the recruitment of N from the ER to Pns11-induced autophagosomes and inhibits N-induced complete autophagic flux, finally facilitating RSMV propagation. In summary, these results demonstrate a previously unappreciated role of autophagy in the regulation of the direct synergistic interaction during co-transmission of two distinct arboviruses by insect vectors and reveal the functional importance of virus-induced autophagosomes in rhabdovirus assembly.
    Keywords:  autophagy; co-transmission; insect vector; rhabdovirus; synergistic interaction
  23. Curr Neuropharmacol. 2023 Mar 14.
      With the advancement in novel drug discovery, biologically active compounds are considered pharmacological tools to understand complex biological mechanisms and the identification of potent therapeutic agents. Mitochondria boast a central role in different integral biological processes and mitochondrial dysfunction is associated with multiple pathologies. It is, therefore, prudent to target mitochondrial quality control mechanisms by using pharmacological approaches. However, there is a scarcity of biologically active molecules, which can interact with mitochondria directly. Currently, the chemical compounds used to induce mitophagy include oligomycin and antimycin A for impaired respiration and acute dissipation of mitochondrial membrane potential by using CCCP/FCCP, the mitochondrial uncouplers. These chemical probes alter the homeostasis of the mitochondria and limit our understanding of the energy regulatory mechanisms. Efforts are underway to find molecules that can bring about selective removal of defective mitochondria without compromising normal mitochondrial respiration. In this report, we have tried to summarize and status of the recently reported modulators of mitophagy.
    Keywords:  Mitophagy; mitochondria; mitochondrial quality control.; neurological disorders; pharmacology; power management system
  24. Stem Cells Dev. 2023 Mar 16.
      Stem cell exhaustion is a hallmark of aging. Klotho-deficient mice (kl/kl mice) is a murine model that mimics human aging with significant bone abnormalities. The aim of this study is using kl/kl mice to investigate the functional change of bone marrow-derived mesenchymal stem cells (BMSCs) and explore the underlying mechanism. We found klotho-deficiency leads to bone abnormalities. In addition, kl/kl BMSCs manifested hyper-active proliferation but functional declined both in vivo and in vitro. mTORC1 activity was higher in freshly isolated kl/kl BMSCs and autophagy in kl/kl BMSCs were significantly decreased, possibly through mTORC1 activation. Conditional medium containing soluble Klotho protein (sKL) rescued hyper-proliferation of kl/kl BMSCs by inhibiting mTORC1 activity and restoring autophagy. Finally, intraperitoneally injection of mTORC1 inhibitor rapamycin restored BMSC quiescence, ameliorated bone phenotype and increased lifespan of kl/kl mice in vivo. This research highlights a therapeutic strategy to maintain the homeostasis of adult stem cell pool for healthy bone aging.
  25. Genes Cancer. 2023 ;14 30-49
      We and others have recently shown that proteins involved in the DNA damage response (DDR) are critical for KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell growth in vitro. However, the CRISPR-Cas9 library that enabled us to identify these key proteins had limited representation of DDR-related genes. To further investigate the DDR in this context, we performed a comprehensive, DDR-focused CRISPR-Cas9 loss-of-function screen. This screen identified valosin-containing protein (VCP) as an essential gene in KRAS-mutant PDAC cell lines. We observed that genetic and pharmacologic inhibition of VCP limited cell growth and induced apoptotic death. Addressing the basis for VCP-dependent growth, we first evaluated the contribution of VCP to the DDR and found that loss of VCP resulted in accumulation of DNA double-strand breaks. We next addressed its role in proteostasis and found that loss of VCP caused accumulation of polyubiquitinated proteins. We also found that loss of VCP increased autophagy. Therefore, we reasoned that inhibiting both VCP and autophagy could be an effective combination. Accordingly, we found that VCP inhibition synergized with the autophagy inhibitor chloroquine. We conclude that concurrent targeting of autophagy can enhance the efficacy of VCP inhibitors in KRAS-mutant PDAC.
    Keywords:  DNA damage response; KRAS; VCP; autophagy; pancreatic cancer
  26. PLoS One. 2023 ;18(3): e0281577
      Evidence shows that chaperone-mediated autophagy (CMA) is involved in cancer cell pathogenesis and progression. However, the potential role of CMA in breast cancer angiogenesis remains unknown. We first manipulated CMA activity by knockdown and overexpressing of lysosome-associated membrane protein type 2A (LAMP2A) in MDA-MB-231, MDA-MB-436, T47D and MCF7 cells. We found that the tube formation, migration and proliferation abilities of human umbilical vein endothelial cells (HUVECs) were inhibited after cocultured with tumor-conditioned medium from breast cancer cells of LAMP2A knockdown. While the above changes were promoted after cocultured with tumor-conditioned medium from breast cancer cells of LAMP2A overexpression. Moreover, we found that CMA could promote VEGFA expression in breast cancer cells and in xenograft model through upregulating lactate production. Finally, we found that lactate regulation in breast cancer cells is hexokinase 2 (HK2) dependent, and knockdown of HK2 can significantly reduce the ability of CMA-mediated tube formation capacity of HUVECs. Collectively, these results indicate that CMA could promote breast cancer angiogenesis via regulation of HK2-dependent aerobic glycolysis, which may serve as an attractive target for breast cancer therapies.
  27. Mol Med. 2023 Mar 14. 29(1): 34
      The homologous to the E6-AP carboxyl terminus (HECT)-type E3 ubiquitin ligases are the selective executers in the protein ubiquitination, playing a vital role in modulation of the protein function and stability. Evidence shows the regulatory role of HECT-type E3 ligases in various steps of the autophagic process. Autophagy is an intracellular digestive and recycling process that controls the cellular hemostasis. Defective autophagy is involved in tumorigenesis and has been detected in various types of cancer cells. A growing body of findings indicates that HECT-type E3 ligases, in particular members of the neural precursor cell expressed developmentally downregulated protein 4 (NEDD4) including NEDD4-1, NEDD4-L, SMURFs, WWPs, and ITCH, play critical roles in dysregulation or dysfunction of autophagy in cancer cells. The present review focuses on NEDD4 E3 ligases involved in defective autophagy in cancer cells and discusses their autophagic function in different cancer cells as well as substrates and the signaling pathways in which they participate, conferring a basis for the cancer treatment through the modulating of these E3 ligases.
    Keywords:  Autophagy; Cancer; E3 ligase; NEDD4; Ubiquitin
  28. EMBO Rep. 2023 Mar 16. e56134
      Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.
    Keywords:  Atg9; Cvt pathway; Drs2; TRAPPIII; vesicle transport
  29. J Ginseng Res. 2023 Mar;47(2): 337-346
      Background: Ginsenoside Rb2, a major active component of Panax ginseng, has various physiological activities, including anticancer and anti-inflammatory effects. However, the mechanisms underlying the rejuvenation effect of Rb2 in human skin cells have not been elucidated.Methods: We performed a senescence-associated β-galactosidase staining assay to confirm cellular senescence in human dermal fibroblasts (HDFs). The regulatory effects of Rb2 on autophagy were evaluated by analyzing the expression of autophagy marker proteins, such as microtubule-associated protein 1A/1B-light chain (LC) 3 and p62, using immunoblotting. Autophagosome and autolysosome formation was monitored using transmission electron microscopy. Autophagic flux was analyzed using tandem-labeled GFP-RFP-LC3, and lysosomal function was assessed with Lysotracker. We performed RNA sequencing to identify potential target genes related to HDF rejuvenation mediated by Rb2. To verify the functions of the target genes, we silenced them using shRNAs.
    Results: Rb2 decreased β-galactosidase activity and altered the expression of cell cycle regulatory proteins in senescent HDFs. Rb2 markedly induced the conversion of LC3-Ⅰ to LC3-Ⅱ and LC3 puncta. Moreover, Rb2 increased lysosomal function and red puncta in tandem-labeled GFP-RFP-LC3, which indicate that Rb2 promoted autophagic flux. RNA sequencing data showed that the expression of DNA damage-regulated autophagy modulator 2 (DRAM2) was induced by Rb2. In autophagy signaling, Rb2 activated the AMPK-ULK1 pathway and inactivated mTOR. DRAM2 knockdown inhibited autophagy and Rb2-restored cellular senescence.
    Conclusion: Rb2 reverses cellular senescence by activating autophagy via the AMPK-mTOR pathway and induction of DRAM2, suggesting that Rb2 might have potential value as an antiaging agent.
    Keywords:  Autophagy; DRAM2; Rb2; Senescence
  30. Res Sq. 2023 Feb 27. pii: [Epub ahead of print]
      Myofiber size regulation is critical in health, disease, and aging. MuSK (muscle-specific kinase) is a BMP (bone morphogenetic protein) co-receptor that promotes and shapes BMP signaling. MuSK is expressed at all neuromuscular junctions and is also present extrasynaptically in the slow soleus muscle. To investigate the role of the MuSK-BMP pathway in vivo we generated mice lacking the BMP-binding MuSK Ig3 domain. These ∆Ig3-MuSKmice are viable and fertile with innervation levels comparable to wild type. In 3-month-old mice myofibers are smaller in the slow soleus, but not in the fast tibialis anterior (TA). Transcriptomic analysis revealed soleus-selective decreases in RNA metabolism and protein synthesis pathways as well as dysregulation of IGF1-Akt-mTOR pathway components. Biochemical analysis showed that Akt-mTOR signaling is reduced in soleus but not TA. We propose that the MuSK-BMP pathway acts extrasynaptically to maintain myofiber size in slow muscle by promoting protein synthetic pathways including IGF1-Akt-mTOR signaling. These results reveal a novel mechanism for regulating myofiber size in slow muscle and introduce the MuSK-BMP pathway as a target for promoting muscle growth and combatting atrophy.
  31. Mitochondrion. 2023 Mar 10. pii: S1567-7249(23)00028-4. [Epub ahead of print]70 20-30
      BACKGROUND AND AIMS: Hemorrhagic shock-resuscitation (HSR) following trauma contributes to organ dysfunction by causing ischemia-reperfusion injury (IRI). We previously showed that 'remote ischemic preconditioning' (RIPC) exerted multi-organ protection from IRI. Maintenance of mitochondrial quality by clearance of dysfunctional mitochondria via mitophagy is vital in restoring organ integrity. We hypothesized that parkin-dependent mitophagy played a role in RIPC-induced hepatoprotection following HSR.METHODS: The hepatoprotective effect of RIPC in a murine model of HSR-IRI was investigated in wild type and parkin-/- animals. Mice were subjected to HSR ± RIPC and blood and organs were collected, followed by cytokine ELISAs, histology, qPCR, Western blots, and transmission electron microscopy.
    RESULTS: HSR increased hepatocellular injury, as measured by plasma ALT and liver necrosis, while antecedent RIPC prevented this injury; in parkin-/- mice, RIPC failed to exert hepatoprotection. The ability of RIPC to lessen HSR-induced rises in plasma IL-6 and TNFα, was lost in parkin-/- mice. While RIPC alone did not induce mitophagy, the application of RIPC prior to HSR caused a synergistic increase in mitophagy, this increase was not observed in parkin-/- mice. RIPC induced shifts in mitochondrial morphology favoring mitophagy in WT but not in parkin-/- animals.
    CONCLUSIONS: RIPC was hepatoprotective in WT mice following HSR but not in parkin-/- mice. Loss of protection in parkin-/- mice corresponded with the failure of RIPC plus HSR to upregulate the mitophagic process. Improving mitochondrial quality by modulating mitophagy, may prove to be an attractive therapeutic target in disease processes caused by IRI.
    Keywords:  Hemorrhagic shock-resuscitation; Ischemia–reperfusion injury; Mitochondria; Mitochondrial quality control; Parkin-dependent mitophagy
  32. Neural Regen Res. 2023 Sep;18(9): 2075-2081
      Recent studies have shown that a 9-hour fast in mice reduces the amount of time spent immobile in the forced swimming test. However, whether 9-hour fasting has therapeutic effects in female mice with depressive symptoms has not been established. Therefore, in this study, we simulated perimenopausal depression via an ovariectomy in mice, and subjected them to a single 9-hour fasting 7 days later. We found that the ovariectomy increased the time spent immobile in the forced swimming test, inhibited expression of the mammalian target of rapamycin complex 1 signaling pathway in the hippocampus and prefrontal cortex, and decreased the density of dendritic spines in the hippocampus. The 9-hour acute fasting alleviated the above-mentioned phenomena. Furthermore, all of the antidepressant-like effects of 9-hour fasting were reversed by an inhibitor of the mammalian target of rapamycin complex 1. Electrophysiology data showed a remarkable increase in long-term potentiation in the hippocampal CA1 of the ovariectomized mice subjected to fasting compared with the findings in the ovariectomized mice not subjected to fasting. These findings show that the antidepressant-like effects of 9-hour fasting may be related to the activation of the mammalian target of the rapamycin complex 1 signaling pathway and synaptic plasticity in the mammalian hippocampus. Thus, fasting may be a potential treatment for depression.
    Keywords:  LTP; antidepressant; brain-derived neurotrophic factor; dendritic spine; fasting; hippocampus; mTOR complex 1; neural plasticity; ovariectomized mice; rapamycin
  33. Vet Res. 2023 Mar 14. 54(1): 23
      Tembusu virus (TMUV) is an emerging flavivirus that has broken out in different regions of China. TMUV infection has been reported to induce autophagy in duck embryo fibroblast cells. However, the molecular mechanisms underlying this autophagy induction remain unclear. Here, we explored the interactions between autophagy and TMUV and the effects of the structural and nonstructural proteins of TMUV on autophagy in vitro. Among our results, TMUV infection enhanced autophagy to facilitate viral replication in HEK293T cells. After pharmacologically inducing autophagy with rapamycin (Rapa), the replication of TMUV increased by a maximum of 14-fold compared with the control group. To determine which TMUV protein primarily induced autophagy, cells were transfected with two structural proteins and seven nonstructural proteins of TMUV. Western blotting showed that nonstructural proteins 2B (NS2B) and 4 A (NS4A) of TMUV significantly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3) from LC3-I to LC3-II in HEK293T cells. In addition, through immunofluorescence assays, we found that NS2B and NS4A significantly increased the punctate fluorescence of GFP-LC3-II. Furthermore, we found that both NS2B and NS4A interacted with polyubiquitin-binding protein sequestosome 1 (SQSTM1/p62) in a coimmunoprecipitation assay. Moreover, the autophagic degradation of p62 and LC3 mediated by NS2B or NS4A was inhibited by treatment with the autophagic flux inhibitor chloroquine (CQ). These results confirmed the vital effects of NS2B and NS4A in TMUV-induced complete autophagy and clarified the importance of complete autophagy for viral replication, providing novel insight into the relationship between TMUV and autophagy.
    Keywords:  Tembusu virus; autophagy; nonstructural protein 2B; nonstructural protein 4A; p62
  34. Cell Death Dis. 2023 Mar 16. 14(3): 199
      During hypoxia, FUNDC1 acts as a mitophagy receptor and accumulates at the ER (endoplasmic reticulum)-mitochondria contact sites (EMC), also called mitochondria-associated membranes (MAM). In mitophagy, the ULK1 complex phosphorylates FUNDC1(S17) at the EMC site. However, how mitochondria sense the stress and send the signal from the inside to the outside of mitochondria to trigger mitophagy is still unclear. Mitochondrial Lon was reported to be localized at the EMC under stress although the function remained unknown. In this study, we explored the mechanism of how mitochondrial sensors of hypoxia trigger and stabilize the FUNDC1-ULK1 complex by Lon in the EMC for cell survival and cancer progression. We demonstrated that Lon is accumulated in the EMC and associated with FUNDC1-ULK1 complex to induce mitophagy via chaperone activity under hypoxia. Intriguingly, we found that Lon-induced mitophagy is through binding with mitochondrial Na+/Ca2+ exchanger (NCLX) to promote FUNDC1-ULK1-mediated mitophagy at the EMC site in vitro and in vivo. Accordingly, our findings highlight a novel mechanism responsible for mitophagy initiation under hypoxia by chaperone Lon in mitochondria through the interaction with FUNDC1-ULK1 complex at the EMC site. These findings provide a direct correlation between Lon and mitophagy on cell survival and cancer progression.
  35. Rev Neurol (Paris). 2023 Mar 09. pii: S0035-3787(23)00867-6. [Epub ahead of print]
      Dysregulation of the mTOR pathway is now well documented in several neurodevelopmental disorders associated with epilepsy. Mutations of mTOR pathway genes are involved in tuberous sclerosis complex (TSC) as well as in a range of cortical malformations from hemimegalencephaly (HME) to type II focal cortical dysplasia (FCD II), leading to the concept of "mTORopathies" (mTOR pathway-related malformations). This suggests that mTOR inhibitors (notably rapamycin (sirolimus), and everolimus) could be used as antiseizure medication. In this review, we provide an overview of pharmacological treatments targeting the mTOR pathway for epilepsy based on lectures from the ILAE French Chapter meeting in October 2022 in Grenoble. There is strong preclinical evidence for the antiseizure effects of mTOR inhibitors in TSC and cortical malformation mouse models. There are also open studies on the antiseizure effects of mTOR inhibitors, as well as one phase III study showing the antiseizure effect of everolimus in TSC patients. Finally, we discuss to which extent mTOR inhibitors might have properties beyond the antiseizure effect on associated neuropsychiatric comorbidities. We also discuss a new way of treatment on the mTOR pathways.
    Keywords:  Epilepsy; Everolimus; Focal cortical dysplasia; Rapamycin; Sirolimus; Tuberous sclerosis complex; mTOR signaling pathway
  36. Front Cell Dev Biol. 2023 ;11 1127618
      Mitochondria are central hubs for energy production, metabolism and cellular signal transduction in eukaryotic cells. Maintenance of mitochondrial homeostasis is important for cellular function and survival. In particular, cellular metabolic state is in constant communication with mitochondrial homeostasis. One of the most important metabolic processes that provide energy in the cell is amino acid metabolism. Almost all of the 20 amino acids that serve as the building blocks of proteins are produced or degraded in the mitochondria. The synthesis of the amino acids aspartate and arginine depends on the activity of the respiratory chain, which is essential for cell proliferation. The degradation of branched-chain amino acids mainly occurs in the mitochondrial matrix, contributing to energy metabolism, mitochondrial biogenesis, as well as protein quality control in both mitochondria and cytosol. Dietary supplementation or restriction of amino acids in worms, flies and mice modulates lifespan and health, which has been associated with changes in mitochondrial biogenesis, antioxidant response, as well as the activity of tricarboxylic acid cycle and respiratory chain. Consequently, impaired amino acid metabolism has been associated with both primary mitochondrial diseases and diseases with mitochondrial dysfunction such as cancer. Here, we present recent observations on the crosstalk between amino acid metabolism and mitochondrial homeostasis, summarise the underlying molecular mechanisms to date, and discuss their role in cellular functions and organismal physiology.
    Keywords:  TCA cycle; amino acid metabolism; amino acid recycling; lifespan; mitochondrial homeostasis; proteasome; respiratory chain
  37. Small. 2023 Mar 18. e2207898
      Chemotherapeutics can induce immunogenic cell death (ICD) by triggering autophagy and mediate antitumor immunotherapy. However, using chemotherapeutics alone can only cause mild cell-protective autophagy and be incapable of inducing sufficient ICD efficacy. The participation of autophagy inducer is competent to enhance autophagy, so the level of ICD is promoted and the effect of antitumor immunotherapy is highly increased. Herein, tailor-made autophagy cascade amplification polymeric nanoparticles STF@AHPPE are constructed to enhance tumor immunotherapy. Arginine (Arg), polyethyleneglycol-polycaprolactone, and epirubicin (EPI) are grafted onto hyaluronic acid (HA) via disulfide bond to form the AHPPE nanoparticles and autophagy inducer STF-62247 (STF) is loaded. When STF@AHPPE nanoparticles target to tumor tissues and efficiently enter into tumor cells with the help of HA and Arg, the high glutathione concentration leads to the cleavage of disulfide bond and the release of EPI and STF. Finally, STF@AHPPE induces violent cytotoxic autophagy and strong ICD efficacy. As compared to AHPPE nanoparticles, STF@AHPPE nanoparticles kill the most tumor cells and show the more obvious ICD efficacy and immune activation ability. This work provides a novel strategy for combining tumor chemo-immunotherapy with autophagy induction.
    Keywords:  autophagy; chemo-immunotherapy; immunogenic cell death; polymeric nanoparticles; reduction-responsive
  38. Mol Psychiatry. 2023 Mar 13.
      Recent studies based on animal models of various neurological disorders have indicated that mitophagy, a selective autophagy that eliminates damaged and superfluous mitochondria through autophagic degradation, may be involved in various neurological diseases. As an important mechanism of cellular stress response, much less is known about the role of mitophagy in stress-related mood disorders. Here, we found that tumor necrosis factor-α (TNF-α), an inflammation cytokine that plays a particular role in stress responses, impaired the mitophagy in the medial prefrontal cortex (mPFC) via triggering degradation of an outer mitochondrial membrane protein, NIP3-like protein X (NIX). The deficits in the NIX-mediated mitophagy by TNF-α led to the accumulation of damaged mitochondria, which triggered synaptic defects and behavioral abnormalities. Genetic ablation of NIX in the excitatory neurons of mPFC caused passive coping behaviors to stress, and overexpression of NIX in the mPFC improved TNF-α-induced synaptic and behavioral abnormalities. Notably, ketamine, a rapid on-set and long-lasting antidepressant, reversed the TNF-α-induced behavioral abnormalities through activation of NIX-mediated mitophagy. Furthermore, the downregulation of NIX level was also observed in the blood of major depressive disorder patients and the mPFC tissue of animal models. Infliximab, a clinically used TNF-α antagonist, alleviated both chronic stress- and inflammation-induced behavioral abnormalities via restoring NIX level. Taken together, these results suggest that NIX-mediated mitophagy links inflammation signaling to passive coping behaviors to stress, which underlies the pathophysiology of stress-related emotional disorders.
  39. J Child Psychol Psychiatry. 2023 Mar 15.
      BACKGROUND: Larger than average head and brain sizes are often observed in individuals with autism spectrum disorders (ASDs). ASD and brain volume are both highly heritable, with multiple genetic variants contributing. However, it is unclear whether ASD and brain volume share any genetic mechanisms. Genes from the mammalian target of rapamycin (mTOR) pathway influence brain volume, and variants are found in rare genetic syndromes that include ASD features. Here we investigated whether variants in mTOR-related genes are also associated with ASD and if they constitute a genetic link between large brains and ASD.METHODS: We extended our analyses between large heads (macrocephaly) and rare de novo mTOR-related variants in an intellectual disability cohort (N = 2,258). Subsequently using Fisher's exact tests we investigated the co-occurrence of mTOR-related de novo variants and ASD in the de-novo-db database (N = 23,098). We next selected common genetic variants within a set of 96 mTOR-related genes in genome-wide genetic association data of ASD (N = 46,350) to test gene-set association using MAGMA. Lastly, we tested genetic correlation between genome-wide genetic association data of ASD (N = 46,350) and intracranial volume (N = 25,974) globally using linkage disequilibrium score regression as well as mTOR specific by restricting the genetic correlation to the mTOR-related genes using GNOVA.
    RESULTS: Our results show that both macrocephaly and ASD occur above chance level in individuals carrying rare de novo variants in mTOR-related genes. We found a significant mTOR gene-set association with ASD (p = .0029) and an mTOR-stratified positive genetic correlation between ASD and intracranial volume (p = .027), despite the absence of a significant genome-wide correlation (p = .81).
    CONCLUSIONS: This work indicates that both rare and common variants in mTOR-related genes are associated with brain volume and ASD and genetically correlate them in the expected direction. We demonstrate that genes involved in mTOR signalling are potential mediators of the relationship between having a large brain and having ASD.
    Keywords:  Autism spectrum disorders; brain volume; genetics; mammalian target of rapamycin; stratified genetic correlation
  40. Mol Cancer Res. 2023 Mar 17. pii: MCR-22-0825. [Epub ahead of print]
      Recent studies have shown that autophagy plays an important role in gynecological tumours, and ubiquitin modification of autophagy regulatory components is essential to regulate autophagic. In this study, we found that UBE2C affects endometrial cancer cell apoptosis and proliferation by inhibiting autophagy. Electron microscopy observation of cell ultrastructure and experimental biochemical analysis showed that EC cells with UBE2C expression knocked down display typical autophagic characteristics. Cells were cotreated with the autophagy pharmacological inhibitors chloroquine and/or bafilomycin A1, and mRFP-GFP-LC3 assays were performed to monitor autophagic flux and determine whether UBE2C suppresses the autophagy program. Investigation of the corresponding mechanism by which UBE2C inhibits autophagy revealed that UBE2C induces K48-linked SIRT1 ubiquitination and promotes ubiquitination-dependent degradation of SIRT1, subsequently reducing H4K16 deacetylation levels and epigenetically inhibiting the expression of autophagy-related genes. The results of CCK-8, Hoechst staining, and immunofluorescence assays further indicated that deletion of the autophagy-related gene BECN1 significantly attenuates UBE2C knockdown-induced cell apoptosis. Moreover, overexpression of UBE2C promoted tumor growth in the xenograft mice model. While, the introduction of rapamycin, an agonist of autophagy, successfully reversed tumor growth and apoptosis inhibition mediated by UBE2C overexpression in vitro and in vivo. Taken together, our results suggested that UBE2C-mediated ubiquitination and degradation of SIRT1 contribute to the malignant progression of endometrial cancer through epigenetic inhibition of autophagy. Implications: Our study highlights the tumorigenic role and regulatory mechanism of UBE2C in EC; UBE2C inhibits EC cell apoptosis through autophagy-related mechanisms and our findings provide new insights into the treatment of endometrial cancer.
  41. bioRxiv. 2023 Mar 01. pii: 2023.02.28.530351. [Epub ahead of print]
      Pptc7 is a resident mitochondrial phosphatase essential for maintaining proper mitochondrial content and function. Newborn mice lacking Pptc7 exhibit aberrant mitochondrial protein phosphorylation, suffer from a range of metabolic defects, and fail to survive beyond one day after birth. Using an inducible knockout model, we reveal that loss of Pptc7 in adult mice causes marked reduction in mitochondrial mass concomitant with elevation of the mitophagy receptors Bnip3 and Nix. Consistently, Pptc7 -/- mouse embryonic fibroblasts (MEFs) exhibit a major increase in mitophagy that is reversed upon deletion of these receptors. Our phosphoproteomics analyses reveal a common set of elevated phosphosites between perinatal tissues, adult liver, and MEFsâ€" including multiple sites on Bnip3 and Nix. These data suggest that Pptc7 deletion causes mitochondrial dysfunction via dysregulation of several metabolic pathways and that Pptc7 may directly regulate mitophagy receptor function or stability. Overall, our work reveals a significant role for Pptc7 in the mitophagic response and furthers the growing notion that management of mitochondrial protein phosphorylation is essential for ensuring proper organelle content and function.
  42. Biochem Pharmacol. 2023 Mar 14. pii: S0006-2952(23)00094-1. [Epub ahead of print] 115503
      Conventional glucocorticoid (GC) treatment has a long-term influence on T-cell immunity, resulting in an increased risk of opportunistic infection after drug withdrawal. The underlying mechanisms remain ambiguous. This study demonstrated that long-term GC treatment induced persistent lymphopenia in patients with primary glomerular disease. GCs continuously suppressed the proportion of CD4+ T cells even after the daily dose was tapered down to the physiologic equivalences, leading to a significant decline of the CD4/CD8 ratio. Meanwhile, GCs impaired CD4+ T cell biology, leading to enhanced apoptotic cell death, reduced proliferative capacity, downregulated pro-inflammatory genes, and upregulated immunoregulatory genes. Specifically, GCs altered FOXP3 expression pattern in CD4+ T cells and favored their acquisition of an active T regulatory (Treg) cell phenotype with enhanced IL-10 production upon stimulation. Mechanistically, GCs tampered with the transcriptional regulation of mechanistic target of rapamycin complex 1 (mTORC1) pathway, resulting in an inhibitory impact on the signaling activity. Targeting mTORC1 signaling by siRNAs could sufficiently modify the viability of GC-exposed CD4+ T cells. By high-throughput sequencing of genome-wide DNA methylation and mRNA, we further uncovered a causal relationship between the altered DNA methylation level and transcription activity in a subset of mTORC1 pathway genes in long-term GC exposure. Taken together, this study reveals a novel regulation of mTORC1 signaling, which might dominate the long-term influence of GC on CD4+ T cell biology in a dose-independent manner.
    Keywords:  DNA methylation; FOXP3; Glucocorticoid; Mechanistic target of rapamycin complex 1; T cell
  43. Mol Metab. 2023 Mar 11. pii: S2212-8778(23)00039-X. [Epub ahead of print] 101705
      OBJECTIVE: In brown adipose tissue (iBAT), the balance between lipid/glucose uptake and lipolysis is tightly regulated by insulin signaling. Downstream of the insulin receptor, PDK1 and mTORC2 phosphorylate AKT, which activates glucose uptake and lysosomal mTORC1 signaling. The latter requires the late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR/Ragulator) complex, which serves to translate the nutrient status of the cell to the respective kinase. However, the role of LAMTOR in metabolically active iBAT has been elusive.METHODS: Using an AdipoqCRE-transgenic mouse line, we deleted LAMTOR2 (and thereby the entire LAMTOR complex) in adipose tissue (LT2 AKO). To examine the metabolic consequences, we performed metabolic and biochemical studies in iBAT isolated from mice housed at different temperatures (30 °C, room temperature and 5 °C), after insulin treatment, or in fasted and refed condition. For mechanistic studies, mouse embryonic fibroblasts (MEFs) lacking LAMTOR 2 were analyzed.
    RESULTS: Deletion of the LAMTOR complex in mouse adipocytes resulted in insulin-independent AKT hyperphosphorylation in iBAT, causing increased glucose and fatty acid uptake, which led to massively enlarged lipid droplets. As LAMTOR2 was essential for the upregulation of de novo lipogenesis, LAMTOR2 deficiency triggered exogenous glucose storage as glycogen in iBAT. These effects are cell autonomous, since AKT hyperphosphorylation was abrogated by PI3K inhibition or by deletion of the mTORC2 component Rictor in LAMTOR2-deficient MEFs.
    CONCLUSIONS: We identified a homeostatic circuit for the maintenance of iBAT metabolism that links the LAMTOR-mTORC1 pathway to PI3K-mTORC2-AKT signaling downstream of the insulin receptor.
    Keywords:  AKT; Brown adipose tissue; LAMTOR; Lysosome; Ragulator; mTORC1/2
  44. Mitochondrion. 2023 Mar 10. pii: S1567-7249(23)00027-2. [Epub ahead of print]70 8-19
      Huntington's disease (HD) is an autosomal dominant neurodegenerative disease. It is caused by the expansion of the CAG trinucleotide repeat sequence in the HTT gene. HD mainly manifests as involuntary dance-like movements and severe mental disorders. As it progresses, patients lose the ability to speak, think, and even swallow. Although the pathogenesis is unclear, studies have found that mitochondrial dysfunctions occupy an important position in the pathogenesis of HD. Based on the latest research advances, this review sorts out and discusses the role of mitochondrial dysfunction on HD in terms of bioenergetics, abnormal autophagy, and abnormal mitochondrial membranes. This review provides researchers with a more complete perspective on the mechanisms underlying the relationship between mitochondrial dysregulation and HD.
    Keywords:  Bioenergetics; Huntington's disease; Mitochondrial Dynamics; Mitochondrial dysfunction; Mitophagy
  45. Biochimie. 2023 Mar 11. pii: S0300-9084(23)00063-9. [Epub ahead of print]211 57-67
      The Wnt/β-catenin signaling pathway is a highly conserved pathway that is involved in cell development, proliferation, differentiation, apoptosis and autophagy. Among these processes, apoptosis and autophagy occur physiologically during host defense and the maintenance of intracellular homeostasis. Mounting evidence suggests that the crosstalk between Wnt/β-catenin-regulated apoptosis and autophagy has broad functional significance in various diseases. Herein, we summarize the recent studies in understanding the role of the Wnt/β-catenin signaling pathway in apoptosis and autophagy, and draw the following conclusions: a) For apoptosis, the regulation of Wnt/β-catenin is generally positive. However, a small amount of evidence indicates the presence of a negatively regulated relationship between Wnt/β-catenin and apoptosis; b) Wnt/β-catenin influences the occurrence and development of autophagy by regulating autophagy-related factors, and these factors in turn affect Wnt/β-catenin pathway; c) Wnt/β-catenin always balances the molecular damage caused by the crosstalk between autophagy and apoptosis in a compensatory manner. Understanding the specific role of the Wnt/β-catenin signaling pathway during different stages of autophagy and apoptosis may provide new insights into the progression of related diseases regulated by the Wnt/β-catenin signaling pathway.
    Keywords:  Apoptosis; Autophagy; Crosstalk; Wnt/β-catenin signaling
  46. Ecotoxicol Environ Saf. 2023 Mar 14. pii: S0147-6513(23)00276-2. [Epub ahead of print]255 114772
      Chronic fluoride exposure can cause developmental neurotoxicity, however the precise mechanisms remain unclear. To explore the mechanism of mitophagy in fluoride-induced developmental neurotoxicity, specifically focusing on PRKAA1 in regulating the PINK1/Parkin pathway, we established a Sprage Dawley rat model with continuous sodium fluoride (NaF) exposure and an NaF-treated SH-SY5Y cell model. We found that NaF exposure increased the levels of LC3-Ⅱ and p62, impaired autophagic degradation, and subsequently blocked autophagic flux. Additionally, NaF exposure increased the expression of PINK1, Parkin, TOMM-20, and Cyt C and cleaved PARP in vivo and in vitro, indicating NaF promotes mitophagy and neuronal apoptosis. Meanwhile, phosphoproteomics and western blot analysis showed that NaF treatment enhanced PRKAA1 phosphorylation. Remarkably, the application of both 3-methyladenosine (3-MA; autophagy inhibitor) and dorsomorphin (DM; AMPK inhibitor) suppressed NaF-induced neuronal apoptosis by restoring aberrant mitophagy. In addition, 3-MA attenuated an increase in p62 protein levels and NaF-induced autophagic degradation. Collectively, our findings indicated that NaF causes aberrant mitophagy via PRKAA1 in a PINK1/Parkin-dependent manner, which triggers neuronal apoptosis. Thus, regulating PRKAA1-activated PINK1/Parkin-dependent mitophagy may be a potential treatment for NaF-induced developmental neurotoxicity.
    Keywords:  Aberrant mitophagy; Autophagic degradation; Developmental neurotoxicity; Fluoride; Neuronal apoptosis; PRKAA1
  47. EMBO Rep. 2023 Mar 17. e56114
      Vesicular transport is a means of communication. While cells can communicate with each other via secretion of extracellular vesicles, less is known regarding organelle-to organelle communication, particularly in the case of mitochondria. Mitochondria are responsible for the production of energy and for essential metabolic pathways in the cell, as well as fundamental processes such as apoptosis and aging. Here, we show that functional mitochondria isolated from Saccharomyces cerevisiae release vesicles, independent of the fission machinery. We isolate these mitochondrial-derived vesicles (MDVs) and find that they are relatively uniform in size, of about 100 nm, and carry selective protein cargo enriched for ATP synthase subunits. Remarkably, we further find that these MDVs harbor a functional ATP synthase complex. We demonstrate that these vesicles have a membrane potential, produce ATP, and seem to fuse with naive mitochondria. Our findings reveal a possible delivery mechanism of ATP-producing vesicles, which can potentially regenerate ATP-deficient mitochondria and may participate in organelle-to-organelle communication.
    Keywords:  ATP synthase; membrane potential; mitochondria; mitochondrial-derived vesicles; protein distribution
  48. Ecotoxicol Environ Saf. 2023 Mar 11. pii: S0147-6513(23)00255-5. [Epub ahead of print]254 114751
      Arsenic, a serious environmental poison to human health, is widely distributed in nature. As the main organ of arsenic metabolism, liver is easily damaged. In the present study, we found that arsenic exposure can cause liver injury in vivo and in vitro, to date the underlying mechanism of which is yet unclear. Autophagy is a process that depends on lysosomes to degrade damaged proteins and organelles. Here, we reported that oxidative stress can be induced and then activated the SESTRIN2/AMPK/ULK1 pathway, damaged lysosomes, and finally induced necrosis upon arsenic exposure in rats and primary hepatocytes, which was characterized by lipidation of LC3II, the accumulation of P62 and the activation of RIPK1 and RIPK3. Similarly, lysosomes function and autophagy can be damaged under arsenic exposure, which can be alleviated after NAC treatment and aggravated by Leupeptin treatment in primary hepatocytes. Moreover, we also found that the transcription and protein expressions of necrotic-related indicators RIPK1 and RIPK3 in primary hepatocytes were decreased after P62 siRNA. Taken together, the results revealed that arsenic can induce oxidative stress, activate SESTRIN2/AMPK/ULK1 pathway to damage lysosomes and autophagy, and eventually induce necrosis to damage liver.
    Keywords:  Arsenic; Liver injury; Lysosomes; Necrosis; Oxidative stress; SESTRIN2/AMPK/ULK1 pathway
  49. Int J Biol Sci. 2023 ;19(4): 1192-1210
      Cisplatin is widely recommended in combination for the treatment of tumors, thus inevitably increasing the incidence of cisplatin-induced acute kidney injury. Mitophagy is a type of mitochondrial quality control mechanism that degrades damaged mitochondria and maintains cellular homeostasis. Ferroptosis, a new modality of programmed cell death, is characterized by iron-dependent phospholipid peroxidation and oxidative membrane damage. However, the role of mitophagy in ferroptosis in kidney disease is unclear. Here, we investigated the mechanism underlying both BNIP3-mediated and PINK1-PARK2-mediated mitophagy-induced attenuation of ferroptosis in cisplatin-induced acute kidney injury. The results showed that cisplatin induced mitochondrial injury, ROS release, intracellular iron accumulation, lipid peroxidation and ferroptosis in the kidney, which were aggravated in Bnip3 knockout, Pink1 knockout or Park2 knockout cisplatin-treated mice. Ferrstatin-1, a synthetic antioxidative ferroptosis inhibitor, rescued iron accumulation, lipid peroxidation and ferroptosis caused by inhibition of mitophagy. Thus, the present study elucidated a novel mechanism by which both BNIP3-mediated and PINK1-PARK2-mediated mitophagy protects against cisplatin-induced renal tubular epithelial cell ferroptosis through the ROS/HO1/GPX4 axis.
    Keywords:  Acute kidney injury; cisplatin nephrotoxicity; ferroptosis; mitophagy