bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2022‒09‒25
fifty-one papers selected by
Viktor Korolchuk
Newcastle University

  1. Brain Behav Immun Health. 2022 Nov;25 100510
      Autophagy is a lysosomal degradative pathway essential for maintaining cellular homeostasis and is also implicated in multiple aspects of both innate and adaptive immunity. Neuroinflammation, along with demyelination and axonal loss, is an important component of multiple sclerosis (MS). Induction of autophagy ameliorated disease progression in experimental autoimmune encephalomyelitis (EAE), a mouse model for MS, underlying a possible link between autophagy and MS pathology. However, it is still unclear how autophagy is affected during different stages of MS. Here, we show a decreased expression of the autophagy-related (ATG) genes during the acute phase of EAE development in mice as well as in mixed active/inactive lesions of post-mortem human MS brain tissues. Using spatial transcriptomics, we observed that this decreased ATG gene expression is most prominent in the core of mixed active/inactive lesions. Furthermore, we observed a hyper-activation of the mammalian target of rapamycin complex 1 (mTORC1) in lesions, which could inhibit both the initiation of autophagy and the transcription factors that regulate the expression of the ATG genes. Thus, based on our data, we propose a negative regulation of autophagy in MS, possibly through persistent mTORC1 activation, which depends on the lesion stage. Our results contribute to the understanding of the role of autophagy in different stages of MS pathology and point to the mTORC1 pathway as a potential modulator that likely regulates central nervous system (CNS) homeostasis and neuroinflammation in MS.
    Keywords:  Autophagy; Experimental autoimmune encephalomyelitis; Mammalian target of rapamycin complex 1; Multiple sclerosis; Neuroinflammation
  2. Exp Cell Res. 2022 Sep 15. pii: S0014-4827(22)00350-0. [Epub ahead of print] 113357
      Neurodegenerative diseases are progressive disorders of the nervous system primarily affecting the loss of neuronal cells present in the brain. Although most neurodegenerative cases are sporadic, some familial genes are found to be involved in the neurodegenerative diseases. The extensively studied parkin and pink1 gene products are known to be involved in the removal of damaged mitochondria via autophagy (mitophagy), a quality control process. If the function of any of these genes is somehow disrupted, accumulation of damaged mitochondria occurs in the forms of protein aggregates in the cytoplasm, leading to formation of the Lewy-bodies. Autophagy is an important catabolic process where the endosomal Rab proteins are seen to be involved. Rab11, an endosomal recycling protein, serves as an ATG9A carrier that helps in autophagosome formation and maturation. Earlier studies have reported that loss of Rab11 prevents the fusion of autophagosomes with the late endosomes hampering the autophagy pathway resulting in apoptosis of cells. In this study, we have emphasized on the importance and functional role of Rab11 in the molecular pathway of Parkin/Pink1 in Parkinson's disease.
    Keywords:  ATG9A; Alpha-synuclein; Autophagosomes; Dopaminergic neuron; Drosophila; Mitophagy; Neurodegenerative diseases; Parkin/Pink1; Parkinson's disease; Rab proteins; Rab11; Recycling endosome; Vesicle trafficking
  3. Biochemistry. 2022 Sep 21.
      In the scope of targeted protein degradation (TPD), proteolysis-targeting chimeras (PROTACs), leveraging the ubiquitin-proteasome system, have been extensively studied. However, they are limited to the degradation of soluble and membrane proteins, excluding the aggregated and extracellular proteins and dysfunctional organelles. As an alternative protein degradation pathway, lysosomes serve as a feasible tool for accessing these untouched proteins and/or organelles by proteosomes. Here, we focus on reviewing the emerging lysosome-mediated TPD, such as AUTAC, ATTEC, AUTOTAC, LYTAC, and MoDE-A. Intracellular targets, such as soluble and aggregated proteins and organelles, can be degraded via the autophagy-lysosome pathway. Extracellular targets, such as membrane proteins, and secreted extracellular proteins can be degraded via the endosome-lysosome pathway. In addition, we summarize the mechanism and regulation of autophagy, available methods and assays for monitoring the autophagy process, and the recently developed chemical probes for perturbing the autophagy pathways.
  4. Ann Neurol. 2022 Sep 23.
      OBJECTIVE: The mechanistic target of rapamycin (mTOR) kinase is one of the master coordinators of cellular stress responses, regulating metabolism, autophagy, and apoptosis. We recently reported that Staufen1 (STAU1), a stress granule (SG) protein, was overabundant in fibroblast cell lines from patients with spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis, frontotemporal degeneration, Huntington's, Alzheimer's, and Parkinson's diseases as well as animal models, and patient tissues. STAU1 overabundance is associated with mTOR hyperactivation and links SG formation with autophagy. Our objective was to determine the mechanism of mTOR regulation by STAU1.METHODS: We determined STAU1 abundance with disease- and chemical-induced cellular stressors in patient cells and animal models. We also used RNA binding assays to contextualize STAU1 interaction with MTOR mRNA.
    RESULTS: STAU1 and mTOR were overabundant in BAC-C9ORF72, ATXN2Q127 , and Thy1-TDP-43 transgenic mouse models. Reducing STAU1 levels in these mice normalized mTOR levels and activity and autophagy-related marker proteins. We also saw increased STAU1 levels in HEK293 cells transfected to express C9ORF72-relevant dipeptide repeats (DPRs). Conversely, DPR accumulations were not observed in cells treated by STAU1 RNAi. Overexpression of STAU1 in HEK293 cells increased mTOR levels through direct MTOR mRNA interaction, activating downstream targets and impairing autophagic flux. Targeting mTOR by rapamycin or RNAi normalized STAU1 abundance in a SCA2 cellular model.
    INTERPRETATION: STAU1 interaction with mTOR drives its hyperactivation and inhibits autophagic flux in multiple models of neurodegeneration. Staufen, therefore, constitutes a novel target to modulate mTOR activity, autophagy, and for the treatment of neurodegenerative diseases. This article is protected by copyright. All rights reserved.
  5. Autophagy. 2022 Sep 18.
      The acidic environment within lysosomes is maintained within a narrow pH range (pH 4.5-5.0) optimal for digesting autophagic cargo macromolecules so that the resulting building block metabolites can be reused. This pH homeostasis is a consequence of proton influx produced by a V-type H+-translocating ATPase (V-ATPase) and rapid proton efflux through an unidentified "leak" pathway. By performing a candidate expression screening, we discovered that the TMEM175 gene encodes a proton-activated, proton-selective channel (LyPAP) that is required for lysosomal H+ "leak" currents. The activity of LyPAP is most active when lysosomes are hyper-acidified, and cells lacking TMEM175 exhibit lysosomal hyper-acidification and impaired proteolytic degradation, both of which can be restored by optimizing lysosomal pH using pharmacological agents. Variants of TMEM175 that are associated with susceptibility to Parkinson disease (PD) cause a reduction in TMEM175-dependent LyPAP currents and lysosomal hyper-acidification. Hence, our studies not only reveal an essential H+-dissipating pathway in lysosomes, but also provide a molecular target to regulate pH-dependent lysosomal functions and associated pathologies.
    Keywords:  H+ leak; Proton channel; TMEM175; acidification; lysosome
  6. Autophagy. 2022 Sep 21. 1-16
      How macroautophagy/autophagy influences neurofilament (NF) proteins in neurons, a frequent target in neurodegenerative diseases and injury, is not known. NFs in axons have exceptionally long half-lives in vivo enabling formation of large stable supporting networks, but they can be rapidly degraded during Wallerian degeneration initiated by a limited calpain cleavage. Here, we identify autophagy as a previously unrecognized pathway for NF subunit protein degradation that modulates constitutive and inducible NF turnover in vivo. Levels of NEFL/NF-L, NEFM/NF-M, and NEFH/NF-H subunits rise substantially in neuroblastoma (N2a) cells after blocking autophagy either with the phosphatidylinositol 3-kinase (PtdIns3K) inhibitor 3-methyladenine (3-MA), by depleting ATG5 expression with shRNA, or by using both treatments. In contrast, activating autophagy with rapamycin significantly lowers NF levels in N2a cells. In the mouse brain, NF subunit levels increase in vivo after intracerebroventricular infusion of 3-MA. Furthermore, using tomographic confocal microscopy, immunoelectron microscopy, and biochemical fractionation, we demonstrate the presence of NF proteins intra-lumenally within autophagosomes (APs), autolysosomes (ALs), and lysosomes (LYs). Our findings establish a prominent role for autophagy in NF proteolysis. Autophagy may regulate axon cytoskeleton size and responses of the NF cytoskeleton to injury and disease.
    Keywords:  3-MA; Con A; MG115; calpeptin; proteasome; rapamycin
  7. Trends Cell Biol. 2022 Sep 20. pii: S0962-8924(22)00208-2. [Epub ahead of print]
      Biomolecular condensates are membraneless compartments formed by liquid-liquid phase separation. They can phase transit into gel-like and solid states. The amount and state of biomolecular condensates must be tightly regulated to maintain normal cellular function. Autophagy targets biomolecular condensates to the lysosome for degradation or other purposes, which we term biocondensophagy. In biocondensophagy, autophagy receptors recognize biomolecular condensates and target them to the autophagosome, the vesicle carrier of autophagy. Multiple types of autophagy receptors have been identified and they are specifically involved in targeting biomolecular condensates with different phase transition states. The receptors also organize the phase transition of biomolecular condensate to facilitate biocondensophagy. Here, we briefly discuss the latest discoveries regarding how biomolecular condensates are recognized by autophagy receptors.
    Keywords:  aggrephagy; autophagy; autophagy receptor; biocondensophagy; biomolecular condensate; phase separation; ubiquitin
  8. Methods Mol Biol. 2023 ;2566 37-43
      Lysosomes play key roles in different cellular processes such as autophagy, phagocytosis, and apoptosis. Lysosomal dysfunction is related to many diseases. Fluorescence lysosome staining strategy is valuable for the researches on the lysosome involvement in different pathological diagnosis. Here we describe fluorescence lysosome staining methods with carbon dots for the identification of lysosomes in living and fixed cells.
    Keywords:  Carbon dots; Cell imaging; Confocal fluorescence microscopy; Lysosome
  9. Cells. 2022 Sep 13. pii: 2847. [Epub ahead of print]11(18):
      In the basal, proliferative layer of healthy skin, the mTOR complex 1 (mTORC1) is activated, thus regulating proliferation while preventing differentiation. When cells leave the proliferative, basal compartment, mTORC1 signaling is turned off, which allows differentiation. Under inflammatory conditions, this switch is hijacked by cytokines and prevents proper differentiation. It is currently unknown how mTORC1 is regulated to mediate these effects on keratinocyte differentiation. In other tissues, mTORC1 activity is controlled through various pathways via the tuberous sclerosis complex (TSC). Thus, we investigated whether the TS complex is regulated by proinflammatory cytokines and contributes to the pathogenesis of psoriasis. TNF-α as well as IL-1β induced the phosphorylation of TSC2, especially on S939 via the PI3-K/AKT and MAPK pathway. Surprisingly, increased TSC2 phosphorylation could not be detected in psoriasis patients. Instead, TSC2 was strongly downregulated in lesional psoriatic skin compared to non-lesional skin of the same patients or healthy skin. In vitro inflammatory cytokines induced dissociation of TSC2 from the lysosome, followed by destabilization of the TS complex and degradation. Thus, we assume that in psoriasis, inflammatory cytokines induce strong TSC2 phosphorylation, which in turn leads to its degradation. Consequently, chronic mTORC1 activity impairs ordered keratinocyte differentiation and contributes to the phenotypical changes seen in the psoriatic epidermis.
    Keywords:  cytokines; inflammation; mTORC1; psoriasis; tuberous sclerosis complex
  10. Metabolites. 2022 Aug 25. pii: 788. [Epub ahead of print]12(9):
      Lysine (Lys) is essential for skeletal muscle growth and protein synthesis in mammals. However, the regulatory network underlying Lys-regulated skeletal muscle development is unknown. To determine whether any cross-talk occurs among mammalian targets of rapamycin complex 1 (mTORC1) and Lys in the regulation of muscle satellite cells (SCs) proliferation, we applied the treatment rapamycin (a mTORC1 inhibitor) and MHY1485 (a mTORC1 activator) on Lys-added or -deficient SCs. The results show Lys deprivation significantly decreases SCs viability, protein synthesis, and cell cycling, increases autophagy and apoptosis, and inhibits the mTORC1 signaling pathway. Restoration of Lys content significantly attenuates this effect. mTORC1 signaling pathway activation during Lys deprivation or mTORC1 signaling pathway inhibition during Lys addition attenuates the effect of Lys deprivation or addition on SCs viability, protein synthesis, cell cycling, autophagy, and apoptosis. In conclusion, Lys could improve SCs proliferation, and inhibit SCs apoptosis and autophagy, via the mTORC1 signaling pathway.
    Keywords:  apoptosis; autophagy; lysine; mTORC1 signaling pathway; proliferation; satellite cells
  11. Int J Biol Sci. 2022 ;18(14): 5329-5344
      It is emerging that autophagy-related proteins regulate the adaptive response to DNA damage in maintaining genome stability at multiple pathways. Here, we discuss recent insights into how autophagy-related proteins participate in DNA damage repair processes, influence chromosomal instability, and regulate the cell cycle through autophagy-dependent and independent actions. There is increasing awareness of the importance of these pathways mediated by autophagy-related proteins to DNA damage response (DDR), and disturbances in these regulatory connections may be linked to genomic instability participated in various human diseases, such as cancer and aging.
    Keywords:  Autophagy; Autophagy-related proteins; Genome Stability; Non-autophagic functions
  12. Methods Mol Biol. 2023 ;2566 141-147
      Autophagy is crucial for maintaining cellular homeostasis and its deregulation is involved in disease development, including cancer. The key players of chaperone-mediated autophagy (CMA), a particular selective subtype of autophagy, are HSPA8 and LAMP2A. Both proteins can be immunohistochemically detected in formalin-fixed paraffin-embedded (FFPE) tissue. LAMP2A is frequently overexpressed in a variety of cancers where it likely supports cancer cell survival and resistance to anti-cancer therapies in a context-dependent manner. Here we present the immunohistochemical staining protocol of antibodies against LAMP2A and HSPA8, using an automated staining system, suitable for routine diagnostics. Additionally, we also suggest a staining evaluation method.
    Keywords:  CMA; Chaperone-mediated autophagy; HSPA8; Immunohistochemistry; LAMP2A
  13. Am J Respir Cell Mol Biol. 2022 Sep 23.
      Idiopathic pulmonary fibrosis (IPF) is an age-related disease. Failure of the proteostasis network with age, including insufficient autophagy, contributes to the pathology of IPF. Mechanisms underlying autophagy disruption in IPF are unclear and may involve regulation of ubiquitin-specific protease (USP) by post-translational modifications. To expand our previous observation of low USP13 expression in IPF, this study evaluated the role of USP13 in age-related lung fibrosis. Here, we demonstrated that Usp13-deficient aged mice exhibited impaired autophagic activity and increased vulnerability to bleomycin-induced fibrosis. Mechanistically, USP13 interacted with and deubiquitinated Beclin 1, and Beclin 1 overexpression abolished the effects of USP13 disruption. Additionally, Beclin 1 inhibition resulted in insufficient autophagy and more severe lung fibrosis after BLM injury, consistent with the phenotype of aged Usp13-deficient mice. Collectively, we show a protective role of USP13 in age-related pulmonary fibrosis. Ageing-mediated USP13 loss impairs autophagic activity and facilitates lung fibrosis through Beclin 1 deubiquitination. Our findings support the notion that age-dependent dysregulation of autophagic regulators enhances vulnerability to lung fibrosis.
    Keywords:  Ubiquitin-specific protease 13, Idiopathic pulmonary fibrosis, Age, Autophagy, Beclin 1
  14. Autophagy. 2022 Sep 24. 1-18
      Macroautophagy/autophagy is a conserved recycling process that maintains cellular homeostasis during environmental stress. Autophagy is negatively regulated by TOR (target of rapamycin), a nutrient-regulated protein kinase that in plants is activated by several phytohormones, leading to increased growth. However, the detailed molecular mechanisms by which TOR integrates autophagy and hormone signaling are poorly understood. Here, we show that TOR modulates brassinosteroid (BR)-regulated plant growth and stress-response pathways. Active TOR was required for full BR-mediated growth in Arabidopsis thaliana. Autophagy was constitutively up-regulated upon blocking BR biosynthesis or signaling, and down-regulated by increasing the activity of the BR pathway. BIN2 (brassinosteroid-insensitive 2) kinase, a GSK3-like kinase functioning as a negative regulator in BR signaling, directly phosphorylated RAPTOR1B (regulatory-associated protein of TOR 1B), a substrate-recruiting subunit in the TOR complex, at a conserved serine residue within a typical BIN2 phosphorylation motif. Mutation of RAPTOR1B serine 916 to alanine, to block phosphorylation by BIN2, repressed autophagy and increased phosphorylation of the TOR substrate ATG13a (autophagy-related protein 13a). By contrast, this mutation had only a limited effect on growth. We present a model in which RAPTOR1B is phosphorylated and inhibited by BIN2 when BRs are absent, activating the autophagy pathway. When BRs signal and inhibit BIN2, RAPTOR1B is thus less inhibited by BIN2 phosphorylation. This leads to increased TOR activity and ATG13a phosphorylation, and decreased autophagy activity. Our studies define a new mechanism by which coordination between BR and TOR signaling pathways helps to maintain the balance between plant growth and stress responses.
    Keywords:  Arabidopsis thaliana; BIN2; BR response; TOR; autophagosome; signaling
  15. Technol Cancer Res Treat. 2022 Jan-Dec;21:21 15330338221114178
      Chaperone-mediated autophagy (CMA) plays an important role in regulating a variety of cellular functions by selectively degrading damaged or functional proteins in the cytoplasm. One of the cellular processes in which CMA participates is the oxidative stress response. Oxidative stress regulates CMA activity, while CMA protects cells from oxidative damage by degrading oxidized proteins and preventing the accumulation of excessive reactive oxygen species (ROS). Changes in CMA activity have been found in many human diseases, and oxidative stress is also involved. Therefore, understanding the interaction mechanism of ROS and CMA will provide new targets for disease treatment. In this review, we discuss the role of CMA in combatting oxidative stress during the development of different conditions, such as aging, neurodegeneration, liver diseases, infections, pulmonary disorders, and cancers.
    Keywords:  autophagy; immunity; metabolism; neurodegeneration; pulmonary disease; reactive oxygen species; tumor
  16. Nanoscale Adv. 2022 Sep 13. 4(18): 3676-3688
      Autophagy is an evolutionarily conserved catabolic process that can degrade cytoplasmic materials and recycle energy to maintain metabolite homeostasis in cells. Autophagy is closely related to various physiological or pathological processes. Macromolecular materials are widely used in drug delivery systems and disease treatments due to their intrinsic effects, such as altered pharmacokinetics and biodistribution. Interaction of autophagic flux or the signal pathway with macromolecules may cause autophagy inhibition or autophagy cell death. This review covers autophagy regulation pathways and macromolecular materials (including functional micelles, biodegradable and pH-sensitive polymers, biomacromolecules, dendrimers, coordination polymers, and hybrid nanoparticles) mediated autophagy modulation.
  17. Cells. 2022 Sep 14. pii: 2862. [Epub ahead of print]11(18):
      The control of exosome release is associated with numerous physiological and pathological activities, and that release is often indicative of health, disease, and environmental nutrient stress. Tuberous sclerosis complex (TSC) regulates the cell viability via the negative regulation of the mammalian target of rapamycin complex (mTORC1) during glucose deprivation. However, the mechanism by which viability of TSC-null cells is regulated by mTORC1 inhibition under glucose deprivation remains unclear. Here, we demonstrated that exosome release regulates cell death induced by glucose deprivation in TSC-null cells. The mTORC1 inhibition by rapamycin significantly increased the exosome biogenesis, exosome secretion, and cell viability in TSC-null cells. In addition, the increase in cell viability by mTORC1 inhibition was attenuated by two different types of inhibitors of exosome release under glucose deprivation. Taken together, we suggest that exosome release inhibition might be a novel way for regression of cell growth in TSC-null cells showing lack of cell death by mTORC1 inhibition.
    Keywords:  TSC; cell viability; exosome; glucose deprivation; mTORC1
  18. Nutrition. 2022 Jul 17. pii: S0899-9007(22)00210-6. [Epub ahead of print]103-104 111797
      Maintaining muscle quality throughout life is crucial to human health and well-being. Muscle is the most extensive form of protein storage in the human body; skeletal muscle mass is determined by the balance between muscle protein synthesis (MPS) and muscle protein breakdown (MPB). MPB provides amino acids needed by various organs; however, excessive MPB, especially with aging, may cause loss of muscle mass and a decline in motor function, even threatening life. The turnover of muscle protein is vital to the health of humans. Thus, although the study of MPS and MPB has theoretical and practical significance, the network that controls MPS is very complicated and we cannot discuss both MPS and MPB in a single review. Therefore, the aim of this review is to discuss the regulation of MPS, especially by amino acids. Amino acids regulate protein synthesis in cell and animal models, but compelling evidence for amino acids promoting protein synthesis in human muscles is ambiguous. Studies on the stimulation of human MPS by branched-chain amino acids (BCAAs) have been inconsistent. Amino acids other than BCAAs such as threonine and tryptophan may also have MPS-stimulating effects, and alternatives to BCAAs, such as β-hydroxy-β-methyl butyrate and branched-chain keto acids are also worthy of further investigation explore. Amino acids coordinate protein synthesis and degradation through the mechanistic target of rapamycin complex 1 (mTORC1); however, the amino acid-mTORC1-protein synthesis pathway is complex, and new insights into amino acid control continue to emerge. Understanding how amino acids control MPS is of forward-looking significance for treating muscle mass loss during human aging.
    Keywords:  Amino acid; Muscle protein synthesis; mTORC1
  19. Methods Mol Biol. 2023 ;2566 133-139
      Autophagy is a highly conserved cellular mechanism of "self-digestion," ensuring cellular homeostasis and playing a role in many diseases including cancer. As a stress response mechanism, it may also be involved in cellular response to therapy. LC3 and Sequestosome 1 (p62/SQSTM1) are among the most widely used markers to monitor autophagy and can be visualized in formalin-fixed and paraffin-embedded tissue by immunohistochemistry. Here we describe a validated staining protocol using an automated staining system available in many routine pathology laboratories, enabling high-throughput staining under standardized conditions.
    Keywords:  Autophagy; Immunohistochemistry; LC3; Sequestosome 1; p62/SQSTM1
  20. Antioxidants (Basel). 2022 Sep 14. pii: 1807. [Epub ahead of print]11(9):
      Selective regional iron accumulation is a hallmark of several neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. The underlying mechanisms of neuronal iron dyshomeostasis have been studied, mainly in a gene-by-gene approach. However, recent high-content phenotypic screens using CRISPR/Cas9-based gene perturbations allow for the identification of new pathways that contribute to iron accumulation in neuronal cells. Herein, we perform a bioinformatic analysis of a CRISPR-based screening of lysosomal iron accumulation and the functional genomics of human neurons derived from induced pluripotent stem cells (iPSCs). Consistent with previous studies, we identified mitochondrial electron transport chain dysfunction as one of the main mechanisms triggering iron accumulation, although we substantially expanded the gene set causing this phenomenon, encompassing mitochondrial complexes I to IV, several associated assembly factors, and coenzyme Q biosynthetic enzymes. Similarly, the loss of numerous genes participating through the complete macroautophagic process elicit iron accumulation. As a novelty, we found that the impaired synthesis of glycophosphatidylinositol (GPI) and GPI-anchored protein trafficking also trigger iron accumulation in a cell-autonomous manner. Finally, the loss of critical components of the iron transporters trafficking machinery, including MON2 and PD-associated gene VPS35, also contribute to increased neuronal levels. Our analysis suggests that neuronal iron accumulation can arise from the dysfunction of an expanded, previously uncharacterized array of molecular pathways.
    Keywords:  CRISPR interference; autophagy; bioinformatics; glycosylphosphatidylinositol; iron; mitochondria; neurons
  21. Nanoscale Adv. 2021 Mar 23. 3(6): 1656-1673
      In recent years, with the increasing understanding of the role of autophagy in tumorigenesis and development, a steady stream of studies have demonstrated that both excessive induction and inhibition of autophagy could effectively improve the therapeutic efficacy against tumors during cytotoxic or molecularly targeted drug therapy. Among them, autophagy inhibition mediated by nanomaterials has become an appealing notion in nanomedicine therapeutics, since it can be exploited as an effective adjuvant in chemotherapy or as a potential anti-tumor agent. Herein, we constructed a pH-sensitive nanoplatform loaded with epirubicin (EPI) (mPEG-b-P(DPA-b-DMAEMA)/EPI), enabling effective autophagy inhibition in the process of tumor-targeting therapy and further sensitized the tumors to EPI. It was found that polycationic nanomicelles (PEDD-Ms) displayed specific localization in lysosomes after entering tumor cells and caused the impairment of lysosomal degradation capacity through lysosomal alkalization in a dose-dependent manner. HepG2 cells treated with PEDD-Ms displayed a large-scale accumulation of autophagosomes and LC3 (an autophagosome marker protein), and the degradation of the autophagy substrate p62 was also blocked, which indicated that these functional nanomicelles could significantly inhibit autophagy. Meanwhile, the typical morphological characteristics of autophagosomes were directly visualized by TEM. In vivo results also showed that the tumor-targeted and autophagy inhibition-associated nanoplatform therapy could effectively improve the therapeutic efficiency of EPI, which may be partially attributed to the fact that autophagy inhibition could enhance the sensitivity of tumor cells to EPI. Overall, we revealed the effect of polycationic nanomicelles on autophagic processes in tumor cells and explored their possible molecular mechanism, also considering the synergistic outcome between autophagy mediated by nanomaterials and chemotherapeutic drugs to improve the therapeutic effect on tumors.
  22. Cells. 2022 Sep 15. pii: 2886. [Epub ahead of print]11(18):
      Charcot-Marie-Tooth (CMT) disease is the most common inherited neurodegenerative disorder with selective degeneration of peripheral nerves. Despite advances in identifying CMT-causing genes, the underlying molecular mechanism, particularly of selective degeneration of peripheral neurons remains to be elucidated. Since peripheral neurons are sensitive to multiple stresses, we hypothesized that daily repeated stress might be an essential contributor to the selective degeneration of peripheral neurons induced by CMT-causing mutations. Here, we mainly focused on the biological effects of the dominant missense mutation (S135F) in the 27-kDa small heat-shock protein HSPB1 under repeated heat shock. HSPB1S135F presented hyperactive binding to both α-tubulin and acetylated α-tubulin during repeated heat shock when compared with the wild type. The aberrant interactions with tubulin prevented microtubule-based transport of heat shock-induced misfolded proteins for the formation of perinuclear aggresomes. Furthermore, the transport of autophagosomes along microtubules was also blocked. These results indicate that the autophagy pathway was disrupted, leading to an accumulation of ubiquitinated protein aggregates and a significant decrease in cell adaptation to repeated stress. Our findings provide novel insights into the molecular mechanisms of HSPB1S135F-induced selective degeneration of peripheral neurons and perspectives for targeting autophagy as a promising therapeutic strategy for CMT neuropathy.
    Keywords:  Charcot-Marie-Tooth; HSPB1; autophagy; axonal transport; misfolded proteins; repeated stress
  23. Metabolites. 2022 Sep 17. pii: 879. [Epub ahead of print]12(9):
      Autophagy is an important cellular process, involving the transportation of cytoplasmic contents in the double membrane vesicles to lysosomes for degradation. Autophagy disorder contributes to many diseases, such as immune dysfunction, cancers and nervous system diseases. Hydrogen sulfide (H2S) is a volatile and toxic gas with a rotten egg odor. For a long time, it was considered as an environmental pollution gas. In recent years, H2S is regarded as the third most important gas signal molecule after NO and CO. H2S has a variety of biological functions and can play an important role in a variety of physiological and pathological processes. Increasingly more evidences show that H2S can regulate autophagy to play a protective role in the nervous system, but the mechanism is not fully understood. In this review, we summarize the recent literatures on the role of H2S in the pathological process of the nervous system by regulating autophagy, and analyze the mechanism in detail, hoping to provide the reference for future related research.
    Keywords:  Parkinson’s disease; autophagy; cognitive impairment; diabetes depression; hydrogen sulfide; traumatic brain injury
  24. Cells. 2022 Sep 16. pii: 2897. [Epub ahead of print]11(18):
      AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolic and immune functions mainly through the inhibition of the mechanistic target of rapamycin (mTOR)-dependent anabolic pathways and the activation of catabolic processes such as autophagy. The AMPK/mTOR signaling pathway and autophagy markers were analyzed by immunoblotting in blood mononuclear cells of 20 healthy control subjects and 23 patients with an acute demyelinating form of Guillain-Barré syndrome (GBS). The activation of the liver kinase B1 (LKB1)/AMPK/Raptor signaling axis was significantly reduced in GBS compared to control subjects. In contrast, the phosphorylated forms of mTOR activator AKT and mTOR substrate 4EBP1, as well as the levels of autophagy markers LC3-II, beclin-1, ATG5, p62/sequestosome 1, and NBR1 were similar between the two groups. The downregulation of LKB1/AMPK signaling, but not the activation status of the AKT/mTOR/4EBP1 pathway or the levels of autophagy markers, correlated with higher clinical activity and worse outcomes of GBS. A retrospective study in a diabetic cohort of GBS patients demonstrated that treatment with AMPK activator metformin was associated with milder GBS compared to insulin/sulphonylurea therapy. In conclusion, the impairment of the LKB1/AMPK pathway might contribute to the development/progression of GBS, thus representing a potential therapeutic target in this immune-mediated peripheral polyneuropathy.
    Keywords:  AMP-activated protein kinase; Guillain–Barré syndrome; autophagy; mTOR; metformin; peripheral blood mononuclear cells
  25. Life (Basel). 2022 Sep 08. pii: 1396. [Epub ahead of print]12(9):
      Glycogen storage diseases (GSDs) are rare metabolic monogenic disorders characterized by an excessive accumulation of glycogen in the cell. However, monogenic disorders are not simple regarding genotype-phenotype correlation. Genes outside the major disease-causing locus could have modulatory effect on GSDs, and thus explain the genotype-phenotype inconsistencies observed in these patients. Nowadays, when the sequencing of all clinically relevant genes, whole human exomes, and even whole human genomes is fast, easily available and affordable, we have a scientific obligation to holistically analyze data and draw smarter connections between genotype and phenotype. Recently, the importance of glycogen-selective autophagy for the pathophysiology of disorders of glycogen metabolism have been described. Therefore, in this manuscript, we review the potential role of genes involved in glycogen-selective autophagy as modifiers of GSDs. Given the small number of genes associated with glycogen-selective autophagy, we also include genes, transcription factors, and non-coding RNAs involved in autophagy. A cross-link with apoptosis is addressed. All these genes could be analyzed in GSD patients with unusual discrepancies between genotype and phenotype in order to discover genetic variants potentially modifying their phenotype. The discovery of modifier genes related to glycogen-selective autophagy and autophagy will start a new chapter in understanding of GSDs and enable the usage of autophagy-inducing drugs for the treatment of this group of rare-disease patients.
    Keywords:  apoptosis; autophagy; autophagy-inducing drugs; glycogen storage diseases; glycogen-selective autophagy; modifier genes
  26. Alzheimers Dement. 2022 Sep 21.
      INTRODUCTION: Lysosomes are degradative organelles that maintain cellular homeostasis and protein quality control. Transcription factor EB (TFEB)-mediated lysosome biogenesis enhances lysosome-dependent degradation and alleviates neurodegenerative diseases, but the mechanisms underlying TFEB regulation and modification are still poorly understood.METHODS: By screening novel small-molecule compounds, we identified a group of lysosome-enhancing compounds (LYECs) that promote TFEB activation and lysosome biogenesis.
    RESULTS: One of these compounds, LH2-051, significantly inhibited the function of the dopamine transporter (DAT) and subsequently promoted lysosome biogenesis. We uncovered cyclin-dependent kinase 9 (CDK9) as a novel regulator of DAT-mediated lysosome biogenesis and identified six novel CDK9-phosphorylated sites on TFEB. We observed that signal transduction by the DAT-CDK9-TFEB axis occurs on lysosomes. Finally, we found that LH2-051 enhanced the degradation of amyloid beta plaques and improved the memory of amyloid precursor protein (APP)/Presenilin 1 (PS1) mice.
    DISCUSSION: We identified the DAT-CDK9-TFEB signaling axis as a novel regulator of lysosome biogenesis. Our study sheds light on the mechanisms of protein quality control under pathophysiological conditions.
    Keywords:  Alzheimer's disease; CDK9; TFEB; dopamine transporter; lysosome biogenesis
  27. Int J Immunopathol Pharmacol. 2022 Jan-Dec;36:36 3946320221125090
      Burn healing should be regarded as a dynamic process consisting of two main, interrelated phases: (a) the inflammatory phase when neutrophils and monocytes infiltrate the injury site, through localized vasodilation and fluid extravasation, and (b) the proliferative-remodeling phase, which represents a key event in wound healing. In the skin, both canonical autophagy (induced by starvation, oxidative stress, and environmental aggressions) and non-canonical or selective autophagy have evolved to play a discrete, but, essential, "housekeeping" role, for homeostasis, immune tolerance, and survival. Experimental data supporting the pro-survival roles of autophagy, highlighting its Yang, luminous and positive feature of this complex but insufficient explored molecular pathway, have been reported. Autophagic cell death describes an "excessive" degradation of important cellular components that are necessary for normal cell function. This deadly molecular mechanism brings to light the darker, concealed, Yin feature of autophagy. Autophagy seems to perform dual, conflicting roles in the angiogenesis context, revealing once again, its Yin-Yang features. Autophagy with its Yin-Yang features remains the shadow player, able to decide quietly whether the cell survives or dies.
    Keywords:  autophagy; burn wound healing; burns; oxidative stress
  28. Biochem J. 2022 Sep 30. 479(18): 1917-1940
      As first demonstrated in budding yeast (Saccharomyces cerevisiae), all eukaryotic cells contain two, distinct multi-component protein kinase complexes that each harbor the TOR (Target Of Rapamycin) polypeptide as the catalytic subunit. These ensembles, dubbed TORC1 and TORC2, function as universal, centrally important sensors, integrators, and controllers of eukaryotic cell growth and homeostasis. TORC1, activated on the cytosolic surface of the lysosome (or, in yeast, on the cytosolic surface of the vacuole), has emerged as a primary nutrient sensor that promotes cellular biosynthesis and suppresses autophagy. TORC2, located primarily at the plasma membrane, plays a major role in maintaining the proper levels and bilayer distribution of all plasma membrane components (sphingolipids, glycerophospholipids, sterols, and integral membrane proteins). This article surveys what we have learned about signaling via the TORC2 complex, largely through studies conducted in S. cerevisiae. In this yeast, conditions that challenge plasma membrane integrity can, depending on the nature of the stress, stimulate or inhibit TORC2, resulting in, respectively, up-regulation or down-regulation of the phosphorylation and thus the activity of its essential downstream effector the AGC family protein kinase Ypk1. Through the ensuing effect on the efficiency with which Ypk1 phosphorylates multiple substrates that control diverse processes, membrane homeostasis is maintained. Thus, the major focus here is on TORC2, Ypk1, and the multifarious targets of Ypk1 and how the functions of these substrates are regulated by their Ypk1-mediated phosphorylation, with emphasis on recent advances in our understanding of these processes.
    Keywords:  contact sites; lipids; membrane proteins; phosphorylation; plasma membrane; protein kinases
  29. Nature. 2022 Sep 21.
      Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus1. Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs)2-4. For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)-the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism.
  30. Genes Dis. 2022 Nov;9(6): 1594-1607
      Autophagy is an evolutionarily conserved process where long-lived and damaged or-ganelles are degraded. Autophagy has been widely associated with several ageing-process as well in diseases such as neurodegeneration, cancer and fibrosis, and is now being utilised as a target in these diseases. Idiopathic pulmonary fibrosis (IPF) is a progressive, interstitial lung disease with limited treatment options available. It is characterised by abnormal extracellular matrix (ECM) deposition by activated myofibroblasts. It is understood that repetitive micro-injuries to aged-alveolar epithelium combined with genetic factors drive the disease. Several groups have demonstrated that autophagy is altered in IPF although whether autophagy has a protective effect or not is yet to be determined. Autophagy has also been shown to influence many other processes including epithelial-mesenchymal transition (EMT) and endothelial-mesenchymal transition (EndMT) which are known to be key in the pathogenesis of IPF. In this review, we summarise the findings of evidence of altered autophagy in IPF lungs, as well as examine its roles within lung fibrosis. Given these findings, together with the growing use of autophagy manipulation in a clinical setting, this is an exciting area for further research in the study of lung fibrosis.
    Keywords:  Ageing; Autophagy; EMT; Fibrosis; IPF
  31. Proc Natl Acad Sci U S A. 2022 Sep 27. 119(39): e2209823119
      Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.
    Keywords:  autophagosome biogenesis; autophagy; cryo-electron tomography; membrane structure; organelle contact sites
  32. Autophagy. 2022 Sep 21.
      Macroautophagy/autophagy, an evolutionarily conserved degradative process essential for cell homeostasis and development in eukaryotes, involves autophagosome formation and fusion with a lysosome/vacuole. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play important roles in regulating autophagy in mammals and yeast, but relatively little is known about SNARE function in plant autophagy. Here we identified and characterized two Arabidopsis SNAREs, AT4G15780/VAMP724 and AT1G04760/VAMP726, involved in plant autophagy. Phenotypic analysis showed that mutants of VAMP724 and VAMP726 are sensitive to nutrient-starved conditions. Live-cell imaging on mutants of VAMP724 and VAMP726 expressing YFP-ATG8e showed the formation of abnormal autophagic structures outside the vacuoles and compromised autophagic flux. Further immunogold transmission electron microscopy and electron tomography (ET) analysis demonstrated a direct connection between the tubular autophagic structures and the endoplasmic reticulum (ER) in vamp724-1 vamp726-1 double mutants. Further transient co-expression, co-immunoprecipitation and double immunogold TEM analysis showed that ATG9 (autophagy related 9) interacts and colocalizes with VAMP724 and VAMP726 in ATG9-positive vesicles during autophagosome formation. Taken together, VAMP724 and VAMP726 regulate autophagosome formation likely working together with ATG9 in Arabidopsis.
    Keywords:  ATG9; Arabidopsis; SNARE; VAMP724; VAMP726; autophagy
  33. Aging Cell. 2022 Sep 18. e13713
      Autophagy is essential for protein quality control and regulation of the functional proteome. Failure of autophagy pathways with age contributes to loss of proteostasis in aged organisms and accelerates the progression of age-related diseases. In this work, we show that activity of endosomal microautophagy (eMI), a selective type of autophagy occurring in late endosomes, declines with age and identify the sub-proteome affected by this loss of function. Proteomics of late endosomes from old mice revealed an aberrant glycation signature for Hsc70, the chaperone responsible for substrate targeting to eMI. Age-related Hsc70 glycation reduces its stability in late endosomes by favoring its organization into high molecular weight protein complexes and promoting its internalization/degradation inside late endosomes. Reduction of eMI with age associates with an increase in protein secretion, as late endosomes can release protein-loaded exosomes upon plasma membrane fusion. Our search for molecular mediators of the eMI/secretion switch identified the exocyst-RalA complex, known for its role in exocytosis, as a novel physiological eMI inhibitor that interacts with Hsc70 and acts directly at the late endosome membrane. This inhibitory function along with the higher exocyst-RalA complex levels detected in late endosomes from old mice could explain, at least in part, reduced eMI activity with age. Interaction of Hsc70 with components of the exocyst-RalA complex places this chaperone in the switch from eMI to secretion. Reduced intracellular degradation in favor of extracellular release of undegraded material with age may be relevant to the spreading of proteotoxicity associated with aging and progression of proteinopathies.
    Keywords:  aging; autophagy; chaperones; endosomal microautophagy; exocyst complex; late endosomes; protein secretion; proteostasis
  34. Nat Commun. 2022 Sep 17. 13(1): 5469
      Oncogenic RAS mutations are common in multiple myeloma (MM), an incurable malignancy of plasma cells. However, the mechanisms of pathogenic RAS signaling in this disease remain enigmatic and difficult to inhibit therapeutically. We employ an unbiased proteogenomic approach to dissect RAS signaling in MM. We discover that mutant isoforms of RAS organize a signaling complex with the amino acid transporter, SLC3A2, and MTOR on endolysosomes, which directly activates mTORC1 by co-opting amino acid sensing pathways. MM tumors with high expression of mTORC1-dependent genes are more aggressive and enriched in RAS mutations, and we detect interactions between RAS and MTOR in MM patient tumors harboring mutant RAS isoforms. Inhibition of RAS-dependent mTORC1 activity synergizes with MEK and ERK inhibitors to quench pathogenic RAS signaling in MM cells. This study redefines the RAS pathway in MM and provides a mechanistic and rational basis to target this mode of RAS signaling.
  35. Med Sci (Basel). 2022 Sep 10. pii: 51. [Epub ahead of print]10(3):
      The polyamines putrescine, spermidine and spermine are nutrient-like polycationic molecules involved in metabolic processes and signaling pathways linked to cell growth and cancer. One important pathway is the PI3K/Akt pathway where studies have shown that polyamines mediate downstream growth effects. Downstream of PI3K/Akt is the mTOR signaling pathway, a nutrient-sensing pathway that regulate translation initiation through 4EBP1 and p70S6K phosphorylation and, along with the PI3K/Akt, is frequently dysregulated in breast cancer. In this study, we investigated the effect of intracellular polyamine modulation on mTORC1 downstream protein and general translation state in two breast cancer cell lines, MCF-7 and MDA-MB-231. The effect of mTORC1 pathway inhibition on the growth and intracellular polyamines was also measured. Results showed that polyamine modulation alters 4EBP1 and p70S6K phosphorylation and translation initiation in the breast cancer cells. mTOR siRNA gene knockdown also inhibited cell growth and decreased putrescine and spermidine content. Co-treatment of inhibitors of polyamine biosynthesis and mTORC1 pathway induced greater cytotoxicity and translation inhibition in the breast cancer cells. Taken together, these data suggest that polyamines promote cell growth in part through interaction with mTOR pathway. Similarly intracellular polyamine content appears to be linked to mTOR pathway regulation. Finally, dual inhibition of polyamine and mTOR pathways may provide therapeutic benefits in some breast cancers.
    Keywords:  4E-BP1; growth; mTOR pathway; p70SK1 and translation initiation; phosphorylation; polyamines
  36. Cells. 2022 Sep 12. pii: 2839. [Epub ahead of print]11(18):
      Although autophagy regulates the quality and quantity of cellular compartments, the regulatory mechanisms underlying peroxisomal autophagy (pexophagy) remain largely unknown. In this study, we identified several BRD4 inhibitors, including molibresib, a novel pexophagy inducer, via chemical library screening. Treatment with molibresib promotes loss of peroxisomes selectively, but not mitochondria, ER, or Golgi apparatus in HeLa cells. Consistently, depletion of BRD4 expression also induced pexophagy in RPE cells. In addition, the inhibition of BRD4 by molibresib increased autophagic degradation of peroxisome ATG7-dependency. We further found that molibresib produced reactive oxygen species (ROS), which potentiates ATM activation. Inhibition of ROS or ATM suppressed the loss of peroxisomes in molibresib-treated cells. Taken together, our data suggest that inhibition of BRD4 promotes pexophagy by increasing ROS and ATM activation.
    Keywords:  BRD4; ROS; molibresib; peroxisome; pexophagy
  37. Stem Cell Reports. 2022 Sep 09. pii: S2213-6711(22)00419-2. [Epub ahead of print]
      We have developed an efficient approach to generate functional induced dopaminergic (DA) neurons from adult human dermal fibroblasts. When performing DA neuronal conversion of patient fibroblasts with idiopathic Parkinson's disease (PD), we could specifically detect disease-relevant pathology in these cells. We show that the patient-derived neurons maintain age-related properties of the donor and exhibit lower basal chaperone-mediated autophagy compared with healthy donors. Furthermore, stress-induced autophagy resulted in an age-dependent accumulation of macroautophagic structures. Finally, we show that these impairments in patient-derived DA neurons leads to an accumulation of phosphorylated alpha-synuclein, the classical hallmark of PD pathology. This pathological phenotype is absent in neurons generated from induced pluripotent stem cells from the same patients. Taken together, our results show that direct neural reprogramming can be used for obtaining patient-derived DA neurons, which uniquely function as a cellular model to study age-related pathology relevant to idiopathic PD.
    Keywords:  Parkinson's disease; alpha-synuclein; autophagy; direct neural reprogramming; dopaminergic neurons; induced neurons; induced pluripotent stem cells
  38. Sci Rep. 2022 Sep 23. 12(1): 15870
      Leucine (Leu) regulates protein synthesis and degradation via activation of mammalian target of rapamycin complex 1 (mTORC1). Glutamine (Gln) synergistically promotes mTORC1 activation with Leu via glutaminolysis and Leu absorption via an antiporter. However, Gln has also been shown to inhibit mTORC1 activity. To resolve this paradox, we aimed to elucidate the effects of Gln on Leu-mediated mTORC1 activation. We administered Leu, Gln, tryptophan, Leu + Gln, or Leu + tryptophan to mice after 24-h fasting. The mice were then administered puromycin to evaluate protein synthesis and the gastrocnemius muscle was harvested 30 min later. Phosphorylated eukaryotic initiation factor 4E-binding protein 1, 70-kDa ribosomal protein S6 kinase 1, and Unc-51 like kinase 1 levels were the highest in the Leu + Gln group and significantly increased compared with those in the control group; however, Gln alone did not increase the levels of phosphorylated proteins. No difference in glutamate dehydrogenase activity was observed between the groups. Leu concentrations in the gastrocnemius muscle were similar in the Leu-intake groups. Our study highlights a novel mechanism underlying the promotive effect of Gln on Leu-mediated mTORC1 activation, providing insights into the pathway through which amino acids regulate muscle protein metabolism.
  39. J Fungi (Basel). 2022 Sep 18. pii: 974. [Epub ahead of print]8(9):
      Camellia oleifera is one of the most valuable woody edible-oil crops, and anthracnose seriously afflicts its yield and quality. We recently showed that the CfSnt2 regulates the pathogenicity of Colletotrichum fructicola, the dominant causal agent of anthracnose on C. oleifera. However, the molecular mechanisms of CfSnt2-mediated pathogenesis remain largely unknown. Here, we found that CfSnt2 is localized to the nucleus to regulate the deacetylation of histone H3. The further transcriptomic analysis revealed that CfSnt2 mediates the expression of global genes, including most autophagy-related genes. Furthermore, we provided evidence showing that CfSnt2 negatively regulates autophagy and is involved in the responses to host-derived ROS and ER stresses. These combined functions contribute to the pivotal roles of CfSnt2 on pathogenicity. Taken together, our studies not only illustrate how CfSnt2 functions in the nucleus, but also link its roles on the autophagy and responses to host-derived stresses with pathogenicity in C. fructicola.
    Keywords:  C. fructicola; autophagy; histone deacetylation; pathogenicity
  40. PLoS Genet. 2022 Sep 20. 18(9): e1010400
      Women's reproductive cessation is the earliest sign of human aging and is caused by decreasing oocyte quality. Similarly, C. elegans' reproduction declines in mid-adulthood and is caused by oocyte quality decline. Aberrant mitochondrial morphology is a hallmark of age-related dysfunction, but the role of mitochondrial morphology and dynamics in reproductive aging is unclear. We examined the requirements for mitochondrial fusion and fission in oocytes of both wild-type worms and the long-lived, long-reproducing insulin-like receptor mutant daf-2. We find that normal reproduction requires both fusion and fission, but that daf-2 mutants utilize a shift towards fission, but not fusion, to extend their reproductive span and oocyte health. daf-2 mutant oocytes' mitochondria are punctate (fissioned) and this morphology is primed for mitophagy, as loss of the mitophagy regulator PINK-1 shortens daf-2's reproductive span. daf-2 mutants maintain oocyte mitochondria quality with age at least in part through a shift toward punctate mitochondrial morphology and subsequent mitophagy. Supporting this model, Urolithin A, a metabolite that promotes mitophagy, extends reproductive span in wild-type mothers-even in mid-reproduction-by maintaining youthful oocytes with age. Our data suggest that promotion of mitophagy may be an effective strategy to maintain oocyte health with age.
  41. Am J Cancer Res. 2022 ;12(8): 3760-3779
      Osteosarcoma (OS) is the most common primary malignant bone tumor in adolescents and children. The pathogenesis of this disease is complex and the mechanisms involved have not been fully elucidated. Thioredoxin-interacting protein (TXNIP), as a member of the α-rhodopsin inhibitory protein family, can combine with thioredoxin to inhibit its antioxidant function. This process inhibits glucose absorption and metabolic rearrangement necessary for the regulation of cellular growth. In recent years, TXNIP has emerged as a new candidate target for tumors. However, the biological function and role of TXNIP in OS remains unclear. This study confirmed the low expression of TXNIP in OS tissues and cells, which was significantly related to the poor survival rate and clinical characteristics of patients with OS. Various cell phenotype experiments have shown that TXNIP inhibits the proliferation, migration, and invasion of OS cells, and promotes their apoptosis. Further studies found that the tumor suppressor effect of TXNIP was mediated by upregulating DNA damage-inducible transcript 4 (DDIT4) and inhibiting the phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) downstream substrate S6. Based on the above, our study explored the key role of TXNIP/DDIT4/mTORC1 suppression as a regulatory axis in the progression of OS, and laid the foundation for precise targeted therapy for OS.
    Keywords:  DDIT4; Osteosarcoma; TXNIP; mTORC1; tumor progression
  42. J Mol Endocrinol. 2022 Sep 01. pii: JME-22-0060. [Epub ahead of print]
      Proteolytic cleavage of thyroglobulin (Tg) for thyroid hormone (TH) liberation is followed by TH release from thyroid follicles into the circulation, enabled by TH transporters. The existence of a functional link between Tg-processing cathepsin proteases and TH transporters has been shown to be independent of the hypothalamus-pituitary-thyroid axis. Thus, lack of cathepsin K, combined with genetic defects in the TH transporters Mct8 and Mct10, i.e. the Ctsk‑/-/Mct8-/y/Mct10-/- genotype, results in persistent Tg proteolysis due to autophagy induction. Because amino acid transport by L-type amino acid transporter 2 (Lat2) has been described to regulate autophagy, we asked whether Lat2 availability is affected in Ctsk‑/-/Mct8-/y/Mct10-/- thyroid glands. Our data revealed that while mRNA amounts and sub-cellular localization of Lat2 remained unaltered in thyroid tissue of Ctsk‑/-/Mct8-/y/Mct10-/- mice in comparison to wild-type (WT) controls, the Lat2 protein amounts were significantly reduced. These data suggest a direct link between Lat2 function and autophagy induction in Ctsk‑/-/Mct8-/y/Mct10‑/‑ mice. Indeed, thyroid tissue of Lat2-/- mice showed enhanced endo-lysosomal cathepsin activities, increased autophagosome formation, and enhanced autophagic flux. Collectively, these results suggest a mechanistic link between insufficient Lat2 protein function and autophagy induction in the thyroid gland of male mice.
  43. EMBO Rep. 2022 Sep 19. e55209
      The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here, we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic vesicle content predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineage-agnostic marker for the prospective identification of facultative stem cells.
    Keywords:  autophagy; facultative stem cell; organoid formation; paligenosis; regeneration
  44. Clin Exp Rheumatol. 2022 Sep 22.
      Autophagy, a vital mechanism restricted in tissues, exerts its cytoprotective role through the degradation mechanism of damaged or aging organelles, harmful protein aggregates and intracellular pathogens, followed by energy furnishment. However, dysfunctional autophagy is associated with the development of autoimmune diseases such as rheumatoid arthritis (RA). In pathological conditions, autophagy may be involved in the maturation, survival and proliferation of various immune and non-immune cells and plays a key role in the pathogenesis of RA. Furthermore, autophagy appears to be involved in the citrullination of T lymphocytes and the presentation of citrullinated peptides, which are presented to T lymphocytes via the major histocompatibility complex, causing immune responses and chronic inflammation, as well as bone and cartilage destruction associated with apoptosis resistance of RA fibroblast-like synoviocyte (RAFLS) and osteoclastogenesis. In this review, we have summarised the roles of autophagy in the pathogenesis of RA including citrullination, immune tolerance break, osteoclastogenesis, RA FLS cell dysplasia, apoptosis resistance, together with the therapeutic potentials of autophagy regulators.
  45. Bioorg Chem. 2022 Sep 11. pii: S0045-2068(22)00547-8. [Epub ahead of print]129 106141
      Regulatory T (Treg) cells play an instrumental role in coordinating immune homeostasis via potent inhibitory effects. Defects in Treg cells lead to autoimmunity, but an overwhelming proportion of Treg cells encourages cancer progression. Hence, targeting Treg cells has emerged as a promising approach for mitigating disease severity. Recent studies have revealed that kinases are a critical component for tuning the fate of Treg cells, but the entire network of Treg-modulating kinases is still unclear. Here, we propose that the autophagy-activating UNC-51-like kinase 1 (ULK1) is a candidate for Treg cell modulation. While accumulating evidence has highlighted the role of autophagy-related kinases in Treg cells, the ULK1-Treg cell axis is yet to be examined. In this review, we predicted the potential role of ULK1 in Treg cell modulation. Furthermore, we summarized current ULK1 activators and inhibitors that can be investigated as Treg-targeting strategies, which might have beneficial outcomes in autoimmunity and cancer.
    Keywords:  Autophagy; Regulatory T cells; Treg-targeting therapy; ULK1 modulator; UNC-51-like kinase 1
  46. Int J Mol Sci. 2022 Sep 12. pii: 10573. [Epub ahead of print]23(18):
      Mitochondrial dysfunction has been recognised a major contributory factor to the pathophysiology of a number of lysosomal storage disorders (LSDs). The cause of mitochondrial dysfunction in LSDs is as yet uncertain, but appears to be triggered by a number of different factors, although oxidative stress and impaired mitophagy appear to be common inhibitory mechanisms shared amongst this group of disorders, including Gaucher's disease, Niemann-Pick disease, type C, and mucopolysaccharidosis. Many LSDs resulting from defects in lysosomal hydrolase activity show neurodegeneration, which remains challenging to treat. Currently available curative therapies are not sufficient to meet patients' needs. In view of the documented evidence of mitochondrial dysfunction in the neurodegeneration of LSDs, along with the reciprocal interaction between the mitochondrion and the lysosome, novel therapeutic strategies that target the impairment in both of these organelles could be considered in the clinical management of the long-term neurodegenerative complications of these diseases. The purpose of this review is to outline the putative mechanisms that may be responsible for the reported mitochondrial dysfunction in LSDs and to discuss the new potential therapeutic developments.
    Keywords:  Gaucher disease; Niemann–Pick disease; lysosomal storage diseases; mucopolysaccharidosis; neurodegeneration; secondary mitochondrial dysfunction; type C
  47. Cell Cycle. 2022 Sep 19. 1-23
      Self-sustained quiescence (SSQ) has been characterized as a stable but reversible non-proliferative cellular state that limits the cloning of cultured cancer cells. By developing refined clonogenic assays, we showed here that cancer cells in SSQ can be selected with anticancer agents and that culture at low cell density induced SSQ in pancreas and prostate adenocarcinoma cells. Pre-culture of cells in 3D or their pretreatment with pharmacological inhibitors of mechanistic target of rapamycin (mTOR) synergize with low cell density for induction of SSQ in a Beclin-1-dependent manner. Dissociated pancreatic adenocarcinoma (PAAD) cells rendered defective for SSQ by down-regulating Beclin-1 expression exhibit higher tumor growth rate when injected subcutaneously into mice. Conversely, dissociated PAAD cells in SSQ promote the formation of small indolent tumors that eventually transitioned to a rapid growth phase. Ex vivo clonogenic assays showed that up to 40% of clonogenic cancer cells enzymatically dissociated from resected fast-growing tumors could enter SSQ, suggesting that SSQ could significantly impact the proliferation of cancer cells that are naturally dispersed from tumors. Remarkably, the kinetics of clinical metastatic recurrence in 124 patients with pancreatic adenocarcinoma included in the TGCA-PAAD project could be predicted from Beclin-1 and Cyclin-A2 mRNA levels in their primary tumor, Cyclin A2 mRNA being a marker of both cell proliferation and mTOR complex 1 activity. Overall, our data show that SSQ is likely to promote the late development of clinical metastases and suggest that identifying new agents targeting cancer cells in SSQ could help improve patient survival.
    Keywords:  Cancer cell persistence; clonogenicity; drug resistance; epithelial-mesenchymal transition; pancreas adenocarcinoma; prostate adenocarcinoma
  48. Antioxidants (Basel). 2022 Sep 02. pii: 1747. [Epub ahead of print]11(9):
      Cerebrovascular disease is highly prevalent and has a complex etiology and variable pathophysiological activities. It thus poses a serious threat to human life and health. Currently, pathophysiological research on cerebrovascular diseases is gradually improving, and oxidative stress and autophagy have been identified as important pathophysiological activities that are gradually attracting increasing attention. Many studies have found some effects of oxidative stress and autophagy on cerebrovascular diseases, and studies on the crosstalk between the two in cerebrovascular diseases have made modest progress. However, further, more detailed studies are needed to determine the specific mechanisms. This review discusses nuclear factor erythroid 2-related factor 2 (Nrf2) molecules, which are closely associated with oxidative stress and autophagy, and the crosstalk between them, with the aim of providing clues for studying the two important pathophysiological changes and their crosstalk in cerebrovascular diseases as well as exploring new target treatments.
    Keywords:  Nrf2; autophagy; crosstalk; ischemic cerebral vascular diseases; oxidative stress
  49. Cells. 2022 Sep 08. pii: 2807. [Epub ahead of print]11(18):
      Lysosomal storage diseases (LSDs) resulting from inherited gene mutations constitute a family of disorders that disturb lysosomal degradative function leading to abnormal storage of macromolecular substrates. In most LSDs, central nervous system (CNS) involvement is common and leads to the progressive appearance of neurodegeneration and early death. A growing amount of evidence suggests that ion channels in the endolysosomal system play a crucial role in the pathology of neurodegenerative LSDs. One of the main basic mechanisms through which the endolysosomal ion channels regulate the function of the endolysosomal system is Ca2+ release, which is thought to be essential for intracellular compartment fusion, fission, trafficking and lysosomal exocytosis. The intracellular TRPML (transient receptor potential mucolipin) and TPC (two-pore channel) ion channel families constitute the main essential Ca2+-permeable channels expressed on endolysosomal membranes, and they are considered potential drug targets for the prevention and treatment of LSDs. Although TRPML1 activation has shown rescue effects on LSD phenotypes, its activity is pH dependent, and it is blocked by sphingomyelin accumulation, which is characteristic of some LSDs. In contrast, TPC2 activation is pH-independent and not blocked by sphingomyelin, potentially representing an advantage over TRPML1. Here, we discuss the rescue of cellular phenotypes associated with LSDs such as cholesterol and lactosylceramide (LacCer) accumulation or ultrastructural changes seen by electron microscopy, mediated by the small molecule agonist of TPC2, TPC2-A1-P, which promotes lysosomal exocytosis and autophagy. In summary, new data suggest that TPC2 is a promising target for the treatment of different types of LSDs such as MLIV, NPC1, and Batten disease, both in vitro and in vivo.
    Keywords:  BK; COPD; TRPA1; TRPM2; TRPML; TRPML3; TRPV2; asthma; cystic fibrosis; emphysema; lung injury