bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2024–12–01
sixty-six papers selected by
Viktor Korolchuk, Newcastle University
  1. Structural basis for growth factor and nutrient signal integration on the lysosomal membrane by mTORC1.
  2. Nuclear mTORC1 Live-Cell Sensor nTORSEL Reports Differential Nuclear mTORC1 Activity in Cell Lines.
  3. The nutrient-sensing Rag-GTPase complex in B cells controls humoral immunity via TFEB/TFE3-dependent mitochondrial fitness.
  4. Salmonella-induced cholesterol accumulation in infected macrophages suppresses autophagy via mTORC1 activation.
  5. Transcription Factor Deformed Wings Is an Atg8a-Interacting Protein That Regulates Autophagy.
  6. Impaired chaperone-mediated autophagy leads to abnormal SORT1 (sortilin 1) turnover and CES1-dependent triglyceride hydrolysis.
  7. Functional Role of Hepatitis C Virus NS5A in the Regulation of Autophagy.
  8. Signaling Regulation of FAM134-Dependent ER-Phagy in Cells.
  9. Rapamycin improves satellite cells autophagy and muscle regeneration during hypercapnia.
  10. ER quality control through reticulophagy and protein secretion.
  11. TSC2-mTORC1 axis regulates morphogenesis and neurological function of Gli1+ adult-born dentate granule cells.
  12. 14-3-3 proteins inhibit autophagy by regulating SINAT-mediated proteolysis of ATG6 in Arabidopsis.
  13. Dual Role of Lysosome in Cancer Development and Progression.
  14. Targeting the mTOR-Autophagy Axis: Unveiling Therapeutic Potentials in Osteoporosis.
  15. Autophagy degradation: a promising dimension in drug discovery for neurodegenerative diseases.
  16. Autophagy in cholangiocarcinoma: a comprehensive review about roles and regulatory mechanisms.
  17. RNF144A inhibits autophagy by targeting BECN1 for degradation during L. monocytogenes infection.
  18. Current Understanding of the Role of Autophagy in the Treatment of Myeloid Leukemia.
  19. Selective autophagy impedes KSHV entry after recruiting the membrane damage sensor galectin-8 to virus-containing endosomes.
  20. BCL2 regulates antibacterial autophagy in the intestinal epithelium.
  21. TgATG9 is required for autophagosome biogenesis and maintenance of chronic infection in Toxoplasma gondii.
  22. MicroRNAs regulating autophagy: opportunities in treating neurodegenerative diseases.
  23. Reveal the autophagic degradation of glutaredoxin Grx4 in Schizosaccharomyces pombe.
  24. RICTOR-mediated activation of AKT/mTOR signaling and autophagy inhibition promote osteoarthritis.
  25. Unveiling the physiological impact of ESCRT-dependent autophagosome closure by targeting the VPS37A ubiquitin E2 variant-like domain.
  26. Autophagy in cancer development, immune evasion, and drug resistance.
  27. Mitochondrial Quality Control in Alzheimer's Disease: Insights from Caenorhabditis elegans Models.
  28. Autophagy Involvement in Non-Neoplastic and Neoplastic Endometrial Pathology: The State of the Art with a Focus on Carcinoma.
  29. PIKFYVE inhibition induces endosome- and lysosome-derived vacuole enlargement via ammonium accumulation.
  30. α-Synuclein ubiquitination - functions in proteostasis and development of Lewy bodies.
  31. The Parkinson's disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons.
  32. Mitochondrial dysfunction in Parkinson's disease.
  33. Emerging Role of Ubiquitin Proteasome System and Autophagy in Pediatric Demyelinating Leukodystrophies and Therapeutic Opportunity.
  34. Autophagy in Muscle Regeneration: Mechanisms, Targets, and Therapeutic Perspective.
  35. FAAH Inhibition Counteracts Neuroinflammation via Autophagy Recovery in AD Models.
  36. Advancements in Autophagy Modulation for the Management of Oral Disease: A Focus on Drug Targets and Therapeutics.
  37. Thrombospondin 1 Mediates Autophagy Upon Inhibition of the Rho-Associated Protein Kinase Inhibitor.
  38. Cryo-EM Structure of AAA + ATPase Thorase Reveals Novel Helical Filament Formation.
  39. The effect of high-intensity exercise in temperate and hot ambient conditions on autophagy and the cellular stress response in young and older females.
  40. UBAC2 serves as a reticulophagy receptor to suppress inflammatory responses.
  41. Obesity induced by a high-fat diet changes p62 protein levels in mouse reproductive organs.
  42. Asparagine prevents intestinal stem cell aging via the autophagy-lysosomal pathway.
  43. AMPK regulates Bcl2-L-13-mediated mitophagy induction for cardioprotection.
  44. Protective effects of PIK3CG knockdown against OGD/R-induced neuronal damage via inhibition of autophagy through the AMPK/mTOR pathway.
  45. Small molecule modulation of p75NTR engages the autophagy-lysosomal pathway and reduces huntingtin aggregates in cellular and mouse models of Huntington's disease.
  46. High-density lipoprotein protects normotensive and hypertensive rats against ischemia-reperfusion injury through differential regulation of mTORC1 and mTORC2 signaling.
  47. Schaftoside improves HFpEF through regulation the autophagy-lysosome pathway by allosterically targeting CaMKII-δ.
  48. Critical assessment of LC3/GABARAP ligands used for degrader development and ligandability of LC3/GABARAP binding pockets.
  49. C/EBPβ-dependent autophagy inhibition hinders NK cell function in cancer.
  50. Augmenter of liver regeneration inhibits renal fibrosis during acute kidney injury to chronic kidney disease transition by regulating autophagic flux.
  51. LSD1 Demethylates and Destabilizes Autophagy Protein LC3B in Ovarian Cancer.
  52. The role of ferritinophagy and ferroptosis in Alzheimer's disease.
  53. mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different Manners.
  54. Zinc Ions Facilitate Metabolic Bioenergetic Recovery Post Spinal Cord Injury by Activating Microglial Mitophagy through the STAT3-FOXO3a-SOD2 Pathway.
  55. The Mechanistic Link Between Tau-Driven Proteotoxic Stress and Cellular Senescence in Alzheimer's Disease.
  56. Enhanced Parkin-mediated mitophagy mitigates adverse left ventricular remodelling after myocardial infarction: role of PR-364.
  57. CARMIL1-AA selectively inhibits macropinocytosis while sparing autophagy.
  58. Linking Phosphoinositides to Proteins: A Novel Signaling PIPeline.
  59. S-acylation of PNPLA2/ATGL: a necessity for triacylglycerol lipolysis and lipophagy in hepatocytes.
  60. Piezo1 Modulates Neuronal Autophagy and Apoptosis in Cerebral Ischemia-Reperfusion Injury Through the AMPK-mTOR Signaling Pathway.
  61. The Mitochondrial Integrated Stress Response: A novel approach to anti-aging and pro-longevity.
  62. Quercetin's Potential in MASLD: Investigating the Role of Autophagy and Key Molecular Pathways in Liver Steatosis and Inflammation.
  63. Haploinsufficiency of lysosomal enzyme genes in Alzheimer's disease.
  64. p21Waf1/Cip1 Is a Novel Downstream Target of 40S Ribosomal S6 Kinase 2.
  65. Lipid droplet protein Perilipin 2 is critical for the regulation of insulin secretion through beta cell lipophagy and glucagon expression in pancreatic islets.
  66. Investigating p.Ala1035Val in NPC1: New Cellular Models for Niemann-Pick Type C Disease.
  1. bioRxiv. 2024 Nov 15. pii: 2024.11.15.623810. [Epub ahead of print]
    Structural basis for growth factor and nutrient signal integration on the lysosomal membrane by mTORC1.
    Zhicheng Cui, Alessandra Esposito, Gennaro Napolitano, Andrea Ballabio, James H Hurley.
      Mechanistic target of rapamycin complex 1 (mTORC1), which consists of mTOR, Raptor, and mLST8, receives signaling inputs from growth factor signals and nutrients. These signals are mediated by the Rheb and Rag small GTPases, respectively, which activate mTORC1 on the cytosolic face of the lysosome membrane. We biochemically reconstituted the activation of mTORC1 on membranes by physiological submicromolar concentrations of Rheb, Rags, and Ragulator. We determined the cryo-EM structure and found that Raptor and mTOR directly interact with the membrane at anchor points separated by up to 230 Å across the membrane surface. Full engagement of the membrane anchors is required for maximal activation, which is brought about by alignment of the catalytic residues in the mTOR kinase active site. The observations show at the molecular and atomic scale how converging signals from growth factors and nutrients drive mTORC1 recruitment to and activation on the lysosomal membrane in a three-step process, consisting of (1) Rag-Ragulator-driven recruitment to within ∼100 Å of the lysosomal membrane, (2) Rheb-driven recruitment to within ∼40 Å, and finally (3) direct engagement of mTOR and Raptor with the membrane. The combination of Rheb and membrane engagement leads to full catalytic activation, providing a structural explanation for growth factor and nutrient signal integration at the lysosome.
    DOI:  https://doi.org/10.1101/2024.11.15.623810
  2. Int J Mol Sci. 2024 Nov 12. pii: 12117. [Epub ahead of print]25(22):
    Nuclear mTORC1 Live-Cell Sensor nTORSEL Reports Differential Nuclear mTORC1 Activity in Cell Lines.
    Yifan Wang, Canrong Li, Yingyi Ouyang, Xiaoduo Xie.
      The mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is activated on the surface of lysosomes and phosphorylates substrates at various subcellular locations, including the lysosome, cytosol, and nucleus. However, the signaling and biological functions of nuclear mTORC1 (nmTORC1) are not well understood, primarily due to limited tools for monitoring mTORC1 activity in the nucleus. In this study, we developed a genetically encoded nmTORC1 sensor, termed nTORSEL, based on the phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4EBP1) by mTORC1 within the nucleus. nTORSEL, like its predecessor TORSEL, exhibits a fluorescent punctate pattern in the nucleus through multivalent protein-protein interactions between oligomerized 4EBP1 and eIF4E when nmTORC1 activity is low. We validated nTORSEL using biochemical analyses and imaging techniques across representative cell lines with varying levels of nmTORC1 activity. Notably, nTORSEL specifically detects physiological, pharmacological, and genetic inhibition of nmTORC1 in mouse embryonic fibroblast (MEF) cells but not in HEK293T cells. Therefore, nTORSEL is an effective tool for investigating nuclear mTORC1 signaling in cell lines.
    Keywords:  PI3K-AKT-mTOR pathway; amino acid; fluorescent reporter; live-cell sensor; nuclear mTORC1
    DOI:  https://doi.org/10.3390/ijms252212117
  3. Nat Commun. 2024 Nov 23. 15(1): 10163
    The nutrient-sensing Rag-GTPase complex in B cells controls humoral immunity via TFEB/TFE3-dependent mitochondrial fitness.
    Xingxing Zhu, Yue Wu, Yanfeng Li, Xian Zhou, Jens O Watzlawik, Yin Maggie Chen, Ariel L Raybuck, Daniel D Billadeau, Virginia Smith Shapiro, Wolfdieter Springer, Jie Sun, Mark R Boothby, Hu Zeng.
      Germinal center (GC) formation, which is an integrant part of humoral immunity, involves energy-consuming metabolic reprogramming. Rag-GTPases are known to signal amino acid availability to cellular pathways that regulate nutrient distribution such as the mechanistic target of rapamycin complex 1 (mTORC1) pathway and the transcription factors TFEB and TFE3. However, the contribution of these factors to humoral immunity remains undefined. Here, we show that B cell-intrinsic Rag-GTPases are critical for the development and activation of B cells. RagA/RagB deficient B cells fail to form GCs, produce antibodies, and to generate plasmablasts during both T-dependent (TD) and T-independent (TI) humoral immune responses. Deletion of RagA/RagB in GC B cells leads to abnormal dark zone (DZ) to light zone (LZ) ratio and reduced affinity maturation. Mechanistically, the Rag-GTPase complex constrains TFEB/TFE3 activity to prevent mitophagy dysregulation and maintain mitochondrial fitness in B cells, which are independent of canonical mTORC1 activation. TFEB/TFE3 deletion restores B cell development, GC formation in Peyer's patches and TI humoral immunity, but not TD humoral immunity in the absence of Rag-GTPases. Collectively, our data establish the Rag GTPase-TFEB/TFE3 pathway as a likely mTORC1 independent mechanism to coordinating nutrient sensing and mitochondrial metabolism in B cells.
    DOI:  https://doi.org/10.1038/s41467-024-54344-5
  4. Mol Biol Cell. 2024 Nov 27. mbcE24060283
    Salmonella-induced cholesterol accumulation in infected macrophages suppresses autophagy via mTORC1 activation.
    Holly M Torsilieri, Clint M Upchurch, Norbert Leitinger, James E Casanova.
      Salmonella enterica serovar Typhimurium is a Gram-negative bacillus that infects the host intestinal epithelium and resident macrophages. Many intracellular pathogens induce an autophagic response in host cells but have evolved mechanisms to subvert that response. Autophagy is closely linked to cellular cholesterol levels; mTORC1 senses increased cholesterol in lysosomal membranes, leading to its hyperactivity and suppression of autophagy. Previous studies indicate that Salmonella infection induces dramatic accumulation of cholesterol in macrophages, a fraction of which localizes to Salmonella containing vacuoles (SCVs). We previously reported that the bacterial effector protein SseJ triggers cholesterol accumulation through a signaling cascade involving Focal Adhesion Kinase (FAK) and Akt. Here we show that mTORC1 is recruited to SCVs and is hyperactivated in a cholesterol-dependent manner. If cholesterol accumulation is prevented pharmacologically or through mutation of sseJ, autophagy is induced and bacterial survival is attenuated. Notably, the host lipid transfer protein OSBP is also recruited to SCVs and its activity is necessary for both cholesterol transfer to SCVs and mTORC1 activation during infection. Finally, lipidomic analysis of Salmonella-infected macrophages revealed new insights into how Salmonella may manipulate lipid homeostasis to benefit its survival. We propose that S. Typhimurium induces cholesterol accumulation through SseJ to activate mTORC1, preventing autophagic clearance of bacteria. [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E24-06-0283
  5. Cells. 2024 Nov 17. pii: 1897. [Epub ahead of print]13(22):
    Transcription Factor Deformed Wings Is an Atg8a-Interacting Protein That Regulates Autophagy.
    Marta Kołodziej, Panagiotis Tsapras, Alexander D Cameron, Ioannis P Nezis.
      LC3 (microtubule-associated protein 1 light chain 3, called Atg8 in yeast and Drosophila) is one of the most well-studied autophagy-related proteins. LC3 controls the selectivity of autophagic degradation by interacting with LIR (LC3-interacting region) motifs also known as AIM (Atg8-interacting motifs) on selective autophagy receptors that carry cargo for degradation. Although the function of Atg8 family proteins is primarily cytoplasmic, they are also enriched in the nucleus. Despite the accumulating evidence indicating the presence of Atg8 proteins in the nucleus, the mechanisms by which they are targeted to the nucleus, their interactions with nuclear components, and their nuclear role in remain poorly understood. Here, we used yeast two-hybrid screening, and we identified transcription factor Deformed wings (Dwg) as an Atg8a-interacting protein in Drosophila. Dwg-Atg8a interaction is LIR motif-dependent. We have created Dwg Y129A/I132A LIR mutant flies and shown that they exhibit elevated autophagy, improved resistance to oxidative stress, and starvation. Our results provide novel insights into the transcriptional regulation of autophagy in Drosophila.
    Keywords:  LIR motif; autophagy; transcription factors
    DOI:  https://doi.org/10.3390/cells13221897
  6. Autophagy. 2024 Nov 29.
    Impaired chaperone-mediated autophagy leads to abnormal SORT1 (sortilin 1) turnover and CES1-dependent triglyceride hydrolysis.
    You-Jin Choi, Yoon Ah Nam, Ji Ye Hyun, Jihyeon Yu, Yewon Mun, Sung Ho Yun, Wonseok Lee, Cheon Jun Park, Byung Woo Han, Byung-Hoon Lee.
      SORT1 (sortilin 1), a member of the the Vps10 (vacuolar protein sorting 10) family, is involved in hepatic lipid metabolism by regulating very low-density lipoprotein (VLDL) secretion and facilitating the lysosomal degradation of CES1 (carboxylesterase 1), crucial for triglyceride (TG) breakdown in the liver. This study explores whether SORT1 is targeted for degradation by chaperone-mediated autophagy (CMA), a selective protein degradation pathway that directs proteins containing KFERQ-like motifs to lysosomes via LAMP2A (lysosomal-associated membrane protein 2A). Silencing LAMP2A or HSPA8/Hsc70 with siRNA increased cytosolic SORT1 protein levels. Leupeptin treatment induced lysosomal accumulation of SORT1, unaffected by siLAMP2A co-treatment, indicating CMA-dependent degradation. Human SORT1 contains five KFERQ-like motifs (658VVTKQ662, 730VREVK734, 733VKDLK737, 734KDLKK738, and 735DLKKK739), crucial for HSPA8 recognition; mutating any single amino acid within these motifs decreased HSPA8 binding. Furthermore, compromised CMA activity resulted in elevated SORT1-mediated degradation of CES1, contributing to increased lipid accumulation in hepatocytes. Consistent with in vitro findings, LAMP2A knockdown in mice exacerbated high-fructose diet-induced fatty liver, marked by increased SORT1 and decreased CES1 levels. Conversely, LAMP2A overexpression promoted SORT1 degradation and CES1D accumulation, counteracting fasting-induced CES1D suppression through CMA activation. Our findings reveal that SORT1 is a substrate of CMA, highlighting its crucial role in directing CES1 to lysosomes. Consequently, disrupting CMA-mediated SORT1 degradation significantly affects CES1-dependent TG hydrolysis, thereby affecting hepatic lipid homeostasis.
    Keywords:  Carboxylesterase 1; chaperone-mediated autophagy; nonalcoholic fatty liver; sortilin 1; triglyceride hydorolysis
    DOI:  https://doi.org/10.1080/15548627.2024.2435234
  7. Pathogens. 2024 Nov 08. pii: 980. [Epub ahead of print]13(11):
    Functional Role of Hepatitis C Virus NS5A in the Regulation of Autophagy.
    Po-Yuan Ke, Chau-Ting Yeh.
      Many types of RNA viruses, including the hepatitis C virus (HCV), activate autophagy in infected cells to promote viral growth and counteract the host defense response. Autophagy acts as a catabolic pathway in which unnecessary materials are removed via the lysosome, thus maintaining cellular homeostasis. The HCV non-structural 5A (NS5A) protein is a phosphoprotein required for viral RNA replication, virion assembly, and the determination of interferon (IFN) sensitivity. Recently, increasing evidence has shown that HCV NS5A can induce autophagy to promote mitochondrial turnover and the degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) and diacylglycerol acyltransferase 1 (DGAT1). In this review, we summarize recent progress in understanding the detailed mechanism by which HCV NS5A triggers autophagy, and outline the physiological significance of the balance between host-virus interactions.
    Keywords:  HCV; autophagy; chaperone-mediated autophagy; microautophagy; selective autophagy
    DOI:  https://doi.org/10.3390/pathogens13110980
  8. J Cell Physiol. 2024 Nov 25. e31492
    Signaling Regulation of FAM134-Dependent ER-Phagy in Cells.
    Alessandro Palma, Alessio Reggio.
      The endoplasmic reticulum (ER) is a pivotal organelle responsible for protein and lipid synthesis, calcium homeostasis, and protein quality control within eukaryotic cells. To maintain cellular health, damaged or excess portions of the ER must be selectively degraded via a process known as selective autophagy, or ER-phagy. This specificity is driven by a network of protein receptors and regulatory mechanisms. In this review, we explore the molecular mechanisms governing ER-phagy, with a focus on the FAM134 family of ER-resident ER-phagy receptors. We discuss the molecular pathways and Posttranslational modifications that regulate receptor activation and clustering, and how these modifications fine-tune ER-phagy in response to stress. This review provides a concise understanding of how ER-phagy contributes to cellular homeostasis and highlights the need for further studies in models where ER stress and autophagy are dysregulated.
    Keywords:  ER‐phagy; FAM134B; autophagy; endoplasmic reticulum; ubiquitination
    DOI:  https://doi.org/10.1002/jcp.31492
  9. JCI Insight. 2024 Nov 26. pii: e182842. [Epub ahead of print]
    Rapamycin improves satellite cells autophagy and muscle regeneration during hypercapnia.
    Joseph Balnis, Emily L Jackson, Lisa A Drake, Diane V Singer, Ramon Bossardi Ramos, Harold A Singer, Ariel Jaitovich.
      Both CO2 retention, or hypercapnia, and skeletal muscle dysfunction predict higher mortality in critically ill patients. Mechanistically, muscle injury and reduced myogenesis contribute to critical illness myopathy, and while hypercapnia causes muscle wasting, no research has been conducted on hypercapnia-driven dysfunctional myogenesis in vivo. Autophagy flux regulates myogenesis by supporting muscle stem cell -satellite cell- activation, and previous data suggests that hypercapnia inhibits autophagy. We tested whether hypercapnia worsens satellite cell autophagy flux and myogenic potential, and if autophagy induction reverses these deficits. Satellite cell transplantation and lineage tracing experiments showed that hypercapnia undermines satellite cells activation, replication, and myogenic capacity. Bulk and single cell sequencing analyses indicated that hypercapnia disrupts autophagy, senescence, and other satellite cells programs. Autophagy activation was reduced in hypercapnic cultured myoblasts, and autophagy genetic knockdown phenocopied these changes in vitro. Rapamycin stimulation led to AMPK activation and downregulation of the mTOR pathway, which are both associated with accelerated autophagy flux and cell replication. Moreover, hypercapnic mice receiving rapamycin showed improved satellite cells autophagy flux, activation, replication rate, and post transplantation myogenic capacity. In conclusion, we have shown that hypercapnia interferes with satellite cell activation, autophagy flux and myogenesis, and systemic rapamycin administration improved these outcomes.
    Keywords:  Autophagy; Mouse stem cells; Pulmonology; Respiration
    DOI:  https://doi.org/10.1172/jci.insight.182842
  10. Autophagy. 2024 Nov 27. 1-2
    ER quality control through reticulophagy and protein secretion.
    Cathena Meiling Li, Yong-Keun Jung.
      The endoplasmic reticulum (ER) is the site of multiple cellular events and maintaining its quality control is thus crucial for cell homeostasis. Through a morphology-based gain-of-function screen, we identified the cytosolic protein FKBPL as a regulator of reticulophagy. With multiple protein-binding domains, FKBPL binds to the ER-resident CKAP4, acting as a bridge that connects the ER to the phagophore and facilitating the delivery of ER contents for lysosomal degradation. The FKBPL-CKAP4 axis is essential for both basal and stress-induced reticulophagy. Loss of the FKBPL-CKAP4 interaction attenuates reticulophagy and enhances protein secretion via microvesicle shedding. Here, we propose a dual role for the FKBPL-CKAP4 axis in regulating reticulophagy and protein secretion.
    Keywords:  ER quality control; ER stress; microvesicle shedding; protein secretion; reticulophagy
    DOI:  https://doi.org/10.1080/15548627.2024.2431340
  11. Mol Biol Cell. 2024 Nov 27. mbcE24080366
    TSC2-mTORC1 axis regulates morphogenesis and neurological function of Gli1+ adult-born dentate granule cells.
    Max Kowalczyk, Yu-Ju Lee, Wei-Hsiang Huang.
      Aberrant adult hippocampal neurogenesis is implicated in neurological and mood disorders associated with dysregulation of the mechanistic target of rapamycin (mTOR). Understanding how the mTOR pathway shapes the functional development of different subpopulations of adult-born hippocampal neural stem cells will enable insight into potential therapeutic pathways for these disorders. Here we study how loss of TSC2, a regulator of mTOR pathway and a causal gene for Tuberous Sclerosis Complex (TSC), affects dentate gyrus granule cell (dGC) morphogenesis and hippocampal-dependent function. We found that Tsc2KO mice with TSC2 specifically ablated from Gli1+ adult-born neural stem cells showed neuronal hypertrophy, reduced NEUN expression, increased dendritic arborization, premature cellular senescence, and hypervascularization of the dentate gyrus (DG). Neurologically, Tsc2KO mice showed altered exploratory behavior, impaired spatial learning, abnormal contextual recall, and hypersensitivity to kainic acid-induced seizures. Importantly, genetic reduction of Raptor, essential for mTORC1 signaling, rebalanced mTORC1 signaling and mitigated molecular, cellular, and neurological deficits in Tsc2KO mice. This study uncovered functions of TSC2 in Gli1+ adult-born neural stem cells and highlights RAPTOR as a potential therapeutic target for reversing disease features associated with TSC2 mutations.
    DOI:  https://doi.org/10.1091/mbc.E24-08-0366
  12. BMC Plant Biol. 2024 Nov 29. 24(1): 1148
    14-3-3 proteins inhibit autophagy by regulating SINAT-mediated proteolysis of ATG6 in Arabidopsis.
    Ting Liu, Yuping Zheng, Shunkang Zhou, Yao Wang, Xue Lei, Lijuan Xie, Qingqi Lin, Changqing Chang, Shi Xiao, Rongliang Qiu, Hua Qi.
       BACKGROUND: Autophagy is a conserved cellular process crucial for recycling cytoplasmic components and maintaining cellular homeostasis in eukaryotes. During autophagy, the formation of a protein complex involving AUTOPHAGY-RELATED PROTEIN 6 (ATG6) and phosphatidylinositol 3-kinase is pivotal for recruiting proteins involved in phagophore expansion. However, the intricate molecular mechanism regulating this protein complex in plants remains elusive.
    RESULTS: Here, we aimed to unravel the molecular regulation of autophagy dynamics in Arabidopsis thaliana by investigating the involvement of the scaffold proteins 14-3-3λ and 14-3-3κ in regulating the proteolysis of ATG6. Phenotypic analyses revealed that 14-3-3λ and 14-3-3κ overexpression lines exhibited increased sensitivity to nutrient starvation, premature leaf senescence, and a decrease in starvation-induced autophagic vesicles, resembling the phenotypes of autophagy-defective mutants, suggesting the potential roles of 14-3-3 proteins in regulating autophagy in plants. Furthermore, our investigation unveiled the involvement of 14-3-3λ and 14-3-3κ in the RING finger E3 ligase SINAT1-mediated ubiquitination and destabilization of ATG6 in vivo. We also observed repressed turnover of ATG6 and translocation of GFP-ATG6 to mCherry-ATG8a-labelled punctate structures in the autophagy-defective mutant, which suggesting that ATG6 is probably a target of autophagy. Additionally, 14-3-3λ and 14-3-3κ interacted with Tumor necrosis factor Receptor Associated Factor 1a (TRAF1a) to promote the stability of TRAF1a in vivo under nutrient-rich conditions, suggesting a feedback regulation of autophagy. These findings demonstrate that 14-3-3λ and 14-3-3κ serve as scaffold proteins to regulate autophagy by facilitating the SINAT1-mediated proteolysis of ATG6, involving both direct and indirect mechanisms, in plants.
    CONCLUSIONS: 14-3-3 proteins regulate autophagy by directly or indirectly binding to ATG6 and SINAT1 to promote ubiquitination and degradation of ATG6. 14-3-3 proteins are involved in modulating autophagy dynamics by facilitating SINAT1-mediated ubiquitination and degradation of ATG6.
    Keywords:   Arabidopsis thaliana ; 14-3-3 proteins; Autophagy; Autophagy-related protein 6 (ATG6); Ubiquitination
    DOI:  https://doi.org/10.1186/s12870-024-05854-3
  13. Front Biosci (Landmark Ed). 2024 Nov 20. 29(11): 393
    Dual Role of Lysosome in Cancer Development and Progression.
    Xiao-Qiong Chen, Quan Yang, Wei-Min Chen, Zi-Wei Chen, Guang-Hui Guo, Xuan Zhang, Xiao-Ming Sun, Tao Shen, Fu-Hui Xiao, Yun-Feng Li.
      Lysosomes are essential intracellular catabolic organelles that contain digestive enzymes involved in the degradation and recycle of damaged proteins, organelles, etc. Thus, they play an important role in various biological processes, including autophagy regulation, ion homeostasis, cell death, cell senescence. A myriad of studies has shown that the dysfunction of lysosome is implicated in human aging and various age-related diseases, including cancer. However, what is noteworthy is that the modulation of lysosome-based signaling and degradation has both the cancer-suppressive and cancer-promotive functions in diverse cancers depending on stage, biology, or tumor microenvironment. This dual role limits their application as targets in cancer therapy. In this review, we provide an overview of lysosome and autophagy-lysosomal pathway and outline their critical roles in many cellular processes, including cell death. We highlight the different functions of autophagy-lysosomal pathway in cancer development and progression, underscoring its potential as a target for effective cancer therapies.
    Keywords:  autophagy; cancer; cell death; cell senescence; lysosome
    DOI:  https://doi.org/10.31083/j.fbl2911393
  14. Biomolecules. 2024 Nov 15. pii: 1452. [Epub ahead of print]14(11):
    Targeting the mTOR-Autophagy Axis: Unveiling Therapeutic Potentials in Osteoporosis.
    Rongjin Chen, Chenhui Yang, Fei Yang, Ao Yang, Hefang Xiao, Bo Peng, Changshun Chen, Bin Geng, Yayi Xia.
      Osteoporosis (OP) is a widespread age-related disorder marked by decreased bone density and increased fracture risk, presenting a significant public health challenge. Central to the development and progression of OP is the dysregulation of the mechanistic target of the rapamycin (mTOR)-signaling pathway, which plays a critical role in cellular processes including autophagy, growth, and proliferation. The mTOR-autophagy axis is emerging as a promising therapeutic target due to its regulatory capacity in bone metabolism and homeostasis. This review aims to (1) elucidate the role of mTOR signaling in bone metabolism and its dysregulation in OP, (2) explore the interplay between mTOR and autophagy in the context of bone cell activity, and (3) assess the therapeutic potential of targeting the mTOR pathway with modulators as innovative strategies for OP treatment. By examining the interactions among autophagy, mTOR, and OP, including insights from various types of OP and the impact on different bone cells, this review underscores the complexity of mTOR's role in bone health. Despite advances, significant gaps remain in understanding the detailed mechanisms of mTOR's effects on autophagy and bone cell function, highlighting the need for comprehensive clinical trials to establish the efficacy and safety of mTOR inhibitors in OP management. Future research directions include clarifying mTOR's molecular interactions with bone metabolism and investigating the combined benefits of mTOR modulation with other therapeutic approaches. Addressing these challenges is crucial for developing more effective treatments and improving outcomes for individuals with OP, thereby unveiling the therapeutic potentials of targeting the mTOR-autophagy axis in this prevalent disease.
    Keywords:  autophagy; bone metabolism; mTOR signaling pathway; osteoporosis; therapeutic targeting
    DOI:  https://doi.org/10.3390/biom14111452
  15. Future Med Chem. 2024 Nov 27. 1-3
    Autophagy degradation: a promising dimension in drug discovery for neurodegenerative diseases.
    Shuai-Jiang Liu, Chenxi Cai, Hong-Ping Zhu, Xiang Li, Bo Han.
      
    Keywords:  Neurodegenerative diseases; autophagy; lysosome; organelles; protein degradation
    DOI:  https://doi.org/10.1080/17568919.2024.2431477
  16. Clin Transl Oncol. 2024 Nov 25.
    Autophagy in cholangiocarcinoma: a comprehensive review about roles and regulatory mechanisms.
    Yuxia Yang, Qiuyan Li, Lok Ting Chu, Xiaocong Lin, Helian Chen, Linsong Chen, Jinjing Tang, Tao Zeng.
      The role of autophagy in cholangiocarcinogenesis and its development is intricate. Autophagy has a dual role in cholangiocarcinoma, and understanding the function and mechanism of autophagy in cholangiocarcinoma is pivotal in guiding therapeutic approaches to its treatment in clinical settings. Recent studies have revealed that autophagy is involved in the complex biological behavior of cholangiocarcinoma. In this review, we have summarized the genes and drugs that would promote or inhibit autophagy, leading to change in cellular behaviors of cholangiocarcinoma, including apoptosis, proliferation, invasion and migration, and influence its cellular drug resistance. In addition, we concluded the signaling pathways modulating autophagy in cholangiocarcinoma cells, including PI3K/AKT/mTOR,p38MAPK,AMPK/mTOR,LKB1-AMPK, and AKT/WNK1, and ERK signaling pathways, which subsequently impacting apoptosis, death, migration, invasion, and proliferation. In conclusion, we would like that we can provide ideas for future cholangiocarcinoma treatment by comprehensively summarizing the latest studies on the relationship between autophagy and cholangiocarcinoma, including the factors affecting autophagy and related signaling pathways.
    Keywords:  Apoptosis; Autophagy; Cholangiocarcinoma; Mechanisms; Metastasis; Proliferation
    DOI:  https://doi.org/10.1007/s12094-024-03797-7
  17. Autophagy. 2024 Nov 28. 1-18
    RNF144A inhibits autophagy by targeting BECN1 for degradation during L. monocytogenes infection.
    Bo Yang, Mengyang Shen, Chen Lu, Yi Wang, Xin Zhao, Qunmei Zhang, Xiao Qin, Jinyong Pei, Hui Wang, Jie Wang.
      Listeria monocytogenes (L. monocytogenes, Lm) is widely used in the laboratory as an infection model for the research on pathogenesis and host defense against gram-positive intracellular bacteria. Macroautophagy (called simply "autophagy" hereafter), is important in the host defense against pathogens, such as bacteria, viruses, and parasites. BECN1 plays a pivotal role in the initiation of autophagy and accumulating evidence indicates that post-translational modifications of BECN1 provide multiple strategies for autophagy regulation. In this study, we demonstrated that the RING1-IBR-RING2 (RBR) family member RNF144A (ring finger protein 144A), which was induced by Lm infection, promoted Lm infection in an autophagy-dependent but STING1-independent pattern. rnf144a deficiency in mice protected mice from Lm infection with inhibited innate immune responses. Interestingly, RNF144A decreased Lm-induced autophagosome accumulation. Mechanistic investigation indicated that RNF144A interacted with BECN1 and promoted its K48-linked ubiquitination, leading to the subsequent proteasome-dependent degradation of BECN1 and reduced autophagosome accumulation. Further study demonstrated that RNF144A promoted the ubiquitination of BECN1 at K117 and K427, and these two ubiquitination sites were essential to the role of BECN1 in autophagy and Lm infection. Thus, our findings suggested a new regulator in intracellular bacterial infection and autophagy, which may contribute to our understanding of host defense against intracellular bacterial infection via autophagy.Abbreviations: ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG10: autophagy related 10; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; Baf A1: bafilomycin A1; BECN1: beclin 1; BMDC: bone marrow-derived dendritic cell; BMDM: bone marrow-derived macrophage; CFUs: colony-forming units; CHX: cycloheximide; CQ: chloroquine; CXCL10/IP-10: C-X-C motif chemokine ligand 10; EBSS: Earle's balanced salt solution; ELISA: enzyme-linked immunosorbent assay; IFIT1/ISG56: interferon induced protein with tetratricopeptide repeats 1; IFNB/IFN-β: interferon beta; IL6: interleukin 6; IRF3, interferon regulatory factor 3; Lm: L. monocytogenes; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MOI: multiplicity of infection; PLA: proximity ligation assay; PMA: phorbol myristate acetate; PMA-THP1, PMA-differentiated THP1; PMs: peritoneal macrophages; PTMs: posttranslational modifications; RBR: RING1-IBR-RING2; RNF144A: ring finger protein 144A; STING1, stimulator of interferon response cGAMP interactor 1; TBK1, TANK binding kinase 1; TNF/TNF-α: tumor necrosis factor.
    Keywords:  Autophagosome accumulation; E3 ligase; autophagy-related genes; intracellular bacterial; post-translational modification; ring finger proteins
    DOI:  https://doi.org/10.1080/15548627.2024.2429380
  18. Int J Mol Sci. 2024 Nov 14. pii: 12219. [Epub ahead of print]25(22):
    Current Understanding of the Role of Autophagy in the Treatment of Myeloid Leukemia.
    Yasushi Kubota, Shinya Kimura.
      The most important issues in acute myeloid leukemia are preventing relapse and treating relapse. Although the remission rate has improved to approximately 80%, the 5-year survival rate is only around 30%. The main reasons for this are the high relapse rate and the limited treatment options. In chronic myeloid leukemia patients, when a deep molecular response is achieved for a certain period of time through tyrosine kinase inhibitor treatment, about half of them will reach treatment-free remission, but relapse is still a problem. Therefore, potential therapeutic targets for myeloid leukemias are eagerly awaited. Autophagy suppresses the development of cancer by maintaining cellular homeostasis; however, it also promotes cancer progression by helping cancer cells survive under various metabolic stresses. In addition, autophagy is promoted or suppressed in cancer cells by various genetic mutations. Therefore, the development of therapies that target autophagy is also being actively researched in the field of leukemia. In this review, studies of the role of autophagy in hematopoiesis, leukemogenesis, and myeloid leukemias are presented, and the impact of autophagy regulation on leukemia treatment and the clinical trials of autophagy-related drugs to date is discussed.
    Keywords:  acute myeloid leukemia; chronic myeloid leukemia; clinical trial; cyclodextrin; folate receptor; folic acid; hydroxypropyl-β-cyclodextrin; mitophagy
    DOI:  https://doi.org/10.3390/ijms252212219
  19. Cell Rep. 2024 Nov 26. pii: S2211-1247(24)01370-6. [Epub ahead of print]43(12): 115019
    Selective autophagy impedes KSHV entry after recruiting the membrane damage sensor galectin-8 to virus-containing endosomes.
    Katarina Wendy Schmidt, Charlotte Montespan, Danielle Thompson, Miriam S Lucas, Laure-Anne Ligeon, Harald Wodrich, Alexander S Hahn, Urs F Greber, Christian Münz.
      Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus. Autophagy during KSHV entry has remained unexplored. We show that LC3 lipidation as a hallmark of autophagy is induced shortly after KSHV entry. LC3 co-localizes with KSHV in amphisomes during entry and loss of LC3 lipidation increases infection. Accordingly, NDP52, a receptor of selective autophagy, was recruited to endocytosed viral particles, and its reduction increased KSHV infection. Additionally, virus particles co-localized with the endolysosome damage sensor galectin-8 upon KSHV entry and depletion of galectin-8 promoted KSHV infection. Compared with herpes simplex virus, listeriolysin, adenovirus, and influenza virus, and in contrast to what was previously thought about enveloped viruses, KSHV binding to EphA2 by its envelope protein gH causes endolysosomal membrane damage, akin to non-enveloped viruses and bacteria. Taken together, our study identifies an important anti-viral role for galectin-8, NDP52, and the autophagy machinery at virus-damaged endosomes, restricting KSHV entry by selective autophagy.
    Keywords:  CP: Immunology; CP: Microbiology; KSHV; Kaposi sarcoma-associated herpesvirus; NDP52; endosomal damage; galectin-8; macroautophagy
    DOI:  https://doi.org/10.1016/j.celrep.2024.115019
  20. Proc Natl Acad Sci U S A. 2024 Dec 03. 121(49): e2410205121
    BCL2 regulates antibacterial autophagy in the intestinal epithelium.
    Yun Li, Shai Bel, Jamaal L Benjamin, Kelly A Ruhn, Brian Hassell, Cassie L Behrendt, Zheng Kuang, Lora V Hooper.
      Autophagy is a key innate immune defense mechanism in intestinal epithelial cells. Bacterial invasion of epithelial cells activates antibacterial autophagy through a process that requires the innate immune adaptor protein MYD88, yet how MYD88 signaling connects to the autophagy machinery is unknown. Here, we show that the mouse intestinal pathogen Salmonella enterica Serovar Typhimurium (Salmonella Typhimurium) triggers MYD88 signaling that regulates binding of the anti-autophagy factor B cell lymphoma 2 (BCL2) to the essential autophagy protein Beclin1 (BECN1) in small intestinal enterocytes, a key epithelial cell lineage. Salmonella infection activated the kinase c-Jun N-terminal protein kinase 1 (JNK1) downstream of MYD88. JNK1 induced enterocyte BCL2 phosphorylation, promoting dissociation of the inhibitory BCL2-BECN1 complex and releasing BECN1 to initiate autophagy. Mice with BCL2 phosphorylation site mutations that prevent BCL2-BECN1 dissociation showed increased Salmonella invasion of enterocytes and dissemination to extraintestinal sites. These findings reveal that BCL2 links MYD88 signaling to enterocyte autophagy initiation, providing mechanistic insight into how invading bacteria trigger autophagy in the intestinal epithelium.
    Keywords:  autophagy; enterocyte; innate immunity; intestinal epithelium; pathogenic bacteria
    DOI:  https://doi.org/10.1073/pnas.2410205121
  21. Autophagy Rep. 2024 ;pii: 2418256. [Epub ahead of print]3(1):
    TgATG9 is required for autophagosome biogenesis and maintenance of chronic infection in Toxoplasma gondii.
    Pariyamon Thaprawat, Zhihai Zhang, Eric C Rentchler, Fengrong Wang, Shreya Chalasani, Christopher J Giuliano, Sebastian Lourido, Manlio Di Cristina, Daniel J Klionsky, Vern B Carruthers.
      Toxoplasma gondii is a ubiquitous protozoan parasite that can reside long-term within hosts as intracellular tissue cysts comprised of chronic stage bradyzoites. To perturb chronic infection requires a better understanding of the cellular processes that mediate parasite persistence. Macroautophagy/autophagy is a catabolic and homeostatic pathway that is required for T. gondii chronic infection, although the molecular details of this process remain poorly understood. A key step in autophagy is the initial formation of the phagophore that sequesters cytoplasmic components and matures into a double-membraned autophagosome for delivery of the cargo to a cell's digestive organelle for degradative recycling. While T. gondii appears to have a reduced repertoire of autophagy proteins, it possesses a putative phospholipid scramblase, TgATG9. Through structural modeling and complementation assays, we show herein that TgATG9 can partially rescue bulk autophagy in atg9Δ yeast. We demonstrated the importance of TgATG9 for proper autophagosome dynamics at the subcellular level using three-dimensional live cell lattice light sheet microscopy. Conditional knockdown of TgATG9 in T. gondii after bradyzoite differentiation resulted in markedly reduced parasite viability. Together, our findings provide insights into the molecular dynamics of autophagosome biogenesis within an early-branching eukaryote and pinpoint the indispensable role of autophagy in maintaining T. gondii chronic infection.
    Keywords:  Apicomplexa; autophagy; bradyzoite; conditional knockdown; lattice light sheet microscopy; yeast complementation
    DOI:  https://doi.org/10.1080/27694127.2024.2418256
  22. Front Neurosci. 2024 ;18 1397106
    MicroRNAs regulating autophagy: opportunities in treating neurodegenerative diseases.
    Mahdi Mohseni, Ghazal Behzad, Arezoo Farhadi, Javad Behroozi, Hamraz Mohseni, Behnaz Valipour.
      Neurodegenerative diseases (NDs) are increasingly prevalent in our aging population, imposing significant social and economic burdens. Currently, most ND patients receive only symptomatic treatment due to limited understanding of their underlying causes. Consequently, there is a pressing need for comprehensive research into the pathological mechanisms of NDs by both researchers and clinicians. Autophagy, a cellular mechanism responsible for maintaining cellular equilibrium by removing dysfunctional organelles and misfolded proteins, plays a vital role in cell health and is implicated in various diseases. MicroRNAs (miRNAs) exert influence on autophagy and hold promise for treating these diseases. These small oligonucleotides bind to the 3'-untranslated region (UTR) of target mRNAs, leading to mRNA silencing, degradation, or translation blockade. This review explores recent findings on the regulation of autophagy and autophagy-related genes by different miRNAs in various pathological conditions, including neurodegeneration and inflammation-related diseases. The recognition of miRNAs as key regulators of autophagy in human diseases has spurred investigations into pharmacological compounds and traditional medicines targeting these miRNAs in disease models. This has catalyzed a new wave of therapeutic interventions aimed at modulating autophagy.
    Keywords:  autophagy; autophagy-related genes; microRNA; neurodegenerative diseases; therapeutic interventions
    DOI:  https://doi.org/10.3389/fnins.2024.1397106
  23. Arch Biochem Biophys. 2024 Nov 25. pii: S0003-9861(24)00349-7. [Epub ahead of print] 110227
    Reveal the autophagic degradation of glutaredoxin Grx4 in Schizosaccharomyces pombe.
    Rong Li, Ying Huang.
      Glutaredoxins (Grxs) are small, heat-stable proteins that serve as multi-functional glutathione-dependent thiol transferases. Recent studies have elucidated their role in regulating cellular iron and copper homeostasis. To further elucidate their functions, we employed a combination of bioinformatics and experimental analyses. In S. pombe, five Grxs have been identified. Our study utilized multiple sequence alignment and conserved domain prediction, revealing that Grx4 and its homologs possess a glutaredoxin domain (GRX domain) at the C-terminal and a thioredoxin-like domain (TRX domain) exclusively at the N-terminal. The functional roles of the GRX domain and TRX domain were investigated by constructing strains that express a truncated Grx4 under the regulation of either a constitutive cam1 promoter or its native promoter. Our findings indicated that two Atg8 interacting motifs (AIM), FLKI and FQEI, located within the TRX domain of Grx4, are sufficient to induce autophagic degradation under nitrogen- or iron-starvation conditions, respectively. This represents a significant advancement in understanding TRX domain function within Grxs for the first time. Moreover, the altered expression level of Pcl1 in Δatg5 or Δatg8 strains under iron starvation suggests that autophagy is essential for maintaining iron homeostasis. Further investigations revealed that Grx4 is required for cellular survival and endoplasmic reticulum autophagy (ER-phagy) during DTT treatment, implying a potential correlation between Grxs and the endoplasmic reticulum (ER). Additionally, the loss of Grx4 disrupted nuclear integrity during ER stress, highlighting the versatility and importance of further investigations into the functions of Grx4.
    Keywords:  Autophagy; ER stress; Glutaredoxins; S. pombe; iron homeostasis
    DOI:  https://doi.org/10.1016/j.abb.2024.110227
  24. Int Immunopharmacol. 2024 Nov 25. pii: S1567-5769(24)02203-3. [Epub ahead of print]144 113681
    RICTOR-mediated activation of AKT/mTOR signaling and autophagy inhibition promote osteoarthritis.
    Jingting Xu, Genchun Wang, Yanjun Hou, Kai Sun, Zehang Zheng, Zhou Guo, Liangcai Hou, Xiong Zhang, Zhaoxuan Ruan, Yaping Ye, Fengjing Guo.
      The most common joint disease in the elderly is osteoarthritis (OA), which is characterized by synovitis, cartilage degeneration, and osteophytes, for which there are currently no effective therapies. Chondrocytes, responsible for extracellular matrix (ECM) synthesis and degradation, undergo changes in OA, leading to ECM disruption and disease progression. There is no clear role for the Mechanistic target of rapamycin complex 2 (mTORC2) in OA, but it is known to regulate cellular functions, such as proliferation, metabolism, motility, and apoptosis. The purpose of this study was to determine the molecular mechanism by which Rapamycin-insensitive companion of mTOR (RICTOR), a component of mTORC2, contributes to OA progression. The results demonstrate that IL-1β induces high expression of RICTOR in chondrocytes, promoting downregulation of collagen II expression and impairing autophagy. Silencing RICTOR reverses IL-1β-induced downregulating of collagen II expression and mitochondrial dysfunction. RICTOR inhibits chondrocyte autophagy by inhibiting autophagosome formation and preventing autophagosome-lysosome fusion. Additionally, RICTOR promotes oxidative stress in chondrocytes, leading to disruption of normal mitochondrial structure and disturbance of the articular cartilage microenvironment. This study reveals the potential of RICTOR to treat OA. Specifically, blocking mTORC2 might be an effective treatment strategy.
    Keywords:  Autophagy; Chondrocyte; Osteoarthritis; RICTOR; mTOR
    DOI:  https://doi.org/10.1016/j.intimp.2024.113681
  25. Cell Rep. 2024 Nov 27. pii: S2211-1247(24)01367-6. [Epub ahead of print]43(12): 115016
    Unveiling the physiological impact of ESCRT-dependent autophagosome closure by targeting the VPS37A ubiquitin E2 variant-like domain.
    Kouta Hamamoto, Xinwen Liang, Ayako Ito, Matthew Lanza, Van Bui, Jiawen Zhang, David M Opozda, Tatsuya Hattori, Longgui Chen, David Haddock, Fumiaki Imamura, Hong-Gang Wang, Yoshinori Takahashi.
      Macroautophagy (autophagy) involves the formation of phagophores that mature into autophagosomes. The impact of inhibiting autophagosome closure remains unclear. Here, we report the generation and analysis of mice with impaired autophagosome closure by targeting the ubiquitin E2 variant-like (UEVL) β strands of the endosomal sorting complex required for transport (ESCRT) I subunit VPS37A. The VPS37A UEVL mutation (Δ43-139) impairs bulk autophagic flux without disrupting ESCRT-I complex assembly and endosomal function. Homozygous mutant mice exhibit signs of autophagy impairment, including p62/SQSTM1 and ubiquitinated protein accumulation, neuronal dysfunction, growth retardation, antioxidant gene upregulation, and tissue abnormalities. However, about half of the mutant neonates survive to adulthood without severe liver injury. LC3 proximity proteomics reveals that the VPS37A UEVL mutation leads to active TANK-binding kinase 1 (TBK1) accumulation on phagophores, resulting in increased p62 phosphorylation and inclusion formation. These findings reveal a previously unappreciated role of LC3-conjugated phagophores in facilitating protein aggregation and sequestration, potentially alleviating proteotoxicity.
    Keywords:  CP: Cell biology; LC3-conjugated phagophore; TBK1; UEVL; VPS37A; autophagosome closure; mouse; neonatal survival; p62 phosphorylation; protein aggregation; tissue abnormalities; ubiquitin E2 variant-like domain
    DOI:  https://doi.org/10.1016/j.celrep.2024.115016
  26. Drug Resist Updat. 2024 Nov 15. pii: S1368-7646(24)00128-6. [Epub ahead of print]78 101170
    Autophagy in cancer development, immune evasion, and drug resistance.
    Xuegang Niu, Qi You, Kaijian Hou, Yu Tian, Penghui Wei, Yang Zhu, Bin Gao, Milad Ashrafizadeh, Amir Reza Aref, Alireza Kalbasi, Israel Cañadas, Gautam Sethi, Vinay Tergaonkar, Lingzhi Wang, Yuanxiang Lin, Dezhi Kang, Daniel J Klionsky.
      Macroautophagy/autophagy is a highly conserved evolutionary mechanism involving lysosomes for the degradation of cytoplasmic components including organelles. The constitutive, basal level of autophagy is fundamental for preserving cellular homeostasis; however, alterations in autophagy can cause disease pathogenesis, including cancer. The role of autophagy in cancer is particularly complicated, since this process acts both as a tumor suppressor in precancerous stages but facilitates tumor progression during carcinogenesis and later stages of cancer progression. This shift between anti-tumor and pro-tumor roles may be influenced by genetic and environmental factors modulating key pathways such as those involving autophagy-related proteins, the PI3K-AKT-MTOR axis, and AMPK, which often show dysregulation in tumors. Autophagy regulates various cellular functions, including metabolism of glucose, glutamine, and lipids, cell proliferation, metastasis, and several types of cell death (apoptosis, ferroptosis, necroptosis and immunogenic cell death). These multifaceted roles demonstrate the potential of autophagy to affect DNA damage repair, cell death pathways, proliferation and survival, which are critical in determining cancer cells' response to chemotherapy. Therefore, targeting autophagy pathways presents a promising strategy to combat chemoresistance, as one of the major reasons for the failure in cancer patient treatment. Furthermore, autophagy modulates immune evasion and the function of immune cells such as T cells and dendritic cells, influencing the tumor microenvironment and cancer's biological behavior. However, the therapeutic targeting of autophagy is complex due to its dual role in promoting survival and inducing cell death in cancer cells, highlighting the need for strategies that consider both the beneficial and detrimental effects of autophagy modulation in cancer therapy. Hence, both inducers and inhibitors of autophagy have been introduced for the treatment of cancer. This review emphasizes the intricate interplay between autophagy, tumor biology, and immune responses, offering insights into potential therapeutic approaches that deploy autophagy in the cancer suppression.
    Keywords:  Autophagy; cancer drug resistance; immunotherapy; therapeutic approaches; tumor microenvironment remodeling
    DOI:  https://doi.org/10.1016/j.drup.2024.101170
  27. Antioxidants (Basel). 2024 Nov 01. pii: 1343. [Epub ahead of print]13(11):
    Mitochondrial Quality Control in Alzheimer's Disease: Insights from Caenorhabditis elegans Models.
    Upasana Ganguly, Trae Carroll, Keith Nehrke, Gail V W Johnson.
      Alzheimer's disease (AD) is a complex neurodegenerative disorder that is classically defined by the extracellular deposition of senile plaques rich in amyloid-beta (Aβ) protein and the intracellular accumulation of neurofibrillary tangles (NFTs) that are rich in aberrantly modified tau protein. In addition to aggregative and proteostatic abnormalities, neurons affected by AD also frequently possess dysfunctional mitochondria and disrupted mitochondrial maintenance, such as the inability to eliminate damaged mitochondria via mitophagy. Decades have been spent interrogating the etiopathogenesis of AD, and contributions from model organism research have aided in developing a more fundamental understanding of molecular dysfunction caused by Aβ and toxic tau aggregates. The soil nematode C. elegans is a genetic model organism that has been widely used for interrogating neurodegenerative mechanisms including AD. In this review, we discuss the advantages and limitations of the many C. elegans AD models, with a special focus and discussion on how mitochondrial quality control pathways (namely mitophagy) may contribute to AD development. We also summarize evidence on how targeting mitophagy has been therapeutically beneficial in AD. Lastly, we delineate possible mechanisms that can work alone or in concert to ultimately lead to mitophagy impairment in neurons and may contribute to AD etiopathology.
    Keywords:  Alzheimer’s disease; Caenorhabditis elegans; aging; mitochondria; mitochondria quality control; mitophagy; model organism; neurodegeneration
    DOI:  https://doi.org/10.3390/antiox13111343
  28. Int J Mol Sci. 2024 Nov 12. pii: 12118. [Epub ahead of print]25(22):
    Autophagy Involvement in Non-Neoplastic and Neoplastic Endometrial Pathology: The State of the Art with a Focus on Carcinoma.
    Cristina Pizzimenti, Vincenzo Fiorentino, Chiara Ruggeri, Mariausilia Franchina, Alfredo Ercoli, Giovanni Tuccari, Antonio Ieni.
      Autophagy is a cellular process crucial for maintaining homeostasis by degrading damaged proteins and organelles. It is stimulated in response to stress, recycling nutrients and generating energy for cell survival. In normal endometrium, it suppresses tumorigenesis by preventing toxic accumulation and maintaining cellular homeostasis. It is involved in the cyclic remodelling of the endometrium during the menstrual cycle and contributes to decidualisation for successful pregnancy. Such a process is regulated by various signalling pathways, including PI3K/AKT/mTOR, AMPK/mTOR, and p53. Dysregulation of autophagy has been associated with benign conditions like endometriosis and endometrial hyperplasia but also with malignant neoplasms such as endometrial carcinoma. In fact, it has emerged as a crucial player in endometrial carcinoma biology, exhibiting a dual role in both tumour suppression and tumour promotion, providing nutrients during metabolic stress and allowing cancer cell survival. It also regulates cancer stem cells, metastasis and therapy resistance. Targeting autophagy is therefore a promising therapeutic strategy in endometrial carcinoma and potential for overcoming resistance to standard treatments. The aim of this review is to delve into the intricate details of autophagy's role in endometrial pathology, exploring its mechanisms, signalling pathways and potential therapeutic implications.
    Keywords:  autophagy; endometrial carcinoma; endometriosis; targeted therapies
    DOI:  https://doi.org/10.3390/ijms252212118
  29. J Cell Sci. 2024 Nov 26. pii: jcs.262236. [Epub ahead of print]
    PIKFYVE inhibition induces endosome- and lysosome-derived vacuole enlargement via ammonium accumulation.
    Junsuke Uwada, Hitomi Nakazawa, Takeshi Kiyoi, Takashi Yazawa, Ikunobu Muramatsu, Takayoshi Masuoka.
      FYVE-type zinc finger-containing phosphoinositide kinase (PIKFYVE), that is essential for PtdIns(3,5)P2 production, is an important regulator of lysosomal homeostasis. PIKFYVE dysfunction leads to cytoplasmic vacuolization; however, the underlying mechanism remains unknown. In this study, we explored the cause of vacuole enlargement upon PIKFYVE inhibition in DU145 prostate cancer cells. Enlargement of vacuoles by PIKFYVE inhibition required glutamine and its metabolism by glutaminases. Addition of ammonia, a metabolite of glutamine, was sufficient to enlarge vacuoles via PIKFYVE inhibition. Moreover, PIKFYVE inhibition led to intracellular ammonium accumulation. Endosome-lysosome permeabilization resulted in ammonium leakage from the cells, indicating ammonium accumulation in the endosomes and lysosomes. Ammonium accumulation and vacuole expansion were suppressed by the lysosomal lumen neutralization. It is therefore assumed that PIKFYVE inhibition interferes with the efflux of NH4+, which is protonated NH3 in the lysosomal lumen, leading to osmotic swelling of vacuoles. Notably, glutamine or ammonium is required for PIKFYVE inhibition-induced suppression of lysosomal function and autophagic flux. In conclusion, this study showed that PIKfyve inhibition disrupts lysosomal homeostasis via ammonium accumulation.
    Keywords:  Ammonium; Endosome; Glutamine; Lysosome; PIKFYVE; TRPML1
    DOI:  https://doi.org/10.1242/jcs.262236
  30. Front Mol Neurosci. 2024 ;17 1498459
    α-Synuclein ubiquitination - functions in proteostasis and development of Lewy bodies.
    Hung-Hsiang Ho, Simon S Wing.
      Synucleinopathies are neurodegenerative disorders characterized by the accumulation of α-synuclein containing Lewy bodies. Ubiquitination, a key post-translational modification, has been recognized as a pivotal regulator of α-synuclein's cellular dynamics, influencing its degradation, aggregation, and associated neurotoxicity. This review examines comprehensively the current understanding of α-synuclein ubiquitination and its role in the pathogenesis of synucleinopathies, particularly in the context of Parkinson's disease. We explore the molecular mechanisms responsible for α-synuclein ubiquitination, with a focus on the roles of E3 ligases and deubiquitinases implicated in the degradation process which occurs primarily through the endosomal lysosomal pathway. The review further discusses how the dysregulation of these mechanisms contributes to α-synuclein aggregation and LB formation and offers suggestions for future investigations into the role of α-synuclein ubiquitination. Understanding these processes may shed light on potential therapeutic avenues that can modulate α-synuclein ubiquitination to alleviate its pathological impact in synucleinopathies.
    Keywords:  Lewy body; Parkinson’s disease; autophagy; endosome; lysosome; proteasome; ubiquitin; α-synuclein
    DOI:  https://doi.org/10.3389/fnmol.2024.1498459
  31. Mol Neurodegener. 2024 Nov 25. 19(1): 88
    The Parkinson's disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons.
    Jace Jones-Tabah, Kathy He, Nathan Karpilovsky, Konstantin Senkevich, Ghislaine Deyab, Isabella Pietrantonio, Thomas Goiran, Yuting Cousineau, Daria Nikanorova, Taylor Goldsmith, Esther Del Cid Pellitero, Carol X-Q Chen, Wen Luo, Zhipeng You, Narges Abdian, Jamil Ahmad, Jennifer A Ruskey, Farnaz Asayesh, Dan Spiegelman, Stanley Fahn, Cheryl Waters, Oury Monchi, Yves Dauvilliers, Nicolas Dupré, Irina Miliukhina, Alla Timofeeva, Anton Emelyanov, Sofya Pchelina, Lior Greenbaum, Sharon Hassin-Baer, Roy N Alcalay, Austen Milnerwood, Thomas M Durcan, Ziv Gan-Or, Edward A Fon.
       BACKGROUND: Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson's disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified.
    METHODS: Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines, induced pluripotent stem cell-derived dopaminergic neurons and midbrain organoids and assessed lysosomal activity and the handling of aggregated synuclein fibrils.
    RESULTS: We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein.
    CONCLUSIONS: These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.
    DOI:  https://doi.org/10.1186/s13024-024-00779-9
  32. J Neural Transm (Vienna). 2024 Dec;131(12): 1415-1428
    Mitochondrial dysfunction in Parkinson's disease.
    Nobutaka Hattori, Shigeto Sato.
      The exact cause of nigral cell death in Parkinson's disease (PD) is still unknown. However, research on MPTP-induced experimental parkinsonism has significantly advanced our understanding. In this model, it is widely accepted that mitochondrial respiratory failure is the primary mechanism of cell death. Studies have shown that a toxic metabolite of MPTP inhibits Complex I and alpha-ketoglutarate dehydrogenase activities in mitochondria. Since then, many research groups have focused on mitochondrial dysfunction in PD, identifying deficiencies in Complex I or III in PD patients' brains, skeletal muscle, and platelets. There is some debate about the decline in mitochondrial function in peripheral organs. However, since α-synuclein, the main component protein of Lewy bodies, accumulates in peripheral organs, it is reasonable to consider PD a systemic disease. Additionally, mutant mitochondrial DNA with a 4,977 base pair deletion has been found in the brains of PD patients, suggesting that age-related accumulation of deleted mtDNA is accelerated in the striatum and may contribute to the pathophysiology of PD. While the cause of PD remains unknown, mitochondrial dysfunction is undoubtedly a factor in cell death in PD. In addition, the causative gene for familial PD, parkin (now PRKN), and PTEN-induced putative kinase 1 (PINK1), both gene products are also involved in mitochondrial quality control. Moreover, we have successfully isolated and identified CHCHD2, which is involved in the mitochondrial electron transfer system. There is no doubt that mitochondrial dysfunction contributes to cell death in PD.
    Keywords:   PARK2 ; Autophagy-lysosome pathway; Mitochondria; Mitophagy; Parkin; Ubiquitin-proteasome pathway
    DOI:  https://doi.org/10.1007/s00702-024-02863-2
  33. Cells. 2024 Nov 12. pii: 1873. [Epub ahead of print]13(22):
    Emerging Role of Ubiquitin Proteasome System and Autophagy in Pediatric Demyelinating Leukodystrophies and Therapeutic Opportunity.
    Dar-Shong Lin, Che-Sheng Ho.
      Leukodystrophies represent a heterogeneous group of disorders characterized by specific genetic mutations, metabolic abnormalities, and degeneration of white matter in the central nervous system. These disorders are classified into several categories, with X-linked adrenoleukodystrophy (X-ALD), metachromatic leukodystrophy (MLD), and globoid cell leukodystrophy (GLD) being the most prevalent demyelinating leukodystrophies in pediatric populations. Maintaining proteostasis, which is critical for normal cellular function, relies fundamentally on the ubiquitin-proteasome system (UPS) and autophagy for the degradation of misfolded and damaged proteins. Compelling evidence has highlighted the critical roles of UPS and autophagy dysfunction in the pathogenesis of neurodegenerative diseases. Given the complex and poorly understood pathomechanisms underlying demyelinating leukodystrophies, coupled with the pressing need for effective therapeutic strategies, this review aims to systemically analyze the molecular and pathological evidence linking UPS and autophagy dysfunction to demyelinating leukodystrophies, specifically X-ALD and GLD. Furthermore, we will assess the therapeutic potential of autophagy modulators in the management of X-ALD and GLD, with the objective to inspire further research into therapeutic approaches that target autophagy and UPS pathways. Novel therapies that enhance autophagy and UPS function hold promise as complementary regimens in combination therapies aimed at achieving comprehensive correction of the pathogenic mechanisms in demyelinating leukodystrophies.
    Keywords:  X-linked adrenoleukodystrophy; autophagy; demyelinating leukodystrophies; globoid cell leukodystrophy; ubiquitin–proteasome system; white matter diseases
    DOI:  https://doi.org/10.3390/cells13221873
  34. Int J Mol Sci. 2024 Nov 06. pii: 11901. [Epub ahead of print]25(22):
    Autophagy in Muscle Regeneration: Mechanisms, Targets, and Therapeutic Perspective.
    Yun Chu, Xinrun Yuan, Yiming Tao, Bin Yang, Jinlong Luo.
      Autophagy maintains the stability of eukaryotic cells by degrading unwanted components and recycling nutrients and plays a pivotal role in muscle regeneration by regulating the quiescence, activation, and differentiation of satellite cells. Effective muscle regeneration is vital for maintaining muscle health and homeostasis. However, under certain disease conditions, such as aging, muscle regeneration can fail due to dysfunctional satellite cells. Dysregulated autophagy may limit satellite cell self-renewal, hinder differentiation, and increase susceptibility to apoptosis, thereby impeding muscle regeneration. This review explores the critical role of autophagy in muscle regeneration, emphasizing its interplay with apoptosis and recent advances in autophagy research related to diseases characterized by impaired muscle regeneration. Additionally, we discuss new approaches involving autophagy regulation to promote macrophage polarization, enhancing muscle regeneration. We suggest that utilizing cell therapy and biomaterials to modulate autophagy could be a promising strategy for supporting muscle regeneration. We hope that this review will provide new insights into the treatment of muscle diseases and promote muscle regeneration.
    Keywords:  apoptosis; autophagy; cell therapy; muscle regeneration; satellite cells
    DOI:  https://doi.org/10.3390/ijms252211901
  35. Int J Mol Sci. 2024 Nov 09. pii: 12044. [Epub ahead of print]25(22):
    FAAH Inhibition Counteracts Neuroinflammation via Autophagy Recovery in AD Models.
    Federica Armeli, Roberto Coccurello, Giacomo Giacovazzo, Beatrice Mengoni, Ilaria Paoletti, Sergio Oddi, Mauro Maccarrone, Rita Businaro.
      Endocannabinoids have attracted great interest for their ability to counteract the neuroinflammation underlying Alzheimer's disease (AD). Our study aimed at evaluating whether this activity was also due to a rebalance of autophagic mechanisms in cellular and animal models of AD. We supplied URB597, an inhibitor of Fatty-Acid Amide Hydrolase (FAAH), the degradation enzyme of anandamide, to microglial cultures treated with Aβ25-35, and to Tg2576 transgenic mice, thus increasing the endocannabinoid tone. The addition of URB597 did not alter cell viability and induced microglia polarization toward an anti-inflammatory phenotype, as shown by the modulation of pro- and anti-inflammatory cytokines, as well as M1 and M2 markers; moreover microglia, after URB597 treatment released higher levels of Bdnf and Nrf2, confirming the protective role underlying endocannabinoids increase, as shown by RT-PCR and immunofluorescence experiments. We assessed the number and area of amyloid plaques in animals administered with URB597 compared to untreated animals and the expression of autophagy key markers in the hippocampus and prefrontal cortex from both groups of mice, via immunohistochemistry and ELISA. After URB597 supply, we detected a reduction in the number and areas of amyloid plaques, as detected by Congo Red staining and a reshaping of microglia activation as shown by M1 and M2 markers' modulation. URB597 administration restored autophagy in Tg2576 mice via an increase in BECN1 (Beclin1), ATG7 (Autophagy Related 7), LC3 (light chain 3) and SQSTM1/p62 (sequestrome 1) as well as via the activation of the ULK1 (Unc-51 Like Autophagy Activating Kinase 1) signaling pathway, suggesting that it targets mTOR/ULK1-dependent autophagy pathway. The potential of endocannabinoids to rebalance autophagy machinery may be considered as a new perspective for therapeutic intervention in AD.
    Keywords:  ATG7; Alzheimer’s disease (AD); BDNF; URB597; autophagy; endocannabinoids; fatty acid amide hydrolase; mTOR; microglia; neuroinflammation
    DOI:  https://doi.org/10.3390/ijms252212044
  36. Biomedicines. 2024 Nov 19. pii: 2645. [Epub ahead of print]12(11):
    Advancements in Autophagy Modulation for the Management of Oral Disease: A Focus on Drug Targets and Therapeutics.
    Md Ataur Rahman, Mushfiq Hassan Shaikh, Rajat Das Gupta, Nazeeba Siddika, Muhammad Saad Shaikh, Muhammad Sohail Zafar, Bonglee Kim, Ehsanul Hoque Apu.
      Autophagy is an intrinsic breakdown system that recycles organelles and macromolecules, which influences metabolic pathways, differentiation, and thereby cell survival. Oral health is an essential component of integrated well-being, and it is critical for developing therapeutic interventions to understand the molecular mechanisms underlying the maintenance of oral homeostasis. However, because of the complex dynamic relationship between autophagy and oral health, associated treatment modalities have not yet been well elucidated. Determining how autophagy affects oral health at the molecular level may enhance the understanding of prevention and treatment of targeted oral diseases. At the molecular level, hard and soft oral tissues develop because of complex interactions between epithelial and mesenchymal cells. Aging contributes to the progression of various oral disorders including periodontitis, oral cancer, and periapical lesions during aging. Autophagy levels decrease with age, thus indicating a possible association between autophagy and oral disorders with aging. In this review, we critically review various aspects of autophagy and their significance in the context of various oral diseases including oral cancer, periapical lesions, periodontal conditions, and candidiasis. A better understanding of autophagy and its underlying mechanisms can guide us to develop new preventative and therapeutic strategies for the management of oral diseases.
    Keywords:  autophagy; molecular biology; oral cancer; oral health; periodontitis; preventive mechanism; therapeutic target
    DOI:  https://doi.org/10.3390/biomedicines12112645
  37. Cells. 2024 Nov 18. pii: 1907. [Epub ahead of print]13(22):
    Thrombospondin 1 Mediates Autophagy Upon Inhibition of the Rho-Associated Protein Kinase Inhibitor.
    Kirk Patrick Carreon Catral, Choi-Yee Tse, Wei-Ying Yang, Choi-Ying Ling, Oi-Lam Kwok, Kit-Ying Choy, Da-Qian Lu, Jing-Fang Bian, Thomas Chuen Lam, Dennis Yan-Yin Tse, Samantha Sze-Wan Shan.
      Age-related macular degeneration (AMD) is a degenerative eye disease leading to central vision loss and is characterized by dysregulated autophagy of the retinal pigment epithelium (RPE) layer. Recent studies have suggested that rho-associated protein kinase (ROCK) inhibitors may enhance autophagy in neurodegenerative diseases and promote the survival of RPE cells. This study investigated the effect of ROCK inhibitors on autophagy gene expression and autophagic vacuole formation in a human RPE (ARPE-19) cell line. The highly selective and potent ROCK inhibitor Y-39983 enhanced the expression of autophagy genes in ARPE-19 cells and increased autophagic vacuole formation. A proteomic analysis using mass spectrometry was performed to further characterize the effects of ROCK inhibition at the protein level. Y-39983 downregulated thrombospondin-1 (THBS1), and suppression of THBS1 in ARPE-19 cells resulted in an increase in autophagic vacuole formation. Our data showed that ROCK inhibitor-induced autophagy was mediated by THBS1 downregulation. We identified ROCK and THBS1 as potential novel therapeutic targets in AMD.
    Keywords:  AMD; ROCK inhibitor; Thrombospondin-1; autophagy; signaling pathway
    DOI:  https://doi.org/10.3390/cells13221907
  38. bioRxiv. 2024 Nov 22. pii: 2024.11.22.624887. [Epub ahead of print]
    Cryo-EM Structure of AAA + ATPase Thorase Reveals Novel Helical Filament Formation.
    Mohamad Aasif Dar, Robert Louder, Marisol Cortes, Rong Chen, Qianqian Ma, Mayukh Chakrabarti, George K E Umanah, Ted M Dawson, Valina L Dawson.
      The AAA+ ( A TPases a ssociated with a variety of cellular a ctivities) ATPase, Thorase, also known as ATAD1, plays multiple roles in synaptic plasticity, mitochondrial quality control and mTOR signaling through disassembling protein complexes like AMPAR and mTORC1 in an ATP-dependent manner. The Oligomerization of Thorase is crucial for its disassembly and remodeling functions. We show that wild-type Thorase forms long helical filaments in vitro , dependent on ATP binding but not hydrolysis. We report the Cryogenic Electron Microscopy (cryo-EM) structure of the Thorase filament at a resolution of 4 Å, revealing the dimeric arrangement of the basic repeating unit that is formed through a distinct interface compared to the hexameric MSP1/ATAD1E193Q assembly. Structure-guided mutagenesis confirms the role of critical amino acid residues required for filament formation, oligomerization and disassembly of mTORC1 protein complex. Together, our data reveals a novel filament structure of Thorase and provides critical information that elucidates the mechanism underlying Thorase filament formation and Thorase-mediated disassembly of the mTORC1 complex.
    DOI:  https://doi.org/10.1101/2024.11.22.624887
  39. Am J Physiol Regul Integr Comp Physiol. 2024 Nov 27.
    The effect of high-intensity exercise in temperate and hot ambient conditions on autophagy and the cellular stress response in young and older females.
    James J McCormick, Nicholas Goulet, Kelli E King, Naoto Fujii, Tatsuro Amano, Glen P Kenny.
      The process of autophagy is vital in maintaining normal cellular function, especially during exposure to elevated states of physiological stress associated with exercise and hot ambient temperatures. While prior observations are primarily limited to responses in males, the autophagic response to acute physiological stress in females represents a considerable knowledge gap. Therefore, we assessed autophagy and related pathways of cellular stress in peripheral blood mononuclear cells (PBMCs) from 20 healthy young (n=10, mean [SD]: aged 23 years [3]) and older (n=10, aged 69 years [3]) females in response to 30 minutes of semi-recumbent high-intensity cycling exercise (70% of pre-determined maximal oxygen consumption) in temperate (25°C) and hot (40°C) ambient conditions (15% relative humidity). Mean body temperature (rectal and skin) was measured throughout, while cellular responses were evaluated before and after exercise, including up to 6 hours of seated recovery. Proteins associated with autophagy and related pathways were assessed via Western blot. Mean body temperature was elevated after exercise in both conditions, with significant elevations observed after exercise in the heat (all, p≤0.05). While young females displayed signs of elevated autophagic activity (elevations in LC3-II and beclin-2) in response to exercise performed in both temperate and hot ambient conditions (all, p≤0.05), responses were attenuated in older females. This was accompanied by elevations in chaperone mediated autophagy in young but not older females in response to exercise independent of ambient temperature. Our findings indicate exercise, with and without ambient heat exposure may stimulate the autophagic response in young but not older females.
    Keywords:  apoptosis; chaperone-mediated autophagy; exercise; female; heat shock response; heat stress; inflammation; macroautophagy
    DOI:  https://doi.org/10.1152/ajpregu.00178.2024
  40. Autophagy. 2024 Nov 27. 1-2
    UBAC2 serves as a reticulophagy receptor to suppress inflammatory responses.
    Xing He, Shouheng Jin.
      Reticulophagy selectively degrades fragments of the endoplasmic reticulum (ER) through macroautophagy/autophagy to maintain ER homeostasis. The deficiency of reticulophagy results in the unfolded protein response (UPR), which is a crucial clue to the pathogenesis of inflammatory diseases. However, the detailed mechanism underlying the cross-regulation between reticulophagy and inflammatory diseases remains largely unclear. Recently, we have revealed that UBAC2 (UBA domain containing 2) is essential for controlling ER homeostasis as a novel reticulophagy receptor. MARK2 catalyzes the phosphorylation of UBAC2 at serine (S) 223, hence facilitating the progression of reticulophagy and inhibiting ER stress-induced inflammatory responses.
    Keywords:  Inflammatory responses; MARK2; UBAC2; UPR; reticulophagy
    DOI:  https://doi.org/10.1080/15548627.2024.2431341
  41. J Mol Histol. 2024 Nov 29. 56(1): 13
    Obesity induced by a high-fat diet changes p62 protein levels in mouse reproductive organs.
    Nazlican Bozdemir, Tuba Kablan, Gozde Sukur, Ozgur Cinar, Fatma Uysal.
      Obesity is one of the major risk factor for infertility since it causes decreased quality and quantity of gametes and a disrupted uterine environment which might result in miscarriage, stillbirth, and fetal abnormal growth. Obesity induces oxidative stress which is strongly associated with infertility. The clearing of oxidative stress by autophagy is maintained through the p62/ Keap1/Nrf2 pathway. In this pathway, oxidative stress induces p62 for binding to Keap1, thereby Keap1 cannot bind to the Nrf2 transcription factor. Then, Nrf2 translocates into the nucleus and initiates antioxidant-related gene expression. While p62, bound to Keap1, acts as an adaptor protein between autophagosome and damaged substrates which needs to be degraded for homeostasis. Up to date, obesity is strongly linked to abnormal autophagy activity. However, p62 protein expression has not been investigated in the obese ovary, testis, and uterus in detail. Thus, in the present study, we aimed to evaluate the effects of a high-fat diet (HFD)-induced obesity on p62 protein levels of the ovary, testis, and uterus in mice. Our results demonstrated that the p62 expression level was significantly altered by HFD in uterine glands, epithelium, myometrium, and stroma, and in the ovarian corpus luteum, testicular spermatogonium and spermatocytes.
    Keywords:  Autophagy; Obesity; Reproductive organs; p62
    DOI:  https://doi.org/10.1007/s10735-024-10310-5
  42. Aging Cell. 2024 Nov 25. e14423
    Asparagine prevents intestinal stem cell aging via the autophagy-lysosomal pathway.
    Ting Luo, Liusha Zhao, Chenxi Feng, Jinhua Yan, Yu Yuan, Haiyang Chen.
      The age-associated decline in intestinal stem cell (ISC) function is a key factor in intestinal aging in organisms, resulting in impaired intestinal function and increased susceptibility to age-related diseases. Consequently, it is imperative to develop effective therapeutic strategies to prevent ISC aging and functional decline. In this study, we utilized an aging Drosophila model screening of amino acids and found that asparagine (Asn), a nonessential amino acid in vivo, exhibits its profound anti-aging properties on ISCs. Asn inhibits the hyperproliferation of aging ISCs in Drosophila, maintains intestinal homeostasis, and extends the lifespan of aging flies. Complementarily, Asn promotes the growth and branching of elderly murine intestinal organoids, indicating its anti-aging capacity to enhance ISC function. Mechanistic analyses have revealed that Asn exerts its effects via the activation of the autophagic signaling pathway. In summary, this study has preliminarily explored the potential supportive role of Asn in ameliorating intestinal aging, providing a foundation for further research into therapeutic interventions targeting age-related intestinal dysfunction.
    Keywords:   Drosophila ; aging; asparagine; autophagy; gut; intestinal stem cell
    DOI:  https://doi.org/10.1111/acel.14423
  43. Cell Rep. 2024 Nov 23. pii: S2211-1247(24)01352-4. [Epub ahead of print]43(12): 115001
    AMPK regulates Bcl2-L-13-mediated mitophagy induction for cardioprotection.
    Tomokazu Murakawa, Jumpei Ito, Mara-Camelia Rusu, Manabu Taneike, Shigemiki Omiya, Javier Moncayo-Arlandi, Chiaki Nakanishi, Ryuta Sugihara, Hiroki Nishida, Kentaro Mine, Roland Fleck, Min Zhang, Kazuhiko Nishida, Ajay M Shah, Osamu Yamaguchi, Yasushi Sakata, Kinya Otsu.
      The accumulation of damaged mitochondria in the heart is associated with heart failure. Mitophagy is an autophagic degradation system that specifically targets damaged mitochondria. We have reported previously that Bcl2-like protein 13 (Bcl2-L-13) mediates mitophagy and mitochondrial fission in mammalian cells. However, the in vivo function of Bcl2-L-13 remains unclear. Here, we demonstrate that Bcl2-L-13-deficient mice and knockin mice, in which the phosphorylation site (Ser272) on Bcl2-L-13 was changed to Ala, showed left ventricular dysfunction in response to pressure overload. Attenuation of mitochondrial fission and mitophagy led to impairment of ATP production in these mouse hearts. In addition, we identified AMPKα2 as the kinase responsible for the phosphorylation of Bcl2-L-13 at Ser272. These results indicate that Bcl2-L-13 and its phosphorylation play an important role in maintaining cardiac function. Furthermore, the amplitude of stress-stimulated mitophagic activity could be modulated by AMPKα2.
    Keywords:  Bcl2-L-13; CP: Cell biology; heart failure; mitochondria; mitophagy
    DOI:  https://doi.org/10.1016/j.celrep.2024.115001
  44. Neuroscience. 2024 Nov 25. pii: S0306-4522(24)00653-5. [Epub ahead of print]
    Protective effects of PIK3CG knockdown against OGD/R-induced neuronal damage via inhibition of autophagy through the AMPK/mTOR pathway.
    Luting Lv, Jiayi Qian, Junzhi Sang, Jie Li, Tingting Liu.
       BACKGROUND: Ischemic stroke represents an urgent need for more efficacious therapies owing to modest effectiveness of current treatment.
    METHODS: Download data from stroke patients and collect blood samples from clinical patients to analyze phosphatidylinositol-3 kinase catalytic subunit γ (PIK3CG) expression. To establish a brain damage model, oxygen glucose deprivation/reperfusion (OGD/R) was applied to SH-SY5Y cells. Impact of PIK3CG on AMPK/mTOR autophagy pathway was verified treating cells with AMPK activator metformin. Proliferation and apoptosis were identified by CCK8 and flow cytometry.
    RESULTS: Differential expression analysis and clinical testing show that PIK3CG is highly expressed in patients. Prolonged ODG/R exposure increased PIK3CG levels, supressed cell proliferation, and induced apoptosis. KEGG pathway analysis implicated PIK3CG in autophagy pathway. Knockdown of PIK3CG supressed OGD/R-induced reductions in cell proliferation and OGD/R-induced increases in apoptosis and expressions of Beclin 1 and LC3 II. Following OGD/R, AMPK phosphorylation was upregulated while mammalian target of rapamycin (mTOR) phosphorylation was downregulated, indicating AMPK/mTOR autophagy activation. Knockdown of PIK3CG opposed metformin-induced rises in Beclin 1, LC3 II and apoptosis along with decreases in proliferation.
    CONCLUSION: PIK3CG knockdown protects neuronal cells by inhibiting AMPK/mTOR autophagy pathway and further inhibiting autophagy.
    Keywords:  AMPK/mTOR; Autophagy; Ischemic stroke; OGD/R; PIK3CG
    DOI:  https://doi.org/10.1016/j.neuroscience.2024.11.064
  45. Neurotherapeutics. 2024 Nov 25. pii: S1878-7479(24)00182-X. [Epub ahead of print] e00495
    Small molecule modulation of p75NTR engages the autophagy-lysosomal pathway and reduces huntingtin aggregates in cellular and mouse models of Huntington's disease.
    Danielle A Simmons, Namitha Alexander, Gloria Cao, Ido Rippin, Yarine Lugassy, Hagit Eldar-Finkelman, Frank M Longo.
      Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene encoding a mutant huntingtin (mHtt) protein. mHtt aggregates within neurons causing degeneration primarily in the striatum. There is currently a need for disease-modifying treatments for HD. Many therapeutic studies have focused on lowering mHtt levels by reducing its production or enhancing its clearance. One way to clear mHtt aggregates is to promote autophagy, which is disrupted in HD. Our previous studies showed that the small molecule p75 neurotrophin receptor (p75NTR) ligand, LM11A-31, prevented HD-related neuropathologies and behavioral deficits in multiple HD mouse models. This study investigated whether modulating p75NTR with LM11A-31, would reduce mHtt aggregates via autophagic/lysosomal mechanisms in HD models. LM11A-31 decreased mHtt aggregates in human neuroblastoma SH-SY5Y cells expressing mHtt (exon 1 with 74 CAG repeats) and in the striatum of R6/2 and zQ175dn mouse models of HD. The LM11A-31 associated decrease in mHtt aggregates in vitro was accompanied by increased autophagic/lysosomal activity as indicated by altered levels of relevant markers including p62/SQSTM1 and the lysosomal protease, mature cathepsin D, and increased autophagy flux. In R6/2 and/or zQ175dn striatum, LM11A-31 increased AMPK activation, normalized p62/SQSTM1 and LC3II levels, and enhanced LAMP1 and decreased LC3B association with mHtt. Thus, LM11A-31 reduces mHtt aggregates and may do so via engaging autophagy/lysosomal systems. LM11A-31 has successfully completed a Phase 2a clinical trial for mild-to-moderate Alzheimer's disease and our results here strengthen its potential as a candidate for HD clinical testing.
    Keywords:  Autophagy; Huntingtin inclusions; Neurodegeneration; Neurotrophin; p75(NTR)
    DOI:  https://doi.org/10.1016/j.neurot.2024.e00495
  46. Front Pharmacol. 2024 ;15 1398630
    High-density lipoprotein protects normotensive and hypertensive rats against ischemia-reperfusion injury through differential regulation of mTORC1 and mTORC2 signaling.
    Reham Al-Othman, Aishah Al-Jarallah, Fawzi Babiker.
       Background: High-density lipoprotein (HDL) protects against myocardial ischemia-reperfusion (I/R) injury. Mammalian target of rapamycin complexes 1 and 2 (mTORC1 and mTORC2) play opposing roles in protecting against I/R injury, whereby mTORC1 appears to be detrimental while mTORC2 is protective. However, the role of HDL and mTORC signaling in protecting against I/R in hypertensive rodents is not clearly understood. In this study, we investigated the involvement of mTORC1 and mTORC2 in HDL-mediated protection against myocardial I/R injury in normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR).
    Methods: Hearts from WKY and SHR were subjected to I/R injury using a modified Langendorff system. Hemodynamics data were collected, and infarct size was measured. Rapamycin and JR-AB2-011 were used to test the role of mTORC1 and mTORC2, respectively. MK-2206 was used to test the role of Akt in HDL-mediated cardiac protection. The expression levels and the activation states of mediators of mTORC1 and mTORC2 signaling and myocardial apoptosis were measured by immunoblotting and/or enzyme-linked immunosorbent assay (ELISA).
    Results: HDL protected hearts from WKY and SHR against I/R injury as indicated by significant improvements in cardiac hemodynamics and reduction in infarct size. HDL induced greater protection in WKY compared to SHR. HDL treatment attenuated mTORC1 signaling in WKY by reducing the phosphorylation of P70S6K (mTORC1 substrate). In SHR however, HDL attenuated mTORC1 signaling by reducing the levels of phospho-mTORC1, Rag C (mTORC1 activator), and phospho-PRAS40 (mTORC1 inhibitor). HDL increased the phosphorylation of mTORC2 substrate Akt, specifically the Akt2 isoform in SHR and to a greater extent in WKY. HDL-induced protection was abolished in the presence of Akt antagonist and involved attenuation of GSK, caspases 7 and 8 activation, and cytochrome C release.
    Conclusion: HDL mediates cardiac protection via attenuation of mTORC1, activation of mTORC2-Akt2, and inhibition of myocardial apoptosis. HDL regulates mTORC1 and mTORC2 signaling via distinct mechanisms in normotensive and hypertensive rats. HDL attenuation of mTORC1 and activation of mTORC2-Akt2 signaling could be a mechanism by which HDL protects against myocardial I/R injury in hypertension.
    Keywords:  Akt; HDL; apoptosis; hypertension; ischemia/reperfusion injury; mTOR
    DOI:  https://doi.org/10.3389/fphar.2024.1398630
  47. Redox Biol. 2024 Nov 13. pii: S2213-2317(24)00402-6. [Epub ahead of print]78 103424
    Schaftoside improves HFpEF through regulation the autophagy-lysosome pathway by allosterically targeting CaMKII-δ.
    Haiying Zhang, Yanan Gao, Min Zhang, Zhexin Yuan, Yu Chen, Aiping Wang, Xinxing Liu, Shunchang Ji, Jianfeng Jin, Jingwei Liang, Yan Liu.
      Heart failure with preserved ejection fraction (HFpEF) presents a significant challenge to global healthcare systems due to its complex presentation. HFpEF presents with a normal or near-normal left ventricular ejection fraction, cardiac diastolic dysfunction, and a metabolic profile characterized by impaired inflammation and oxidative stress. There have been few valuable drug targets reported for HFpEF to date. Here, we discovered that schaftoside, an active component from licorice, has a significant protective effect on the cardiac remodeling induced by continuous infusion of angiotensin II (AngII), which leads to the HFpEF phenotype. Mechanistically, schaftoside has demonstrated the ability to ameliorate lysosomal dysfunction in both in vitro and in vivo models, thereby activating autophagy. Bioinformatic analyses based on proteome and phosphoproteome suggested that Ca2+/calmodulin-dependent protein kinase II (CaMKII) was a potential target for schaftoside. It was confirmed that schaftoside allosterically mediated CaMKII-δ conformation via targeting a unique active pocket near the ATP-binding site to inhibit protein phosphorylation and regulate the lysosomal autophagy pathway. Therefore, schaftoside represents the first small molecule identified to inhibit CaMKII-δ activity through allosteric inhibition, providing a novel candidate for alleviating cardiac metabolic imbalance in HFpEF.
    Keywords:  Allosteric inhibition; Autophagy; CaMKII-δ; HFpEF; Schaftoside
    DOI:  https://doi.org/10.1016/j.redox.2024.103424
  48. Nat Commun. 2024 Nov 25. 15(1): 10204
    Critical assessment of LC3/GABARAP ligands used for degrader development and ligandability of LC3/GABARAP binding pockets.
    Martin P Schwalm, Johannes Dopfer, Adarsh Kumar, Francesco A Greco, Nicolas Bauer, Frank Löhr, Jan Heering, Sara Cano-Franco, Severin Lechner, Thomas Hanke, Ivana Jaser, Viktoria Morasch, Christopher Lenz, Daren Fearon, Peter G Marples, Charles W E Tomlinson, Lorene Brunello, Krishna Saxena, Nathan B P Adams, Frank von Delft, Susanne Müller, Alexandra Stolz, Ewgenij Proschak, Bernhard Kuster, Stefan Knapp, Vladimir V Rogov.
      Recent successes in developing small molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the recruitment of targets to LC3/GABARAP, a family of ubiquitin-like proteins that presumably bind to the autophagosome membrane and tether cargo-loaded autophagy receptors into the autophagosome. In this work, we rigorously tested the target engagement of the reported ATTECs to validate the existing LC3/GABARAP ligands. Surprisingly, we were unable to detect interaction with their designated target LC3 using a diversity of biophysical methods. Intrigued by the idea of developing ATTECs, we evaluated the ligandability of LC3/GABARAP by in silico docking and large-scale crystallographic fragment screening. Data based on approximately 1000 crystal structures revealed that most fragments bound to the HP2 but not to the HP1 pocket within the LIR docking site, suggesting a favorable ligandability of HP2. Through this study, we identified diverse validated LC3/GABARAP ligands and fragments as starting points for chemical probe and ATTEC development.
    DOI:  https://doi.org/10.1038/s41467-024-54409-5
  49. Nat Commun. 2024 Nov 28. 15(1): 10343
    C/EBPβ-dependent autophagy inhibition hinders NK cell function in cancer.
    Federica Portale, Roberta Carriero, Marta Iovino, Paolo Kunderfranco, Marta Pandini, Giulia Marelli, Nicolò Morina, Massimo Lazzeri, Paolo Casale, Piergiuseppe Colombo, Gabriele De Simone, Chiara Camisaschi, Enrico Lugli, Gianluca Basso, Javier Cibella, Sergio Marchini, Matteo Bordi, Greta Meregalli, Anna Garbin, Monica Dambra, Elena Magrini, Wiebke Rackwitz, Francesco Cecconi, Alessandro Corbelli, Fabio Fiordaliso, Jiri Eitler, Torsten Tonn, Diletta Di Mitri.
      NK cells are endowed with tumor killing ability, nevertheless most cancers impair NK cell functionality, and cell-based therapies have limited efficacy in solid tumors. How cancers render NK cell dysfunctional is unclear, and overcoming resistance is an important immune-therapeutic aim. Here, we identify autophagy as a central regulator of NK cell anti-tumor function. Analysis of differentially expressed genes in tumor-infiltrating versus non-tumor NK cells from our previously published scRNA-seq data of advanced human prostate cancer shows deregulation of the autophagic pathway in tumor-infiltrating NK cells. We confirm this by flow cytometry in patients and in diverse cancer models in mice. We further demonstrate that exposure of NK cells to cancer deregulates the autophagic process, decreases mitochondrial polarization and impairs effector functions. Mechanistically, CCAAT enhancer binding protein beta (C/EBPβ), downstream of CXCL12-CXCR4 interaction, acts as regulator of NK cell metabolism. Accordingly, inhibition of CXCR4 and C/EBPβ restores NK cell fitness. Finally, genetic and pharmacological activation of autophagy improves NK cell effector and cytotoxic functions, which enables tumour control by NK and CAR-NK cells. In conclusion, our study identifies autophagy as an intracellular checkpoint in NK cells and introduces autophagy regulation as an approach to strengthen NK-cell-based immunotherapies.
    DOI:  https://doi.org/10.1038/s41467-024-54355-2
  50. Arch Biochem Biophys. 2024 Nov 27. pii: S0003-9861(24)00340-0. [Epub ahead of print] 110218
    Augmenter of liver regeneration inhibits renal fibrosis during acute kidney injury to chronic kidney disease transition by regulating autophagic flux.
    Chunxia Wang, Xujia Zeng, Pengfei Yang, Gang Wang, Zheng Zhang, Xiaohui Liao.
       BACKGROUND: Augmenter of liver regeneration (ALR) is believed to protect against acute kidney injury (AKI). The objective of this study was to investigate the mechanisms of ALR in the transition from AKI to chronic kidney disease (CKD).
    METHODS: ALR Conditional Knockout (CKO) mice were bilateral renal artery clamped to induce AKI and CKD. Serum creatinine, blood urea nitrogen, and uric acid were measured to reflect renal function. Renal histology was used to assess kidney damage. Transcriptome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to identify differentially expressed genes (DEGs) and related pathways. TUNEL assay was conducted to assess apoptosis. Polymerase chain reaction and immunohistology were used to analyze autophagy-related factors and kidney fibrosis. AAV9-mRFP-GFP-LC3 was injected to observe autophagy flux.
    RESULTS: In the murine models of AKI and CKD, loss of ALR led to markedly reduced renal function and renal tubular pathology injury. Multiple autophagy-related pathways were found to be enriched in up-regulated DEGs in transcriptome sequencing of ALR CKO and control groups with AKI. Renal fibrosis was evident in ALR CKO mice, with marked suppression of Beclin-1, a factor associated with the initiation phase of autophagy, and ATG5, an important factor in the extension phase of autophagosomes. The marked accumulation of LC3 and SQSTM1/P62, which is associated with the formation of autophagosomes, was also observed, suggesting an impairment of autophagic processes. Correspondingly, the AAV9-mRFP-GFP-LC3 results indicated that decreased ALR led to the accumulation of autophagosomes and impaired autophagic lysosome generation.
    CONCLUSIONS: Collectively, these results suggested that ALR deficiency led to apoptosis and enhanced renal fibrosis by impairing autophagic flux, which in turn led to the transition of AKI to CKD.
    Keywords:  Acute kidney injury; Augmenter of liver regeneration; Autophagy; Chronic kidney disease; Fibrosis
    DOI:  https://doi.org/10.1016/j.abb.2024.110218
  51. Biomolecules. 2024 Oct 29. pii: 1377. [Epub ahead of print]14(11):
    LSD1 Demethylates and Destabilizes Autophagy Protein LC3B in Ovarian Cancer.
    Mingyang Li, Jie Feng, Kangrong Zhao, Ting Huang, Bowen Zhang, Yifan Yang, Aiqin Sun, Qiong Lin, Genbao Shao.
      Autophagy is a complex cellular process that can either promote or inhibit cancer progression and development, depending on the context and molecular regulation involved. This study investigates how LSD1 regulates autophagy in ovarian cancer by interacting with the autophagy protein LC3B. Utilizing the bioinformatic analysis of TCGA, CPTAC, and GEO datasets, as well as immunohistochemistry in ovarian cancer patients, we explored the expression association between LSD1 and LC3B. Molecular mechanisms were further analyzed using Western blotting, immunoprecipitation, and GST pull-down assays. Our findings reveal that LSD1 binds to LC3B via its SWIRM domain, and high levels of LSD1 are closely associated with aggressive ovarian cancer and poor patient outcomes. Mechanistically, LSD1 demethylates LC3B, leading to decreased LC3B stability. The observed inverse correlation between LSD1 expression and LC3B protein levels in clinical samples underscores the need for further investigation to elucidate how reduced LC3B protein levels induced by LSD1 demethylation may contribute to ovarian cancer.
    Keywords:  autophagy; demethylation; epigenetic modification; prognosis; proteasome degradation
    DOI:  https://doi.org/10.3390/biom14111377
  52. Brain Res. 2024 Nov 23. pii: S0006-8993(24)00595-X. [Epub ahead of print] 149340
    The role of ferritinophagy and ferroptosis in Alzheimer's disease.
    Ziwen Chen, Nan Zheng, Fuwei Wang, Qiong Zhou, Zihao Chen, Lihua Xie, Qiang Sun, Li Li, Baohong Li.
      Iron is a crucial mineral element within human cells, serving as a pivotal cofactor for diverse biological enzymes. Ferritin plays a crucial role in maintaining iron homeostasis within the body through its ability to sequester and release iron. Ferritinophagy is a selective autophagic process in cells that specifically facilitates the degradation of ferritin and subsequent release of free iron, thereby regulating intracellular iron homeostasis. The nuclear receptor coactivator 4 (NCOA4) serves as a pivotal regulator in the entire process of ferritinophagy, facilitating its binding to ferritin and subsequent delivering to lysosomes for degradation, thereby enabling the release of free iron. The free iron ions within the cell undergo catalysis through the Fenton reaction, resulting in a substantial generation of reactive oxygen species (ROS). This process induces lipid peroxidation, thereby stimulating a cascade leading to cellular tissue damage and subsequent initiation of ferroptosis. Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive deterioration of emotional memory and cognitive function, accompanied by mental and behavioral aberrations. The pathology of the disease is characterized by aberrant deposition of amyloid β-protein (Aβ) and hyperphosphorylated tau protein. It has been observed that evident iron metabolism disorders and accumulation of lipid peroxides occur in AD, indicating a significant impact of ferritinophagy and ferroptosis on the pathogenesis and progression of AD. This article elucidates the process and mechanism of ferritinophagy and ferroptosis, investigating their implications in AD to identify novel targets for therapeutic intervention.
    Keywords:  AD; Ferritinophagy; Ferroptosis; Lipid peroxidation; NCOA4
    DOI:  https://doi.org/10.1016/j.brainres.2024.149340
  53. Biomedicines. 2024 Nov 14. pii: 2604. [Epub ahead of print]12(11):
    mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different Manners.
    Megumi Watanabe, Tatsuya Sato, Toshiyuki Yano, Megumi Higashide, Toshifumi Ogawa, Nami Nishikiori, Masato Furuhashi, Hiroshi Ohguro.
      Background/Objectives: Mammalian target of rapamycin (mTOR) inhibition may have been suggested to have a beneficial effect on the glaucomatous human trabecular meshwork (HTM). To study the effects of the mTOR inhibitors rapamycin (Rapa) and Torin1 on the glaucomatous HTM, transforming growth factor-β2 (TGF-β2)-treated two-dimensionally (2D) and three-dimensionally (3D) cultured HTM cells were used. Methods: We evaluated (1) the levels of autophagy via Western blot analysis using a specific antibody against microtubule-associated protein 1 light chain 3 (LC3), (2) barrier capacity based on transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) permeability (2D), (3) cellular metabolic functions (2D), (4) the size and stiffness of spheroids, and (5) the mRNA expression of ECM proteins. Results: TGF-β2-induced inhibition of autophagy was significantly inhibited by Rapa and Torin1. Rapa and Torin1 substantially decreased barrier capacity in both TGF-β2-untreated and TGF-β2-treated HTM cells. Cellular metabolic analysis indicated that Rapa, but not Torin1, substantially enhanced both mitochondrial and glycolytic functions of TGF-β2-untreated HTM cells. In the physical properties of spheroids, TGF-β2 resulted in the formation of down-sized and stiffened spheroids. mTOR inhibitors decreased the size but not the stiffness of TGF-β2-untreated spheroids and significantly reduced the TGF-β2-related increase in the stiffness but not the size of spheroids. The diverse effects of mTOR inhibitors on TGF-β2-untreated and TGF-β2-treated spheroids were also observed in the mRNA expression of extracellular matrix proteins. Conclusions: The results taken together suggest that mTOR inhibitors significantly influence the biological aspects of both a single layer and multiple layers of the TGF-β2-treated HTM and untreated HTM.
    Keywords:  3D culture; TGF-β2; autophagy; human trabecular meshwork; mTOR; rapamycin
    DOI:  https://doi.org/10.3390/biomedicines12112604
  54. Free Radic Biol Med. 2024 Nov 27. pii: S0891-5849(24)01079-7. [Epub ahead of print]
    Zinc Ions Facilitate Metabolic Bioenergetic Recovery Post Spinal Cord Injury by Activating Microglial Mitophagy through the STAT3-FOXO3a-SOD2 Pathway.
    Yang Cui, Mingyu Bai, Shuang Gao, Haosen Zhao, Xifan Mei.
      Spinal cord injury (SCI) is a devastating condition of the central nervous system (CNS) with high global rates of disability and mortality, and no effective cure currently available. Microglia play a critical role in the progression of SCI, and enhancing their metabolic function may facilitate tissue repair and recovery. Mitochondrial dysfunction is a key feature of metabolic impairment, with the regulation of autophagy being essential for maintaining mitochondrial homeostasis and cell survival. The transcription factor Forkhead box O3a (FOXO3a) is integral to cellular metabolism, mitochondrial dysfunction, and oxidative stress responses, yet its role in post-SCI microglial metabolism remains underexplored. In this study, single-cell RNA sequencing reveals the crucial involvement of the FOXO signaling pathway in zinc ion-mediated enhancement of microglial metabolism. Mechanistically, oxidative stress-induced reactive oxygen species (ROS) accumulation exacerbates metabolic dysfunction by promoting excessive mitochondrial fission and impairing mitophagy. Importantly, zinc ions induce the nuclear translocation of FOXO3a, leading to its activation as a transcription factor. This activation enhances mitochondrial autophagy and fusion processes, thereby restoring microglial metabolic capacity. Our findings suggest that the zinc ion regulation of the STAT3-FOXO3a-SOD2 axis is pivotal in modulating mitochondrial gene expression, which governs microglial energy homeostasis and improves the spinal cord microenvironment, potentially enhancing neuronal survival. These insights highlight a promising therapeutic target for SCI.
    Keywords:  Microglia; Mitophagy; Oxidative stress; Spinal cord injury; Zinc
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2024.11.045
  55. Int J Mol Sci. 2024 Nov 17. pii: 12335. [Epub ahead of print]25(22):
    The Mechanistic Link Between Tau-Driven Proteotoxic Stress and Cellular Senescence in Alzheimer's Disease.
    Karthikeyan Tangavelou, Kiran Bhaskar.
      In Alzheimer's disease (AD), tau dissociates from microtubules (MTs) due to hyperphosphorylation and misfolding. It is degraded by various mechanisms, including the 20S proteasome, chaperone-mediated autophagy (CMA), 26S proteasome, macroautophagy, and aggrephagy. Neurofibrillary tangles (NFTs) form upon the impairment of aggrephagy, and eventually, the ubiquitin chaperone valosin-containing protein (VCP) and heat shock 70 kDa protein (HSP70) are recruited to the sites of NFTs for the extraction of tau for the ubiquitin-proteasome system (UPS)-mediated degradation. However, the impairment of tau degradation in neurons allows tau to be secreted into the extracellular space. Secreted tau can be monomers, oligomers, and paired helical filaments (PHFs), which are seeding competent pathological tau that can be endocytosed/phagocytosed by healthy neurons, microglia, astrocytes, oligodendrocyte progenitor cells (OPCs), and oligodendrocytes, often causing proteotoxic stress and eventually triggers senescence. Senescent cells secrete various senescence-associated secretory phenotype (SASP) factors, which trigger cellular atrophy, causing decreased brain volume in human AD. However, the molecular mechanisms of proteotoxic stress and cellular senescence are not entirely understood and are an emerging area of research. Therefore, this comprehensive review summarizes pertinent studies that provided evidence for the sequential tau degradation, failure, and the mechanistic link between tau-driven proteotoxic stress and cellular senescence in AD.
    Keywords:  Alzheimer’s disease; aggrephagy; autophagy; cellular senescence; nucleophagy; proteotoxic stress; senolytic drugs; tau; tauopathy; ubiquitin–proteasome system
    DOI:  https://doi.org/10.3390/ijms252212335
  56. Eur Heart J. 2024 Nov 27. pii: ehae782. [Epub ahead of print]
    Enhanced Parkin-mediated mitophagy mitigates adverse left ventricular remodelling after myocardial infarction: role of PR-364.
    Lizhuo Ai, Juliana de Freitas Germano, Chengqun Huang, Marianne Aniag, Savannah Sawaged, Jon Sin, Reetu Thakur, Deepika Rai, Christopher Rainville, David E Sterner, Yang Song, Honit Piplani, Suresh Kumar, Tauseef R Butt, Robert M Mentzer, Aleksandr Stotland, Roberta A Gottlieb, Jennifer E Van Eyk.
       BACKGROUND AND AIMS: Almost 30% of survivors of myocardial infarction (MI) develop heart failure (HF), in part due to damage caused by the accumulation of dysfunctional mitochondria. Organelle quality control through Parkin-mediated mitochondrial autophagy (mitophagy) is known to play a role in mediating protection against HF damage post-ischaemic injury and remodelling of the subsequent deteriorated myocardium.
    METHODS: This study has shown that a single i.p. dose (2 h post-MI) of the selective small molecule Parkin activator PR-364 reduced mortality, preserved cardiac ejection fraction, and mitigated the progression of HF. To reveal the mechanism of PR-364, a multi-omic strategy was deployed in combination with classical functional assays using in vivo MI and in vitro cardiomyocyte models.
    RESULTS: In vitro cell data indicated that Parkin activation by PR-364 increased mitophagy and mitochondrial biogenesis, enhanced adenosine triphosphate production via improved citric acid cycle, altered accumulation of calcium localization to the mitochondria, and initiated translational reprogramming with increased expression of mitochondrial translational proteins. In mice, PR-364 administered post-MI resulted in widespread proteome changes, indicating an up-regulation of mitochondrial metabolism and mitochondrial translation in the surviving myocardium.
    CONCLUSIONS: This study demonstrates the therapeutic potential of targeting Parkin-mediated mitophagy using PR-364 to protect surviving cardiac tissue post-MI from progression to HF.
    Keywords:  Heart failure; Mitochondrial function; Multi-omics; Myocardial infarction; Parkin-dependent mitophagy; Proteomics; Translational reprogramming
    DOI:  https://doi.org/10.1093/eurheartj/ehae782
  57. Mol Biol Cell. 2024 Nov 27. mbcE24090434
    CARMIL1-AA selectively inhibits macropinocytosis while sparing autophagy.
    Rebecca M Lim, Alexa Lu, Brennan M Chuang, Cecily Anaraki, Brandon Chu, Christopher J Halbrook, Aimee L Edinger.
      Macropinocytosis is reported to fuel tumor growth and drug resistance by allowing cancer cells to scavenge extracellular macromolecules. However, accurately defining the role of macropinocytosis in cancer depends on our ability to selectively block this process. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) is widely used to inhibit macropinocytosis but affects multiple Na+/H+ exchangers (NHE) that regulate cytoplasmic and organellar pH. Consistent with this, we report that EIPA slows proliferation to a greater extent than can be accounted for by macropinocytosis inhibition and triggers conjugation of ATG8 to single membranes (CASM). Knocking down only NHE1 would not avoid macropinocytosis-independent effects on pH. Moreover, contrary to published reports, NHE1 loss did not block macropinocytosis in multiple cell lines. Knocking down CARMIL1 with CRISPR-Cas9 editing limited macropinocytosis, but only by 50%. In contrast, expressing the CARMIL1-AA mutant inhibits macropinocytosis induced by a wide range of macropinocytic stimuli to a similar extent as EIPA. CARMIL1-AA expression did not inhibit proliferation, highlighting the shortcomings of EIPA as a macropinocytosis inhibitor. Importantly, autophagy, another actin dependent, nutrient-producing process, was not affected by CARMIL1-AA expression. In sum, constitutive or inducible CARMIL1-AA expression reduced macropinocytosis without affecting proliferation, RAC activation, or autophagy, other processes that drive tumor initiation and progression. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E24-09-0434
  58. J Cell Signal. 2024 ;5(3): 114-121
    Linking Phosphoinositides to Proteins: A Novel Signaling PIPeline.
    Noah D Carrillo, Poorwa Awasthi, Jeong Hyo Lee, Tianmu Wen, Mo Chen, Colin Sterling, Trevor J Wolfe, Vincent L Cryns, Richard A Anderson.
      Phosphoinositide (PIPn) signaling plays pivotal roles in myriad biological processes and is altered in many diseases including cancer. Canonical PIPn signaling involves membrane-associated PIPn lipid second messengers that modulate protein recruitment and activity at membrane focal points. In the nucleus, PIPn signaling operates separately from membranous compartments defining the paradigm of non-canonical PIPn signaling. However, the mechanisms by which this non-membranous nuclear PIPn pool is established and mediates stress signaling is poorly understood. The recent discovery of a p53-signalosome by Chen et al. (Nature Cell Biology 2022) represents a new PIPn signaling axis that operates independently from membrane structures where PIPns are dynamically linked to nuclear p53 and modified by PIPn kinases and phosphatases, allowing the activation of a nuclear PI 3-kinase/Akt pathway that is entirely distinct from the canonical membrane-localized pathway. Here, we will discuss emerging insights about the non-canonical PIPn pathway, which links PIPns to a growing number of cellular targets and highlight the similarities/differences with its canonical counterpart. We will also discuss potential therapeutic targets in this non-canonical PIPn pathway, which is likely to be deregulated in many diseases.
    Keywords:  PI3K; PIPn linked proteins; Phosphoinositide; cancer; nucleus; signalosome
    DOI:  https://doi.org/10.33696/signaling.5.118
  59. Autophagy. 2024 Nov 29.
    S-acylation of PNPLA2/ATGL: a necessity for triacylglycerol lipolysis and lipophagy in hepatocytes.
    Zheng Yuping, Dante Neculai, Gregory D Fairm.
      The intricate balance between lipolysis and lipophagy in cellular lipid homeostasis has fascinated researchers for years. A growing body of evidence highlights the critical roles of PNPLA2/ATGL (patatin like phospholipase domain containing 2) in both lipolysis and lipophagy. Here, we discuss our recent study, which revealed that PNPLA2 must be S-acylated on Cys15 for its robust catalytic activity. Additionally, we discuss the results highlighting that genetic inactivation of the ZDHHC11 acyltransferase or expression of S-acylation deficient PNPLA2 mutants impairs not only lipolysis but also lipophagy. This finding suggests that the mere presence of PNPLA2 with its LC3-interacting region (LIR) motifs is insufficient to drive lipophagy without triacylglycerol breakdown. Our study provides insights into yet another mode of regulation of PNPLA2 activity with implications for understanding lipid droplet catabolism, lipophagy, and cellular energy homeostasis.
    Keywords:  Fatty liver disease; PNPLA2; S-acylation; lipid droplet; lipophagy; triacylglycerol
    DOI:  https://doi.org/10.1080/15548627.2024.2435873
  60. Neurochem Res. 2024 Nov 25. 50(1): 32
    Piezo1 Modulates Neuronal Autophagy and Apoptosis in Cerebral Ischemia-Reperfusion Injury Through the AMPK-mTOR Signaling Pathway.
    Yingjie Yue, Pingping Chen, Chongwen Ren.
      Cerebral ischemia-reperfusion (I/R) injury is a complex pathophysiological process involving multiple mechanisms, including apoptosis and autophagy, which can lead to significant neuronal damage. PIEZO1, a stretch-activated ion channel, has recently emerged as a potential regulator of cellular responses to ischemic conditions. However, its role in neuronal cell survival and death during ischemic events is not well elucidated. This study aimed to ascertain the regulatory function of PIEZO1 in neuronal cell apoptosis and autophagy in an in vitro model of hypoxia-reoxygenation and an in vivo model of brain I/R injury. HT22 hippocampal neuronal cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate ischemic conditions, with subsequent reoxygenation. In vitro, PIEZO1 expression was silenced using small interfering RNA (si-RNA) transfection. The effects on cell viability, apoptosis, and autophagy were assessed using CCK-8 assays, PI-Annexin/V staining combined with flow cytometry, and Western blot analysis. Additionally, intracellular Ca2+ levels in HT22 cells were measured using a Ca2+ probe. The involvement of the AMPK-mTOR pathway was investigated using rapamycin. For in vivo validation, middle cerebral artery occlusion/reperfusion (MCAO/R) in rats was employed. To determine the neuroprotective role of PIEZO1 silencing, sh-PIEZO1 adeno-associated virus was stereotaxically injected into the cerebral ventricle, and neurological and histological outcomes were assessed using neurological scoring, TTC staining, H&E staining, Nissl staining, and immunofluorescence. In HT22 cells, OGD/R injury notably upregulated PIEZO1 expression and intracellular Ca2+ levels. Silencing PIEZO1 significantly diminished OGD/R-induced Ca2+ influx, apoptosis, and autophagy, as indicated by lower levels of pro-apoptotic and autophagy-related proteins and improved cell viability. Additionally, PIEZO1 modulated the AMPK-mTOR signaling pathway, an effect that was counteracted by rapamycin treatment, implying its regulatory role. In vivo, PIEZO1 silencing ameliorated brain I/R injury in MCAO/R rats, demonstrated by improved neurological function scores and reduced neuronal apoptosis and autophagy. However, these neuroprotective effects were reversed through rapamycin treatment. Our findings indicate that PIEZO1 is upregulated following ischemic injury and facilitates Ca2+ influx, apoptosis, and autophagy via the AMPK-mTOR pathway. Silencing PIEZO1 confers neuroprotection against I/R injury both in vitro and in vivo, highlighting its potential as a therapeutic target for stroke management.
    Keywords:  AMPK-mTOR pathway; Apoptosis; Autophagy; Ischemia–reperfusion injury; Neuroprotection; PIEZO1
    DOI:  https://doi.org/10.1007/s11064-024-04291-w
  61. Ageing Res Rev. 2024 Nov 26. pii: S1568-1637(24)00421-5. [Epub ahead of print] 102603
    The Mitochondrial Integrated Stress Response: A novel approach to anti-aging and pro-longevity.
    Xiaoding Wang, Guangyu Zhang.
      The ISR is a cellular signaling pathway that responds to various physiological changes and types of stimulation. The mitochondrial integrated stress response (ISRmt) is a stress response specific to mitochondria which is initiated by eIF2α phosphorylation and is responsive to mitochondrial stressors. The ISRmt triggers diverse metabolic responses reliant on activating transcription factor 4 (ATF4). The preliminary phases of ISRmt can provoke an adaptive stress response that antagonizes age-related diseases and promotes longevity. In this review, we provide an overview of the molecular mechanisms of the ISRmt, with a particular focus on its potential as a therapeutic target for age-related disease and the promotion of longevity.
    Keywords:  FGF21; Mitochondrial integrated stress response; aging; longevity
    DOI:  https://doi.org/10.1016/j.arr.2024.102603
  62. Nutrients. 2024 Nov 05. pii: 3789. [Epub ahead of print]16(22):
    Quercetin's Potential in MASLD: Investigating the Role of Autophagy and Key Molecular Pathways in Liver Steatosis and Inflammation.
    Ioannis Katsaros, Maria Sotiropoulou, Michail Vailas, Emmanouil Ioannis Kapetanakis, Georgia Valsami, Alexandra Tsaroucha, Dimitrios Schizas.
      Metabolic dysfunction-associated fatty liver disease (MASLD) is a widespread liver disorder characterized by excessive fat accumulation in the liver, commonly associated with metabolic syndrome components such as obesity, diabetes, and dyslipidemia. With a global prevalence of up to 30%, MASLD is projected to affect over 100 million people in the U.S. and 20 million in Europe by 2030. The disease ranges from Steatotic Lived Disease (SLD) to more severe forms like metabolic dysfunction-associated steatohepatitis (MASH), which can progress to cirrhosis and hepatocellular carcinoma. Autophagy, a cellular process crucial for lipid metabolism and homeostasis, is often impaired in MASLD, leading to increased hepatic lipid accumulation and inflammation. Key autophagy-related proteins, such as Beclin1, LC3A, SQSTM1 (p62), CD36, and Perilipin 3, play significant roles in regulating this process. Disruption in these proteins contributes to the pathogenesis of MASLD. Quercetin, a natural polyphenolic flavonoid with antioxidant and anti-inflammatory properties, has promising results in mitigating MASLD. It may reduce hepatic lipid accumulation, improve mitochondrial function, and enhance autophagy. However, further research is needed to elucidate its mechanisms and validate its therapeutic potential in clinical settings. This underscores the need for continued investigation into autophagy and novel treatments for MASLD.
    Keywords:  Beclin1; CD36; LC3A; MASLD; NAFLD; Perilipin3; SQSTM1; autophagy; quercetin
    DOI:  https://doi.org/10.3390/nu16223789
  63. bioRxiv. 2024 Nov 18. pii: 2024.11.16.623962. [Epub ahead of print]
    Haploinsufficiency of lysosomal enzyme genes in Alzheimer's disease.
    Bruno A Benitez, Clare E Wallace, Maulikkumar Patel, Niko-Petteri Nykanen, Carla M Yuede, Samantha L Eaton, Cyril Pottier, Arda Cetin, Matthew Johnson, Mia T Bevan, Woodrow D Gardiner, Hannah M Edwards, Brookelyn M Doherty, Ryan T Harrigan, Dominic Kurian, Thomas M Wishart, Colin Smith, John R Cirrito, Mark S Sands.
      There is growing evidence suggesting that the lysosome or lysosome dysfunction is associated with Alzheimer's disease (AD). Pathway analysis of post mortem brain-derived proteomic data from AD patients shows that the lysosomal system is perturbed relative to similarly aged unaffected controls. However, it is unclear if these changes contributed to the pathogenesis or are a response to the disease. Consistent with the hypothesis that lysosome dysfunction contributes to AD pathogenesis, whole genome sequencing data indicate that heterozygous pathogenic mutations and predicted protein-damaging variants in multiple lysosomal enzyme genes are enriched in AD patients compared to matched controls. Heterozygous loss-of-function mutations in the palmitoyl protein thioesterase-1 ( PPT1 ), α-L-iduronidase ( IDUA ), β-glucuronidase ( GUSB ), N-acetylglucosaminidase ( NAGLU ), and galactocerebrosidase ( GALC ) genes have a gene-dosage effect on Aβ 40 levels in brain interstitial fluid in C57BL/6 mice and significantly increase Aβ plaque formation in the 5xFAD mouse model of AD, thus providing in vivo validation of the human genetic data. A more detailed analysis of PPT1 heterozygosity in 18-month-old mice revealed changes in α-, β-, and γ-secretases that favor an amyloidogenic pathway. Proteomic changes in brain tissue from aged PPT1 heterozygous sheep are consistent with both the mouse data and the potential activation of AD pathways. Finally, CNS-directed, AAV-mediated gene therapy significantly decreased Aβ plaques, increased life span, and improved behavioral performance in 5xFAD/PPT1+/- mice. Collectively, these data strongly suggest that heterozygosity of multiple lysosomal enzyme genes represent risk factors for AD and may identify precise therapeutic targets for a subset of genetically-defined AD patients.
    Significance Statement: Lysosomes play a role in the degradation of aggregation-prone proteins such as amyloid β (Aβ). Homozygous lysosomal enzyme gene defects result in fatal pediatric lysosomal storage diseases and, historically, carriers were considered normal. However, a human genetic analysis identified deleterious heterozygous variants in multiple lysosomal enzyme genes that are enriched in Alzheimer's disease (AD) patients. Those findings were validated in vivo by demonstrating that heterozygous loss-of-function (LoF) mutations in five different lysosomal enzyme genes affect Aβ processing and exacerbate Aβ plaque formation. CNS-directed gene therapy ameliorated the effects of a heterozygous LoF mutation in one of those genes in a mouse model of AD. These findings provide insights into the role of lysosomes in AD and have important therapeutic implications.
    DOI:  https://doi.org/10.1101/2024.11.16.623962
  64. Cancers (Basel). 2024 Nov 10. pii: 3783. [Epub ahead of print]16(22):
    p21Waf1/Cip1 Is a Novel Downstream Target of 40S Ribosomal S6 Kinase 2.
    Alakananda Basu, Zhenyu Xuan.
      Background/Objectives: The ribosomal S6 kinase 2 (S6K2) acts downstream of the mechanistic target of rapamycin complex 1 and is a homolog of S6K1 but little is known about its downstream effectors. The objective of this study was to use an unbiased transcriptome profiling to uncover how S6K2 promotes breast cancer cell survival. Methods: RNA-Seq analysis was performed to identify novel S6K2 targets. Cells were transfected with siRNAs or plasmids containing genes of interest. Western blot analyses were performed to quantify total and phosphorylated proteins. Apoptosis was monitored by treating cells with different concentrations of doxorubicin. Results: Silencing of S6K2, but not S6K1, decreased p21 in MCF-7 and T47D breast cancer cells. Knockdown of Akt1 but not Akt2 decreased p21 in MCF-7 cells whereas both Akt1 and Akt2 knockdown attenuated p21 in T47D cells. While Akt1 overexpression enhanced p21 and partially reversed the effect of S6K2 deficiency on p21 downregulation in MCF-7 cells, it had little effect in T47D cells. S6K2 knockdown increased JUN mRNA and knockdown of cJun enhanced p21. Low concentrations of doxorubicin increased, and high concentrations decreased p21 levels in T47D cells. Silencing of S6K2 or p21 sensitized T47D cells to doxorubicin via c-Jun N-terminal kinase (JNK)-mediated downregulation of Mcl-1. Conclusions: S6K2 knockdown enhanced doxorubicin-induced apoptosis by downregulating the cell cycle inhibitor p21 and the anti-apoptotic protein Mcl-1 via Akt and/or JNK.
    Keywords:  Akt1; Akt2; JNK; S6K1; S6K2/RPS6KB2; apoptosis; breast cancer; cJun/JUN; chemoresistance; p21/CDKN1A
    DOI:  https://doi.org/10.3390/cancers16223783
  65. bioRxiv. 2024 Nov 18. pii: 2024.11.17.624030. [Epub ahead of print]
    Lipid droplet protein Perilipin 2 is critical for the regulation of insulin secretion through beta cell lipophagy and glucagon expression in pancreatic islets.
    Siming Liu, Israel Wipf, Aditya Joglekar, Aidan Freshly, Corinne E Bovee, Lucy Kim, Syreine L Richtsmeier, Spencer Peachee, Shayla Kopriva, Anamika Vikram, Dalal El Ladiki, Fatma Ilerisoy, Beyza Ilerisoy, Gianna Sagona, Claudia Jun, Michelle Giedt, Tina L Tootle, James Ankrum, Yumi Imai.
      Knockdown (KD) of lipid droplet (LD) protein perilipin 2 (PLIN2) in beta cells impairs glucose-stimulated insulin secretion (GSIS) and mitochondrial function. Here, we addressed a pathway responsible for compromised mitochondrial integrity in PLIN2 KD beta cells. In PLIN2 KD human islets, mitochondria were fragmented in beta cells but not in alpha cells. Glucagon but not insulin level was elevated. While the formation of early LDs followed by fluorescent fatty acids (FA) analog Bodipy C12 (C12) was preserved, C12 accumulated in mitochondria over time in PLIN2 KD INS-1 cells. A lysosomal acid lipase inhibitor Lali2 prevented C12 transfer to mitochondria, mitochondrial fragmentation, and the impairment of GSIS. Direct interactions between LD-lysosome and lysosome-mitochondria were increased in PLIN2 KD INS-1 cells. Thus, FA released from LDs by microlipophagy cause mitochondrial changes and impair GSIS in PLIN2 KD beta cells. Interestingly, glucolipotoxic condition (GLT) caused C12 accumulation and mitochondrial fragmentation similar to PLIN2 KD in beta cells. Moreover, Lali2 reversed mitochondrial fragmentation and improved GSIS in human islets under GLT. In summary, PLIN2 regulates microlipophagy to prevent excess FA flux to mitochondria in beta cells. This pathway also contributes to GSIS impairment when LD pool expands under nutrient load in beta cells.
    DOI:  https://doi.org/10.1101/2024.11.17.624030
  66. Int J Mol Sci. 2024 Nov 13. pii: 12186. [Epub ahead of print]25(22):
    Investigating p.Ala1035Val in NPC1: New Cellular Models for Niemann-Pick Type C Disease.
    Hugo David, Jlenia Monfregola, Isaura Ribeiro, Maria Teresa Cardoso, Ana Catarina Sandiares, Luciana Moreira, Maria Francisca Coutinho, Dulce Quelhas, Andrea Ballabio, Sandra Alves, Marisa Encarnação.
      Niemann-Pick type C (NPC) is a lysosomal storage disorder (LSD) caused by pathogenic variants in either the NPC1 or NPC2 genes, which encode proteins involved in the lysosomal export of unesterified cholesterol. In patients of Western European descent, the p.Ile1061Thr variant in NPC1 is especially prevalent. However, mounting evidence has positioned p.Ala1035Val as the most common variant in Portugal and the second most prevalent variant worldwide. By analyzing 10 Portuguese NPC patients homozygous for p.Ala1035Val, we found an SNP in cis on position 858 (p.Ile858Val), which we hypothesize could have a disease-modifying effect. To address this query, we created variant-specific in vitro models of NPC by stably transducing NPC1-/- ARPE-19 cells with constructs encoding different fluorescently-tagged variants of NPC1, which we used, alongside patient-derived skin fibroblasts, to investigate lysosomal positioning and the trafficking routes elicited by p.Ile1061Thr and p.Ala1035Val (with and without the p.Ile858Val SNP in cis). Our results corroborate the previously described decrease in p.Ile1061Thr-NPC1 trafficking to the lysosome and suggest a similar, if not worse, scenario for the p.Ala1035Val variant, especially when in cis with p.Ile858Val. This is the first reported functional study addressing the impact of the p.Ala1035Val variant at the cellular level, paving the way for novel therapeutic options.
    Keywords:  ARPE-19; NPC1; Niemann–Pick type C; cell models; complex alleles; lysosomal storage disorders; p.Ala1035Val; p.Ile1061Thr; p.Ile858Val; phenotypic variability
    DOI:  https://doi.org/10.3390/ijms252212186
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