bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2022‒08‒28
67 papers selected by
Viktor Korolchuk, Newcastle University

  1. Nucleus. 2022 Dec;13(1): 203-207
      Autophagy has emerged as a key regulator of cell metabolism. Recently, we have demonstrated that autophagy is involved in RNA metabolism by regulating ribosomal RNA (rRNA) synthesis. We found that autophagy-deficient cells display much higher 47S precursor rRNA level, which is caused by the accumulation of SQSTM1/p62 (sequestosome 1) but not other autophagy receptors. Mechanistically, SQSTM1 accumulation potentiates the activation of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) signaling, which facilitates the assembly of RNA polymerase I pre-initiation complex at ribosomal DNA (rDNA) promoter regions and leads to the activation of rDNA transcription. Finally, we showed that SQSTM1 accumulation is responsible for the increase in protein synthesis, cell growth and cell proliferation in autophagy-deficient cells. Taken together, our findings reveal a regulatory role of autophagy and autophagy receptor SQSTM1 in rRNA synthesis and may provide novel mechanisms for the hyperactivated rDNA transcription in autophagy-related human diseases.Abbreviations: 5-FUrd: 5-fluorouridine; LAP: MAP1LC3/LC3-associated phagocytosis; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PIC: pre-initiation complex; POLR1: RNA polymerase I; POLR1A: RNA polymerase I subunit A; rDNA: ribosomal DNA; RRN3: RRN3 homolog, RNA polymerase I transcription factor; rRNA: ribosomal RNA; SQSTM1/p62: sequestosome 1; TP53INP2: tumor protein p53 inducible nuclear protein 2; UBTF: upstream binding transcription factor.
    Keywords:  Autophagy; MTORC1; SQSTM1/p62; rDNA; rRNA
  2. Front Mol Biosci. 2022 ;9 930223
      Autophagy is an evolutionary conserved catabolic pathway that uses a unique double-membrane vesicle, called autophagosome, to sequester cytosolic components, deliver them to lysosomes and recycle amino-acids. Essentially, autophagy acts as a cellular cleaning system that maintains metabolic balance under basal conditions and helps to ensure nutrient viability under stress conditions. It is also an important quality control mechanism that removes misfolded or aggregated proteins and mediates the turnover of damaged and obsolete organelles. In this regard, the idea that autophagy is a non-selective bulk process is outdated. It is now widely accepted that forms of selective autophagy are responsible for metabolic rewiring in response to cellular demand. Given its importance, autophagy plays an essential role during tumorigenesis as it sustains malignant cellular growth by acting as a coping-mechanisms for intracellular and environmental stress that occurs during malignant transformation. Cancer development is accompanied by the formation of a peculiar tumor microenvironment that is mainly characterized by hypoxia (oxygen < 2%) and low nutrient availability. Such conditions challenge cancer cells that must adapt their metabolism to survive. Here we review the regulation of autophagy and selective autophagy by hypoxia and the crosstalk with other stress response mechanisms, such as UPR. Finally, we discuss the emerging role of ER-phagy in sustaining cellular remodeling and quality control during stress conditions that drive tumorigenesis.
    Keywords:  ER stress; ER-phagy; UPR; autophagy; cancer; endoplasmic reticulum; hypoxia
  3. Cells. 2022 Aug 17. pii: 2562. [Epub ahead of print]11(16):
      Autophagy is a central mechanism for maintaining cellular homeostasis in health and disease as it provides the critical energy through the breakdown and recycling of cellular components and molecules within lysosomes. One of the three types of autophagy is chaperone-mediated autophagy (CMA), a degradation pathway selective for soluble cytosolic proteins that contain a targeting motif related to KFERQ in their amino acid sequence. This motif marks them as CMA substrate and is, in the initial step of CMA, recognised by the heat shock protein 70 (Hsc70). The protein complex is then targeted to the lysosomal membrane where the interaction with the splice variant A of the lysosomal-associated membrane protein-2 (LAMP-2A) results in its unfolding and translocation into the lysosome for degradation. Altered levels of CMA have been reported in a wide range of pathologies including many cancer types that upregulate CMA as part of the pro-tumorigenic phenotype, while in aging a decline is observed and associated with a decrease of LAMP-2 expression. The potential of altering CMA to modify a physiological or pathological process has been firmly established through genetic manipulation in animals and chemical interference with this pathway. However, its use for therapeutic purposes has remained limited. Compounds used to target and modify CMA have been applied successfully to gain a better understanding of its cellular mechanisms, but they are mostly not specific, also influence other autophagic pathways and are associated with high levels of toxicity. Here, we will focus on the molecular mechanisms involved in CMA regulation as well as on potential ways to intersect them, describe modulators successfully used, their mechanism of action and therapeutic potential. Furthermore, we will discuss the potential benefits and drawbacks of CMA modulation in diseases such as cancer.
    Keywords:  KFERQ; LAMP-2A; autophagy; cancer; chaperone; chaperone-mediated autophagy; lysosome; protein degradation
  4. Biochem Biophys Res Commun. 2022 Aug 12. pii: S0006-291X(22)01140-8. [Epub ahead of print]626 175-186
      Parkinson's disease (PD) is a common neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. The pathophysiology of this disease is the formation of the Lewy body, mostly consisting of alpha-synuclein and dysfunctional mitochondria. There are two common PD-associated genes, Pink1 (encoding a mitochondrial ser/thr kinase) and Parkin (encoding cytosolic E3-ubiquitin ligase), involved in the mitochondrial quality control pathway. They assist in removing damaged mitochondria via selective autophagy (mitophagy) which if unchecked, results in the formation of protein aggregates in the cytoplasm. The role of Rab11, a small Ras-like GTPase associated with recycling endosomes, in PD is still unclear. In the present study, we used the PD model of Drosophila melanogaster and found that Rab11 has a crucial role in the regulation of mitochondrial quality control and endo-lysosomal pathways in association with Parkin and Pink1 and Rab11 acting downstream of Parkin. Additionally, overexpression of Rab11 in parkin mutant rescued the mitochondrial impairment, suggesting the therapeutic potential of Rab11 in PD pathogenesis.
    Keywords:  Dopaminergic neurons; Mitochondria; Mitophagy; Parkinson's disease; Rab11; Vesicle trafficking
  5. Autophagy. 2022 Aug 26.
      Macroautophagy/autophagy acts to promote homeostasis and is increasingly understood to selectively target cargo for degradation. The LC3-family of proteins mediate diverse yet distinct cargo recruitment to phagophores. However, what underlies specificity for cargo engagement among LC3 proteins is poorly understood. Using an unbiased protein interaction screen of LC3B and LC3C we uncover a novel LC3C-endocytic-associated-pathway (LEAP) that recruits selective plasma membrane (PM) cargo to phagophores. We show LC3C but not LC3B localizes to peripheral endosomes and engages proteins that traffic between the PM, endosomes and autophagosomes. We establish that endocytic LC3C binds cargo internalized from the PM, including MET receptor tyrosine kinase and TFRC (transferrin receptor), and targets them towards autophagic degradation. These findings identify LEAP as an unexpected LC3C-dependent pathway, providing new understanding of selective coupling of PM signaling and autophagic degradation with important implications in cancer and other disease states.
    Keywords:  Atg8-orthologs; LC3C; MET-RTK; autophagy; endocytic trafficking; selective cargo recruitment; signalophagy
  6. Autophagy. 2022 Aug 26.
      Macroautophagy/autophagy is a multistep degradative process that is essential for maintaining cellular homeostasis and is often dysregulated during disease. Systematically quantifying flux through this pathway is critical for gaining fundamental insights and effectively modulating this process. Established methods to quantify flux use steady state measurements, which provide limited information about the perturbation and the cellular response. We present a theoretical and experimental framework to measure autophagic steps in the form of rates under non-steady state conditions. We use this approach to measure temporal responses to rapamycin and wortmannin treatments, two commonly used autophagy modulators. We quantified changes in autophagy rates in as little as 10 min, which can establish direct mechanisms for autophagy perturbation before feedback begins. We identified concentration-dependent effects of rapamycin on the initial and temporal progression of autophagy rates. We also found variable recovery time from wortmannin's inhibition of autophagy, which is further accelerated by rapamycin. Furthermore, we applied this approach to study the effect of serum and glutamine starvation on autophagy. Serum starvation led to a rapid and transient increase in all the rates. Glutamine starvation led to a decrease in the rates on a longer timescale. In summary, this new approach enables the quantification of autophagy flux with high sensitivity and temporal resolution and facilitates a comprehensive understanding of this process.
    Keywords:  Autophagy flux; [10 maximum]; cargo; dynamic; glutamine starvation; live cell fluorescence microscopy; puncta; rapamycin; serum starvation; temporal dynamics; wortmannin
  7. Science. 2022 Aug 25. eabg6621
      Lysosomes coordinate cellular metabolism and growth upon sensing of essential nutrients, including cholesterol. Through bioinformatic analysis of lysosomal proteomes, we identified LYsosomal CHOlesterol Signaling (LYCHOS, previously annotated as G-protein coupled receptor 155), a multidomain transmembrane protein that enables cholesterol-dependent activation of the master growth regulator, the protein kinase mechanistic Target of Rapamycin Complex 1 (mTORC1). Cholesterol bound to the N-terminal permease-like region of LYCHOS, and mutating this site impaired mTORC1 activation. At high cholesterol concentrations, LYCHOS bound to the GATOR1 complex, a GTPase-activating protein for the Rag guanosine triphosphatases, through a conserved cytoplasm-facing loop. By sequestering GATOR1, LYCHOS promotes cholesterol- and Rag-dependent recruitment of mTORC1 to lysosomes. Thus, LYCHOS functions in a lysosomal pathway for cholesterol sensing, and couples cholesterol concentrations to mTORC1-dependent anabolic signaling.
  8. Autophagy. 2022 Aug 21. 1-9
      Macroautophagy/autophagy is necessary for lifespan extension in multiple model organisms and autophagy dysfunction impacts age-related phenotypes and diseases. Introduction of an F121A mutation into the essential autophagy protein BECN1 constitutively increases basal autophagy in young mice and reduces cardiac and renal age-related changes in longer lived Becn1F121A mutant mice. However, both autophagic and lysosomal activities decline with age. Thus, whether autophagic flux is maintained during aging and whether it is enhanced in Becn1F121A mice is unknown. Here, we demonstrate that old wild-type mice maintained functional autophagic flux in heart, kidney and skeletal muscle but not liver, and old Becn1F121A mice had increased autophagic flux in those same organs compared to wild type. In parallel, Becn1F121A mice were not protected against age-associated hepatic phenotypes but demonstrated reduced skeletal muscle fiber atrophy. These findings identify an organ-specific role for the ability of autophagy to impact organ aging phenotypes.
    Keywords:  Aging; BECN1; autophagic flux; liver; mouse; skeletal muscle
  9. Front Nutr. 2022 ;9 965771
      The mechanistic target of rapamycin complex 1 (mTORC1) is involved in nutrient-induced signaling and is a master regulator of cell growth and metabolism. Amino acid-deficient conditions affect mTORC1 activity; however, its upstream regulators warrant further investigation. MicroRNAs are key regulators of nutrient-related responses; therefore, the present study aimed to assess the leucine starvation-induced microRNA profile and its impact on mTORC1 activity. Transcriptome analysis of human hepatocellular carcinoma cells (HepG2) under leucine deprivation revealed that hsa-miR-663a and hsa-miR-1469 were altered in a transcription factor 4-dependent manner. Overexpression of these microRNAs induced phosphorylation of the ribosomal protein S6 kinase beta-1, a mTORC1 downstream target. Furthermore, hsa-miR-663a downregulated proline-rich Akt1 substrate of 40 kDa (PRAS40), one of the mTORC1 components. In summary, this study provides new insights into the regulatory role of microRNAs in amino acid metabolism and demonstrates alterations in microRNA profile under leucine deprivation in human hepatocytes.
    Keywords:  ATF4; hsa-miR-663a; leucine deprivation; mTORC1; microRNAs
  10. J Vis Exp. 2022 Aug 04.
      Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans. The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.
  11. Antioxidants (Basel). 2022 Aug 18. pii: 1599. [Epub ahead of print]11(8):
      All processes in human physiology relies on homeostatic mechanisms which require the activation of specific control circuits to adapt the changes imposed by external stimuli. One of the critical modulators of homeostatic balance is autophagy, a catabolic process that is responsible of the destruction of long-lived proteins and organelles through a lysosome degradative pathway. Identification of the mechanism underlying autophagic flux is considered of great importance as both protective and detrimental functions are linked with deregulated autophagy. At the mechanistic and regulatory levels, autophagy is activated in response to diverse stress conditions (food deprivation, hyperthermia and hypoxia), even a novel perspective highlight the potential role of physical forces in autophagy modulation. To understand the crosstalk between all these controlling mechanisms could give us new clues about the specific contribution of autophagy in a wide range of diseases including vascular disorders, inflammation and cancer. Of note, any homeostatic control critically depends in at least two additional and poorly studied interdependent components: a receptor and its downstream effectors. Addressing the selective receptors involved in autophagy regulation is an open question and represents a new area of research in this field. G-protein coupled receptors (GPCRs) represent one of the largest and druggable targets membrane receptor protein superfamily. By exerting their action through G proteins, GPCRs play fundamental roles in the control of cellular homeostasis. Novel studies have shown Gαq, a subunit of heterotrimeric G proteins, as a core modulator of mTORC1 and autophagy, suggesting a fundamental contribution of Gαq-coupled GPCRs mechanisms in the control of this homeostatic feedback loop. To address how GPCR-G proteins machinery integrates the response to different stresses including oxidative conditions and mechanical stimuli, could provide deeper insight into new signaling pathways and open potential and novel therapeutic strategies in the modulation of different pathological conditions.
    Keywords:  GPCR; Gq; autophagy; extracellular matrix; mechanotransduction; oxidative stress
  12. Autophagy. 2022 Aug 25. 1-17
      Macroautophagy/autophagy proteins have been linked with the development of immune-mediated diseases including lupus, but the mechanisms for this are unclear due to the complex roles of these proteins in multiple immune cell types. We have previously shown that a form of noncanonical autophagy induced by ITGAV/alpha(v) integrins regulates B cell activation by viral and self-antigens, in mice. Here, we investigate the involvement of this pathway in B cells from human tissues. Our data reveal that autophagy is specifically induced in the germinal center and memory B cell subpopulations of human tonsils and spleens. Transcriptomic analysis show that the induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of ITGAV/alpha(v) integrin-dependent autophagy in human B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells, we found that ITGAV/alpha(v)-dependent autophagy limits activation of specific pathways related to B cell responses, while promoting others. These data provide new mechanistic links for autophagy and B-cell-mediated immune dysregulation in diseases such as lupus.
    Keywords:  Autophagy; B cells; TLR signaling; cell-cycle; mitophagy; non-canonical autophagy
  13. Autophagy. 2022 Aug 21. 1-17
      The endosomal system maintains cellular homeostasis by coordinating multiple vesicular trafficking events, and the retromer complex plays a critical role in endosomal cargo recognition and sorting. Here, we demonstrate an essential role for the small GTPase RAB21 in regulating retromer-mediated recycling of the glucose transporter SLC2A1/GLUT1 and macroautophagy/autophagy. RAB21 depletion mis-sorts SLC2A1 to lysosomes and affects glucose uptake, thereby activating the AMPK-ULK1 pathway to increase autophagic flux. RAB21 depletion also increases lysosome function. Notably, RAB21 depletion does not overtly affect retrograde transport of IGF2R/CI-M6PR or WLS from endosomes to the trans-Golgi network. We speculate that RAB21 regulates fission of retromer-decorated endosomal tubules, as RAB21 depletion causes accumulation of the SNX27-containing retromer complex on enlarged endosomes at the perinuclear region. Functionally, RAB21 depletion sensitizes cancer cells to energy stress and inhibits tumor growth in vivo, suggesting an oncogenic role for RAB21. Overall, our study illuminates the role of RAB21 in regulating endosomal dynamics and maintaining cellular energy homeostasis and suggests RAB21 as a potential metabolic target for cancer therapy.
    Keywords:  Autophagic flux; glucose uptake; membrane trafficking; retromer complex; small GTPases
  14. Autophagy. 2022 Aug 25.
      As a new emerging severe coronavirus, the knowledge on the SARS-CoV-2 and COVID-19 remains very limited, whereas many concepts can be learned from the homologous coronaviruses. Macroautophagy/autophagy is finely regulated by SARS-CoV-2 infection and plays important roles in SARS-CoV-2 infection and pathogenesis. This review will explore the subversion and mechanism of the autophagy-related machinery, vacuoles and organelle-specific autophagy during infection of SARS-CoV-2 and coronaviruses, to provide meaningful insights into the autophagy-related therapeutic strategies for infectious diseases of SARS-CoV-2 and coronaviruses.
    Keywords:  Autophagy; SARS-CoV-2; coronaviruses; infection; organelle-specific autophagy; replication
  15. Brain. 2022 Aug 27. pii: awac308. [Epub ahead of print]
      SPG15 is a hereditary spastic paraplegia (HSP) subtype caused by mutations in Spastizin, a protein encoded by the ZFYVE26 gene. Spastizin is involved in autophagosome maturation and autophagic lysosome reformation (ALR) and SPG15-related mutations lead to ALR defects with lysosome enlargement, free lysosomes depletion and autophagosome accumulation. Symptomatic and rehabilitative treatments are the only therapy currently available for patients. Here, we targeted autophagy and lysosomes in SPG15 patient-derived cells by using a library of autophagy-modulating compounds. We identified a rose of compounds, affecting intracellular calcium levels, the calcium-calpain pathway, or lysosomal functions, that reduced autophagosome accumulation. The six most effective compounds were tested in vivo in a new SPG15 loss of function Drosophila model that mimicked the reported SPG15 phenotype, with autophagosome accumulation, enlarged lysosomes, reduced free lysosomes, ALR defects and locomotor deficit. These compounds, namely verapamil, Bay K8644, 2',5'-dideoxyadenosine, trehalose, Small Molecule Enhancer of Rapamycin 28 (SMER28) and trifluoperazine, improved lysosome biogenesis and function in vivo, demonstrating that lysosomes are a key pharmacological target to rescue SPG15 phenotype. Among the others, the small molecule enhancer of autophagy SMER28 was the most effective, rescuing both ALR defects and locomotor deficit, and could be considered as a potential therapeutic compound for this HSP subtype.
    Keywords:  ALR; SMER28; SPG15; autophagy; lysosomes
  16. Cell Signal. 2022 Aug 20. pii: S0898-6568(22)00203-0. [Epub ahead of print]99 110441
      N-(3-oxododecanoyl) homoserine lactone (3oc) is a Pseudomonas aeruginosa secreted quorum-sensing signal molecule playing a crucial role in regulating quorum-sensing (QS) dependent biofilm formation and secretion of virulence factors. In addition to regulating quorum sensing, 3oc also plays an immunomodulatory role in the host by triggering regulated cell death in immune cells. The molecular mechanisms of 3oc in modulating macrophage pathologies are still unclear. In this study, we hypothesized the novel 3oc mediated crosstalk between autophagy and apoptosis at the interphase of calcium signaling in human macrophages. The study showed that 3oc induces mitochondrial dysfunction and apoptosis in macrophages through elevating cytosolic Ca+2 ([Ca+2]cyt) levels. Pre-treatment with the calcium-specific chelator BAPTA-AM effectively abrogated 3oc-induced apoptotic events, like mitochondrial ROS generation (mROS), mitochondrial membrane potential (MMP) drop, and phosphatidylserine (PS) exposure. The study also showed that 3oc induces autophagy, as assessed by the accumulation of autophagic vacuoles, induction of lysosomal biogenesis, upregulation of autophagy genes (LC3, BECLIN 1, STX17, PINK1, and TFEB), autophagosomes formation, and LC3 lipidation. Mechanistically, our study proved that 3oc-induced autophagy was [Ca+2]cyt dependent as BAPTA-AM pre-treatment reduced autophagosome formation. Furthermore, inhibiting autophagy with chloroquine attenuated 3oc-induced apoptosis, while autophagy induction with rapamycin aggravated cell death, suggesting autophagy plays a role in cell death in 3oc-treated macrophages. In conclusion, our findings indicate that 3oc activates a multifaceted death signaling by activating autophagy and apoptosis through Ca+2 signaling, and we propose pharmacological modulation of Ca+2 signaling may act as a combinatorial therapeutic intervention in patients with Pseudomonas aeruginosa-associated infections.
    Keywords:  Apoptosis; Autophagy; Calcium signaling; N-(3-oxododecanoyl) homoserine lactone; Pseudomonas aeruginosa
  17. Biochem Biophys Res Commun. 2022 Aug 15. pii: S0006-291X(22)01153-6. [Epub ahead of print]627 5-11
      CDK2 forms a complex with cyclin A and cyclin E to promote the progress of cell cycle, but when cyclin A and cyclin E are dissociated from the complex and degraded by the ubiquitin proteasome pathway, the fate of the inactive CDK2 is unclear. In this study, we found that the inactive CDK2 protein was degraded by autophagy-lysosome pathway. In the classic model of G0/G1 phase arrest induced by serum starvation, we found that the mRNA level in CDK2 did not change but the protein level decreased. Subsequently, using PI3K and AKT inhibitors and gene knockout methods, it was found that CDK2 degradation was mediated by the inhibition of PI3Kα/AKTT308. In addition, P62/SQSTM1 was found to bind to the inactivated CDK2 protein to help it enter autophagy-lysosome degradation in a CTSB-dependent manner. Taken together, these results confirm that the PI3Kα/AKTT308 inhibition leads to degradation of CDK2 protein in the autophagy-lysosome pathway. These data reveal a new molecular mechanism of CDK2 protein degradation and provide a new strategy and method for regulating CDK2 protein.
    Keywords:  Autophagy–lysosome; CDK2; Degradation; P62/SQSTM1; PI3Kα/AKT(T308)
  18. Mol Cell Biol. 2022 Aug 25. e0024122
      Upon pathogen infection, intricate innate signaling cascades are induced to initiate the transcription of immune effectors, including cytokines and chemokines. Transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy genes, was found recently to be a novel regulator of innate immunity in both Caenorhabditis elegans and mammals. Despite TFEB participating in critical mechanisms of pathogen recognition and in the transcriptional response to infection in mammalian macrophages, little is known about its roles in the infected epithelium or infected nonimmune cells in general. Here, we demonstrate that TFEB is activated in nonimmune cells upon infection with bacterial pathogens through a pathway dependent on mTORC1 inhibition and RAG-GTPase activity, reflecting the importance of membrane damage and amino acid starvation responses during infection. Additionally, we present data demonstrating that although TFEB does not affect bacterial killing or load in nonimmune cells, it alters the host transcriptome upon infection, thus promoting an antibacterial transcriptomic landscape. Elucidating the roles of TFEB in infected nonimmune cells and the upstream signaling cascade provides critical insight into understanding how cells recognize and respond to bacterial pathogens.
    Keywords:  Shigella; TFEB; amino acid starvation; host-microbe interactions; mTOR
  19. Front Endocrinol (Lausanne). 2022 ;13 903836
      Autophagy is an intracellular degradation mechanism that allows recycling of organelles and macromolecules. Autophagic function increases metabolite availability modulating metabolic pathways, differentiation and cell survival. The oral environment is composed of several structures, including mineralized and soft tissues, which are formed by complex interactions between epithelial and mesenchymal cells. With aging, increased prevalence of oral diseases such as periodontitis, oral cancer and periapical lesions are observed in humans. These aging-related oral diseases are chronic conditions that alter the epithelial-mesenchymal homeostasis, disrupting the oral tissue architecture affecting the quality of life of the patients. Given that autophagy levels are reduced with age, the purpose of this review is to discuss the link between autophagy and age-related oral diseases.
    Keywords:  aging; autophagy; oral cancer; oral diseases; periapical lesions; periodontitis
  20. Antioxidants (Basel). 2022 Jul 28. pii: 1476. [Epub ahead of print]11(8):
      Doxorubicin (DOX), a widely used chemotherapeutic agent, has been linked to an increased risk of bone damage in human patients and induces bone loss in mice. DOX induces autophagy, which contributes to bone homeostasis and excess autophagy in osteoclasts (OCs), resulting in bone loss. We hypothesized that DOX-induced bone loss is caused by the induction of autophagy in OCs. In vitro, DOX significantly increased the area of OCs and bone resorption activity, whereas it decreased OC number through apoptosis. DOX enhanced the level of LC3II and acidic vesicular organelles-containing cells in OCs, whereas an autophagy inhibitor, 3-methyladenine (3-MA), reversed these, indicating that enhanced autophagy was responsible for the effects of DOX. Increased mitochondrial reactive oxygen species (mROS) by DOX oxidized transient receptor potential mucolipin 1 (TRPML1) on the lysosomal membrane, which led to nuclear localization of transcription factor EB (TFEB), an autophagy-inducing transcription factor. In vivo, micro-computerized tomography analysis revealed that the injection of 3-MA reversed DOX-induced bone loss, and tartrate-resistant acid phosphatase staining showed that 3-MA reduced the area of OCs on the bone surface, which was enhanced upon DOX administration. Collectively, DOX-induced bone loss is at least partly attributable to autophagy upregulation in OCs via an mROS/TRPML1/TFEB axis.
    Keywords:  autophagy; bone loss; doxorubicin; osteoclast; reactive oxygen species
  21. Nat Commun. 2022 Aug 22. 13(1): 4918
      Considerable evidence supports the release of pathogenic aggregates of the neuronal protein α-Synuclein (αSyn) into the extracellular space. While this release is proposed to instigate the neuron-to-neuron transmission and spread of αSyn pathology in synucleinopathies including Parkinson's disease, the molecular-cellular mechanism(s) remain unclear. To study this, we generated a new mouse model to specifically immunoisolate neuronal lysosomes, and established a long-term culture model where αSyn aggregates are produced within neurons without the addition of exogenous fibrils. We show that neuronally generated pathogenic species of αSyn accumulate within neuronal lysosomes in mouse brains and primary neurons. We then find that neurons release these pathogenic αSyn species via SNARE-dependent lysosomal exocytosis. The released aggregates are non-membrane enveloped and seeding-competent. Additionally, we find that this release is dependent on neuronal activity and cytosolic Ca2+. These results propose lysosomal exocytosis as a central mechanism for the release of aggregated and degradation-resistant proteins from neurons.
  22. Antioxidants (Basel). 2022 Aug 14. pii: 1571. [Epub ahead of print]11(8):
      Intervertebral disc degeneration (IVDD) is a prevalent cause of low back pain. IVDD is characterized by abnormal expression of extracellular matrix components such as collagen and aggrecan. In addition, it results in dysfunctional growth, senescence, and death of intervertebral cells. The biological pathways involved in the development and progression of IVDD are not fully understood. Therefore, a better understanding of the molecular mechanisms underlying IVDD could aid in the development of strategies for prevention and treatment. Autophagy is a cellular process that removes damaged proteins and dysfunctional organelles, and its dysfunction is linked to a variety of diseases, including IVDD and osteoarthritis. In this review, we describe recent research findings on the role of autophagy in IVDD pathogenesis and highlight autophagy-targeting molecules which can be exploited to treat IVDD. Many studies exhibit that autophagy protects against and postpones disc degeneration. Further research is needed to determine whether autophagy is required for cell integrity in intervertebral discs and to establish autophagy as a viable therapeutic target for IVDD.
    Keywords:  ECM degradation; autophagy; autophagy-targeting compounds; intervertebral disc degeneration; low back pain
  23. Acta Pharmacol Sin. 2022 Aug 25.
      Mitochondrial dynamics, including mitochondrial fission and fusion, are critical for maintaining mitochondrial functions. Evidence shows that TANK-binding kinase 1 (TBK1) regulates mitochondrial fusion and fission and then mitophagy. Since a previous study demonstrates a strong correlation between mitophagy and osteoarthritis (OA), we herein investigated the potential role of TBK1 in OA process and mitochondrial functions. We demonstrated a strong correlation between TBK1 and OA, evidenced by significantly downregulated expression of TBK1 in cartilage tissue samples of OA patients and in the chondrocytes of aged mice, as well as TNF-α-stimulated phosphorylation of TBK1 in primary mouse chondrocytes. TBK1 overexpression significantly attenuated TNF-α-induced apoptosis and abnormal mitochondrial function in primary mouse chondrocytes. Furthermore, TBK1 overexpression induced remodeling of mitochondrial morphology by directly phosphorylating dynamin-related protein 1 (DRP1) at Ser637, abolishing the fission of DRP1 and preventing its fragmentation function. Moreover, TBK1 recruitment and DRP1 phosphorylation at Ser637 was necessary for engulfing damaged mitochondria by autophagosomal membranes during mitophagy. Moreover, we demonstrated that APMK/ULK1 signaling contributed to TBK1 activation. In OA mouse models established by surgical destabilization of the medial meniscus, intraarticular injection of lentivirus-TBK1 significantly ameliorated cartilage degradation via regulation of autophagy and alleviation of cell apoptosis. In conclusion, our results suggest that the TBK1/DRP1 pathway is involved in OA and pharmacological targeting of the TBK1-DRP1 cascade provides prospective therapeutic benefits for the treatment of OA.
    Keywords:  APMK/ULK1; TBK1/DRP1 axis; malvidin-3-O-arabinoside; mitochondria morphology; mitophagy; osteoarthritis
  24. Metabolites. 2022 Aug 12. pii: 740. [Epub ahead of print]12(8):
      GPRC6A, an important member of the G-protein-coupled receptor superfamily, has been widely studied in body health maintenance and related diseases. However, it is still controversial whether GPRC6A plays a vital role in glucose homeostasis, and the role of GPRC6A on amino acid homeostasis has not been reported. In this study, GPRC6A was knocked out in C57BL6 mice, and we found that GPRC6A plays an important role in the glucose metabolism, mainly affecting the glucose clearance capacity and gluconeogenesis in mice. GPRC6A plays an important role in maintaining amino acid homeostasis under dietary restrictions, and this may be realized by participating in the regulation of autophagy. Since a large amount of amino acid is lost from urine in aged GPRC6A-/- mice, it is possible that GPRC6A regulates amino acid homeostasis by affecting the integrity of tissue structure. GPRC6A is involved in the regulation of mTORC1 activation but is not necessary for mTORC1 activation under sufficient nutritional supply. In the absence of exogenous amino acids, the loss of GPRC6A induces the GCN2 pathway activation and excessive autophagy of cells, leading to the overactivation of mTORC1, which may be detrimental to body health and cell survival. In summary, this study provides a theoretical and experimental basis for the metabolic process of GPRC6A in body growth and health.
    Keywords:  GPRC6A; amino acids; autophagy; glucose; mTORC1
  25. Medicina (Kaunas). 2022 Aug 20. pii: 1129. [Epub ahead of print]58(8):
      Background and Objectives: Age-related macular degeneration is a slow-progressing disease in which lipofuscin accumulates in the retina, causing inflammation and apoptosis of retinal pigment epithelial (RPE) cells. This study aimed to identify N-methyl-D-aspartate (NMDA) signaling as a novel mechanism for scavenging N-retinylidene-N-retinylethanolamine (A2E), a component of ocular lipofuscin, in human RPE cells. Materials and Methods: A2E degradation assays were performed in ARPE-19 cells using fluorescently labeled A2E. The autophagic activity in ARPE-19 cells was measured upon blue light (BL) exposure, after A2E treatment. Autophagy flux was determined by measuring LC3-II formation using immunoblotting and confocal microscopy. To determine whether autophagy via the NMDA receptor is involved in A2E clearance, ATG5-deficient cells were used. Results: Ro 25-6981, an NR2B-selective NMDA receptor antagonist, effectively cleared A2E. Ro 25-6981 reduced A2E accumulation in the lysosomes of ARPE-19 cells at sub-cytotoxic concentrations, while increasing the formation of LC3-II and decreasing p62 protein levels in a concentration-dependent manner. The autophagic flux monitored by RFP-GFP-LC3 and bafilomycin A1 assays was significantly increased by Ro 25-6981. A2E clearance by Ro 25-6981 was abolished in ATG5-depleted ARPE-19 cells, suggesting that A2E degradation by Ro 25-6981 was mediated by autophagy. Furthermore, treatment with other NMDA receptor antagonists, CP-101,606 and AZD6765, showed similar effects on autophagy activation and A2E degradation in ARPE-19 cells. In contrast, glutamate, an NMDA receptor agonist, exhibited a contrasting effect, suggesting that both the activation of autophagy and the degradation of A2E by Ro 25-6981 in ARPE-19 cells occur through inhibition of the NMDA receptor pathway. Conclusions: This study demonstrates that NMDA receptor antagonists degrade lipofuscin via autophagy in human RPE cells and suggests that NMDA receptor antagonists could be promising new therapeutics for retinal degenerative diseases.
    Keywords:  N-methyl-D-aspartate (NMDA); N-retinylidene-N-retinylethanolamine (A2E); autophagy; drusen; retinal pigment epithelial cells
  26. Stem Cell Res Ther. 2022 Aug 20. 13(1): 427
      BACKGROUND: The therapeutic effects of adipose-derived mesenchymal stromal cells (ADSCs) may be mainly mediated by their paracrine effects. The ADSC-secretome can ameliorate hepatic ischemia-reperfusion injury (IRI). We explored the therapeutic effect of the ADSC-secretome from the perspective of excessive hepatocyte autophagy induced by hepatic IRI.METHODS: We established a miniature pig model of hepatic ischemia-reperfusion (I/R) and hepatectomy using a laparoscopic technique and transplanted ADSCs and the ADSC-secretome into the liver parenchyma immediately after surgery. Liver injury and hepatocyte autophagy were evaluated by histopathological examination and assessment of relevant cytokines and other factors.
    RESULTS: The results showed that the ADSC-secretome alleviated the pathological changes of liver tissue and the microstructural damage of hepatocytes after IRI. Moreover, the expression levels of autophagy-related markers including Beclin-1, ATG5, ATG12, and LC3II/LC3I decreased, whereas those of p62 increased during phagophore expansion. Furthermore, the expression levels of markers related to the autophagy inhibition pathway phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR), including PI3K, Akt, and mTOR, increased.
    CONCLUSION: The ADSC-secretome attenuates hepatic I/R and hepatectomy-induced liver damage by inhibiting autophagy, which is possibly mediated by activation of the PI3K/Akt/mTOR signaling pathway. In addition, there was no significant difference between ADSCs and the ADSC-secretome in the regulation of hepatocyte autophagy. Therefore, ADSCs may improve the excessive autophagy-induced injury of hepatocytes in hepatic I/R and hepatectomy through paracrine effect. Our findings provide new insight into the therapeutic potential of cell-free products, which could replace cell therapy in liver diseases.
    Keywords:  ADSC-secretome; Autophagy; IRI; Laparoscopy; Miniature pig
  27. Geroscience. 2022 Aug 26.
      Lysosomes are crucial for degradation and recycling of damaged proteins and cellular components. Therapeutic strategies enhancing lysosomal function are a promising approach for aging and age-related neurodegenerative diseases. Here, we show that an FDA approved drug sodium polystyrene sulfonate (SPS), used to reduce high blood potassium in humans, enhances lysosomal function both in C. elegans and in human neuronal cells. Enhanced lysosomal function following SPS treatment is accompanied by the suppression of proteotoxicity caused by expression of the neurotoxic peptides Aβ and TAU. Additionally, treatment with SPS imparts health benefits as it significantly increases lifespan in C. elegans. Overall our work supports the potential use of SPS as a prospective geroprotective intervention.
    Keywords:  Amyloid-β; Autophagy; C. elegans; Lifespan; Lysosome; Neurodegeneration; Potassium restriction; Proteotoxicity; SH-SY5Y neuronal cells
  28. Leukemia. 2022 Aug 23.
      Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25 % of acute myeloid leukemia (AML) patients, drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and occurs in the absence of identifiable genetic causes. This suggests that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. Here, we systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD + AML using recently developed functional translatome proteomics (measuring changes in the nascent proteome) with phosphoproteomics. This pinpointed AKT-mTORC1-ULK1-dependent autophagy as a dominant resistance mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD + cells in vitro and in primary human AML cells ex vivo. Pharmacological or genetic inhibition of autophagy increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In mice xenografted with FLT3-ITD + AML cells, co-treatment with oral FLT3 and autophagy inhibitors synergistically impaired leukemia progression and extended overall survival. Our findings identify a molecular mechanism responsible for primary FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.
  29. Autophagy. 2022 Aug 23.
      NPPA/atrial natriuretic peptide (natriuretic peptide type A) exerts critical pleiotropic effects in the cardiovascular system, limiting cardiomyocyte hypertrophy and death, reducing cardiac fibrosis and promoting vascular integrity. However, the molecular mechanisms underlying these beneficial effects still need to be clarified. We demonstrated for the first time that macroautophagy/autophagy is involved in the local protective effects of NPPA in cardiomyocytes (CMs), both in vitro and in vivo. Exogenous NPPA rapidly activates autophagy in CMs through NPR1/type A natriuretic peptide receptor and PRKG/protein kinase G signalling and also increases cardiac autophagy in mice. Remarkably, endogenous NPPA is secreted by CMs in response to glucose deprivation or hypoxia, thereby stimulating autophagy through autocrine/paracrine mechanisms. NPPA preserves cell viability and reduces hypertrophy in response to stress through autophagy activation. In vivo, we found that Nppa knockout mice undergoing ischemia-reperfusion (I/R) show increased infarct size and reduced autophagy. Reactivation of autophagy by Tat-Beclin D11 limits I/R injury. We also found that the protective effects of NPPA in reducing infarct size are abrogated in the presence of autophagy inhibition. Mechanistically, we found that NPPA stimulates autophagy through the activation of TFEB (transcription factor EB). Our data suggest that NPPA is a novel extracellular regulator of autophagy in the heart.
    Keywords:  Atrial natriuretic peptide; autophagy; cardiomyocytes; ischemia-reperfusion; transcription factor EB
  30. Cell Death Dis. 2022 Aug 25. 13(8): 733
      Ammonium tetrathiomolybdate (TTM) is a copper chelator in clinical trials for treatment of Wilson's disease, tumors and other diseases. In the current study, we innovatively discovered that TTM is a novel NRF2 activator and illustrated that autophagy contributed to TTM-induced NRF2 activation. We showed that TTM treatment promoted NRF2 nuclear translocation and upregulated transcription level of NRF2 target genes including HMOX1, GCLM, and SLC7A11 in vascular endothelial cells (HUVECs). Moreover, NRF2 deficiency directly hindered TTM-mediated antioxidative effects. Followingly, we revealed that overexpression of KEAP1, a negative regulator of NRF2, significantly repressed NRF2 activation induced by TTM. Further mutation analysis revealed that KEAP1 Cys151 is a major sensor responsible for TTM-initiated NRF2 signaling, suggesting that KEAP1 is involved in TTM-mediated NRF2 activation. Notably, we found that TTM can trigger autophagy as evidenced by accumulation of autophagosomes, elevation of LC3BI-II/I, increase of LC3 puncta and activation of AMPK/mTOR/ULK1 pathway. Autophagic flux assay indicated that TTM significantly enhanced autophagic flux in HUVECs. Inhibition of autophagy with knockout of autophagy key gene ATG5 resulted in suppression of TTM-induced NRF2 activation. TTM also induced phosphorylation of autophagy receptor SQSTM1 at Ser349, while SQSTM1-deficiency inhibited KEAP1 degradation and blocked NRF2 signaling pathway, suggesting that TTM-induced NRF2 activation is autophagy dependent. As the novel NRF2 activator, TTM protected against sodium arsenite (NaAsO2)-induced oxidative stress and cell death, while NRF2 deficiency weakened TTM antioxidative effects. Finally, we showed that autophagy-dependent NRF2 activation contributed to the protective effects of TTM against NaAsO2-induced oxidative injury, because of ATG5 or SQSTM1 knockout aggravated NaAsO2-induced elevation of HMOX1, cleaved PARP and γH2AX. Taken together, our findings highlight copper chelator TTM is a novel autophagy-dependent NRF2 activator and shed a new light on the cure for oxidative damage-related diseases.
  31. Front Mol Neurosci. 2022 ;15 934820
      Gaucher disease (GD) is an inherited disorder caused by recessive mutations in the GBA1 gene that encodes the lysosomal enzyme β-glucocerebrosidase (β-GC). β-GC hydrolyzes glucosylceramide (GluCer) into glucose and ceramide in the lysosome, and the loss of its activity leads to GluCer accumulation in different tissues. In severe cases, enzymatic deficiency triggers inflammation, organomegaly, bone disease, and neurodegeneration. Neuronopathic Gaucher disease (nGD) encompasses two different forms of the disease, characterized by chronic or acute damage to the central nervous system (CNS). The cellular and molecular studies that uncover the pathological mechanisms of nGD mainly focus on lysosomal dysfunction since the lysosome is the key organelle affected in GD. However, new studies show alterations in other organelles that contribute to nGD pathology. For instance, abnormal accumulation of GluCer in lysosomes due to the loss of β-GC activity leads to excessive calcium release from the endoplasmic reticulum (ER), activating the ER-associated degradation pathway and the unfolded protein response. Recent evidence indicates mitophagy is altered in nGD, resulting in the accumulation of dysfunctional mitochondria, a critical factor in disease progression. Additionally, nGD patients present alterations in mitochondrial morphology, membrane potential, ATP production, and increased reactive oxygen species (ROS) levels. Little is known about potential dysfunction in other organelles of the secretory pathway, such as the Golgi apparatus and exosomes. This review focuses on collecting evidence regarding organelle dysfunction beyond lysosomes in nGD. We briefly describe cellular and animal models and signaling pathways relevant to uncovering the pathological mechanisms and new therapeutic targets in GD.
    Keywords:  Golgi apparatus; Neuronopathic Gaucher disease (nGD); Parkinson's disease (PD); autophagy; endoplasmic reticulum; lysosomal storage disorders (LSD); lysosome; mitochondria
  32. Pharmacol Res. 2022 Aug 18. pii: S1043-6618(22)00353-X. [Epub ahead of print] 106408
      The incidence of diabetes has been increasing in recent decades which is affecting the population of both, developed and developing countries. Diabetes is associated with micro and macrovascular complications which predominantly result from hyperglycemia and disrupted metabolic pathways. Persistent hyperglycemia leads to increased reactive oxygen species (ROS) generation, formation of misfolded and abnormal proteins, and disruption of normal cellular functioning. The inability to maintain metabolic homeostasis under excessive energy and nutrient input, which induces insulin resistance, is a crucial feature during the transition from obesity to diabetes. According to various study reports, redox alterations, intracellular stress and chronic inflammation responses have all been linked to dysregulated energy metabolism and insulin resistance. Autophagy has been considered a cleansing mechanism to prevent these anomalies and restore cellular homeostasis. However, disrupted autophagy has been linked to the pathogenesis of metabolic disorders such as obesity and diabetes. Recent studies have reported that the regulation of autophagy has a beneficial role against these conditions. When there is plenty of food, nutrient-sensing pathways activate anabolism and storage, but the shortage of food activates homeostatic mechanisms like autophagy, which mobilises internal stockpiles. These nutrient-sensing pathways are well conserved in eukaryotes and are involved in the regulation of autophagy which includes SIRT1, mTOR and AMPK. The current review focuses on the role of SIRT1, mTOR and AMPK in regulating autophagy and suggests autophagy along with these nutrient-sensing pathways as potential therapeutic targets in reducing the progression of various diabetic complications.
    Keywords:  AMPK; SIRT1; diabetic cardiomyopathy; diabetic nephropathy; diabetic neuropathy; diabetic retinopathy; mTOR
  33. Plants (Basel). 2022 Aug 19. pii: 2157. [Epub ahead of print]11(16):
      The physiological implications of autophagy in plant cells have not been fully elucidated. Therefore, we investigated the consequences of autophagy in the moss Physcomitrella by measuring biochemical parameters (fresh and dry weights; starch, amino acid, carbohydrate, and NH3 content) in wild-type (WT) and autophagy-deficient atg5&amp;nbsp;Physcomitrella cells. We found higher starch levels and a higher net starch synthesis rate in WT cells than in atg5 cells cultured in a glucose-containing culture medium, whereas net starch degradation was similar in the two strains cultured in a glucose-deficient culture medium. Additionally, the treatment of cells with the autophagy inhibitor 3-methyladenine suppressed starch synthesis. Loading bovine serum albumin into atg5 cells through endocytosis, i.e., supplying proteins to vacuoles in the same way as through autophagy, accelerated starch synthesis, whereas loading glutamine through the plasma membrane had no such effect, suggesting that Physcomitrella cells distinguish between different amino acid supply pathways. After net starch synthesis, NH3 levels increased in WT cells, although the change in total amino acid content did not differ between WT and atg5 cells, indicating that autophagy-produced amino acids are oxidized rapidly. We conclude that autophagy promotes starch synthesis in Physcomitrella by supplying the energy obtained by oxidizing autophagy-produced amino acids.
    Keywords:  Physcomitrella (Physcomitrium); amino acid; autophagy; endocytosis; starch synthesis
  34. Proc Natl Acad Sci U S A. 2022 Aug 30. 119(35): e2203742119
      Vacuolar-type adenosine triphosphatases (V-ATPases) not only function as rotary proton pumps in cellular organelles but also serve as signaling hubs. To identify the endogenous binding partners of V-ATPase, we collected a large dataset of human V-ATPases and did extensive classification and focused refinement of human V-ATPases. Unexpectedly, about 17% of particles in state 2 of human V-ATPases display additional density with an overall resolution of 3.3 Å. Structural analysis combined with artificial intelligence modeling enables us to identify this additional density as mEAK-7, a protein involved in mechanistic target of rapamycin (mTOR) signaling in mammals. Our structure shows that mEAK-7 interacts with subunits A, B, D, and E of V-ATPases in state 2. Thus, we propose that mEAK-7 may regulate V-ATPase function through binding to V-ATPases in state 2 as well as mediate mTOR signaling.
    Keywords:  V-ATPase; lysosomal signaling; mEAK-7; mTOR
  35. Biomedicines. 2022 Jul 28. pii: 1817. [Epub ahead of print]10(8):
      Autophagy is a major degradation pathway that removes harmful intracellular substances to maintain homeostasis. Various stressors, such as starvation and oxidative stress, upregulate autophagy, and the dysregulation of autophagy is associated with various human diseases, including cancer and skin diseases. The skin is the first defense barrier against external environmental hazards such as invading pathogens, ultraviolet rays, chemical toxins, and heat. Although the skin is exposed to various stressors that can activate autophagy, the roles of autophagy in the skin have not yet been fully elucidated. Accumulating evidence suggests that autophagy is closely associated with pathogenesis and the treatment of immune-related skin diseases. In this study, we review how autophagy interacts with skin cells, including keratinocytes and immune cells, enabling them to successfully perform their protective functions by eliminating pathogens and maintaining skin homeostasis. Furthermore, we discuss the implications of autophagy in immune-related skin diseases, such as alopecia areata, psoriasis, and atopic dermatitis, and suggest that a combination of autophagy modulators with conventional therapies may be a better strategy for the treatment of these diseases.
    Keywords:  alopecia areata; atopic dermatitis; autophagy; keloid; psoriasis; skin homeostasis
  36. J Neurogenet. 2022 Aug 24. 1-10
      Tau is a microtubule-associated protein that forms insoluble filaments that accumulate as neurofibrillary tangles in neurodegenerative diseases such as Alzheimer's disease and other related tauopathies. A relationship between abnormal Tau accumulation and ubiquitin-proteasome system impairment has been reported. However, the molecular mechanism linking Tau accumulation and ubiquitin proteasome system (UPS) dysfunction remains unclear. Here, we show that overexpression of wild-type or mutant (P301L) Tau increases the abundance of polyubiquitinated proteins and activates the autophagy-lysosome pathway in mammalian neuronal cells. Previous studies found that PTK2 inhibition mitigates toxicity induced by UPS impairment. Thus, we investigated whether PTK2 inhibition can attenuate Tau-induced UPS impairment and cell toxicity. We found that PTK2 inhibition significantly reduces Tau-induced death in mammalian neuronal cells. Moreover, overexpression of WT or mutant Tau increased the phosphorylation levels of PTK2 and p62. We also confirmed that PTK2 inhibition suppresses Tau-induced phosphorylation of PTK2 and p62. Furthermore, PTK2 inhibition significantly attenuated the climbing defect and shortened the lifespan in the Drosophila model of tauopathy. In addition, we observed that phosphorylation of p62 is markedly increased in Alzheimer's disease patients with tauopathies. Taken together, our results indicate that the UPS dysfunction induced by Tau accumulation might contribute directly to neurodegeneration in tauopathies and that PTK2 could be a promising therapeutic target for tauopathies.
    Keywords:  Alzheimer’s disease; MAPT/Tau; PTK2; p62; ubiquitin-proteasome system
  37. Autophagy. 2022 Aug 26.
      Lymphatic endothelial cells (LECs) exploit fatty acid oxidation (FAO) to grow and to maintain lymphatic vessel identity through the epigenetic regulation of the essential transcription factor PROX1. In our recent study, we found that LEC-specific loss of ATG5 prevents injury-induced lymphangiogenesis in vivo. Inadequate degradation of lipid droplets (LDs) caused by genetic ablation of ATG5 in LECs disturbs mitochondrial fitness, and reduces mitochondrial FAO and acetyl-CoA levels, ultimately affecting PROX1-mediated epigenetic regulation of CPT1A and key lymphatic markers, most importantly FLT4/VEGFR3. Supplementing the fatty acid precursor acetate rescues defective inflammation-driven lymphangiogenesis in LEC-specific atg5 knockout mice. Thus, efficient macroautophagy/autophagy-mediated LD breakdown is critical to maintain mitochondrial metabolism and acetyl-CoA levels, which sustain a PROX1-mediated lymphatic gene program required for LEC identity and inflammation-driven lymphangiogenesis.
    Keywords:  autophagy; lipid metabolism; lipophagy; lymphangiogenesis; lymphatic endothelial cells; mitochondria
  38. Curr Issues Mol Biol. 2022 Aug 11. 44(8): 3611-3620
      Both mechanical loading and autophagy play important roles in regulating bone growth and remodeling, but the relationship between the two remains unclear. In this study, we examined bone structure with micro-CT imaging and measured bone mechanical properties with three-point bending experiments using bones from wild-type (WT) mice and conditional knockout (cKO) mice with Atg7 deletion in their osteoblasts. We found that the knockout mice had significantly less bone volume, bone thickness, bone ultimate breaking force, and bone stiffness compared to wild-type mice. Additionally, bone marrow cells from knockout mice had reduced differentiation and mineralization capacities in terms of alkaline phosphatase and calcium secretion, as well as Runx2 and osteopontin expression. Knockout mice also had significantly less relative bone formation rate due to mechanical loading. Furthermore, we found that the osteoblasts from wild-type mice had stronger responses to mechanical stimulation compared to autophagy-deficient osteoblasts from knockout mice. When inhibiting autophagy with 3 MA in wild-type osteoblasts, we found similar results as we did in autophagy-deficient osteoblasts. We also found that mechanical loading-induced ATP release is able to regulate ERK1/2, Runx2, alkaline phosphatase, and osteopontin activities. These results suggest that the ATP pathway may play an important role in the possible involvement of autophagy in osteoblast mechanobiology.
    Keywords:  ATP; autophagy; mechanotransduction; osteoblast
  39. Cell Signal. 2022 Aug 19. pii: S0898-6568(22)00206-6. [Epub ahead of print]99 110444
      The exact role of autophagy in myocardial ischemia/reperfusion (I/R) injury is still controversial. Excessive or insufficient autophagy may lead to cell death. Therefore, how to regulate autophagic balance during myocardial ischemia/reperfusion is critical to the treatment of myocardial I/R injury. Raptor is an mTOR regulatory related protein and closely related to the induction of autophagy. ZNF143 is widely expressed in various cells and acts as a transcription factor, which is involved in the regulation of autophagy, cell growth and development. In this study, we aimed to explore the mechanism by which ZNF143 regulated autophagy in myocardial I/R injury and the relationship between ZNF143 and Raptor. In our results, we found that ZNF143 expression was down-regulated in myocardial I/R. Inhibition of ZNF143 expression further enhanced autophagy and restored the deficiency of autophagic flux caused by myocardial I/R, subsequently alleviating myocardial I/R injury. On the other hand, overexpression of ZNF143 up-regulated Raptor expression and reduced autophagic activity, consequently exacerbating myocardial I/R injury. Taken together, our study revealed that ZNF143 might be a key target of the regulation of autophagy and a novel therapeutic target of myocardial I/R injury.
    Keywords:  Raptor; ZNF143; autophagic flux; autophagy; myocardial ischemia/reperfusion injury
  40. Nat Metab. 2022 Aug;4(8): 1022-1040
      Cholesterol contributes to the structural basis of biological membranes and functions as a signaling molecule, whose dysregulation has been associated with various human diseases. Here, we report that the long non-coding RNA (lncRNA) SNHG6 increases progression from non-alcoholic fatty liver disease (NAFLD) to hepatocellular carcinoma (HCC) by modulating cholesterol-induced mTORC1 activation. Mechanistically, cholesterol binds ER-anchored FAF2 protein to promote the formation of a SNHG6-FAF2-mTOR complex. As a putative cholesterol effector, SNHG6 enhances cholesterol-dependent mTORC1 lysosomal recruitment and activation via enhancing FAF2-mTOR interaction at ER-lysosome contacts, thereby coordinating mTORC1 kinase cascade activation with cellular cholesterol biosynthesis in a self-amplified cycle to accelerate cholesterol-driven NAFLD-HCC development. Notably, loss of SNHG6 inhibits mTORC1 signaling and impairs growth of patient-derived xenograft liver cancer tumors, identifyifng SNHG6 as a potential target for liver cancer treatment. Together, our findings illustrate the crucial role of organelle-associated lncRNA in organelle communication, nutrient sensing, and kinase cascades.
  41. Cells. 2022 Aug 12. pii: 2514. [Epub ahead of print]11(16):
      Recent studies have highlighted the importance of autophagy and particularly non-canonical autophagy in the development and progression of acute pancreatitis (a frequent disease with considerable morbidity and significant mortality). An important early event in the development of acute pancreatitis is the intrapancreatic activation of trypsinogen, (i.e., formation of trypsin) leading to the autodigestion of the organ. Another prominent phenomenon associated with the initiation of this disease is vacuolisation and specifically the formation of giant endocytic vacuoles in pancreatic acinar cells. These organelles develop in acinar cells exposed to several inducers of acute pancreatitis (including taurolithocholic acid and high concentrations of secretagogues cholecystokinin and acetylcholine). Notably, early trypsinogen activation occurs in the endocytic vacuoles. These trypsinogen-activating organelles undergo activation, long-distance trafficking, and non-canonical autophagy. In this review, we will discuss the role of autophagy in acute pancreatitis and particularly focus on the recently discovered LAP-like non-canonical autophagy (LNCA) of endocytic vacuoles.
    Keywords:  ATG8; CASM; LAP; LAP-like non-canonical autophagy; LC3; LC3-associated phagocytosis; LNCA; acute pancreatitis; autophagy; cholecystokinin; endocytic vacuole; non-canonical autophagy; pancreatic acinar cell; trypsin; trypsinogen; zymogen granule
  42. J Cell Sci. 2022 Aug 24. pii: jcs.259994. [Epub ahead of print]
      TORC1 is a critical controller of cell growth in eukaryotes. In yeast, the presence of nutrients is signaled to TORC1 by several upstream regulatory sensors that together coordinate TORC1 activity. TORC1 localizes to both vacuolar and endosomal membranes, where differential signaling occurs. This localization is mimicked by Pib2, a key upstream TORC1 regulator that is essential for TORC1 reactivation after nutrient starvation or pharmacological inhibition. Pib2 has both positive and negative effects on TORC1 activity, but the mechanisms remain poorly understood. Here, we pinpoint the Pib2 inhibitory function on TORC1 to residues within short, conserved N-terminal regions. We also show that Pib2 C-terminal regions, helical region E and tail, are essential for TORC1 reactivation. Further, the Pib2 FYVE domain plays a role in vacuolar localization but it is surprisingly unnecessary for recovery from rapamycin exposure. Using chimeric Pib2 targeting constructs, we show that endosomal localization is not necessary for TORC1 reactivation and cell growth after rapamycin treatment. Thus, a comprehensive molecular dissection of Pib2 demonstrates that each of its conserved regions differentially contribute to Pib2 regulation of TORC1 activity.
    Keywords:  FYVE; Pib2; Rapamycin; TORC1
  43. Carbohydr Polym. 2022 Nov 01. pii: S0144-8617(22)00746-9. [Epub ahead of print]295 119841
      Mitophagy can selectively remove damaged mitochondria, which is critical in regulating mitochondrial homeostasis in diseases, such as cancer. Herein, we found that Aloe gel glucomannan (AGP) significantly inhibited the proliferation of colon cancer cells. RNA-seq analysis revealed that AGP upregulated autophagy, lysosome and mitochondrial fission signal pathways in colon cancer cell line CT26. Notably, AGP induced the accumulation of impaired and reactive oxygen species (ROS)-generating mitochondria, which triggered excessive mitophagy. Interestingly, the mitophagy activator enhanced AGP-induced mitophagy and cytotoxicity, whereas the mitophagy inhibitor reversed the influence of AGP. Furthermore, activation of PINK1/Parkin mitophagy pathway and transcription factor EB (TFEB) signaling was dependent on ROS overproduction. Taken together, these results indicated that AGP induced cytotoxic mitophagy through ROS-related PINK1/Parkin pathway and TFEB activation in CT26 cells. The research would provide theoretical basis for the development of AGP as a promising anticancer agent.
    Keywords:  Aloe gel glucomannan; Ammonium sulfate (PubChem CID: 6097028); Crystal Violet (PubChem CID: 11057); DAPI (PubChem CID: 2954); Ethanol (PubChem CID: 702); FCCP (PubChem CID: 3330); Hoechst 33342 (PubChem CID: 1464); Mdivi-1 (PubChem CID: 3825829); MitoTracker Red (PubChem CID: 22613925); Mitophagy; N-Acetyl-L-cysteine (PubChem CID: 12035); Phenol (PubChem CID: 996); ROS; Rapamycin (PubChem CID: 5284616); Sulfuric Acid (PubChem CID: 1118); TFEB; Triton X-100 (PubChem CID: 5590); colon cancer
  44. Bio Protoc. 2022 Apr 05. 12(7): e4369
      Ultra-precise stimulation solely to individual mitochondria, without any influence to the whole cell, is quite difficult by traditional biochemical reagents. In mitophagy research, the mitochondria and even the whole cell usually suffer irreversible and great damage caused by treatment with potent chemicals. In this protocol, we present the technical procedures of our developed noninvasive ultra- precise laser stimulation (UPLaS) technology, which introduces precise stimulation to individual mitochondria, to excite mitochondrial Ca 2+ (mitoCa 2+ ) oscillations, with little perturbation to mitochondrial membrane potential (MMP), or mitochondrial reactive oxygen species (mitoROS). The mitoCa 2+ oscillation by UPLaS was able to initiate the PINK1/Parkin pathway for mitophagy. This protocol has good potential to benefit researches on mitophagy and mitochondrial diseases. Graphic abstract: Figure 1.Flowchart of the UPLaS technology.The femtosecond laser (1030 nm, 1 MHz, 220 fs) can stimulate individual mitochondria (1 μm 2 ) for a short period (0.1 s), whereas confocal microscopy (CM) provides continuous cell imaging to monitor molecular dynamics in real time, before and after UPLaS.
    Keywords:  Ca 2+; Confocal microscope; Femtosecond laser; Mitophagy; PINK1; Parkin; Ultra-precise laser stimulation
  45. Int J Mol Sci. 2022 Aug 17. pii: 9228. [Epub ahead of print]23(16):
      The beneficial effects of diet and exercise on brain function are traditionally attributed to the enhancement of autophagy, which plays a key role in neuroprotection via the degradation of potentially harmful intracellular structures. The molecular machinery of autophagy has also been suggested to influence synaptic signaling via interaction with trafficking and endocytosis of synaptic vesicles and proteins. Still, the role of autophagy in the regulation of synaptic plasticity remains elusive, especially in the mammalian brain. We explored the impact of autophagy on synaptic transmission and homeostatic and acute synaptic plasticity using transgenic mice with induced deletion of the Beclin1 protein. We observed down-regulation of glutamatergic and up-regulation of GABAergic synaptic currents and impairment of long-term plasticity in the neocortex and hippocampus of Beclin1-deficient mice. Beclin1 deficiency also significantly reduced the effects of environmental enrichment, caloric restriction and its pharmacological mimetics (metformin and resveratrol) on synaptic transmission and plasticity. Taken together, our data strongly support the importance of autophagy in the regulation of excitatory and inhibitory synaptic transmission and synaptic plasticity in the neocortex and hippocampus. Our results also strongly suggest that the positive modulatory actions of metformin and resveratrol in acute and homeostatic synaptic plasticity, and therefore their beneficial effects on brain function, occur via the modulation of autophagy.
    Keywords:  AMPA receptor; Beclin1; GABAA receptor; LTP; VPS-34; autophagy; endocytosis; synaptic current; synaptic scaling; trafficking
  46. Int J Mol Sci. 2022 Aug 10. pii: 8886. [Epub ahead of print]23(16):
      Glioblastoma (GB) cells physically interact with peritumoral pericytes (PCs) present in the brain microvasculature. These interactions facilitate tumor cells to aberrantly increase and benefit from chaperone-mediated autophagy (CMA) in the PC. GB-induced CMA leads to major changes in PC immunomodulatory phenotypes, which, in turn, support cancer progression. In this review, we focus on the consequences of the GB-induced up-regulation of CMA activity in PCs and evaluate how manipulation of this process could offer new strategies to fight glioblastoma, increasing the availability of treatments for this cancer that escapes conventional therapies. We finally discuss the use of modified PCs unable to increase CMA in response to GB as a cell therapy alternative to minimize undesired off-target effects associated with a generalized CMA inhibition.
    Keywords:  autophagy; cell therapy; chaperone-mediated autophagy; glioblastoma; glioblastoma therapy; pericytes; perivascular niche
  47. Int J Mol Sci. 2022 Aug 11. pii: 8936. [Epub ahead of print]23(16):
      Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, of the so-called minority diseases, due to its low prevalence. It is caused by an abnormally long track of glutamines (polyQs) in mutant huntingtin (mHtt), which makes the protein toxic and prone to aggregation. Many pathways of clearance of badly-folded proteins are disrupted in neurons of patients with HD. In this work, we show that one Mn(II) quinone complex (4QMn), designed to work as an artificial superoxide dismutase, is able to activate both the ubiquitin-proteasome system and the autophagy pathway in vitro and in vivo models of HD. Activation of these pathways degrades mHtt and other protein-containing polyQs, which restores proteostasis in these models. Hence, we propose 4QMn as a potential drug to develop a therapy to treat HD.
    Keywords:  Caenorhabditis elegans; Huntington’s disease; Mn(II) complexes; autophagy; polyQ toxicity; proteasome
  48. Antioxid Redox Signal. 2022 Aug 26.
      SIGNIFICANCE: Liver disease is one of the biggest threats to public health, affecting as much as 5.5 million people worldwide. Mitochondrial dysfunction is associated with various acute and chronic liver diseases. Mitophagy plays a key role either in the pathogenesis or in maintaining hepatic homeostasis in response to various liver diseases.RECENT ADVANCES: Significant progress has been achieved to ascertain the causes of liver disease. The conserved pathways for mitochondrial degradation via mitophagy, the deregulation of mitophagy in liver diseases, and pharmacological or genetic maneuvers that alter the mitophagic flux for liver disease treatment have been widely studied but yet to be comprehensively reviewed.
    CRITICAL ISSUES: Liver disease is considered a leading cause of mortality globally, causing the heavy burden of disability and the increased health care utilization that needs to be settled urgently. Mitophagy plays an important role in protecting liver from tissue damage. Elaborating mitophagy implicated in the pathogenesis of liver disease, as well as potential therapeutic regimens by targeting mitophagy is of great significance for the understanding and treating of liver disease.
    FUTURE DIRECTIONS: This review comprehensively describes the distinct mitophagy signaling pathways and their interplay with various liver diseases. A deeper understanding of how to modulate mitophagy could provide innovative avenues for precise therapy. Future studies based on pharmacologically or genetically targeting mitophagy-relevant factors will uncover the links between intact mitophagic responses and hepatic homeostasis in physiological and pathological settings. This will allow us to overcome obstacles of applying mitophagy as the therapeutic target in clinic.
  49. Neurobiol Dis. 2022 Aug 22. pii: S0969-9961(22)00235-2. [Epub ahead of print] 105843
  50. Front Pharmacol. 2022 ;13 956501
      Autophagy, originally described as a mechanism for intracellular waste disposal and recovery, has been becoming a crucial biological process closely related to many types of human tumors, including breast cancer, osteosarcoma, glioma, etc., suggesting that intervention of autophagy is a promising therapeutic strategy for cancer drug development. Therefore, a high-quality database is crucial for unraveling the complicated relationship between autophagy and human cancers, elucidating the crosstalk between the key autophagic pathways, and autophagic modulators with their remarkable antitumor activities. To achieve this goal, a comprehensive database of autophagic modulators (AMTDB) was developed. AMTDB focuses on 153 cancer types, 1,153 autophagic regulators, 860 targets, and 2,046 mechanisms/signaling pathways. In addition, a variety of classification methods, advanced retrieval, and target prediction functions are provided exclusively to cater to the different demands of users. Collectively, AMTDB is expected to serve as a powerful online resource to provide a new clue for the discovery of more candidate cancer drugs.
    Keywords:  AMTDB; anti-tumor drug; autophagic modulator; autophagy; database
  51. Adv Exp Med Biol. 2022 Aug 23.
      Heat shock factor 1 (HSF1) is a master transcription regulator that mediates the induction of heat shock protein chaperones for quality control (QC) of the proteome and maintenance of proteostasis as a protective mechanism in response to stress. Research in this particular area has accelerated dramatically over the past three decades following successful isolation, cloning, and characterization of HSF1. The intricate multi-protein complexes and transcriptional activation orchestrated by HSF1 are fundamental processes within the cellular QC machinery. Our primary focus is on the regulation and function of HSF1 in aging and neurodegenerative diseases (ND) which represent physiological and pathological states of dysfunction in protein QC. This chapter presents an overview of HSF1 structural, functional, and energetic properties in healthy cells while addressing the deterioration of HSF1 function viz-à-viz age-dependent and neuron-specific vulnerability to ND. We discuss the structural domains of HSF1 with emphasis on the intrinsically disordered regions and note that disease proteins associated with ND are often structurally disordered and exquisitely sensitive to changes in cellular environment as may occur during aging. We propose a hypothesis that age-dependent changes of the intrinsically disordered proteome likely hold answers to understand many of the functional, structural, and organizational changes of proteins and signaling pathways in aging - dysfunction of HSF1 and accumulation of disease protein aggregates in ND included.Structured AbstractsIntroduction: Heat shock factor 1 (HSF1) is a master transcription regulator that mediates the induction of heat shock protein chaperones for quality control (QC) of the proteome as a cyto-protective mechanism in response to stress. There is cumulative evidence of age-related deterioration of this QC mechanism that contributes to disease vulnerability.OBJECTIVES: Herein we discuss the regulation and function of HSF1 as they relate to the pathophysiological changes of protein quality control in aging and neurodegenerative diseases (ND).
    METHODS: We present an overview of HSF1 structural, functional, and energetic properties in healthy cells while addressing the deterioration of HSF1 function vis-à-vis age-dependent and neuron-specific vulnerability to neurodegenerative diseases.
    RESULTS: We examine the impact of intrinsically disordered regions on the function of HSF1 and note that proteins associated with neurodegeneration are natively unstructured and exquisitely sensitive to changes in cellular environment as may occur during aging.
    CONCLUSIONS: We put forth a hypothesis that age-dependent changes of the intrinsically disordered proteome hold answers to understanding many of the functional, structural, and organizational changes of proteins - dysfunction of HSF1 in aging and appearance of disease protein aggregates in neurodegenerative diseases included.
    Keywords:  Aging; HSF1, Heat shock factor 1; HSP, heat shock protein family; Hsp, Specific heat shock protein; Intrinsically disordered proteome; Neurodegeneration; Protein homeostasis
  52. Hum Mol Genet. 2022 Aug 22. pii: ddac201. [Epub ahead of print]
      Dominant mutations in ubiquitously expressed Mitofusin 2 gene (MFN2) cause Charcot-Marie-Tooth type 2A (CMT2A; OMIM 609260), an inherited sensory-motor neuropathy that affects peripheral nerve axons. Mitofusin 2 protein has been found to take part in mitochondrial fusion, mitochondria-endoplasmic reticulum tethering, mitochondrial trafficking along axons, mitochondrial quality control, and various types of cancer, in which MFN2 has been indicated as a tumor suppressor gene. Discordant data on the mitochondrial altered phenotypes in patient-derived fibroblasts harboring MFN2 mutations and in animal models have been reported. We addressed some of these issues by focusing on mitochondria behavior during autophagy and mitophagy in fibroblasts derived from a CMT2AMFN2 patient with an MFN2650G > T/C217F mutation in the GTPase domain. This study investigated mitochondrial dynamics, respiratory capacity, and autophagy/mitophagy, to tackle the multifaceted MFN2 contribution to CMT2A pathogenesis. We found that MFN2 mutated fibroblasts showed impairment of mitochondrial morphology, bioenergetics capacity, and impairment of the early stages of autophagy, but not mitophagy. Unexpectedly, transcriptomic analysis of mutated fibroblasts highlighted marked differentially expressed pathways related to cell population proliferation and extracellular matrix organization. We consistently found the activation of mTORC2/AKT signaling and accelerated proliferation in the CMT2AMFN2 fibroblasts. In conclusion, our evidence indicates that MFN2 mutation can positively drive cell proliferation in CMT2AMFN2 fibroblasts.
  53. Molecules. 2022 Aug 19. pii: 5295. [Epub ahead of print]27(16):
      The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase (PIKK) family. The kinase exists in the forms of two complexes, mTORC1 and mTORC2, and it participates in cell growth, proliferation, metabolism, and survival. The kinase activity is closely related to the occurrence and development of multiple human diseases. Inhibitors of mTOR block critical pathways to produce antiviral, anti-inflammatory, antiproliferative and other effects, and they have been applied to research in cancer, inflammation, central nervous system diseases and viral infections. Existing mTOR inhibitors are commonly divided into mTOR allosteric inhibitors, ATP-competitive inhibitors and dual binding site inhibitors, according to their sites of action. In addition, there exist several dual-target mTOR inhibitors that target PI3K, histone deacetylases (HDAC) or ataxia telangiectasia mutated and Rad-3 related (ATR) kinases. This review focuses on the structure of mTOR protein and related signaling pathways as well as the structure and characteristics of various mTOR inhibitors. Non-rapalog allosteric inhibitors will open new directions for the development of new therapeutics specifically targeting mTORC1. The applications of ATP-competitive inhibitors in central nervous system diseases, viral infections and inflammation have laid the foundation for expanding the indications of mTOR inhibitors. Both dual-binding site inhibitors and dual-target inhibitors are beneficial in overcoming mTOR inhibitor resistance.
    Keywords:  dual inhibitors; human diseases; mTOR; mTOR inhibitors
  54. Biomolecules. 2022 Aug 02. pii: 1069. [Epub ahead of print]12(8):
      Alternative splicing allows the synthesis of different protein variants starting from a single gene. Human Beclin 1 (BECN1) is a key autophagy regulator that acts as haploinsufficient tumor suppressor since its decreased expression correlates with tumorigenesis and poor prognosis in cancer patients. Recent studies show that BECN1 mRNA undergoes alternative splicing. Here, we report on the isolation and molecular and functional characterization of three BECN1 transcript variants (named BECN1-α, -β and -γ) in human cancer cells. In ovarian cancer NIHOVCAR3, these splicing variants were found along with the canonical wild-type. BECN1-α lacks 143 nucleotides at its C-terminus and corresponds to a variant previously described. BECN1-β and -γ lack the BCL2 homology 3 domain and other regions at their C-termini. Following overexpression in breast cancer cells MDA-MB231, we found that BECN1-α stimulates autophagy. Specifically, BECN1-α binds to Parkin and stimulates mitophagy. On the contrary, BECN1-β reduces autophagy with a dominant negative effect over the endogenous wild-type isoform. BECN1-γ maintains its ability to interact with the vacuolar protein sorting 34 and only has a slight effect on autophagy. It is possible that cancer cells utilize the alternative splicing of BECN1 for modulating autophagy and mitophagy in response to environmental stresses.
    Keywords:  BH3 domain breast cancers; Bcl-2; Beclin 1; alternative splicing; autophagy; isoforms; mitophagy; ovarian cancers
  55. Front Physiol. 2022 ;13 960480
      Left atrial appendage (LAA) thrombus detachment resulting in intracranial embolism is a major complication of atrial fibrillation (AF). Endocardial endothelial cell (EEC) injury leads to thrombosis, whereas autophagy protects against EEC dysfunction. However, the role and underlying mechanisms of autophagy in EECs during AF have not been elucidated. In this study, we isolated EECs from AF model mice and observed reduced autophagic flux and intracellular calcium concentrations in EECs from AF mice. In addition, we detected an increased expression of the mechanosensitive protein PLXND1 in the cytomembranes of EECs. PLXND1 served as a scaffold protein to bind with ORAI1 and further decreased ORAI1-mediated calcium influx. The decrease in the calcium influx-mediated phosphorylation of CAMK2 is associated with the inhibition of autophagy, which results in EEC dysfunction in AF. Our study demonstrated that the change in PLXND1 expression contributes to intracellular calcium dyshomeostasis, which inhibits autophagy flux and results in EEC dysfunction in AF. This study provides a potential intervention target for EEC dysfunction to prevent and treat intracardiac thrombosis in AF and its complications.
    Keywords:  PLXND1; atrial fibrillation; autophagy; calcium flux; endocardial endothelial cell
  56. 3 Biotech. 2022 Sep;12(9): 230
      Organelle crosstalk is significant in regulating their respective functions and subsequent cell fate. Mitochondria and lysosomes are amongst the essential organelles in maintaining cellular homeostasis. Mitochondria-lysosome connections, which may develop dynamically in the human neurons, have been identified as sites of bidirectional communication. Aberrancies are often associated with neurodegenerative disorders like Parkinson's disease (PD), suggesting the physical and functional link between these two organelles. PD is often linked with genetic mutations of several mutations discovered in the familial forms of the disease; some are considered risk factors. Many of these genes are either associated with mitochondrial function or belong to endo-lysosomal pathways. The recent investigations have indicated that neurons with mutant glucosylceramidase beta (GBA1) exhibit extended mitochondria-lysosome connections in individuals with PD. This may be due to impaired control of the untethering protein, which aids in the hydrolysis of Rab7 GTP required for contact untethering. A GCase modulator may be used to augment the reduced GBA1 lysosomal enzyme activity in the neurons of PD patients. This review focuses on how GBA1 mutation in PD is interlinked with mitochondria-lysosome (ML) crosstalk, exploring the pathways governing these interactions and mechanistically comprehending the mitochondrial and lysosomal miscommunication in the pathophysiology of PD. This review is based on the limited literature available on the topic and hence may be subject to bias in its views. Our estimates may be conservative and limited due to the lack of studies under the said discipline due to its inherent complex nature. The current association of GBA1 to PD pathogenesis is based on the limited scope of study and further research is necessary to explore the risk factors further and identify the relationship with more detail.
    Keywords:  Glucosylceramidase Beta (GBA); Lysosome; ML crosstalk; Mitochondria; Neuronal dysfunction; Parkinson's disease
  57. Biomedicines. 2022 Aug 11. pii: 1944. [Epub ahead of print]10(8):
      Autophagy is a highly conserved dynamic process by which cells deliver their contents to lysosomes for degradation, thus ensuring cell homeostasis. In response to environmental stress, the induction of autophagy is crucial for cell survival. The dysregulation of this degradative process has been implicated in a wide range of pathologies, including lung diseases, representing a relevant potential target with significant clinical outcomes. During lung disease progression and infections, autophagy may exert both protective and harmful effects on cells. In this review, we will explore the implications of autophagy and its selective forms in several lung infections, such as SARS-CoV-2, Respiratory Syncytial Virus (RSV) and Mycobacterium tuberculosis (Mtb) infections, and different lung diseases such as Cystic Fibrosis (CF), Chronic Obstructive Pulmonary Disease (COPD), and Malignant Mesothelioma (MM).
    Keywords:  COPD; Cystic Fibrosis; Malignant Mesothelioma; Mycobacterium tuberculosis; Respiratory Syncytial Virus; autophagy; lung disease; lung infectious; xenophagy SARS-CoV-2
  58. Front Microbiol. 2022 ;13 944006
      Exogenous pathogen infection can induce autophagy in cells. Autophagy is essential for cell survival, development, and homeostasis. It not only regulates cell defense and stress, but also has a close relationship with innate and adaptive immunity. Complement is an important part of innate immunity, which could be activated by three approaches, including classic, alternative, and lectin pathways. All the three pathways result in the activation of C3, and generate anaphylatoxin fragments C3a and C5a, and formation of the membrane attack complex. Either C3a or C5a induces the inflammatory cytokines through binding to C3aR or C5aR, respectively. However, it is still unknown whether the complement could regulate the autophagy of intracellular microorganisms or not. In this study, we constructed a Toxoplasma gondii (T. gondii) and macrophages co-culture experimental model using T. gondii expressing enhanced green fluorescence protein (EGFP) fluorescence and C3-/-C57BL/6 J mice for that T. gondii invaded peritoneal macrophages in mice. Western blot, laser confocal microscopy (LCM), and transmission electron microscopy (TEM) were used to observe the changes of autophagy between the macrophages from wild-type (WT) and C3-/- mice. Flow cytometry and LCM were used to investigate the effect of autophagy on the killing ability of macrophages against T. gondii. Here, we found that local C3 could suppress not only the canonical autophagy of macrophage, but also the xenophagy to T. gondii. Interestingly, the inhibition of C3 on host cell autophagy could significantly suppress the clearance of T. gondii by the IFN-γ-primed macrophage. Finally, we investigated the mechanism of the autophagy regulation of C3 that the effect of C3 on the macrophage-specific autophagy against T. gondii depends on mTOR. And, there is C3a but not C5a/C5aR involved in regulating macrophage xenophagy against T. gondii. Collectively, our findings suggest locally generated C3 regulates the clearance of T. gondii by Macrophage through the regulation of the non-canonical IFN-γ-dependent autophagy pathway, and paint a clearer picture in the regulation of autophagy by innate immune components.
    Keywords:  C3; Toxoplasma; autophagy; macrophage; xenophagy
  59. Cell Mol Neurobiol. 2022 Aug 25.
      After restoration of spontaneous circulation (ROSC) following cardiac arrest, complements can be activated and excessive autophagy can contribute to the brain ischemia-reperfusion (I/R) injury. Mild hypothermia (HT) protects against brain I/R injury after ROSC, but the mechanisms have not been fully elucidated. Here, we found that HT significantly inhibited the increases in serum NSE, S100β, and C5a, as well as neurologic deficit scores, TUNEL-positive cells, and autophagic vacuoles in the pig brain cortex after ROSC. The C5a receptor 1 (C5aR1) mRNA and the C5a, C5aR1, Beclin 1, LC3-II, and cleaved caspase-3 proteins were significantly increased, but the P62 protein and the PI3K/Akt/mTOR pathway-related proteins were significantly reduced in pigs after ROSC or neuronal oxygen-glucose deprivation/reoxygenation. HT could significantly attenuate the above changes in NT-treated neurons. Furthermore, C5a treatment induced autophagy and apoptosis and reduced the PI3K/Akt/mTOR pathway-related proteins in cultured neurons, which could be reversed by C5aR1 antagonist PMX205. Our findings demonstrated that C5a could bind to C5aR1 to induce neuronal autophagy during the brain I/R injury, which was associated with the inhibited PI3K/Akt/mTOR pathway. HT could inhibit C5a-induced neuronal autophagy by regulating the C5a-C5aR1 interaction and the PI3K/Akt/mTOR pathway, which might be one of the neuroprotective mechanisms underlying I/R injury. The C5a receptor 1 (C5aR1) mRNA and the C5a, C5aR1, Beclin 1, LC3-II, and cleaved caspase-3 proteins were significantly increased, but the P62 protein and the PI3K/Akt/mTOR pathway-related proteins were significantly reduced in pigs after ROSC or neuronal oxygen-glucose deprivation/reoxygenation. Mild hypothermia (HT) could significantly attenuate the above changes in NT-treated neurons. Furthermore, C5a treatment induced autophagy and apoptosis and reduced the PI3K/Akt/mTOR pathway-related proteins in cultured neurons, which could be reversed by C5aR1 antagonist PMX205. Proposed mechanism by which HT protects against brain I/R injury by repressing C5a-C5aR1-induced excessive autophagy. Complement activation in response to brain I/R injury generates C5a that can interact with C5aR1 to inactivate mTOR, probably through the PI3K-AKT pathway, which can finally lead to autophagy activation. The excessively activated autophagy ultimately contributes to cell apoptosis and brain injury. HT may alleviate complement activation and then reduce C5a-induced autophagy to protect against brain I/R injury. HT, mild hypothermia; I/R, ischemia reperfusion.
    Keywords:  Autophagy; C5a; Cardiopulmonary resuscitation; Ischemia; Mild hypothermia; Reperfusion injury
  60. Life Sci. 2022 Aug 17. pii: S0024-3205(22)00588-4. [Epub ahead of print]307 120888
      AIM: Excess cholesterol deposition in lysosomes may result in lysosomal stress and dysfunction. Here, we focus on the role of lysosome membrane protein 2 (LIMP2) in relieving the lysosomal stress caused by excess cholesterol and the mechanism that regulate its expression.MATERIAL AND METHODS: Cholesterol enrichment in lamprey liver tissue was evaluated by RNA transcriptome data analysis, RT-qPCR, H&E, and Oil Red O staining. Gene markers of autophagy and cholesterol synthesis were determined by western blot or RT-qPCR. Lysosomal morphology and pH value was measured by confocal observation or flow cytometry. Dual-Luciferase reporter assay was performed to test the expression regulation relationship.
    KEY FINDINGS: We report that lamprey limp2 (L-limp2) is evolutionarily highly conserved with human LIMP2 (H-LIMP2). The biological function of L-limp2, consistent with H-LIMP2, includes maintaining lysosomal morphology, modulating autophagy, and aiding cholesterol efflux from lysosomes. Furthermore, we find that both L-limp2 and H-limp2 can restore cholesterol-induced elevation of lysosomal pH and impaired autophagic flux. We demonstrate that lamprey transcription factor binding to IGHM enhancer 3 (L-TFE3) can bind with coordinated lysosomal expression and regulation (CLEAR) elements on the L-limp2 promoter and regulate its expression. Moreover, this regulatory relationship is also available in humans. Taken together, the present study demonstrates that the evolutionarily conserved TFE3-LIMP2 axis may have a protective role against the impaired lysosomal function caused by excess cholesterol.
    SIGNIFICANCE: The protective effect of TFE3-LIMP2 axis against cholesterol-triggered lysosomal stress may provide a new target for the treatment of diseases caused by excessive cholesterol accumulation in lysosomes.
    Keywords:  LIMP2; Lamprey; Lysosomal stress; TFE3
  61. Int J Mol Sci. 2022 Aug 16. pii: 9196. [Epub ahead of print]23(16):
      The global prevalence of nonalcoholic fatty liver disease (NAFLD) continues to rise, yet effective treatments are lacking due to the complex pathogenesis of this disease. Although recent research has provided evidence for the "multiple strikes" theory, the classic "two strikes" theory has not been overturned. Therefore, there is a crucial need to identify multiple targets in NAFLD pathogenesis for the development of diagnostic markers and targeted therapeutics. Since its discovery, the mechanistic target of rapamycin (mTOR) has been recognized as the central node of a network that regulates cell growth and development and is closely related to liver lipid metabolism and other processes. This paper will explore the mechanisms by which mTOR regulates lipid metabolism (SREBPs), insulin resistance (Foxo1, Lipin1), oxidative stress (PIG3, p53, JNK), intestinal microbiota (TLRs), autophagy, inflammation, genetic polymorphisms, and epigenetics in NAFLD. The specific influence of mTOR on NAFLD was hypothesized to be divided into micro regulation (the mechanism of mTOR's influence on NAFLD factors) and macro mediation (the relationship between various influencing factors) to summarize the influence of mTOR on the developmental process of NAFLD, and prove the importance of mTOR as an influencing factor of NAFLD regarding multiple aspects. The effects of crosstalk between mTOR and its upstream regulators, Notch, Hedgehog, and Hippo, on the occurrence and development of NAFLD-associated hepatocellular carcinoma are also summarized. This analysis will hopefully support the development of diagnostic markers and new therapeutic targets in NAFLD.
    Keywords:  hedgehog; hippo; mTOR; nonalcoholic fatty liver disease; notch; targeted therapy
  62. Dig Liver Dis. 2022 Aug 21. pii: S1590-8658(22)00631-4. [Epub ahead of print]
      Long-term colitis in people with inflammatory bowel disease (IBD) may lead to colon cancer called colitis-associated colorectal cancer (CAC). Since the advent of preclinical prototypes of CAC, various immunological messaging cascades have been identified as implicated in developing this disease. The toll-like receptor (TLR)s, Janus kinase (JAK)-signal transducer and activator of transcription (STAT), Nuclear factor-kappa B (NF-κB), mammalian target of rapamycin complex (mTOR), autophagy, and oxidative stress are only a few of the molecular mechanisms that have been recognized as major components to CAC progression. These pathways may also represent attractive medicinal candidates for the prevention and management of CAC. CAC signaling mechanisms at the molecular level and how their dysregulation may cause illness are summarized in this comprehensive overview.
    Keywords:  Colitis; Colorectal cancer; Crohn's disease; IBD; Signaling
  63. Mol Metab. 2022 Aug 23. pii: S2212-8778(22)00149-1. [Epub ahead of print] 101580
      OBJECTIVE: Adipose tissue, via sympathetic and possibly sensory neurons, communicates with the central nervous system (CNS) to mediate energy homeostasis. In contrast to the sympathetic nervous system, the morphology, role and regulation of the sensory nervous system in adipose tissue are poorly characterized.METHODS AND RESULTS: Taking advantage of recent progress in whole-mount three-dimensional imaging, we identified a network of calcitonin gene-related protein (CGRP)-positive sensory neurons in murine white adipose tissue (WAT). We found that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, is required for arborization of sensory, but not of sympathetic neurons. Time course experiments revealed that adipose mTORC2 is required for maintenance of sensory neurons. Furthermore, loss of sensory innervation in WAT coincided with systemic insulin resistance. Finally, we established that neuronal protein growth-associated protein 43 (GAP43) is a marker for sensory neurons in adipose tissue.
    CONCLUSION: Our findings indicate that adipose mTORC2 is necessary for sensory innervation in WAT. In addition, our results also suggest that WAT may affect whole-body energy homeostasis via sensory neurons.
    Keywords:  CGRP; adipose tissue; diabetes; mTORC2; neuropathy; sensory nervous system; whole-body energy homeostasis
  64. Cells. 2022 Aug 12. pii: 2508. [Epub ahead of print]11(16):
      Phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB/AKT) and mechanistic target of rapamycin (mTOR) (PAM) pathways play important roles in breast tumorigenesis and confer worse prognosis in breast cancer patients. The inhibitors targeting three key nodes of these pathways, PI3K, AKT and mTOR, are continuously developed. For breast cancer patients to truly benefit from PAM pathway inhibitors, it is necessary to clarify the frequency and mechanism of abnormal alterations in the PAM pathway in different breast cancer subtypes, and further explore reliable biomarkers to identify the appropriate population for precision therapy. Some PI3K and mTOR inhibitors have been approved by regulatory authorities for the treatment of specific breast cancer patient populations, and many new-generation PI3K/mTOR inhibitors and AKT isoform inhibitors have also been shown to have good prospects for cancer therapy. This review summarizes the changes in the PAM signaling pathway in different subtypes of breast cancer, and the latest research progress about the biomarkers and clinical application of PAM-targeted inhibitors.
    Keywords:  AKT; PI3K; biomarker; breast cancer; cancer therapy; mTOR
  65. Curr Drug Targets. 2022 ;23(11): 1057-1071
      Obesity and type 2 diabetes mellitus (T2DM), as common metabolic diseases, are pathologically characterized by overnutrition and insulin resistance (IR), which subsequently lead to glucose and lipid metabolism disorders. The liver, a major metabolic organ of the body, integrates hormone and metabolic signals to regulate the synthesis of lipids and glucose as well as their transport to peripheral tissues, hence playing an essential role in the development of obesity and T2DM. Adenosine 5'-monophosphate-activated protein kinase (AMPK) is a central regulator involved in cellular and organismal metabolism in eukaryotes, which activates processes that produce ATP and diminishes its consumption. In addition, AMPK also regulates mitochondrial homeostasis and promotes autophagy, both of which are associated with the pathogenesis of IR. Therefore, increasing AMPK activity is considered a promising therapeutic strategy to prevent obesity and T2DM. In this review, we summarize the role of hepatic AMPK in obesity and T2DM and the potential of using AMPK activators as therapeutics for metabolic disorders.
    Keywords:  AMPK; AMPK activators; hyperglycemia; liver; obesity; type 2 diabetes mellitus
  66. Front Physiol. 2022 ;13 956344
      Diabetes is a common metabolic disease whose hyperglycemic state can induce diverse complications and even threaten human health and life security. Currently, the treatment of diabetes is restricted to drugs that regulate blood glucose and have certain accompanying side effects. Autophagy, a research hotspot, has been proven to be involved in the occurrence and progression of the chronic complications of diabetes. Autophagy, as an essential organismal defense mechanism, refers to the wrapping of cytoplasmic proteins, broken organelles or pathogens by vesicles, which are then degraded by lysosomes to maintain the stability of the intracellular environment. Here, we review the relevant aspects of autophagy and the molecular mechanisms of autophagy in diabetic chronic complications, and further analyze the impact of improving autophagy on diabetic chronic complications, which will contribute to a new direction for further prevention and treatment of diabetic chronic complications.
    Keywords:  autophagy; chronic complications; diabetes drug; molecular mechanism; regulation
  67. Pathogens. 2022 Jul 30. pii: 863. [Epub ahead of print]11(8):
      LC3-associated phagocytosis (LAP) is a noncanonical autophagy process reported in recent years and is one of the effective mechanisms of host defense against bacterial infection. During LAP, bacteria are recognized by pattern recognition receptors (PRRs), enter the body, and then recruit LC3 onto a single-membrane phagosome to form a LAPosome. LC3 conjugation can promote the fusion of the LAPosomes with lysosomes, resulting in their maturation into phagolysosomes, which can effectively kill the identified pathogens. However, to survive in host cells, bacteria have also evolved strategies to evade killing by LAP. In this review, we summarized the mechanism of LAP in resistance to bacterial infection and the ways in which bacteria escape LAP. We aim to provide new clues for developing novel therapeutic strategies for bacterial infectious diseases.
    Keywords:  LC3-associated phagocytosis; bacterial infection; immune evasion; phagocyte