bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2023‒05‒28
sixty-two papers selected by
Viktor Korolchuk, Newcastle University
  1. Redefining the role of AMPK in autophagy and the energy stress response.
  2. MAP4K3 inhibits Sirtuin-1 to repress the LKB1-AMPK pathway to promote amino acid-dependent activation of the mTORC1 complex.
  3. Mitophagy restricts BAX/BAK-independent, Parkin-mediated apoptosis.
  4. mTORC1 activity negatively regulates human hair follicle growth and pigmentation.
  5. Mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is involved in phagocytosis activation during THP-1 cell differentiation.
  6. PROX1 induction by autolysosomal activity stabilizes persister-like state of colon cancer via feedback repression of the NOX1-mTORC1 pathway.
  7. Distinct Roles of DRP1 in Conventional and Alternative Mitophagy in Obesity Cardiomyopathy.
  8. The lysosome-mitochondrion crosstalk engaged in silver nanoparticles-disturbed mitochondrial homeostasis.
  9. Response to endoplasmic reticulum stress in arginine vasopressin neurons.
  10. The role of lysosomal membrane proteins in autophagy and related diseases.
  11. Autophagic Degradation Is Involved in Cell Protection against Ricin Toxin.
  12. Metamorphic proteins at the basis of human autophagy initiation and lipid transfer.
  13. Crosstalk between the mTOR and Hippo pathways.
  14. Autophagy regulates sex steroid hormone synthesis through lysosomal degradation of lipid droplets in human ovary and testis.
  15. The KEAP1-NRF2 pathway regulates TFEB/TFE3-dependent lysosomal biogenesis.
  16. Enhanced expression of the autophagosomal marker LC3-II in detergent-resistant protein lysates from a CLN3 patient's post-mortem brain.
  17. Unbiased evaluation of rapamycin's specificity as an mTOR inhibitor.
  18. "Dirty Dancing" of Calcium and Autophagy in Alzheimer's Disease.
  19. Hsp70-Bim interaction facilitates mitophagy by recruiting parkin and TOMM20 into a complex.
  20. The versatile defender: exploring the multifaceted role of p62 in intracellular bacterial infection.
  21. Autophagy, innate immunity, and cardiac disease.
  22. Autophagy modulation in breast cancer utilizing nanomaterials and nanoparticles.
  23. Advances in the mTOR signaling pathway and its inhibitor rapamycin in epilepsy.
  24. The Autophagy Nucleation Factor ATG9 Forms Nanoclusters with the HIV-1 Receptor DC-SIGN and Regulates Early Antiviral Autophagy in Human Dendritic Cells.
  25. FOXK1 promotes nonalcoholic fatty liver disease by mediating mTORC1-dependent inhibition of hepatic fatty acid oxidation.
  26. Cellular Metabolism: A Fundamental Component of Degeneration in the Nervous System.
  27. Phosphorylation of STAT3 at Tyr705 contributes to TFEB-mediated autophagy-lysosomal pathway dysfunction and leads to ischemic injury in rats.
  28. mTORC1/ERK1/2 Interplay Regulates Protein Synthesis and Survival in Acute Myeloid Leukemia Cell Lines.
  29. Handelin protects human skin keratinocytes against ultraviolet B-induced photodamage via autophagy activation by regulating the AMPK-mTOR signaling pathway.
  30. Sites of sub-cellular photodamage: Apoptotic and autophagic responses.
  31. The mTOR signalling in corneal diseases: a recent update.
  32. The Effect of Calorie Restriction on Protein Quality Control in Yeast.
  33. Autophagy enables microglia to engage amyloid plaques and prevents microglial senescence.
  34. Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.
  35. Transmembrane Protein 175, a Lysosomal Ion Channel Related to Parkinson's Disease.
  36. Parkin Precipitates on Mitochondria via Aggregation and Autoubiquitination.
  37. Inhibition of endolysosome fusion increases exosome secretion.
  38. The STAT3-Regulated Autophagy Pathway in Glioblastoma.
  39. Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.
  40. Longitudinal modeling of human neuronal aging identifies RCAN1-TFEB pathway contributing to neurodegeneration of Huntington's disease.
  41. Autophagic activation in porcine oocytes is independent of meiotic progression.
  42. Autophagy blockage and lysosomal dysfunction are involved in diallyl sulfide-induced inhibition of malignant growth in hepatocellular carcinoma cells.
  43. LncRP11-675F6.3 responds to rapamycin treatment and reduces triglyceride accumulation via interacting with HK1 in hepatocytes by regulating autophagy and VLDL-related proteins.
  44. Monitoring H2S fluctuation during autophagic fusion of lysosomes and mitochondria using a lysosome-targeting fluorogenic probe.
  45. Macrophages influence Schwann cell myelin autophagy after nerve injury and in a model of Charcot-Marie-Tooth disease.
  46. Autophagy modulators influence the content of important signalling molecules in PS-positive extracellular vesicles.
  47. Phospholipase D3 degrades mitochondrial DNA to regulate nucleotide signaling and APP metabolism.
  48. The Essential Role of Light-Induced Autophagy in the Inner Choroid/Outer Retinal Neurovascular Unit in Baseline Conditions and Degeneration.
  49. The role of autophagy in regulating metabolism in the tumor microenvironment.
  50. Anomanolide C suppresses tumor progression and metastasis by ubiquitinating GPX4-driven autophagy-dependent ferroptosis in triple negative breast cancer.
  51. Engineered bacterial outer membrane vesicles encapsulating oncolytic adenoviruses enhance the efficacy of cancer virotherapy by augmenting tumor cell autophagy.
  52. Hippo (YAP)-autophagy axis protects against hepatic ischemia-reperfusion injury through JNK signaling.
  53. Epigenetic dysregulation of autophagy in sepsis-induced acute kidney injury: the underlying mechanisms for renoprotection.
  54. Phospholipase A2-activating protein induces mitophagy trough anti-apoptotic MCL1-mediated NLRX1 oligomerization.
  55. Dysfunctional Autophagy, Proteostasis, and Mitochondria as a Prelude to Age-Related Macular Degeneration.
  56. Regulation of hematopoietic stem cells differentiation, self-renewal, and quiescence through the mTOR signaling pathway.
  57. Comprehensive Profiling of Rapamycin Interacting Proteins with Multiple Mass Spectrometry-Based Omics Techniques.
  58. Neuroprotection of Andrographolide against Neurotoxin MPP+-Induced Apoptosis in SH-SY5Y Cells via Activating Mitophagy, Autophagy, and Antioxidant Activities.
  59. Deacidification of endolysosomes by neuronal aging drives synapse loss.
  60. Studying lysosomal function and dysfunction using LysoIP.
  61. Host cell egress of Brucella abortus requires BNIP3L-mediated mitophagy.
  62. Spermidine improves angiogenic capacity of senescent endothelial cells, and enhances ischemia-induced neovascularization in aged mice.
  1. Nat Commun. 2023 May 24. 14(1): 2994
    Redefining the role of AMPK in autophagy and the energy stress response.
    Ji-Man Park, Da-Hye Lee, Do-Hyung Kim.
      Autophagy maintains cellular homeostasis during low energy states. According to the current understanding, glucose-depleted cells induce autophagy through AMPK, the primary energy-sensing kinase, to acquire energy for survival. However, contrary to the prevailing concept, our study demonstrates that AMPK inhibits ULK1, the kinase responsible for autophagy initiation, thereby suppressing autophagy. We found that glucose starvation suppresses amino acid starvation-induced stimulation of ULK1-Atg14-Vps34 signaling via AMPK activation. During an energy crisis caused by mitochondrial dysfunction, the LKB1-AMPK axis inhibits ULK1 activation and autophagy induction, even under amino acid starvation. Despite its inhibitory effect, AMPK protects the ULK1-associated autophagy machinery from caspase-mediated degradation during energy deficiency, preserving the cellular ability to initiate autophagy and restore homeostasis once the stress subsides. Our findings reveal that dual functions of AMPK, restraining abrupt induction of autophagy upon energy shortage while preserving essential autophagy components, are crucial to maintain cellular homeostasis and survival during energy stress.
    DOI:  https://doi.org/10.1038/s41467-023-38401-z
  2. Life Sci Alliance. 2023 08;pii: e202201525. [Epub ahead of print]6(8):
    MAP4K3 inhibits Sirtuin-1 to repress the LKB1-AMPK pathway to promote amino acid-dependent activation of the mTORC1 complex.
    Mary Rose Branch, Cynthia L Hsu, Kohta Ohnishi, Wen-Chuan Shen, Elian Lee, Jill Meisenhelder, Brett Winborn, Bryce L Sopher, J Paul Taylor, Tony Hunter, Albert R La Spada.
      mTORC1 is the key rheostat controlling the cellular metabolic state. Of the various inputs to mTORC1, the most potent effector of intracellular nutrient status is amino acid supply. Despite an established role for MAP4K3 in promoting mTORC1 activation in the presence of amino acids, the signaling pathway by which MAP4K3 controls mTORC1 activation remains unknown. Here, we examined the process of MAP4K3 regulation of mTORC1 and found that MAP4K3 represses the LKB1-AMPK pathway to achieve robust mTORC1 activation. When we sought the regulatory link between MAP4K3 and LKB1 inhibition, we discovered that MAP4K3 physically interacts with the master nutrient regulatory factor sirtuin-1 (SIRT1) and phosphorylates SIRT1 to repress LKB1 activation. Our results reveal the existence of a novel signaling pathway linking amino acid satiety with MAP4K3-dependent suppression of SIRT1 to inactivate the repressive LKB1-AMPK pathway and thereby potently activate the mTORC1 complex to dictate the metabolic disposition of the cell.
    DOI:  https://doi.org/10.26508/lsa.202201525
  3. Sci Adv. 2023 May 24. 9(21): eadg8156
    Mitophagy restricts BAX/BAK-independent, Parkin-mediated apoptosis.
    Giovanni Quarato, Luigi Mari, Nicholas J Barrows, Mao Yang, Sebastian Ruehl, Mark J Chen, Cliff S Guy, Jonathan Low, Taosheng Chen, Douglas R Green.
      Degradation of defective mitochondria is an essential process to maintain cellular homeostasis and it is strictly regulated by the ubiquitin-proteasome system (UPS) and lysosomal activities. Here, using genome-wide CRISPR and small interference RNA screens, we identified a critical contribution of the lysosomal system in controlling aberrant induction of apoptosis following mitochondrial damage. After treatment with mitochondrial toxins, activation of the PINK1-Parkin axis triggered a BAX- and BAK-independent process of cytochrome c release from mitochondria followed by APAF1 and caspase 9-dependent apoptosis. This phenomenon was mediated by UPS-dependent outer mitochondrial membrane (OMM) degradation and was reversed using proteasome inhibitors. We found that the subsequent recruitment of the autophagy machinery to the OMM protected cells from apoptosis, mediating the lysosomal degradation of dysfunctional mitochondria. Our results underscore a major role of the autophagy machinery in counteracting aberrant noncanonical apoptosis and identified autophagy receptors as key elements in the regulation of this process.
    DOI:  https://doi.org/10.1126/sciadv.adg8156
  4. EMBO Rep. 2023 May 22. e56574
    mTORC1 activity negatively regulates human hair follicle growth and pigmentation.
    Takahiro Suzuki, Jérémy Chéret, Fernanda Dinelli Scala, Aysun Akhundlu, Jennifer Gherardini, Dana-Lee Demetrius, James D B O'Sullivan, Gorana Kuka Epstein, Alan J Bauman, Constantinos Demetriades, Ralf Paus.
      Dysregulation of the activity of the mechanistic target of rapamycin complex 1 (mTORC1) is commonly linked to aging, cancer, and genetic disorders such as tuberous sclerosis (TS), a rare neurodevelopmental multisystemic disease characterized by benign tumors, seizures, and intellectual disability. Although patches of white hair on the scalp (poliosis) are considered as early signs of TS, the underlying molecular mechanisms and potential involvement of mTORC1 in hair depigmentation remain unclear. Here, we have used healthy, organ-cultured human scalp hair follicles (HFs) to interrogate the role of mTORC1 in a prototypic human (mini-)organ. Gray/white HFs exhibit high mTORC1 activity, while mTORC1 inhibition by rapamycin stimulated HF growth and pigmentation, even in gray/white HFs that still contained some surviving melanocytes. Mechanistically, this occurred via increased intrafollicular production of the melanotropic hormone, α-MSH. In contrast, knockdown of intrafollicular TSC2, a negative regulator of mTORC1, significantly reduced HF pigmentation. Our findings introduce mTORC1 activity as an important negative regulator of human HF growth and pigmentation and suggest that pharmacological mTORC1 inhibition could become a novel strategy in the management of hair loss and depigmentation disorders.
    Keywords:  alpha-MSH/MC1R; mTORC1; melanocyte; rapamycin; tuberous sclerosis
    DOI:  https://doi.org/10.15252/embr.202256574
  5. J Vet Med Sci. 2023 May 25.
    Mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is involved in phagocytosis activation during THP-1 cell differentiation.
    Hiroka Murokawa, Karin Egusa, Shusaku Shibutani, Hiroyuki Iwata.
      Mechanistic/mammalian target of rapamycin complex 1 (mTORC1) is a serine/threonine kinase that plays a major role in cell metabolism. Although mTORC1 inhibitors are known to exert immunosuppressive effects, their effects on immune cells are not fully understood. In the present study, we examined the involvement of mTORC1 in the differentiation and functions of macrophages using THP-1 cells, which are derived from human monocytic leukemia and differentiate into macrophage-like cells upon treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We also examined the effects of two mTOR inhibitors, Torin 1 and rapamycin, on TPA-stimulated THP-1 cells. Although mTORC1 activation was observed upon TPA stimulation, mTOR inhibitors did not affect TPA-induced morphological changes or expression of the general macrophage marker, CD11b. In contrast, phagocytosis and fluid endocytosis were significantly impaired by the mTOR inhibitors. Endocytosis suppression was observed when mTOR inhibitors were added during differentiation, but not before or after differentiation, suggesting that endocytosis suppression altered the direction of differentiation. Furthermore, mTOR inhibitors altered the expression of M1/M2 polarization markers. These results suggest that the immunosuppressive effects of mTOR inhibitors may involve the suppression of macrophage endocytosis caused by abnormal cell differentiation.
    Keywords:  THP-1; macrophage; mechanistic/mammalian target of rapamycin (mTOR); mechanistic/mammalian target of rapamycin complex 1 (mTORC1); phagocytosis
    DOI:  https://doi.org/10.1292/jvms.22-0504
  6. Cell Rep. 2023 May 17. pii: S2211-1247(23)00530-2. [Epub ahead of print] 112519
    PROX1 induction by autolysosomal activity stabilizes persister-like state of colon cancer via feedback repression of the NOX1-mTORC1 pathway.
    Hirokazu Ohata, Daisuke Shiokawa, Hiroaki Sakai, Yusuke Kanda, Yoshie Okimoto, Syuzo Kaneko, Ryuji Hamamoto, Hitoshi Nakagama, Koji Okamoto.
      Cancer chemoresistance is often attributed to slow-cycling persister populations with cancer stem cell (CSC)-like features. However, how persister populations emerge and prevail in cancer remains obscure. We previously demonstrated that while the NOX1-mTORC1 pathway is responsible for proliferation of a fast-cycling CSC population, PROX1 expression is required for chemoresistant persisters in colon cancer. Here, we show that enhanced autolysosomal activity mediated by mTORC1 inhibition induces PROX1 expression and that PROX1 induction in turn inhibits NOX1-mTORC1 activation. CDX2, identified as a transcriptional activator of NOX1, mediates PROX1-dependent NOX1 inhibition. PROX1-positive and CDX2-positive cells are present in distinct populations, and mTOR inhibition triggers conversion of the CDX2-positive population to the PROX1-positive population. Inhibition of autophagy synergizes with mTOR inhibition to block cancer proliferation. Thus, mTORC1 inhibition-mediated induction of PROX1 stabilizes a persister-like state with high autolysosomal activity via a feedback regulation that involves a key cascade of proliferating CSCs.
    Keywords:  CP: Cancer; NOX1; PROX1; autophagy; colon cancer; mTORC1; persister cells
    DOI:  https://doi.org/10.1016/j.celrep.2023.112519
  7. Circ Res. 2023 May 26.
    Distinct Roles of DRP1 in Conventional and Alternative Mitophagy in Obesity Cardiomyopathy.
    Mingming Tong, Risa Mukai, Satvik Mareedu, Peiyong Zhai, Shin-Ichi Oka, Chun-Yang Huang, Chiao-Po Hsu, Fawad A K Yousufzai, Luke Fritzky, Wataru Mizushima, Gopal J Babu, Junichi Sadoshima.
      RATIONALE: Obesity induces cardiomyopathy characterized by hypertrophy and diastolic dysfunction. Whereas mitophagy mediated through an Atg7-dependent mechanism serves as an essential mechanism to maintain mitochondrial quality during the initial development of obesity cardiomyopathy, Rab9-dependent alternative mitophagy takes over the role during the chronic phase. Although it has been postulated that DRP1 (dynamin-related protein 1)-mediated mitochondrial fission and consequent separation of the damaged portions of mitochondria are essential for mitophagy, the involvement of DRP1 in mitophagy remains controversial.OBJECTIVE: We investigated whether endogenous DRP1 is essential in mediating the 2 forms of mitophagy during high-fat diet (HFD)-induced obesity cardiomyopathy and, if so, what the underlying mechanisms are.
    METHODS AND RESULTS: Mice were fed either a normal diet or an HFD (60 kcal %fat). Mitophagy, evaluated with Mito-Keima, was increased after 3 weeks of HFD consumption. The induction of mitophagy by HFD consumption was completely abolished in tamoxifen-inducible cardiac-specific Drp1knockout (Drp1 MCM) mouse hearts, in which both diastolic and systolic dysfunction were exacerbated. The increase in LC3-dependent general autophagy and colocalization between LC3 and mitochondrial proteins was abolished in Drp1 MCM mice. Activation of alternative mitophagy was also completely abolished in Drp1 MCM mice during the chronic phase of HFD consumption. DRP1 was phosphorylated at Ser616, localized at the mitochondria-associated membranes, and associated with Rab9 and Fis1 only during the chronic, but not acute, phase of HFD consumption.
    CONCLUSIONS: DRP1 is an essential factor in mitochondrial quality control during obesity cardiomyopathy that controls multiple forms of mitophagy. Although DRP1 regulates conventional mitophagy through a mitochondria-associated membrane-independent mechanism during the acute phase, it acts as a component of the mitophagy machinery at the mitochondria-associated membranes in alternative mitophagy during the chronic phase of HFD consumption.
    Keywords:  diet, high-fat; mice, knockout; mitochondria; mitophagy; obesity cardiomyopathy
    DOI:  https://doi.org/10.1161/CIRCRESAHA.123.322512
  8. Sci Total Environ. 2023 May 18. pii: S0048-9697(23)02699-2. [Epub ahead of print]889 164078
    The lysosome-mitochondrion crosstalk engaged in silver nanoparticles-disturbed mitochondrial homeostasis.
    Jia Liu, Zhendong Huang, Suhan Yin, Xinru Zhou, Yanping Jiang, Longquan Shao.
      Given their increasing industrial and biomedical applications, silver nanoparticles (AgNPs) have become widely present in the environment. However, to date, studies on their potential health risks have been far from sufficient, especially those regarding their neurotoxic effects. This study investigated the neurotoxic effects of AgNPs on neural PC-12 cells in the context of mitochondria, which play an important role in AgNP-induced cellular metabolism disturbance and even cell death. Our results show that the endocytosed AgNPs, and not extracellular Ag+, appear to directly determine cell fate. Importantly, endocytosed AgNPs led to mitochondrial swelling and vacuolation without direct interaction. Although mitophagy, a selective autophagy process, was invoked to rescue damaged mitochondria, it failed to function in mitochondrial degradation and recycling. Discovery of the underlying mechanism showed that the endocytosed AgNPs could directly translocate into lysosomes and then cause lysosome perturbation, which is the main factor leading to mitophagy blockade and the subsequent accumulation of defective mitochondria. After lysosomal reacidification via cyclic adenosine monophosphate (cAMP), AgNP-induced dysfunctional autolysosome formation and disturbed mitochondrial homeostasis were reversed. In summary, this study reveals that lysosome-mitochondrion crosstalk is a main mechanism for AgNP-induced neurotoxic effects, offering an inspiring perspective on the neurotoxic effects of AgNPs.
    Keywords:  Lysosome impairment; Mitochondrial dysfunction; Mitophagy; Neurotoxic effects; Silver nanoparticles
    DOI:  https://doi.org/10.1016/j.scitotenv.2023.164078
  9. Endocr J. 2023 May 19.
    Response to endoplasmic reticulum stress in arginine vasopressin neurons.
    Daisuke Hagiwara, Yoshinori Azuma, Yohei Kawaguchi, Takashi Miyata, Hiroshi Arima.
      Arginine vasopressin (AVP) is an antidiuretic hormone synthesized principally in the hypothalamic supraoptic and paraventricular nuclei. The immunoglobulin heavy chain binding protein (BiP), one of the most abundant endoplasmic reticulum (ER) chaperones, is highly expressed in AVP neurons, even under basal conditions. Moreover, its expression is upregulated in proportion to the increase in AVP expression under dehydration. These data suggest that AVP neurons are constantly exposed to ER stress. BiP knockdown in AVP neurons induces ER stress and autophagy, resulting in AVP neuronal loss, indicating that BiP is pivotal in maintaining the AVP neuron system. Furthermore, inhibition of autophagy after BiP knockdown exacerbates AVP neuronal loss, suggesting that autophagy induced under ER stress is a protective cellular mechanism by which AVP neurons cope with ER stress. Familial neurohypophysial diabetes insipidus (FNDI) is an autosomal dominant disorder caused by mutations in the AVP gene. It is characterized by delayed-onset progressive polyuria and eventual AVP neuronal loss. In AVP neurons of FNDI model mice, mutant protein aggregates are confined to a specific compartment of the ER, called the ER-associated compartment (ERAC). The formation of ERACs contributes to maintaining the function of the remaining intact ER, and mutant protein aggregates in ERACs undergo autophagic-lysosomal degradation without isolation or translocation from the ER, representing a novel protein degradation system in the ER.
    Keywords:  Arginine vasopressin; Autophagy; Endoplasmic reticulum stress; Endoplasmic reticulum-associated compartment; Familial neurohypophysial diabetes insipidus
    DOI:  https://doi.org/10.1507/endocrj.EJ23-0193
  10. FEBS J. 2023 May 23.
    The role of lysosomal membrane proteins in autophagy and related diseases.
    Jiahao Xu, Jing Gu, Wenjun Pei, Yao Zhang, Lizhuo Wang, Jialin Gao.
      As a self-degrading and highly conserved survival mechanism, autophagy plays an important role in maintaining cell survival and recycling. The discovery of autophagy-related (ATG) genes has revolutionized our understanding of autophagy. Lysosomal membrane proteins (LMPs) are important executors of lysosomal function, and increasing evidence has demonstrated their role in the induction and regulation of autophagy. In addition, the functional dysregulation of the process mediated by LMPs at all stages of autophagy is closely related to neurodegenerative diseases and cancer. Here, we review the role of LMPs in autophagy, focusing on their roles in vesicle nucleation, vesicle elongation and completion, the fusion of autophagosomes and lysosomes, and degradation, as well as their broad association with related diseases.
    Keywords:  Autophagy; Autophagy-related gene; Disease; Lysosomal membrane protein; Lysosome
    DOI:  https://doi.org/10.1111/febs.16820
  11. Toxins (Basel). 2023 Apr 23. pii: 304. [Epub ahead of print]15(5):
    Autophagic Degradation Is Involved in Cell Protection against Ricin Toxin.
    Yu Wu, Clémence Taisne, Nassim Mahtal, Alison Forrester, Marion Lussignol, Jean-Christophe Cintrat, Audrey Esclatine, Daniel Gillet, Julien Barbier.
      Autophagy is a complex and highly regulated degradative process, which acts as a survival pathway in response to cellular stress, starvation and pathogen infection. Ricin toxin is a plant toxin produced by the castor bean and classified as a category B biothreat agent. Ricin toxin inhibits cellular protein synthesis by catalytically inactivating ribosomes, leading to cell death. Currently, there is no licensed treatment for patients exposed to ricin. Ricin-induced apoptosis has been extensively studied; however, whether its intoxication via protein synthesis inhibition affects autophagy is not yet resolved. In this work, we demonstrated that ricin intoxication is accompanied by its own autophagic degradation in mammalian cells. Autophagy deficiency, by knocking down ATG5, attenuates ricin degradation, thus aggravating ricin-induced cytotoxicity. Additionally, the autophagy inducer SMER28 (Small Molecule Enhancer 28) partially protects cells against ricin cytotoxicity, an effect not observed in autophagy-deficient cells. These results demonstrate that autophagic degradation acts as a survival response of cells against ricin intoxication. This suggests that stimulation of autophagic degradation may be a strategy to counteract ricin intoxication.
    Keywords:  ATG5; SMER28; autophagy; degradation; ricin toxin
    DOI:  https://doi.org/10.3390/toxins15050304
  12. Mol Cell. 2023 May 12. pii: S1097-2765(23)00321-0. [Epub ahead of print]
    Metamorphic proteins at the basis of human autophagy initiation and lipid transfer.
    Anh Nguyen, Francesca Lugarini, Céline David, Pouya Hosnani, Çağla Alagöz, Annabelle Friedrich, David Schlütermann, Barbora Knotkova, Anoshi Patel, Iwan Parfentev, Henning Urlaub, Michael Meinecke, Björn Stork, Alex C Faesen.
      Autophagy is a conserved intracellular degradation pathway that generates de novo double-membrane autophagosomes to target a wide range of material for lysosomal degradation. In multicellular organisms, autophagy initiation requires the timely assembly of a contact site between the ER and the nascent autophagosome. Here, we report the in vitro reconstitution of a full-length seven-subunit human autophagy initiation supercomplex built on a core complex of ATG13-101 and ATG9. Assembly of this core complex requires the rare ability of ATG13 and ATG101 to switch between distinct folds. The slow spontaneous metamorphic conversion is rate limiting for the self-assembly of the supercomplex. The interaction of the core complex with ATG2-WIPI4 enhances tethering of membrane vesicles and accelerates lipid transfer of ATG2 by both ATG9 and ATG13-101. Our work uncovers the molecular basis of the contact site and its assembly mechanisms imposed by the metamorphosis of ATG13-101 to regulate autophagosome biogenesis in space and time.
    Keywords:  autophagy; autophagy initiation; lipid transfer; membrane-contact site; metabolism; protein metamorphosis
    DOI:  https://doi.org/10.1016/j.molcel.2023.04.026
  13. Dev Growth Differ. 2023 May 20.
    Crosstalk between the mTOR and Hippo pathways.
    Daichi Honda, Misako Okumura, Takahiro Chihara.
      Cell behavior changes in response to multiple stimuli, such as growth factors, nutrients, and cell density. The mechanistic target of rapamycin (mTOR) pathway is activated by growth factors and nutrient stimuli to regulate cell growth and autophagy, whereas the Hippo pathway has negative effects on cell proliferation and tissue growth in response to cell density, DNA damage, and hormonal signals. These two signaling pathways must be precisely regulated and integrated for proper cell behavior. This integrative mechanism is not completely understood; nevertheless, recent studies have suggested that components of the mTOR and Hippo pathways interact with each other. Herein, as per contemporary knowledge, we review the molecular mechanisms of the interaction between the mTOR and Hippo pathways in mammals and Drosophila. Moreover, we discuss the advantage of this interaction in terms of tissue growth and nutrient consumption. This article is protected by copyright. All rights reserved.
    Keywords:  Hippo; Insulin; YAP; Yki; mTOR
    DOI:  https://doi.org/10.1111/dgd.12867
  14. Cell Death Dis. 2023 May 26. 14(5): 342
    Autophagy regulates sex steroid hormone synthesis through lysosomal degradation of lipid droplets in human ovary and testis.
    Yashar Esmaeilian, Francesko Hela, Gamze Bildik, Ece İltumur, Sevgi Yusufoglu, Ceren Sultan Yildiz, Kayhan Yakin, Yakup Kordan, Ozgur Oktem.
      Autophagy is an evolutionarily conserved process that aims to maintain the energy homeostasis of the cell by recycling long-lived proteins and organelles. Previous studies documented the role of autophagy in sex steroid hormone biosynthesis in different animal models and human testis. Here we demonstrate in this study that sex steroid hormones estrogen and progesterone are produced through the same autophagy-mediated mechanism in the human ovary in addition to the human testis. In brief, pharmacological inhibition and genetic interruption of autophagy through silencing of autophagy genes (Beclin1 and ATG5) via siRNA and shRNA technologies significantly reduced basal and gonadotropin-stimulated estradiol (E2), progesterone (P4) and testosterone (T) production in the ex vivo explant tissue culture of ovary and testis and primary and immortalized granulosa cells. Consistent with the findings of the previous works, we observed that lipophagy, a special form of autophagy, mediates the association of the lipid droplets (LD)s with lysosome to deliver the lipid cargo within the LDs to lysosomes for degradation in order to release free cholesterol required for steroid synthesis. Gonadotropin hormones are likely to augment the production of sex steroid hormones by upregulating the expression of autophagy genes, accelerating autophagic flux and promoting the association of LDs with autophagosome and lysosome. Moreover, we detected some aberrations at different steps of lipophagy-mediated P4 production in the luteinized GCs of women with defective ovarian luteal function. The progression of autophagy and the fusion of the LDs with lysosome are markedly defective, along with reduced P4 production in these patients. Our data, together with the findings of the previous works, may have significant clinical implications by opening a new avenue in understanding and treatment of a wide range of diseases, from reproductive disorders to sex steroid-producing neoplasms, sex steroid-dependent malignancies (breast, endometrium, prostate) and benign disorders (endometriosis).
    DOI:  https://doi.org/10.1038/s41419-023-05864-3
  15. Proc Natl Acad Sci U S A. 2023 05 30. 120(22): e2217425120
    The KEAP1-NRF2 pathway regulates TFEB/TFE3-dependent lysosomal biogenesis.
    Athena Jessica S Ong, Cerys E Bladen, Tara A Tigani, Anthony P Karamalakis, Kimberley J Evason, Kristin K Brown, Andrew G Cox.
      The maintenance of redox and metabolic homeostasis is integral to embryonic development. Nuclear factor erythroid 2-related factor 2 (NRF2) is a stress-induced transcription factor that plays a central role in the regulation of redox balance and cellular metabolism. Under homeostatic conditions, NRF2 is repressed by Kelch-like ECH-associated protein 1 (KEAP1). Here, we demonstrate that Keap1 deficiency induces Nrf2 activation and postdevelopmental lethality. Loss of viability is preceded by severe liver abnormalities characterized by an accumulation of lysosomes. Mechanistically, we demonstrate that loss of Keap1 promotes aberrant activation of transcription factor EB (TFEB)/transcription factor binding to IGHM Enhancer 3 (TFE3)-dependent lysosomal biogenesis. Importantly, we find that NRF2-dependent regulation of lysosomal biogenesis is cell autonomous and evolutionarily conserved. These studies identify a role for the KEAP1-NRF2 pathway in the regulation of lysosomal biogenesis and suggest that maintenance of lysosomal homeostasis is required during embryonic development.
    Keywords:  KEAP1; NRF2; TFEB/TFE3; lysosome; zebrafish
    DOI:  https://doi.org/10.1073/pnas.2217425120
  16. Biochim Biophys Acta Mol Basis Dis. 2023 May 19. pii: S0925-4439(23)00122-9. [Epub ahead of print]1869(6): 166756
    Enhanced expression of the autophagosomal marker LC3-II in detergent-resistant protein lysates from a CLN3 patient's post-mortem brain.
    Francesco Pezzini, Michele Fiorini, Stefano Doccini, Filippo Maria Santorelli, Gianluigi Zanusso, Alessandro Simonati.
      • Neuronal Ceroido Lipofuscinoses (NCL) are inherited, neurodegenerative disorders associated with lysosomal storage. • Impaired autophagy plays a pathogenetic role in several NCL forms, including CLN3 disease, but study on human brains are lacking. • In post-mortem brain samples of a CLN3 patient the LC3-I to LC3-II shift was consistent with activated autophagy. However, the autophagic process seemed to be ineffective due to the presence of lysosomal storage markers. • After fractionation with buffers of increasing detergent-denaturing strength, a peculiar solubility pattern of LC3-II was observed in CLN3 patient's samples, suggesting a different lipid composition of the membranes where LC3-II is stacked.
    Keywords:  Autophagy markers; Differential detergent fractioning; Human brain; LC3B; Lysosomal storage; Neuronal ceroid Lipofuscinoses (CLN3 disease)
    DOI:  https://doi.org/10.1016/j.bbadis.2023.166756
  17. Aging Cell. 2023 May 24. e13888
    Unbiased evaluation of rapamycin's specificity as an mTOR inhibitor.
    Filippo Artoni, Nina Grützmacher, Constantinos Demetriades.
      Rapamycin is a macrolide antibiotic that functions as an immunosuppressive and anti-cancer agent, and displays robust anti-ageing effects in multiple organisms including humans. Importantly, rapamycin analogues (rapalogs) are of clinical importance against certain cancer types and neurodevelopmental diseases. Although rapamycin is widely perceived as an allosteric inhibitor of mTOR (mechanistic target of rapamycin), the master regulator of cellular and organismal physiology, its specificity has not been thoroughly evaluated so far. In fact, previous studies in cells and in mice hinted that rapamycin may be also acting independently from mTOR to influence various cellular processes. Here, we generated a gene-edited cell line that expresses a rapamycin-resistant mTOR mutant (mTORRR ) and assessed the effects of rapamycin treatment on the transcriptome and proteome of control or mTORRR -expressing cells. Our data reveal a striking specificity of rapamycin towards mTOR, demonstrated by virtually no changes in mRNA or protein levels in rapamycin-treated mTORRR cells, even following prolonged drug treatment. Overall, this study provides the first unbiased and conclusive assessment of rapamycin's specificity, with potential implications for ageing research and human therapeutics.
    Keywords:  ageing; mTORC1; proteomics; rapamycin; sirolimus
    DOI:  https://doi.org/10.1111/acel.13888
  18. Life (Basel). 2023 May 15. pii: 1187. [Epub ahead of print]13(5):
    "Dirty Dancing" of Calcium and Autophagy in Alzheimer's Disease.
    Hua Zhang, Ilya Bezprozvanny.
      Alzheimer's disease (AD) is the most common cause of dementia. There is a growing body of evidence that dysregulation in neuronal calcium (Ca2+) signaling plays a major role in the initiation of AD pathogenesis. In particular, it is well established that Ryanodine receptor (RyanR) expression levels are increased in AD neurons and Ca2+ release via RyanRs is augmented in AD neurons. Autophagy is important for removing unnecessary or dysfunctional components and long-lived protein aggregates, and autophagy impairment in AD neurons has been extensively reported. In this review we discuss recent results that suggest a causal link between intracellular Ca2+ signaling and lysosomal/autophagic dysregulation. These new results offer novel mechanistic insight into AD pathogenesis and may potentially lead to identification of novel therapeutic targets for treating AD and possibly other neurodegenerative disorders.
    Keywords:  Alzheimer’s disease; autophagy; calcineurin; calcium signaling; ryanodine receptor
    DOI:  https://doi.org/10.3390/life13051187
  19. Cell Mol Biol Lett. 2023 May 26. 28(1): 46
    Hsp70-Bim interaction facilitates mitophagy by recruiting parkin and TOMM20 into a complex.
    Ting Song, Fangkui Yin, Ziqian Wang, Hong Zhang, Peng Liu, Yafei Guo, Yao Tang, Zhichao Zhang.
      BACKGROUND: For cancer therapy, the identification of both selective autophagy targets and small molecules that specifically regulate autophagy is greatly needed. Heat shock protein 70 (Hsp70) is a recently discovered BH3 receptor that forms a protein‒protein interaction (PPI) with Bcl-2-interacting mediator of cell death (Bim). Herein, a specific inhibitor of the Hsp70-Bim PPI, S1g-2, and its analog S1, which is a Bcl-2-Bim disruptor, were used as chemical tools to explore the role of Hsp70-Bim PPI in regulating mitophagy.METHODS: Co-immunoprecipitation and immunofluorescence assays were used to determine protein interactions and colocalization patterns. Organelle purification and immunodetection of LC3-II/LC3-I on mitochondria, endoplasmic reticulum (ER) and Golgi were applied to identify specific types of autophagy. Cell-based and in vitro ubiquitination studies were used to study the role of the Hsp70-Bim PPI in parkin-mediated ubiquitination of outer mitochondrial membrane 20 (TOMM20).
    RESULTS: We found that after the establishment of their PPI, Hsp70 and Bim form a complex with parkin and TOMM20, which in turn facilitates parkin translocation to mitochondria, TOMM20 ubiquitination and mitophagic flux independent of Bax/Bak. Moreover, S1g-2 selectively inhibits stress-induced mitophagy without interfering with basal autophagy.
    CONCLUSIONS: The findings highlight the dual protective function of the Hsp70-Bim PPI in regulating both mitophagy and apoptosis. S1g-2 is thus a newly discovered antitumor drug candidate that drives both mitophagy and cell death via apoptosis.
    Keywords:  Apoptosis; Hsp70-Bim; Mitophagy; Parkin; TOMM20
    DOI:  https://doi.org/10.1186/s11658-023-00458-5
  20. Front Cell Infect Microbiol. 2023 ;13 1180708
    The versatile defender: exploring the multifaceted role of p62 in intracellular bacterial infection.
    Yuhao Zhou, Shucheng Hua, Lei Song.
      As a highly conserved, multifunctional protein with multiple domains, p62/SQSTM1 plays a crucial role in several essential cellular activities, particularly selective autophagy. Recent research has shown that p62 is crucial in eradicating intracellular bacteria by xenophagy, a selective autophagic process that identifies and eliminates such microorganisms. This review highlights the various roles of p62 in intracellular bacterial infections, including both direct and indirect, antibacterial and infection-promoting aspects, and xenophagy-dependent and independent functions, as documented in published literature. Additionally, the potential applications of synthetic drugs targeting the p62-mediated xenophagy mechanism and unresolved questions about p62's roles in bacterial infections are also discussed.
    Keywords:  bacterial protein; intracellular bacterial infection; microdomain; p62 (sequestosome 1(SQSTM1); xenophagy
    DOI:  https://doi.org/10.3389/fcimb.2023.1180708
  21. Front Cell Dev Biol. 2023 ;11 1149409
    Autophagy, innate immunity, and cardiac disease.
    Donato Santovito, Sabine Steffens, Serena Barachini, Rosalinda Madonna.
      Autophagy is an evolutionarily conserved mechanism of cell adaptation to metabolic and environmental stress. It mediates the disposal of protein aggregates and dysfunctional organelles, although non-conventional features have recently emerged to broadly extend the pathophysiological relevance of autophagy. In baseline conditions, basal autophagy critically regulates cardiac homeostasis to preserve structural and functional integrity and protect against cell damage and genomic instability occurring with aging. Moreover, autophagy is stimulated by multiple cardiac injuries and contributes to mechanisms of response and remodeling following ischemia, pressure overload, and metabolic stress. Besides cardiac cells, autophagy orchestrates the maturation of neutrophils and other immune cells, influencing their function. In this review, we will discuss the evidence supporting the role of autophagy in cardiac homeostasis, aging, and cardioimmunological response to cardiac injury. Finally, we highlight possible translational perspectives of modulating autophagy for therapeutic purposes to improve the care of patients with acute and chronic cardiac disease.
    Keywords:  aging; autophagy; cardiac function; diabetic cardiomiopathy; heterophagy; immune response; myocardial infarction
    DOI:  https://doi.org/10.3389/fcell.2023.1149409
  22. Front Oncol. 2023 ;13 1150492
    Autophagy modulation in breast cancer utilizing nanomaterials and nanoparticles.
    Azar Gharoonpour, Dorsa Simiyari, Ali Yousefzadeh, Fatemeh Badragheh, Marveh Rahmati.
      Autophagy regenerates cellular nutrients, recycles metabolites, and maintains hemostasis through multistep signaling pathways, in conjunction with lysosomal degradation mechanisms. In tumor cells, autophagy has been shown to play a dual role as both tumor suppressor and tumor promoter, leading to the discovery of new therapeutic strategies for cancer. Therefore, regulation of autophagy is essential during cancer progression. In this regard, the use of nanoparticles (NPs) is a promising technique in the clinic to modulate autophagy pathways. Here, we summarized the importance of breast cancer worldwide, and we discussed its classification, current treatment strategies, and the strengths and weaknesses of available treatments. We have also described the application of NPs and nanocarriers (NCs) in breast cancer treatment and their capability to modulate autophagy. Then the advantages and disadvantaged of NPs in cancer therapy along with future applications will be disscussed. The purpose of this review is to provide up-to-date information on NPs used in breast cancer treatment and their impacts on autophagy pathways for researchers.
    Keywords:  autophagy; breast cancer; drug resistance; nanomaterial; nanoparticles
    DOI:  https://doi.org/10.3389/fonc.2023.1150492
  23. Brain Behav. 2023 May 23. e2995
    Advances in the mTOR signaling pathway and its inhibitor rapamycin in epilepsy.
    Wei Zhao, Cong Xie, Xu Zhang, Ju Liu, Jinzhi Liu, Zhangyong Xia.
      INTRODUCTION: Epilepsy is one of the most common and serious brain syndromes and has adverse consequences on a patient's neurobiological, cognitive, psychological, and social wellbeing, thereby threatening their quality of life. Some patients with epilepsy experience poor treatment effects due to the unclear pathophysiological mechanisms of the syndrome. Dysregulation of the mammalian target of the rapamycin (mTOR) pathway is thought to play an important role in the onset and progression of some epilepsies.METHODS: This review summarizes the role of the mTOR signaling pathway in the pathogenesis of epilepsy and the prospects for the use of mTOR inhibitors.
    RESULTS: The mTOR pathway functions as a vital mediator in epilepsy development through diverse mechanisms, indicating that the it has great potential as an effective target for epilepsy therapy. The excessive activation of mTOR signaling pathway leads to structural changes in neurons, inhibits autophagy, exacerbates neuron damage, affects mossy fiber sprouting, enhances neuronal excitability, increases neuroinflammation, and is closely associated with tau upregulation in epilepsy. A growing number of studies have demonstrated that mTOR inhibitors exhibit significant antiepileptic effects in both clinical applications and animal models. Specifically, rapamycin, a specific inhibitor of TOR, reduces the intensity and frequency of seizures. Clinical studies in patients with tuberous sclerosis complex have shown that rapamycin has the function of reducing seizures and improving this disease. Everolimus, a chemically modified derivative of rapamycin, has been approved as an added treatment to other antiepileptic medicines. Further explorations are needed to evaluate the therapeutic efficacy and application value of mTOR inhibitors in epilepsy.
    CONCLUSIONS: Targeting the mTOR signaling pathway provides a promising prospect for the treatment of epilepsy.
    Keywords:  epilepsy; mTOR; rapamycin; signaling pathway
    DOI:  https://doi.org/10.1002/brb3.2995
  24. Int J Mol Sci. 2023 May 19. pii: 9008. [Epub ahead of print]24(10):
    The Autophagy Nucleation Factor ATG9 Forms Nanoclusters with the HIV-1 Receptor DC-SIGN and Regulates Early Antiviral Autophagy in Human Dendritic Cells.
    Laure Papin, Martin Lehmann, Justine Lagisquet, Ghizlane Maarifi, Véronique Robert-Hebmann, Christophe Mariller, Yann Guerardel, Lucile Espert, Volker Haucke, Fabien P Blanchet.
      Dendritic cells (DC) are critical cellular mediators of host immunity, notably by expressing a broad panel of pattern recognition receptors. One of those receptors, the C-type lectin receptor DC-SIGN, was previously reported as a regulator of endo/lysosomal targeting through functional connections with the autophagy pathway. Here, we confirmed that DC-SIGN internalization intersects with LC3+ autophagy structures in primary human monocyte-derived dendritic cells (MoDC). DC-SIGN engagement promoted autophagy flux which coincided with the recruitment of ATG-related factors. As such, the autophagy initiation factor ATG9 was found to be associated with DC-SIGN very early upon receptor engagement and required for an optimal DC-SIGN-mediated autophagy flux. The autophagy flux activation upon DC-SIGN engagement was recapitulated using engineered DC-SIGN-expressing epithelial cells in which ATG9 association with the receptor was also confirmed. Finally, Stimulated emission depletion (STED) microscopy performed in primary human MoDC revealed DC-SIGN-dependent submembrane nanoclusters formed with ATG9, which was required to degrade incoming viruses and further limit DC-mediated transmission of HIV-1 infection to CD4+ T lymphocytes. Our study unveils a physical association between the Pattern Recognition Receptor DC-SIGN and essential components of the autophagy pathway contributing to early endocytic events and the host's antiviral immune response.
    Keywords:  DC-SIGN; HIV-1; autophagy; dendritic cells; innate immunity
    DOI:  https://doi.org/10.3390/ijms24109008
  25. Cell Rep. 2023 May 18. pii: S2211-1247(23)00541-7. [Epub ahead of print]42(5): 112530
    FOXK1 promotes nonalcoholic fatty liver disease by mediating mTORC1-dependent inhibition of hepatic fatty acid oxidation.
    Shun Fujinuma, Hirokazu Nakatsumi, Hideyuki Shimizu, Shigeaki Sugiyama, Akihito Harada, Takeshi Goya, Masatake Tanaka, Motoyuki Kohjima, Masatomo Takahashi, Yoshihiro Izumi, Mikako Yagi, Dongchon Kang, Mari Kaneko, Mayo Shigeta, Takeshi Bamba, Yasuyuki Ohkawa, Keiichi I Nakayama.
      Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disorder caused by overnutrition and can lead to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). The transcription factor Forkhead box K1 (FOXK1) is implicated in regulation of lipid metabolism downstream of mechanistic target of rapamycin complex 1 (mTORC1), but its role in NAFLD-NASH pathogenesis is understudied. Here, we show that FOXK1 mediates nutrient-dependent suppression of lipid catabolism in the liver. Hepatocyte-specific deletion of Foxk1 in mice fed a NASH-inducing diet ameliorates not only hepatic steatosis but also associated inflammation, fibrosis, and tumorigenesis, resulting in improved survival. Genome-wide transcriptomic and chromatin immunoprecipitation analyses identify several lipid metabolism-related genes, including Ppara, as direct targets of FOXK1 in the liver. Our results suggest that FOXK1 plays a key role in the regulation of hepatic lipid metabolism and that its inhibition is a promising therapeutic strategy for NAFLD-NASH, as well as for HCC.
    Keywords:  CP: Metabolism; FOXK1; Forkhead box K1; HCC; NAFLD; NASH; conditional knockout mouse; hepatocellular carcinoma; lipid metabolism; mTORC1; mechanistic target of rapamycin complex 1; nonalcoholic fatty liver disease; nonalcoholic steatohepatitis
    DOI:  https://doi.org/10.1016/j.celrep.2023.112530
  26. Biomolecules. 2023 May 11. pii: 816. [Epub ahead of print]13(5):
    Cellular Metabolism: A Fundamental Component of Degeneration in the Nervous System.
    Kenneth Maiese.
      It is estimated that, at minimum, 500 million individuals suffer from cellular metabolic dysfunction, such as diabetes mellitus (DM), throughout the world. Even more concerning is the knowledge that metabolic disease is intimately tied to neurodegenerative disorders, affecting both the central and peripheral nervous systems as well as leading to dementia, the seventh leading cause of death. New and innovative therapeutic strategies that address cellular metabolism, apoptosis, autophagy, and pyroptosis, the mechanistic target of rapamycin (mTOR), AMP activated protein kinase (AMPK), growth factor signaling with erythropoietin (EPO), and risk factors such as the apolipoprotein E (APOE-ε4) gene and coronavirus disease 2019 (COVID-19) can offer valuable insights for the clinical care and treatment of neurodegenerative disorders impacted by cellular metabolic disease. Critical insight into and modulation of these complex pathways are required since mTOR signaling pathways, such as AMPK activation, can improve memory retention in Alzheimer's disease (AD) and DM, promote healthy aging, facilitate clearance of β-amyloid (Aß) and tau in the brain, and control inflammation, but also may lead to cognitive loss and long-COVID syndrome through mechanisms that can include oxidative stress, mitochondrial dysfunction, cytokine release, and APOE-ε4 if pathways such as autophagy and other mechanisms of programmed cell death are left unchecked.
    Keywords:  AMP activated protein kinase (AMPK); Alzheimer’s disease; COVID-19; apolipoprotein E (APOE-ε4); autophagy; dementia; diabetes mellitus; erythropoietin; mechanistic target of rapamycin (mTOR); pyroptosis
    DOI:  https://doi.org/10.3390/biom13050816
  27. Cell Mol Life Sci. 2023 May 20. 80(6): 160
    Phosphorylation of STAT3 at Tyr705 contributes to TFEB-mediated autophagy-lysosomal pathway dysfunction and leads to ischemic injury in rats.
    Yueyang Liu, Xiaohang Che, Xiangnan Yu, Hanxiao Shang, Peirui Cui, Xiaoxiao Fu, Xianda Lu, Yuhuan Liu, Chunfu Wu, Jingyu Yang.
      We previously reported that permanent ischemia induces marked dysfunction of the autophagy-lysosomal pathway (ALP) in rats, which is possibly mediated by the transcription factor EB (TFEB). However, it is still unclear whether signal transducer and activator of transcription 3 (STAT3) is responsible for the TFEB-mediated dysfunction of ALP in ischemic stroke. In the present study, we used AAV-mediated genetic knockdown and pharmacological blockade of p-STAT3 to investigate the role of p-STAT3 in regulating TFEB-mediated ALP dysfunction in rats subjected to permanent middle cerebral occlusion (pMCAO). The results showed that the level of p-STAT3 (Tyr705) in the rat cortex increased at 24 h after pMCAO and subsequently led to lysosomal membrane permeabilization (LMP) and ALP dysfunction. These effects can be alleviated by inhibitors of p-STAT3 (Tyr705) or by STAT3 knockdown. Additionally, STAT3 knockdown significantly increased the nuclear translocation of TFEB and the transcription of TFEB-targeted genes. Notably, TFEB knockdown markedly reversed STAT3 knockdown-mediated improvement in ALP function after pMCAO. This is the first study to show that the contribution of p-STAT3 (Tyr705) to ALP dysfunction may be partly associated with its inhibitory effect on TFEB transcriptional activity, which further leads to ischemic injury in rats.
    Keywords:  Autophagy-lysosomal pathway; Ischemic stroke; Lysosomal membrane permeabilization; Signal transducer and activator of transcription 3; Transcription factor EB
    DOI:  https://doi.org/10.1007/s00018-023-04792-x
  28. Biology (Basel). 2023 May 02. pii: 676. [Epub ahead of print]12(5):
    mTORC1/ERK1/2 Interplay Regulates Protein Synthesis and Survival in Acute Myeloid Leukemia Cell Lines.
    Concetta Anna Germano, Giuseppe Clemente, Antonello Storniolo, Maria Anele Romeo, Elisabetta Ferretti, Mara Cirone, Livia Di Renzo.
      mTOR is constitutively activated in acute myeloid leukemia (AML) cells, as indicated by the phosphorylation of its substrates, 4EBP1 and P70S6K. Here, we found that quercetin (Q) and rapamycin (Rap) inhibited P70S6K phosphorylation, partially dephosphorylated 4EBP1, and activated ERK1/2 in U937 and THP1, two leukemia cell lines. ERK1/2 inhibition by U0126 induced a stronger dephosphorylation of mTORC1 substrates and activated AKT. The concomitant inhibition of ERK1/2 and AKT further dephosphorylated 4EBP1 and further increased Q- or Rap-mediated cytotoxicity, compared to the single ERK1/2 or AKT inhibition in cells undergoing Q- or Rap-treatments. Moreover, quercetin or rapamycin reduced autophagy, particularly when used in combination with the ERK1/2 inhibitor, U0126. This effect was not dependent on TFEB localization in nuclei or cytoplasm or on the transcription of different autophagy genes, but did correlate with the reduction in protein translation due to a strong eIF2α-Ser51 phosphorylation. Thus, ERK1/2, by limiting 4EBP1 de-phosphorylation and eIF2α phosphorylation, behaves as a paladin of protein synthesis. Based on these findings, the combined inhibition of mTORC1, ERK1/2, and AKT should be considered in treatment of AML.
    Keywords:  4EBP1; AKT; AML; ERK1/2; P70S6K; autophagy; eIF2α; mTORC1; quercetin; rapamycin
    DOI:  https://doi.org/10.3390/biology12050676
  29. Arch Biochem Biophys. 2023 May 22. pii: S0003-9861(23)00145-5. [Epub ahead of print] 109646
    Handelin protects human skin keratinocytes against ultraviolet B-induced photodamage via autophagy activation by regulating the AMPK-mTOR signaling pathway.
    Jimin Chu, Yang Xiang, Xianghong Lin, Miao He, Yan Wang, Qiong Ma, Jingxian Duan, Sunjiao Sun.
      Handelin is a natural ingredient extracted from Chrysanthemum boreale flowers that has been shown to decrease stress-related cell death, prolong lifespan, and promote anti-photoaging. However, whether handelin inhibits ultraviolet (UV) B stress-induced photodamage remains unclear. In the present study, we investigate whether handelin has protective properties on skin keratinocytes under UVB irradiation. Human immortalized keratinocytes (HaCaT keratinocytes) were pretreated with handelin for 12 h before UVB irradiation. The results indicated that handelin protects keratinocytes against UVB-induced photodamage by activating autophagy. However, the photoprotective effect of handelin was suppressed by an autophagic inhibitor (wortmannin) or the transfection of keratinocytes with a small interfering RNA targeting ATG5. Notably, handelin reduced mammalian target of rapamycin (mTOR) activity in UVB-irradiated cells in a manner similar to that shown by the mTOR inhibitor rapamycin. Adenosine monophosphate-activated protein kinase (AMPK) activity was also induced by handelin in UVB-damaged keratinocytes. Finally, certain effects of handelin, including autophagy induction, mTOR activity inhibition, AMPK activation, and reduction of cytotoxicity, were suppressed by an AMPK inhibitor (compound C). Our data suggest that handelin effectively prevents photodamage by protecting skin keratinocytes against UVB-induced cytotoxicity via the regulation of AMPK/mTOR-mediated autophagy. These findings provide novel insights that can aid the development of therapeutic agents against UVB-induced keratinocyte photodamage.
    Keywords:  AMPK; Autophagy; Handelin; Photodamaging; UVB; mTOR
    DOI:  https://doi.org/10.1016/j.abb.2023.109646
  30. Photochem Photobiol. 2023 May 22.
    Sites of sub-cellular photodamage: Apoptotic and autophagic responses.
    David Kessel.
      Log dose-response curves relating to the effect of mitochondrial photodamage on clonogenic survival using benzoporphyrin derivative. With wild-type cells, autophagy produces a "shoulder" on the curve which is absent when an ATG5 knockdown is examined. Loss of ATG5 prevents the process of autophagy, which is seen to be cytoprotective.
    Keywords:  apoptosis; autophagy; photodynamic
    DOI:  https://doi.org/10.1111/php.13815
  31. Biochem Pharmacol. 2023 May 20. pii: S0006-2952(23)00211-3. [Epub ahead of print] 115620
    The mTOR signalling in corneal diseases: a recent update.
    Xiang Li, Kuangqi Chen, Zixi Wang, Jiayuan Li, Xiawei Wang, Chen Xie, Jianping Tong, Ye Shen.
      Corneal diseases affect 4.2 million people worldwide and are a leading cause of vision impairment and blindness. Current treatments for corneal diseases, such as antibiotics, steroids, and surgical interventions, have numerous disadvantages and challenges. Thus, there is an urgent need for more effective therapies. Although the pathogenesis of corneal diseases is not fully understood, it is known that injury caused by various stresses and postinjury healing, such as epithelial renewal, inflammation, stromal fibrosis, and neovascularization, are highly involved. Mammalian target of rapamycin (mTOR) is a key regulator of cell growth, metabolism, and the immune response. Recent studies have revealed that activation of mTOR signalling extensively contributes to the pathogenesis of various corneal diseases, and inhibition of mTOR with rapamycin achieves promising outcomes, supporting the potential of mTOR as a therapeutic target. In this review, we detail the function of mTOR in corneal diseases and how these characteristics contribute to disease treatment using mTOR-targeted drugs.
    Keywords:  Immunosuppression; Inflammation; Rapamycin; autophagy; mTOR, cornea
    DOI:  https://doi.org/10.1016/j.bcp.2023.115620
  32. Biomolecules. 2023 May 15. pii: 841. [Epub ahead of print]13(5):
    The Effect of Calorie Restriction on Protein Quality Control in Yeast.
    Petter Uvdal, Sviatlana Shashkova.
      Initially, protein aggregates were regarded as a sign of a pathological state of the cell. Later, it was found that these assemblies are formed in response to stress, and that some of them serve as signalling mechanisms. This review has a particular focus on how intracellular protein aggregates are related to altered metabolism caused by different glucose concentrations in the extracellular environment. We summarise the current knowledge of the role of energy homeostasis signalling pathways in the consequent effect on intracellular protein aggregate accumulation and removal. This covers regulation at different levels, including elevated protein degradation and proteasome activity mediated by the Hxk2 protein, the enhanced ubiquitination of aberrant proteins through Torc1/Sch9 and Msn2/Whi2, and the activation of autophagy mediated through ATG genes. Finally, certain proteins form reversible biomolecular aggregates in response to stress and reduced glucose levels, which are used as a signalling mechanism in the cell, controlling major primary energy pathways related to glucose sensing.
    Keywords:  Hsp104; Saccharomyces cerevisiae; age-related diseases; autophagy; calorie restriction; carbon metabolism; degradation; misfolded proteins; neurodegenerative diseases; protein aggregation; protein quality control; stress response; yeast
    DOI:  https://doi.org/10.3390/biom13050841
  33. Nat Cell Biol. 2023 May 25.
    Autophagy enables microglia to engage amyloid plaques and prevents microglial senescence.
    Insup Choi, Minghui Wang, Seungyeul Yoo, Peng Xu, Steven P Seegobin, Xianting Li, Xian Han, Qian Wang, Junmin Peng, Bin Zhang, Zhenyu Yue.
      Dysfunctional autophagy has been implicated in the pathogenesis of Alzheimer's disease (AD). Previous evidence suggested disruptions of multiple stages of the autophagy-lysosomal pathway in affected neurons. However, whether and how deregulated autophagy in microglia, a cell type with an important link to AD, contributes to AD progression remains elusive. Here we report that autophagy is activated in microglia, particularly of disease-associated microglia surrounding amyloid plaques in AD mouse models. Inhibition of microglial autophagy causes disengagement of microglia from amyloid plaques, suppression of disease-associated microglia, and aggravation of neuropathology in AD mice. Mechanistically, autophagy deficiency promotes senescence-associated microglia as evidenced by reduced proliferation, increased Cdkn1a/p21Cip1, dystrophic morphologies and senescence-associated secretory phenotype. Pharmacological treatment removes autophagy-deficient senescent microglia and alleviates neuropathology in AD mice. Our study demonstrates the protective role of microglial autophagy in regulating the homeostasis of amyloid plaques and preventing senescence; removal of senescent microglia is a promising therapeutic strategy.
    DOI:  https://doi.org/10.1038/s41556-023-01158-0
  34. Nature. 2023 May 24.
    Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.
    Hector Foronda, Yangxue Fu, Adriana Covarrubias-Pinto, Hartmut T Bocker, Alexis González, Eric Seemann, Patricia Franzka, Andrea Bock, Ramachandra M Bhaskara, Lutz Liebmann, Marina E Hoffmann, Istvan Katona, Nicole Koch, Joachim Weis, Ingo Kurth, Joseph G Gleeson, Fulvio Reggiori, Gerhard Hummer, Michael M Kessels, Britta Qualmann, Muriel Mari, Ivan Dikić, Christian A Hübner.
      Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.
    DOI:  https://doi.org/10.1038/s41586-023-06090-9
  35. Biomolecules. 2023 May 09. pii: 802. [Epub ahead of print]13(5):
    Transmembrane Protein 175, a Lysosomal Ion Channel Related to Parkinson's Disease.
    Tuoxian Tang, Boshuo Jian, Zhenjiang Liu.
      Lysosomes are membrane-bound organelles with an acidic lumen and are traditionally characterized as a recycling center in cells. Lysosomal ion channels are integral membrane proteins that form pores in lysosomal membranes and allow the influx and efflux of essential ions. Transmembrane protein 175 (TMEM175) is a unique lysosomal potassium channel that shares little sequence similarity with other potassium channels. It is found in bacteria, archaea, and animals. The prokaryotic TMEM175 consists of one six-transmembrane domain that adopts a tetrameric architecture, while the mammalian TMEM175 is comprised of two six-transmembrane domains that function as a dimer in lysosomal membranes. Previous studies have demonstrated that the lysosomal K+ conductance mediated by TMEM175 is critical for setting membrane potential, maintaining pH stability, and regulating lysosome-autophagosome fusion. AKT and B-cell lymphoma 2 regulate TMEM175's channel activity through direct binding. Two recent studies reported that the human TMEM175 is also a proton-selective channel under normal lysosomal pH (4.5-5.5) as the K+ permeation dramatically decreased at low pH while the H+ current through TMEM175 greatly increased. Genome-wide association studies and functional studies in mouse models have established that TMEM175 is implicated in the pathogenesis of Parkinson's disease, which sparks more research interests in this lysosomal channel.
    Keywords:  Parkinson’s disease; TMEM175; lysosome; potassium channel; proton channel
    DOI:  https://doi.org/10.3390/biom13050802
  36. Int J Mol Sci. 2023 May 19. pii: 9027. [Epub ahead of print]24(10):
    Parkin Precipitates on Mitochondria via Aggregation and Autoubiquitination.
    Mustafa T Ardah, Nada Radwan, Engila Khan, Tohru Kitada, M Emdadul Haque.
      The loss of the E3 ligase Parkin, in a familial form of Parkinson's disease, is thought to cause the failure of both the polyubiquitination of abnormal mitochondria and the consequent induction of mitophagy, resulting in abnormal mitochondrial accumulation. However, this has not been confirmed in patient autopsy cases or animal models. More recently, the function of Parkin as a redox molecule that directly scavenges hydrogen peroxide has attracted much attention. To determine the role of Parkin as a redox molecule in the mitochondria, we overexpressed various combinations of Parkin, along with its substrates FAF1, PINK1, and ubiquitin in cell culture systems. Here, we observed that the E3 Parkin monomer was surprisingly not recruited to abnormal mitochondria but self-aggregated with or without self-ubiquitination into the inner and outer membranes, becoming insoluble. Parkin overexpression alone generated aggregates without self-ubiquitination, but it activated autophagy. These results suggest that for damaged mitochondria, the polyubiquitination of Parkin substrates on the mitochondria is not indispensable for mitophagy.
    Keywords:  Parkin; Parkinson’s disease; neurodegeneration; ubiquitin
    DOI:  https://doi.org/10.3390/ijms24109027
  37. J Cell Biol. 2023 06 05. pii: e202209084. [Epub ahead of print]222(6):
    Inhibition of endolysosome fusion increases exosome secretion.
    Ganesh Vilas Shelke, Chad D Williamson, Michal Jarnik, Juan S Bonifacino.
      Exosomes are small vesicles that are secreted from cells to dispose of undegraded materials and mediate intercellular communication. A major source of exosomes is intraluminal vesicles within multivesicular endosomes that undergo exocytic fusion with the plasma membrane. An alternative fate of multivesicular endosomes is fusion with lysosomes, resulting in degradation of the intraluminal vesicles. The factors that determine whether multivesicular endosomes fuse with the plasma membrane or with lysosomes are unknown. In this study, we show that impairment of endolysosomal fusion by disruption of a pathway involving the BLOC-one-related complex (BORC), the small GTPase ARL8, and the tethering factor HOPS increases exosome secretion by preventing the delivery of intraluminal vesicles to lysosomes. These findings demonstrate that endolysosomal fusion is a critical determinant of the amount of exosome secretion and suggest that suppression of the BORC-ARL8-HOPS pathway could be used to boost exosome yields in biotechnology applications.
    DOI:  https://doi.org/10.1083/jcb.202209084
  38. Pharmaceuticals (Basel). 2023 Apr 29. pii: 671. [Epub ahead of print]16(5):
    The STAT3-Regulated Autophagy Pathway in Glioblastoma.
    Ronald Nicholas Laribee, Andrew B Boucher, Saivikram Madireddy, Lawrence M Pfeffer.
      Glioblastoma (GBM) is the most common primary brain malignancy in adults with a dismal prognosis. Despite advances in genomic analysis and surgical technique and the development of targeted therapeutics, most treatment options are ineffective and mainly palliative. Autophagy is a form of cellular self-digestion with the goal of recycling intracellular components to maintain cell metabolism. Here, we describe some recent findings that suggest GBM tumors are more sensitive to the excessive overactivation of autophagy leading to autophagy-dependent cell death. GBM cancer stem cells (GSCs) are a subset of the GBM tumor population that play critical roles in tumor formation and progression, metastasis, and relapse, and they are inherently resistant to most therapeutic strategies. Evidence suggests that GSCs are able to adapt to a tumor microenvironment of hypoxia, acidosis, and lack of nutrients. These findings have suggested that autophagy may promote and maintain the stem-like state of GSCs as well as their resistance to cancer treatment. However, autophagy is a double-edged sword and may have anti-tumor properties under certain conditions. The role of the STAT3 transcription factor in autophagy is also described. These findings provide the basis for future research aimed at targeting the autophagy-dependent pathway to overcome the inherent therapeutic resistance of GBM in general and to specifically target the highly therapy-resistant GSC population through autophagy regulation.
    Keywords:  GBM; GBM cancer stem cell; GSC; STAT3; autophagy; glioblastoma; glioma
    DOI:  https://doi.org/10.3390/ph16050671
  39. Nature. 2023 May 24.
    Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.
    Alexis González, Adriana Covarrubias-Pinto, Ramachandra M Bhaskara, Marius Glogger, Santosh K Kuncha, Audrey Xavier, Eric Seemann, Mohit Misra, Marina E Hoffmann, Bastian Bräuning, Ashwin Balakrishnan, Britta Qualmann, Volker Dötsch, Brenda A Schulman, Michael M Kessels, Christian A Hübner, Mike Heilemann, Gerhard Hummer, Ivan Dikić.
      The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.
    DOI:  https://doi.org/10.1038/s41586-023-06089-2
  40. Res Sq. 2023 May 09. pii: rs.3.rs-2815300. [Epub ahead of print]
    Longitudinal modeling of human neuronal aging identifies RCAN1-TFEB pathway contributing to neurodegeneration of Huntington's disease.
    Andrew Yoo, Seong Won Lee, Young Mi Oh, Matheus Victor, Ilya Strunilin, Shawei Chen, Sonika Dahiya, Roland Dolle, Stephen Pak, Gary Silverman, David Perlmutter.
      Aging is a common risk factor in neurodegenerative disorders and the ability to investigate aging of neurons in an isogenic background would facilitate discovering the interplay between neuronal aging and onset of neurodegeneration. Here, we perform direct neuronal reprogramming of longitudinally collected human fibroblasts to reveal genetic pathways altered at different ages. Comparative transcriptome analysis of longitudinally aged striatal medium spiny neurons (MSNs), a primary neuronal subtype affected in Huntington's disease (HD), identified pathways associated with RCAN1, a negative regulator of calcineurin. Notably, RCAN1 undergoes age-dependent increase at the protein level detected in reprogrammed MSNs as well as in human postmortem striatum. In patient-derived MSNs of adult-onset HD (HD-MSNs), counteracting RCAN1 by gene knockdown (KD) rescued HD-MSNs from degeneration. The protective effect of RCAN1 KD was associated with enhanced chromatin accessibility of genes involved in longevity and autophagy, mediated through enhanced calcineurin activity, which in turn dephosphorylates and promotes nuclear localization of TFEB transcription factor. Furthermore, we reveal that G2-115 compound, an analog of glibenclamide with autophagy-enhancing activities, reduces the RCAN1-Calcineurin interaction, phenocopying the effect of RCAN1 KD. Our results demonstrate that RCAN1 is a potential genetic or pharmacological target whose reduction-of-function increases neuronal resilience to neurodegeneration in HD through chromatin reconfiguration.
    DOI:  https://doi.org/10.21203/rs.3.rs-2815300/v1
  41. Zygote. 2023 May 22. 1-6
    Autophagic activation in porcine oocytes is independent of meiotic progression.
    SeungHoon Lee, Haeyun Jeong, Hayeon Wi, Jin-Gu No, Whi-Cheul Lee, Seokho Kim, Hyeon Yang, Sung June Byun, Sejin Park, Jong Gug Kim.
      In this study, we built on our previous research that discovered that autophagy activated the metaphase I stage during porcine oocytes in vitro maturation. We investigated the relationship between autophagy and oocyte maturation. First, we confirmed whether autophagy was activated differently by different media (TCM199 and NCSU-23) during maturation. Then, we investigated whether oocyte maturation affected autophagic activation. In addition, we examined whether the inhibition of autophagy affected the nuclear maturation rate of porcine oocytes. As for the main experiment, we measured LC3-II levels using western blotting after inhibition of nuclear maturation via cAMP treatment in an in vitro culture to clarify whether nuclear maturation affected autophagy. After autophagy inhibition, we also counted matured oocytes by treating them with wortmannin or a E64d and pepstatin A mixture. Both groups, which had different treatment times of cAMP, showed the same levels of LC3-II, while the maturation rates were about four times higher after cAMP 22 h treatment than that of the 42 h treatment group. This indicated that neither cAMP nor nuclear status affected autophagy. Autophagy inhibition during in vitro oocyte maturation with wortmannin treatment reduced oocyte maturation rates by about half, while autophagy inhibition by the E64d and pepstatin A mixture treatment did not significantly affect the oocyte maturation. Therefore, wortmannin itself, or the autophagy induction step, but not the degradation step, is involved in the oocyte maturation of porcine oocytes. Overall, we propose that oocyte maturation does not stand upstream of autophagy activation, but autophagy may exist upstream of oocyte maturation.
    Keywords:  Autophagy; In vitro; LC3-II; Oocyte maturation; Porcine
    DOI:  https://doi.org/10.1017/S0967199423000102
  42. Environ Toxicol. 2023 May 20.
    Autophagy blockage and lysosomal dysfunction are involved in diallyl sulfide-induced inhibition of malignant growth in hepatocellular carcinoma cells.
    Haiyan Yu, Zhiwei Hao, Xuemin Liu, Zhixuan Wei, Renming Tan, Xiaotian Liu, Qiongxia Chen, Ying Chen, Hongyan Zhou, Yuchen Liu, Zhengqi Fu.
      Diallyl sulfide (DAS), as a major component of garlic extracts, has been shown to inhibit growth of hepatocellular carcinoma cells (HCC), but the underlying mechanism is still elusive. In this study, we aimed to explore the involvement of autophagy in DAS-induced growth inhibition of HepG2 and Huh7 hepatocellular carcinoma cells. We studied growth of DAS-treated HepG2 and Huh7 cells using the MTS and clonogenic assays. Autophagic flux was examined by immunofluorescence and confocal microscopy. The expression levels of autophagy-related proteins AMPK, mTOR, p62, LC3-II, LAMP1, and cathepsin D in the HepG2 and Huh7 cells treated with DAS as well as the tumors formed by HepG2 cells in the nude mice in the presence or absence of DAS were examined using western blotting and immunohistochemistry analysis. We found that DAS treatment induced activation of AMPK/mTOR, and accumulation of LC3-II and p62 both in vivo and in vitro. DAS inhibited autophagic flux through blocking the fusion of autophagosomes with lysosomes. Furthermore, DAS induced an increase in lysosomal pH and inhibition of Cathepsin D maturation. Co-treatment with an autophagy inhibitor (Chloroquine, CQ) further enhanced the growth inhibitory activity of DAS in HCC cells. Thus, our findings indicate that autophagy is involved in DAS-mediated growth inhibition of HCC cells both in vitro and in vivo.
    Keywords:  autophagic flux; diallyl sulfide; hepatocellular carcinoma cells; lysosomal dysfunction; lysosomes
    DOI:  https://doi.org/10.1002/tox.23834
  43. Acta Biochim Biophys Sin (Shanghai). 2023 May 24.
    LncRP11-675F6.3 responds to rapamycin treatment and reduces triglyceride accumulation via interacting with HK1 in hepatocytes by regulating autophagy and VLDL-related proteins.
    Lingling Wang, Xiaojuan Fang, Ziyou Yang, Xueling Li, Mengdi Cheng, Liang Cheng, Ganglin Wang, Wei Li, Lin Liu.
      Long noncoding RNAs (lncRNAs) have been widely proven to be involved in liver lipid homeostasis. Herein, we identify an upregulated lncRNA named lncRP11-675F6.3 in response to rapamycin treatment using a microarray in HepG2 cells. Knockdown of lncRP11-675F6. 3 leads to a significant reduction in apolipoprotein 100 (ApoB100), microsomal triglyceride transfer protein (MTTP), ApoE and ApoC3 with increased cellular triglyceride level and autophagy. Furthermore, we find that ApoB100 is obviously colocalized with GFP-LC3 in autophagosomes when lncRP11-675F6. 3 is knocked down, indicating that elevated triglyceride accumulation likely related to autophagy induces the degradation of ApoB100 and impairs very low-density lipoprotein (VLDL) assembly. We then identify and validate that hexokinase 1 (HK1) acts as the binding protein of lncRP11-675F6.3 and mediates triglyceride regulation and cell autophagy. More importantly, we find that lncRP11-675F6.3 and HK1 attenuate high fat diet induced nonalcoholic fatty liver disease (NAFLD) by regulating VLDL-related proteins and autophagy. In conclusion, this study reveals that lncRP11-675F6.3 is potentially involved in the downstream of mTOR signaling pathway and the regulatory network of hepatic triglyceride metabolism in cooperation with its interacting protein HK1, which may provide a new target for fatty liver disorder treatment.
    Keywords:  autophagy; hexokinase 1; lipoprotein; lncRP11-675F6.3; nonalcoholic fatty liver disease; triglycerides
    DOI:  https://doi.org/10.3724/abbs.2023091
  44. Anal Chim Acta. 2023 Jul 18. pii: S0003-2670(23)00577-9. [Epub ahead of print]1265 341356
    Monitoring H2S fluctuation during autophagic fusion of lysosomes and mitochondria using a lysosome-targeting fluorogenic probe.
    Taoyun Wang, Xu Huang, Sheng Yang, Shan Hu, Xianglan Zheng, Guojiang Mao, Yi Li, Yibo Zhou.
      Hydrogen sulfide (H2S) plays a cytoprotective role during mitophagy by detoxifying superfluous reactive oxygen species (ROS), and its concentration fluctuates in this process. However, no work has been reported to reveal the variation in H2S levels during autophagic fusion of lysosomes and mitochondria. Herein, we present a lysosome-targeted fluorogenic probe, named NA-HS, for real-time monitoring of H2S fluctuation for the first time. The newly synthesized probe exhibits good selectivity and high sensitivity (detection limit of 23.6 nM). Fluorescence imaging results demonstrated that NA-HS could image exogenous and endogenous H2S in living cells. Interestingly, the colocalization results revealed that the level of H2S was upregulated after autophagy began because of the cytoprotective effect, and was finally gradually reduced during subsequent autophagic fusion. This work not only affords a powerful fluorescence tool to monitor the variations in H2S levels during mitophagy, but also offers new insights into targeting small molecules for elaborating the complex cellular signal pathways.
    Keywords:  Fluorogenic probe; Hydrogen sulfide fluctuation; Mitophagy; Subcellular imaging
    DOI:  https://doi.org/10.1016/j.aca.2023.341356
  45. J Peripher Nerv Syst. 2023 May 20.
    Macrophages influence Schwann cell myelin autophagy after nerve injury and in a model of Charcot-Marie-Tooth disease.
    Eva Maria Weiß, Miriam Geldermann, Rudolf Martini, Dennis Klein.
      BACKGROUND AND AIMS: The complex cellular and molecular interactions between Schwann cells and macrophages during Wallerian degeneration are a prerequisite to allow rapid uptake and degradation of myelin debris and axonal regeneration after peripheral nerve injury. In contrast, in non-injured nerves of Charcot-Marie-Tooth 1 neuropathies, aberrant macrophage activation by Schwann cells carrying myelin gene defects is a disease amplifier that drives nerve damage and subsequent functional decline. Consequently, targeting nerve macrophages might be a translatable treatment strategy to mitigate disease outcome in CMT1 patients. Indeed, in previous approaches, macrophage targeting alleviated the axonopathy and promoted sprouting of damaged fibers. Surprisingly, this was still accompanied by robust myelinopathy in a model for CMT1X, suggesting additional cellular mechanisms of myelin degradation in mutant peripheral nerves. We here investigated the possibility of an increased Schwann cell-related myelin autophagy upon macrophage targeting in Cx32def mice.METHODS: Combining ex vivo and in vivo approaches, macrophages were targeted by PLX5622 treatment. Schwann cell autophagy was investigated by immunohistochemical and electron microscopical techniques.
    RESULTS: We demonstrate a robust upregulation of markers for Schwann cell autophagy after injury and in genetically-mediated neuropathy when nerve macrophages are pharmacologically depleted. Corroborating these findings, we provide ultrastructural evidence for increased Schwann cell myelin autophagy upon treatment in vivo.
    INTERPRETATION: These findings reveal a novel communication and interaction between Schwann cells and macrophages. This identification of alternative pathways of myelin degradation may have important implications for a better understanding of therapeutic mechanisms of pharmacological macrophage targeting in diseased peripheral nerves. This article is protected by copyright. All rights reserved.
    Keywords:  Schwann cell autophagy; Wallerian degeneration; colony stimulating factor 1 receptor inhibitor; inherited peripheral neuropathy; macrophage; neuroinflammation
    DOI:  https://doi.org/10.1111/jns.12561
  46. Cell Commun Signal. 2023 May 24. 21(1): 120
    Autophagy modulators influence the content of important signalling molecules in PS-positive extracellular vesicles.
    Klara Hanelova, Martina Raudenska, Monika Kratochvilova, Jiri Navratil, Tomas Vicar, Maria Bugajova, Jaromir Gumulec, Michal Masarik, Jan Balvan.
      Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. Many studies suggest that cancer cells release higher amounts of EVs exposing phosphatidylserine (PS) at the surface. There are lots of interconnections between EVs biogenesis and autophagy machinery. Modulation of autophagy can probably affect not only the quantity of EVs but also their content, which can deeply influence the resulting pro-tumourigenic or anticancer effect of autophagy modulators. In this study, we found that autophagy modulators autophinib, CPD18, EACC, bafilomycin A1 (BAFA1), 3-hydroxychloroquine (HCQ), rapamycin, NVP-BEZ235, Torin1, and starvation significantly alter the composition of the protein content of phosphatidylserine-positive EVs (PS-EVs) produced by cancer cells. The greatest impact had HCQ, BAFA1, CPD18, and starvation. The most abundant proteins in PS-EVs were proteins typical for extracellular exosomes, cytosol, cytoplasm, and cell surface involved in cell adhesion and angiogenesis. PS-EVs protein content involved mitochondrial proteins and signalling molecules such as SQSTM1 and TGFβ1 pro-protein. Interestingly, PS-EVs contained no commonly determined cytokines, such as IL-6, IL-8, GRO-α, MCP-1, RANTES, and GM-CSF, which indicates that secretion of these cytokines is not predominantly mediated through PS-EVs. Nevertheless, the altered protein content of PS-EVs can still participate in the modulation of the fibroblast metabolism and phenotype as p21 was accumulated in fibroblasts influenced by EVs derived from CPD18-treated FaDu cells. The altered protein content of PS-EVs (data are available via ProteomeXchange with identifier PXD037164) also provides information about the cellular compartments and processes that are affected by the applied autophagy modulators. Video Abstract.
    Keywords:  3-hydroxychloroquine; Autophagy; Autophagy modulation; Autophinib; Bafilomycin A1; CPD18; EACC; Extracellular vesicles; Head and neck cancer; NVP-BEZ235; Phosphatidylserine-positive extracellular vesicles; Proteomics; Rapamycin; SQSTM1; Senescence; Starvation; Torin1; p21
    DOI:  https://doi.org/10.1186/s12964-023-01126-z
  47. Nat Commun. 2023 May 24. 14(1): 2847
    Phospholipase D3 degrades mitochondrial DNA to regulate nucleotide signaling and APP metabolism.
    Zoë P Van Acker, Anika Perdok, Ruben Hellemans, Katherine North, Inge Vorsters, Cedric Cappel, Jonas Dehairs, Johannes V Swinnen, Ragna Sannerud, Marine Bretou, Markus Damme, Wim Annaert.
      Phospholipase D3 (PLD3) polymorphisms are linked to late-onset Alzheimer's disease (LOAD). Being a lysosomal 5'-3' exonuclease, its neuronal substrates remained unknown as well as how a defective lysosomal nucleotide catabolism connects to AD-proteinopathy. We identified mitochondrial DNA (mtDNA) as a major physiological substrate and show its manifest build-up in lysosomes of PLD3-defective cells. mtDNA accretion creates a degradative (proteolytic) bottleneck that presents at the ultrastructural level as a marked abundance of multilamellar bodies, often containing mitochondrial remnants, which correlates with increased PINK1-dependent mitophagy. Lysosomal leakage of mtDNA to the cytosol activates cGAS-STING signaling that upregulates autophagy and induces amyloid precursor C-terminal fragment (APP-CTF) and cholesterol accumulation. STING inhibition largely normalizes APP-CTF levels, whereas an APP knockout in PLD3-deficient backgrounds lowers STING activation and normalizes cholesterol biosynthesis. Collectively, we demonstrate molecular cross-talks through feedforward loops between lysosomal nucleotide turnover, cGAS-STING and APP metabolism that, when dysregulated, result in neuronal endolysosomal demise as observed in LOAD.
    DOI:  https://doi.org/10.1038/s41467-023-38501-w
  48. Int J Mol Sci. 2023 May 19. pii: 8979. [Epub ahead of print]24(10):
    The Essential Role of Light-Induced Autophagy in the Inner Choroid/Outer Retinal Neurovascular Unit in Baseline Conditions and Degeneration.
    Roberto Pinelli, Michela Ferrucci, Caterina Berti, Francesca Biagioni, Elena Scaffidi, Violet Vakunseth Bumah, Carla L Busceti, Paola Lenzi, Gloria Lazzeri, Francesco Fornai.
      The present article discusses the role of light in altering autophagy, both within the outer retina (retinal pigment epithelium, RPE, and the outer segment of photoreceptors) and the inner choroid (Bruch's membrane, BM, endothelial cells and the pericytes of choriocapillaris, CC). Here autophagy is needed to maintain the high metabolic requirements and to provide the specific physiological activity sub-serving the process of vision. Activation or inhibition of autophagy within RPE strongly depends on light exposure and it is concomitant with activation or inhibition of the outer segment of the photoreceptors. This also recruits CC, which provides blood flow and metabolic substrates. Thus, the inner choroid and outer retina are mutually dependent and their activity is orchestrated by light exposure in order to cope with metabolic demand. This is tuned by the autophagy status, which works as a sort of pivot in the cross-talk within the inner choroid/outer retina neurovascular unit. In degenerative conditions, and mostly during age-related macular degeneration (AMD), autophagy dysfunction occurs in this area to induce cell loss and extracellular aggregates. Therefore, a detailed analysis of the autophagy status encompassing CC, RPE and interposed BM is key to understanding the fine anatomy and altered biochemistry which underlie the onset and progression of AMD.
    Keywords:  Bruch’s membrane; age-related macular degeneration; choriocapillaris; endothelium; light exposure; nutraceuticals; pericytes; photo-biomodulation; photo-sensitive phytochemicals; retinal pigment epithelium
    DOI:  https://doi.org/10.3390/ijms24108979
  49. Genes Dis. 2023 Mar;10(2): 447-456
    The role of autophagy in regulating metabolism in the tumor microenvironment.
    Panpan Zhang, Shanshan Cheng, Xiaonan Sheng, Huijuan Dai, Kang He, Yueyao Du.
      Autophagy, as a special programmed cell death, is a critical degradative process that eliminates intracellular abnormal proteins or damage organelles to balance cell energy and favor cell metabolism with autophagy-related (ATG) proteins. Autophagy activation is being increasingly recognized as an essential hallmark in tumorigenesis through influencing the metabolism of stromal cells in the tumor microenvironment (TME) which comprises of tumor cells, cancer-associated fibroblasts (CAFs), cancer-associated endothelial cells (CAEs), immune cells and adipocytes. Tumor cells can reuse autophagy-involved recycling to maintain mitochondrial function and energy supply to meet the metabolic demand of their growth and proliferation. However, the mechanism through which autophagy can promote a crosstalk between tumor and stroma cells is not clear. Reprogramed metabolism is one of the main characteristics of TME leading to higher adaptability of tumor cells with diverse mechanisms. The activation of autophagy has expanded our understanding on the interaction between tumor metabolism and TME. The aim of this review is to report recent advances on the metabolic cross-talk between stromal cells and solid tumor cells induced by autophagy in TME and revealed potential therapeutic targets.
    Keywords:  Autophagy; Metabolism; Therapeutic targets; Tumor; Tumor microenvironment
    DOI:  https://doi.org/10.1016/j.gendis.2021.10.010
  50. Int J Biol Sci. 2023 ;19(8): 2531-2550
    Anomanolide C suppresses tumor progression and metastasis by ubiquitinating GPX4-driven autophagy-dependent ferroptosis in triple negative breast cancer.
    Yan-Mei Chen, Wei Xu, Yang Liu, Jia-Hui Zhang, Yuan-Yuan Yang, Zhi-Wen Wang, De-Juan Sun, Hua Li, Bo Liu, Li-Xia Chen.
      Anomanolide C (AC), a natural withanolide isolated from Tubocapsicum anomalum, has been reported to have exhibits remarkable anti-tumour activities in several types of human cancers, particularly triple-negative breast cancer (TNBC). However, its intricate mechanisms still remain need to be clarified. Here, we evaluated whether AC could inhibit cell proliferation and the role of AC in ferroptosis induction and autophagy activation. Subsequently, the anti-migration potential of AC was found via autophagy-dependent ferroptosis. Additionally, we found that AC reduced the expression of GPX4 by ubiquitination and inhibited TNBC proliferation and metastasis in vitro and in vivo. Moreover, we demonstrated that AC induced autophagy-dependent ferroptosis, and led to Fe2+ accumulation via ubiquitinating GPX4. Moreover, AC was shown to induce autophagy-dependent ferroptosis as well as to inhibit TNBC proliferation and migration via GPX4 ubiquitination. Together, these results demonstrated that AC inhibited the progression and metastasis of TNBC by inducing autophagy-dependent ferroptosis via ubiquitinating GPX4, which might shed light on exploiting AC as a new drug candidate for the future TNBC therapy.
    Keywords:  Anomanolide C; Autophagy; Ferroptosis; GPX4; Triple negative breast cancer; Ubiquitination
    DOI:  https://doi.org/10.7150/ijbs.82120
  51. Nat Commun. 2023 May 22. 14(1): 2933
    Engineered bacterial outer membrane vesicles encapsulating oncolytic adenoviruses enhance the efficacy of cancer virotherapy by augmenting tumor cell autophagy.
    Weiyue Ban, Mengchi Sun, Hanwei Huang, Wanxu Huang, Siwei Pan, Pengfei Liu, Bingwu Li, Zhenguo Cheng, Zhonggui He, Funan Liu, Jin Sun.
      Oncolytic adenovirus (Ad) infection promotes intracellular autophagy in tumors. This could kill cancer cells and contribute to Ads-mediated anticancer immunity. However, the low intratumoral content of intravenously delivered Ads could be insufficient to efficiently activate tumor over-autophagy. Herein, we report bacterial outer membrane vesicles (OMVs)-encapsulating Ads as microbial nanocomposites that are engineered for autophagy-cascade-augmented immunotherapy. Biomineral shells cover the surface antigens of OMVs to slow their clearance during in vivo circulation, enhancing intratumoral accumulation. After entering tumor cells, there is excessive H2O2 accumulation through the catalytic effect of overexpressed pyranose oxidase (P2O) from microbial nanocomposite. This increases oxidative stress levels and triggers tumor autophagy. The autophagy-induced autophagosomes further promote Ads replication in infected tumor cells, leading to Ads-overactivated autophagy. Moreover, OMVs are powerful immunostimulants for remolding the immunosuppressive tumor microenvironment, facilitating antitumor immune response in preclinical cancer models in female mice. Therefore, the present autophagy-cascade-boosted immunotherapeutic method can expand OVs-based immunotherapy.
    DOI:  https://doi.org/10.1038/s41467-023-38679-z
  52. Chin Med J (Engl). 2023 May 26.
    Hippo (YAP)-autophagy axis protects against hepatic ischemia-reperfusion injury through JNK signaling.
    Shuguang Zhu, Xiaowen Wang, Haoqi Chen, Wenfeng Zhu, Xuejiao Li, Ruiwen Cui, Xiaomeng Yi, Xiaolong Chen, Hua Li, Genshu Wang.
      BACKGROUND: Hepatic ischemia-reperfusion injury (HIRI) remains a common complication during liver transplantation (LT) in patients. As a key downstream effector of the Hippo pathway, Yes-associated protein (YAP) has been reported to be involved in various physiological and pathological processes. However, it remains elusive whether and how YAP may control autophagy activation during ischemia-reperfusion.METHODS: Human liver tissues from patients who had undergone LT were obtained to evaluate the correlation between YAP and autophagy activation. Both an in vitro hepatocyte cell line and in vivo liver-specific YAP knockdown mice were used to establish the hepatic ischemia-reperfusion models to determine the role of YAP in the activation of autophagy and the mechanism of regulation.
    RESULTS: Autophagy was activated in the post-perfusion liver grafts during LT in patients, and the expression of YAP positively correlated with the autophagic level of hepatocytes. Liver-specific knockdown of YAP inhibited hepatocytes autophagy upon hypoxia-reoxygenation and HIRI (P <0.05). YAP deficiency aggravated HIRI by promoting the apoptosis of hepatocytes both in the in vitro and in vivo models (P <0.05). Attenuated HIRI by overexpression of YAP was diminished after the inhibition of autophagy with 3-methyladenine. In addition, inhibiting autophagy activation by YAP knockdown exacerbated mitochondrial damage through increasing reactive oxygen species (P <0.05). Moreover, the regulation of autophagy by YAP during HIRI was mediated by AP1 (c-Jun) N-terminal kinase (JNK) signaling through binding to the transcriptional enhanced associate domain (TEAD).
    CONCLUSIONS: YAP protects against HIRI by inducing autophagy via JNK signaling that suppresses the apoptosis of hepatocytes. Targeting Hippo (YAP)-JNK-autophagy axis may provide a novel strategy for the prevention and treatment of HIRI.
    DOI:  https://doi.org/10.1097/CM9.0000000000002727
  53. Front Immunol. 2023 ;14 1180866
    Epigenetic dysregulation of autophagy in sepsis-induced acute kidney injury: the underlying mechanisms for renoprotection.
    Shankun Zhao, Jian Liao, Maolei Shen, Xin Li, Mei Wu.
      Sepsis-induced acute kidney injury (SI-AKI), a common critically ill, represents one of the leading causes of global death. Emerging evidence reveals autophagy as a pivotal modulator of SI-AKI. Autophagy affects the cellular processes of renal lesions, including cell death, inflammation, and immune responses. Herein, we conducted a systematic and comprehensive review on the topic of the proposed roles of autophagy in SI-AKI. Forty-one relevant studies were finally included and further summarized and analyzed. This review revealed that a majority of included studies (24/41, 58.5%) showed an elevation of the autophagy level during SI-AKI, while 22% and 19.5% of the included studies reported an inhibition and an elevation at the early stage but a declination of renal autophagy in SI-AKI, respectively. Multiple intracellular signaling molecules and pathways targeting autophagy (e.g. mTOR, non-coding RNA, Sirtuins family, mitophagy, AMPK, ROS, NF-Kb, and Parkin) involved in the process of SI-AKI, exerting multiple biological effects on the kidney. Multiple treatment modalities (e.g. small molecule inhibitors, temsirolimus, rapamycin, polydatin, ascorbate, recombinant human erythropoietin, stem cells, Procyanidin B2, and dexmedetomidine) have been found to improve renal function, which may be attributed to the elevation of the autophagy level in SI-AKI. Though the exact roles of autophagy in SI-AKI have not been well elucidated, it may be implicated in preventing SI-AKI through various molecular pathways. Targeting the autophagy-associated proteins and pathways may hint towards a new prospective in the treatment of critically ill patients with SI-AKI, but more preclinical studies are still warranted to validate this hypothesis.
    Keywords:  acute kidney injury; autophagy; mechanism; protection; sepsis
    DOI:  https://doi.org/10.3389/fimmu.2023.1180866
  54. Biochim Biophys Acta Mol Cell Res. 2023 May 19. pii: S0167-4889(23)00059-9. [Epub ahead of print] 119487
    Phospholipase A2-activating protein induces mitophagy trough anti-apoptotic MCL1-mediated NLRX1 oligomerization.
    Zhilong Zheng, Lu Han, Yuanbo Li, Zhen Chen, Wangju Yang, Chunyue Liu, Mengdan Tao, Yueqing Jiang, Xiaoyan Ke, Yan Liu, Xing Guo.
      Mitochondrial protein homeostasis is fine-tuned by diverse physiological processes such as mitochondria-associated degradation (MAD), which is regulated by valosin-containing protein (VCP) and its cofactors. As a cofactor of VCP, the mutation of phospholipase A-2-activating protein (PLAA) is the genetic cause of PLAA-associated neurodevelopmental disorder (PLAAND). However, the physiological and pathological roles of PLAA in mitochondria remain unclear. Here, we demonstrate that PLAA partially associates with mitochondria. Deficiency in PLAA increases mitochondrial reactive oxygen species (ROS) production, reduces mitochondrial membrane potential, inhibits mitochondrial respiratory activity and causes excessive mitophagy. Mechanically, PLAA interacts with myeloid cell leukemia-1 (MCL1) and facilitates its retro-translocation and proteasome-dependent degradation. The upregulation of MCL1 promotes the oligomerization of NLR family member X1 (NLRX1) and activation of mitophagy. Whereas downregulating NLRX1 abolishes MCL1 induced mitophagy. In summary, our data identify PLAA as a novel mediator of mitophagy by regulating MCL1-NLRX1 axis. We propose mitophagy as a target for therapeutic intervention in PLAAND.
    Keywords:  Mitochondria; Mitochondria-associated degradation; Mitophagy; Myeloid cell leukemia 1; NLR family member X1; Phospholipase A-2-activating protein
    DOI:  https://doi.org/10.1016/j.bbamcr.2023.119487
  55. Int J Mol Sci. 2023 May 15. pii: 8763. [Epub ahead of print]24(10):
    Dysfunctional Autophagy, Proteostasis, and Mitochondria as a Prelude to Age-Related Macular Degeneration.
    Raji Rajesh Lenin, Yi Hui Koh, Zheting Zhang, Yan Zhuang Yeo, Bhav Harshad Parikh, Ivan Seah, Wendy Wong, Xinyi Su.
      Retinal pigment epithelial (RPE) cell dysfunction is a key driving force of AMD. RPE cells form a metabolic interface between photoreceptors and choriocapillaris, performing essential functions for retinal homeostasis. Through their multiple functions, RPE cells are constantly exposed to oxidative stress, which leads to the accumulation of damaged proteins, lipids, nucleic acids, and cellular organelles, including mitochondria. As miniature chemical engines of the cell, self-replicating mitochondria are heavily implicated in the aging process through a variety of mechanisms. In the eye, mitochondrial dysfunction is strongly associated with several diseases, including age-related macular degeneration (AMD), which is a leading cause of irreversible vision loss in millions of people globally. Aged mitochondria exhibit decreased rates of oxidative phosphorylation, increased reactive oxygen species (ROS) generation, and increased numbers of mitochondrial DNA mutations. Mitochondrial bioenergetics and autophagy decline during aging because of insufficient free radical scavenger systems, the impairment of DNA repair mechanisms, and reductions in mitochondrial turnover. Recent research has uncovered a much more complex role of mitochondrial function and cytosolic protein translation and proteostasis in AMD pathogenesis. The coupling of autophagy and mitochondrial apoptosis modulates the proteostasis and aging processes. This review aims to summarise and provide a perspective on (i) the current evidence of autophagy, proteostasis, and mitochondrial dysfunction in dry AMD; (ii) current in vitro and in vivo disease models relevant to assessing mitochondrial dysfunction in AMD, and their utility in drug screening; and (iii) ongoing clinical trials targeting mitochondrial dysfunction for AMD therapeutics.
    Keywords:  age-related macular degeneration; aging; autophagy; clinical trials; mitochondrial dysfunction; retinal pigment epithelium
    DOI:  https://doi.org/10.3390/ijms24108763
  56. Front Cell Dev Biol. 2023 ;11 1186850
    Regulation of hematopoietic stem cells differentiation, self-renewal, and quiescence through the mTOR signaling pathway.
    Bai Ling, Yunyang Xu, Siyuan Qian, Ze Xiang, Shihai Xuan, Jian Wu.
      Hematopoietic stem cells (HSCs) are important for the hematopoietic system because they can self-renew to increase their number and differentiate into all the blood cells. At a steady state, most of the HSCs remain in quiescence to preserve their capacities and protect themselves from damage and exhaustive stress. However, when there are some emergencies, HSCs are activated to start their self-renewal and differentiation. The mTOR signaling pathway has been shown as an important signaling pathway that can regulate the differentiation, self-renewal, and quiescence of HSCs, and many types of molecules can regulate HSCs' these three potentials by influencing the mTOR signaling pathway. Here we review how mTOR signaling pathway regulates HSCs three potentials, and introduce some molecules that can work as the regulator of HSCs' these potentials through the mTOR signaling. Finally, we outline the clinical significance of studying the regulation of HSCs three potentials through the mTOR signaling pathway and make some predictions.
    Keywords:  differentiation; hematopoietic stem cells (HSCS); mTOR; quiescence; self-renewal
    DOI:  https://doi.org/10.3389/fcell.2023.1186850
  57. Anal Chem. 2023 May 22.
    Comprehensive Profiling of Rapamycin Interacting Proteins with Multiple Mass Spectrometry-Based Omics Techniques.
    Yao Xu, Mengmeng Zheng, Li Gong, Guizhen Liu, Shanshan Qian, Ying Han, Jingwu Kang.
      Profiling drug-protein interactions is critical for understanding a drug's mechanism of action and predicting the possible adverse side effects. However, to comprehensively profile drug-protein interactions remains a challenge. To address this issue, we proposed a strategy that integrates multiple mass spectrometry-based omics analysis to provided global drug-protein interactions, including physical interactions and functional interactions, with rapamycin (Rap) as a model. Chemoproteomics profiling reveals 47 Rap binding proteins including the known target protein FKBP12 with high confidence. Gen Ontology enrichment analysis suggested that the Rap binding proteins are implicated in several important cellular processes, such as DNA replication, immunity, autophagy, programmed cell death, aging, transcription modulation, vesicle-mediated transport, membrane organization, and carbohydrate and nucleobase metabolic processes. The phosphoproteomics profiling revealed 255 down-regulated and 150 up-regulated phosphoproteins responding to Rap stimulation; they mainly involve the PI3K-Akt-mTORC1 signaling axis. Untargeted metabolomic profiling revealed 22 down-regulated metabolites and 75 up-regulated metabolites responding to Rap stimulation; they are mainly associated with the synthesis processes of pyrimidine and purine. The integrative multiomics data analysis provides deep insight into the drug-protein interactions and reveals Rap's complicated mechanism of action.
    DOI:  https://doi.org/10.1021/acs.analchem.3c00867
  58. Int J Mol Sci. 2023 May 10. pii: 8528. [Epub ahead of print]24(10):
    Neuroprotection of Andrographolide against Neurotoxin MPP+-Induced Apoptosis in SH-SY5Y Cells via Activating Mitophagy, Autophagy, and Antioxidant Activities.
    Prachayaporn Prasertsuksri, Pichnaree Kraokaew, Kanta Pranweerapaiboon, Prasert Sobhon, Kulathida Chaithirayanon.
      Parkinson's disease (PD) is associated with dopaminergic neuron loss and alpha-synuclein aggregation caused by ROS overproduction, leading to mitochondrial dysfunction and autophagy impairment. Recently, andrographolide (Andro) has been extensively studied for various pharmacological properties, such as anti-diabetic, anti-cancer, anti-inflammatory, and anti-atherosclerosis. However, its potential neuroprotective effects on neurotoxin MPP+-induced SH-SY5Y cells, a cellular PD model, remain uninvestigated. In this study, we hypothesized that Andro has neuroprotective effects against MPP+-induced apoptosis, which may be mediated through the clearance of dysfunctional mitochondria by mitophagy and ROS by antioxidant activities. Herein, Andro pretreatment could attenuate MPP+-induced neuronal cell death that was reflected by reducing mitochondrial membrane potential (MMP) depolarization, alpha-synuclein, and pro-apoptotic proteins expressions. Concomitantly, Andro attenuated MPP+-induced oxidative stress through mitophagy, as indicated by increasing colocalization of MitoTracker Red with LC3, upregulations of the PINK1-Parkin pathway, and autophagy-related proteins. On the contrary, Andro-activated autophagy was compromised when pretreated with 3-MA. Furthermore, Andro activated the Nrf2/KEAP1 pathway, leading to increasing genes encoding antioxidant enzymes and activities. This study elucidated that Andro exhibited significant neuroprotective effects against MPP+-induced SH-SY5Y cell death in vitro by enhancing mitophagy and clearance of alpha-synuclein through autophagy, as well as increasing antioxidant capacity. Our results provide evidence that Andro could be considered a potential supplement for PD prevention.
    Keywords:  MPP+; Nrf2; Parkinson’s disease; ROS; SH-SY5Y cells; andrographolide; mitophagy
    DOI:  https://doi.org/10.3390/ijms24108528
  59. Traffic. 2023 May 23.
    Deacidification of endolysosomes by neuronal aging drives synapse loss.
    Tatiana Burrinha, César Cunha, Michael J Hall, Mafalda Lopes-da-Silva, Miguel C Seabra, Cláudia Guimas Almeida.
      Previously, we found that age-dependent accumulation of beta-amyloid is not sufficient to cause synaptic decline. Late-endocytic organelles (LEOs) may be driving synaptic decline as lysosomes (Lys) are a target of cellular aging and relevant for synapses. We found that LAMP1-positive LEOs increased in size and number and accumulated near synapses in aged neurons and brains. LEOs' distal accumulation might relate to the increased anterograde movement in aged neurons. Dissecting the LEOs, we found that late-endosomes accumulated while there are fewer terminal Lys in aged neurites, but not in the cell body. The most abundant LEOs were degradative Lys or endolysosomes (ELys), especially in neurites. ELys activity was reduced because of acidification defects, supported by the reduction in v-ATPase subunit V0a1 with aging. Increasing the acidification of aged ELys recovered degradation and reverted synaptic decline, while alkalinization or v-ATPase inhibition, mimicked age-dependent Lys and synapse dysfunction. We identify ELys deacidification as a neuronal mechanism of age-dependent synapse loss. Our findings suggest that future therapeutic strategies to address endolysosomal defects might be able to delay age-related synaptic decline.
    Keywords:  aging; endolysosomal system; lysosome; neuron; synapses
    DOI:  https://doi.org/10.1111/tra.12889
  60. Nat Rev Mol Cell Biol. 2023 May 26.
    Studying lysosomal function and dysfunction using LysoIP.
    Nouf N Laqtom.
      
    DOI:  https://doi.org/10.1038/s41580-023-00619-6
  61. EMBO J. 2023 May 25. e112817
    Host cell egress of Brucella abortus requires BNIP3L-mediated mitophagy.
    Jérémy Verbeke, Youri Fayt, Lisa Martin, Oya Yilmaz, Jaroslaw Sedzicki, Angéline Reboul, Michel Jadot, Patricia Renard, Christoph Dehio, Henri-François Renard, Jean-Jacques Letesson, Xavier De Bolle, Thierry Arnould.
      The facultative intracellular pathogen Brucella abortus interacts with several organelles of the host cell to reach its replicative niche inside the endoplasmic reticulum. However, little is known about the interplay between the intracellular bacteria and the host cell mitochondria. Here, we showed that B. abortus triggers substantive mitochondrial network fragmentation, accompanied by mitophagy and the formation of mitochondrial Brucella-containing vacuoles during the late steps of cellular infection. Brucella-induced expression of the mitophagy receptor BNIP3L is essential for these events and relies on the iron-dependent stabilisation of the hypoxia-inducible factor 1α. Functionally, BNIP3L-mediated mitophagy appears to be advantageous for bacterial exit from the host cell as BNIP3L depletion drastically reduces the number of reinfection events. Altogether, these findings highlight the intricate link between Brucella trafficking and the mitochondria during host cell infection.
    Keywords:   Brucella ; BNIP3L; intracellular trafficking; iron; mitophagy
    DOI:  https://doi.org/10.15252/embj.2022112817
  62. Sci Rep. 2023 May 23. 13(1): 8338
    Spermidine improves angiogenic capacity of senescent endothelial cells, and enhances ischemia-induced neovascularization in aged mice.
    Daisuke Ueno, Koji Ikeda, Ekura Yamazaki, Akiko Katayama, Ryota Urata, Satoaki Matoba.
      Aging is closely associated with the increased morbidity and mortality of ischemic cardiovascular disease, at least partially through impaired angiogenic capacity. Endothelial cells (ECs) play a crucial role in angiogenesis, and their angiogenic capacity declines during aging. Spermidine is a naturally occurring polyamine, and its dietary supplementation has exhibited distinct anti-aging and healthy lifespan-extending effects in various species such as yeast, worms, flies, and mice. Here, we explore the effects of spermidine supplementation on the age-related decline in angiogenesis both in vitro and in vivo. Intracellular polyamine contents were reduced in replicative senescent ECs, which were subsequently recovered by spermidine supplementation. Our findings reveal that spermidine supplementation improved the declined angiogenic capacity of senescent ECs, including migration and tube-formation, without affecting the senescence phenotypes. Mechanistically, spermidine enhanced both autophagy and mitophagy, and improved mitochondrial quality in senescent ECs. Ischemia-induced neovascularization was assessed using the hind-limb ischemia model in mice. Limb blood flow recovery and neovascularization in the ischemic muscle were considerably impaired in aged mice compared to young ones. Of note, dietary spermidine significantly enhanced ischemia-induced angiogenesis, and improved the blood flow recovery in the ischemic limb, especially in aged mice. Our results reveal novel proangiogenic functions of spermidine, suggesting its therapeutic potential against ischemic disease.
    DOI:  https://doi.org/10.1038/s41598-023-35447-3
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