bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2025–10–19
fifty-nine papers selected by
Viktor Korolchuk, Newcastle University
  1. Interplay Between mTORC1 Signaling and Iron Homeostasis in Muscle Atrophy.
  2. Monitoring Autophagosome Biogenesis and Degradation in Mammalian Cells Using LC3B Blotting.
  3. Crosstalk Between Allergic Inflammation and Autophagy.
  4. Rapamycin and Autophagy: Potential Therapeutic Approach for Parkinson's Disease Treatment.
  5. Vesicular transport in the autophagic route: RAB GTPases as pivotal regulators of autophagy.
  6. The emerging roles of autophagy in the homeostasis of lysosome-related organelles.
  7. Autophagy and Neuropsychiatric Disorders: Unraveling Molecular Mechanisms and Signaling Pathways.
  8. Preservation of Autophagy May Be a Mechanism Behind Healthy Aging.
  9. Autophagy in doxorubicin resistance: basic concepts, therapeutic perspectives and clinical translation.
  10. HUWE1 stimulates mTORC1 activity by enhancing Rheb interaction with mTORC1 and supports de novo pyrimidine synthesis.
  11. Measurement of Physiological Autophagic Flux in the Human Peripheral Blood Mononuclear Cell Pool.
  12. Global conformation of the Rag GTPase heterodimer governs eukaryotic amino acid sensing.
  13. Chaperone-mediated autophagy regulates neuronal activity by sex-specific remodelling of the synaptic proteome.
  14. NAD+ Homeostasis and Autophagy: Integrated Control Through Nutrient Signaling in Yeast and Mammals.
  15. The space-time continuum in neurological disorders of the autophagosome-lysosome fusion machinery.
  16. Oxidative Stress, Mitochondrial Quality Control, Autophagy, and Sirtuins in Heart Failure.
  17. Genetic and proteomic analysis identifies BAG3 as an amyloid-responsive regulator of neuronal proteostasis.
  18. The crosstalk between MiRNAs and autophagy in leukemia: mechanisms and therapeutic insights.
  19. Remimazolam alleviates hippocampal neuronal injury in an in vitro Alzheimer's disease model by promoting PINK1/Parkin-mediated mitophagy.
  20. Lysosomal Immunoprecipitation (LysoIP) from Cells and Tissues for Metabolomic and Proteomic Analyses.
  21. Modulation of mTOR Within Retinal Pigment Epithelium Affects Cell Viability and Mitochondrial Pathology.
  22. Autophagy regulation by signaling.
  23. Visualization of lysosomal membrane proteins by cryo electron tomography.
  24. Linking autophagy to endothelial cell immunogenicity in transplant-associated ischemia-reperfusion injury.
  25. Acetylation promotes mutant (MUT) TP53-HSPA8 and HSPA8-BAG3 interactions, facilitating MUT TP53 lysosomal degradation preferentially via CASA.
  26. Using CMAC Staining for Vacuole Characterization in Yeast.
  27. Proteomics Insights Into Lysosome Biogenesis and Maturation.
  28. BNIP3L/BNIP3-Mediated Mitophagy Contributes to the Maintenance of Ovarian Cancer Stem Cells.
  29. The EGR1/miR-199a-3p/mTOR axis: A crucial pathway in the amplification of myocardial damage under intermittent hypoxic conditions.
  30. Label-Free Imaging of Lysosomes in Living Cells by Photothermal Microscopy.
  31. Isoginkgetin antagonizes ALS pathologies in its animal and patient iPSC models via PINK1-Parkin-dependent mitophagy.
  32. Analyzing Endolysosome-Mitochondria Membrane Contact Sites by Thin Section TEM of Human Fibroblasts.
  33. Autophagy in the lung: guardian of homeostasis or driver of disease.
  34. Modeling Lysosomal Disease in Dictyostelium discoideum: Examining the Trafficking and Secretion of Lysosomal Enzymes.
  35. Implication of LAMP proteins and autophagy markers in colorectal cancer aggressiveness.
  36. PINK1 overexpression suppresses p38 MAPK/NF‑κB signaling to attenuate chondrocyte senescence in osteoarthritis.
  37. Autophagy acts as a brake on obesity-related fibrosis by controlling purine nucleoside signalling.
  38. mTORC2-mediated cell-cell interactions promote BMP4-induced WNT activation and mesoderm differentiation.
  39. Influence of skeletal muscle heterogeneity on autophagic signaling and response.
  40. Determining Lysosomal Enzyme Activity Using Fluorogenic Probes.
  41. Targeting Autophagy via Mitochondrial Uncoupling: Discovery of Novel Serratin Derivative as a Potential Therapeutic for Parkinson's Disease.
  42. Cdk5 Contributes to Diabetic Islet β Cell and Kidney Injury by Impairing Autophagy.
  43. METTL3 Affects Endometrial Receptivity Through Modulation of P62-Mediated Autophagic Flux.
  44. Lysosomal proteomics reveals mechanisms of neuronal APOE4-associated lysosomal dysfunction.
  45. The autophagy-recessive tissue hormone DBI/ACBP (diazepam binding inhibitor, acyl-CoA binding protein) contributes to the pathogenesis of osteoarthritis.
  46. Assessing Perturbations in Lysosome Health by Flow Cytometry in iPSC-Derived Human Neuron Cultures.
  47. MAPL regulates gasdermin-mediated release of mtDNA from lysosomes to drive pyroptotic cell death.
  48. Role of AAA-ATPase Cdc48p in peroxisomal quality control.
  49. 18β-Glycyrrhetinic Acid Regulates Endoplasmic Reticulum Stress and Autophagy Dysregulation in the MPTP/p-Induced Model of Parkinson Disease.
  50. Pramipexole improves cognitive deficits and synaptic plasticity impairments in 3xTg-AD mice through enhancing autophagy.
  51. Lysosomal Network Defects in Early-Onset Parkinson's Disease Patients Carrying Rare Variants in Lysosomal Hydrolytic Enzyme Genes.
  52. Suppression of OTUD4 protects against myocardial ischemia-reperfusion injury by increasing autophagic flux and inhibiting apoptosis in cardiomyocytes.
  53. Dual-responsive diazo probe for labeling of aggrephagic compartments in live cells.
  54. Immunostaining Methods for Lysosomal Storage Disorder Research.
  55. Novel links of ATG4 proteases to cytoskeletal adapters of the obscurin protein family.
  56. Dietary and Pharmacological Modulation of Aging-Related Metabolic Pathways: Molecular Insights, Clinical Evidence, and a Translational Model.
  57. Interaction proteomics of Polycystins 1 and 2 reveal a novel role for the BLOC-1/BORC lysosomal positioning complex.
  58. Capturing disease severity in LIS1-lissencephaly reveals proteostasis dysregulation in patient-derived forebrain organoids.
  59. OPTN protects retinal ganglion cells and ameliorates neuroinflammation in optic neuropathies.
  1. Free Radic Biol Med. 2025 Oct 12. pii: S0891-5849(25)01271-7. [Epub ahead of print]
    Interplay Between mTORC1 Signaling and Iron Homeostasis in Muscle Atrophy.
    Eunbin Jee, Walaa Ouf, Jonghan Kim, Yuho Kim.
      The mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in maintaining skeletal muscle homeostasis by regulating cell growth, protein degradation, and nutrient sensing. Beyond its role in muscle growth and atrophy, recent findings suggest that mTORC1 also regulates cellular iron metabolism. Although iron is essential for energy production and mitochondrial function in skeletal muscle, both iron deficiency and overload contribute to muscle degeneration through dysfunctional mitochondria, oxidative stress, and activation of catabolic pathways. In this review, while focusing on the role of mTORC1 in iron-dependent muscle disorders such as cancer cachexia, sarcopenia, and Duchenne muscle dystrophy, we explore how altered mTORC1 activity affects major protein degradation systems, including the ubiquitin-proteasome system, autophagy, and mitophagy, under iron imbalance in skeletal muscle. We also highlight the emerging evidence linking mTORC1 signaling to iron metabolism in skeletal muscle, with a focus on the regulation of iron transport, ferritinophagy, and the autophagosome-lysosome system. The crosstalk between mTORC1 and iron metabolism in muscle atrophy provides novel insights into the molecular mechanisms underlying muscle disorders. Collectively, these perspectives suggest new therapeutic strategies for treating muscle diseases associated with disrupted iron homeostasis and impaired mTORC1 signaling.
    Keywords:  Duchenne muscle dystrophy; autophagy; cachexia; iron deficiency; iron overload; mTORC1; mitophagy; sarcopenia; ubiquitin-proteasome system
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2025.10.257
  2. Methods Mol Biol. 2026 ;2976 47-60
    Monitoring Autophagosome Biogenesis and Degradation in Mammalian Cells Using LC3B Blotting.
    Jennifer E Palmer, David C Rubinsztein.
      Autophagy is a conserved lysosomal degradation pathway that recycles protein aggregates and damaged organelles to maintain cytoplasmic quality control. Measuring the amount of the lipid-conjugated autophagic protein LC3B-II is a useful way to test whether a particular perturbation affects autophagy. However, the level of LC3B-II is affected by factors that alter either the rate of autophagosome biogenesis or degradation. Consequently, the same steady-state LC3B-II level can be reached by opposing autophagic fluxes. It is thus essential when measuring LC3B-II to perform the assay both in the absence and presence of a lysosomal inhibitor, enabling measurement of the rate of synthesis independent of its degradation. LC3B-II is also a small protein that can be challenging to detect by western blotting. In this chapter, we will provide a method for the efficient western blotting of LC3B-II and guidance as to the interpretation of the results.
    Keywords:  ATG8; Autophagy; Bafilomycin A1; LC3B; SDS-PAGE; Western blotting
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_5
  3. Int J Mol Sci. 2025 Oct 07. pii: 9765. [Epub ahead of print]26(19):
    Crosstalk Between Allergic Inflammation and Autophagy.
    Jaewhoon Jeoung, Wonho Kim, Dooil Jeoung.
      Autophagy is a conserved process that involves the degradation of damaged proteins and organelles to restore cellular homeostasis. Autophagy plays a critical role in cell differentiation, immune responses, and protection against pathogens, as well as the development and progression of allergic inflammation. Crosstalk between autophagy and signaling pathways modulates immune responses to inflammatory signals. Here, we discuss the regulatory roles of autophagy in allergic inflammation. Autophagy can promote allergic inflammation by enhancing the secretion of inflammatory mediators. Impaired autophagy resulting from the accumulation of autophagosomes can exacerbate allergic inflammation. Mast cell degranulation and activation require energy provided by mitochondrial respiration. Mast cell activation is accompanied by morphological changes and mitochondrial fragmentation. Mitochondrial fragmentation (mitophagy) induced by oxidative stress involves the degradation of defective mitochondria. Therefore, we discuss the relationship between mitophagy and allergic inflammation. Targeting autophagy and oxidative stress can be a strategy for developing anti-allergy therapeutics. In this review, we also discuss future research directions to better understand allergic diseases with respect to autophagy and develop effective anti-allergy drugs.
    Keywords:  allergy; autophagy; crosstalk; mitochondria; mitophagy
    DOI:  https://doi.org/10.3390/ijms26199765
  4. CNS Neurol Disord Drug Targets. 2025 Oct 14.
    Rapamycin and Autophagy: Potential Therapeutic Approach for Parkinson's Disease Treatment.
    Ahsas Goyal, Anshika Kumari, Aanchal Verma, Neetu Agrawal.
      Parkinson's disease (PD) is a chronic, progressive neurodegenerative disorder marked by the degeneration of dopaminergic neurons in the substantia nigra, leading to characteristic motor symptoms such as bradykinesia, tremor, and rigidity, as well as a range of non-motor manifestations including cognitive impairment, mood disturbances and autonomic dysfunction. Among the multiple cellular mechanisms implicated in PD, the dysregulation of autophagy has gained significant attention in recent years. Autophagy is a crucial intracellular degradation pathway responsible for the removal of misfolded proteins and damaged organelles, processes that are particularly relevant in neurodegenerative diseases. Impairment of autophagic flux contributes to the accumulation of toxic protein aggregates and cellular stress in PD. Rapamycin, a compound originally isolated from Streptomyces hygroscopicus, is a well-established inhibitor of the mechanistic target of rapamycin (mTOR), a central regulator of autophagy. Preclinical studies have shown that rapamycin can stimulate autophagic pathways by suppressing mTOR signalling, leading to increased expression of autophagy markers. These effects have been associated with reduced neuronal damage, improved motor performance and decreased accumulation of pathological proteins in PD models. This review provides an overview of current preclinical research on rapamycin's neuroprotective potential in PD through autophagy enhancement. Although findings are promising, translating these outcomes into clinical practice necessitates a thorough understanding of rapamycin's pharmacodynamics, optimal dosing strategies, potential side effects and long-term safety. Further research is essential to establish its therapeutic viability in human populations.
    Keywords:  Rapamycin; autophagy; mTOR and Parkinson's disease.; neuroprotection
    DOI:  https://doi.org/10.2174/0118715273401017250918141227
  5. Biochem J. 2025 Oct 17. 482(20): 1531-1544
    Vesicular transport in the autophagic route: RAB GTPases as pivotal regulators of autophagy.
    Romina Abba, María Isabel Colombo.
      Autophagy is recognized as one of the two main intracellular recycling pathways that play an essential role in cellular homeostasis by maintaining accurate energy levels and carrying out quality control functions. One of the major autophagic mechanisms, the so-called macroautophagy, is involved in the lysosomal degradation of different cytoplasmic components, such as long-lived proteins and damaged or dysfunctional organelles. Numerous studies have demonstrated that participation of intracellular membrane trafficking events is key for the progression of autophagy. In this review, we will focus on the small GTPases of the RAS-related in brain protein family, which have a crucial role in vesicular transport.
    Keywords:  RAB GTPases; autophagy; membrane traffic
    DOI:  https://doi.org/10.1042/BCJ20253092
  6. Front Cell Dev Biol. 2025 ;13 1638905
    The emerging roles of autophagy in the homeostasis of lysosome-related organelles.
    Xinran Wang, Luan Zhang, Xueqin Cao, Yuting Zhao.
      
    Keywords:  LC3-associated phagocytosis; autophagy; lysosome-related organelle; melanophagy; non-canonical autophagy; secretory autophagy; selective autophagy; selective autophagy receptor
    DOI:  https://doi.org/10.3389/fcell.2025.1638905
  7. CNS Neurol Disord Drug Targets. 2025 Oct 09.
    Autophagy and Neuropsychiatric Disorders: Unraveling Molecular Mechanisms and Signaling Pathways.
    Amirhossein Habibzadeh, Zeinab Shirvani-Farsani, Vahid Ehsanian Mofrad, Fateme Derijani.
      Autophagy is a catabolic process that helps maintain cellular homeostasis by degrading damaged proteins and organelles while recycling essential biomolecules. Neuropsychiatric disorders, such as schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders, have been linked to autophagy dysregulation. In this manuscript, we review the complex role of autophagy in the neurobiology of these disorders, encompassing neuronal function, neurodevelopment, and neuroplasticity. The molecular mechanisms by which autophagy dysregulation contributes to the manifestation and progression of neuropsychiatric diseases, including those related to autophagy genes and pathways, are also discussed. Additionally, potential entry points for autophagytargeted therapy in these disorders, such as modulating mTOR and combining autophagy modulators with existing treatments, are also explored. We also specifically examine the neuroprotective effects of lithium, a mood stabilizer, through its influence on autophagy pathways. Overall, understanding the intricate relationship between autophagy and neuropsychiatric disorders provides new avenues for developing new treatments for these devastating conditions.
    Keywords:  Autophagy; bipolar disorder; depression; lithium.; neuropsychiatric diseases; schizophrenia; substance abuse
    DOI:  https://doi.org/10.2174/0118715273384110250915073216
  8. Aging Cell. 2025 Oct 13. e70246
    Preservation of Autophagy May Be a Mechanism Behind Healthy Aging.
    Arsun Bektas, Shepherd H Schurman, Julián Candia, Olaya Santiago-Fernández, Susmita Kaushik, Ana Maria Cuervo, Luigi Ferrucci.
      Autophagy is intricately linked with protective cellular processes, including mitochondrial function, proteostasis, and cellular senescence. Animal studies have indicated that autophagy becomes dysfunctional with aging and may contribute to T cell immunosenescence. In humans, it remains unclear whether autophagy is impaired in CD4+ T cells as people age. To answer this question, we examined basal and inducible autophagic activity in a series of experiments comparing CD4+ T cells from younger (23-35 years old) and older (67-93 years old) healthy donors. We used immunofluorescence to detect LC3 (a marker of autophagosomes and autolysosomes) and LAMP2 (a marker of endolysosomes) in conjunction with bafilomycin A1 (which inhibits the acidification of lysosomes) and CCCP (a mitochondrial uncoupler) to manipulate autophagic flux. We found a significantly higher autophagy flux in CD4+ T cells from older compared to younger donors and a higher number of LC3+ compartments among older donors. Since the overall amount of autophagosomes degraded was comparable between the two groups, we concluded that autophagosome biogenesis was reduced in the older group. Rather than a decline, our findings in healthy older donors point toward a compensatory enhancement of human CD4+ T cell autophagy with age, which may be a mechanism behind healthy aging.
    Keywords:  CD4+ T cells; autophagy; healthy aging
    DOI:  https://doi.org/10.1111/acel.70246
  9. Front Immunol. 2025 ;16 1642050
    Autophagy in doxorubicin resistance: basic concepts, therapeutic perspectives and clinical translation.
    Yantao Zhang, Yanqin Ji, Yanyang Tu, Yi Li.
      Doxorubicin (DOX) is still one of the leading compounds for cancer chemotherapy, but its clinical application has been restricted by the drug resistance. The emerging evidence has demonstrated that autophagy is a meticulously regulated by the lysosomal degradation as a regulator of this drug resistance. Autophagy can exert a pro-survival strategy under therapeutic stress through recycling cellular components, inhibiting apoptosis and remodelling metabolism, thereby enhancing carcinogenesis. The present review aims to highlight the interaction between autophagy and DOX resistance, providing the molecular machinery of autophagy and its control by genetic factors, microenvironmental factors and non-coding RNAs. Mechanistically, autophagy can be considered as protective or cytotoxic, relying on the cellular context, but in most cases, autophagy serves as a survival pathway promoting chemoresistance. The present review will also discuss about the function of DOX in autophagy induction through ROS generation, DNA damage response and AMPK/mTOR axis, whereas providing context-specific adaptations including mitophagy in cancer stem cells and lysosomal remodelling. The pre-clinical studies have highlighted the function of pharmacological compounds and nanoparticles for the regulation of autophagy for improving DOX sensitivity in cancer, accelerating therapeutic index. The strategies have focused on the application of small-molecule inhibitors, natural compounds, nanocarrier-mediated co-delivery of DOX with autophagy modulators and the development of combination therapeites providing the crosstalk of autophagy and cell death mechanisms in DOX resistance. The clinical translation depends on the development of more effective autophagy-targeted drugs in combination therapies. Hence, the present review highlights the role of autophagy as a biomarker and therapeutic factors in reversing DOX resistance. By elucidating the complex biology linking autophagy to drug resistance, it is emphasized that tailored approaches integrating autophagy modulation may yield more effective and less toxic cancer treatments.
    Keywords:  autophagy; cancer therapy; clinical translation; doxorubicin; drug resistance
    DOI:  https://doi.org/10.3389/fimmu.2025.1642050
  10. Cell Rep. 2025 Oct 09. pii: S2211-1247(25)01153-2. [Epub ahead of print]44(10): 116382
    HUWE1 stimulates mTORC1 activity by enhancing Rheb interaction with mTORC1 and supports de novo pyrimidine synthesis.
    Takayuki Ikeda, Sungki Hong, Shota Yoshida, Swayam Prakash Srivastava, Abhiram Kunamneni, Yao Yao, Om Khuperkar, Venkatesha Basrur, Henry Kuang, Maureen Kachman, Abraham Raskind, Heidi Baum, Arya Joshi, Vinamra Swaroop, Aaron Thurman, Oliviamae Kopasz-Gemmen, Siddharth Kandukuri, F N U Pradeepa, Hideto Yonekura, Jiandie D Lin, Ken Inoki.
      The mechanistic target of rapamycin complex 1 (mTORC1), a central regulator of cell growth, is activated by Rheb small GTPase. Our recent studies have demonstrated that polyubiquitinated Rheb enhances its interaction with mTORC1, resulting in the activation of mTORC1. Here, we demonstrate that the HECT, UBA, and WWE domain containing E3 ubiquitin protein ligase 1 (HUWE1), an E3 ubiquitin ligase, preferentially interacts with ubiquitinated Rheb and facilitates Rheb's binding to mTORC1 and its subsequent activation. The ablation of HUWE1 results in reduced ubiquitination of Rheb and decreased mTORC1 activity in cultured cells and mouse liver. HUWE1 is also necessary for Rheb to interact with carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase (CAD), a key enzyme in pyrimidine biosynthesis, and for CAD activation through the activation of the mTORC1-S6K1 pathway. Moreover, HUWE1 maintains CAD expression by increasing its transcript in cells and liver tissues. Therefore, HUWE1 acts as a key organizer of the ubiquitinated Rheb complex, playing a vital role in enhancing mTORC1 activity and pyrimidine synthesis by increasing both CAD activity and expression.
    Keywords:  CAD; CP: Cell biology; CP: Molecular biology; HUWE1; Rheb; mTOR; mTORC1; pyrimidine; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2025.116382
  11. Methods Mol Biol. 2026 ;2976 61-72
    Measurement of Physiological Autophagic Flux in the Human Peripheral Blood Mononuclear Cell Pool.
    Leanne K Hein, Kathryn J Hattersley, Julien Bensalem, Timothy J Sargeant.
      Autophagic flux, a crucial cellular process that is often overlooked in its significance for human health, has been linked to various chronic diseases, including cancer, heart disease, and dementia. Despite its importance, direct measurement in humans has been lacking, which has hindered translational efforts. Here, we describe a protocol for the measurement of human autophagic flux in blood. By treating whole blood with chloroquine and isolating peripheral blood mononuclear cells, autophagic flux can be measured by quantifying the autophagy protein LC3BII. This approach preserves individual genetic and nutritional parameters. This method can be used to identify factors influencing autophagic flux in humans, facilitating clinical translation and potentially serving as a biomarker for age-related chronic diseases. In this protocol, we describe detailed methodology, along with risks and limitations of this technique.
    Keywords:  Autophagic flux; Autophagy; Blood; Human; Lysosome; PBMC
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_6
  12. Proc Natl Acad Sci U S A. 2025 Oct 21. 122(42): e2517050122
    Global conformation of the Rag GTPase heterodimer governs eukaryotic amino acid sensing.
    Dylan D Doxsey, Kuang Shen.
      The Rag GTPase heterodimer is a central mediator of amino acid sensing in eukaryotic cells. When amino acids are abundant, it binds to the mechanistic target of rapamycin complex 1 to activate cellular programs for growth and proliferation. In its functional cycle, besides local conformational changes near the nucleotides that are commonly observed in monomeric signaling GTPases, the relative positioning of the two Rag subunits, i.e., the global conformation, is unique due to the heterodimeric architecture. Although various global conformations have been captured in static structural models, dynamic transitions between these conformations and their biological relevance remain unclear. Here, we visualize the global conformation of the Rag GTPase heterodimer using single-molecule Förster resonance energy transfer. By tracking the movement of individual protein molecules, we found that the two subunits explore a wide conformational space, which is strictly dictated by the bound nucleotides, regulators, and mutations. Additionally, we demonstrate that proper modulation of the global conformation is crucial for correctly interpreting amino acid signals. Our results defined a checkpoint of amino acid sensing in eukaryotic cells.
    Keywords:  Rag GTPase; amino acid sensing; mTORC1; protein conformation; single-molecule FRET
    DOI:  https://doi.org/10.1073/pnas.2517050122
  13. Nat Cell Biol. 2025 Oct;27(10): 1688-1707
    Chaperone-mediated autophagy regulates neuronal activity by sex-specific remodelling of the synaptic proteome.
    Rabia R Khawaja, Ernesto Griego, Kristen Lindenau, Asma Salek, Jessica Gambardella, Aurora Scrivo, Hannah R Monday, Mathieu Bourdenx, Jesús Madero-Pérez, Zohaib N Khan, Bhakti Chavda, Ronald Cutler, Sarah Graff, Simone Sidoli, Gaetano Santulli, Laura Santambrogio, Inmaculada Tasset, Susmita Kaushik, Li Gan, Pablo E Castillo, Ana Maria Cuervo.
      Chaperone-mediated autophagy (CMA) declines in ageing and neurodegenerative diseases. Loss of CMA in neurons leads to neurodegeneration and behavioural changes in mice but the role of CMA in neuronal physiology is largely unknown. Here we show that CMA deficiency causes neuronal hyperactivity, increased seizure susceptibility and disrupted calcium homeostasis. Pre-synaptic neurotransmitter release and NMDA receptor-mediated transmission were enhanced in CMA-deficient females, whereas males exhibited elevated post-synaptic AMPA-receptor activity. Comparative quantitative proteomics revealed sexual dimorphism in the synaptic proteins degraded by CMA, with preferential remodelling of the pre-synaptic proteome in females and the post-synaptic proteome in males. We demonstrate that genetic or pharmacological CMA activation in old mice and an Alzheimer's disease mouse model restores synaptic protein levels, reduces neuronal hyperexcitability and seizure susceptibility, and normalizes neurotransmission. Our findings unveil a role for CMA in regulating neuronal excitability and highlight this pathway as a potential target for mitigating age-related neuronal decline.
    DOI:  https://doi.org/10.1038/s41556-025-01771-1
  14. Cells. 2025 Sep 24. pii: 1495. [Epub ahead of print]14(19):
    NAD+ Homeostasis and Autophagy: Integrated Control Through Nutrient Signaling in Yeast and Mammals.
    Matilda McDaniel, Lan-Hsuan Lee, Su-Ju Lin.
      Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite facilitating redox and biochemical reactions in many cellular processes. Maintaining NAD+ homeostasis is critical for proper cellular function, and abnormalities in NAD+ metabolism have been associated with various human diseases. However, the mechanisms underlying its regulation and interconnection with nutrient-sensing pathways remain incompletely understood. Recent studies show that autophagy, a conserved catabolic pathway essential for cellular homeostasis, plays an important role in maintaining the NAD+ pool. NAD+ may also impact autophagy through its regulation of cellular metabolism and sirtuins, a family of NAD+-dependent deacetylases. Given the complexity of these pathways, their mechanistic interconnection is not fully understood. Here, we discuss studies examining the interactions of NAD+ metabolism, autophagy, and nutrient-sensing pathways, with a focus on the budding yeast Saccharomyces cerevisiae and connections to mammalian systems. We also discuss the role of sirtuins in these pathways and the impacts of NAD+ precursor supplementation. This review provides insights into how nutrient-sensing pathways may mediate the co-regulation of NAD+, autophagy, and cellular homeostasis. The studies discussed provide the basis for the development of future research directions that may inform potential therapeutic targets for human disorders associated with the dysregulation of NAD+ metabolism and autophagy.
    Keywords:  NAD+; autophagy; nutrient sensing; sirtuin
    DOI:  https://doi.org/10.3390/cells14191495
  15. Autophagy Rep. 2025 ;4(1): 2560903
    The space-time continuum in neurological disorders of the autophagosome-lysosome fusion machinery.
    Hormos Salimi Dafsari, Juliane Schuler, Emil Schober, Birk Möller, Adam Antebi, Manolis Fanto, Heinz Jungbluth.
      Autophagy is a highly conserved cellular pathway for the degradation and recycling of defective intracellular cargo and plays a vital role in the homeostasis of post-mitotic tissues, particularly the nervous system. Autophagosome-lysosome fusion represents the final critical step in macroautophagy with a tightly regulated process mediated by a complex molecular machinery of tethering vesicles for degradation. Since the first reports of human autophagy disorders, the scientific and clinical focus condensed on severe phenotypes with biallelic-truncating genotypes as monogenic models of near-complete autophagy perturbation. Recent reports suggest a much wider disease spectrum with defective autophagy, ranging from neurodevelopmental disorders to neurodegenerative phenotypes with later manifestation due to "milder" genotypes, including Alzheimer's disease (AD), Parkinson's disease (PD), and Amyotrophic Lateral Sclerosis-Frontotemporal Dementia (ALS-FTD). In addition, recent evidence identified molecular connections between physiological autophagy regulation during normal aging and pathophysiological hallmarks of aging-related disorders. These translational observations led to a more comprehensive understanding of autophagy at health and disease, in particular: 1) genetic location and allelism of pathogenic variants ("genomic space"); 2) protein-protein interaction in functional protein complexes ("proteomic space"); 3) metabolic autophagic flux with positive and negative regulators ("metabolomic space"); 4) age-related phenotypic progression over time. Here, we review the autophagosome-lysosome fusion machinery as a key structure both on the molecular level and with regards to the pathogenesis of the autophagy-related disease spectrum. We highlight the clinicopathological signature of disorders in the autophagosome-lysosome fusion machinery, in particular features warranting awareness from clinicians and geneticists to inform adequate diagnosis, surveillance, and patient guidance.
    Keywords:  Autophagy; aging; autophagosome-lysosome fusion machinery; neurodegenerative disorders; neurodevelopmental disorders
    DOI:  https://doi.org/10.1080/27694127.2025.2560903
  16. Int J Mol Sci. 2025 Oct 09. pii: 9826. [Epub ahead of print]26(19):
    Oxidative Stress, Mitochondrial Quality Control, Autophagy, and Sirtuins in Heart Failure.
    Jan Krekora, Marcin Derwich, Jarosław Drożdż, Elzbieta Pawlowska, Janusz Blasiak.
      Heart failure (HF) has become an emerging problem, especially in regions where life expectancy is increasing. Despite its prevalence, the mechanisms behind HF development are not well understood, which is reflected in the lack of curative therapies. Mitochondria, autophagy, and sirtuins form a crucial triad involved in HF pathogenesis, interconnected by oxidative stress. Identifying a common pathway involving these three components could be valuable in developing new treatment strategies. Since HF is the end result of several cardiovascular diseases, this review highlights the main HF precursors and explores the roles of mitochondrial quality control (mtQC), autophagy, and sirtuins in HF development. Dysfunctional mitochondria may play a key role by enhancing oxidative stress and influencing autophagy and sirtuins, both of which possess antioxidant properties. The dual nature of autophagy-its pro-life and pro-death roles-may contribute to different outcomes in HF related to oxidative stress. As mtQC, autophagy, and sirtuins may interact, we present data on their mutual dependencies in HF. However, the specificity of these interactions remains unclear and needs further investigation, which could help identify new therapeutic targets. In conclusion, the interplay between mtQC, autophagy, and sirtuins may be crucial in HF pathogenesis and could be leveraged in developing HF treatments.
    Keywords:  autophagy; heart failure; mitochondria; oxidative stress; sirtuins
    DOI:  https://doi.org/10.3390/ijms26199826
  17. Acta Neuropathol. 2025 Oct 14. 150(1): 43
    Genetic and proteomic analysis identifies BAG3 as an amyloid-responsive regulator of neuronal proteostasis.
    Zachary M Augur, Garrett M Fogo, Mason R Arbery, Yi-Chen Hsieh, Nalini R Rao, Kritika Goyal, Emily Dexter, David A Bennett, Jeffrey N Savas, Andrew M Stern, Tracy L Young-Pearse.
      The autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS) are the primary protein degradative mechanisms maintaining proteostasis in neurons. However, the impact of human genetic variation on these pathways and the role of BAG3 are poorly understood, particularly in the context of Alzheimer's disease, where proteostatic dysfunction is a defining hallmark. We utilized a large panel of iPSCs from deeply phenotyped cohorts to interrogate genetic contributions to baseline autophagic flux and UPS activity in human neurons, and protein turnover was assessed using SILAC-based quantitative proteomics. Across this panel of neurons, we observed substantial inter-individual differences in autophagic flux, which was inversely correlated with UPS activity. This reciprocal relationship extended to tau homeostasis, where higher autophagic flux resulted in reduced accumulation of aggregated, phosphorylated tau. Proteomic analyses revealed that global protein turnover dynamics stratified based on degradation pathway activity and could predict pathway-specific substrate dependencies. Interestingly, Bcl-2-associated athanogene 3 (BAG3), an important member of the chaperone-assisted selective autophagy pathway, emerged as a dynamically regulated autophagy chaperone, responsive to pharmacological inhibition of both the UPS and ALP. BAG3 knockout in neurons decreased autophagic flux and increased levels of high-molecular-weight phosphorylated tau. Notably, familial AD mutations and Aβ exposure induced BAG3 expression in neurons, while elevated BAG3 levels in human brain tissue were associated with higher neuropathological burden and disease progression. Our findings identify BAG3 as a key modulator of proteostasis in human neurons. Its regulation across genetic backgrounds and pathological stimuli suggests a central role in maintaining degradation activities in Alzheimer's disease and related disorders.
    Keywords:  Alzheimer’s disease; Amyloid-beta; Autophagy; BAG3; Neurons; Ubiquitin proteasome system; iPSC
    DOI:  https://doi.org/10.1007/s00401-025-02947-7
  18. Discov Oncol. 2025 Oct 17. 16(1): 1917
    The crosstalk between MiRNAs and autophagy in leukemia: mechanisms and therapeutic insights.
    Alireza Faghiri Beyrami, Ali Akbar Movasaghpour Akbari, Leili Aghebati-Maleki, Sanam Dolati, Mehdi Yousefi.
      Autophagy, a process of cellular degradation and recycling, plays a biphasic role in leukemia; it acts either as a protective process that promotes the cancer cell to survive, or as a pathway that triggers cell death depending on certain conditions. MicroRNAs (miRNAs) regulate many important genes of autophagy, and these involve autophagy's role in leukemia progression, drug resistance and therapy response. In this review, we will lay out the framework of how autophagy and miRNAs interact in that context and concentrate upon studies representative of how certain miRNAs such as miR-21, miR-155, or miR-29, regulate autophagy signaling pathways. We will also reflect on the potential of targeting the autophagy-miRNA axis to overcome drug resistance and enhance the outcome with treatment of leukemia. Although the utilization of miRNA-therapy and inhibitors have shown preclinical viability in leukemia, issues of delivery, specificity and toxicity create many obstacles. A greater comprehension of the molecular mechanisms underlying miRNA-mediated regulation of autophagy could lead to effective and novel therapeutic strategies for leukemia. This review highlights a clinical rationale to employ miRNAs and target autophagy modulation as a possible strategy to combat drug resistance leukemia.
    Keywords:  Autophagy; Autophagy-related (ATG) proteins; Leukemia; miRNA
    DOI:  https://doi.org/10.1007/s12672-025-03686-7
  19. Eur J Med Res. 2025 Oct 13. 30(1): 962
    Remimazolam alleviates hippocampal neuronal injury in an in vitro Alzheimer's disease model by promoting PINK1/Parkin-mediated mitophagy.
    Wei Zhang, Bowen Shi, Yuanyuan Xie, Yan Li, Jianke Yang, Ke Zhao.
      Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the pathological accumulation of amyloid-β (Aβ) plaques and hyperphosphorylated tau proteins. Remimazolam (RMZ), a novel ultra-short-acting benzodiazepine, exhibits neuroprotective effects by enhancing mitochondrial autophagy independently of traditional GABAergic mechanisms. This study investigates the protective role of RMZ against Aβ1-42-induced neuronal damage through PINK1/Parkin-mediated mitophagy. In hippocampal HT22 cells, RMZ significantly attenuated Aβ1-42-induced cytotoxicity, reduced apoptosis, suppressed reactive oxygen species (ROS) production, and decreased lactate dehydrogenase (LDH) release. Moreover, RMZ ameliorated mitochondrial membrane depolarization and tau hyperphosphorylation, while enhancing mitophagy, evidenced by an increased LC3-II/LC3-I ratio, elevated Beclin-1 expression, and decreased P62 levels. Mechanistically, RMZ upregulated PINK1 and Parkin expression, facilitating mitochondrial recruitment and clearance of damaged mitochondria. Importantly, knockdown of PINK1 abolished RMZ's protective effects, confirming the pathway's specificity. These findings suggest that RMZ promotes mitochondrial homeostasis and offers a promising strategy for AD therapy via PINK1/Parkin-mediated mitophagy.
    Keywords:  Alzheimer’s disease; Mitophagy; PINK1/Parkin pathway; Remimazolam
    DOI:  https://doi.org/10.1186/s40001-025-03179-x
  20. Methods Mol Biol. 2026 ;2976 85-102
    Lysosomal Immunoprecipitation (LysoIP) from Cells and Tissues for Metabolomic and Proteomic Analyses.
    Nouf N Laqtom, Monther Abu-Remaileh.
      Lysosomes, known for degrading biomolecules and damaged cellular components, are now recognized as signaling hubs for nutrient sensing and metabolic adaptation, and their dysfunction is implicated in diseases including cancer and neurodegeneration. To understand the composition of the lysosome, the dynamic behavior of its contents, and its specific roles in health and disease, we describe a lysosomal immunoprecipitation method, termed "LysoIP," that enables the isolation of intact lysosomes from cultured cells and mouse tissues. This method utilizes a lysosome-localized 3xHA epitope tag (LysoTag) and a simple, yet robust organelle immunoprecipitation workflow. Isolated lysosomes are extracted with optimized buffers to allow the efficient retrieval of lysosomal proteins, polar metabolites, and lipids, maintaining compatibility with downstream liquid chromatography and mass spectrometry (LC-MS) analyses.
    Keywords:  LC-MS analyses; LysoIP; LysoTag; LysoTag mouse; Lysosomes; Metabolomics; Proteomics; TMEM192
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_8
  21. Int J Mol Sci. 2025 Sep 26. pii: 9442. [Epub ahead of print]26(19):
    Modulation of mTOR Within Retinal Pigment Epithelium Affects Cell Viability and Mitochondrial Pathology.
    Gloria Lazzeri, Michela Ferrucci, Paola Lenzi, Maria Anita Giambelluca, Francesca Biagioni, Carla Letizia Busceti, Alessandro Frati, Francesco Fornai.
      The relevance of well-structured mitochondria in sustaining the integrity of the retinal pigment epithelium (RPE) is increasingly evident. Conversely, altered mitochondria are a culprit of age-related macular degeneration (AMD), which is influenced by the activity of mechanistic target of rapamycin (mTOR). In the present manuscript, the mitochondrial status of RPE cells was investigated by light and electron microscopy following the administration of various doses of compounds, which modulate mTOR. The study combines MitoTracker dyes and mitochondrial immunohistochemistry with in situ mitochondrial morphometry. Various doses of 3-methyladenine (3-MA), curcumin, and rapamycin were administered alone or in combination. The activity of autophagy and mTOR was quantified following each treatment. Administration of 3-MA led to activation of mTOR, which was associated with severe cell death, altered membrane permeability, and altered ZO-1 expression. In this condition, mitochondrial mass was reduced, despite a dramatic increase in damaged mitochondria being reported. The decrease in healthy mitochondria was concomitant with alterations in key mitochondria-related antigens such as Tomm20, Pink1, and Parkin. Specific mitochondrial alterations were quantified through in situ ultrastructural morphometry. Both curcumin and rapamycin counteract mTOR activation and rescue mitochondrial status, while preventing RPE cell loss and misplacement of decreased ZO-1 expression. Mitigation of mTOR may protect mitochondria in retinal degeneration.
    Keywords:  MitoTracker Green; MitoTracker Red; PINK1; Parkin; Tomm20; ZO-1; autophagy; curcumin; mitochondrial morphology; mitochondrial ultrastructure; rapamycin
    DOI:  https://doi.org/10.3390/ijms26199442
  22. Curr Opin Cell Biol. 2025 Oct 13. pii: S0955-0674(25)00133-4. [Epub ahead of print]97 102595
    Autophagy regulation by signaling.
    Tor Erik Rusten.
      The first identified regulators of autophagy were the hormones Glucagon and Insulin, long before the recognition that they act through receptor-mediated cell signaling pathways. More than two decades since the identification of the autophagy molecular machinery, we appreciate that regulation of autophagy is complex, operates at several levels, and serves many functions to maintain cellular and organismal health. TORC1 remains the best-defined gatekeeper, serving to sense nutrient and energy sufficiency while also integrating systemic growth factor signaling to control cell growth and autophagy autonomously. This review summarizes the current understanding of autophagy regulation by signaling through TORC1-dependent and independent mechanisms and discusses the emerging understanding of control and coordinated response of autophagy at the organism level.
    DOI:  https://doi.org/10.1016/j.ceb.2025.102595
  23. Nat Commun. 2025 Oct 17. 16(1): 9234
    Visualization of lysosomal membrane proteins by cryo electron tomography.
    Bridget M McVeigh, José J De Jesús-Pérez, Dirk H Siepe, Prerana Gogoi, Shrawan Kumar Mageswaran, Marian Kalocsay, Elaine M Mihelc, Vera Y Moiseenkova-Bell.
      Lysosomes are essential organelles for cellular homeostasis and signaling, with dysfunction linked to neurological disorders, lysosomal storage diseases, and cancer. While proteomics has advanced our understanding of lysosomal composition, the structural characterization of lysosomal membrane proteins in their native environment remains a significant challenge. Here, we developed a cryo electron tomography workflow to visualize lysosomal membrane proteins within intact, native lysosomal membranes. We isolated endolysosomes by independently targeting two lysosomal membrane proteins, transient receptor potential mucolipin 1 and transmembrane protein 192, enriching organelles that exhibited the expected morphology and proteomic composition of the endolysosomal system. Sub-tomogram averaging enabled the structural refinement of key membrane and membrane-associated proteins, including V-ATPase, Flotillin, and Clathrin, directly within the lysosomal membrane, revealing their heterogeneous distribution across endolysosomal organelles. By integrating proteomics with structural biology, our workflow establishes a powerful platform for studying lysosomal membrane protein function in health and disease, paving the way for future discoveries in membrane-associated lysosomal mechanisms.
    DOI:  https://doi.org/10.1038/s41467-025-64314-0
  24. Transpl Immunol. 2025 Oct 11. pii: S0966-3274(25)00143-1. [Epub ahead of print]93 102315
    Linking autophagy to endothelial cell immunogenicity in transplant-associated ischemia-reperfusion injury.
    Meredith E Taylor, Dinesh Jaishankar, Krishnaraju Madavaraju, Simseok A Yuk, Kaijie Zhang, Hongzhang Mei, Connor W Lantz, Luisa H Andrade da Silva, Yu Min Lee, Jacqueline A Burke, Carl Atkinson, Bowen Wang, Evan A Scott, Satish N Nadig.
       BACKGROUND: Cold storage (CS) and ischemia-reperfusion injury (IRI) are inevitable consequences of heart transplantation (HTx), primarily affecting the microvascular endothelial cells (ECs) of donor hearts. The nutrient deprivation and oxidative stresses sustained by ECs during CS and IRI activate and trigger alloimmune recognition. ECs adapt to these conditions by utilizing autophagy to maintain cellular homeostasis. Whether autophagy plays a role in EC activation and immunogenicity during CS and IRI in HTx remains unknown.
    METHODS: Autophagy in murine microvascular cardiac endothelial cells (MCEC) was modulated by genetic (knockout of the autophagy-related gene 5 (Atg5) by CRISPR/Cas9 technology) or pharmacological (rapamycin to induce autophagy and chloroquine to inhibit autophagy) approaches or by nanocarriers encapsulating rapamycin. MCECs were subjected to a previously established cold storage-warm reperfusion (CS-WR) model to mimic CS and IRI. We evaluated a) changes in autophagy by immunoblotting and confocal imaging, b) MCEC activation via pro-inflammatory cytokine and chemokine secretion by ELISA, and c) MCEC immunogenicity by determining T-cell activation in EC-T-cell co-cultures with MCEC-sensitized CD8+ T cells. The human relevance of EC autophagy was demonstrated by reanalyzing a recently published single-nucleus RNA sequencing dataset obtained from baseline and cold-preserved human donor hearts.
    RESULTS: Autophagy was reduced in MCECs when subjected to CS alone in organ preservation solution and increased early on during WR. The absence of the autophagic machinery in Atg5-/- MCECs significantly increased MCEC activation and immunogenicity, while pharmacological induction of autophagy with rapamycin significantly mitigated this effect. Additionally, treating MCECs with an organ preservation solution supplemented with rapamycin-loaded nanocarriers during hypothermic CS demonstrated a protective effect in mitigating MCEC immunogenicity. Reanalysis of the single-nucleus RNA sequencing dataset in EC subclusters revealed that cold storage induced an activated EC state characterized by reduced expression of autophagy-related genes, aligning with our in vitro findings.
    CONCLUSIONS: A novel role of autophagy in MCEC immunogenicity during CS and IRI is implicated. A viable preconditioning approach has also been demonstrated to bolster MCEC autophagy with nanocarriers during CS and mitigate its immunogenicity.
    Keywords:  Antigen presentation; Autophagy; Endothelial cells; Heart transplantation; Ischemia-reperfusion injury
    DOI:  https://doi.org/10.1016/j.trim.2025.102315
  25. Autophagy. 2025 Oct 16.
    Acetylation promotes mutant (MUT) TP53-HSPA8 and HSPA8-BAG3 interactions, facilitating MUT TP53 lysosomal degradation preferentially via CASA.
    Michele Di Crosta, Francesca Chiara Ragone, Rossella Benedetti, Gabriella D'Orazi, Roberta Santarelli, Roberta Gonnella, Maria Saveria Gilardini Montani, Mara Cirone.
      Targeting mutant (MUT) TP53 is crucial in anticancer therapy, given the oncogenic properties that these proteins often acquire. Therefore, it is of paramount importance to unravel strategies and mechanisms through which this goal can be achieved. Valproic acid (VPA) downregulates the expression of MUT TP53 in several tumor cells, although the mechanisms involved remain to be explored. Here, we demonstrate for the first time that acetylation induced by VPA promotes the lysosomal degradation of MUT TP53 and that it occurs preferentially through chaperone-assisted selective autophagy (CASA). Indeed, acetylation of MUT TP53 increases its interaction with STUB1 (STIP1 homology and U-box containing protein 1), HSPB8 (heat shock protein family B (small) member 8) and HSPA8 (heat shock protein family A (Hsp70) member 8) and the latter, itself acetylated by VPA, binds to BAG3 (BAG cochaperone 3), facilitating the recruitment of MUT TP53 into the CASA pathway. These findings elucidate the mechanisms through which acetylation leads to the selective lysosomal clearance of MUT TP53, highlighting a potential therapeutic vulnerability of aggressive tumors expressing this oncoprotein.
    Keywords:  Acetylation; Lysosomes; MUT TP53; Macroautophagy; Pancreatic cancer; VPA
    DOI:  https://doi.org/10.1080/15548627.2025.2576615
  26. Methods Mol Biol. 2026 ;2976 175-188
    Using CMAC Staining for Vacuole Characterization in Yeast.
    José Ángel Clemente-Ramos, Sara E Mole.
      CMAC (7-amino-4-chloromethylcoumarin) staining is a valuable tool for visualizing acidic organelles, including vacuoles, in yeast cells. By selectively accumulating in acidic compartments, CMAC allows specific labeling and visualization of vacuoles in living or fixed cells, aiding in the investigation of vacuole dynamics, morphology, and distribution under various conditions. This staining method provides a clear contrast against cellular autofluorescence, enhancing imaging clarity. It can be combined with automated imaging capture for faster throughput and analysis.Characterizing vacuole phenotypes using CMAC staining contributes to our understanding of cellular homeostasis and disease mechanisms in yeast. It has applications in studying fundamental cellular processes, including endocytic trafficking and autophagy, as well as in drug screening assays, and can be used in yeast models of disease to offer insights into potential therapeutic targets for diseases affecting the lysosome, including neurodegenerative diseases.
    Keywords:  7-amino-4-chloromethylcoumarin; CMAC staining; Fission yeast; Homeostasis; Lysosome; Schizosaccharomyces pombe; Vacuole
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_13
  27. Proteomics. 2025 Oct 15. e70058
    Proteomics Insights Into Lysosome Biogenesis and Maturation.
    Katharina Hirn, Sofía Fajardo-Callejón, Dominic Winter.
      Lysosomes constitute the main degradative organelle of most eukaryotic cells and are capable of breaking down a wide spectrum of biomolecules, including proteins, lipids, glycans, and DNA/RNA. They play crucial roles in the regulation of cellular homeostasis, acting as metabolic signaling centers for the correlation of nutrient availability and biosynthetic processes. The lysosome's importance is highlighted by several human diseases associated with its dysfunction, including both early- and late-onset conditions, dependent on the level of functional impairment. Lysosomal biogenesis presents a multi-step process consisting of various delivery routes for its individual constituents, enabling strict activity control of the currently known ∼60 lysosomal hydrolases to prevent cellular self-digestion and proper assembly of the lysosomal membrane. In this review, we recapitulate the contribution of mass spectrometry (MS)-based proteomics to the characterization of lysosomal biogenesis in the last two decades. The enrichment and proteomic analysis of lysosomes and lysosomal proteins played an invaluable role for the investigation of lysosomes, encompassing the control of lysosomal gene expression, the characterization of sorting/trafficking processes, and the assignment of lysosomal proteins. This has resulted so far in the definition of ∼350 proteins which have been identified to be located in/at lysosomes or are of crucial importance for their function.
    Keywords:  biogenesis; lysosomal proteins; lysosome; mass spectrometry; maturation; protein trafficking; proteomics; vesicular transport
    DOI:  https://doi.org/10.1002/pmic.70058
  28. J Cell Mol Med. 2025 Oct;29(19): e70704
    BNIP3L/BNIP3-Mediated Mitophagy Contributes to the Maintenance of Ovarian Cancer Stem Cells.
    Na Li, Tejinder Pal Khaket, Yajing Yang, Linzhou Wang, Shurui Cai, Aidan Li, Elsa Wani, Jessica Miao, Nan Zhang, Qingfei Zheng, Junran Zhang, Xuefeng Liu, Selvendiran Karuppaiyah, Dehua Pei, Qi-En Wang.
      Ovarian cancer remains the most lethal gynaecological malignancy, with tumour recurrence and chemoresistance posing significant therapeutic challenges. Emerging evidence suggests that cancer stem cells (CSCs), a rare subpopulation within tumours with self-renewal and differentiation capacities, contribute to these hurdles. Therefore, elucidating the mechanisms that sustain CSCs is critical for improving treatment strategies. Mitophagy, a selective process for eliminating damaged mitochondria, plays a key role in maintaining cellular homeostasis, including CSC survival. Our study demonstrates that ovarian CSCs exhibit enhanced mitophagy, accompanied by elevated expression of the mitochondrial outer membrane receptors BNIP3 and BNIP3L. Knockdown of BNIP3 or BNIP3L significantly reduces mitophagy and impairs CSC self-renewal, indicating that receptor-mediated mitophagy is essential for CSC maintenance. Mechanistically, we identify that hyperactivated NF-κB signalling drives the upregulation of BNIP3 and BNIP3L in ovarian CSCs. Inhibition of NF-κB signalling, either via p65 knockdown or pharmacological inhibitors, effectively suppresses mitophagy. Furthermore, we demonstrate that elevated DNA-PK expression contributes to the constitutive activation of NF-κB signalling, thereby promoting mitophagy in ovarian CSCs. In summary, our findings establish that BNIP3/BNIP3L-mediated mitophagy, driven by DNA-PK-dependent NF-κB hyperactivation, is essential for CSC maintenance. Targeting the DNA-PK/NF-κB/BNIP3L-BNIP3 axis to disrupt mitochondrial quality control in CSCs represents a promising therapeutic strategy to prevent ovarian cancer recurrence and metastasis.
    Keywords:  BNIP3; BNIP3L; DNA‐PK; NF‐κB; cancer stem cells; mitophagy; ovarian cancer
    DOI:  https://doi.org/10.1111/jcmm.70704
  29. Eur J Pharmacol. 2025 Oct 15. pii: S0014-2999(25)01005-2. [Epub ahead of print] 178251
    The EGR1/miR-199a-3p/mTOR axis: A crucial pathway in the amplification of myocardial damage under intermittent hypoxic conditions.
    Na Dong, Hongmei Yue.
      Intermittent hypoxia (IH) is a hallmark pathological feature of obstructive sleep apnea (OSA) and a key contributor to cardiovascular and myocardial injury in affected patients. Despite its clinical importance, the molecular mechanisms linking OSA to cardiovascular diseases remain poorly understood. Early Growth Response 1 (EGR1) has been shown to aggravate myocardial injury by regulating autophagy and apoptosis during hypoxia/reoxygenation (H/R) and ischemia-reperfusion (IR) injury. However, its role in IH-induced myocardial injury has not been elucidated. miR-199a-3p, a microRNA implicated in the regulation of cardiovascular disease progression and myocardial repair, has not been investigated under IH conditions. In this study, We explored the functions of EGR1 and miR-199a-3p in IH-induced myocardial injury using an in vitro model. We found that silencing EGR1 promoted autophagy, suppressed apoptosis, and alleviating IH-induced myocardial injury. Upregulation of miR-199a-3p inhibited mTOR signaling and enhanced autophagy, thereby mitigating the detrimental effects of IH on myocardial cells. Moreover, Our findings demonstrate that EGR1, as a transcription factor, regulates the miR-199a-3p/mTOR pathway to exacerbate IH-induced myocardial injury. These results suggest that the EGR1/miR-199a-3p/mTOR axis represents a promising therapeutic target for OSA associated myocardial injury.
    Keywords:  EGR1 Intermittent hypoxia miR-199a-3p mTOR Autophagy
    DOI:  https://doi.org/10.1016/j.ejphar.2025.178251
  30. Methods Mol Biol. 2026 ;2976 35-45
    Label-Free Imaging of Lysosomes in Living Cells by Photothermal Microscopy.
    Jun Miyazaki, Ryotaro Wagatsuma.
      Photothermal microscopy is an optical imaging technique used to visualize the distribution of trace amounts of endogenous dyes in living cells. This technique facilitates lysosomal observation and functional analysis by detecting and identifying accumulated substances within them, eliminating the need for molecular labeling. Here, we describe the fundamental principles and system configuration of a photothermal microscope. We also present a detailed procedure for performing photothermal measurements in living cells.
    Keywords:  Autophagy; Label-free; Live-cell imaging; Optical absorption; Photothermal microscope
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_4
  31. EMBO Mol Med. 2025 Oct 15.
    Isoginkgetin antagonizes ALS pathologies in its animal and patient iPSC models via PINK1-Parkin-dependent mitophagy.
    Ang Li, Sen Huang, Shu-Qin Cao, Jinyi Lin, Linping Zhao, Feng Yu, Miaodan Huang, Lele Yang, Jiaqi Xin, Jing Wen, Lingli Yan, Ke Zhang, Maoyuan Jiang, Weidong Le, Peng Li, Yong U Liu, Dajiang Qin, Jiahong Lu, Guang Lu, Hanming Shen, Xiaoli Yao, Evandro F Fang, Huanxing Su.
      Damaged mitochondria initiate mitochondrial dysfunction-associated senescence, which is considered to be a critical cause for amyotrophic lateral sclerosis (ALS). Thus, mitophagic elimination of damaged mitochondria provides a promising strategy in ALS treatment. Here, through screening of a large natural compound library (n = 9555), we have identified isoginkgetin (ISO), a bioflavonoid from Ginkgo biloba, as a robust and specific mitophagy inducer. ISO enhances PINK1-Parkin-dependent mitophagy via stabilization of the PINK1/TOM complex. In a translational perspective, ISO antagonizes ALS pathology in C. elegans and mouse models; intriguingly, ISO improves mitochondrial function and antagonizes motor neuron pathologies in three ALS patient-derived induced pluripotent stem cell systems (C9, SOD1, and TDP-43), highlighting a potential broad application to ALS patients of different genetic background. At the molecular level, ISO inhibits ALS pathologies in a PINK1-Parkin-dependent manner, as depletion or inhibition of PINK1 or Parkin blunts its benefits. These results support the hypothesis that mitochondrial dysfunction is a driver of ALS pathology and that defective mitophagy is a druggable therapeutic target for ALS.
    Keywords:  Amyotrophic Lateral Sclerosis; Drug Screening; Isoginkgetin; Mitophagy; PINK1-Parkin
    DOI:  https://doi.org/10.1038/s44321-025-00323-2
  32. Methods Mol Biol. 2026 ;2976 119-134
    Analyzing Endolysosome-Mitochondria Membrane Contact Sites by Thin Section TEM of Human Fibroblasts.
    Claudia Parra-Ruiz, Carlos Enrich, Silvana Zanlungo.
      Membrane contact sites (MCS) are dynamic nanoregions of close apposition between two different organelles, functioning as discrete lipid or ion transfer sites. This new concept in cell biology involves unique proteins at both membrane sites, named tethers, and emerges in early observations by transmission electron microscopy (TEM). Currently, this technique still constitutes a valuable tool for MCS visualization and quantification. In the last decade, Lysosomal Storage Diseases (LSD) have been instrumental in studying the MCS between lysosomes (Ly), or endolysosomes (EL), and other organelles in close proximity such as mitochondria or the endoplasmic reticulum (ER). At present, the analysis of composition, functioning, and alterations/rewiring of MCS in health and disease represents an innovative area of research for designing therapeutic strategies in a variety of pathologies. Here, we describe procedures for chemical fixation using the Flat Embedding technique to characterize and quantify the MCS between LE/Lys and mitochondria in human fibroblasts by thin-section TEM.
    Keywords:  Flat embedding; Image analysis; Lysosomal storage diseases; Lysosomes; Membrane contact sites; Mitochondria; Transmission electron microscopy-chemical fixation
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_10
  33. Autophagy Rep. 2025 ;4(1): 2568537
    Autophagy in the lung: guardian of homeostasis or driver of disease.
    Hyungsin Kim, Wenping Wang, Ioana Dobrescu, Joel Lee, Joshua Martorelli, Samuel Wang, Jessie Yanxiang Guo.
      Autophagy is a lysosome-directed recycling program that preserves lung homeostasis yet, when dysregulated, can cause disease. This review organizes current evidence by lung compartment and disease phase, proposing that autophagy polarity is determined by cell identity, micro-niche, and timing along the injury-repair continuum. In chronic obstructive pulmonary disease, epithelial autophagy is initially cytoprotective, but chronic smoke exposure reveals a lysosome bottleneck and stalled flux, while alveolar macrophages show impaired xenophagy and poor acidification. In idiopathic pulmonary fibrosis, autophagy is suppressed in type II epithelial cells and fibroblasts downstream of transforming growth factor beta (TGF-β) and mTORC1, which promotes epithelial stress programs and collagen translation. In acute lung injury and respiratory distress syndrome, timely autophagy activation limits cGAS-STING and NLRP3 signaling, preserves barrier integrity, and supports recovery. In asthma, autophagy supports mucin biogenesis in epithelial cells but is reduced in antigen-presenting cells, while eosinophil and mast cell effector functions rely on autophagy. In infection, xenophagy clears microbes but is actively subverted by bacteria and respiratory viruses. In non-small cell lung cancer (NSCLC), tumor-intrinsic autophagy maintains energy metabolism, redox balance, and enables immune evasion, whereas host autophagy can alternately support antitumor immunity or supply nutrients. We summarize small-molecule modulators, delivery strategies, and flux-aware tools that enable precise, cell- and phase-resolved modulation of autophagy to guide patient selection and improve therapy in respiratory disease.
    Keywords:  Autophagy; lung disease; lung disease treatment; lung homeostasis; metabolism
    DOI:  https://doi.org/10.1080/27694127.2025.2568537
  34. Methods Mol Biol. 2026 ;2976 189-207
    Modeling Lysosomal Disease in Dictyostelium discoideum: Examining the Trafficking and Secretion of Lysosomal Enzymes.
    William D Kim, Robert J Huber.
      Non-mammalian models are powerful systems for enhancing our understanding of lysosomal function and lysosomal storage diseases. The social amoeba Dictyostelium discoideum is an excellent model organism for studying lysosomal function, as its genome encodes many proteins associated with lysosomal disease. Methods for gene knockout are straightforward in D. discoideum and include restriction enzyme-mediated integration (REMI) mutagenesis, homologous recombination via the Cre-loxP system, and CRISPR/Cas9-mediated gene editing, which collectively allow researchers to study protein function (e.g., lysosomal enzymes) in a genetically tractable biomedical model system. Additionally, activity assays for conserved lysosomal enzymes are well-established in D. discoideum. In this chapter, we outline methods for studying the intracellular localization and secretion of conserved lysosomal proteins in D. discoideum.
    Keywords:  Dictyostelium discoideum; Enzyme; Immunofluorescence; Lysosomes; Model organism; Neurodegeneration; Secretion; Trafficking; Western blotting
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_14
  35. Front Immunol. 2025 ;16 1662830
    Implication of LAMP proteins and autophagy markers in colorectal cancer aggressiveness.
    Tsvetomira Ivanova, Dorian Dikov, Diana Molander, Angel M Dzhambov, Yordan Sbirkov, Nikolay Mehterov, Nikolay Belev, Boyko Atanasov, Maria Kazakova, Victoria Sarafian.
      Lysosome-associated membrane proteins (LAMPs) play a critical role in various cellular processes, including phagocytosis, lipid transport, neoangiogenesis, and tissue remodeling. Recent discussions have focused on their involvement in autophagy related to tumor progression. Emerging studies have underscored the connection between tumorigenesis and autophagy, highlighting that dysregulation of this process is associated with the resistance of tumor cells to conventional chemotherapy across numerous malignancies, including colorectal cancer (CRC). Currently, there is a notable absence of reliable prognostic biomarkers for effectively stratifying and predicting treatment responses in CRC patients. Our study examines the expression of LAMP1 and LAMP2 proteins and genes alongside key autophagy markers such as BECLIN1 and LC3B, investigating their relationships with CRC invasiveness. We present original data illustrating the association of LAMP molecules with standard autophagy markers and tumor budding. Our findings provide novel insights into the significance of these markers in CRC invasiveness, their relationship with survival rates, and their potential role as prognostic biomarkers.
    Keywords:  BECLIN1; LAMP1; LAMP2; LC3B; liquid biopsy; tumor budding
    DOI:  https://doi.org/10.3389/fimmu.2025.1662830
  36. Int J Mol Med. 2025 Dec;pii: 224. [Epub ahead of print]56(6):
    PINK1 overexpression suppresses p38 MAPK/NF‑κB signaling to attenuate chondrocyte senescence in osteoarthritis.
    Lishi Jie, Yuanhui Zhang, Jiangyu Liu, Houyu Fu, Zaishi Zhu, Zeling Huang, Xiaoqing Shi, Peimin Wang, Songjiang Yin.
      PTEN‑induced putative kinase 1 (PINK1), a master regulator of mitophagy, is implicated in mitochondrial homeostasis, yet its role in knee osteoarthritis (OA) pathogenesis remains unclear. The present study investigated the mechanisms by which PINK1 modulates chondrocyte senescence during OA progression. Utilizing a destabilization of the medial meniscus‑induced OA murine model, decreased PINK1 expression, impaired mitochondrial function and suppressed mitophagy were observed in OA cartilage. In vitro, lipopolysaccharide‑induced chondrocyte senescence was exacerbated by PINK1 knockdown but mitigated by PINK1 overexpression, which restored mitophagy and reduced senescence‑associated β‑galactosidase activity, reactive oxygen species accumulation and mitochondrial membrane potential collapse. RNA sequencing and mechanistic studies identified the p38 MAPK/NF‑κB pathway as a downstream target; PINK1 knockdown amplified the phosphorylation of p38 MAPK/NF‑κB, promoting mitochondrial dysfunction and senescence. By contrast, pharmacological inhibition of p38 MAPK/NF‑κB rescued these effects in PINK1‑deficient chondrocytes. Collectively, PINK1 attenuated OA progression by suppressing chondrocyte senescence via inhibition of the p38 MAPK/NF‑κB pathway, highlighting its potential as a therapeutic target for OA management.
    Keywords:  PTEN‑induced putative kinase 1; chondrocyte senescence; mitophagy; osteoarthritis; p38 MAPK/NF‑κB signaling
    DOI:  https://doi.org/10.3892/ijmm.2025.5665
  37. Nat Commun. 2025 Oct 17. 16(1): 9220
    Autophagy acts as a brake on obesity-related fibrosis by controlling purine nucleoside signalling.
    Klara Piletic, Amir H Kayvanjoo, Felix Clemens Richter, Mariana Borsa, Ana V Lechuga-Vieco, Oliver Popp, Sacha Grenet, Jacky Ka Long Ko, Lin Luo, Kristina Zec, Maria Kyriazi, Harriet K Haysom, Lada Koneva, Stephen Sansom, Philipp Mertins, Fiona Powrie, Ghada Alsaleh, Anna Katharina Simon.
      A hallmark of obesity is a pathological expansion of white adipose tissue (WAT), accompanied by marked tissue dysfunction and fibrosis. Autophagy promotes adipocyte differentiation and lipid homeostasis, but its role in obese adipocytes and adipose tissue dysfunction remains incompletely understood. Using a mouse model, we demonstrate that autophagy is a key tissue-specific regulator of WAT remodelling in diet-induced obesity. Importantly, loss of adipocyte autophagy substantially exacerbates pericellular fibrosis in visceral WAT. Change in WAT architecture correlates with increased infiltration of macrophages with tissue-reparative, fibrotic features. We uncover that autophagy restrains purine nucleoside metabolism in obese adipocytes. This ultimately leads to a reduced release of the purine catabolites xanthine and hypoxanthine. Purines signal cell-extrinsically for fibrosis by driving macrophage polarisation towards a tissue reparative phenotype. Our findings in mice reveal a role for adipocyte autophagy in regulating tissue purine nucleoside metabolism, thereby limiting obesity-associated fibrosis and maintaining the functional integrity of visceral WAT. Purine signals may serve as a critical balance checkpoint and therapeutic target in fibrotic diseases.
    DOI:  https://doi.org/10.1038/s41467-025-64266-5
  38. Stem Cell Reports. 2025 Oct 16. pii: S2213-6711(25)00284-X. [Epub ahead of print] 102680
    mTORC2-mediated cell-cell interactions promote BMP4-induced WNT activation and mesoderm differentiation.
    Li Tong, Faiza Batool, Yueh-Ho Chiu, Priscilla Di Wu, Yudong Zhou, Xiaolun Ma, Santosh Atanur, Wei Cui.
      The mechanistic target of rapamycin complex 2 (mTORC2) is essential for embryonic development, but its underlying molecular mechanisms remain unclear. Here, we show that disruption of mTORC2 in human embryonic stem cells (hESCs) considerably alters the Rho/Rac signaling dynamics and reduces E-cadherin expression and cell adhesion. Despite this, mTORC2-deficient hESCs maintain self-renewal and expression of pluripotent markers when cultured in mouse embryonic fibroblast conditioned medium (MEF-CM) supplemented with bFGF. However, these hESCs exhibit significantly impaired mesoderm and endoderm differentiation in response to BMP4 and Activin treatment, respectively, possibly due to reduced WNT activation mediated by cell-cell interactions. Direct activation of the WNT pathway using a GSK3 inhibitor restores mesendoderm differentiation in mTORC2-deficient hESCs. Our study uncovers a novel mechanism by which mTORC2 regulates cell fate determination and highlights a critical link between the intercellular adhesion and the activation of canonical WNT genes.
    Keywords:  BMP4; WNT; cell adhesion; cell-cell interactions; gastrulation; human embryonic stem cells; mTORC2; mesoderm/endoderm differentiation
    DOI:  https://doi.org/10.1016/j.stemcr.2025.102680
  39. Autophagy Rep. 2025 ;4(1): 2562429
    Influence of skeletal muscle heterogeneity on autophagic signaling and response.
    Fasih A Rahman, Joe Quadrilatero.
      Skeletal muscle is a heterogeneous tissue composed of fibers with distinct contractile, metabolic, and molecular characteristics. This intrinsic heterogeneity influences how individual fibers respond to physiological stimuli, pathological stress, and cellular remodeling processes such as autophagy. Skeletal muscle autophagy is essential for maintaining proteostasis and organelle quality, particularly in high-demand tissues like skeletal muscle. However, emerging evidence indicates that autophagy is not uniformly regulated across all muscles and fibers within a skeletal muscle. Fast/glycolytic fibers, characterized by faster contractile speed and high glycolytic capacity, exhibit greater autophagic flux potentially driven by activation of energy signals, calcium, and redox-sensitive pathways. In contrast, slow/oxidative fibers, characterized by slow contractile speed and higher oxidative metabolism, show lower inducible autophagy despite elevated basal expression of autophagy-related proteins. These differences are compounded by fiber type - specific organelle architecture, recruitment patterns during activity and disuse, and substrate availability and utilization. Further, pathological conditions such as disuse, chronic disease, and myopathies often induce fiber type alterations as well as changes to organelle content and function that are closely associated with changes in autophagy signaling. Additionally, species and strain variability add another layer of complexity, complicating both the interpretation and translational relevance of autophagy studies in skeletal muscle. This review synthesizes current evidence linking skeletal muscle phenotype to autophagy regulation and highlights the need to consider skeletal muscle heterogeneity as a central variable in skeletal muscle autophagy research. A deeper understanding of skeletal muscle type/fiber-specific autophagy will improve our ability to interpret experimental findings and develop targeted interventions for skeletal muscle dysfunction.
    Keywords:  Skeletal muscle; autophagy; fiber type; heterogeneity; metabolic phenotype; muscle plasticity
    DOI:  https://doi.org/10.1080/27694127.2025.2562429
  40. Methods Mol Biol. 2026 ;2976 1-10
    Determining Lysosomal Enzyme Activity Using Fluorogenic Probes.
    Etienne Sauvageau, Stephane Lefrancois.
      Lysosomes are responsible for a number of cellular functions, including the degradation of various biological molecules. Soluble enzymes within the lysosomal lumen are required to perform this function. Lysosomal activity can be disrupted in a variety of diseases, and measuring the activity of specific enzymes can be performed. In this chapter, we detail how lysosomal enzyme activity can be measured either in cell lysates or intact cells. This can be used to study fundamental cell biology or the effect of therapeutics targeting lysosomal function.
    Keywords:  Batten disease; Fluorogenic substrates; Lysosomal enzyme activity; Lysosomes
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_1
  41. Biochimie. 2025 Oct 15. pii: S0300-9084(25)00235-4. [Epub ahead of print]
    Targeting Autophagy via Mitochondrial Uncoupling: Discovery of Novel Serratin Derivative as a Potential Therapeutic for Parkinson's Disease.
    Alexander Yu Rudenko, Ekaterina А Guseva, Ratislav M Ozhiganov, Boris P Myasnikov, Maria V Belopolskaya, Olga V Fadeeva, Elena О Morgun, Olga A Averina, Elena A Tukhovskaya, Gulsara A Slashcheva, Igor A Dyachenko, Elena I Marusich, Mariia Mokhina, Rose-Lys Zaranaina, Yuriy A Ikhalaynen, Igor A Rodin, Ljudmila S Khailova, Yuri N Antonenko, Vladimir I Polshakov, Maxim L Lovat, Viktor G Kartsev, Arkady N Murashev, Petr V Sergiev.
      Autophagy, a highly conserved cellular degradation pathway, plays a critical role in maintaining cellular homeostasis across eukaryotes. Dysregulation of autophagy has been implicated in numerous diseases, including neurodegenerative disorders such as Parkinson's disease. Although natural compounds like urolithin A and its synthetic analogue serratin (AN2) have been shown to induce autophagy, their limited potency and safety profiles necessitate the development of improved alternatives. In this study, a library of 27 novel AN2 analogues was synthesized and screened for autophagy-inducing activity. Among them, ORA471 emerged as a lead compound, exhibiting superior autophagy and mitophagy activation compared to AN2, along with reduced cytotoxicity in human fibroblast (VA-13) and neuroblastoma (SH-SY5Y) cell lines. Mechanistic investigations revealed that ORA471 induces autophagy primarily via the AMPK/ULK1 signaling pathway and acts as a mitochondrial uncoupler, dissipating membrane potential and enhancing respiration in isolated rat liver mitochondria. In vivo, ORA471 demonstrated low toxicity in Caenorhabditis elegans, zebrafish (Danio rerio), and mice (maximum tolerated dose: 300 mg/kg). Notably, it significantly improved motor function in a zebrafish model of MPTP-induced Parkinson's disease without eliciting adverse effects. These findings highlight ORA471 as a promising therapeutic candidate for the treatment of autophagy-related disorders, particularly Parkinson's disease.
    Keywords:  Autophagy; Parkinson's disease; mitochondrial dysfunction; mitophagy; serratin
    DOI:  https://doi.org/10.1016/j.biochi.2025.10.011
  42. FASEB J. 2025 Oct 31. 39(20): e71134
    Cdk5 Contributes to Diabetic Islet β Cell and Kidney Injury by Impairing Autophagy.
    Yuejia Tao, Li Wang, Yipeng Liu, Yong Wei, Ruixue Wang, Sha Sha, Shanshan Zheng, Shijie Hou, Shunyao Liu.
      Insufficient insulin secretion due to islet β cell damage is a hallmark of diabetes mellitus. Diabetic nephropathy (DN) is a common microvascular complication of diabetes, and podocyte damage is the main cause of proteinuria in patients with DN. Over-activation of cyclin-dependent kinase 5 (Cdk5) is involved in the development of diabetes and its complications; however, the specific mechanism remains to be elucidated. The aim of this study was to investigate the role of Cdk5 in diabetic islet β cells and renal injury. Our results showed that Cdk5 expression was upregulated in diabetic mice, which induced attenuated autophagy and increased apoptosis of islet β cells, as well as decreased insulin secretion. Similarly, Cdk5 activation impaired autophagy and apoptosis of podocytes. Decreasing the expression of Cdk5 in diabetes and DN partially restored the autophagy of islet β cells and podocytes and reduced the damage to islet β cells and podocytes. In conclusion, Cdk5 is involved in islet β cell and podocyte damage in a high glucose environment; thus, targeting Cdk5 may be a significant therapeutic option for diabetes and its complications.
    Keywords:  Cdk5; autophagy; diabetes mellitus; diabetic nephropathy; islet β cell; podocyte
    DOI:  https://doi.org/10.1096/fj.202500483RRRRR
  43. FASEB J. 2025 Oct 31. 39(20): e71154
    METTL3 Affects Endometrial Receptivity Through Modulation of P62-Mediated Autophagic Flux.
    Ming Zhang, Xiaolong Ren, Xiujuan Hu, Jincheng Li, Wenjuan Xia, Jiafeng Lu, Qingxia Meng, Boxian Huang.
      Endometrial receptivity is a crucial factor in the successful implantation of embryos. Our previous study has proved that METTL3 acted as a key regulatory factor in endometrial receptivity. However, the regulatory relationship between METTL3-mediated modifications to m6A and autophagy in endometrial receptivity is not yet elucidated. Here, this study investigated the functions and essential processes of METTL3 in autophagy. In this study, METTL3 loss is linked to autophagy dysregulation in endometrial tissue with recurrent implantation failure, and METTL3 deletion reduced the attachment efficiency of BeWo cell spheres. Mechanically, down-expression of METTL3 increased H3K9me3 modification at the gene body of P62 by upregulating SUV39H1 expression, leading to the upregulation of P62 mRNA expression. Meanwhile, METTL3 absence enhanced the mRNA stability of P62 in a m6A-dependent manner. These alterations collectively lead to increased P62 expression, enhancing senescence, inflammation, apoptosis, and autophagy in endometrial cells. These findings elucidate the molecular underpinnings of endometrial receptivity and highlight potential therapeutic targets for recurrent implantation failure.
    Keywords:  METTL3; P62; RIF; autophagy; endometrial receptivity; m6A
    DOI:  https://doi.org/10.1096/fj.202501383RR
  44. Autophagy. 2025 Oct 16.
    Lysosomal proteomics reveals mechanisms of neuronal APOE4-associated lysosomal dysfunction.
    Einar K Krogsaeter, Justin McKetney, Leopoldo Valiente-Banuet, Angelica Marquez, Alexandra Willis, Zeynep Cakir, Erica Stevenson, Gwendolyn M Jang, Antara Rao, Emmy Li, Anton Zhou, Anjani Attili, Timothy S Chang, Martin Kampmann, Yadong Huang, Nevan J Krogan, Danielle L Swaney.
      APOE4 is the primary risk factor for Alzheimer disease (AD). Early AD pathological events first affect the neuronal endolysosomal system, which in turn causes neuronal protein aggregation and cell death. Despite the crucial influence of lysosomes upon AD pathophysiology, and that APOE4 localizes to lysosomes, the influence of APOE4 on lysosomal function remains unexplored. We find that expression of APOE4 in neuronal cell lines results in lysosomal alkalinization and impaired lysosomal function. To identify driving factors for these defects, we performed quantitative lysosomal proteome profiling. This revealed that APOE4 expression results in differential regulation of numerous lysosomal proteins, correlating with APOE allele status and disease severity in AD brains. In particular, APOE4 expression results in the depletion of lysosomal LGALS3BP and the accumulation of lysosomal TMED5. We additionally validated that these lysosomal protein changes can be targeted to modulate lysosomal function. Taken together, this work thereby reveals that APOE4 causes widespread lysosomal defects through remodeling the lysosomal proteome, with the lysosomal TMED5 accumulation and LGALS3BP depletion manifesting as lysosomal alkalinization in APOE4 neurons.
    Keywords:  APOE4; Alzheimer disease; LGALS3BP; LysoIP; TMED5; lysosomes; pH
    DOI:  https://doi.org/10.1080/15548627.2025.2576613
  45. Autophagy. 2025 Oct 15. 1-3
    The autophagy-recessive tissue hormone DBI/ACBP (diazepam binding inhibitor, acyl-CoA binding protein) contributes to the pathogenesis of osteoarthritis.
    Uxía Nogueira-Recalde, Beatriz Caramés, Isabelle Martins, Guido Kroemer.
      Osteoarthritis (OA) is the most common form of arthritis and a leading cause of disability in the elderly, characterized by the progressive destruction of cartilage, synovial inflammation, and subchondral bone remodeling. While mechanical stress, metabolic derangements, and systemic inflammation are recognized contributors, accumulating evidence underscores the pivotal role of impaired macroautophagy/autophagy in disease pathogenesis. Autophagy declines with age, depriving chondrocytes and synovial cells of their cytoprotective capacity and rendering them vulnerable to apoptosis, matrix degradation, and inflammatory activation. Recent work has identified DBI/ACBP (diazepam binding inhibitor, acyl-CoA binding protein) as an extracellular hormone that represses autophagy through binding to the GABRG2/GABAARγ2 (gamma-aminobutyric type A receptor subunit gamma2) subunit of the GABA type A receptor. Plasma levels of DBI/ACBP are elevated in metabolic syndrome, obesity, diabetes, and aging, all known OA risk factors, and are upregulated in patients at risk of severe OA. In murine models of experimental OA, genetic deletion or antibody-mediated neutralization of DBI/ACBP mitigates joint inflammation, reduces cartilage destruction, and improves functional outcomes. These findings establish DBI/ACBP as a central pathogenic mediator linking aging-associated autophagy decline to osteoarthritis progression. Targeting DBI/ACBP represents a promising strategy to restore tissue homeostasis and modify the natural course of OA, with direct translational potential.
    Keywords:  Aging; autophagy; immunosurveillance; inflammation; senescence
    DOI:  https://doi.org/10.1080/15548627.2025.2573220
  46. Methods Mol Biol. 2026 ;2976 25-34
    Assessing Perturbations in Lysosome Health by Flow Cytometry in iPSC-Derived Human Neuron Cultures.
    Kirstin O McDonald, Stephanie M Hughes, Indranil Basak.
      Fluorescent molecular probes have frequently been used to monitor lysosomal health, localization, abundance, and movement through the detection of acidic organelles and lysosomal enzyme activity. Flow cytometry technology provides rapid and accurate analysis of single cells (neurons) or particles (lysosomes) in suspension through laser detection. Herein, we describe how to detect lysosomes via LysoTracker™ and Magic Red® Cathepsin Activity assays in iPSC-derived human neuron cultures by flow cytometry.
    Keywords:  Flow cytometry; Fluorescent probes; Lysosome acidity; Lysosome function; iPSC-derived neuron cultures
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_3
  47. Nat Cell Biol. 2025 Oct;27(10): 1708-1724
    MAPL regulates gasdermin-mediated release of mtDNA from lysosomes to drive pyroptotic cell death.
    Mai Nguyen, Jack J Collier, Olesia Ignatenko, Genevieve Morin, Vanessa Goyon, Alexandre Janer, Camila Tiefensee Ribeiro, Austen J Milnerwood, Sidong Huang, Michel Desjardins, Heidi M McBride.
      Mitochondrial control of cell death is of central importance to disease mechanisms from cancer to neurodegeneration. Mitochondrial anchored protein ligase (MAPL) is an outer mitochondrial membrane small ubiquitin-like modifier ligase that is a key determinant of cell survival, yet how MAPL controls the fate of this process remains unclear. Combining genome-wide functional genetic screening and cell biological approaches, we found that MAPL induces pyroptosis through an inflammatory pathway involving mitochondria and lysosomes. MAPL overexpression promotes mitochondrial DNA trafficking in mitochondrial-derived vesicles to lysosomes, which are permeabilized in a process requiring gasdermin pores. This triggers the release of mtDNA into the cytosol, activating the DNA sensor cGAS, required for cell death. Additionally, multiple Parkinson's disease-related genes, including VPS35 and LRRK2, also regulate MAPL-induced pyroptosis. Notably, depletion of MAPL, LRRK2 or VPS35 inhibited inflammatory cell death in primary macrophages, placing MAPL and the mitochondria-lysosome pathway at the nexus of immune signalling and cell death.
    DOI:  https://doi.org/10.1038/s41556-025-01774-y
  48. Cell Rep. 2025 Oct 10. pii: S2211-1247(25)01176-3. [Epub ahead of print]44(10): 116405
    Role of AAA-ATPase Cdc48p in peroxisomal quality control.
    Ismaila Francis Yusuf, Tomasz Jeziorek, Andrea Droste, Wolfgang Girzalsky, Ralf Erdmann.
      Most peroxisomal matrix proteins contain a type 1 peroxisomal targeting signal (PTS1), which is recognized by the cytosolic receptor Pex5p for delivery into peroxisomes. Following cargo translocation, the receptor is monoubiquitinated and recycled to the cytosol by the AAA-ATPases Pex1p and Pex6p. Defects in recycling trigger a quality control process by which the receptor is polyubiquitinated, extracted, and targeted to the proteasome for degradation by the RADAR (receptor accumulation and degradation in the absence of recycling) pathway. Although the RADAR pathway is evolutionarily conserved, it is unknown whether it is active in Saccharomyces cerevisiae. Here, we identify and characterize the RADAR pathway in S. cerevisiae and discover that the AAA-ATPases Msp1p and predominantly Cdc48p, together with its cofactors Ufd1p/Npl4p, are constituents of this pathway. We conclude that peroxisomes contain an endoplasmic reticulum-associated degradation-like protein quality control system (RADAR) in which Cdc48p and Ufd1p/Npl4p extract misfolded, polyubiquitinated receptors from the peroxisomal membrane for proteasomal degradation.
    Keywords:  AAA-ATPase; ATPases associated with diverse cellular activities; CHX; CP: Cell biology; CP: Molecular biology; RADAR; UPS; cycloheximide chase; peroxins; peroxisomal import receptor; peroxisome; protein degradation; protein stability; quality control; ubiquitin proteasomal system
    DOI:  https://doi.org/10.1016/j.celrep.2025.116405
  49. Mol Neurobiol. 2025 Oct 14.
    18β-Glycyrrhetinic Acid Regulates Endoplasmic Reticulum Stress and Autophagy Dysregulation in the MPTP/p-Induced Model of Parkinson Disease.
    Priyanka Kumari Keshri, Aaina Singh Rathore, Richa Singh, Hagera Dilnashin, Shekhar Singh, Nitesh Kumar Gupta, Singh Ankit Satyaprakash, Kumud Tiwari, Surya Pratap Singh.
      Parkinson disease (PD) is marked by a significant reduction in dopaminergic neurons in the substantia nigra pars compacta region of the brain. This neuronal loss is accompanied by aggregation of the α-synuclein protein, persistent endoplasmic reticulum (ER) stress, and disruption in the autophagy process. 18β-Glycyrrhetinic acid (18βGA), an oleanolic acid-type triterpenoid, has been shown to exhibit anti-inflammatory properties and neuroprotective effects. This study is the first to explore the potential neuroprotective effects of 18βGA in a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid (MPTP/p)-induced mouse model of PD, focusing on the role of ER stress and autophagy and examining the potential underlying mechanisms. MPTP/p-treated mice exhibited impaired motor function and elevated levels of α-synuclein and ER stress markers such as BiP, protein kinase RNA-like ER kinase (p-PERK), phosphorylated inositol-requiring enzyme 1 (p-IRE1), phosphorylated eukaryotic initiation factor α (p-eIF2α), and C/EBP homologous binding protein (CHOP). It also shows autophagy dysregulation, marked by increased phosphorylated c-Jun N-terminal kinase 1 (p-JNK-1), Beclin-1, and microtubule-associated protein 1 light chain 3 (LC3)-II, as well as autophagic vacuoles, and decreased B-cell lymphoma 2 (BCL-2) and p62. Treatment with 18βGA significantly improved motor performance, reduced α-synuclein accumulation, and restored tyrosine hydroxylase (TH) expression. It also attenuated ER stress markers, including BiP, p-PERK, p-IRE1, p-eIF2α, and CHOP. Moreover, 18βGA normalized autophagy-related alterations by decreasing p-JNK-1, Beclin-1, LC3-II, and autophagic vacuole formation, while increasing BCL-2 and p62 expression. These findings suggest that 18βGA confers neuroprotection by suppressing ER stress (via PERK and IRE1α pathways) and modulating autophagy through the BCL-2/Beclin-1 axis. Thus, 18βGA holds promise as a therapeutic candidate for Parkinson disease.
    Keywords:  18β-Glycyrrhetinic acid; Autophagy dysfunction; ER stress; IRE1α-JNK1; PERK-EIF2α; Parkinson disease; α-Synuclein
    DOI:  https://doi.org/10.1007/s12035-025-05274-w
  50. Exp Neurol. 2025 Oct 13. pii: S0014-4886(25)00368-1. [Epub ahead of print]395 115503
    Pramipexole improves cognitive deficits and synaptic plasticity impairments in 3xTg-AD mice through enhancing autophagy.
    Chunmei Tuo, Xiaofan Sui, Chenglong Cui, Feifei Han, Juanjuan Jiao, Hongyan Cai, Meina Wu.
      Autophagy dysfunction plays important roles in the pathogenesis of Alzheimer's disease (AD), and activation of autophagy might be a potential strategy in AD treatment. Although some autophagy inducers play beneficial roles in AD, no autophagy inducer has been available in AD clinical treatment due to lack of efficacy or obvious side effects. A recent study demonstrated that pramipexole (PPX) can activate autophagy in the brain without interfering with protein synthesis, thereby avoiding the side effects associated with prolonged use of an autophagy inducer. However, whether PPX can exert neuroprotective effects on AD and whether its potential mechanism is related to autophagy remains unclear. In the present study, the effects of PPX on the cognitive function of 3xTg-AD mice were observed using multiple behavioral tests, then synaptic plasticity was evaluated through in vivo hippocampal electrophysiological recordings and by measuring synaptic proteins, while Aβ and tau pathologies were detected using immunofluorescence staining and western blot. Finally, autophagy was detected and the potential target was predicted using bioinformatics analysis, qRT-PCR and molecular docking. Results demonstrated that PPX significantly improved cognitive deficits and hippocampal long-term potentiation (LTP) depression, increased the expression of PSD95, reduced Aβ deposition and tau hyperphosphorylation, activated autophagy by increasing LC3-II/LC3-I ratios and decreasing p62 levels, and restored Beclin1 expression. Bioinformatics analysis identified MAPK1 as a key target in PPX-ameliorated AD by enhancing autophagy. These findings suggest that PPX alleviates AD-related cognitive deficits and neuropathologies through increased autophagy, emphasising its potential as a disease-modifying therapeutic strategy for AD.
    Keywords:  Alzheimer's disease; Autophagy; Cognitive function; Pramipexole
    DOI:  https://doi.org/10.1016/j.expneurol.2025.115503
  51. Int J Mol Sci. 2025 Sep 27. pii: 9454. [Epub ahead of print]26(19):
    Lysosomal Network Defects in Early-Onset Parkinson's Disease Patients Carrying Rare Variants in Lysosomal Hydrolytic Enzyme Genes.
    Alba Pascual, Thaleia Moulka, Oriol de Fàbregues, Roberta Repossi, Pedro J García-Ruiz, Saida Ortolano, Marisel De Lucca, Lydia Vela-Desojo, Marta Alves-Villar, Marcos Frías, Cici Feliz-Feliz, Mònica Roldán, Jonathan Olival, Guerau Fernàndez, Francesc Palau, Jordi Pijuan, Janet Hoenicka.
      Despite significant advances in understanding the genetics of Parkinson's disease (PD) and Parkinsonism, the diagnostic yield remains low. Pathogenic variants of GBA1, which encodes the lysosomal enzyme β-glucocerebrosidase and causes recessive Gaucher dis-ease, are recognized as the most important genetic risk factor for PD in heterozygous carriers. This study focuses on the functional genomics of rare genetic variations in other lysosomal hydrolytic enzymes genes in patient-derived fibroblasts. We examined 49 early-onset PD patients using whole exome sequencing and in silico panel analysis based on a curated PD gene list. Two patients were found to carry the p.Asp313Tyr variant in the X-linked GLA gene (encoding GALA, typically associated with Fabry disease), and one patient carried the p.Arg419Gln variant in GLB1 (encoding β-Gal, linked to the recessive GM1 gangliosidosis and mucopolysaccharidosis type IVB). The in silico study of both variants supports a potentially damaging impact on the encoded protein function and structural destabilization. Additional candidate variants were found related to lysosomes, Golgi apparatus and neurodegeneration, suggesting a multifactorial contribution to the disease. However, none of these variants met diagnostic standards. Functional assays showed a significant decrease in GALA expression and partial retention of the enzyme in the trans-Golgi network in fibroblasts with GLA:p.Asp313Tyr, while altered Golgi morphology was observed in fibroblasts with GLB1:p.Arg419Gln. Moreover, all patients exhibited abnormalities in lysosomal morphology, altered lysosomal pH, and impaired autophagic flux. Our findings suggest that rare, heterozygous variants in lysosomal-related genes, even when individually insufficient for monogenic disease, can converge to impair lysosomal homeostasis and autophagic flux in EOPD. The underlying genetic and cellular heterogeneity among patients emphasizes the importance of combining genetic and functional approaches to better understand the mechanisms behind the EOPD, which could enhance both diagnosis and future treatments.
    Keywords:  GLA; GLB1; early-onset Parkinson’s disease (EOPD); lysosomal dysfunction; α-galactosidase A; β-galactosidase
    DOI:  https://doi.org/10.3390/ijms26199454
  52. J Mol Med (Berl). 2025 Oct 18.
    Suppression of OTUD4 protects against myocardial ischemia-reperfusion injury by increasing autophagic flux and inhibiting apoptosis in cardiomyocytes.
    Xin-Xin Sun, Lin Qi, Ming-Jun Dai, Fang-Lin Lu, Yi-Min Xiao, Wei-Jin Sun, Yu Zhuang.
      Dysregulated autophagic flux plays a critical role in myocardial ischemia-reperfusion injury (MIRI), complicating cardiac reperfusion therapy. In this study, we identified OTUD4 as a potential regulator of autophagic flux in MIRI using CRISPR/Cas9 sgRNA sequencing. However, the underlying mechanism is poorly understood. The purpose of this study is to investigate the effects of OTUD4 on autophagic flux in OGD-R treated AC16 cells (IRI model in vitro) and LAD artery ligation induced myocardial ischemia-reperfusion mice (MIRI model in vivo). In the in vitro IRI cell model, OTUD4 knockdown significantly reversed impaired autophagic flux, increased mitochondrial membrane potential, and decreased LDH activity, ROS production, autophagy and apoptosis. Overexpression of OTUD4 showed the opposite result. In the in vivo MIRI model, OTUD4 knockdown also significantly decreased infarct area, improved cardiac structure and function, reduced serum BNP and LDH levels, attenuated cardiac tissue injury/fibrosis/myocardial hypertrophy, and ultimately exerted myocardial protective effects against ischemia-reperfusion injury. Importantly, OTUD4 knockdown inhibited autophagosome-associated markers (LC3II/LC3I, Beclin1, ATG9), autophagy substrate p62, increased lysosomal activity marker LAMP2, and activated the autophagy pathway (AKT/mTOR), thereby promoting the recovery of impaired autophagic flux in the MIRI model. Moreover, OTUD4 showed strong interaction with UBAC1, and OTUD4 deficiency decreases UBAC1 protein expression by impairing its deubiquitination, thereby regulating autophagy. In short, blocking OTUD4 restored damaged autophagic flux in I/R induced myocardial injury both in vivo and in vitro, inhibited myocardial cell apoptosis, and greatly improved cardiac function in ischemia-reperfusion mice. KEY MESSAGES: OTUD4 was identified as a key negative regulator of autophagy flux in myocardial ischemia-reperfusion injury (MIRI) via genome-wide CRISPR/Cas9 screening. OTUD4 knockdown exerts cardioprotective effects by reducing apoptosis and ROS generation and improving heart function in both in vitro and in vivo models. The interaction between OTUD4 and UBAC1 was confirmed, and OTUD4 maintains UBAC1 stability through deubiquitination, providing new insights into the ubiquitination regulatory mechanism in myocardial injury. Targeting OTUD4 has therapeutic potential for MIRI, as OTUD4 knockdown alleviated MIRI in both in vitro and in vivo models, suggesting the possibility of developing OTUD4 inhibitors for cardiac reperfusion treatment.
    Keywords:  Apoptosis; Autophagic flux; Myocardial ischemia-reperfusion injury; OTUD4; UBAC1
    DOI:  https://doi.org/10.1007/s00109-025-02605-1
  53. Talanta. 2025 Oct 15. pii: S0039-9140(25)01481-X. [Epub ahead of print]298(Pt B): 128990
    Dual-responsive diazo probe for labeling of aggrephagic compartments in live cells.
    Xiaomeng Jia, Hao Jin, Rui Sun, Huaiyue Zhang, Yongquan Xiao, Yiyang Luo, Hongliang Yu, Jintai Deng, Di Shen, Man Li.
      Aggrephagy, a selective form of autophagy pathway for degrading misfolded and aggregated proteins, plays a crucial role in maintaining cellular proteostasis. Despite its biological significance, covalent labeling strategies for aggrephagy-related aggregates remain limited, primarily due to the challenges posed by the acidic and degradative environment of lysosomes. Herein, we developed a dual-responsive diazo probe (P1, λex = 506 nm, λem = 609 nm) for labeling of aggrephagy-related aggregates in living cells. P1 integrates three functional components: an aggregation-targeting moiety, a lysosome-directing unit, and a diazo group for covalent modification. The probe selectively binds and labels aggregated proteins over their properly folded counterparts. Notably, P1 activation requires the concurrent presence of visible light (λ = 300-800 nm) and an acidic microenvironment (pH = 4.4-6.23), ensuring high spatial and conditional specificity. We demonstrate that P1 enables the visualization and enrichment of aggregated proteins involved in the aggrephagy pathway. This tool is potentially useful for capturing and profiling protein factors participating cellular aggrephagy involving in neurodegeneration and cancer progression.
    Keywords:  Aggrephagy; Covalent labeling; Diazo probe; Fluorescent sensor; Protein aggregation
    DOI:  https://doi.org/10.1016/j.talanta.2025.128990
  54. Methods Mol Biol. 2026 ;2976 227-235
    Immunostaining Methods for Lysosomal Storage Disorder Research.
    Ewa A Ziółkowska, Sophie H Wang, Hemanth R Nelvagal, Jonathan D Cooper.
      Immunostaining enables the detection of proteins and antigens in histological preparations due to specific antigen-antibody interactions. Such visualization can be performed by immunoperoxidase methods, but more recently, we have switched to immunofluorescence methods that offer superior sensitivity and simultaneous detection of multiple antigens. Key to this approach is a counterstaining method that masks tissue histofluorescence and the autofluorescent storage material that accumulates in the disorders we study. Such methods allow the analysis of the structural organization of lysosomes and the impact of their dysfunction on cells and tissues. In this chapter, we present an optimized staining protocol that can serve as a standard procedure in histological examinations, ensuring high quality and consistency of quantitative results.
    Keywords:  Histology; Immunofluorescence; Immunohistochemistry; Primary antibody; Secondary antibody; Staining; TrueBlack®
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_16
  55. Autophagy. 2025 Oct 15. 1-3
    Novel links of ATG4 proteases to cytoskeletal adapters of the obscurin protein family.
    Virginia Actis Dato, Kyohei Fujita, Stephan Lange.
      The obscurin family, containing the giant protein OBSCN (obscurin, cytoskeletal calmodulin and titin-interacting RhoGEF) and its closely related OBSL1 (obscurin like cytoskeletal adaptor 1) as well as SPEG (striated muscle enriched protein kinase) are a group of intracellular proteins that contain serially linked immunoglobulin (Ig) and fibronectin type III (Fn3) domains, along with signaling modules such as protein kinase domains. Hence, obscurins harbor multi-faceted roles for the architecture and organization of cell- and organelle membrane proteins. Besides mediating cellular signaling and promoting protein homeostasis, obscurin proteins are also proposed to act as versatile cytoskeletal linkers. Due to their close homology, many functions for OBSCN are evolutionary conserved in OBSL1 and SPEG. However, their expression patterns differ widely, with OBSL1 being ubiquitously expressed in all cell types, while OBSCN and SPEG are more restricted to cross-striated muscles. Recent evidence indicates that autophagy-linked peptidases of the ATG4 family interact with the cytoskeletal adapter proteins OBSL1 and OBSCN. Peptidases of the ATG4 family process Atg8-family proteins (e.g. LC3) in their immature state (i.e. as pro-peptides like pro-LC3) or their bioactive lipidated state (i.e. LC3-II) and facilitate their conversion to the delipidated state (i.e. LC3-I). Loss of interaction between ATG4 peptidases and obscurin family proteins affects cellular macroautophagy/autophagy and mitophagy, leading in situations of cellular stress to depletion of ATG4 and accumulation of the lipidated state for Atg8-family proteins (e.g. LC3-II).
    Keywords:  ATG4; ATG8; OBSCN; OBSL1; mitophagy
    DOI:  https://doi.org/10.1080/15548627.2025.2572530
  56. Int J Mol Sci. 2025 Oct 02. pii: 9643. [Epub ahead of print]26(19):
    Dietary and Pharmacological Modulation of Aging-Related Metabolic Pathways: Molecular Insights, Clinical Evidence, and a Translational Model.
    Antonio Fernando Murillo-Cancho, David Lozano-Paniagua, Bruno José Nievas-Soriano.
      Advances in geroscience suggest that aging is modulated by molecular pathways that are amenable to dietary and pharmacological intervention. We conducted an integrative critical review of caloric restriction (CR), intermittent fasting (IF), and caloric restriction mimetics (CR-mimetics) to compare shared mechanisms, clinical evidence, limitations, and translational potential. Across modalities, CR and IF consistently activate AMP-activated protein kinase and sirtuins, inhibit mTOR (mechanistic target of rapamycin) signaling, and enhance autophagy, aligning with improvements in insulin sensitivity, lipid profile, low-grade inflammation, and selected epigenetic aging measures in humans. CR-mimetics, such as metformin, resveratrol, rapamycin, and spermidine, partially reproduce these effects; however, long-term safety and efficacy in healthy populations remain incompletely defined. Methodological constraints-short trial duration, selective samples, intermediate (nonclinical) endpoints, and limited adherence monitoring-impede definitive conclusions on hard outcomes (frailty, disability, hospitalization, mortality). We propose the Active Management of Aging and Longevity (AMAL) model, a three-level biomarker-guided framework that integrates personalized diet, chrono-nutrition, exercise, and the selective use of CR-mimetics, along with digital monitoring and decision support. AMAL emphasizes epigenetic clocks, multi-omics profiling, inflammatory and microbiome metrics, and adaptive protocols to enhance adherence and clinical relevance. Overall, CR, IF, and CR mimetics constitute promising, complementary strategies to modulate biological aging; rigorous long-term trials with standardized biomarkers and clinically meaningful endpoints are needed to enable their scalable implementation.
    Keywords:  AMPK; autophagy; caloric restriction; caloric restriction mimetics; healthy longevity; intermittent fasting; mTOR; precision nutrition; sirtuins
    DOI:  https://doi.org/10.3390/ijms26199643
  57. Mol Cell Proteomics. 2025 Oct 12. pii: S1535-9476(25)00190-2. [Epub ahead of print] 101091
    Interaction proteomics of Polycystins 1 and 2 reveal a novel role for the BLOC-1/BORC lysosomal positioning complex.
    Fatima Lukmani, Jonathan M Shillingford, Mackenzie Brauer, Dhairya Patel, Jonathan St- Germain, James A Shayman, Brian Raught, Gagan D Gupta.
      PKD1 and PKD2 are the most commonly mutated genes in autosomal dominant polycystic kidney disease (ADPKD). However, the precise roles of the encoded Polycystin 1/2 (PC1, PC2) proteins, and how their functions are disrupted in ADPKD, remain unclear. Here, we characterize the protein interaction networks of PC1 and PC2 in cycling and ciliated cells using proximity-dependent biotinylation (BioID), identifying a common set of 172 proteins that interact with the C-terminus of PC1 and the full length PC2 protein, enriched in autophagy regulators, endoplasmic reticulum (ER) tethers, ER stress proteins, and other proteins previously linked to ADPKD. Notably, we also find that PC1 specifically interacts with ciliary and lysosomal proteins, including components of the biogenesis of lysosome-related organelles complex (BLOC-1) and BLOC-one-related-complex (BORC). BLOC-1/BORC co-localizes with PC1 at lysosomes and cilia, and is required for proper ciliary PC1 localization. Additionally, PC1 mutant kidney cells derived from an ADPKD patient display defects in BLOC-1/BORC distribution. Renal cells depleted of PC1 exhibit abnormal lysosomal distribution, similar to those depleted of BLOC-1/BORC components. Lastly, shRNA knockdown of BLOC-1/BORC components promoted cystogenesis in a 3D in vitro cyst model, and this could be attenuated by heterologous expression of the C-terminus of PC1. This rich dataset thus links the BLOC-1/BORC complex to PC1 function and can be further mined for additional mechanistic insights into the PC1/2 ADPKD proteins.
    DOI:  https://doi.org/10.1016/j.mcpro.2025.101091
  58. Nat Commun. 2025 Oct 13. 16(1): 9091
    Capturing disease severity in LIS1-lissencephaly reveals proteostasis dysregulation in patient-derived forebrain organoids.
    Lea Zillich, Matteo Gasparotto, Andrea Carlo Rossetti, Olivia Fechtner, Camille Maillard, Anne Hoffrichter, Eric Zillich, Ammar Jabali, Fabio Marsoner, Annasara Artioli, Ruven Wilkens, Christina B Schroeter, Andreas Hentschel, Stephanie H Witt, Nico Melzer, Sven G Meuth, Tobias Ruck, Philipp Koch, Andreas Roos, Nadia Bahi-Buisson, Fiona Francis, Julia Ladewig.
      LIS1-lissencephaly is a neurodevelopmental disorder marked by reduced cortical folding and severe neurological impairment. Although all cases result from heterozygous mutations in the LIS1 gene, patients present a broad spectrum of severity. Here, we use patient-derived forebrain organoids representing mild, moderate, and severe LIS1-lissencephaly to uncover mechanisms underlying this variability. We show that LIS1 protein levels vary across patient lines and partly correlate with clinical severity, indicating mutation-specific effects on protein function. Integrated morphological, transcriptomic, and proteomic analyses reveal progressive changes in neural progenitor homeostasis and neurogenesis that scale with severity. Mechanistically, microtubule destabilization disrupts cell-cell junctions and impairs WNT signaling, and defects in protein homeostasis, causing stress from misfolded proteins, emerge as key severity-linked pathways. Pharmacological inhibition of mTORC1 partially rescues these defects. Our findings demonstrate that patient-derived organoids can model disease severity, enabling mechanistic dissection and guiding targeted strategies in neurodevelopmental disorders.
    DOI:  https://doi.org/10.1038/s41467-025-64980-0
  59. Commun Biol. 2025 Oct 16. 8(1): 1475
    OPTN protects retinal ganglion cells and ameliorates neuroinflammation in optic neuropathies.
    Qinglong Wang, Yiqi Wang, Yi Da Douglas Jiang, Ryan Donahue, Gaby Cao, Weixuan Yan, Hong Guo, Zhenghui Li, Jenna Liang, Jin Hao, Yi Lu, Fengfeng Bei, Qianbin Wang, Feng Tian.
      Optineurin (OPTN) is an adaptor protein that plays a crucial role in many cellular pathways, including NF-κB signaling, programmed cell death, and vesicular trafficking. OPTN dysfunction has been implicated in the pathogenesis of several diseases, such as primary open angle glaucoma (POAG), amyotrophic lateral sclerosis (ALS). While mutations of OPTN seem to be predominantly loss-of-function in ALS, only gain-of-function mechanisms have been reported in POAG. Here, we demonstrate that OPTN knockout in the retina contributes to short-term astrogliosis, retinal ganglion cell (RGC) loss and long-term microglial activation. Moreover, OPTN loss of function does not exacerbate RGC death induced by ocular hypertension. Integrated bioinformatics and immunofluorescence analyses reveal that OPTN dysfunction leads to neuropeptide Y (NPY) downregulation and CHOP upregulation. Overexpression of wild-type OPTN in a hypertension glaucoma model prevents the RGC loss and attenuates microglial activation. Together, our findings highlight a neuroprotective role for OPTN as a key neuroimmune modulator.
    DOI:  https://doi.org/10.1038/s42003-025-08534-6
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