bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2023‒06‒04
fifty papers selected by
Viktor Korolchuk, Newcastle University
  1. Alpha-synuclein-associated changes in PINK1-PRKN-mediated mitophagy are disease context dependent.
  2. Wild-type and pathogenic forms of ubiquilin 2 differentially modulate components of the autophagy-lysosome pathways.
  3. Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels using Live Imaging in HeLa Cells.
  4. Immune modulation through secretory autophagy.
  5. Aggrephagy at a glance.
  6. Membrane association of the ATG8 conjugation machinery emerges as a key regulatory feature for autophagosome biogenesis.
  7. Enhancement of oligodendrocyte autophagy alleviates white matter injury and cognitive impairment induced by chronic cerebral hypoperfusion in rats.
  8. Selective autophagy regulates chloroplast protein import and promotes plant stress tolerance.
  9. Restoration of sleep and circadian behavior by autophagy modulation in Huntington's disease.
  10. PINK1-Dependent Mitophagy Inhibits Elevated Ubiquitin Phosphorylation Caused by Mitochondrial Damage.
  11. Exercise-Induced Autophagy Ameliorates Motor Symptoms Progressivity in Parkinson's Disease Through Alpha-Synuclein Degradation: A Review.
  12. TFEB - at the crossroads of host-pathogen interactions.
  13. Intermittent inhibition of FYVE finger-containing phosphoinositide kinase induces melanosome degradation in B16F10 melanoma cells.
  14. Autophagy in the commercial arena: a conversation with Dr. Leon Murphy, Chief Scientific Officer at Casma therapeutics.
  15. Bruchpiloting synaptic connections: Egfr in autophagy-mediated neuronal circuit development.
  16. HSF4 Transcriptionally Activates Autophagy by Regulating ATG9a During Lens Terminal Differentiation.
  17. Advancement of targeted protein degradation strategies as therapeutics for undruggable disease targets.
  18. Deubiquitinase USP9X loss sensitizes renal cancer cells to mTOR inhibition.
  19. Nucleolin promotes autophagy through PGC-1α in LPS-induced myocardial injury.
  20. mTOR/α-ketoglutarate signaling: impact on brain cell homeostasis under ischemic conditions.
  21. Inactivation of Minar2 in Mice Hyperactivates mTOR Signaling and Results in Obesity.
  22. Concomitant Neuronal Tau Deposition and FKBP52 Decrease Is an Early Feature of Different Human and Experimental Tauopathies.
  23. The interrelation of galectins and autophagy.
  24. ER-to-Lysosome Associated Degradation (ERLAD) in a Nutshell: mammalian, yeast and plant ER-phagy as induced by misfolded proteins.
  25. Proteasome and p62/SQSTM1 are involved in methylmercury toxicity mitigation in mouse embryonic fibroblast cells.
  26. mTOR-inhibitors and post-transplant diabetes mellitus: a link still debated in kidney transplantation.
  27. A metabolic shift toward glycolysis enables cancer cells to maintain survival upon concomitant glutamine deprivation and V-ATPase inhibition.
  28. Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health.
  29. Unbalanced Redox With Autophagy in Cardiovascular Disease.
  30. Semisynthetic LC3 Probes for Autophagy Pathways Reveal a Noncanonical LC3 Interacting Region Motif Crucial for the Enzymatic Activity of Human ATG3.
  31. A bi-steric mTORC1-selective inhibitor overcomes drug resistance in breast cancer.
  32. An emerging role of non-canonical conjugation of ATG8 proteins in plant response to heat stress.
  33. 'Candidatus Liberibacter asiaticus' secretory protein SDE3 inhibits host autophagy to promote Huanglongbing disease in citrus.
  34. B cell activation via immunometabolism in systemic lupus erythematosus.
  35. Cellular redox homeostasis maintained by malic enzyme 2 is essential for MYC-driven T cell lymphomagenesis.
  36. Methods for Monitoring Mitochondrial Biogenesis and Turnover in Cultured Hepatocytes and Mouse Liver Using MitoTimer Reporter Assay.
  37. LC3-associated phagocytosis promotes glial degradation of axon debris after injury in Drosophila models.
  38. Clearance of lipid droplets by chimeric autophagy-tethering compound ameliorates the age-related macular degeneration phenotype in mice lacking APOE.
  39. Monitoring Stub1-Mediated Pexophagy.
  40. Promotion of degradative autophagy by 6-bromoindirubin-3'-oxime attenuates neuropathy.
  41. Disruption of axonal transport in neurodegeneration.
  42. The role of TSC2 in breast cancer: a literature review.
  43. Activation of TLR7-mediated autophagy increases epileptic susceptibility via reduced KIF5A-dependent GABAA receptor transport in a murine model.
  44. The mechanisms to dispose of misfolded proteins in the endoplasmic reticulum of adipocytes.
  45. Oleic acid improves hepatic lipotoxicity injury by alleviating autophagy dysfunction.
  46. Mitophagy in atherosclerosis: from mechanism to therapy.
  47. The crosstalk between PTEN-induced kinase 1-mediated mitophagy and the inflammasome in the pathogenesis of alopecia areata.
  48. The hepatitis B virus promotes the progression of non-alcoholic fatty liver disease through incomplete autophagy.
  49. A Turn-On Fluorescent Amino Acid Sensor Reveals Chloroquine's Effect on Cellular Amino Acids via Inhibiting Cathepsin L.
  50. ULK1-mediated metabolic reprogramming regulates Vps34 lipid kinase activity by its lactylation.
  1. Brain Pathol. 2023 May 31. e13175
    Alpha-synuclein-associated changes in PINK1-PRKN-mediated mitophagy are disease context dependent.
    Xu Hou, Taylor Hsuan-Yu Chen, Shunsuke Koga, Jenny M Bredenberg, Ayman H Faroqi, Marion Delenclos, Guojun Bu, Zbigniew K Wszolek, Jonathan A Carr, Owen A Ross, Pamela J McLean, Melissa E Murray, Dennis W Dickson, Fabienne C Fiesel, Wolfdieter Springer.
      Alpha-synuclein (αsyn) aggregates are pathological features of several neurodegenerative conditions including Parkinson disease (PD), dementia with Lewy bodies, and multiple system atrophy (MSA). Accumulating evidence suggests that mitochondrial dysfunction and impairments of the autophagic-lysosomal system can contribute to the deposition of αsyn, which in turn may interfere with health and function of these organelles in a potentially vicious cycle. Here we investigated a potential convergence of αsyn with the PINK1-PRKN-mediated mitochondrial autophagy pathway in cell models, αsyn transgenic mice, and human autopsy brain. PINK1 and PRKN identify and selectively label damaged mitochondria with phosphorylated ubiquitin (pS65-Ub) to mark them for degradation (mitophagy). We found that disease-causing multiplications of αsyn resulted in accumulation of the ubiquitin ligase PRKN in cells. This effect could be normalized by starvation-induced autophagy activation and by CRISPR/Cas9-mediated αsyn knockout. Upon acute mitochondrial damage, the increased levels of PRKN protein contributed to an enhanced pS65-Ub response. We further confirmed increased pS65-Ub-immunopositive signals in mouse brain with αsyn overexpression and in postmortem human disease brain. Of note, increased pS65-Ub was associated with neuronal Lewy body-type αsyn pathology, but not glial cytoplasmic inclusions of αsyn as seen in MSA. While our results add another layer of complexity to the crosstalk between αsyn and the PINK1-PRKN pathway, distinct mechanisms may underlie in cells and brain tissue despite similar outcomes. Notwithstanding, our finding suggests that pS65-Ub may be useful as a biomarker to discriminate different synucleinopathies and may serve as a potential therapeutic target for Lewy body disease.
    Keywords:  Lewy body disease; PINK1; PRKN; Parkinson disease; alpha-synuclein; autophagy; mitochondria; mitophagy; multiple system atrophy; phosphorylated ubiquitin
    DOI:  https://doi.org/10.1111/bpa.13175
  2. J Pharmacol Sci. 2023 Jul;pii: S1347-8613(23)00032-4. [Epub ahead of print]152(3): 182-192
    Wild-type and pathogenic forms of ubiquilin 2 differentially modulate components of the autophagy-lysosome pathways.
    Akiko Idera, Lisa M Sharkey, Yuki Kurauchi, Keiichi Kadoyama, Henry L Paulson, Hiroshi Katsuki, Takahiro Seki.
      Missense mutations of ubiquilin 2 (UBQLN2) have been identified to cause X-linked amyotrophic lateral sclerosis (ALS). Proteasome-mediated protein degradation is reported to be impaired by ALS-associated mutations of UBQLN2. However, it remains unknown how these mutations affect autophagy-lysosome protein degradation, which consists of macroautophagy (MA), microautophagy (mA), and chaperone-mediated autophagy (CMA). Using a CMA/mA fluorescence reporter we found that overexpression of wild-type UBQLN2 impairs CMA. Conversely, knockdown of endogenous UBQLN2 increases CMA activity, suggesting that normally UBQLN2 negatively regulates CMA. ALS-associated mutant forms of UBQLN2 exacerbate this impairment of CMA. Using cells stably transfected with wild-type or ALS-associated mutant UBQLN2, we further determined that wild-type UBQLN2 increased the ratio of LAMP2A (a CMA-related protein) to LAMP1 (a lysosomal protein). This could represent a compensatory reaction to the impairment of CMA by wild-type UBQLN2. However, ALS-associated mutant UBQLN2 failed to show this compensation, exacerbating the impairment of CMA by mutant UBQLN2. We further demonstrated that ALS-associated mutant forms of UBQLN2 also impair MA, but wild-type UBQLN2 does not. These results support the view that ALS-associated mutant forms of UBQLN2 impair both CMA and MA which may contribute to the neurodegeneration observed in patients with UBQLN2-mediated ALS.
    Keywords:  Amyotrophic lateral sclerosis; Chaperone-mediated autophagy; Macroautophagy; Ubiquilin 2
    DOI:  https://doi.org/10.1016/j.jphs.2023.05.002
  3. J Vis Exp. 2023 05 12.
    Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels using Live Imaging in HeLa Cells.
    Mohammad Fazli, Chantell S Evans.
      Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.
    DOI:  https://doi.org/10.3791/65304
  4. J Cell Biochem. 2023 Jun 01.
    Immune modulation through secretory autophagy.
    Andreas Weigert, Lina Herhaus.
      Autophagy is a central mechanism of cellular homeostasis through the degradation of a wide range of cellular constituents. However, recent evidence suggests that autophagy actively provides information to neighboring cells via a process called secretory autophagy. Secretory autophagy couples the autophagy machinery to the secretion of cellular content via extracellular vesicles (EVs). EVs carry a variety of cargo, that reflect the pathophysiological state of the originating cells and have the potential to change the functional profile of recipient cells, to modulate cell biology. The immune system has evolved to maintain local and systemic homeostasis. It is able to sense a wide array of molecules signaling disturbed homeostasis, including EVs and their content. In this review, we explore the emerging concept of secretory autophagy as a means to communicate cellular, and in total tissue pathophysiological states to the immune system to initiate the restoration of tissue homeostasis. Understanding how autophagy mediates the secretion of immunogenic factors may hold great potential for therapeutic intervention.
    Keywords:  autophagy; cancer; extracellular vesicles; immunity; infection; inflammation; secretion; secretory autophagy
    DOI:  https://doi.org/10.1002/jcb.30427
  5. J Cell Sci. 2023 May 15. pii: jcs260888. [Epub ahead of print]136(10):
    Aggrephagy at a glance.
    Bernd Bauer, Sascha Martens, Luca Ferrari.
      Cells keep their proteome functional by the action of the proteostasis network, composed of the chaperones, the ubiquitin-proteasome system and autophagy. The decline of this network results in the accumulation of protein aggregates and is associated with aging and disease. In this Cell Science at a Glance and accompanying poster, we provide an overview of the molecular mechanisms of the removal of protein aggregates by a selective autophagy pathway, termed aggrephagy. We outline how aggrephagy is regulated by post-translational modifications and via auxiliary proteins. We further describe alternative aggrephagy pathways in physiology and their disruption in pathology. In particular, we discuss aggrephagy pathways in neurons and accumulation of protein aggregates in a wide range of diseases. Finally, we highlight strategies to reprogram aggrephagy to treat protein aggregation diseases.
    Keywords:  Autophagy; Neurodegeneration; Proteostasis network
    DOI:  https://doi.org/10.1242/jcs.260888
  6. FEBS Lett. 2023 Jun 01.
    Membrane association of the ATG8 conjugation machinery emerges as a key regulatory feature for autophagosome biogenesis.
    Sharon A Tooze, Wenxin Zhang, Gianmarco Lazzeri, Deepanshi Gahlot, Lipi Thukral, Roberto Covino, Taki Nishimura.
      Autophagy is a highly conserved intracellular pathway which is essential for survival in all eukaryotes. In healthy cells, autophagy is used to remove damaged intracellular components, which can be as simple as unfolded proteins or as complex as whole mitochondria. Once the damaged component is captured, the autophagosome engulfs it and closes, isolating the content from the cytoplasm. The autophagosome then fuses with the late endosome and/or lysosome to deliver its content to the lysosome for degradation. Formation of the autophagosome, sequestration or capture of content, and closure all require the ATG proteins, which constitute the essential core autophagy protein machinery. This brief "nutshell" will highlight recent data revealing the importance of small membrane associated domains in the ATG proteins. In particular, recent findings from two parallel studies reveal the unexpected key role of α-helical structures in the ATG8 conjugation machinery and ATG8s. These studies illustrate how unique membrane association modules can control the formation of autophagosomes.
    Keywords:  ATG3; ATG8 lipidation; Amphipathic α-helix; Autophagy; MD simulation; autophagosome size; cis-membrane association; membrane expansion
    DOI:  https://doi.org/10.1002/1873-3468.14676
  7. Acta Pharm Sin B. 2023 May;13(5): 2107-2123
    Enhancement of oligodendrocyte autophagy alleviates white matter injury and cognitive impairment induced by chronic cerebral hypoperfusion in rats.
    Huiyang Wang, Yueyang Liu, Zhenkun Guo, Minghui Cui, Peng Pang, Jingyu Yang, Chunfu Wu.
      Cognitive impairment caused by chronic cerebral hypoperfusion (CCH) is associated with white matter injury (WMI), possibly through the alteration of autophagy. Here, the autophagy-lysosomal pathway (ALP) dysfunction in white matter (WM) and its relationship with cognitive impairment were investigated in rats subjected to two vessel occlusion (2VO). The results showed that cognitive impairment occurred by the 28th day after 2VO. Injury and autophagy activation of mature oligodendrocytes and neuronal axons sequentially occurred in WM by the 3rd day. By the 14th day, abnormal accumulation of autophagy substrate, lysosomal dysfunction, and the activation of mechanistic target of rapamycin (MTOR) pathway were observed in WM, paralleled with mature oligodendrocyte death. This indicates autophagy activation was followed by ALP dysfunction caused by autophagy inhibition or lysosomal dysfunction. To target the ALP dysfunction, enhanced autophagy by systemic rapamycin treatment or overexpression of Beclin1 (BECN1) in oligodendrocytes reduced mature oligodendrocyte death, and subsequently alleviated the WMI and cognitive impairment after CCH. These results reveal that early autophagy activation was followed by ALP dysfunction in WM after 2VO, which was associated with the aggravation of WMI and cognitive impairment. This study highlights that alleviating ALP dysfunction by enhancing oligodendrocyte autophagy has benefits for cognitive recovery after CCH.
    Keywords:  Autophagy-lysosomal pathway; Chronic cerebral hypoperfusion; Cognitive impairment; Mechanistic target of rapamycin; Myelin sheath; Oligodendrocyte; Two vessel occlusion; White matter
    DOI:  https://doi.org/10.1016/j.apsb.2023.03.014
  8. EMBO J. 2023 May 30. e112534
    Selective autophagy regulates chloroplast protein import and promotes plant stress tolerance.
    Chen Wan, Hui Zhang, Hongying Cheng, Robert G Sowden, Wenjuan Cai, R Paul Jarvis, Qihua Ling.
      Chloroplasts are plant organelles responsible for photosynthesis and environmental sensing. Most chloroplast proteins are imported from the cytosol through the translocon at the outer envelope membrane of chloroplasts (TOC). Previous work has shown that TOC components are regulated by the ubiquitin-proteasome system (UPS) to control the chloroplast proteome, which is crucial for the organelle's function and plant development. Here, we demonstrate that the TOC apparatus is also subject to K63-linked polyubiquitination and regulation by selective autophagy, potentially promoting plant stress tolerance. We identify NBR1 as a selective autophagy adaptor targeting TOC components, and mediating their relocation into vacuoles for autophagic degradation. Such selective autophagy is shown to control TOC protein levels and chloroplast protein import and to influence photosynthetic activity as well as tolerance to UV-B irradiation and heat stress in Arabidopsis plants. These findings uncover the vital role of selective autophagy in the proteolytic regulation of specific chloroplast proteins, and how dynamic control of chloroplast protein import is critically important for plants to cope with challenging environments.
    Keywords:  NBR1; autophagy; chloroplast; protein import; stress response
    DOI:  https://doi.org/10.15252/embj.2022112534
  9. J Neurosci. 2023 Jun 01. pii: JN-RM-1894-22. [Epub ahead of print]
    Restoration of sleep and circadian behavior by autophagy modulation in Huntington's disease.
    Ankit Sharma, Kavyashree N, Ravi Manjithaya, Vasu Sheeba.
      Circadian and sleep defects are well documented in Huntington's disease (HD). Modulation of the autophagy pathway has been shown to mitigate toxic effects of mutant Huntingtin protein. However, it is not clear whether autophagy induction can also rescue circadian and sleep defects. Using a genetic approach, we expressed human mutant HTT protein in a subset of Drosophila circadian neurons and sleep center neurons. In this context, we examined the contribution of autophagy in mitigating toxicity caused by mutant HTT protein. We found that targeted overexpression of an autophagy gene - Atg8a in male flies induces autophagy pathway and partially rescues several HTT-induced behavioral defects including sleep fragmentation, a key hallmark of many neurodegenerative disorders. Using cellular markers and genetic approaches, we demonstrate that indeed the autophagy pathway is involved in behavioral rescue. Surprisingly, despite behavioral rescue and evidence for the involvement of the autophagy pathway, the large aggregates of mutant HTT protein were not eliminated. We show that the rescue in behavior is associated with increased mutant protein aggregation and enhanced output from the targeted neurons, resulting in the strengthening of downstream circuits. Overall, our study suggests that in presence of mutant HTT protein, Atg8a induces autophagy and improves the functioning of circadian and sleep circuits.Significance statement:Defects in sleep and circadian rhythms are well documented in Huntington's disease. Recent literature suggests that circadian and sleep disturbances exacerbate neurodegenerative phenotypes. Hence, identifying potential modifiers that can improve the functioning of these circuits could greatly improve disease management. We used a genetic approach to enhance cellular proteostasis and found that overexpression of a crucial autophagy gene - Atg8a, induces the autophagy pathway in the Drosophila circadian and sleep neurons and rescues sleep and activity rhythm. We demonstrate that the Atg8a improves synaptic function of these circuits by enhancing the aggregation of the mutant protein in neurons. Further, our results suggest that differences in basal levels of protein homeostatic pathways is a factor that determines selective susceptibility of neurons.
    DOI:  https://doi.org/10.1523/JNEUROSCI.1894-22.2023
  10. J Med Chem. 2023 May 29.
    PINK1-Dependent Mitophagy Inhibits Elevated Ubiquitin Phosphorylation Caused by Mitochondrial Damage.
    Olivia A Lambourne, Shane Bell, Léa P Wilhelm, Erika B Yarbrough, Gabriel G Holly, Oliver M Russell, Arwa M Alghamdi, Azeza M Fdel, Carmine Varricchio, Emma L Lane, Ian G Ganley, Arwyn T Jones, Matthew S Goldberg, Youcef Mehellou.
      Ubiquitin phosphorylation by the mitochondrial protein kinase PTEN-induced kinase 1 (PINK1), upon mitochondrial depolarization, is an important intermediate step in the recycling of damaged mitochondria via mitophagy. As mutations in PINK1 can cause early-onset Parkinson's disease (PD), there has been a growing interest in small-molecule activators of PINK1-mediated mitophagy as potential PD treatments. Herein, we show that N6-substituted adenosines, such as N6-(2-furanylmethyl)adenosine (known as kinetin riboside) and N6-benzyladenosine, activate PINK1 in HeLa cells and induce PINK1-dependent mitophagy in primary mouse fibroblasts. Interestingly, pre-treatment of HeLa cells and astrocytes with these compounds inhibited elevated ubiquitin phosphorylation that is induced by established mitochondrial depolarizing agents, carbonyl cyanide m-chlorophenyl-hydrazine and niclosamide. Together, this highlights N6-substituted adenosines as progenitor PINK1 activators that could potentially be developed, in the future, as treatments for aged and sporadic PD patients who have elevated phosphorylated ubiquitin levels in the brain.
    DOI:  https://doi.org/10.1021/acs.jmedchem.3c00555
  11. Neuropsychiatr Dis Treat. 2023 ;19 1253-1262
    Exercise-Induced Autophagy Ameliorates Motor Symptoms Progressivity in Parkinson's Disease Through Alpha-Synuclein Degradation: A Review.
    Hanna Goenawan, Shabrina Kiasati, Nova Sylviana, Imam Megantara, Ronny Lesmana.
      This study reviews the molecular mechanism of exercise-induced autophagy/mitophagy and its possible mechanism in delaying motor symptoms progressivity in Parkinson's disease (PD). Relevant articles obtained from PubMed and EBSCOhost were reviewed. After analyzing the articles, it was found that autophagy can be induced by exercise and can possibly be activated through the AMPK-ULK1 pathway. Mitophagy can also be induced by exercise and can possibly be activated through PINK1/Parkin pathway and AMPK-dependent pathway. Moreover, exercise-induced autophagy can decrease the accumulation of toxic α-synuclein aggregates in PD and therefore can delay motor symptoms progressivity.
    Keywords:  Parkinson’s disease; alpha-synuclein; autophagy; exercise
    DOI:  https://doi.org/10.2147/NDT.S401416
  12. J Cell Sci. 2021 Aug 01. pii: jcs252981. [Epub ahead of print]134(15):
    TFEB - at the crossroads of host-pathogen interactions.
    Keerti, Aarti Pant, Ravi Manjithaya.
      The existence of constantly evolving dynamic interactions between the host and the pathogen determines their fate in this continuous arms race. Hence, identifying the molecular basis of processes that reinforce host defensive strategies to eliminate intracellular pathogens is of utmost significance. Pathogenic intrusion activates autophagy and phagocytic pathways that culminate in the lysosome, a vital organelle responsible for pathogen clearance. The transcription factor TFEB plays a pivotal role in autophagy-lysosomal function. Although TFEB is an emerging transcription factor in the field of immune signaling pathways, its role in infectious diseases remains contentious. Recent evidence suggests that infection with certain bacterial and viral pathogens causes TFEB, which is normally located in the cytoplasm, to translocate to the nucleus. There, it activates the transcription of genes that trigger the autophagy-lysosomal and inflammatory pathways to target intracellular pathogens. It is known that some pathogens modulate TFEB to establish themselves inside the host; in some cases, pathogens restrict TFEB to the cytoplasm, whereas in others, functional TFEB fuels pathogen survival and replication. However, the key regulators and molecular mechanisms that decide the outcome of TFEB function during intracellular infection are not clear. In this Review, we attempt to dissect the complex functions of TFEB in host-pathogen interactions and explore the suitability of TFEB as a therapeutic target of clinical relevance.
    Keywords:  Autophagy; Host-directed therapy; Infection; Inflammatory; Innate immunity; Lysosome; Nuclear translocation; TFEB; Therapeutics
    DOI:  https://doi.org/10.1242/jcs.252981
  13. Mol Biol Rep. 2023 May 29.
    Intermittent inhibition of FYVE finger-containing phosphoinositide kinase induces melanosome degradation in B16F10 melanoma cells.
    Kyoka Kawaguchi, Miyu Watanabe, Saho Furukawa, Kenichi Koga, Hiromitsu Kanamori, Mitsushi J Ikemoto, Shigeo Takashima, Miwa Maeda, Kentaro Oh-Hashi, Yoko Hirata, Kyoji Furuta, Hiroshi Takemori.
      BACKGROUND: Melanosomes are lysosome-related organelles that contain melanogenic factors and synthesize melanin as they mature. FYVE finger-containing phosphoinositide kinase (PIKfyve) regulates late endosome and lysosome morphology, vesicle trafficking, and autophagy. In melanocytes, PIKfyve inhibition has been reported to induce hypopigmentation due to impairments in the metabolism of early-stage melanosomes.METHODS AND RESULTS: Here, we report a new type of melanosome metabolism: post-PIKfyve inhibition, which was found during the characterization of the endosome/lysosome fluoroprobe GIF-2250. In B16F10 mouse melanoma cells, GIF-2250 highlighted vesicles positive for lysosomal-associated membrane protein 1 (lysosome marker) and other endosome/lysosome markers (CD63 and Rab7/9). When cells were continuously treated with PIKfyve inhibitors, intracellular vacuoles formed, while GIF-2250 fluorescence signals diminished and were diffusely distributed in the vacuoles. After removal of the PIKfyve inhibitors, the GIF-2250 signal intensity was restored, and some GIF-2250-positive vesicles wrapped the melanosomes, which spun at high speed. In addition, intermittent PIKfyve inhibition caused melanin diffusion in the vacuoles and possible leakage into the cytoplasmic compartments, and melanosome degradation was detected by a transmission electron microscope. Melanosome degradation was accompanied by decreased levels of melanin synthesis enzymes and increased levels of the autophagosome maker LC3BII, which is also associated with early melanosomes. However, the protein levels of p62, which is degraded during autophagy, were increased, suggesting an impairment in autophagy flux during intermittent PIKfyve inhibition. Moreover, the autophagy inhibitor 3-methyladenine does not affect these protein levels, suggesting that the melanosome degradation by the intermittent inhibition of PIKfyve is not mediated by canonical autophagy.
    CONCLUSIONS: In conclusion, disturbance of PIKfyve activity induces melanosome degradation in a canonical autophagy-independent manner.
    Keywords:  Autophagy; Endosome; Lysosome; Melanosome; PIKfyve
    DOI:  https://doi.org/10.1007/s11033-023-08536-9
  14. Autophagy. 2023 May 31. 1-4
    Autophagy in the commercial arena: a conversation with Dr. Leon Murphy, Chief Scientific Officer at Casma therapeutics.
    Leon O Murphy, Nicholas T Ktistakis.
      As a maturing field that continues to provide fundamental insights into cell physiology, autophagy is also beginning to attract considerable interest from the biotechnology/pharmaceutical sector. For this Editor's corner, I thought it would be both useful and interesting to talk with somebody who has spent a lot of time in the commercial sphere, working on autophagy and related processes. I was fortunate that Dr. Leon Murphy, Chief Scientific Officer at Casma therapeutics, was willing and able to answer my questions. In addition to his insights on the commercial interest for autophagy, Dr. Murphy also shared his personal experience on the scientific life working in large and small pharmaceutical companies.
    Keywords:  Autophagy activation; CASMA; MTOR; biotechnology; chemical tools
    DOI:  https://doi.org/10.1080/15548627.2023.2217023
  15. Autophagy. 2023 May 27. 1-2
    Bruchpiloting synaptic connections: Egfr in autophagy-mediated neuronal circuit development.
    Yashveer Chohan, Emily A Eitzman, Wayne D Hawkins, Daniel J Klionsky.
      Macroautophagy/autophagy is involved in many aspects of human development including the formation of neuronal circuits. A recent study from Dutta et al. found that the recruitment of Egfr (Epidermal growth factor receptor) to synapses suppresses autophagic degradation of presynaptic proteins, a requirement for proper neuronal circuit development. The findings suggest that Egfr inactivation during a distinct critical interval in late development results in increased levels of autophagy in the brain and decreased neuronal circuit development. Furthermore, the presence of brp (bruchpilot) in the synapse is critical for proper neuronal functioning over this same period. Dutta and colleagues found that increased autophagy due to Egfr inactivation results in decreased brp levels and, therefore, reduced neuronal connectivity. Through live cell imaging, it was determined that only the synaptic branches that accumulate both Egfr and brp are stabilized, allowing for the persistence of active zones, further supporting the importance of both Egfr and brp in the brain. While Dutta and colleagues collected these data based on studies conducted on Drosophila brains, the findings provide great insight as to how these different proteins may be implicated in human neurology.
    Keywords:  EGFR; macroautophagy; neuronal circuit development; synapse; synaptic pruning
    DOI:  https://doi.org/10.1080/15548627.2023.2217015
  16. Invest Ophthalmol Vis Sci. 2023 Jun 01. 64(7): 5
    HSF4 Transcriptionally Activates Autophagy by Regulating ATG9a During Lens Terminal Differentiation.
    Jing Zhang, Ning Jiang, Chunxiao Du, Hongzan Guo, Ranqi Meng, Xinyu Hou, Mugen Liu, Yanzhong Hu, Xiukun Cui.
      Purpose: HSF4 mutations are responsible for congenital cataract formation. Dysfunction of HSF4 leads to defects in lens terminal differentiation. We aimed to study the mechanism of how HSF4 promotes organelle degradation during lens differentiation.Methods: HSF4del42 mutant mice that developed congenital cataracts were employed. The organelle degradation and autophagic function in lens fibers were detected by immunofluorescence and Immunoblotting. Transcriptome analysis was performed to investigate the differentially expressed genes in HSF4del42 lenses, whereas luciferase report assay and ChIP assay were used to confirm the directly transcriptional regulation of ATG9a by HSF4.
    Results: HSF4del42 mice displayed delayed organelle clearance and impaired autophagic degradation function in lens fibers. Activation of autophagy by rapamycin ameliorated the defects in organelle clearance in HSF4del42 lenses ex vivo and in vivo. Depletion of HSF4 attenuated autophagic flux by disrupting autophagosome biogenesis and maturation in lens epithelial cells. HSF4 directly transcriptionally activated the core autophagy protein ATG9a. Instead of the canonical ATG9a isoform, the ATG9a-X2 isoform was predominantly expressed in the lens and alleviated autophagic defects in HSF4 KO lens epithelial cells. The ATG9a-X2 protein displayed a short half-life, and rapamycin treatment restored its levels in HSF4 KO lens epithelial cells and HSF4del42 lenses.
    Conclusions: Our findings demonstrate that HSF4 facilitates organelle degradation probably by transcriptionally activating autophagy during lens terminal differentiation. We first report the involvement of HSF4 in autophagy and the tissue specific splicing of ATG9a. Our study indicates that autophagy activation is a possible therapeutic strategy for HSF4-related congenital cataracts.
    DOI:  https://doi.org/10.1167/iovs.64.7.5
  17. Future Med Chem. 2023 May 31.
    Advancement of targeted protein degradation strategies as therapeutics for undruggable disease targets.
    Mayuri P Kannan, Sarojini Sreeraman, Chaitanya S Somala, Raja Bs Kushwah, Saravanan K Mani, Vickram Sundaram, Anand Thirunavukarasou.
      Targeted protein degradation (TPD) aids in developing novel bifunctional small-molecule degraders and eliminates proteins of interest. The TPD approach shows promising results in oncological, neurogenerative, cardiovascular and gynecological drug development. We provide an overview of technology advancements in TPD, including molecular glues, proteolysis-targeting chimeras (PROTACs), lysosome-targeting chimeras, antibody-based PROTAC, GlueBody PROTAC, autophagy-targeting chimera, autophagosome-tethering compound, autophagy-targeting chimera and chaperone-mediated autophagy-based degraders. Here we discuss the development and evolution of the TPD field, the variety of proteins that PROTACs target and the biological repercussions of their degradation. We particularly highlight the recent improvements in TPD research that utilize autophagy or the endolysosomal pathway, which enables the targeting of undruggable targets.
    Keywords:  ligase; lysosome; molecular glues; targeted protein degradation; ubiquitination
    DOI:  https://doi.org/10.4155/fmc-2023-0072
  18. Int J Cancer. 2023 Jun 01.
    Deubiquitinase USP9X loss sensitizes renal cancer cells to mTOR inhibition.
    Juan M Roldán-Romero, Carlos Valdivia, Maria Santos, Javier Lanillos, Pablo Maroto, Georgia Anguera, Bruna Calsina, Angel Martinez-Montes, María Monteagudo, Sara Mellid, Luis J Leandro-García, Cristina Montero-Conde, Alberto Cascón, Giovanna Roncador, Javier Coloma, Mercedes Robledo, Cristina Rodriguez-Antona.
      Mammalian target of rapamycin (mTOR) is a central regulator of mammalian metabolism and physiology. Aberrant hyperactivation of the mTOR pathway promotes tumor growth and metastasis, and can also promote tumor resistance to chemotherapy and cancer drugs; this makes mTOR an attractive cancer therapeutic target. mTOR inhibitors have been approved to treat cancer; however, the mechanisms underlying drug sensitivity remain poorly understood. Here, whole exome sequencing of three chromophobe renal cell carcinoma (chRCC) patients with exceptional mTOR inhibitor sensitivity revealed that all three patients shared somatic mutations in the deubiquitinase gene USP9X. The clonal characteristics of the mutations, which were amassed by studying multiple patients' primary and metastatic samples from various years, together with the low USP9X mutation rate in unselected chRCC series, reinforced a causal link between USP9X and mTOR inhibitor sensitivity. Rapamycin treatment of USP9X-depleted HeLa and renal cancer 786-O cells, along with the pharmacological inhibition of USP9X, confirmed that this protein plays a role in patients' sensitivity to mTOR inhibitors. USP9X was not found to exert a direct effect on mTORC1, but subsequent ubiquitylome analyses identified p62 as a direct USP9X target. Increased p62 ubiquitination and the augmented rapamycin effect upon bortezomib treatment, together with the results of p62 and LC3 immunofluorescence assays, suggested that dysregulated autophagy in USP9X-depleted cells can have a synergistic effect with mTOR inhibitors. In summary, we show that USP9X constitutes a potential novel marker of sensitivity to mTOR inhibitors in chRCC patients, and represents a clinical strategy for increasing the sensitivity to these drugs.
    Keywords:  USP9X; mTOR signaling; p62; renal cell carcinoma; ubiquitination
    DOI:  https://doi.org/10.1002/ijc.34575
  19. Shock. 2023 May 31.
    Nucleolin promotes autophagy through PGC-1α in LPS-induced myocardial injury.
    Leijing Yin, Ludong Yuan, Yuting Tang, Zhengyang Luo, Xiaofang Lin, Shuxin Wang, Pengfei Liang, Bimei Jiang.
      ABSTRACT: As a multifunctional protein, nucleolin can participate in a variety of cellular processes. Nucleolin also has multiple protective effects on heart disease. Previous studies have shown that nucleolin could not only resist oxidative stress damage and inflammatory damage, but also regulate autophagy to play a protective role in cardiac ischemia. However, the specific mechanism has not been fully elucidated in LPS-induced myocardial injury. Therefore, the aim of this study is to explore the underlying mechanism by which nucleolin regulates autophagy to protect against LPS-induced myocardial injury in vivo and in vitro. In our study, we found that nucleolin could bind to PGC-1α, and we predicted that this interaction could promote autophagy and played a role in inhibiting cardiomyocyte apoptosis. Downregulation of nucleolin in H9C2 cells resulted in decreased autophagy and increased cell apoptosis during LPS-induced myocardial injury, while up-regulation of PGC-1α had the opposite protective effect. Up-regulation of nucleolin expression in cardiomyocytes could increase the level of autophagy during LPS-induced myocardial injury. In contrast, interference with PGC-1α expression resulted in a decrease in the protective effect of nucleolin, leading to reduced autophagy and thus increasing apoptosis. By using tandem fluorescent-tagged LC3 autophagic flux detection system, we observed autophagic flux and determined that PGC-1α interference could block autophagic lysosomal progression. We further tested our hypothesis in the nucleolin cardiac-specific knockout mice. Finally, we also found that inhibition of autophagy can reduce mitochondrial biogenesis as well as increase apoptosis, which demonstrated the importance of autophagy. Therefore, we can speculate that nucleolin can protect LPS-induced myocardial injury by regulating autophagy, and this protective effect may be mediated by the interaction with PGC-1α, which can positively regulate the ULK1, an autophagy-related protein. Our study provides a new clue for the cardioprotective effect of nucleolin, and may provide new evidence for the treatment of LPS-induced myocardial injury through the regulation of autophagy.
    DOI:  https://doi.org/10.1097/SHK.0000000000002152
  20. Front Cell Neurosci. 2023 ;17 1132114
    mTOR/α-ketoglutarate signaling: impact on brain cell homeostasis under ischemic conditions.
    Iryna Lushnikova, Olha Kostiuchenko, Magdalena Kowalczyk, Galyna Skibo.
      The multifunctional molecules mechanistic target of rapamycin (mTOR) and α-ketoglutarate (αKG) are crucial players in the regulatory mechanisms that maintain cell homeostasis in an ever-changing environment. Cerebral ischemia is associated primarily with oxygen-glucose deficiency (OGD) due to circulatory disorders. Upon exceeding a threshold of resistance to OGD, essential pathways of cellular metabolism can be disrupted, leading to damage of brain cells up to the loss of function and death. This mini-review focuses on the role of mTOR and αKG signaling in the metabolic homeostasis of brain cells under OGD conditions. Integral mechanisms concerning the relative cell resistance to OGD and the molecular basis of αKG-mediated neuroprotection are discussed. The study of molecular events associated with cerebral ischemia and endogenous neuroprotection is relevant for improving the effectiveness of therapeutic strategies.
    Keywords:  autophagy; brain ischemia; homeostasis; mTOR; neuroprotection; α-ketoglutarate (αKG)
    DOI:  https://doi.org/10.3389/fncel.2023.1132114
  21. Mol Metab. 2023 May 26. pii: S2212-8778(23)00078-9. [Epub ahead of print] 101744
    Inactivation of Minar2 in Mice Hyperactivates mTOR Signaling and Results in Obesity.
    Saran Lotfollahzadeh, Chaoshuang Xia, Razie Amraei, Ning Hua, Konstantin V Kandror, Stephen R Farmer, Wenyi Wei, Catherine E Costello, Vipul Chitalia, Nader Rahimi.
      Obesity is a complex disorder and is linked to chronic diseases such as type 2 diabetes. Major intrinsically disordered NOTCH2-associated receptor2 (MINAR2) is an understudied protein with an unknown role in obesity and metabolism. To examine its physiological role in adipocytes, we generated Minar2 knockout (KO) mice and demonstrated that its inactivation results in increased body fat with hypertrophic adipocytes. Minar2 KO mice on a high-fat diet develop obesity and impaired glucose tolerance and metabolism. Mechanistically, Minar2 interacts with raptor, a specific and essential component of mammalian TOR complex 1 (mTORC1) and inhibits mTOR activation. mTOR is hyperactivated in the adipocytes deficient for Minar2 and over-expression of Minar2 in HEK-293 cells inhibited mTOR activation and phosphorylation of mTORC1 substrates, including S6 kinase, and 4E-BP1. These results suggest that Minar2 is a physiological negative regulator of mTORC1 with a key role in obesity and metabolic disorders. Impaired expression of MINAR2 could lead to obesity and obesity-associated diseases.
    Keywords:  Minar2; Raptor; mTOR; mTORC1; obesity
    DOI:  https://doi.org/10.1016/j.molmet.2023.101744
  22. J Alzheimers Dis. 2023 May 20.
    Concomitant Neuronal Tau Deposition and FKBP52 Decrease Is an Early Feature of Different Human and Experimental Tauopathies.
    Geri Meduri, Kevin Guillemeau, Corentin Daguinot, Omar Dounane, Melanie Genet, Luigi Ferrara, Beatrice Chambraud, Etienne Emile Baulieu, Julien Giustiniani.
      BACKGROUND: Pathological tau proteins constitute neurofibrillary tangles that accumulate in tauopathies including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and familial frontotemporal lobar degeneration (FTLD-Tau). We previously showed that the FKBP52 immunophilin interacts functionally with tau and strongly decreases in AD brain neurons in correlation with tau deposition. We also reported that FKBP52 co-localizes with autophagy-lysosomal markers and an early pathological tau isoform in AD neurons, suggesting its involvement in autophagic tau clearance.OBJECTIVE: Our objective was to evaluate if differences in neuronal FKBP52 expression levels and subcellular localization might be detected in AD, PSP, familial FTLD-Tau, and in the hTau-P301 S mouse model compared to controls.
    METHODS: Cell by cell immunohistofluorescence analyses and quantification of FKBP52 were performed on postmortem brain samples of some human tauopathies and on hTau-P301 S mice spinal cords.
    RESULTS: We describe a similar FKBP52 decrease and its localization with early pathological tau forms in the neuronal autophagy-lysosomal pathway in various tauopathies and hTau-P301 S mice. We find that FKBP52 decreases early during the pathologic process as it occurs in rare neurons with tau deposits in the marginally affected frontal cortex region of AD Braak IV brains and in the spinal cord of symptomless 1-month-old hTau-P301 S mice.
    CONCLUSION: As FKBP52 plays a significant role in cellular signaling and conceivably in tau clearance, our data support the idea that the prevention of FKBP52 decrease or the restoration of its normal expression at early pathologic stages might represent a new potential therapeutic approach in tauopathies including AD, familial FTLD-Tau, and PSP.
    Keywords:  Alzheimer’s disease; FK506-binding protein; FKBP52; FTLD-Tau; caspase-cleaved tau; lysosome; progressive supranuclear palsy; tau protein; tauopathy
    DOI:  https://doi.org/10.3233/JAD-230127
  23. Int Immunopharmacol. 2023 May 30. pii: S1567-5769(23)00659-8. [Epub ahead of print]120 110336
    The interrelation of galectins and autophagy.
    Lujuan Zheng, Jing Xia, Pengyu Ge, Yuhan Meng, Weili Li, Mingming Li, Min Wang, Chengcheng Song, Yuying Fan, Yifa Zhou.
      Autophagy is a vital physiological process that maintains intracellular homeostasis by removing damaged organelles and senescent or misfolded molecules. However, excessive autophagy results in cell death and apoptosis, which will lead to a variety of diseases. Galectins are a type of animal lectin that binds to β-galactosides and can bind to the cell surface or extracellular matrix glycans, affecting a variety of immune processes in vivo and being linked to the development of many diseases. In many cases, galectins and autophagy both play important regulatory roles in the cellular life course, yet our understanding of the relationship between them is still incomplete. Galectins and autophagy may share common etiological cofactors for some diseases. Hence, we summarize the relationship between galectins and autophagy, aiming to draw attention to the existence of multiple associations between galectins and autophagy in a variety of physiological and pathological processes, which provide new ideas for etiological diagnosis, drug development, and therapeutic targets for related diseases.
    Keywords:  Autophagy; Gal-1; Gal-3; Gal-8; Gal-9
    DOI:  https://doi.org/10.1016/j.intimp.2023.110336
  24. FEBS Lett. 2023 Jun 01.
    ER-to-Lysosome Associated Degradation (ERLAD) in a Nutshell: mammalian, yeast and plant ER-phagy as induced by misfolded proteins.
    Mikhail Rudinskiy, Maurizio Molinari.
      Conserved catabolic pathways operate to remove aberrant polypeptides from the endoplasmic reticulum (ER), the major biosynthetic organelle of eukaryotic cells. The best known are the ER-associated degradation (ERAD) pathways that control retro-translocation of terminally misfolded proteins across the ER membrane for clearance by the cytoplasmic ubiquitin/proteasome system. In this review, we catalogue folding-defective mammalian, yeast, and plant proteins that fail to engage ERAD machineries. We describe that they rather segregate in ER subdomains that eventually vesiculate. These ER-derived vesicles are captured by double membrane autophagosomes, engulfed by endolysosomes/vacuoles, or fuse with degradative organelles to clear cells from their toxic cargo. These client-specific, mechanistically diverse ER-phagy pathways are grouped under the umbrella term of ER-to-Lysosome-Associated Degradation (ERLAD) for description in this essay.
    Keywords:  ER-Associated Degradation (ERAD); ER-phagy; ER-to-Lysosome-Associated degradation (ERLAD); Lysosome/vacuole; Ubiquitin/proteasome system
    DOI:  https://doi.org/10.1002/1873-3468.14674
  25. J Toxicol Sci. 2023 ;48(6): 355-361
    Proteasome and p62/SQSTM1 are involved in methylmercury toxicity mitigation in mouse embryonic fibroblast cells.
    Yasukazu Takanezawa, Ryosuke Nakamura, Yuka Ohshiro, Shimpei Uraguchi, Masako Kiyono.
      Methylmercury (MeHg), an environmental pollutant, disrupts and impairs cellular function. MeHg binds to various cellular proteins, causing dysfunction and misfolding, which are considered underlying causes of MeHg toxicity. The p62 protein, also termed SQSTM1, is a ubiquitin-binding protein that targets ubiquitinated substrates to undergo autophagy and plays a key role in ameliorating MeHg toxicity. p62 also delivers ubiquitinated substrates to proteasomes. However, the role of these degradation systems in mitigating MeHg toxicity remains unknown. Herein, we explored the impact of the proteasome inhibitor MG132 on MeHg toxicity and examined the toxicity of co-treatment with MG132 and MeHg in p62KO mouse embryonic fibroblasts (MEFs) by analyzing cell viability, immunoblotting, mRNA levels, immunofluorescence, and the mercury content. The proteasome inhibitor MG132 enhanced MeHg-induced cytotoxicity while reducing intracellular mercury levels in MEFs. Co-treatment with MG132 and MeHg markedly increased levels of p62 and ubiquitinated proteins. Furthermore, co-treatment with MG132 and MeHg reduced p62KO MEF viability compared to that of wild-type MEFs. Our findings suggest that the proteasome participates in mitigating MeHg cytotoxicity, while p62 may play an important role in transporting MeHg-induced ubiquitinated proteins to the proteasome, as well as in autophagy. Collectively, these results imply that p62, and proteasome, and autophagy are vital for cytoprotection against MeHg toxicity.
    Keywords:  Cell death; MG132; Methylmercury; Proteasome; p62/SQSTM1
    DOI:  https://doi.org/10.2131/jts.48.355
  26. Front Med (Lausanne). 2023 ;10 1168967
    mTOR-inhibitors and post-transplant diabetes mellitus: a link still debated in kidney transplantation.
    Simona Granata, Silvia Mercuri, Dario Troise, Loreto Gesualdo, Giovanni Stallone, Gianluigi Zaza.
      The mammalian target of rapamycin inhibitors (mTOR-Is, Sirolimus, and Everolimus) are immunosuppressive drugs widely employed in kidney transplantation. Their main mechanism of action includes the inhibition of a serine/threonine kinase with a pivotal role in cellular metabolism and in various eukaryotic biological functions (including proteins and lipids synthesis, autophagy, cell survival, cytoskeleton organization, lipogenesis, and gluconeogenesis). Moreover, as well described, the inhibition of the mTOR pathway may also contribute to the development of the post-transplant diabetes mellitus (PTDM), a major clinical complication that may dramatically impact allograft survival (by accelerating the development of the chronic allograft damage) and increase the risk of severe systemic comorbidities. Several factors may contribute to this condition, but the reduction of the beta-cell mass, the impairment of the insulin secretion and resistance, and the induction of glucose intolerance may play a pivotal role. However, although the results of several in vitro and in animal models, the real impact of mTOR-Is on PTDM is still debated and the entire biological machinery is poorly recognized. Therefore, to better elucidate the impact of the mTOR-Is on the risk of PTDM in kidney transplant recipients and to potentially uncover future research topics (particularly for the clinical translational research), we decided to review the available literature evidence regarding this important clinical association. In our opinion, based on the published reports, we cannot draw any conclusion and PTDM remains a challenge. However, also in this case, the administration of the lowest possible dose of mTOR-I should also be recommended.
    Keywords:  allograft co-morbidities; immunosuppressive drugs; kidney transplantation; mTOR-inhibitor; post-transplant diabetes mellitus
    DOI:  https://doi.org/10.3389/fmed.2023.1168967
  27. Front Nutr. 2023 ;10 1124678
    A metabolic shift toward glycolysis enables cancer cells to maintain survival upon concomitant glutamine deprivation and V-ATPase inhibition.
    Florian Lengauer, Franz Geisslinger, Antje Gabriel, Karin von Schwarzenberg, Angelika M Vollmar, Karin Bartel.
      It is widely known that most cancer cells display an increased reliance on glutaminolysis to sustain proliferation and survival. Combining glutamine deprivation with additional anti-cancer therapies is an intensively investigated approach to increase therapeutic effectiveness. In this study, we examined a combination of glutamine deprivation by starvation or pharmacological tools, with the anti-cancer agent archazolid, an inhibitor of the lysosomal V-ATPase. We show that glutamine deprivation leads to lysosomal acidification and induction of pro-survival autophagy, which could be prevented by archazolid. Surprisingly, a combination of glutamine deprivation with archazolid did not lead to synergistic induction of cell death or reduction in proliferation. Investigating the underlying mechanisms revealed elevated expression and activity of amino acid transporters SLC1A5, SLC38A1 upon starvation, whereas archazolid had no additional effect. Furthermore, we found that the export of lysosomal glutamine derived from exogenous sources plays no role in the phenotype as knock-down of SLC38A7, the lysosomal glutamine exporter, could not increase V-ATPase inhibition-induced cell death or reduce proliferation. Analysis of the cellular metabolic phenotype revealed that glutamine deprivation led to a significant increase in glycolytic activity, indicated by an elevated glycolytic capacity and reserve, when V-ATPase function was inhibited concomitantly. This was confirmed by increased glutamine uptake, augmented lactate production, and an increase in hexokinase activity. Our study, therefore, provides evidence, that glutamine deprivation induces autophagy, which can be prevented by simultaneous inhibition of V-ATPase function. However, this does not lead to a therapeutic benefit, as cells are able to circumvent cell death and growth inhibition by a metabolic shift toward glycolysis.
    Keywords:  V-ATPase; cancer; cancer metabolism; glutamine; glycolysis
    DOI:  https://doi.org/10.3389/fnut.2023.1124678
  28. Nat Commun. 2023 May 29. 14(1): 3086
    Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health.
    James L Daly, Chris M Danson, Philip A Lewis, Lu Zhao, Sara Riccardo, Lucio Di Filippo, Davide Cacchiarelli, Daehoon Lee, Stephen J Cross, Kate J Heesom, Wen-Cheng Xiong, Andrea Ballabio, James R Edgar, Peter J Cullen.
      Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling maturation of the endo-lysosomal network. Retromer dysfunction, which is linked to neurodegenerative disorders including Parkinson's and Alzheimer's diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform an integrated multi-omics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify widespread changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide a holistic view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection.
    DOI:  https://doi.org/10.1038/s41467-023-38719-8
  29. J Lipid Atheroscler. 2023 May;12(2): 132-151
    Unbalanced Redox With Autophagy in Cardiovascular Disease.
    Se-Jin Jeong, Goo Taeg Oh.
      Precise redox balance is essential for the optimum health and physiological function of the human body. Furthermore, an unbalanced redox state is widely believed to be part of numerous diseases, ultimately resulting in death. In this review, we discuss the relationship between redox balance and cardiovascular disease (CVD). In various animal models, excessive oxidative stress has been associated with increased atherosclerotic plaque formation, which is linked to the inflammation status of several cell types. However, various antioxidants can defend against reactive oxidative stress, which is associated with an increased risk of CVD and mortality. The different cardiovascular effects of these antioxidants are presumably due to alterations in the multiple pathways that have been mechanistically linked to accelerated atherosclerotic plaque formation, macrophage activation, and endothelial dysfunction in animal models of CVD, as well as in in vitro cell culture systems. Autophagy is a regulated cell survival mechanism that removes dysfunctional or damaged cellular organelles and recycles the nutrients for the generation of energy. Furthermore, in response to atherogenic stress, such as the generation of reactive oxygen species, oxidized lipids, and inflammatory signaling between cells, autophagy protects against plaque formation. In this review, we characterize the broad spectrum of oxidative stress that influences CVD, summarize the role of autophagy in the content of redox balance-associated pathways in atherosclerosis, and discuss potential therapeutic approaches to target CVD by stimulating autophagy.
    Keywords:  Antioxidants; Autophagy; Cardiovascular disease; Inflammation; Oxidative stress
    DOI:  https://doi.org/10.12997/jla.2023.12.2.132
  30. ACS Cent Sci. 2023 May 24. 9(5): 1025-1034
    Semisynthetic LC3 Probes for Autophagy Pathways Reveal a Noncanonical LC3 Interacting Region Motif Crucial for the Enzymatic Activity of Human ATG3.
    Jakob Farnung, Matthias Muhar, Jin Rui Liang, Kateryna A Tolmachova, Roger M Benoit, Jacob E Corn, Jeffrey W Bode.
      Macroautophagy is one of two major degradation systems in eukaryotic cells. Regulation and control of autophagy are often achieved through the presence of short peptide sequences called LC3 interacting regions (LIR) in autophagy-involved proteins. Using a combination of new protein-derived activity-based probes prepared from recombinant LC3 proteins, along with protein modeling and X-ray crystallography of the ATG3-LIR peptide complex, we identified a noncanonical LIR motif in the human E2 enzyme responsible for LC3 lipidation, ATG3. The LIR motif is present in the flexible region of ATG3 and adopts an uncommon β-sheet structure binding to the backside of LC3. We show that the β-sheet conformation is crucial for its interaction with LC3 and used this insight to design synthetic macrocyclic peptide-binders to ATG3. CRISPR-enabled in cellulo studies provide evidence that LIRATG3 is required for LC3 lipidation and ATG3∼LC3 thioester formation. Removal of LIRATG3 negatively impacts the rate of thioester transfer from ATG7 to ATG3.
    DOI:  https://doi.org/10.1021/acscentsci.3c00009
  31. Oncogene. 2023 Jun 01.
    A bi-steric mTORC1-selective inhibitor overcomes drug resistance in breast cancer.
    Delong Meng, Xin Zhao, Yu Chi Yang, Albertas Navickas, Ciara Helland, Hani Goodarzi, Mallika Singh, Sourav Bandyopadhyay.
      Activation of the PI3K-mTOR pathway is central to breast cancer pathogenesis including resistance to many targeted therapies. The mTOR kinase forms two distinct complexes, mTORC1 and mTORC2, and understanding which is required for the survival of malignant cells has been limited by tools to selectively and completely impair either subcomplex. To address this, we used RMC-6272, a bi-steric molecule with a rapamycin-like moiety linked to an mTOR active-site inhibitor that displays >25-fold selectivity for mTORC1 over mTORC2 substrates. Complete suppression of mTORC1 by RMC-6272 causes apoptosis in ER+/HER2- breast cancer cell lines, particularly in those that harbor mutations in PIK3CA or PTEN, due to inhibition of the rapamycin resistant, mTORC1 substrate 4EBP1 and reduction of the pro-survival protein MCL1. RMC-6272 reduced translation of ribosomal mRNAs, MYC target genes, and components of the CDK4/6 pathway, suggesting enhanced impairment of oncogenic pathways compared to the partial mTORC1 inhibitor everolimus. RMC-6272 maintained efficacy in hormone therapy-resistant acquired cell lines and patient-derived xenografts (PDX), showed increased efficacy in CDK4/6 inhibitor treated acquired resistant cell lines versus their parental counterparts, and was efficacious in a PDX from a patient experiencing resistance to CDK4/6 inhibition. Bi-steric mTORC1-selective inhibition may be effective in overcoming multiple forms of therapy-resistance in ER+ breast cancers.
    DOI:  https://doi.org/10.1038/s41388-023-02737-z
  32. Autophagy. 2023 May 31. 1-3
    An emerging role of non-canonical conjugation of ATG8 proteins in plant response to heat stress.
    Xuanang Zheng, Siyu Chen, Caiji Gao, Jun Zhou.
      Members of the ATG8 (autophagy-related protein 8) protein family can be non-canonically conjugated to single membrane-bound organelles. The exact function of ATG8 on these single membranes remains poorly understood. Recently, using Arabidopsis thaliana as a model system, we identified a non-canonical conjugation of ATG8 pathway involved in the reconstruction of the Golgi apparatus upon heat stress. Short acute heat stress resulted in rapid vesiculation of the Golgi, which was accompanied with the translocation of ATG8 proteins (ATG8a to ATG8i) to the dilated cisternae. More importantly, we found that ATG8 proteins can recruit clathrin to facilitate Golgi reassembly by stimulating the budding of ATG8-positive vesicles from dilated cisternae. These findings provide new insight into one of the possible functions of ATG8 translocation onto single membrane organelles, and will contribute to a better understanding of non-canonical conjugation of ATG8 in eukaryotic cells.Abbreviations: ADS, AIMs docking site; AIM, ATG8-interacting motif; ATG, autophagy-related; CLC2, Clathrin light chain 2; ConcA, concanamycin A; HS, heat stress; PE, phosphatidylethanolamine; PM, plasma membrane; PS, phosphatidylserine; TGN, trans-Golgi network; V-ATPase, vacuolar-type ATPase.
    Keywords:  ATG8; Golgi; clathrin; heat stress; non-canonical autophagy; vacuole
    DOI:  https://doi.org/10.1080/15548627.2023.2219161
  33. Autophagy. 2023 May 30. 1-17
    'Candidatus Liberibacter asiaticus' secretory protein SDE3 inhibits host autophagy to promote Huanglongbing disease in citrus.
    Jinxia Shi, Yinan Gong, Hongwei Shi, Xiaoding Ma, Yuanhong Zhu, Fan Yang, Dan Wang, Yating Fu, Yu Lin, Naiying Yang, Zhuhui Yang, Chunhua Zeng, Weimin Li, Changyong Zhou, Xuefeng Wang, Yongli Qiao.
      Antimicrobial acroautophagy/autophagy plays a vital role in degrading intracellular pathogens or microbial molecules in host-microbe interactions. However, microbes evolved various mechanisms to hijack or modulate autophagy to escape elimination. Vector-transmitted phloem-limited bacteria, Candidatus Liberibacter (Ca. Liberibacter) species, cause Huanglongbing (HLB), one of the most catastrophic citrus diseases worldwide, yet contributions of autophagy to HLB disease proliferation remain poorly defined. Here, we report the identification of a virulence effector in "Ca. Liberibacter asiaticus" (Las), SDE3, which is highly conserved among the "Ca. Liberibacter". SDE3 expression not only promotes the disease development of HLB and canker in sweet orange (Citrus sinensis) plants but also facilitates Phytophthora and viral infections in Arabidopsis, and Nicotiana benthamiana (N. benthamiana). SDE3 directly associates with citrus cytosolic glyceraldehyde-3-phosphate dehydrogenases (CsGAPCs), which negatively regulates plant immunity. Overexpression of CsGAPCs and SDE3 significantly inhibits autophagy in citrus, Arabidopsis, and N. benthamiana. Intriguingly, SDE3 undermines autophagy-mediated immunity by the specific degradation of CsATG8 family proteins in a CsGAPC1-dependent manner. CsATG8 degradation is largely rescued by treatment with an inhibitor of the late autophagic pathway, E64d. Furthermore, ectopic expression of CsATG8s enhances Phytophthora resistance. Collectively, these results suggest that SDE3-CsGAPC interactions modulate CsATG8-mediated autophagy to enhance Las progression in citrus.Abbreviations: ACP: asian citrus psyllid; ACD2: ACCELERATED CELL DEATH 2; ATG: autophagy related; Ca. Liberibacter: Candidatus Liberibacter; CaMV: cauliflower mosaic virus; CMV: cucumber mosaic virus; Cs: Citrus sinensis; EV: empty vector; GAPC: cytosolic glyceraldehyde-3-phosphate dehydrogenase; HLB: huanglongbing; H2O2: hydrogen peroxide; Las: liberibacter asiaticus; Laf: liberibacter africanus; Lam: liberibacter americanus; Pst: Pseudomonas syringae pv. tomato; PVX: potato virus X; ROS: reactive oxygen species; SDE3: sec-delivered effector 3; TEM: transmission electron microscopy; VIVE : virus-induced virulence effector; WT: wild-type; Xcc: Xanthomonas citri subsp. citri.
    Keywords:  ATG8; E64D; GAPC; effector; plant immunity
    DOI:  https://doi.org/10.1080/15548627.2023.2213040
  34. Front Immunol. 2023 ;14 1155421
    B cell activation via immunometabolism in systemic lupus erythematosus.
    Shigeru Iwata, Maiko Hajime Sumikawa, Yoshiya Tanaka.
      Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease involving multiple organs in which B cells perform important functions such as antibody and cytokine production and antigen presentation. B cells are activated and differentiated by the primary B cell receptor, co-stimulatory molecule signals-such as CD40/CD40L-, the Toll-like receptors 7,9, and various cytokine signals. The importance of immunometabolism in the activation, differentiation, and exerting functions of B cells and other immune cells has been widely reported in recent years. However, the regulatory mechanism of immunometabolism in B cells and its involvement in SLE pathogenesis remain elusive. Similarly, the importance of the PI3K-Akt-mTOR signaling pathway, glycolytic system, and oxidative phosphorylation has been demonstrated in the mechanisms of B cell immunometabolic activation, mainly in mouse studies. However, the activation of the mTOR pathway in B cells in patients with SLE, the induction of plasmablast differentiation through metabolic and transcription factor regulation by mTOR, and the involvement of this phenomenon in SLE pathogenesis are unclear. In our studies using activated B cells derived from healthy donors and from patients with SLE, we observed that methionine, an essential amino acid, is important for mTORC1 activation. Further, we observed that splenic tyrosine kinase and mTORC1 activation synergistically induce EZH2 expression and plasmablasts by suppressing BACH2 expression through epigenomic modification. Additionally, we identified another mechanism by which the glutaminolysis-induced enhancement of mitochondrial function promotes plasmablast differentiation in SLE. In this review, we focused on the SLE exacerbation mechanisms related to the activation of immune cells-especially B cells-and immunometabolism and reported the latest findings in the field.
    Keywords:  B-cell; SLE; glutaminolysis; immunometabolism; mitochondria
    DOI:  https://doi.org/10.3389/fimmu.2023.1155421
  35. Proc Natl Acad Sci U S A. 2023 Jun 06. 120(23): e2217869120
    Cellular redox homeostasis maintained by malic enzyme 2 is essential for MYC-driven T cell lymphomagenesis.
    Wei Li, Junjie Kou, Zhenxi Zhang, Haoyue Li, Li Li, Wenjing Du.
      T cell lymphomas (TCLs) are a group of rare and heterogeneous tumors. Although proto-oncogene MYC has an important role in driving T cell lymphomagenesis, whether MYC carries out this function remains poorly understood. Here, we show that malic enzyme 2 (ME2), one of the NADPH-producing enzymes associated with glutamine metabolism, is essential for MYC-driven T cell lymphomagenesis. We establish a CD4-Cre; Myc flox/+transgenic mouse mode, and approximately 90% of these mice develop TCL. Interestingly, knockout of Me2 in Myc transgenic mice almost completely suppresses T cell lymphomagenesis. Mechanistically, by transcriptionally up-regulating ME2, MYC maintains redox homeostasis, thereby increasing its tumorigenicity. Reciprocally, ME2 promotes MYC translation by stimulating mTORC1 activity through adjusting glutamine metabolism. Treatment with rapamycin, an inhibitor of mTORC1, blocks the development of TCL both in vitro and in vivo. Therefore, our findings identify an important role for ME2 in MYC-driven T cell lymphomagenesis and reveal that MYC-ME2 circuit may be an effective target for TCL therapy.
    Keywords:  MYC; T cell lymphomas; glutamine metabolism; malic enzyme 2; redox homeostasis
    DOI:  https://doi.org/10.1073/pnas.2217869120
  36. Methods Mol Biol. 2023 ;2675 97-107
    Methods for Monitoring Mitochondrial Biogenesis and Turnover in Cultured Hepatocytes and Mouse Liver Using MitoTimer Reporter Assay.
    Xiaowen Ma, Wen-Xing Ding.
      Mitochondrial biogenesis and turnover rate are critical to maintain homeostasis of the intracellular mitochondrial pool. Altered mitochondrial biogenesis and mitophagy are closely related to many chronic diseases, highlighting the importance of mitochondrial stasis in various pathological conditions including liver diseases. We describe a detailed protocol for monitoring mitochondrial lifecycle in primary cultured mouse hepatocytes and mouse liver using the dual color fluorescence-based imaging of MitoTimer. Three types of mitochondria were visualized in mouse hepatocytes: green-only mitochondria (newly synthesized mitochondria), red-only mitochondria (old/aging mitochondria), as well as the majority of yellow mitochondria (representing an intermediate stage of mitochondria). The ratio of red/green fluorescence in each cell will be used to track mitochondrial aging. Super-resolution microscopy analysis revealed that majority of mitochondria were spatially heterogeneous with proteins from simultaneous new synthesis, maturation, and turnover in hepatocytes. MitoTimer reporter assay can specifically target to mitochondria and be used to monitor mitochondrial biogenesis and maturation as well as turnover in vitro and in vivo.
    Keywords:  Hepatocytes; Liver; MitoTimer; Mitophagy; Quality control
    DOI:  https://doi.org/10.1007/978-1-0716-3247-5_8
  37. Nat Commun. 2023 May 29. 14(1): 3077
    LC3-associated phagocytosis promotes glial degradation of axon debris after injury in Drosophila models.
    Áron Szabó, Virág Vincze, Aishwarya Sanjay Chhatre, András Jipa, Sarolta Bognár, Katalin Eszter Varga, Poulami Banik, Adél Harmatos-Ürmösi, Lukas J Neukomm, Gábor Juhász.
      Glial engulfment of neuron-derived debris after trauma, during development, and in neurodegenerative diseases supports nervous system functions. However, mechanisms governing the efficiency of debris degradation in glia have remained largely unexplored. Here we show that LC3-associated phagocytosis (LAP), an engulfment pathway assisted by certain autophagy factors, promotes glial phagosome maturation in the Drosophila wing nerve. A LAP-specific subset of autophagy-related genes is required in glia for axon debris clearance, encoding members of the Atg8a (LC3) conjugation system and the Vps34 lipid kinase complex including UVRAG and Rubicon. Phagosomal Rubicon and Atg16 WD40 domain-dependent conjugation of Atg8a mediate proper breakdown of internalized axon fragments, and Rubicon overexpression in glia accelerates debris elimination. Finally, LAP promotes survival following traumatic brain injury. Our results reveal a role of glial LAP in the clearance of neuronal debris in vivo, with potential implications for the recovery of the injured nervous system.
    DOI:  https://doi.org/10.1038/s41467-023-38755-4
  38. Autophagy. 2023 Jun 02.
    Clearance of lipid droplets by chimeric autophagy-tethering compound ameliorates the age-related macular degeneration phenotype in mice lacking APOE.
    Yuelu Zhang, Jiancheng Huang, Yu Liang, Jiaqiu Huang, Yuhua Fu, Ningxie Chen, Boxun Lu, Chen Zhao.
      Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among the elderly, and there is currently no clinical treatment targeting the primary impairment of AMD. The earliest clinical hallmark of AMD is drusen, which are yellowish spots mainly composed of lipid droplets (LDs) accumulated under the retinal pigment epithelium (RPE). However, the potential pathogenic role of this excessive LD accumulation in AMD is yet to be determined, partially due to a lack of chemical tools to manipulate LDs specifically. Here, we employed our recently developed Lipid Droplets·AuTophagy Tethering Compounds (LD∙ATTECs) to degrade LDs and to evaluate its consequence on the AMD-like phenotypes in apoe-/- (apolipoprotein E; B6/JGpt-Apoeem1Cd82/Gpt) mouse model. apoe-/- mice fed with high-fat diet (apoe-/--HFD) exhibited excessive LD accumulation in the retina, particularly with AMD-like phenotypes including RPE degeneration, Bruch's membrane (BrM) thickening, drusen-like deposits, and photoreceptor dysfunction. LD·ATTEC treatment significantly cleared LDs in RPE/choroidal tissues without perturbing lipid synthesis-related proteins and rescued RPE degeneration and photoreceptor dysfunction in apoe-/--HFD mice. This observation implied a causal relationship between LD accumulation and AMD-relevant phenotypes. Mechanically, the apoe-/--HFD mice exhibited elevated oxidative stress and inflammatory signals, both of which were mitigated by the LD·ATTEC treatment. Collectively, this study demonstrated that LD accumulation was a trigger for the process of AMD and provided entry points for the treatment of the initial insult of AMD by degrading LDs.
    Keywords:  B6/JGpt-Apoeem1Cd82/Gpt; age-related macular degeneration; autophagy-tethering compounds; lipid droplets; retinal pigment epithelium
    DOI:  https://doi.org/10.1080/15548627.2023.2220540
  39. J Vis Exp. 2023 05 12.
    Monitoring Stub1-Mediated Pexophagy.
    Bo-Hua Chen, Wei Yuan Yang.
      Mammalian cells can turn over peroxisomes through Stub1-mediated pexophagy. The pathway potentially permits cellular control of the quantity and quality of peroxisomes. During this process, heat shock protein 70 and the ubiquitin E3 ligase, Stub1, translocate onto peroxisomes to be turned over to initiate pexophagy. The Stub1 ligase activity allows the accumulation of ubiquitin and other autophagy-related modules on targeted peroxisomes. Elevating reactive oxygen species (ROS) levels within the peroxisomal lumen can activate Stub1-mediated pexophagy. One can, therefore, use dye-assisted ROS generation to trigger and monitor this pathway. This article outlines the procedures for using two classes of dyes, fluorescent proteins and synthetic fluorophores, to initiate pexophagy within mammalian cell cultures. These dye-assisted ROS generation-based protocols can not only be used to target all the peroxisomes within a cell population globally but can also permit the manipulation of individual peroxisomes within single cells. We also describe how Stub1-mediated pexophagy can be followed using live-cell microscopy.
    DOI:  https://doi.org/10.3791/65010
  40. Biofactors. 2023 May 30.
    Promotion of degradative autophagy by 6-bromoindirubin-3'-oxime attenuates neuropathy.
    Praveen Jaiswar, Mitali Bhate, Avadhesha Surolia.
      Damage to the central or peripheral nervous system causes neuropathic pain. Endoplasmic reticulum (ER) stress plays a role in peripheral neuropathy. Increase in ER stress is seen in diabetic neuropathy. Inducers of ER stress also give rise to peripheral neuropathy. ER stress leads to the formation of autophagosome but as their degradation is also stalled during ER stress accumulation of autophagosomes is seen. Accumulation of autophagosomes has deleterious effects on cells. In the present study, we show that treatment with tunicamycin (TM) (ER stress inducer) in mice leads to peripheral neuropathy as assessed by Von Frey and Hot plate method. Administration of a promoter of autophagy viz. 6-bromoindirubin-3'-oxime (6-BIO) subsequent to ER stress induced by TM exhibits a decrease in peripheral neuropathy. 6-BIO was also effective in reducing diabetic peripheral neuropathy. To understand the type of autophagy activated, SH-SY5Y cells were treated with 6-BIO after TM treatment. Levels of cathepsin D (CTSD), a marker for degradative autophagy was higher in cells treated with 6-BIO after TM treatment compared to only TM-treated SH-SY5Y cells while levels of Rab8A,-a marker for secretory autophagy was reduced. Furthermore, in parallel during ER stress secretory, we noted increased levels of lysozyme in autophagosomes destined for secretion. Cells treated with 6-BIO showed reduction of lysozyme in secretory autophagosomes. This shows that 6-BIO increased degradative autophagy and reduced the secretory autophagy. 6-BIO also reduced the caspase-3 activity in 6-BIO-treated cells. Thus, 6-BIO reduced neuropathy in animals by activating degradative autophagy and reducing the secretory autophagy.
    Keywords:  6-BIO; ER stress; autophagy; diabetic neuropathy; neuropathy
    DOI:  https://doi.org/10.1002/biof.1977
  41. J Clin Invest. 2023 Jun 01. pii: e168554. [Epub ahead of print]133(11):
    Disruption of axonal transport in neurodegeneration.
    Sarah H Berth, Thomas E Lloyd.
      Neurons are markedly compartmentalized, which makes them reliant on axonal transport to maintain their health. Axonal transport is important for anterograde delivery of newly synthesized macromolecules and organelles from the cell body to the synapse and for the retrograde delivery of signaling endosomes and autophagosomes for degradation. Dysregulation of axonal transport occurs early in neurodegenerative diseases and plays a key role in axonal degeneration. Here, we provide an overview of mechanisms for regulation of axonal transport; discuss how these mechanisms are disrupted in neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, hereditary spastic paraplegia, amyotrophic lateral sclerosis, and Charcot-Marie-Tooth disease; and discuss therapeutic approaches targeting axonal transport.
    DOI:  https://doi.org/10.1172/JCI168554
  42. Front Oncol. 2023 ;13 1188371
    The role of TSC2 in breast cancer: a literature review.
    Qiao-Yan Zhu, Zhe-Min He, Wen-Ming Cao, Bei Li.
      TSC2 is a tumor suppressor gene as well as a disease-causing gene for autosomal dominant disorder tuberous sclerosis complex (TSC). Research has found that some tumor tissues have lower TSC2 expression levels than normal tissues. Furthermore, low expression of TSC2 is associated with poor prognosis in breast cancer. TSC2 acts as a convergence point of a complex network of signaling pathways and receives signals from the PI3K, AMPK, MAPK, and WNT pathways. It also regulates cellular metabolism and autophagy through inhibition of a mechanistic target of rapamycin complex, which are processes relevant to the progression, treatment, and prognosis of breast cancer. In-depth study of TSC2 functions provides significant guidance for clinical applications in breast cancer, including improving the treatment efficacy, overcoming drug resistance, and predicting prognosis. In this review, protein structure and biological functions of TSC2 were described and recent advances in TSC2 research in different molecular subtypes of breast cancer were summarized.
    Keywords:  PI3K; TSC2; breast cancer; mTOR; signaling pathways
    DOI:  https://doi.org/10.3389/fonc.2023.1188371
  43. Exp Mol Med. 2023 Jun 01.
    Activation of TLR7-mediated autophagy increases epileptic susceptibility via reduced KIF5A-dependent GABAA receptor transport in a murine model.
    Jing Liu, Pingyang Ke, Haokun Guo, Juan Gu, Yan Liu, Xin Tian, Xuefeng Wang, Fei Xiao.
      The pathophysiological mechanisms underlying epileptogenesis are poorly understood but are considered to actively involve an imbalance between excitatory and inhibitory synaptic transmission. Excessive activation of autophagy, a cellular pathway that leads to the removal of proteins, is known to aggravate the disease. Toll-like receptor (TLR) 7 is an innate immune receptor that regulates autophagy in infectious and noninfectious diseases. However, the relationship between TLR7, autophagy, and synaptic transmission during epileptogenesis remains unclear. We found that TLR7 was activated in neurons in the early stage of epileptogenesis. TLR7 knockout significantly suppressed seizure susceptibility and neuronal excitability. Furthermore, activation of TLR7 induced autophagy and decreased the expression of kinesin family member 5 A (KIF5A), which influenced interactions with γ-aminobutyric acid type A receptor (GABAAR)-associated protein and GABAARβ2/3, thus producing abnormal GABAAR-mediated postsynaptic transmission. Our results indicated that TLR7 is an important factor in regulating epileptogenesis, suggesting a possible therapeutic target for epilepsy.
    DOI:  https://doi.org/10.1038/s12276-023-01000-5
  44. Nat Commun. 2023 May 30. 14(1): 3132
    The mechanisms to dispose of misfolded proteins in the endoplasmic reticulum of adipocytes.
    Shuangcheng Alivia Wu, Chenchen Shen, Xiaoqiong Wei, Xiawei Zhang, Siwen Wang, Xinxin Chen, Mauricio Torres, You Lu, Liangguang Leo Lin, Huilun Helen Wang, Allen H Hunter, Deyu Fang, Shengyi Sun, Magdalena I Ivanova, Yi Lin, Ling Qi.
      Endoplasmic reticulum (ER)-associated degradation (ERAD) and ER-phagy are two principal degradative mechanisms for ER proteins and aggregates, respectively; however, the crosstalk between these two pathways under physiological settings remains unexplored. Using adipocytes as a model system, here we report that SEL1L-HRD1 protein complex of ERAD degrades misfolded ER proteins and limits ER-phagy and that, only when SEL1L-HRD1 ERAD is impaired, the ER becomes fragmented and cleared by ER-phagy. When both are compromised, ER fragments containing misfolded proteins spatially coalesce into a distinct architecture termed Coalescence of ER Fragments (CERFs), consisted of lipoprotein lipase (LPL, a key lipolytic enzyme and an endogenous SEL1L-HRD1 substrate) and certain ER chaperones. CERFs enlarge and become increasingly insoluble with age. Finally, we reconstitute the CERFs through LPL and BiP phase separation in vitro, a process influenced by both redox environment and C-terminal tryptophan loop of LPL. Hence, our findings demonstrate a sequence of events centered around SEL1L-HRD1 ERAD to dispose of misfolded proteins in the ER of adipocytes, highlighting the profound cellular adaptability to misfolded proteins in the ER in vivo.
    DOI:  https://doi.org/10.1038/s41467-023-38690-4
  45. Exp Cell Res. 2023 May 28. pii: S0014-4827(23)00202-1. [Epub ahead of print] 113655
    Oleic acid improves hepatic lipotoxicity injury by alleviating autophagy dysfunction.
    Xiaohong Liu, Xiaoyu Li, Shan Su, Yujia Yuan, Wen Liu, Min Zhu, Qing Zheng, Xin Zeng, Fudong Fu, Yanrong Lu, Younan Chen.
      Lipotoxicity caused by excess free fatty acids, particularly saturated fatty acids (SFAs) such as palmitic acid (PA), is one of the most important pathogenesis of nonalcoholic fatty liver disease (NAFLD). However, unsaturated fatty acids (UFAs), such as oleic acid (OA), are nontoxic and can combat SFA-induced toxicity through alleviation of cell apoptosis, endoplasmic reticulum stress (ER stress) and lipids metabolism disorder. However, whether OA is able to regulate autophagy is largely unknown. So, this study aims to investigate the mechanism underlying OA mediated modulation of autophagy in hepatocytes and mice with NAFLD. In vitro, human hepatoma cell line HepG2 cells, human normal liver cells L-02 and mouse normal liver cells AML12 were treated with palmitic acid (PA)/tunicamycin (TM) or/and OA for 48 h. In vivo, C57/BL6 mice were fed with high fat diet (HFD) to induce NAFLD. And the HFD was partial replaced by olive oil to observe the protective effects of olive oil. We demonstrated that PA/TM impaired cell viability and induced cellular apoptosis in HepG2 cells and L-02 cells. Moreover, PA/TM induced autophagy impairment by reducing the nuclear translocation of transcription factor EB (TFEB) and inhibiting the activity of CTSB. However, OA substantially alleviated PA/TM induced cellular apoptosis and autophagy dysfunction in hepatocytes. Additionally, restoring autophagy function is able to reduce ER stress. Similarly, HFD for 20 weeks successfully established NAFLD model in C57/BL6 mice, and significant autophagy impairment were observed in liver tissues. Noteworthily, 30% replacement of HFD with olive oil had profoundly reversed NAFLD. It significantly impoved steatosis, and reduced autophagy dysfunction, ER stress and apoptosis in liver tissue. Conclusively, these data demonstrated that OA is able to effectively impove autophagy dysfunction under the context of both PA and ER stress inducer induced lipotoxicity, and OA mediated regulation of lysosome dysfunction through TFEB plays an important role, suggesting that the regulation of ER stress-autophagy axis is a critical mechanism in OA driven protection in NAFLD.
    Keywords:  Autophagy; Hepatic lipotoxicity; Nonalcoholic fatty liver disease; Oleic acid
    DOI:  https://doi.org/10.1016/j.yexcr.2023.113655
  46. Front Immunol. 2023 ;14 1165507
    Mitophagy in atherosclerosis: from mechanism to therapy.
    Yanhong Zhang, Jiajun Weng, Luyao Huan, Song Sheng, Fengqin Xu.
      Mitophagy is a type of autophagy that can selectively eliminate damaged and depolarized mitochondria to maintain mitochondrial activity and cellular homeostasis. Several pathways have been found to participate in different steps of mitophagy. Mitophagy plays a significant role in the homeostasis and physiological function of vascular endothelial cells, vascular smooth muscle cells, and macrophages, and is involved in the development of atherosclerosis (AS). At present, many medications and natural chemicals have been shown to alter mitophagy and slow the progression of AS. This review serves as an introduction to the field of mitophagy for researchers interested in targeting this pathway as part of a potential AS management strategy.
    Keywords:  atherosclerosis; different cell types; drug and natural product progression; mechanism progression; mitophagy
    DOI:  https://doi.org/10.3389/fimmu.2023.1165507
  47. Exp Dermatol. 2023 Jun 01.
    The crosstalk between PTEN-induced kinase 1-mediated mitophagy and the inflammasome in the pathogenesis of alopecia areata.
    Jung-Min Shin, Kyung Min Kim, Mi Soo Choi, Sanghyun Park, Dongkyun Hong, Kyung-Eun Jung, Young-Joon Seo, Chang Deok Kim, Hanseul Yang, Young Lee.
      Alopecia areata (AA) is a T-cell-mediated autoimmune disease that causes chronic, relapsing hair loss; however, its precise pathogenesis remains to be elucidated. Recent studies have provided compelling evidence of crosstalk between inflammasomes and mitophagy-a process that contributes to the removal of damaged mitochondria. Our previous studies showed that the NLR family pyrin domain containing 3 (NLRP3) inflammasome is important for eliciting and progressing inflammation in AA. In this study, we detected mitochondrial DNA damage in AA-affected scalp tissues and IFNγ and poly(I:C) treated outer root sheath (ORS) cells. In addition, IFNγ and poly(I:C) treatment increased mitochondrial reactive oxygen species (ROS) levels in ORS cells. Moreover, we showed that mitophagy induction alleviates IFNγ and poly(I:C)-induced NLRP3 inflammasome activation in ORS cells. Lastly, PTEN-induced kinase 1 (PINK1) knockdown increased NLRP3 inflammasome activation, indicating that PINK1-mediated mitophagy plays a critical role in NLRP3 inflammasome activation in ORS cells. This study supports previous studies showing that oxidative stress disrupts immune privilege status and promotes autoimmunity in AA. The results emphasize the significance of crosstalk between mitophagy and inflammasomes in the pathogenesis of AA. Finally, mitophagy factors regulating mitochondrial dysfunction and inhibiting inflammasome activation could be novel therapeutic targets for AA.
    Keywords:  NLRP3; PINK1; alopecia areata; inflammasome; mitophagy; reactive oxygen species
    DOI:  https://doi.org/10.1111/exd.14844
  48. Free Radic Biol Med. 2023 May 25. pii: S0891-5849(23)00436-7. [Epub ahead of print]204 326-336
    The hepatitis B virus promotes the progression of non-alcoholic fatty liver disease through incomplete autophagy.
    Yang Wang, Jiaxi Li, Shanshan Wang, Yuheng Pang, Pengxiang Liu, Bangxiang Xie, Shuangshuang Dou, Tongwang Yang, Xiaoni Liu, Ying Shi, Dexi Chen.
      Hepatitis B virus (HBV) infection is still a serious public health problem. In recent years, with the increasing incidence of chronic hepatitis B (CHB) combined with nonalcoholic fatty liver disease (NAFLD), a more in-depth exploration of the pathogenesis of CHB combined with NAFLD is required. HBV can induce autophagy and use to increase replication. The removal of fat by autophagy, also known as lipophagy, is also currently considered an alternative pathway for lipid metabolism in liver cells. This degradation of autophagy prevents hepatotoxicity and steatosis. However, it is not known whether there is a correlation between HBV-related autophagy and the progression of NAFLD. We explored how HBV affects disease progression in NAFLD should be " and determined whether it is associated with HBV-associated autophagy. In this study, we constructed HBV-TG mouse high-fat diet (HFD) models and controls, and the results showed that the presence of HBV promoted the occurrence of NAFLD. We also demonstrated that HBV promotes lipid droplet accumulation in hepatocytes using HBV-stable expression cell lines HepG2.2.15 and AML12-HBV. In addition, this study also found that exogenous OA supplementation reduced HBV replication. We further studied the mechanism and found that HBV-related autophagy can promote the absorption of liver cells to lipid droplets. It can reduce the decomposition of lipid droplets by inhibiting the function of autophagolysosome, and eventually lead to the accumulation of lipid droplets in hepatocytes. In a word, HBV promotes the progression of NAFLD by increasing lipid accumulation in hepatocytes through incomplete autophagy.
    Keywords:  Hepatitis B virus; High-fat diet; Non-alcoholic fatty liver disease; Oleic acid; autophagy
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2023.05.020
  49. ACS Cent Sci. 2023 May 24. 9(5): 980-991
    A Turn-On Fluorescent Amino Acid Sensor Reveals Chloroquine's Effect on Cellular Amino Acids via Inhibiting Cathepsin L.
    Michael R Smith, Le Zhang, Yizhen Jin, Min Yang, Anusha Bade, Kevin D Gillis, Sadhan Jana, Ramesh Naidu Bypaneni, Timothy E Glass, Hening Lin.
      Maintaining homeostasis of metabolites such as amino acids is critical for cell survival. Dysfunction of nutrient balance can result in human diseases such as diabetes. Much remains to be discovered about how cells transport, store, and utilize amino acids due to limited research tools. Here we developed a novel, pan-amino acid fluorescent turn-on sensor, NS560. It detects 18 of the 20 proteogenic amino acids and can be visualized in mammalian cells. Using NS560, we identified amino acids pools in lysosomes, late endosomes, and surrounding the rough endoplasmic reticulum. Interestingly, we observed amino acid accumulation in large cellular foci after treatment with chloroquine, but not with other autophagy inhibitors. Using a biotinylated photo-cross-linking chloroquine analog and chemical proteomics, we identified Cathepsin L (CTSL) as the chloroquine target leading to the amino acid accumulation phenotype. This study establishes NS560 as a useful tool to study amino acid regulation, identifies new mechanisms of action of chloroquine, and demonstrates the importance of CTSL regulation of lysosomes.
    DOI:  https://doi.org/10.1021/acscentsci.2c01325
  50. Sci Adv. 2023 Jun 02. 9(22): eadg4993
    ULK1-mediated metabolic reprogramming regulates Vps34 lipid kinase activity by its lactylation.
    Mengshu Jia, Xiao Yue, Weixia Sun, Qianjun Zhou, Cheng Chang, Weihua Gong, Jian Feng, Xie Li, Ruonan Zhan, Kemin Mo, Lijuan Zhang, Yajie Qian, Yuying Sun, Aoxue Wang, Yejun Zou, Weicai Chen, Yan Li, Li Huang, Yi Yang, Yuzheng Zhao, Xiawei Cheng.
      Autophagy and glycolysis are highly conserved biological processes involved in both physiological and pathological cellular programs, but the interplay between these processes is poorly understood. Here, we show that the glycolytic enzyme lactate dehydrogenase A (LDHA) is activated upon UNC-51-like kinase 1 (ULK1) activation under nutrient deprivation. Specifically, ULK1 directly interacts with LDHA, phosphorylates serine-196 when nutrients are scarce and promotes lactate production. Lactate connects autophagy and glycolysis through Vps34 lactylation (at lysine-356 and lysine-781), which is mediated by the acyltransferase KAT5/TIP60. Vps34 lactylation enhances the association of Vps34 with Beclin1, Atg14L, and UVRAG, and then increases Vps34 lipid kinase activity. Vps34 lactylation promotes autophagic flux and endolysosomal trafficking. Vps34 lactylation in skeletal muscle during intense exercise maintains muscle cell homeostasis and correlates with cancer progress by inducing cell autophagy. Together, our findings describe autophagy regulation mechanism and then integrate cell autophagy and glycolysis.
    DOI:  https://doi.org/10.1126/sciadv.adg4993
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