bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2024–12–22
57 papers selected by
Viktor Korolchuk, Newcastle University
  1. Blocking autophagosome closure manifests the roles of mammalian Atg8-family proteins in phagophore formation and expansion during nutrient starvation.
  2. A Novel 14mer Peptide Inhibits Autophagic Flux via Selective Activation of the mTORC1 Signalling Pathway: Implications for Alzheimer's Disease.
  3. The cGAS-STING pathway activates transcription factor TFEB to stimulate lysosome biogenesis and pathogen clearance.
  4. A molecular glue for Prkn/parkin.
  5. Dynamic mitophagy trajectories hallmark brain aging.
  6. Autophagy and Protein Quality Control in Parkinson's Disease.
  7. Autophagy induction by piplartine ameliorates axonal degeneration caused by mutant HSPB1 and HSPB8 in charcot-marie-tooth type 2 neuropathies.
  8. Restoration of lysosomal function attenuates autophagic flux impairment in nucleus pulposus cells and protects against mechanical overloading-induced intervertebral disc degeneration.
  9. Chaperone-mediated autophagy as a modulator of aging and longevity.
  10. Unveiling the Role of Mechanistic Target of Rapamycin Kinase (MTOR) Signaling in Cancer Progression and the Emergence of MTOR Inhibitors as Therapeutic Strategies.
  11. Isothiocyanates induce autophagy and inhibit protein synthesis in primary cells via modulation of AMPK-mTORC1-S6K1 signaling pathway, and protect against mutant huntingtin aggregation.
  12. AMPK protects proximal tubular epithelial cells from lysosomal dysfunction and dedifferentiation induced by lipotoxicity.
  13. Polyphenol Mediated Assembly: Tailored Nano-Dredger Unblocks Axonal Autophagosomes Retrograde Transport Traffic Jam for Accelerated Alzheimer's Waste Clearance.
  14. Mitophagosomes induced during EV-D68 infection promote viral nonlytic release.
  15. Rapamycin Abrogates Aggregation of Human α-Synuclein Expressed in Fission Yeast via an Autophagy-Independent Mechanism.
  16. Macronucleophagy maintains cell viability under nitrogen starvation by modulating micronucleophagy.
  17. Review on the role of autophagy in the toxicity of nanoparticles and the signaling pathways involved.
  18. Chaperone Activators.
  19. A Binary siRNA-Loaded Tetrahedral DNA Nanobox for Synergetic Anti-Aging Therapy.
  20. CD40 ligation-induced ERK activation leads to enhanced radiosensitivity in cervical carcinoma cells via promoting autophagy.
  21. Autophagy modulators in type 2 diabetes: A new perspective.
  22. The Estrogen-Autophagy Axis: Insights into Cytoprotection and Therapeutic Potential in Cancer and Infection.
  23. Relationship Between Dietary Nutrient Intake and Autophagy-Related Genes in Obese Humans: A Narrative Review.
  24. Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice.
  25. Blockage of polycystin-2 alleviates myocardial ischemia/reperfusion injury by inhibiting autophagy through the Ca2+/Akt/Beclin 1 pathway.
  26. A role for lysosomal calcium channels in mitigating mitochondrial damage and oxidative stress.
  27. Autophagy and nutrigenomics: a winning team against chronic disease and tumors.
  28. Gene-Silencing Therapeutic Approaches Targeting PI3K/Akt/mTOR Signaling in Degenerative Intervertebral Disk Cells: An In Vitro Comparative Study Between RNA Interference and CRISPR-Cas9.
  29. Trehalose Ameliorates Nonalcoholic Fatty Liver Disease by Regulating IRE1α-TFEB Signaling Pathway.
  30. Mutations in AMBRA1 aggravate β-thalassemia by impairing autophagy-mediated clearance of free α-globin.
  31. BMAL1 upregulates STX17 levels to promote autophagosome-lysosome fusion in hippocampal neurons to ameliorate Alzheimer's disease.
  32. Casein kinase 1 controls components of a TORC2 signaling network in budding yeast.
  33. Astrocytes contribute to toll-like receptor 2-mediated neurodegeneration and alpha-synuclein pathology in a human midbrain Parkinson's model.
  34. Phytochemical-mediated efferocytosis and autophagy in inflammation control.
  35. A kaleidoscopic view of extracellular vesicles in lysosomal storage disorders.
  36. Cordycepin ameliorates autoimmunity by promoting STING degradation via autophagy pathway.
  37. A Reduction in Mitophagy Is Associated with Glaucomatous Neurodegeneration in Rodent Models of Glaucoma.
  38. JAG1 mediates apoptosis in herpes simplex keratitis by suppressing autophagy via ROS/JAG1/NOTCH1/pULK1 signaling pathway.
  39. Interplay between β-propeller subunits WDR26 and muskelin regulates the CTLH E3 ligase supramolecular complex.
  40. LC3B: A microtubule-associated protein influences disease progression and prognosis.
  41. CKAP4 in hepatocellular carcinoma: competitive RETREG1/FAM134B binding, reticulophagy regulation, and cancer progression.
  42. Nek6 regulates autophagy through the mTOR signaling pathway to alleviate cerebral ischemia-reperfusion injury.
  43. Determining ATG2 Localization During Autophagosome Formation in Mammalian Cells.
  44. Paeoniflorin ameliorates reperfusion injury in H9C2 cells through SIRT1-PINK1/parkin-mediated mitochondrial autophagy.
  45. BODIPYS Based Fluorescent Markers To Monitor Autophagic Lysosomes and Lipid Droplets in TNBC.
  46. RAB37 suppresses the EMT, migration and invasion of gastric cancer cells by mediating autophagic degradation of β-catenin.
  47. Coniferaldehyde activates autophagy and enhances oxidative stress resistance and lifespan of Caenorhabditis elegans via par-4/aak-2/skn-1 pathway.
  48. Lithocholic acid binds TULP3 to activate sirtuins and AMPK to slow down ageing.
  49. Age-related TFEB downregulation in proximal tubules causes systemic metabolic disorders and occasional apolipoprotein A4-related amyloidosis.
  50. AXL-TBK1 driven AKT3 activation promotes metastasis.
  51. Spermidine alleviates depression via control of the stress response.
  52. Endogenous interactomes of MFN1 and MFN2 provide novel insights into interorganelle communication and autophagy.
  53. New insights into the role of mitophagy related gene affecting the metastasis of osteosarcoma through scRNA-seq and CRISPR-Cas9 genome editing.
  54. Subcellular NAD+ pools are interconnected and buffered by mitochondrial NAD.
  55. Evi1 governs Kdm6b-mediated histone demethylation to regulate the Laptm4b-driven mTOR pathway in hematopoietic progenitor cells.
  56. LOC730101 improves ovarian cancer drug sensitivity by inhibiting autophagy-mediated DNA damage repair via BECN1.
  57. Reversing Persistent PTEN Activation after Traumatic Brain Injury Fuels Long-Term Axonal Regeneration via Akt/mTORC1 Signaling Cascade.
  1. Autophagy. 2024 Dec 18.
    Blocking autophagosome closure manifests the roles of mammalian Atg8-family proteins in phagophore formation and expansion during nutrient starvation.
    Van Bui, Xinwen Liang, Yansheng Ye, William Giang, Fang Tian, Yoshinori Takahashi, Hong-Gang Wang.
      Macroautophagy/autophagy, an evolutionarily conserved cellular degradation pathway, involves phagophores that sequester cytoplasmic constituents and mature into autophagosomes for subsequent lysosomal delivery. The ATG8 gene family, comprising the MAP1LC3/LC3 and GABARAP/GBR subfamilies in mammals, encodes ubiquitin-like proteins that are conjugated to phagophore membranes during autophagosome biogenesis. A central question in the field is how Atg8-family proteins are precisely involved in autophagosome formation, which remains controversial and challenging, at least in part due to the short lifespan of phagophores. In this study, we depleted the autophagosome closure regulator VPS37A to arrest autophagy at the vesicle completion step and determined the roles of mammalian Atg8-family proteins (mATG8s) in nutrient starvation-induced autophagosome biogenesis. Our investigation revealed that LC3 loss hinders phagophore formation, while GBR loss impedes both phagophore formation and expansion. The defect in membrane expansion by GBR loss appears to be attributed to compromised recruitment of ATG proteins containing an LC3-interacting region (LIR), including ULK1 and ATG3. Moreover, a combined deficiency of both LC3 and GBR subfamilies nearly completely inhibits phagophore formation, highlighting their redundant regulation of this process. Consequently, cells lacking all mATG8 members exhibit defects in downstream events such as ESCRT recruitment and autophagic flux. Collectively, these findings underscore the critical roles of mammalian Atg8-family proteins in phagophore formation and expansion during autophagy.
    Keywords:  Autophagosome closure; ESCRT; LIR; The mammalian Atg8 family of proteins; membrane expansion; phagophore formation
    DOI:  https://doi.org/10.1080/15548627.2024.2443300
  2. Int J Mol Sci. 2024 Nov 29. pii: 12837. [Epub ahead of print]25(23):
    A Novel 14mer Peptide Inhibits Autophagic Flux via Selective Activation of the mTORC1 Signalling Pathway: Implications for Alzheimer's Disease.
    Cloe García Porta, Kashif Mahfooz, Joanna Komorowska, Sara Garcia-Rates, Susan Greenfield.
      During development, a 14mer peptide, T14, modulates cell growth via the α-7 nicotinic acetylcholine receptor (α7 nAChR). However, this process could become excitotoxic in the context of the adult brain, leading to pathologies such as Alzheimer's disease (AD). Recent work shows that T14 acts selectively via the mammalian target of rapamycin complex 1 (mTORC1). This pathway is essential for normal development but is overactive in AD. The triggering of mTORC1 has also been associated with the suppression of autophagy, commonly observed in ageing and neurodegeneration. We therefore investigated the relationship between T14 and autophagic flux in tissue cultures, mouse brain slices, and human Alzheimer's disease hippocampus. Here, we demonstrate that T14 and p-mTOR s2448 expression significantly increases in AD human hippocampus, which was associated with the gradual decrease in the autophagosome number across Braak stages. During development, the reduction in T14 positively correlated with pTau (Ser202, Thr205) and two selective autophagy receptors: p62 and optineurin. In vitro studies also indicated that T14 increases p-mTOR s2448 expression, resulting in the aggregation of polyubiquinated substances. The effective blockade of T14 via its cyclic variant, NBP14, has been validated in vitro, in vivo, and ex vivo. In this study, NBP14 significantly attenuated p-mTOR s2448 expression and restored normal autophagic flux, as seen with rapamycin. We conclude that T14 acts at the α-7 receptor to selectively activate the mTORC1 pathway and consequently inhibit autophagic flux. Hence, this study describes a further step in the process by which T14 could drive neurodegeneration.
    Keywords:  Alzheimer’s disease; NBP14; T14; autophagy; mTORC1; rapamycin
    DOI:  https://doi.org/10.3390/ijms252312837
  3. Immunity. 2024 Dec 12. pii: S1074-7613(24)00532-6. [Epub ahead of print]
    The cGAS-STING pathway activates transcription factor TFEB to stimulate lysosome biogenesis and pathogen clearance.
    Yinfeng Xu, Qian Wang, Jun Wang, Chuying Qian, Yusha Wang, Sheng Lu, Lijiang Song, Zhengfu He, Wei Liu, Wei Wan.
      Induction of autophagy is an ancient function of the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway through which autophagic cargoes are delivered to lysosomes for degradation. However, whether lysosome function is also modulated by the cGAS-STING pathway remains unknown. Here, we discovered that the cGAS-STING pathway upregulated lysosomal activity by stimulating lysosome biogenesis independently of the downstream protein kinase TANK-binding kinase 1 (TBK1). STING activation enhanced lysosome biogenesis through inducing the nuclear translocation of transcription factor EB (TFEB) as well as its paralogs transcription factor E3 (TFE3) and microphthalmia-associated transcription factor (MITF). STING-induced lipidation of GABA type A receptor-associated protein (GABARAP), an autophagy-related protein, on STING vesicles was responsible for TFEB activation. Membrane-bound GABARAP sequestered the GTPase-activating protein folliculin (FLCN) and FLCN-interacting protein (FNIP) complex to block its function toward the Rag GTPases Ras-related GTP-binding C and D (RagC and RagD), abolishing mechanistic target of rapamycin (mTOR) complex 1 (mTORC1)-dependent phosphorylation and inactivation of TFEB. Functionally, STING-induced lysosome biogenesis within cells facilitated the clearance of cytoplasmic DNA and invading pathogens. Thus, our findings reveal that induction of lysosome biogenesis is another important function of the cGAS-STING pathway.
    Keywords:  STING; TFEB; cGAS; innate immunity; lysosome
    DOI:  https://doi.org/10.1016/j.immuni.2024.11.017
  4. Autophagy. 2024 Dec 19.
    A molecular glue for Prkn/parkin.
    Véronique Sauvé, Kalle Gehring.
      Parkinson disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra, primarily due to mitochondria dysfunction. PRKN (parkin RBR E3 ubiquitin protein ligase) and PINK1 (PTEN induced kinase 1) are linked to early-onset cases of PD and essential for the clearance of damaged mitochondria via selective mitochondrial autophagy (mitophagy). In a recent publication, we detail how a small molecule can activate PRKN mutants that are unable to be phosphorylated, restoring mitophagy in cellular assays. These findings offer hope for the design of therapeutic drugs for some forms of PD.
    Keywords:  Activator; PARK2; mitochondria; neurodegeneration; parkinson disease; ubiquitin
    DOI:  https://doi.org/10.1080/15548627.2024.2443232
  5. Autophagy. 2024 Dec 19. 1-3
    Dynamic mitophagy trajectories hallmark brain aging.
    Anna Rappe, Thomas G McWilliams.
      Studies using mitophagy reporter mice have established steady-state landscapes of mitochondrial destruction in mammalian tissues, sparking intense interest in basal mitophagy. Yet how basal mitophagy is modified by healthy aging in diverse brain cell types has remained a mystery. We present a comprehensive spatiotemporal analysis of mitophagy and macroautophagy dynamics in the aging mammalian brain, reporting critical region- and cell-specific turnover trajectories in a longitudinal study. We demonstrate that the physiological regulation of mitophagy in the mammalian brain is cell-specific, dynamic and complex. Mitophagy increases significantly in the cerebellum and hippocampus during midlife, while remaining unchanged in the prefrontal cortex (PFC). Conversely, macroautophagy decreases in the hippocampus and PFC, but remains stable in the cerebellum. We also describe emergent lysosomal heterogeneity, with subsets of differential acidified lysosomes accumulating in the aging brain. We further establish midlife as a critical inflection point for autophagy regulation, which may be important for region-specific vulnerability and resilience to aging. By mapping in vivo autophagy dynamics at the single cell level within projection neurons, interneurons and microglia, to astrocytes and secretory cells, we provide a new framework for understanding brain aging and offer potential targets and timepoints for further study and intervention in neurodegenerative diseases.
    Keywords:  Aging; autophagy; brain; mitochondria; mitophagy
    DOI:  https://doi.org/10.1080/15548627.2024.2426115
  6. Cold Spring Harb Perspect Med. 2024 Dec 18. pii: a041619. [Epub ahead of print]
    Autophagy and Protein Quality Control in Parkinson's Disease.
    Marta Martinez-Vicente, Miquel Vila.
      Autophagy is a vital cellular process responsible for the degradation of proteins, organelles, and other cellular components within lysosomes. In neurons, basal autophagy is indispensable for maintaining cellular homeostasis and protein quality control. Accordingly, lysosomal dysfunction has been proposed to be associated with neurodegeneration, and with Parkinson's disease (PD) in particular. Aging, dopamine metabolism, and PD-linked genetic mutations are thought to impair the autophagic-lysosomal pathway, disrupt cellular proteostasis, and contribute to PD pathogenesis. These alterations represent an opportunity to identify potential new therapeutic targets and disease biomarkers, thus laying the groundwork for the development of novel disease-modifying strategies for PD that are aimed at restoring cellular proteostasis and quality control systems.
    DOI:  https://doi.org/10.1101/cshperspect.a041619
  7. Autophagy. 2024 Dec 19.
    Autophagy induction by piplartine ameliorates axonal degeneration caused by mutant HSPB1 and HSPB8 in charcot-marie-tooth type 2 neuropathies.
    Angela Sisto, Tamira van Wermeskerken, Michael Pancher, Pamela Gatto, Bob Asselbergh, Ágata Sofia Assunção Carreira, Vicky De Winter, Valentina Adami, Alessandro Provenzani, Vincent Timmerman.
      HSPB1 [heat shock protein family B (small) member 1] and HSPB8 are essential molecular chaperones for neuronal proteostasis, as they prevent protein aggregation. Mutant HSPB1 and HSPB8 primarily harm peripheral neurons, resulting in axonal Charcot-Marie-Tooth neuropathies (CMT2). Macroautophagy/autophagy is a shared mechanism by which HSPB1 and HSPB8 mutations cause neuronal dysfunction. Autophagosome formation is reduced in mutant HSPB1-induced pluripotent stem-cell-derived motor neurons from CMT type 2F patients. Likewise, the HSPB8K141N knockin mouse model, mimicking CMT type 2 L, exhibits axonal degeneration and muscle atrophy, with SQSTM1/p62-positive deposits. We show here that mouse embryonic fibroblasts isolated from a HSPB8K141N/green fluorescent protein (GFP)-LC3 model have diminished autophagosome production under conditions of MTOR inhibition. To correct the autophagic deficits in the HSPB1 and HSPB8 models, we screened by high-throughput autophagosome quantification the repurposing Spectrum Collection library for molecules that could boost the autophagic activity above the canonical MTOR inhibition. Hit compounds were validated on motor neurons obtained by differentiation of HSPB1P182L and HSPB8K141N patient-derived induced pluripotent stem cells, focusing on autophagy induction as well as neurite network density, axonal degeneration, and mitochondrial morphology. We identified molecules that specifically stimulate autophagosome formation in the HSPB8K141N cells, without affecting autophagy flux. Two top lead compounds induced autophagy and reduced axonal degeneration, thus promoting neuronal network maturation in the CMT2 patient-derived motor neurons. Based on these findings, the phenotypical screen revealed that piplartine rescued autophagy deficiencies in both the HSPB1 and HSPB8 models, demonstrating autophagy induction as an effective therapeutic strategy for CMT neuropathies and other chaperonopathies.
    Keywords:  Autophagy inducer; drug repurposing; inherited peripheral neuropathy; motor neurons; phenotypical screening; small heat shock proteins
    DOI:  https://doi.org/10.1080/15548627.2024.2439649
  8. Autophagy. 2024 Dec 15.
    Restoration of lysosomal function attenuates autophagic flux impairment in nucleus pulposus cells and protects against mechanical overloading-induced intervertebral disc degeneration.
    Sheng Liu, Yiqiang Hu, Weihua Xu, Weijian Liu, Bingjin Wang, Xianlin Zeng, Zengwu Shao, Cao Yang, Liming Xiong, Xianyi Cai.
      Intervertebral disc degeneration (IVDD) is a leading cause of low back pain that incurs large socioeconomic burdens. Growing evidence reveals that macroautophagy/autophagy dysregulation contributes to IVDD, but the exact role of autophagy and its regulatory mechanisms remain largely unknown. Here, we found that mechanical overloading impaired the autophagic flux of nucleus pulposus (NP) cells in vivo and in vitro. Mechanistically, the impairment of autophagic flux was attributed to lysosomal dysfunction induced by overloading. Overloading could also lead to lysosomal membrane permeabilization and consequent lysosome-dependent cell death. As critical effectors of lysosomal quality control pathways, CHMP4B (charged multivesicular body protein 4B) and TFEB (transcription factor EB) were downregulated in overloading-treated NP cells and degenerative discs. Restoring lysosomal function by CHMP4B or TFEB overexpression attenuated autophagic flux impairment of NP cells and protected against overloading-induced IVDD. Additionally, human IVDD was associated with impaired autophagy, and defective lysosomal quality control was also linked to human IVDD. Collectively, these findings highlighted that lysosomal defects were crucial for mechanical overloading-induced autophagic flux impairment and death of NP cells, suggesting the potential therapeutic relevance of restoring lysosomal function for IVDD.
    Keywords:  Autophagy; CHMP4B; TFEB; intervertebral disc degeneration; lysosome; mechanical overloading
    DOI:  https://doi.org/10.1080/15548627.2024.2440844
  9. Front Aging. 2024 ;5 1509400
    Chaperone-mediated autophagy as a modulator of aging and longevity.
    S Joseph Endicott.
      Chaperone-mediated autophagy (CMA) is the lysosomal degradation of individually selected proteins, independent of vesicle fusion. CMA is a central part of the proteostasis network in vertebrate cells. However, CMA is also a negative regulator of anabolism, and it degrades enzymes required for glycolysis, de novo lipogenesis, and translation at the cytoplasmic ribosome. Recently, CMA has gained attention as a possible modulator of rodent aging. Two mechanistic models have been proposed to explain the relationship between CMA and aging in mice. Both of these models are backed by experimental data, and they are not mutually exclusionary. Model 1, the "Longevity Model," states that lifespan-extending interventions that decrease signaling through the INS/IGF1 signaling axis also increase CMA, which degrades (and thereby reduces the abundance of) several proteins that negatively regulate vertebrate lifespan, such as MYC, NLRP3, ACLY, and ACSS2. Therefore, enhanced CMA, in early and midlife, is hypothesized to slow the aging process. Model 2, the "Aging Model," states that changes in lysosomal membrane dynamics with age lead to age-related losses in the essential CMA component LAMP2A, which in turn reduces CMA, contributes to age-related proteostasis collapse, and leads to overaccumulation of proteins that contribute to age-related diseases, such as Alzheimer's disease, Parkinson's disease, cancer, atherosclerosis, and sterile inflammation. The objective of this review paper is to comprehensively describe the data in support of both of these explanatory models, and to discuss the strengths and limitations of each.
    Keywords:  aging; autophagy; chaperone-mediated autophagy; longevity; metabolism
    DOI:  https://doi.org/10.3389/fragi.2024.1509400
  10. ACS Pharmacol Transl Sci. 2024 Dec 13. 7(12): 3758-3779
    Unveiling the Role of Mechanistic Target of Rapamycin Kinase (MTOR) Signaling in Cancer Progression and the Emergence of MTOR Inhibitors as Therapeutic Strategies.
    Devashish Mehta, Kajal Rajput, Dolly Jain, Avinash Bajaj, Ujjaini Dasgupta.
      The mechanistic target of rapamycin kinase (MTOR) is pivotal for cell growth, metabolism, and survival. It functions through two distinct complexes, mechanistic TORC1 and mechanistic TORC2 (mTORC1 and mTORC2). These complexes function in the development and progression of cancer by regulating different cellular processes, such as protein synthesis, lipid metabolism, and glucose homeostasis. The mTORC1 complex senses nutrients and initiates proliferative signals, and mTORC2 is crucial for cell survival and cytoskeletal rearrangements. mTORC1 and mTORC2 have therefore emerged as potential targets for cancer treatment. Several mTOR inhibitors, including rapamycin and its analogs (rapalogs), primarily target mTORC1 and are effective for specific cancer types. However, these inhibitors often lead to resistance and limited long-term advantages due to the activation of survival pathways through feedback mechanisms. Researchers have created next-generation inhibitors targeting mTORC1 and mTORC2 and dual PI3K/mTOR inhibitors to address these difficulties. These inhibitors demonstrate enhanced anti-tumor effects by simultaneously disrupting multiple signaling pathways and show promise for improved and long-lasting therapies. However, development of resistance and adverse side effects remain a significant obstacle. Recent additions known as RapaLinks have emerged as a boon to counter drug-resistant cancer cells, as they are more potent and provide a more comprehensive blockade of mTOR signaling pathways. This Review combines current research findings and clinical insights to enhance our understanding of the crucial role of mTOR signaling in cancer biology and highlights the evolution of mTOR inhibitors as promising therapeutic approaches.
    DOI:  https://doi.org/10.1021/acsptsci.4c00530
  11. Eur J Nutr. 2024 Dec 16. 64(1): 46
    Isothiocyanates induce autophagy and inhibit protein synthesis in primary cells via modulation of AMPK-mTORC1-S6K1 signaling pathway, and protect against mutant huntingtin aggregation.
    Joanna Brokowska, Anna Herman-Antosiewicz, Aleksandra Hać.
       PURPOSE: Autophagy is a degradation process whose activation underlies beneficial effects of caloric restriction. Isothiocyanates (ITCs) induce autophagy in cancer cells, however, their impact on primary cells remains insufficiently explored, particularly in non-epithelial cells. The aim of this study was to investigate whether ITCs induce autophagy in primary (non-immortalized) mesenchymal cells and if so, to determine the molecular mechanism underlying its activation and consequences on cell functioning.
    METHODS: Primary human dermal fibroblasts (HDFa) and prostate cancer cells (PC3) as well as two ITCs, sulforaphane and phenethyl isothiocyanate, were applied. Cell viability was measured by the MTT test, protein synthesis - by 3H-leucine incorporation, and protein level - by immunoblotting. A number of mutant huntingtin (mHtt) aggregates was assessed by fluorescence microscopy.
    RESULTS: Both ITCs efficiently induced autophagy in fibroblasts which coincided with suppression of mTORC1 - a negative autophagy regulator - and protein synthesis arrest. A dephosphorylation of mTORC1 substrate, S6K1, and ribosomal S6 protein was preceded by activation of AMPK, an inhibitor of mTORC1 and autophagy activator. A similar response was observed in phenethyl isothiocyanate-treated prostate cancer cells. We also showed that ITCs-induced autophagy and/or translation block do not affect cells viability and can protect cells against an accumulation of mHtt aggregates - a main cause of Huntington's disease.
    CONCLUSION: Our study showed that ITCs induce autophagy and inhibit protein synthesis in both primary mesenchymal and cancer cells via modulation of the AMPK-mTORC1-S6K1 pathway. Moreover, it suggests that ITCs might have a potential in developing therapeutics for Huntington's disease.
    Keywords:  AMPK; Autophagy; Huntington’s disease (HD); Isothiocyanates; Mutant huntingtin (mHtt); Primary cells
    DOI:  https://doi.org/10.1007/s00394-024-03539-z
  12. Autophagy. 2024 Dec 15. 1-21
    AMPK protects proximal tubular epithelial cells from lysosomal dysfunction and dedifferentiation induced by lipotoxicity.
    Louise Pierre, Florian Juszczak, Valentine Delmotte, Morgane Decarnoncle, Benjamin Ledoux, Laurent Bultot, Luc Bertrand, Marielle Boonen, Patricia Renard, Thierry Arnould, Anne-Emilie Declèves.
      Renal proximal tubules are a primary site of injury in metabolic diseases. In obese patients and animal models, proximal tubular epithelial cells (PTECs) display dysregulated lipid metabolism, organelle dysfunctions, and oxidative stress that contribute to interstitial inflammation, fibrosis and ultimately end-stage renal failure. Our research group previously pointed out AMP-activated protein kinase (AMPK) decline as a driver of obesity-induced renal disease. Because PTECs display high macroautophagic/autophagic activity and rely heavily on their endo-lysosomal system, we investigated the effect of lipid stress on autophagic flux and lysosomes in these cells. Using a model of highly differentiated primary PTECs challenged with palmitate, our data placed lysosomes at the cornerstone of the lipotoxic phenotype. As soon as 6 h after palmitate exposure, cells displayed impaired lysosomal acidification subsequently leading to autophagosome accumulation and activation of lysosomal biogenesis. We also showed the inability of lysosomal quality control to restore acidic pH which finally drove PTECs dedifferentiation. When palmitate-induced AMPK activity decline was prevented by AMPK activators, lysosomal acidification and the differentiation profile of PTECs were preserved. Our work provided key insights on the importance of lysosomes in PTECs homeostasis and lipotoxicity and demonstrated the potential of AMPK in protecting the organelle from lipid stress.Abbreviation: ACAC: acetyl-CoA carboxylase; ACTB: actin beta; AICAR: 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; AMPK: AMP-activated protein kinase; APQ1: aquaporin 1 (Colton blood group); BSA: bovine serum albumin; CDH16: cadherin 16; CKD: chronic kidney disease; CTSB: cathepsin B; CTSD: cathepsin D; EPB41L5: erythrocyte membrane protein band 4.1 like 5; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; EMT: epithelial-to-mesenchymal transition; FA: fatty acid; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GFP: green fluorescent protein; GUSB: glucuronidase beta; HEXB: hexosaminidase subunit beta; LAMP: lysosomal associated membrane protein; LD: lipid droplet; LGALS3: galectin 3; LLOMe: L-leucyl-L-leucine methyl ester hydrobromide; LMP: lysosomal membrane permeabilization; LRP2: LDL receptor related protein 2; LSD: lysosomal storage disorder; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCOLN1: mucolipin TRP cation channel 1; MG132: N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal; MmPTECs: Mus musculus (mouse) proximal tubular epithelial cells; MTORC1: mechanistic target of rapamycin kinase complex 1; OA: oleate; PA: palmitate; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PTs: proximal tubules; PTECs: proximal tubular epithelial cells; PRKAA: protein kinase AMP-activated catalytic subunit alpha; RFP: red fluorescent protein; RPS6KB: ribosomal protein S6 kinase B; SLC5A2: solute carrier family 5 member 2; SOX9: SRY-box transcription factor 9; SQSTM1: sequestosome 1; TFEB: transcription factor EB; Ub: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; VIM: vimentin.
    Keywords:  AMPK; Autophagy; chronic kidney disease; lipid accumulation; obesity; proximal tubules
    DOI:  https://doi.org/10.1080/15548627.2024.2435238
  13. Adv Mater. 2024 Dec 17. e2413614
    Polyphenol Mediated Assembly: Tailored Nano-Dredger Unblocks Axonal Autophagosomes Retrograde Transport Traffic Jam for Accelerated Alzheimer's Waste Clearance.
    Ran Meng, Yixian Li, Xiyu Yang, Yunlong Cheng, Minjun Xu, LingLing Zhou, Chengqin Wu, Shuai Yu, Wenyi Huang, Tianying Wang, Qizhi Zhang.
      Clear-cut evidence has linked defective autophagy to Alzheimer's disease (AD). Recent studies underscore a unique hurdle in AD neuronal autophagy: impaired retrograde axonal transport of autophagosomes, potent enough to induce autophagic stress and neurodegeneration. Nonetheless, pertinent therapy is unavailable. Here, a novel combinational therapy composed of siROCK2 and lithospermic acid B (LA) is introduced, tailored to dredge blocked axonal autophagy by multi-mitigating microtubule disruption, ATP depletion, oxidative stress, and autophagy initiation impediments in AD. Leveraging the recent discovery of multi-interactions between polyphenol LA and siRNA, ε-Poly-L-lysine, and anionic lipid nanovacuoles, LA and siROCK2 are successfully co-loaded into a fresh nano-drug delivery system, LIP@PL-LA/siRC, via a ratio-flexible and straightforward fabrication process. Further modification with the TPL peptide onto LIP@PL-LA/siRC creates a brain-neuron targeted, biocompatible, and pluripotent nanomedicine, named "Nano-dredger" (T-LIP@PL-LA/siRC). Nano-dredger efficiently accelerates axonal retrograde transport and lysosomal degradation of autophagosomes, thereby facilitating the clearance of neurotoxic proteins, improving neuronal complexity, and alleviating memory defects in 3×Tg-AD transgenic mice. This study provides a fresh and flexible polyphenol/siRNA co-delivery paradigm and furnishes conceptual proof that dredging axonal autophagy represents a promising AD therapeutic avenue.
    Keywords:  alzheimer's disease; axonal retrograde transport; neuronal autophagy; polyphenol/siRNA co‐delivery; supramolecular assembly
    DOI:  https://doi.org/10.1002/adma.202413614
  14. bioRxiv. 2024 Dec 06. pii: 2024.12.05.627125. [Epub ahead of print]
    Mitophagosomes induced during EV-D68 infection promote viral nonlytic release.
    Alagie Jassey, Bimal Paudel, Michael A Wagner, Noah Pollack, I-Ting Cheng, Raquel Godoy-Ruiz, David J Weber, William T Jackson.
      Enterovirus-D68 (EV-D68) is a plus-strand RNA virus that primarily causes infant respiratory infections. In rare pediatric cases, infection with EV-D68 has been associated with acute flaccid myelitis, a polio-like paralytic disease. We have previously demonstrated that EV-D68 induces nonselective autophagy for its benefit. Here, we demonstrate that EV-D68 induces mitophagy, the specific autophagic degradation of mitochondria. EV-D68 infection induces mitophagosome formation and several hallmarks of mitophagy, including mitochondrial fragmentation, mitochondrial membrane potential loss, and Parkin translocation to the mitochondria were observed in EV-D68 infected cells. The 3C protease of EV-D68 cleaves the mitochondrial fusion protein, mitofusin-2, near the C-terminal HR2 domain to induce mitochondrial fragmentation, and these fragmented mitochondria colocalized with double-stranded RNA (dsRNA), which labels viral RNA replication sites after peak viral RNA replication. Depleting components of mitophagy signaling specifically reduced EV-D68 release without impacting viral intracellular titers. Our results suggest that whereas the machinery of macroautophagy supports various stages of enterovirus replication, including viral genomic RNA replication and capsid maturation, mitophagy is the specific form of autophagy that regulates the nonlytic release of enteroviruses from cells.
    Keywords:  BCL2L13; EV-D68; Mitofusin-2; Mitophagy; PINK1; Parkin
    DOI:  https://doi.org/10.1101/2024.12.05.627125
  15. Genes Cells. 2025 Jan;30(1): e13185
    Rapamycin Abrogates Aggregation of Human α-Synuclein Expressed in Fission Yeast via an Autophagy-Independent Mechanism.
    Yoshitaka Sugimoto, Teruaki Takasaki, Ryuga Yamada, Ryo Kurosaki, Tomonari Yamane, Reiko Sugiura.
      Aggregation of alpha-synuclein (α-Syn) is implicated in the pathogenesis of several neurodegenerative disorders, such as Parkinson's disease and Dementia with Lewy bodies, collectively termed synucleinopathies. Thus, tremendous efforts are being made to develop strategies to prevent or inhibit α-Syn aggregation. Here, we genetically engineered fission yeast to express human α-Syn C-terminally fused to green fluorescent protein (GFP) at low and high levels. α-Syn was localized at the cell tips and septa at low-level expression. At high-level expression, α-Syn was observed to form cytoplasmic aggregates. Notably, rapamycin, a natural product that allosterically inhibits the mammalian target of rapamycin (mTOR) by forming a complex with FKBP12, and Torin1, a synthetic mTOR inhibitor that blocks ATP binding to mTOR, markedly reduced the number of cells harboring α-Syn aggregates. These mTOR inhibitors abrogate α-Syn aggregation without affecting α-Syn expression levels. Rapamycin, but not Torin1, failed to reduce α-Syn aggregation in the deletion cells of fkh1+, encoding FKBP12, indicating the requirement of FKBP12 for rapamycin-mediated inhibition of α-Syn aggregation. Importantly, the effect of rapamycin was also observed in the cells lacking atg1+, a key regulator of autophagy. Collectively, rapamycin abrogates human α-Syn aggregation expressed in fission yeast via an autophagy-independent mechanism mediated by FKBP12.
    Keywords:  FKBP12; Parkinson's disease; fission yeast; mTOR; rapamycin; α‐Synuclein
    DOI:  https://doi.org/10.1111/gtc.13185
  16. Nat Commun. 2024 Dec 17. 15(1): 10670
    Macronucleophagy maintains cell viability under nitrogen starvation by modulating micronucleophagy.
    Ziyang Li, Keisuke Mochida, Hitoshi Nakatogawa.
      Lysosome/vacuole-mediated intracellular degradation pathways, collectively known as autophagy, play crucial roles in the maintenance and regulation of various cellular functions. However, little is known about the relationship between different modes of autophagy. In the budding yeast Saccharomyces cerevisiae, nitrogen starvation triggers both macronucleophagy and micronucleophagy, in which nuclear components are degraded via macroautophagy and microautophagy, respectively. We previously revealed that Atg39-mediated macronucleophagy is important for cell survival under nitrogen starvation; however, the underlying mechanism remains unknown. Here, we reveal that defective Atg39-mediated macronucleophagy leads to the hyperactivation of micronucleophagy, resulting in the excessive transport of various nuclear components into the vacuole. Micronucleophagy occurs at the nucleus-vacuole junction (NVJ). We show that nuclear membrane proteins localized to the NVJ, including Nvj1, which is responsible for micronucleophagy, are degraded via macronucleophagy. Therefore, defective Atg39-mediated macronucleophagy results in the accumulation of Nvj1, which contributes to micronucleophagy enhancement. Blocking micronucleophagy almost completely suppresses cell death caused by the absence of Atg39, whereas enhanced micronucleophagy correlates with death in Atg39-mutant cells under nitrogen starvation. These results suggest that macronucleophagy modulates micronucleophagy in order to prevent the excess removal of nuclear components, thereby maintaining nuclear and cellular homeostasis during nitrogen starvation.
    DOI:  https://doi.org/10.1038/s41467-024-55045-9
  17. Chem Biol Interact. 2024 Dec 17. pii: S0009-2797(24)00502-7. [Epub ahead of print] 111356
    Review on the role of autophagy in the toxicity of nanoparticles and the signaling pathways involved.
    Na Liu, Bo Zhang, Nengming Lin.
      As the development of nanotechnology, the application of nanoproducts and the advancement of nanomedicine, the contact of nanoparticles (NPs) with human body is becoming increasingly prevalent. This escalation elevates the risk of NPs exposure for workers, consumers, researchers, and both aquatic and terrestrial organisms throughout the production, usage, and disposal stages. Consequently, evaluating nanotoxicity remains critically important, though standardized assessment criteria are still lacking. The diverse and complex properties of NPs further complicate the understanding of their toxicological mechanisms. Autophagy, a fundamental cellular process, exhibits dual functions-both pro-survival and pro-death. This review offers an updated perspective on the dual roles of autophagy in nanotoxicity and examines the factors influencing autophagic responses. However, no definitive framework exists for predicting NPs-induced autophagy. Beyond the conventional autophagy pathways, the review highlights specific transcription factors activated by NPs and explores metabolic reprogramming. Particular attention is given to NPs-induced selective autophagy, including mitophagy, ER-phagy, ferritinophagy, lysophagy, and lipophagy. Additionally, the review investigates autophagy's involvement in NPs-mediated biological processes such as ferroptosis, inflammation, macrophage polarization, epithelial-mesenchymal transition, tumor cell proliferation and drug resistance, as well as liver and kidney injury, neurotoxicity, and other diseases. In summary, this review presents a novel update on selective autophagy-mediated nanotoxicity and elucidates the broader interactions of autophagy in NPs-induced biological processes. Collectively, these insights offer valuable strategies for mitigating nanotoxicity through autophagy modulation and advancing the development of NPs in biomedical applications.
    Keywords:  Autophagy; Ferritinophagy; Macroautophagy; Mechanisms; Mitophagy; Nanotoxicity
    DOI:  https://doi.org/10.1016/j.cbi.2024.111356
  18. Subcell Biochem. 2024 ;107 43-62
    Chaperone Activators.
    Siarhei A Dabravolski.
      Ageing is a complex yet universal and inevitable degenerative process that results in a decline in the cellular capacity for repair and adaptation to external stresses. Therefore, maintaining the appropriate balance of the cellular proteome is crucial. In addition to the ubiquitin-proteasome and autophagy-lysosomal systems, molecular chaperones play a vital role in a sophisticated protein quality control system. Chaperones are responsible for the correct protein assembly, folding, and translocation of other proteins when cells are subjected to various stresses. The equilibrium of chaperones is pivotal for maintaining health and longevity, as a deficiency in their function and quantity can contribute to the development of various diseases and accelerate the ageing processes. Conversely, their overexpression has been associated with tumour growth and progression. In this work, we discuss recent research focused on the application of various natural and artificial substances, as well as physical and nutritional stresses, to activate molecular chaperones and prolong both life- and healthspan. Furthermore, we emphasise the significance of autophagy, apoptosis, mTOR and inflammation signalling pathways in chaperone-mediated extension of life- and healthspan.
    Keywords:  Ageing; Apoptosis; Autophagy-lysosomal system; Chaperone; Longevity; Proteostasis; Ubiquitin-proteasome system
    DOI:  https://doi.org/10.1007/978-3-031-66768-8_3
  19. Small. 2024 Dec 17. e2408323
    A Binary siRNA-Loaded Tetrahedral DNA Nanobox for Synergetic Anti-Aging Therapy.
    Ziqi Yue, Yichen Yang, Lulingxiao Nie, Yuezhang Sun, Qi Wang, Yunfeng Lin, Yang Gao, Xiaoxiao Cai.
      Extensive accumulation of senescent cells contributes to organismal aging, and slowing down the process of cellular senescence may ameliorate age-related pathologies. Targeted inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) is found to suppress the conversion of cells to senescence. The regulatory-associated protein of mTOR (Raptor), a key component of mTORC1, has been implicated as important in the aging process, and its druggability deserves to be investigated. Due to high efficiency and high convenience in drug construction, siRNA shows great potential in silencing Raptor expression via RNA interfering therapy. Here, we developed a functionalized anti-aging nanoplatform based on tetrahedral DNA nanostructures (TDNs) encapsulating siRNA targeting Raptor for synergistic anti-aging therapy, named siR-TDNbox. Anti-inflammatory and antioxidant properties of TDN beneficially attenuate age-associated inflammation while serving as siRNA nanocarrier, and thus play a binary role. The results suggest that the siR-TDNbox binary therapeutic nanoplatform has demonstrated an excellent ability to delay aging, inhibit mTORC1 signaling, and extend lifespan. This anti-aging nanoplatform may provide a medium for the combined application of traditional senotherapeutic drugs and promote the upgrading of nanomaterials with anti-aging effects.
    Keywords:  DNA nanotechnology; aging; framework nucleic acids; senescence; siRNA delivery
    DOI:  https://doi.org/10.1002/smll.202408323
  20. Acta Biochim Biophys Sin (Shanghai). 2024 Dec 18.
    CD40 ligation-induced ERK activation leads to enhanced radiosensitivity in cervical carcinoma cells via promoting autophagy.
    Baocai Liu, Yadong Zhang, Quan Wang, Qian Wang, Zhixin Wang, Li Feng.
      CD40, a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role not only in the immune system but also in tumor progression. CD40 ligation reportedly promotes autophagy in immune cells. However, the effects of CD40 ligation on autophagy and its mechanism in solid tumor cells are still unclear. In this study, we find that CD40 ligation promotes autophagosome formation and consequently promotes autophagic flux in cervical cancer cells. Mechanistically, this effect relies on ERK contributing to CD40 ligation-induced ATG13 upregulation by p53. Furthermore, we demonstrate that CD40 ligation-induced autophagy increases the radiosensitivity of cervical cancer cells. Taken together, our results provide new evidence for the involvement of the CD40 pathway in autophagy and radiotherapy in cervical cancer cells.
    Keywords:  CD40; autophagy; cervical cancer; radiosensitivity
    DOI:  https://doi.org/10.3724/abbs.2024229
  21. J Diabetes. 2024 Dec;16(12): e70010
    Autophagy modulators in type 2 diabetes: A new perspective.
    Ayah Talal Zaidalkilani, Hayder M Al-Kuraishy, Esraa H Fahad, Ali I Al-Gareeb, Yaser Hosny Ali Elewa, Mahmoud Hosny Zahran, Athanasios Alexiou, Marios Papadakis, Ammar Al-Farga, Gaber El-Saber Batiha.
      Type 2 diabetes (T2D) is a chronic metabolic disorder caused by defective insulin signaling, insulin resistance, and impairment of insulin secretion. Autophagy is a conserved lysosomal-dependent catabolic cellular pathway involved in the pathogenesis of T2D and its complications. Basal autophagy regulates pancreatic β-cell function by enhancing insulin release and peripheral insulin sensitivity. Therefore, defective autophagy is associated with impairment of pancreatic β-cell function and the development of insulin rersistance (IR). However, over-activated autophagy increases apoptosis of pancreatic β-cells leading to pancreatic β-cell dysfunction. Hence, autophagy plays a double-edged sword role in T2D. Therefore, the use of autophagy modulators including inhibitors and activators may affect the pathogenesis of T2D. Hence, this review aims to clarify the potential role of autophagy inhibitors and activators in T2D.
    Keywords:  autophagyinsulin resistancetype 2 diabetes mellitus; β‐cells
    DOI:  https://doi.org/10.1111/1753-0407.70010
  22. Int J Mol Sci. 2024 Nov 22. pii: 12576. [Epub ahead of print]25(23):
    The Estrogen-Autophagy Axis: Insights into Cytoprotection and Therapeutic Potential in Cancer and Infection.
    Ying Zhao, Daniel J Klionsky, Xin Wang, Qiaoying Huang, Zixin Deng, Jin Xiang.
      Macroautophagy, commonly referred to as autophagy, is an essential cytoprotective mechanism that plays a significant role in cellular homeostasis. It has emerged as a promising target for drug development aimed at treating various cancers and infectious diseases. However, the scientific community has yet to reach a consensus on the most effective approach to manipulating autophagy, with ongoing debates about whether its inhibition or stimulation is preferable for managing these complex conditions. One critical factor contributing to the variability in treatment responses for both cancers and infectious diseases is estrogen, a hormone known for its diverse biological effects. Given the strong correlations observed between estrogen signaling and autophagy, this review seeks to summarize the intricate molecular mechanisms that underlie the dual cytoprotective effects of estrogen signaling in conjunction with autophagy. We highlight recent findings from studies that involve various ligands, disease contexts, and cell types, including immune cells. Furthermore, we discuss several factors that regulate autophagy in the context of estrogen's influence. Ultimately, we propose a hypothetical model to elucidate the regulatory effects of the estrogen-autophagy axis on cell fate. Understanding these interactions is crucial for advancing our knowledge of related diseases and facilitating the development of innovative treatment strategies.
    Keywords:  autophagy; cancer; estrogen; immunity; infection
    DOI:  https://doi.org/10.3390/ijms252312576
  23. Nutrients. 2024 Nov 22. pii: 4003. [Epub ahead of print]16(23):
    Relationship Between Dietary Nutrient Intake and Autophagy-Related Genes in Obese Humans: A Narrative Review.
    Martyna Bednarczyk, Nicola Dąbrowska-Szeja, Dariusz Łętowski, Sylwia Dzięgielewska-Gęsiak, Dariusz Waniczek, Małgorzata Muc-Wierzgoń.
      Obesity is one of the world's major public health challenges. Its pathogenesis and comorbid metabolic disorders share common mechanisms, such as mitochondrial or endoplasmic reticulum dysfunction or oxidative stress, gut dysbiosis, chronic inflammation and altered autophagy. Numerous pro-autophagy dietary interventions are being investigated for their potential obesity-preventing or therapeutic effects. We summarize current data on the relationship between autophagy and obesity, and discuss various dietary interventions as regulators of autophagy-related genes in the prevention and ultimate treatment of obesity in humans, as available in scientific databases and published through July 2024. Lifestyle modifications (such as calorie restriction, intermittent fasting, physical exercise), including following a diet rich in flavonoids, antioxidants, specific fatty acids, specific amino acids and others, have shown a beneficial role in the induction of this process. The activation of autophagy through various nutritional interventions tends to elicit a consistent response, characterized by the induction of certain kinases (including AMPK, IKK, JNK1, TAK1, ULK1, and VPS34) or the suppression of others (like mTORC1), the deacetylation of proteins, and the alleviation of inhibitory interactions between BECN1 and members of the Bcl-2 family. Significant health/translational properties of many nutrients (nutraceuticals) can affect chronic disease risk through various mechanisms that include the activation or inhibition of autophagy. The role of nutritional intervention in the regulation of autophagy in obesity and its comorbidities is not yet clear, especially in obese individuals.
    Keywords:  autophagy; dietary intake; humans; nutrients; obesity; overweight
    DOI:  https://doi.org/10.3390/nu16234003
  24. bioRxiv. 2024 Dec 05. pii: 2024.12.04.626665. [Epub ahead of print]
    Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice.
    Isabelle C Becker, Maria N Barrachina, Joshua Lykins, Virginia Camacho, Andrew P Stone, Bernadette A Chua, Robert A J Signer, Kellie R Machlus, Sidney W Whiteheart, Harvey G Roweth, Joseph E Italiano.
      Megakaryocytes (MKs) are large, polyploid cells that contribute to bone marrow homeostasis through the secretion of cytokines such as transforming growth factor β1 (TGFβ1). During neoplastic transformation, immature MKs accumulate in the bone marrow where they induce fibrotic remodeling ultimately resulting in myelofibrosis. Current treatment strategies aim to prevent MK hyperproliferation, however, little is understood about the potential of targeting dysregulated cytokine secretion from neoplastic MKs as a novel therapeutic avenue. Unconventional secretion of TGFβ1 as well as interleukin 1β (IL1β) via secretory autophagy occurs in cells other than MKs, which prompted us to investigate whether similar mechanisms are utilized by MKs. Here, we identified that TGFβ1 strongly co-localized with the autophagy marker light chain 3B in native MKs. Disrupting secretory autophagy by inhibiting the small GTPase RhoA or its downstream effector Rho kinase (ROCK) markedly reduced TGFβ1 and IL1β secretion in vitro . In vivo , conditional deletion of the essential autophagy gene Atg5 from the hematopoietic system limited megakaryocytosis and aberrant cytokine secretion in an MPL W515L -driven transplant model. Similarly, mice with a selective deletion of Rhoa from the MK and platelet lineage were protected from progressive fibrosis. Finally, disease hallmarks in MPL W515L -transplanted mice were attenuated upon treatment with the autophagy inhibitor hydroxychloroquine or the ROCK inhibitor Y27632, either as monotherapy or in combination with the JAK2 inhibitor ruxolitinib. Overall, our data indicate that aberrant cytokine secretion is dependent on secretory autophagy downstream of RhoA, targeting of which represents a novel therapeutic avenue in the treatment of myelofibrosis.
    One Sentence Summary: TGFβ1 is released from megakaryocytes via RhoA-mediated secretory autophagy, and targeting this process can alleviate fibrosis progression in a preclinical mouse model of myelofibrosis.
    DOI:  https://doi.org/10.1101/2024.12.04.626665
  25. Biochim Biophys Acta Mol Cell Res. 2024 Dec 15. pii: S0167-4889(24)00235-0. [Epub ahead of print] 119892
    Blockage of polycystin-2 alleviates myocardial ischemia/reperfusion injury by inhibiting autophagy through the Ca2+/Akt/Beclin 1 pathway.
    Xiao-Dan Qin, Jian-Feng Liang, Lin-Yu Gan, Ke-Shan Peng, Xue-Hong Huang, Xiao-Ting Li, Jin-Li Chen, Wan Li, Lei Zhang, Jie Jian, Jun Lu.
      Autophagy is a well-conserved self-protection process that plays an important role in cardiovascular diseases. Excessive autophagy during myocardial ischemia/reperfusion injury (MIRI) induces calcium overload and the overactivation of an autophagic response, thereby aggravating cardiomyocyte damage. Polycystin-2 (PC2) is a Ca2+-permeable nonselective cation channel implicated in the regulation of autophagy. In the present study, autophagy was upregulated in myocardial ischemia/reperfusion in vivo and in vitro. PC2 knockdown using adeno-associated virus9 particles containing Pkd2 short hairpin RNA infection markedly ameliorated MIRI, evidenced by reduced infarct size, diminished morphological changes, decreased cTnI levels, and improved cardiac function. Silencing PC2 reduced the autophagic flux in H9c2 cells. PC2 overexpression-mediated autophagic flux was inhibited by intracellular Ca2+ chelation with BAPTA-AM. Furthermore, PC2 ablation upregulated p-Akt (Ser473) and downregulated Beclin 1 in H/R. BAPTA-AM downregulated p-Akt(Ser473) and upregulated Beclin 1in PC2-overexpressing H9c2 cells. Moreover, the Akt inhibitor MK2206 abolished the BAPTA-AM-blunted PC2-dependent control of autophagy. Collectively, these results indicated that blockade of PC2 may be associated with the Ca2+/Akt/Beclin 1 signaling, thereby inhibiting excessive autophagy and serving as a potential strategy for mitigating MIRI.
    Keywords:  Autophagy; Calcium; Cardiovascular disease; Myocardial ischemia/reperfusion injury; Polycystin-2
    DOI:  https://doi.org/10.1016/j.bbamcr.2024.119892
  26. Cell Calcium. 2024 Dec 13. pii: S0143-4160(24)00144-1. [Epub ahead of print]125 102986
    A role for lysosomal calcium channels in mitigating mitochondrial damage and oxidative stress.
    Leandro C Clementino, Andrew P Thomas, Emily M Rocha, Sabine Hilfiker.
      Elevated free fatty acids and oxidative stress may function as pathogenic factors in endothelial dysfunction that is associated with various cardiovascular complications. In recent work, Feng and colleagues report that activation of a lysosomal Ca2+ channel may be a viable option to alleviate oxidative damage by boosting lysosome biogenesis and mitophagy.
    Keywords:  Calcium signaling; Lysosome; Mitophagy; Reactive oxygen species; TFEB; TRPML1
    DOI:  https://doi.org/10.1016/j.ceca.2024.102986
  27. Front Nutr. 2024 ;11 1409142
    Autophagy and nutrigenomics: a winning team against chronic disease and tumors.
    Roberto Chiarelli, Fabio Caradonna, Flores Naselli.
      Autophagy, a vital cell process, has garnered attention for its role in various diseases and potential therapeutic interventions. Dysregulation of autophagy contributes to conditions such as metabolic diseases, neurodegenerative disorders, and cancer. In diseases such as diabetes, autophagy plays a crucial role in islet β-cell maintenance and glucose homeostasis, offering potential targets for therapeutic intervention. Nutrigenomics, which explores how dietary components interact with the genome, has emerged as a promising avenue for disease management. It sheds light on how diet influences gene expression and cellular processes, offering personalized approaches to disease prevention and management. Studies have showed the impact of specific dietary components, such as polyphenols and omega-3 fatty acids, on autophagy processes, suggesting their potential therapeutic benefits in neurodegenerative conditions and metabolic disorders. In cancer, autophagy's dual role in either suppressing tumorigenesis or promoting cancer cell survival underscores the importance of understanding its modulation through dietary interventions. Combined with conventional chemotherapy drugs, dietary compounds show synergistic effects in cancer treatment. Furthermore, phytochemicals such as indicaxanthin have been found to epigenetically regulate genes involved in autophagy, offering novel insights into personalized cancer therapies. This comprehensive review has the aim to study the autophagy in a combined view with nutrigenomics effects of some dietary molecules in maintaining cellular homeostasis and responding to pathological stimuli. Overall, the intersection of autophagy and nutrigenomics effect of bioactive compounds holds promise for developing targeted interventions for various diseases, emphasizing the significance of dietary interventions in disease prevention and management.
    Keywords:  autophagy; dietary-genetic interactions; nutrigenomic disease strategies; nutrigenomics; personalized dietary approaches
    DOI:  https://doi.org/10.3389/fnut.2024.1409142
  28. Cells. 2024 Dec 09. pii: 2030. [Epub ahead of print]13(23):
    Gene-Silencing Therapeutic Approaches Targeting PI3K/Akt/mTOR Signaling in Degenerative Intervertebral Disk Cells: An In Vitro Comparative Study Between RNA Interference and CRISPR-Cas9.
    Masao Ryu, Takashi Yurube, Yoshiki Takeoka, Yutaro Kanda, Takeru Tsujimoto, Kunihiko Miyazaki, Hiroki Ohnishi, Tomoya Matsuo, Naotoshi Kumagai, Kohei Kuroshima, Yoshiaki Hiranaka, Ryosuke Kuroda, Kenichiro Kakutani.
      The mammalian target of rapamycin (mTOR), a serine/threonine kinase, promotes cell growth and inhibits autophagy. The following two complexes contain mTOR: mTORC1 with the regulatory associated protein of mTOR (RAPTOR) and mTORC2 with the rapamycin-insensitive companion of mTOR (RICTOR). The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway is important in the intervertebral disk, which is the largest avascular, hypoxic, low-nutrient organ in the body. To examine gene-silencing therapeutic approaches targeting PI3K/Akt/mTOR signaling in degenerative disk cells, an in vitro comparative study was designed between small interfering RNA (siRNA)-mediated RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene editing. Surgically obtained human disk nucleus pulposus cells were transfected with a siRNA or CRISPR-Cas9 plasmid targeting mTOR, RAPTOR, or RICTOR. Both of the approaches specifically suppressed target protein expression; however, the 24-h transfection efficiency differed by 53.8-60.3% for RNAi and 88.1-89.3% for CRISPR-Cas9 (p < 0.0001). Targeting mTOR, RAPTOR, and RICTOR all induced autophagy and inhibited apoptosis, senescence, pyroptosis, and matrix catabolism, with the most prominent effects observed with RAPTOR CRISPR-Cas9. In the time-course analysis, the 168-h suppression ratio of RAPTOR protein expression was 83.2% by CRISPR-Cas9 but only 8.8% by RNAi. While RNAi facilitates transient gene knockdown, CRISPR-Cas9 provides extensive gene knockout. Our findings suggest that RAPTOR/mTORC1 is a potential therapeutic target for degenerative disk disease.
    Keywords:  RNA interference (RNAi); autophagy; clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9); disk degeneration; gene-silencing therapy; intervertebral disk; nucleus pulposus (NP) cells; phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling; small interfering RNA (siRNA); spine
    DOI:  https://doi.org/10.3390/cells13232030
  29. J Agric Food Chem. 2024 Dec 16.
    Trehalose Ameliorates Nonalcoholic Fatty Liver Disease by Regulating IRE1α-TFEB Signaling Pathway.
    Shan Su, Xiaohong Liu, Min Zhu, Wen Liu, Jingyi Liu, Yujia Yuan, Fudong Fu, Zhiyong Rao, Jingping Liu, Yanrong Lu, Younan Chen.
      Nonalcoholic fatty liver disease (NAFLD), characterized by hepatic lipid deposition, is one of the most prevalent chronic metabolic disorders globally, and its pharmaceutical treatments are still limited. Excessive lipid accumulation triggers endoplasmic reticulum (ER) stress and autophagy flux dysfunction, which are important mechanisms for NAFLD. Trehalose (Tre), a natural disaccharide, has been identified to reduce hepatic steatosis and glucose intolerance. However, its underlying mechanisms for NAFLD remain unclear. In this study, a high-fat-diet (HFD)-induced mouse NAFLD model and a saturated fatty acid palmitic acid (PA)-stimulated cell model were constructed. The results indicated that Tre supplementation ameliorated hepatocyte lipid deposition in vitro, as well as hepatic steatosis and hyperlipidemia in vivo. Mechanistically, Tre alleviated both autophagy flux dysfunction and endoplasmic reticulum (ER) stress. Under the stimulation of HFD or PA, Tre remarkably increased the expression and nucleic translocation of the lysosomal master protein transcription factor EB (TFEB), while decreasing the accumulation of p62 and also decreasing the ER stress markers (inositol-requiring enzyme 1 (IRE1α), XBP-1, CHOP, and BIP). Similar results were observed in an ER stressor tunicamycin (TM)-induced in vivo and in vitro models. In addition, the transcriptomic analysis of NAFLD patients revealed significant differences in ER stress-related and autophagy-related biomarkers, including TFEB, ATG7, IRE1α, and CHOP. Molecular docking results demonstrated a strong affinity between Tre and both IRE1α and TFEB. Overall, Tre protected hepatocytes from lipotoxicity-related ER stress and autophagy dysfunction, and its regulatory effect on the IRE1α-TFEB signaling pathway may be a critical mechanism. These findings suggest that Tre, as a bioactive substance with significant medicinal potential, holds considerable promise for drug development and clinical application in treating NAFLD.
    Keywords:  IRE1α; autophagy; endoplasmic reticulum stress; nonalcoholic fatty liver disease; transcriptional factor EB; trehalose
    DOI:  https://doi.org/10.1021/acs.jafc.4c08669
  30. Blood. 2024 Dec 18. pii: blood.2023022688. [Epub ahead of print]
    Mutations in AMBRA1 aggravate β-thalassemia by impairing autophagy-mediated clearance of free α-globin.
    Yong Long, Qianqian Zhang, Ling Ling, Yuan Zhuang, Xiaolei Wei, Haoyang Huang, Zhanping Lu, Yushan Huang, Xianming Chen, Yuhua Ye, Xiaoqin Feng, Haokun Hao Zhang, Binbin Huang, Yueyan Huang, Yidan Liang, Mingyan Fang, Yukio Nakamura, Bin Lin, Xinhua Zhang, Daru Lu, Xin Jin, Xiangmin Xu.
      Accumulation of free α-globin is a critical factor in the pathogenesis of β-thalassemia. Autophagy plays a crucial role in clearing toxic free α-globin, thereby reducing disease severity. However, the impact of natural mutations in autophagy-related genes (ATGs) on the phenotypic variability of β-thalassemia remains unclear. In this study, we systematically investigated the relationship between variants in ATGs and disease phenotypes in a cohort of 1,022 patients with β-thalassemia, identifying four missense mutations in the autophagy and beclin 1 regulator 1 (AMBRA1) gene. Disruption of the Ambra1 gene in β-thalassemic mice was found to reduce autophagic clearance of α-globin in red blood cell precursors, exacerbating disease phenotypes. Functional characterization of the AMBRA1 gene and these mutations in patient-derived CD34+ cells, edited HUDEP-2 cells, and engineered HUDEP-2 β-thalassemic cells confirmed that AMBRA1 facilitates the autophagic clearance of free α-globin in human erythroid cells. Functional studies demonstrated that AMBRA1 missense mutants destabilize ULK1 protein, inhibit LC3 lipidation, and subsequently hinder autophagic flux, leading to increased α-globin deposition. Additionally, these mutations were associated with erythrotoxic effects in vitro, including increased intracellular reactive oxygen species levels, higher apoptosis rates, and impaired erythroid differentiation and maturation. This study sheds light on the molecular association between mutations in ATGs and the exacerbation of β-thalassemia, highlighting the potential role of the AMBRA1 gene as a promising diagnostic and therapeutic target for β-hemoglobinopathies.
    DOI:  https://doi.org/10.1182/blood.2023022688
  31. iScience. 2024 Dec 20. 27(12): 111413
    BMAL1 upregulates STX17 levels to promote autophagosome-lysosome fusion in hippocampal neurons to ameliorate Alzheimer's disease.
    Xiuya Zhou, Kaili Du, Tian Mao, Ning Wang, Lifei Zhang, Yuan Tian, Ting Liu, Li Wang, Xiaohui Wang.
      We aim to investigate muscle ARNT-like protein 1 (BMAL1) regulation of syntaxin17 (STX17) in mouse hippocampal neurons, focusing on autophagy and amyloid-β (Aβ) deposition. Autophagosome-lysosome fusion in APP/PS1 hippocampal tissues was observed using transmission electron microscopy, while mRNA levels of LC3II and P62 were measured via reverse-transcription PCR (RT-PCR) after Amyloid precursor protein (APP) overexpression. STX17, linked to autophagy and differentially expressed in Alzheimer's disease (AD) brains, was knocked down or overexpressed to assess its effects. The results showed that reduced STX17 impairs autophagosome-lysosome fusion, leading to abnormal Aβ deposition. Coimmunoprecipitation (Co-IP) and immunofluorescence confirmed STX17 interaction with SNAP29 and VAMP8 to form SNARE complexes. Furthermore, BMAL1 binding to STX17 was examined using luciferase assays. Circadian rhythm disturbances and decreased BMAL1 expression in APP/PS1 mice were noted, while BMAL1 overexpression upregulated STX17 expression and promoted autophagy to reduce Aβ deposition. Thus, the BMAL1 protein can promote STX17 transcription to induce STX17-SNAP29-VAMP8 complex formation to clear intracellular Aβ through autophagy.
    Keywords:  Cellular neuroscience; Molecular neuroscience; Neuroscience
    DOI:  https://doi.org/10.1016/j.isci.2024.111413
  32. J Cell Sci. 2024 Dec 15. pii: jcs262036. [Epub ahead of print]137(24):
    Casein kinase 1 controls components of a TORC2 signaling network in budding yeast.
    Rafael Lucena, Akshi Jasani, Steph Anastasia, Douglas Kellogg, Maria Alcaide-Gavilan.
      Tor kinases play diverse and essential roles in control of nutrient signaling and cell growth. These kinases are assembled into two multiprotein complexes known as TORC1 and TORC2. In budding yeast, TORC2 relays nutrient-dependent signals that strongly influence growth rate and cell size. However, the mechanisms that control TORC2 signaling are poorly understood. Activation of TORC2 requires Mss4, a phosphatidylinositol 4-phosphate 5-kinase that recruits and activates downstream targets of TORC2. Localization of Mss4 to the plasma membrane is thought to be controlled by phosphorylation, and previous work has suggested that yeast homologs of casein kinase 1, Yck1 and Yck2 (referred to here collectively as Yck1/2), Control phosphorylation of Mss4. Here, we generated a new analog-sensitive allele of YCK2 and used it to test whether Yck1/2 influence localization of Mss4 or signaling in the TORC2 network. We found that Yck1/2 strongly influence Mss4 phosphorylation and localization, as well as influencing regulation of multiple components of the TORC2 network. However, inhibition of Yck1/2 causes mild effects on the best-characterized signaling axis in the TORC2 pathway, suggesting that Yck1/2 might play a larger role in influencing less well-understood aspects of TORC2 signaling.
    Keywords:  Casein kinase 1; Nutrients; PP2A; TORC2 signaling
    DOI:  https://doi.org/10.1242/jcs.262036
  33. Transl Neurodegener. 2024 Dec 16. 13(1): 62
    Astrocytes contribute to toll-like receptor 2-mediated neurodegeneration and alpha-synuclein pathology in a human midbrain Parkinson's model.
    Fiona Weiss, Laura Hughes, Yuhong Fu, Cedric Bardy, Glenda M Halliday, Nicolas Dzamko.
       BACKGROUND: Parkinson's disease (PD) is characterised by degeneration of ventral midbrain dopaminergic (DA) neurons and abnormal deposition of α-synuclein (α-syn) in neurons. Activation of the innate immune pathogen recognition receptor toll-like receptor 2 (TLR2) is associated with exacerbation of α-syn pathology. TLR2 is increased on neurons in the PD brain, and its activation results in the accumulation and propagation of α-syn through autophagy inhibition in neurons. In addition to the aggregation and propagation of pathological α-syn, dysfunction of astrocytes may contribute to DA neuronal death and subsequent clinical progression of PD. However, the role of astrocytes in TLR2-mediated PD pathology is less explored but important to address, given that TLR2 is a potential therapeutic target for PD.
    METHODS: Induced pluripotent stem cells from three controls and three PD patients were differentiated into a midbrain model comprised of neurons (including DA neurons) and astrocytes. Cells were treated with or without the TLR2 agonist Pam3CSK4, and α-syn pathology was seeded using pre-formed fibrils. Confocal imaging was used to assess lysosomal function and α-syn pathology in the different cell types, as well as DA neuron health and astrocyte activation.
    RESULTS: TLR2 activation acutely impaired the autophagy lysosomal pathway, and potentiated α-syn pathology seeded by pre-formed fibrils in PD neurons and astrocytes, leading to degeneration and loss of DA neurons. The astrocytes displayed impaired chaperone-mediated autophagy reducing their ability to clear accumulated α-syn, and increases of A1 neurotoxic phenotypic proteins SerpinG1, complement C3, PSMB8 and GBP2. Moreover, the phenotypic changes in astrocytes correlated with a specific loss of DA neurons.
    CONCLUSIONS: Taken together, these results support a role for astrocyte dysfunction in α-syn accumulation and DA neuronal loss following TLR2 activation in PD.
    Keywords:  Alpha-synuclein; Astrocyte; Lysosome; Parkinson’s disease; Toll-like receptor 2
    DOI:  https://doi.org/10.1186/s40035-024-00448-3
  34. Cell Death Discov. 2024 Dec 18. 10(1): 493
    Phytochemical-mediated efferocytosis and autophagy in inflammation control.
    Asma Vafadar, Amir Tajbakhsh, Fatemeh Hosseinpour-Soleimani, Amir Savardshtaki, Mohammad Hashem Hashempur.
      Efferocytosis, the clearance of apoptotic cells, is a critical process that maintains tissue homeostasis and immune regulation. Defective efferocytosis is linked to the development of chronic inflammatory conditions, including atherosclerosis, neurological disorders, and autoimmune diseases. Moreover, the interplay between autophagy and efferocytosis is crucial for inflammation control, as autophagy enhances the ability of phagocytic cells. Efficient efferocytosis, in turn, regulates autophagic pathways, fostering a balanced cellular environment. Dysregulation of this balance can contribute to the pathogenesis of various disorders. Phytochemicals, bioactive compounds found in plants, have emerged as promising therapeutic agents owing to their diverse pharmacological properties, including antioxidant, anti-inflammatory, and immunomodulatory effects. This review aims to highlight the pivotal role of phytochemicals in enhancing efferocytosis and autophagy and explore their potential in the prevention and treatment of related disorders. This study examines how phytochemicals influence key aspects of efferocytosis, including phagocytic cell activation, macrophage polarization, and autophagy induction. The therapeutic potential of phytochemicals in atherosclerosis and neurological diseases is highlighted, emphasizing their ability to enhance efferocytosis and autophagy and reduce inflammation. This review also discusses innovative approaches, such as nanoformulations and combination therapies to improve the targeting and bioavailability of phytochemicals. Ultimately, this study inspires further research and clinical applications in phytochemical-mediated efferocytosis enhancement for managing chronic inflammatory and autoimmune conditions.
    DOI:  https://doi.org/10.1038/s41420-024-02254-2
  35. Extracell Vesicles Circ Nucl Acids. 2022 ;3(4): 393-421
    A kaleidoscopic view of extracellular vesicles in lysosomal storage disorders.
    Charlotte V Hegeman, Olivier G de Jong, Magdalena J Lorenowicz.
      Extracellular vesicles (EVs) are a heterogeneous population of stable lipid membrane particles that play a critical role in the regulation of numerous physiological and pathological processes. EV cargo, which includes lipids, proteins, and RNAs including miRNAs, is affected by the metabolic status of the parental cell. Concordantly, abnormalities in the autophagic-endolysosomal pathway, as seen in lysosomal storage disorders (LSDs), can affect EV release as well as EV cargo. LSDs are a group of over 70 inheritable diseases, characterized by lysosomal dysfunction and gradual accumulation of undigested molecules. LSDs are caused by single gene mutations that lead to a deficiency of a lysosomal protein or lipid. Lysosomal dysfunction sets off a cascade of alterations in the endolysosomal pathway that can affect autophagy and alter calcium homeostasis, leading to energy imbalance, oxidative stress, and apoptosis. The pathophysiology of these diseases is very heterogenous, complex, and currently incompletely understood. LSDs lead to progressive multisystemic symptoms that often include neurological deficits. In this review, a kaleidoscopic overview will be given on the roles of EVs in LSDs, from their contribution to pathology and diagnostics to their role as drug delivery vehicles. Furthermore, EV cargo and surface engineering strategies will be discussed to show the potential of EVs in future LSD treatment, both in the context of enzyme replacement therapy, as well as future gene editing strategies like CRISPR/Cas. The use of engineered EVs as drug delivery vehicles may mask therapeutic cargo from the immune system and protect it from degradation, improving circulation time and targeted delivery.
    Keywords:  Lysosomal storage disorders; autophagy; diagnostics; extracellular vesicles; therapeutics
    DOI:  https://doi.org/10.20517/evcna.2022.41
  36. Br J Pharmacol. 2024 Dec 15.
    Cordycepin ameliorates autoimmunity by promoting STING degradation via autophagy pathway.
    Daidi Yang, Niannian Peng, Hongqian Zhang, Zuocheng Qiu, Lingxiao Xu, Mingyu Pan.
       BACKGROUND AND PURPOSE: Stimulator of interferon response cGAMP interactor 1 (STING), a central hub protein of cyclic GMP-AMP synthase (cGAS)-STING signalling pathway, has a crucial role in regulating type I interferons (IFNs) production and response. Recent studies indicate that excessive activation of STING is strongly associated with autoimmune diseases, including systemic lupus erythematosus (SLE). Searching immunomodulators that negatively regulate STING might greatly contribute to the suppression of autoimmunity.
    EXPERIMENTAL APPROACH: The peripheral blood mononuclear cells (PBMCs) of SLE patients, Hela cells, L929 cells and bone marrow-derived macrophages (BMDMs) from mice were used as in vitro models. While, Trex1 KO mouse autoimmune disease model was used as in vivo model. After treatment with cordycepin, a nucleoside from Cordyceps mushrooms, type I IFNs production and response were determined by western blotting, real-time polymerase chain reaction (PCR), dual-luciferase assay, enzyme-linked immunosorbent assay (ELISA), haematoxylin-eosin staining and RNA-seq.
    KEY RESULTS: Cordycepin inhibited type I IFNs production and response in human and murine systems following cGAS-STING signalling activation. Importantly, cordycepin markedly attenuates the autoinflammatory and autoimmune responses in Trex1 KO BMDMs and Trex1 KO mice. Furthermore, cordycepin effectively suppressed the production of type I IFNs and interferon-stimulated genes (ISGs) in the PBMCs of SLE patients. Mechanistically, cordycepin promoted STING degradation via autophagy pathway upon DNA stimulation.
    CONCLUSION AND IMPLICATIONS: This study shows that cordycepin promotes STING autophagic degradation to alleviate autoimmunity upon DNA stimulation. Cordycepin might be a potential therapeutic candidate for alleviating aberrant type I IFNs in autoimmune and autoinflammatory diseases.
    Keywords:  STING; autoimmune disease; autophagy; cordycepin; innate immunity
    DOI:  https://doi.org/10.1111/bph.17425
  37. Int J Mol Sci. 2024 Dec 04. pii: 13040. [Epub ahead of print]25(23):
    A Reduction in Mitophagy Is Associated with Glaucomatous Neurodegeneration in Rodent Models of Glaucoma.
    Renuka M Chaphalkar, Bindu Kodati, Prabhavathi Maddineni, Shaoqing He, Calvin D Brooks, Dorota L Stankowska, Shaohua Yang, Gulab Zode, Raghu R Krishnamoorthy.
      Glaucoma is a heterogenous group of optic neuropathies characterized by the degeneration of optic nerve axons and the progressive loss of retinal ganglion cells (RGCs), which could ultimately lead to vision loss. Elevated intraocular pressure (IOP) is a major risk factor in the development of glaucoma, and reducing IOP remains the main therapeutic strategy. Endothelin-1 (ET-1), a potent vasoactive peptide, has been shown to produce neurodegenerative effects in animal models of glaucoma. However, the detailed mechanisms underlying ET-1-mediated neurodegeneration in glaucoma are not completely understood. In the current study, using a Seahorse Mitostress assay, we report that ET-1 treatment for 4 h and 24 h time points causes a significant decline in various parameters of mitochondrial function, including ATP production, maximal respiration, and spare respiratory capacity in cultured RGCs. This compromise in mitochondrial function could trigger activation of mitophagy as a quality control mechanism to restore RGC health. Contrary to our expectation, we observed a decrease in mitophagy following ET-1 treatment for 24 h in cultured RGCs. Using Morrison's model of ocular hypertension in rats, we investigated here, for the first time, changes in mitophagosome formation by analyzing the co-localization of LC-3B and TOM20 in RGCs. We also injected ET-1 (24 h) into transgenic GFP-LC3 mice to analyze the formation of mitophagosomes in vivo. In Morrison's model of ocular hypertension, as well as in ET-1 injected GFP-LC3 mice, we found a decrease in co-localization of LC3 and TOM20, indicating reduced mitophagy. Taken together, these results demonstrate that both ocular hypertension and ET-1 administration in rats and mice lead to reduced mitophagy, thus predisposing RGCs to neurodegeneration.
    Keywords:  glaucoma; mitophagy; neurodegeneration
    DOI:  https://doi.org/10.3390/ijms252313040
  38. Cell Biol Toxicol. 2024 Dec 20. 41(1): 1
    JAG1 mediates apoptosis in herpes simplex keratitis by suppressing autophagy via ROS/JAG1/NOTCH1/pULK1 signaling pathway.
    Jingyao Chang, Yao Yao, Xinghong Sun, Wenzhe Wang, Haochen Qian, Yumeilan Liu, Chunyan Xue, Wei Ye, Feng Jiang.
      Herpes simplex keratitis (HSK), an ocular disease resulted from herpes simplex virus type 1 (HSV-1) infection, leads to the majority of infectious corneal blindness worldwide. The apoptosis of corneal epithelial cells (CECs) resulted from HSV-1 disrupts the epithelial barrier and exacerbates the infection; however, there is no definitive cure for HSK. Jagged1 (JAG1), one of the primary functional ligands for NOTCH receptors, plays a crucial role in regulating apoptosis and autophagy; however, its role in HSK is unclear. Our transcriptome analysis showed JAG1 was significantly upregulated in HSV-1-infected human CECs. We aimed to explore JAG1's role in regulating apoptosis in HSV-1-infected human CECs and in HSK mice. HSV-1 infection induced apoptosis and reactive oxygen species (ROS) generation in CECs. HSV-1 also activated the JAG1/NOTCH1 signaling pathway. The ROS scavenger N-acetylcysteine significantly mitigated these effects. Additionally, inhibiting the JAG1/NOTCH1 pathway with short hairpin RNA against JAG1 or a NOTCH1 inhibitor (N-[N-{3,5-difuorophenacetyl}-1-alanyl]-S-phenylglycine t-butyl ester [DAPT]) alleviated HSV-1-induced CEC apoptosis. Transmission electron microscopy and western blotting revealed that HSV-1 infection suppressed ULK1-mediated autophagy in CECs, while DAPT treatment enhanced autophagy by suppressing ULK1 phosphorylation. The activation of autophagy by rapamycin treatment markedly reduced ROS levels and apoptosis in HSV-1-infected CECs, revealing a synergistic effect between the suppressed autophagy and increased ROS levels, ultimately leading to apoptosis. Thus, HSV-1 induces CEC apoptosis by suppressing autophagy through ROS/JAG1/NOTCH1/pULK1 signaling pathway in vitro and in vivo, providing potential therapeutic targets for HSK.
    Keywords:  Apoptosis; Autophagy; Corneal epithelial cell; Herpes simplex virus; JAG1; Reactive oxygen species
    DOI:  https://doi.org/10.1007/s10565-024-09968-0
  39. Commun Biol. 2024 Dec 19. 7(1): 1668
    Interplay between β-propeller subunits WDR26 and muskelin regulates the CTLH E3 ligase supramolecular complex.
    Matthew E R Maitland, Gabriel Onea, Dominic D G Owens, Brianna C Gonga-Cavé, Xu Wang, Cheryl H Arrowsmith, Dalia Barsyte-Lovejoy, Gilles A Lajoie, Caroline Schild-Poulter.
      The Pro/N-degron recognizing C-terminal to LisH (CTLH) complex is an E3 ligase of emerging interest in the developmental biology field and for targeted protein degradation (TPD) modalities. The human CTLH complex forms distinct supramolecular ring-shaped structures dependent on the multimerization of WDR26 or muskelin β-propeller proteins. Here, we find that, in HeLa cells, CTLH complex E3 ligase activity is dictated by an interplay between WDR26 and muskelin in tandem with muskelin autoregulation. Proteomic experiments revealed that complex-associated muskelin protein turnover is a major ubiquitin-mediated degradation event dependent on the CTLH complex in unstimulated HeLa cells. We observed that muskelin and WDR26 binding to the scaffold of the complex is interchangeable, indicative of the formation of separate WDR26 and muskelin complexes, which correlated with distinct proteomes in WDR26 and muskelin knockout cells. We found that mTOR inhibition-induced degradation of Pro/N-degron containing protein HMGCS1 is distinctly regulated by a muskelin-specific CTLH complex. Finally, we found that mTOR inhibition also activated muskelin degradation, likely as an autoregulatory feedback mechanism to regulate CTLH complex activity. Thus, rather than swapping substrate receptors, the CTLH E3 ligase complex controls substrate selectivity through the differential association of its β-propeller oligomeric subunits WDR26 and muskelin.
    DOI:  https://doi.org/10.1038/s42003-024-07371-3
  40. Cytokine Growth Factor Rev. 2024 Dec 12. pii: S1359-6101(24)00094-7. [Epub ahead of print]
    LC3B: A microtubule-associated protein influences disease progression and prognosis.
    Yan Chen, Hong Yi, Shan Liao, Junyu He, Yanhong Zhou, Yan Lei.
      Microtubule-associated protein 1 light chain 3B (MAP1LC3B, also known as LC3B) is a mammalian homolog of the autophagy-related protein 8 (ATG8) family. It plays a crucial role in cellular autophagy and is involved in several vital biological processes, including apoptosis and differentiation. Additionally, LC3B regulates immune responses. Due to its close association with malignant tumors and neurodegenerative diseases, and its potential as a prognostic indicator and therapeutic target, LC3B has become a significant research focus. This article aims to provide a comprehensive and systematic understanding of LC3B's role and mechanisms in autophagy, its impact on apoptosis and the underlying mechanisms, its regulation of cellular differentiation and transdifferentiation, its modulation of immune and inflammatory responses, the influence of upstream regulatory factors on LC3B's function, and its relevance to disease diagnosis, treatment, and prognosis. The goal is to establish a solid foundation for understanding LC3B's role in cellular processes and its regulatory mechanisms.
    Keywords:  Autophagy; Carcinoma; LC3B; Pathogenesis; Tumor
    DOI:  https://doi.org/10.1016/j.cytogfr.2024.11.006
  41. Autophagy. 2024 Dec 17. 1-20
    CKAP4 in hepatocellular carcinoma: competitive RETREG1/FAM134B binding, reticulophagy regulation, and cancer progression.
    Jie Mo, Chen Su, Pengcheng Li, Zhenhua Yang, Ran Tao, Qiumeng Liu, Chaoyi Yuan, Lei Xu, Qianyun Ge, Deng Ning, Huifang Liang, Haidan Zhu, Yan Luo, Xiaoping Chen, Jin Chen, Bixiang Zhang.
      RETREG1/FAM134B is known for its role as a reticulophagy receptor. Our previous study established that RETREG1 is upregulated in hepatocellular carcinoma (HCC) and contributes to disease progression by activating the AKT signaling pathway. However, the specific mechanisms underlying the elevated expression of RETREG1 in HCC remain unclear. This study unveils the interaction of RETREG1 with CKAP4 and TRIM21. We demonstrated that TRIM21 ubiquitinates RETREG1 at K247 and K252, facilitating its proteasomal degradation. Conversely, CKAP4 shields RETREG1 from degradation by competitively binding to it, revealing a novel post-translational modification mechanism for RETREG1. By modulating RETREG1 expression, CKAP4, and TRIM21 intricately regulate reticulophagy. Additionally, we observed that stress-induced TRIM21 upregulation mitigates the function of RETREG1 to restore ER stress equilibrium. The oncogenic potential of CKAP4 in HCC was demonstrated using various animal models. Clinical sample analyses suggested that CKAP4 is a potential biomarker for HCC prognosis and diagnosis.Abbreviation: AKT: thymoma viral proto-oncogene; aa: amino acid; bp: base pair; CHX: cycloheximide; co-IP: co-Immunoprecipitation; CQ: chloroquine; CKAP4: cytoskeleton-associated protein 4; DKK1: dickkopf WNT signaling pathway inhibitor 1; DUBs: deubiquitinating enzymes; EBSS: Earle's balanced salt solution; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HCC: hepatocellular carcinoma; HFD: high-fat diet; HiTV: hyperdynamic tail vein injection; IF: immunofluorescence; IHC: immunohistochemistry; IP-MS: immunoprecipitation-mass spectrometry; LIR: LC3-interacting region; mAbs: monoclonal antibodies; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mCherry: monomeric cherry; oe: overexpression; PDX: patient-derived tumor xenograft; reticulophagy: endoplasmic reticulum selective autophagy; RETREG1: reticulophagy regulator 1; RHD: reticulon-homology domain; Tg: thapsigargin; Tm: tunicamycin; TRIM21: tripartite motif-containing 21; UB: ubiquitin; WT: wild-type.
    Keywords:  Autophagy; TRIM21; proteasome degradation; reticulophagy; ubiquitination
    DOI:  https://doi.org/10.1080/15548627.2024.2435236
  42. Mol Brain. 2024 Dec 19. 17(1): 96
    Nek6 regulates autophagy through the mTOR signaling pathway to alleviate cerebral ischemia-reperfusion injury.
    Qingzhi Wang, Xinjing Liu, Jing Yuan, Ting Yang, Lan Ding, Bo Song, Yuming Xu.
       OBJECTIVE: Cerebral ischemia-reperfusion injury (CIRI) is a major obstacle to neurological recovery after clinical treatment of ischemic stroke. The aim of this study was to investigate the molecular mechanism of Nek6 alleviating CIRI through autophagy after cerebral ischemia.
    MATERIALS AND METHODS: A mouse model of CIRI was constructed by middle cerebral artery occlusion (MCAO). TUNEL staining was used to observe the apoptosis of neuronal cells. The oxygen glucose deprivation/reoxygenation (OGD/R) model was established by hypoxia and reoxygenation. The cell apoptosis and activity was detected. Western blot was performed to detect the expression of autophagy-related proteins, protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and adenosine 5'-monophosphate-activated protein kinase (AMPK)/mTOR signaling pathway-related proteins. Cellular autophagy flux was observed by fluorometric method. NIMA-related kinase 6 (Nek6) mRNA stability was detected by actinomycin D treatment. Methylation RNA immunoprecipitation technique was used to detect Nek6 methylation level.
    RESULTS: Nek6 expression was increased in both MCAO and OGD/R models. Overexpression of Nek6 in OGD/R inhibited apoptosis, decreased LC3II and Beclin-1 expression, increased p62 expression, and occurred lysosome dysfunction. Interference with Nek6 has opposite results. Nek6 overexpression promoted p-Akt and p-mTOR protein expressions, inhibited p-AMPK and p-UNC-51-like kinase 1 protein expressions and cell apoptosis, while LY294002, Rapamycin or RSVA405 treatment reversed this effect. Abnormal methyltransferase·like protein 3 (METTL3) expression in CIRI enhanced m6A modification and promoted Nek6 expression level.
    CONCLUSION: This study confirmed that Nek6 regulates autophagy and alleviates CIRI through the mTOR signaling pathway, which provides a novel therapeutic strategy for patients with ischemic stroke in the future.
    Keywords:  Autophagy; Cerebral ischemia–reperfusion injury; Nek6; mTOR signaling pathway
    DOI:  https://doi.org/10.1186/s13041-024-01166-7
  43. Methods Mol Biol. 2025 ;2888 193-200
    Determining ATG2 Localization During Autophagosome Formation in Mammalian Cells.
    Shenliang Yu.
      This chapter describes two imaging-based approaches for examining the localization of bridge-like lipid transfer proteins at membrane contact sites during native biological processes. These approaches use multi-color fluorescence imaging, enabling high spatial and temporal resolution and overcoming the limitations of biochemical methods. The first approach involves immunofluorescence in fixed cells, while the second utilizes time-lapse imaging in live cells. These methods are showcased through the example of ATG2, an essential autophagy-related protein, and demonstrate the ability to overcome technical difficulties such as large protein size, lack of high-quality antibodies, and imaging highly dynamic subcellular structures. These described methods provide a powerful tool for understanding protein function and biological processes and can be widely applied to various research questions in cell biology.
    Keywords:  ATG2; Autophagy; Fluorescence microscopy; Lipid transport; Membrane contact site
    DOI:  https://doi.org/10.1007/978-1-0716-4318-1_13
  44. Mol Immunol. 2024 Dec 17. pii: S0161-5890(24)00215-3. [Epub ahead of print]177 32-43
    Paeoniflorin ameliorates reperfusion injury in H9C2 cells through SIRT1-PINK1/parkin-mediated mitochondrial autophagy.
    Xingcan Chen, Tong Sun, Yuxiang Qi, Bingqi Zhu, Lan Li, Jie Yu, Zhishan Ding, Fangmei Zhou.
      Myocardial ischemia-reperfusion injury (MIRI) injury is a serious health problem, which can seriously affect the recovery of patients with myocardial infarction and even lead to death. Paeoniflorin (PF) is a potential therapeutic drug to prevent reperfusion injury. However, the mechanism of PF in MIRI is not clear. Compared with other cells, cardiomyocytes have the largest number of mitochondria. Therefore, this study researched the protective mechanism of paeoniflorin pretreatment on myocardial ischemia-reperfusion injury (AMI) from the perspective of mitochondrial autophagy. Paeoniflorin was given or not given to H9C2 cells 12 h before reperfusion. Pretreatment of paeoniflorin can significantly increase the viability of H9C2 cells and inhibit the increase of ROS secretion induced by OGD/R. The increase of MDC autophagy fluorescence and mitochondrial membrane potential (MMP) suggested that the myocardial protective effect of paeoniflorin may also be related to mitochondrial autophagy. Next, we detected the related signals in the classical mitochondrial autophagy pathway of PINK1/parkin by Q-PCR and Western blots. The results showed that the pretreatment of paeoniflorin could promote the levels of SIRT1, Beclin1, PINK1, parkin and LC3, inhibit the level of P62. In order to further clarify whether paeoniflorin-induced SIRT1 activation is necessary for autophagy and its potential mechanism, we detected the autophagy level of H9C2 cells with SIRT1 inhibitor (EX527). The results showed that after pretreatment of EX527, the protective effect of paeoniflorin on oxidative damage and autophagy pathway was significantly decreased. The mechanism may relate to SIRT1-PINK1/parkin mitochondrial autophagy pathway. In summary, these results suggested that paeoniflorin may protect H9C2 cells from OGD/R damage by activating SIRT1-PINK1/parkin pathway. This provides new experimental basis for paeoniflorin in the treatment of MIRI.
    Keywords:  Mitochondrial autophagy; PINK1/parkin; Paeoniflorin; Reperfusion injury; SIRT1
    DOI:  https://doi.org/10.1016/j.molimm.2024.12.003
  45. ACS Med Chem Lett. 2024 Dec 12. 15(12): 2115-2120
    BODIPYS Based Fluorescent Markers To Monitor Autophagic Lysosomes and Lipid Droplets in TNBC.
    Jaydeepsinh Chavda, Arjun Siwach, Sudhir Sabharwal, Anu Janaagal, Dhiraj Bhatia, Iti Gupta.
      Lysosomal enzymes and high accumulation of lipid droplets are associated with breast cancer. The lysosomes and lipid droplets were monitored by BODIPYs, acting as autophagy activators in cancer cells. BD-1 and BD-2 were synthesized and characterized by Mass, UV-visible, fluorescence, and NMR spectroscopies. In BODIPYs, the effect of carbazole groups was reflected by the large Stokes shifts (2143-1651 cm-1) and red fluorescence. BODIPYs generated ROS and induced autophagy in triple negative breast cancer cells (MDA-MB-231) under white light. Confocal experiments revealed that BD-1 and BD-2 preferentially colocalized in lysosomes and lipid droplets. Autophagic lysosomes and lipid droplets released Ca2+ ions in the cytoplasm, which was evident with blue fluorescence of Fura-2M dye. In combination with an autophagy inhibitor, BD-1 displayed excellent photocytotoxicity (5.57 μM) on triple negative breast cancer cells under white light. This work demonstrates the potential of BODIPYs as theranostic agents for the photodynamic therapy against TNBC.
    DOI:  https://doi.org/10.1021/acsmedchemlett.4c00275
  46. Cell Oncol (Dordr). 2024 Dec 19.
    RAB37 suppresses the EMT, migration and invasion of gastric cancer cells by mediating autophagic degradation of β-catenin.
    Jiangling Duan, Xiuyin Guan, Jiaxin Xue, Jiayu Wang, Zhiwei Wang, Xuan Chen, Wen Jiang, Wannian Sui, Yongfang Song, Tianshu Li, Dewang Rao, Xueyan Wu, Ming Lu.
       BACKGROUND: Gastric cancer, characterized by its high morbidity and mortality rates, exhibits low levels of RAB37. The role and molecular mechanisms of RAB37, a small GTPase, in the pathogenesis of gastric cancer are still unclear.
    METHODS: We assessed RAB37 expression in gastric cancer cells using quantitative Polymerase Chain Reaction (qPCR), Western blot, and immunohistochemical staining (IHC), and analyzed EMT marker proteins and autophagy changes via Western blot, immunofluorescence (IF), and transmission electron microscopy (TEM). Co-immunoprecipitation (co-IP) was used to identify protein-protein interactions. We studied the migration and invasion of gastric cancer cells using wound healing and transwell assays in vitro and a mouse pulmonary metastasis model in vivo.
    RESULTS: Overexpression of RAB37 suppressed EMT, invasion, and migration while enhancing autophagy in gastric cancer cells, which was dependent on its GTPase activity. However, all these effects could be reversed by the autophagy inhibitor chloroquine. Regarding the molecular mechanism, RAB37 strengthened the interaction between p62 and β-catenin, which consequently enhanced the p62-mediated autophagic degradation of β-catenin. Furthermore, RAB37 curbed the pulmonary metastasis of both general and cisplatin-resistant gastric cancer cells.
    CONCLUSION: The low level of RAB37 reduces interaction between p62 and β-catenin and then the autophagic degradation of β-catenin, thereby promoting the EMT, invasion, and migration in gastric cancer cells. The low expression of RAB37 in gastric cancer suggests a potential therapeutic target, especially for cisplatin-resistant gastric cancer.
    Keywords:  Autophagy; Gastric cancer; Metastasis; RAB37; β-catenin
    DOI:  https://doi.org/10.1007/s13402-024-01028-3
  47. Biogerontology. 2024 Dec 14. 26(1): 25
    Coniferaldehyde activates autophagy and enhances oxidative stress resistance and lifespan of Caenorhabditis elegans via par-4/aak-2/skn-1 pathway.
    Mahesh Ramatchandirane, Ponsankaran Rajendran, M P Athira, Kitlangki Suchiang.
      Aging represents the gradual accumulation of alterations within an organism over time. The physical and chemical characteristics of our cells gradually change as we age, making it more difficult for our tissues and organs to self-regulate, regenerate, and maintain their structural and functional integrity. AMP- activated protein kinase (AMPK), a well-known sensor of cellular energy status acts as a central regulator of an integrated signalling network that control homeostasis, metabolism, stress resistance, cell survival and autophagy. Coniferaldehyde (CFA), a phenolic compound found in many edible plants, has multiple biological and pharmacological functions. Our findings demonstrated that 50 µM CFA could significantly activate autophagy and reduce oxidative stress, which enhanced the activity of antioxidant enzymes and increased resistance under oxidative stress. CFA treatment could efficiently decrease reactive oxygen species (ROS) levels and positively enhance the expression of antioxidant genes in Caenorhabditis elegans (C. elegans). On the other hand, CFA did not have any role in the lifespan extension of the several mutants linked to the AAK-2/AMPK pathway and it promotes SKN-1 (Skinhead-1) localization into the nucleus, which modulates downstream gene gst-4 (Glutathione S-transferase). In depth investigations revealed that CFA could lower oxidative stress and enhance the lifespan of C. elegans by activating the PAR-4/LKB-1-AAK-2/AMPK-SKN-1/NRF-2 pathway, with crucial involvement of bec-1 and lgg-1 genes for autophagy mediated lifespan extension. This study might contribute to understanding the interactions and mechanisms that allow natural compounds like CFA to treat age-related disorders among several species.
    Keywords:   Caenorhabditis elegans ; par-4/aak-2/skn-1 pathway; Autophagy; Coniferaldehyde; Lifespan; Oxidative stress
    DOI:  https://doi.org/10.1007/s10522-024-10163-1
  48. Nature. 2024 Dec 18.
    Lithocholic acid binds TULP3 to activate sirtuins and AMPK to slow down ageing.
    Qi Qu, Yan Chen, Yu Wang, Weiche Wang, Shating Long, Heng-Ye Yang, Jianfeng Wu, Mengqi Li, Xiao Tian, Xiaoyan Wei, Yan-Hui Liu, Shengrong Xu, Jinye Xiong, Chunyan Yang, Zhenhua Wu, Xi Huang, Changchuan Xie, Yaying Wu, Zheni Xu, Cixiong Zhang, Baoding Zhang, Jin-Wei Feng, Junjie Chen, Yuanji Feng, Huapan Fang, Liyun Lin, Z K Xie, Beibei Sun, Huayu Tian, Yong Yu, Hai-Long Piao, Xiao-Song Xie, Xianming Deng, Chen-Song Zhang, Sheng-Cai Lin.
      Lithocholic acid (LCA) is accumulated in mammals during calorie restriction and it can activate AMP-activated protein kinase (AMPK) to slow down ageing1. However, the molecular details of how LCA activates AMPK and induces these biological effects are unclear. Here we show that LCA enhances the activity of sirtuins to deacetylate and subsequently inhibit vacuolar H+-ATPase (v-ATPase), which leads to AMPK activation through the lysosomal glucose-sensing pathway. Proteomics analyses of proteins that co-immunoprecipitated with sirtuin 1 (SIRT1) identified TUB-like protein 3 (TULP3), a sirtuin-interacting protein2, as a LCA receptor. In detail, LCA-bound TULP3 allosterically activates sirtuins, which then deacetylate the V1E1 subunit of v-ATPase on residues K52, K99 and K191. Muscle-specific expression of a V1E1 mutant (3KR), which mimics the deacetylated state, strongly activates AMPK and rejuvenates muscles in aged mice. In nematodes and flies, LCA depends on the TULP3 homologues tub-1 and ktub, respectively, to activate AMPK and extend lifespan and healthspan. Our study demonstrates that activation of the TULP3-sirtuin-v-ATPase-AMPK pathway by LCA reproduces the benefits of calorie restriction.
    DOI:  https://doi.org/10.1038/s41586-024-08348-2
  49. JCI Insight. 2024 Dec 19. pii: e184451. [Epub ahead of print]
    Age-related TFEB downregulation in proximal tubules causes systemic metabolic disorders and occasional apolipoprotein A4-related amyloidosis.
    Jun Nakamura, Takeshi Yamamoto, Yoshitsugu Takabatake, Tomoko Namba-Hamano, Atsushi Takahashi, Jun Matsuda, Satoshi Minami, Shinsuke Sakai, Hiroaki Yonishi, Shihomi Maeda, Sho Matsui, Hideaki Kawai, Isao Matsui, Tadashi Yamamuro, Ryuya Edahiro, Seiji Takashima, Akira Takasawa, Yukinori Okada, Tamotsu Yoshimori, Andrea Ballabio, Yoshitaka Isaka.
      With the aging of society, the incidence of chronic kidney disease (CKD), a common cause of death, has been increasing. Transcription factor EB (TFEB), the master transcriptional regulator of the autophagy-lysosomal pathway, is regarded as a promising candidate for preventing various age-related diseases. However, whether TFEB in the proximal tubules plays a significant role in elderly CKD patients remains unknown. First, we found that nuclear TFEB localization in proximal tubular epithelial cells (PTECs) declined with age in both mice and humans. Next, we generated PTEC-specific Tfeb-deficient mice and bred them for up to 24 months. We found that TFEB deficiency in the proximal tubules caused metabolic disorders and occasionally led to apolipoprotein A4 (APOA4) amyloidosis. Supporting this result, we identified markedly decreased nuclear TFEB localization in the proximal tubules of elderly patients with APOA4 amyloidosis. The metabolic disturbances were accompanied with mitochondrial dysfunction due to transcriptional changes involved in fatty acid oxidation and oxidative phosphorylation pathways, as well as decreased mitochondrial clearance reflected by the accumulation of mitochondria-lysosome-related organelles, which depends on lysosomal function. These results shed light on the presumptive mechanisms of APOA4 amyloidosis pathogenesis and provide a therapeutic strategy for CKD-related metabolic disorders and APOA4 amyloidosis.
    Keywords:  Chronic kidney disease; Fatty acid oxidation; Metabolism; Mitochondria; Nephrology
    DOI:  https://doi.org/10.1172/jci.insight.184451
  50. Sci Signal. 2024 Dec 17. 17(867): eado6057
    AXL-TBK1 driven AKT3 activation promotes metastasis.
    Emily N Arner, Dina Alzhanova, Jill M Westcott, Stefan Hinz, Crina Elena Tiron, Magnus Blø, Anja Mai, Reetta Virtakoivu, Natalie Phinney, Silje Nord, Kristina Y Aguilera, Ali Rizvi, Jason E Toombs, Tanner C Reese, Vidal Fey, David Micklem, Gro Gausdal, Johanna Ivaska, James B Lorens, Rolf A Brekken.
      The receptor tyrosine kinase AXL promotes tumor progression, metastasis, and therapy resistance through the induction of epithelial-mesenchymal transition (EMT). Here, we found that activation of AXL resulted in the phosphorylation of TANK-binding kinase 1 (TBK1) and the downstream activation of AKT3 and Snail, a transcription factor critical for EMT. Mechanistically, we showed that TBK1 directly bound to and phosphorylated AKT3 in a manner dependent on the multiprotein complex mTORC1. Upon activation, AKT3 interacted with and promoted the nuclear accumulation of Snail, which led to increased EMT as assessed by marker abundance. In human pancreatic ductal adenocarcinoma tissue, nuclear AKT3 colocalized with Snail and correlated with worse clinical outcomes. Primary mouse pancreatic cancer cells deficient in AKT3 showed reduced metastatic spread in vivo, suggesting selective AKT3 inhibition as a potential therapeutic avenue for targeting EMT in aggressive cancers.
    DOI:  https://doi.org/10.1126/scisignal.ado6057
  51. bioRxiv. 2024 Dec 07. pii: 2024.12.04.626625. [Epub ahead of print]
    Spermidine alleviates depression via control of the stress response.
    Sarah Mackert, Christine Niemeyer, Yara Mecdad, Tim Ebert, Thomas Bajaj, Sylvère Durand, Sebastian J Hofer, Andreas Zellner, Bianca Besteher, Jan Engelmann, Barbara Gisabella, Valerie Kempf, Lily A Heckmann, Marcel Laakmann, Emily L Newman, Clara Sokn, Daniel E Heinz, Ellen Junglas, Andrés Uribe-Marino, Christoph Magnes, Claudia Klengel, Anna Müller, Lydia Opriessnig, Andreas Meinitzer, Martina Lennarz, Katja M Wirtz Martin, Rawan Albatarni, Marieke Brockherde, Klaus Lieb, Henrik Rohner, Birgit Stoffel-Wagner, Alexandra Philipsen, Bernhard Kuster, Markus Kölle, Kerry J Ressler, Nils Opel, Mathias V Schmidt, Harry Pantazopoulos, Marianne B Müller, Guido Kroemer, Tobias Eisenberg, Jakob Hartmann, Frank Madeo, Nils C Gassen.
      Depression is a stress-associated disorder, and it represents a major global health issue. Its pathophysiology is complex and remains insufficiently understood, with current medications often showing limited efficacy and undesirable side effects. Here, we identify imbalanced polyamine levels and dysregulated autophagy as key components of the acute stress response in humans, and as hallmarks of chronic stress and depressive disorders. Moreover, conventional antidepressant pharmacotherapy increases endogenous plasma concentrations of the polyamine spermidine exclusively in patients who respond to the treatment, suggesting a link between spermidine and successful outcomes. In a clinical trial, involving drug-naive depressed individuals, three weeks of spermidine supplementation increased autophagy and alleviated symptoms of depression. Behavioral and mechanistic findings of spermidine supplementation were validated in various mouse stress and depression models. In summary, spermidine supplementation mitigates polyamine dysregulation and stimulates autophagy under pathological stress conditions, offering a novel and well-tolerated treatment approach for stress-related depressive disorders.
    DOI:  https://doi.org/10.1101/2024.12.04.626625
  52. Autophagy. 2024 Dec 15.
    Endogenous interactomes of MFN1 and MFN2 provide novel insights into interorganelle communication and autophagy.
    Isabel Gordaliza-Alaguero, Paula Sànchez-Fernàndez-de-Landa, Dragana Radivojevikj, Laura Villarreal, Gianluca Arauz-Garofalo, Marina Gay, Marta Martinez-Vicente, Jorge Seco, Pau Martin-Malpartida, Marta Vilaseca, María J Macías, Manuel Palacin, Saška Ivanova, Antonio Zorzano.
      MFN1 (mitofusin 1) and MFN2 are key players in mitochondrial fusion, endoplasmic reticulum (ER)-mitochondria juxtaposition, and macroautophagy/autophagy. However, the mechanisms by which these proteins participate in these processes are poorly understood. Here, we studied the interactomes of these two proteins by using CRISPR-Cas9 technology to insert an HA-tag at the C terminus of MFN1 and MFN2, and thus generating HeLa cell lines that endogenously expressed MFN1-HA or MFN2-HA. HA-affinity isolation followed by mass spectrometry identified potential interactors of MFN1 and MFN2. A substantial proportion of interactors were common for MFN1 and MFN2 and were regulated by nutrient deprivation. We validated novel ER and endosomal partners of MFN1 and/or MFN2 with a potential role in interorganelle communication. We characterized RAB5C (RAB5C, member RAS oncogene family) as an endosomal modulator of mitochondrial homeostasis, and SLC27A2 (solute carrier family 27 (fatty acid transporter), member 2) as a novel partner of MFN2 relevant in autophagy. We conclude that MFN proteins participate in nutrient-modulated pathways involved in organelle communication and autophagy.
    Keywords:  Autophagosomes; endosomes; mitochondria; mitochondria-endoplasmic reticulum contact sites; mitochondrial dynamics; nutrient deprivation
    DOI:  https://doi.org/10.1080/15548627.2024.2440843
  53. Cell Commun Signal. 2024 Dec 18. 22(1): 592
    New insights into the role of mitophagy related gene affecting the metastasis of osteosarcoma through scRNA-seq and CRISPR-Cas9 genome editing.
    Sikuan Zheng, Mengliang Luo, Hong Huang, Xuanxuan Huang, Zhidong Peng, Shaowei Zheng, Jianye Tan.
       BACKGROUND: Osteosarcoma (OSA), the most common primary bone malignancy, poses significant challenges due to its aggressive nature and propensity for metastasis, especially in adolescents. Mitophagy analysis can help identify new therapeutic targets and combined treatment strategies.
    METHODS: This study integrates single-cell sequencing (scRNA-seq) data and bulk-seq to identify mitophagy-related genes (MRGs) associated with the progression of OSA metastasis and analyze their clinical significance. scRNA-seq data elucidates the relationship between mitophagy and OSA metastasis, employing "CellChat" R package to explore intercellular communications and report on hundreds of ligand-receptor interactions. Subsequently, the combination of bulk-seq and CRISPR-Cas9 gene editing identifies mitophagy-related biomarker associated with metastatic prognosis. Finally, validation of the relationship between mitophagy and OSA metastasis is achieved through cellular biology experiments and animal studies.
    RESULTS: The distinct mitophagy activity of various mitochondria manifests in diverse spatial localization, cellular developmental trajectories, and intercellular interactions. OSA tissue exhibits notable heterogeneity in mitophagy within osteoblastic OSA cells. However, high mitophagy activity correlates consistently with high metastatic potential. Subsequently, we identified three critical genes associated with mitophagy in OSA, namely RPS27A, TOMM20 and UBB. According to the aforementioned queue of genes, we have constructed a mitophagy_score (MIP_score). We observed that it consistently predicts patient prognosis in both internal and external datasets, demonstrating strong robustness and stability. Furthermore, we have found that MIP_score can also guide chemotherapy, with varying sensitivities to chemotherapeutic agents based on different MIP_score. It is noteworthy that, through the integration of CRISPR-Cas9 genome-wide screening and validation via cellular and animal experiments, we have identified RPS27A as a potential novel biomarker for OSA.
    CONCLUSIONS: Our comprehensive analysis elucidated the profile of mitophagy throughout the OSA metastasis process, forming the basis for a mitophagy-related prognostic model that addresses clinical outcomes and drug sensitivity following OSA metastasis. Additionally, an online interactive platform was established to assist clinicians in decision-making ( https://mip-score.shinyapps.io/labtan/ ). These findings lay the groundwork for developing targeted therapies aimed at improving the prognosis of OSA patients.
    DOI:  https://doi.org/10.1186/s12964-024-01989-w
  54. Nat Metab. 2024 Dec;6(12): 2319-2337
    Subcellular NAD+ pools are interconnected and buffered by mitochondrial NAD.
    Lena E Høyland, Magali R VanLinden, Marc Niere, Øyvind Strømland, Suraj Sharma, Jörn Dietze, Ingvill Tolås, Eva Lucena, Ersilia Bifulco, Lars J Sverkeli, Camila Cimadamore-Werthein, Hanan Ashrafi, Kjellfrid F Haukanes, Barbara van der Hoeven, Christian Dölle, Cédric Davidsen, Ina K N Pettersen, Karl J Tronstad, Svein A Mjøs, Faisal Hayat, Mikhail V Makarov, Marie E Migaud, Ines Heiland, Mathias Ziegler.
      The coenzyme NAD+ is consumed by signalling enzymes, including poly-ADP-ribosyltransferases (PARPs) and sirtuins. Ageing is associated with a decrease in cellular NAD+ levels, but how cells cope with persistently decreased NAD+ concentrations is unclear. Here, we show that subcellular NAD+ pools are interconnected, with mitochondria acting as a rheostat to maintain NAD+ levels upon excessive consumption. To evoke chronic, compartment-specific overconsumption of NAD+, we engineered cell lines stably expressing PARP activity in mitochondria, the cytosol, endoplasmic reticulum or peroxisomes, resulting in a decline of cellular NAD+ concentrations by up to 50%. Isotope-tracer flux measurements and mathematical modelling show that the lowered NAD+ concentration kinetically restricts NAD+ consumption to maintain a balance with the NAD+ biosynthesis rate, which remains unchanged. Chronic NAD+ deficiency is well tolerated unless mitochondria are directly targeted. Mitochondria maintain NAD+ by import through SLC25A51 and reversibly cleave NAD+ to nicotinamide mononucleotide and ATP when NMNAT3 is present. Thus, these organelles can maintain an additional, virtual NAD+ pool. Our results are consistent with a well-tolerated ageing-related NAD+ decline as long as the vulnerable mitochondrial pool is not directly affected.
    DOI:  https://doi.org/10.1038/s42255-024-01174-w
  55. J Clin Invest. 2024 Dec 16. pii: e173403. [Epub ahead of print]134(24):
    Evi1 governs Kdm6b-mediated histone demethylation to regulate the Laptm4b-driven mTOR pathway in hematopoietic progenitor cells.
    Qiong Wu, Chunjie Yu, Fang Yu, Yiran Guo, Yue Sheng, Liping Li, Yafang Li, Yutao Zhang, Chao Hu, Jue Wang, Tong-Chuan He, Yong Huang, Hongyu Ni, Zhiguang Huo, Wenshu Wu, Gang Greg Wang, Jianxin Lyu, Zhijian Qian.
      Ecotropic viral integration site 1 (EVI1/MECOM) is frequently upregulated in myeloid malignancies. Here, we present an Evi1-transgenic mouse model with inducible expression in hematopoietic stem/progenitor cells (HSPCs). Upon induction of Evi1 expression, mice displayed anemia, thrombocytopenia, lymphopenia, and erythroid and megakaryocyte dysplasia with a significant expansion of committed myeloid progenitor cells, resembling human myelodysplastic syndrome/myeloproliferative neoplasm-like (MDS/MPN-like) disease. Evi1 overexpression prompted HSPCs to exit quiescence and accelerated their proliferation, leading to expansion of committed myeloid progenitors while inhibiting lymphopoiesis. Analysis of global gene expression and Evi1 binding site profiling in HSPCs revealed that Evi1 directly upregulated lysine demethylase 6b (Kdm6b). Subsequently, Kdm6b-mediated H3K27me3 demethylation resulted in activation of various genes, including Laptm4b. Interestingly, KDM6B and LAPTM4B are positively correlated with EVI1 expression in patients with MDS. The EVI1/KDM6B/H3K27me3/LAPTM4B signaling pathway was also identified in EVI1hi human leukemia cell lines. We found that hyperactivation of the LAPTM4B-driven mTOR pathway was crucial for the growth of EVI1hi leukemia cells. Knockdown of Laptm4b partially rescued Evi1-induced abnormal hematopoiesis in vivo. Thus, our study establishes a mouse model to investigate EVI1hi myeloid malignancies, demonstrating the significance of the EVI1-mediated KDM6B/H3K27me3/LAPTM4B signaling axis in their maintenance.
    Keywords:  Hematology; Hematopoietic stem cells; Leukemias
    DOI:  https://doi.org/10.1172/JCI173403
  56. Cell Death Dis. 2024 Dec 18. 15(12): 893
    LOC730101 improves ovarian cancer drug sensitivity by inhibiting autophagy-mediated DNA damage repair via BECN1.
    Yancheng Zhong, Yang Shuai, Juan Yang, Mojian Zhang, Tiantian He, Leliang Zheng, Sheng Yang, Shuping Peng.
      Drug resistance and recurrence are still the bottlenecks in the clinical treatment of ovarian cancer (OC), seriously affecting patients' prognosis. Therefore, it is an urgent challenge for OC to be overcome towards precision therapy by studying the mechanism of OC drug resistance, finding new drug resistance targets and developing new effective treatment strategies. In this study, we found that lncRNA LOC730101 played an essential role in attenuating drug resistance in OC. LOC730101 was significantly down-regulated in platinum-resistant ovarian cancer tissues, and ectopic overexpression of LOC730101 substantially increased chemotherapy-induced apoptosis. Mechanistically, LOC730101 specifically binds to BECN1 and inhibits the formation of autophagosome BECN1/VPS34 by reducing phosphorylation of BECN1, thereby inhibiting autophagy and promoting drug sensitivity in ovarian cancer cells following treatment with cisplatin and PARP inhibitors. Moreover, LOC730101 inhibits the expression and activity of RNF168 via p62, which in turn affects H2A ubiquitination-mediated DNA damage repair and promotes drug sensitivity in ovarian cancer cells. Our findings demonstrated that LOC730101 played an important role in regulating the formation of the autophagic complex and that inhibition of autophagy significantly enhances the drug sensitivity of OC. And LOC730101 may be used as a prognostic marker to predict the sensitivity of OC to platinum and PARP inhibitors.
    DOI:  https://doi.org/10.1038/s41419-024-07278-1
  57. Adv Sci (Weinh). 2024 Dec 16. e2410136
    Reversing Persistent PTEN Activation after Traumatic Brain Injury Fuels Long-Term Axonal Regeneration via Akt/mTORC1 Signaling Cascade.
    Ziyu Shi, Leilei Mao, Shuning Chen, Zhuoying Du, Jiakun Xiang, Minghong Shi, Yana Wang, Yuqing Wang, Xingdong Chen, Zhi-Xiang Xu, Yanqin Gao.
      Traumatic brain injury (TBI) often leads to enduring axonal damage and persistent neurological deficits. While PTEN's role in neuronal growth is recognized, its long-term activation changes post-TBI and its effects on sensory-motor circuits are not well understood. Here, it is demonstrated that the neuronal knockout of PTEN (PTEN-nKO) significantly enhances both structural and functional recovery over the long term after TBI. Importantly, in vivo, DTI-MRI revealed that PTEN-nKO promotes white matter repair post-TBI. Additionally, calcium imaging and electromyographic recordings indicated that PTEN-nKO facilitates cortical remapping and restores sensory-motor pathways. Mechanistically, PTEN negatively regulates the Akt/mTOR pathway by inhibiting Akt, thereby suppressing mTOR. Raptor is a key component of mTORC1 and its suppression impedes axonal regeneration. The restoration of white matter integrity and the improvements in neural function observed in PTEN-nKO TBI-treated mice are reversed by a PTEN/Raptor double knockout (PTEN/Raptor D-nKO), suggesting that mTORC1 acts as a key mediator. These findings highlight persistent alterations in the PTEN/Akt/mTORC1 axis are critical for neural circuit remodeling and cortical remapping post-TBI, offering new insights into TBI pathophysiology and potential therapeutic targets.
    Keywords:  PTEN; TBI; axon regeneration; cortical remapping; mTORC1
    DOI:  https://doi.org/10.1002/advs.202410136
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