bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2025–06–08
48 papers selected by
Viktor Korolchuk, Newcastle University



  1. bioRxiv. 2025 May 19. pii: 2025.05.16.654474. [Epub ahead of print]
      In stressed cells, the recessive Parkinson disease (PD) associated gene products PINK1 and parkin mediate the autophagic removal of damaged mitochondria (mitophagy). Upon mitochondrial membrane potential disruption, PINK1 phosphorylation activates the ubiquitin ligase parkin which ubiquitinates various mitochondrial protein substrates. These feed-forward modifications on the mitochondria surface attract ubiquitin-binding autophagy receptors that target ubiquitinated mitochondria to autophagosomes and indirectly contribute to phagophore elongation. Investigating post-translational protein modifications during this process, we detected transient ubiquitination of K549 within the third coiled-coil domain (CC3) of TAX1BP1 in HeLa cells expressing WT but not catalytically inactive parkin. Parkin-dependent ubiquitination did not target TAX1BP1 to proteasomal degradation but was rather indicative of a regulatory modification. In cells with the full complement of autophagy receptors, TAX1BP1 plays only a minor role in mitophagy. However, when expressed as a sole autophagy receptor, both WT and ubiquitination deficient TAX1BP1 were capable of promoting mitophagy, albeit mitochondria degradation was slightly delayed under mutant conditions. Use of the lysosomal inhibitor bafilomycin A indicated classical autophagolysosomal targeting of damaged mitochondria mediated by WT TAX1BP1. However, for the ubiquitination-deficient TAX1BP1, we observed an increased prevalence of enlarged endolysosomal vesicles carrying accumulated TAX1BP1-positive autophagosomes filled with mitochondrial material. Thus, while ubiquitination of the CC3 domain of TAX1BP1 is not essential for complete mitophagy, the lack of CC3 in TAX1BP1 reroutes the degradation flux to a less efficient endolysosmal degradative pathway. Interestingly, the PD gene product VPS35, becomes prominently engaged in this alternative mitophagy pathway.
    DOI:  https://doi.org/10.1101/2025.05.16.654474
  2. Autophagy Rep. 2025 ;4(1): 2472450
      Neurons, as post-mitotic and long-lived cells, rely heavily on autophagy to maintain cellular homoeostasis and ensure proper function. Huntingtin (HTT), a protein central to Huntington's disease (HD), has emerged as a putative multifunctional regulator within the neuronal autophagy-lysosome pathway. This review explores normal HTT's multifaceted role in neuronal autophagy, from its potential involvement in autophagy induction, its capacity to influence cargo recognition and autophagosome formation, and its contribution to autophagosome-lysosome fusion and transport. We also discuss the unique challenges that neurons face in maintaining proteostasis through autophagy, emphasising the need for specialised mechanisms like axonal transport of autophagosomes and distinct regulatory pathways. Furthermore, we highlight the spatial and temporal regulation of neuronal autophagy, particularly in the context of ageing and neuronal maturation, underscoring the importance of understanding HTT's role in different neuronal states. By elucidating the intricate relationship between HTT and neuronal autophagy, this review aims to shed light on specific mechanisms of action in autophagy that can be disrupted in neurodegenerative diseases including HD.
    Keywords:  Neurons; autophagy; huntingtin; huntington’s disease
    DOI:  https://doi.org/10.1080/27694127.2025.2472450
  3. Res Sq. 2025 May 13. pii: rs.3.rs-5073364. [Epub ahead of print]
      Mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-responsive master regulator of metabolism. Amino acids control the recruitment and activation of mTORC1 at the lysosome via the nucleotide loading state of the heterodimeric Rag GTPases. Under low nutrients, including arginine (Arg), the GTPase activating protein (GAP) complex, GATOR1, promotes GTP hydrolysis on RagA/B, inactivating mTORC1. GATOR1 is regulated by the cage-like GATOR2 complex and cytosolic amino acid sensors. To understand how the Arg-sensor CASTOR1 binds to GATOR2 to disinhibit GATOR1 under low cytosolic Arg, we determined the cryo-EM structure of GATOR2 bound to apo-CASTOR1. Two MIOS WD40 domain β-propellers of the GATOR2 cage engage with both subunits of a single CASTOR1 homodimer. Each propeller binds to a negatively charged MIOS-binding interface (MBI) on CASTOR1 that is distal to the Arg pocket. The structure shows how Arg-triggered loop ordering in CASTOR1 blocks the MBI, switches off its binding to GATOR2, and so activates mTORC1.
    DOI:  https://doi.org/10.21203/rs.3.rs-5073364/v1
  4. Nat Commun. 2025 May 31. 16(1): 5073
      Dynamic regulation of metabolic activities in astrocytes is critical to meeting the demands of other brain cells. During neuronal stress, lipids are transferred from neurons to astrocytes, where they are stored in lipid droplets (LDs). However, it is not clear whether and how neuron-derived lipids trigger metabolic adaptation in astrocytes. Here, we uncover an endolysosomal function that mediates neuron-astrocyte transcellular lipid signaling. We identify Tweety homolog 1 (TTYH1) as an astrocyte-enriched endolysosomal protein that facilitates autophagic flux and LD degradation. Astrocyte-specific deletion of mouse Ttyh1 and loss of its Drosophila ortholog lead to brain accumulation of neutral lipids. Computational and experimental evidence suggests that TTYH1 mediates endolysosomal clearance of ceramide 1-phosphate (C1P), a sphingolipid that dampens autophagic flux and LD breakdown in mouse and human astrocytes. Furthermore, neuronal C1P secretion induced by inflammatory cytokine interleukin-1β causes TTYH1-dependent autophagic flux and LD adaptations in astrocytes. These findings reveal a neuron-initiated signaling paradigm that culminates in the regulation of catabolic activities in astrocytes.
    DOI:  https://doi.org/10.1038/s41467-025-60402-3
  5. Blood Neoplasia. 2024 Jun;1(2): 100015
      Glucocorticoids (GCs), such as dexamethasone and prednisone, are crucial components of B-cell precursor acute lymphoblastic leukemia (B-ALL) therapies. However, the molecular basis of GC-induced cell death remains elusive. Here, we show that GC suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling and that, conversely, oncogenic activation of mTORC1 confers resistance to GCs. Our genome-wide CRISPR/CRISPR-associated protein 9 (CRISPR/Cas9) dropout screens reveal that depletion of components of either the gap activity toward Rags 1 or tuberous sclerosis complexes, both negative regulators of mTORC1 signaling, significantly attenuates B-ALL cell sensitivity to dexamethasone. Dexamethasone primarily induces B-ALL cell death by downregulating mTORC1 activity, thus promoting autophagy and impairing protein synthesis. Dexamethasone treatment failed to suppress mTORC1 activity in B-ALL cells expressing mutant GC receptors lacking DNA-binding capacity, suggesting that dexamethasone transcriptionally represses mTORC1 activity. RNA-sequencing analysis identified multiple dexamethasone target genes that negatively regulate mTORC1 activity. Our findings suggest that GC sensitivity is significantly influenced by oncogenic stimuli and/or growth factors that activate the PI3K-AKT-mTORC1 pathway. This is consistent with the frequent GC resistance found in Ph and Ph-like ALLs.
    DOI:  https://doi.org/10.1016/j.bneo.2024.100015
  6. Autophagy Rep. 2025 ;4(1): 2476218
      Hydrolytic enzymes within lysosomes maintain cell and tissue homoeostasis by degrading macromolecules delivered by endocytosis and autophagy. The release of lysosomal enzymes into the cytosol can induce apoptosis and "lysosome-dependent cell death" making it important for damaged lysosomes to be repaired or removed. Extensive lysosome damage exposes luminal sugars to galectin-dependent autophagy pathways that use ATG16L1:ATG5-ATG12 complex to conjugate LC3/ATG8 to autophagosomes to facilitate removal by lysophagy. Sphingomyelin exposed on stressed lysosomes recruits the lysosome tethering protein TECPR1 (tectonin beta propeller repeat-containing protein) allowing an alternative TECRP1:ATG5-ATG12 complex to conjugate LC3 directly to lysosomes. Here we have used cells lacking ATG16L1 to follow the recruitment of TECPR1, galectin-3 and LC3/ATG8 to lysosomes in response to osmotic imbalance induced by chloroquine. TECPR1 was recruited to swollen lysosomes that exposed sphingomyelin. LC3II was absent from swollen lysosomes but located to small puncta that contained the V-ATPase and LAMP1. The presence of galectin-3 and PI4P in the small LC3 puncta suggested that the TECPR1:ATG5-ATG12 complex conjugates LC3 to lysosome remnants that have ruptured in response to osmotic imbalance.
    Keywords:  ATG16L1; Autophagy; LC3/ATG8; TECPR1; chloroquine; galectin 3; lysosome damage; osmotic stress; sphingomyelin
    DOI:  https://doi.org/10.1080/27694127.2025.2476218
  7. Autophagy. 2025 Jun 03. 1-17
      Macroautophagy/autophagy is a biological process that sequesters and degrades cytoplasmic material, damaged organelles, and infectious pathogens in eukaryotic cells via lysosomes. Autophagy is involved in different phases of the viral life cycle and regulates viral replication. Here, we demonstrated that pseudorabies virus (PRV) infection induced incomplete autophagy, and blocking the autophagosome-lysosome fusion facilitated PRV replication. Mechanistically, PRV late envelope glycoprotein M (gM) triggered SQSTM1/p62-dependent selective autophagy. Meanwhile, gM protein was found to inhibit the fusion between autophagosomes and lysosomes by activating CASP3 (caspase 3) to degrade SNAP29, resulting in increased viral replication. Interestingly, we confirmed that the gM homologs from several herpesviruses (herpes simplex virus-1, human cytomegalovirus, equine herpesvirus-1, and varicella-zoster virus) shared the same function of activating CASP3 and inhibiting autophagic flux. Deletion of the CASP3 gene led to an intact autophagic pathway and the increased formation of autolysosomes. Collectively, our results illustrated that blockage of autophagosome-lysosome fusion mediated by PRV gM and its homologs in other herpesviruses protected viral proteins from host autophagic signaling, thus facilitating herpesvirus replication.Abbreviations: 3-MA: 3-methyladenine; Baf A1: bafilomycin A1; CASP3: caspase 3; cl-CASP3: cleaved-CASP3; co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; EHV-1: equine herpesvirus 1; gM: glycoprotein M; HCMV: human cytomegalovirus; HSV-1: herpes simplex virus 1; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; OD: optical density; PCR: polymerase chain reaction; PFU: plaque forming units; PRV: pseudorabies virus; Rap: rapamycin; SNAP29; synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TCID: 50% tissue culture infectious doses; UBA: ubiquitin-binding domain; VAMP8: vesicle associated membrane protein 8; µm, micrometer; VZV: varicella-zoster virus; WT: wild type.
    Keywords:  Caspase 3; SNAP29; gM homologs; inhibit autophagic flux; macroautophagy; protein aggregates
    DOI:  https://doi.org/10.1080/15548627.2025.2511584
  8. J Mol Biol. 2025 Jun 03. pii: S0022-2836(25)00329-8. [Epub ahead of print] 169263
      Mitophagy, the selective degradation of mitochondria, is essential for retinal ganglion cell (RGC) differentiation and retinal homeostasis. However, the specific mitophagy pathways involved and their temporal dynamics during retinal development and maturation remain poorly understood. Using proteomics analysis of isolated mouse retinas across developmental stages and the mitophagy reporter mouse line, mito-QC, we characterized mitophagy throughout retinogenesis. While mitolysosomes were more prevalent in the mature retina, we observed two distinct mitophagy peaks during embryonic development. The first, independent of PTEN-induced kinase 1 (PINK1) activation, was associated with RGCs. The second, PINK1-dependent peak was triggered after an increase in retinal oxidative stress. This PINK1-dependent, oxidative stress-induced mitophagy pathway is conserved in mice and zebrafish, providing the first evidence of programmed, PINK1-dependent mitophagy during development.
    Keywords:  PINK1; autophagy; development; mitophagy; retina
    DOI:  https://doi.org/10.1016/j.jmb.2025.169263
  9. bioRxiv. 2025 May 23. pii: 2025.05.20.655155. [Epub ahead of print]
      The mechanistic target of Rapamycin (mTOR) kinase pathway plays critical roles in neuronal function and synaptic plasticity, and its dysfunction is implicated in numerous neurological and psychiatric disorders. Traditional linear models depict mTOR signaling as a sequential phosphorylation cascade, but accumulating evidence supports a model that includes signaling through dynamic protein-protein interaction networks. To examine how neuronal mTOR signaling discriminates between distinct stimuli, we quantified phosphorylation events and protein co-association networks in primary mouse cortical neurons. Unexpectedly, neuronal mTOR activation by IGF or glutamate triggered dissociation-rather than the anticipated assembly-of protein complexes involving mTOR complex1 (TORC1), mTOR complex 2 (TORC2), and translational machinery, distinguishing neurons from proliferative cells. Applying in vitro homeostatic scaling paradigms revealed distinct combinatorial encoding of synaptic scaling direction: both up- and down-scaling induced dissociation of translational complexes, but downscaling uniquely included dissociation of upstream pathway regulators. Cortical neurons from Shank3B knockout mice, modeling autism-associated Phelan-McDermid Syndrome, displayed baseline hyperactivation of the mTOR network, which reduced the dynamic range of network responses to homeostatic scaling and pharmacological inhibition. These findings reveal that neuronal mTOR signaling employs stimulus-specific combinations of dissociative protein interaction modules to encode opposing forms of synaptic plasticity.
    DOI:  https://doi.org/10.1101/2025.05.20.655155
  10. Dev Cell. 2025 May 27. pii: S1534-5807(25)00318-1. [Epub ahead of print]
      Lysosome damage activates multiple pathways to prevent lysosome-dependent cell death, including a repair mechanism involving endoplasmic reticulum (ER)-lysosome membrane contact sites, phosphatidylinositol 4-kinase-2a (PI4K2A), phosphatidylinositol-4 phosphate (PI4P), and oxysterol-binding protein-like proteins (OSBPLs) lipid transfer proteins. PI4K2A localizes to the trans-Golgi network and endosomes, yet how it is delivered to damaged lysosomes remains unknown. During acute sterile damage and damage caused by intracellular bacteria, we show that ATG9A-containing vesicles perform a critical role in delivering PI4K2A to damaged lysosomes. ADP ribosylation factor interacting protein 2 (ARFIP2), a component of ATG9A vesicles, binds and sequesters PI4P on lysosomes, balancing OSBPL-dependent lipid transfer and promoting the retrieval of ATG9A vesicles through the recruitment of the adaptor protein complex-3 (AP-3). Our results identify a role for mobilized ATG9A vesicles and ARFIP2 in lysosome homeostasis after damage and bacterial infection.
    Keywords:  AP-3; ARFIP2; ATG9A; PI4K2A; PI4P; autophagy; lysosomal damage; lysosome; membrane trafficking
    DOI:  https://doi.org/10.1016/j.devcel.2025.05.007
  11. Nat Commun. 2025 Jun 04. 16(1): 5179
      Mitophagy, the selective autophagic elimination of mitochondria, is essential for maintaining mitochondrial quality and cell homeostasis. Impairment of mitophagy flux, a process involving multiple sequential intermediates, is implicated in the onset of numerous neurodegenerative diseases. Screening mitophagy inducers, particularly understanding their impact on mitophagic intermediates, is crucial for neurodegenerative disease treatment. However, existing techniques do not allow simultaneous visualization of distinct mitophagic intermediates in live cells. Here, we introduce an artificial intelligence-assisted fluorescence microscopic system (AI-FM) that enables the uninterrupted recognition and quantification of key mitophagic intermediates by extracting mitochondrial pH and morphological features. Using AI-FM, we identify a potential mitophagy modulator, Y040-7904, which enhances mitophagy by promoting mitochondria transport to autophagosomes and the fusion of autophagosomes with autolysosomes. Y040-7904 also reduces amyloid-β pathologies in both in vitro and in vivo models of Alzheimer's disease. This work offers an approach for visualizing the entire mitophagy flux, advancing the understanding of mitophagy-related mechanisms and enabling the discovery of mitophagy inducers for neurodegenerative diseases.
    DOI:  https://doi.org/10.1038/s41467-025-60315-1
  12. bioRxiv. 2025 May 14. pii: 2025.05.12.653625. [Epub ahead of print]
      Cell growth checkpoints require coordination between multiple sensing and signaling systems to ensure that cells only proceed with growth and division when conditions are favorable and adequate resources are available. This coordination between nutrient sensing and growth signaling is fundamental to understanding how nutrient supply regulates the cellular metabolic economy. Much of our current understanding is driven by studies that examine the cellular response to nutrient deprivation. For example, TORC1 activity promotes cell growth when amino acids are available, but amino acid deprivation decreases TORC1 activity resulting in activation of catabolic activities. In this study, we examine how cells respond to stimulation with excess amino acids. We report that stimulation with excess Ile, Phe and Met slows cell growth and triggers a G1 cell cycle arrest. Similar to a starvation response, surplus Ile, Phe and Met induce autophagy and trigger decreased TORC1 activity. In the case of stimulation with excess Met, the Gcn2 pathway is required for growth arrest, autophagy induction, and TORC1 dampening. Unexpectedly, Gcn2 is activated by stimulation with excess Met, and this activation requires endocytosis of the methionine transporter Mup1. These results indicate that endocytosis of an amino acid transporter is required to activate the Gcn2 pathway, providing an example for how nutrient transporter trafficking may function as a sensor contributing to cell growth control.
    DOI:  https://doi.org/10.1101/2025.05.12.653625
  13. Clin Exp Med. 2025 Jun 04. 25(1): 187
      Autophagy, a critical cellular process for maintaining homeostasis, involves the degradation and recycling of cellular components through double-membraned vesicles that are transported to lysosomes. This mechanism plays a pivotal role in the immune system by influencing cell fate decisions and functional differentiation. Emerging evidence indicates that autophagy significantly impacts the differentiation and function of T cells and group 3 innate lymphoid cell (ILC3), which are the primary producers of Interleukin-22 (IL-22). IL-22, a key cytokine involved in modulating immune responses and maintaining tissue integrity, is particularly important in combating inflammatory diseases, infections, and cancer. It exerts its effects through a signaling pathway that involves the IL-22R1 and IL-10R2 receptors. Studies have demonstrated that IL-22 can promote autophagy by activating the AMPK pathway and that its intervention can upregulate the expression of autophagy-related genes, underscoring its significant role in the regulation of autophagy. These findings reveal a complex relationship and bidirectional relationship between autophagy and IL-22, highlighting their multifaceted interactions under both physiological and pathological conditions. This review aims to provide a detailed exploration of the dynamic interplay between autophagy and IL-22, with a focus on their mutual regulatory mechanisms, functional significance, and potential for therapeutic interventions. By performing a comprehensive analysis, we sought to clarify the intricate cross talk between autophagy and IL-22, thereby advancing our understanding of their roles in cellular and immune homeostasis and their potential as targets for clinical interventions.
    Keywords:  Autophagy; Autophagy-IL-22 interplay; IL-22; Immune responses; Interleukin-22 (IL-22)
    DOI:  https://doi.org/10.1007/s10238-025-01695-y
  14. bioRxiv. 2025 May 14. pii: 2025.05.13.653863. [Epub ahead of print]
      Meiotic segregation errors in human oocytes are the leading cause of miscarriages and trisomic pregnancies and their frequency increases exponentially for women in their thirties. One factor that contributes to increased segregation errors in aging oocytes is premature loss of sister chromatid cohesion. However, the mechanisms underlying age-dependent deterioration of cohesion are not well-defined. Autophagy, a cellular degradation process critical for cellular homeostasis, is known to decline with age in various organisms and cell types. Here we quantify basal autophagy in Drosophila oocytes and use GAL4/UAS inducible knockdown to ask whether disruption of autophagy in prophase oocytes impacts the fidelity of chromosome segregation. We find that individual knockdown of autophagy proteins in Drosophila oocytes during meiotic prophase causes a significant increase in segregation errors. In addition, Atg8a knockdown in prophase oocytes leads to premature loss of arm cohesion and missegregation of recombinant homologs during meiosis I. Using an oocyte aging paradigm that we have previously described, we show that basal autophagy decreases significantly when Drosophila oocytes undergo aging. Our data support the model that a decline in autophagy during oocyte aging contributes to premature loss of meiotic cohesion and segregation errors.
    DOI:  https://doi.org/10.1101/2025.05.13.653863
  15. PLoS One. 2025 ;20(6): e0325505
      Autophagy is a 'waste-disposal' pathway that protects against age-related pathology. It is widely accepted that autophagy declines with age, yet the role that sex and diet-related obesity play during aging remain unknown. Here, we present the most comprehensive in vivo study of autophagic flux to date. We employed transgenic mice overexpressing tandem-florescent LC3B (RFP-GFP-LC3B) to measure autophagic flux in the blood (PBMCs), heart, and motor cortex neurons of aging mice that were fed regular chow or a high-fat diet for 6-, 12- or 18-months. In male mice, aging decreased autophagic flux in the heart, increased it in the blood, and had no effect in motor cortex neurons. Age-dependent changes autophagic flux were less pronounced in female mice. High-fat diet influenced autophagic flux in the blood and heart of male but not female mice. Overall, we uncovered sexual dimorphisms that underpin how autophagy changes with age across different tissues and in response to a high-fat diet.
    DOI:  https://doi.org/10.1371/journal.pone.0325505
  16. Sci Adv. 2025 Jun 06. 11(23): eadu9605
      The autophagosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein) Syntaxin17 (Syx17) plays a pivotal role in autophagosome-lysosome fusion, yet the broader impact of its loss remains elusive. Our investigation of Syx17 function in Drosophila nephrocytes and salivary gland cells revealed unexpected effects. We find that Syx17 loss induces the formation of autophagosome-lysosome clusters in a HOPS (homotypic fusion and vacuole protein sorting)-dependent manner, entrapping this tether, autophagosomes, and lysosomes. While locked in clusters, these organelles cannot participate in other vesicle fusions, impeding endosomal progression and autophagosome secretion. Therefore, the absence of Syx17 not only inhibits autophagosome-lysosome fusion but also prevents HOPS release from autophagosome-lysosome tethering sites causing a "tethering lock." Preventing autophagosome formation or removing the HOPS adaptor Plekhm1 (pleckstrin homology domain-containing family M member 1) leads to release of HOPS and lysosomes from these clusters, thus rescuing secondary effects of Syx17 loss. Our findings show that a tethering lock can disrupt multiple vesicle trafficking routes.
    DOI:  https://doi.org/10.1126/sciadv.adu9605
  17. J Biol Chem. 2025 May 29. pii: S0021-9258(25)02164-7. [Epub ahead of print] 110314
      O-GlcNAcase (OGA) is the sole eraser for the intracellular O-linked N-acetylglucosamine (O-GlcNAc). OGA has many roles in distinct biological processes, such as cancer and embryonic stem cells, but its precise regulatory mechanism is far from being understood. Herein we studied the small ubiquitin-like modifier (SUMO) modification of OGA, and found that OGA is SUMOylated at K358. SUMOylation targets OGA to the chaperone-mediated autophagy (CMA) pathway, which shunts client proteins to the lysosome for degradation. We demonstrate that SUMOylation increases the association between OGA and the heat shock cognate protein 70 (HSC70), the CMA chaperone, and facilitates OGA further degradation. We further mapped a SUMO-interacting motif (SIM) (VLIFD, aa. 195-199) on HSC70. Notably, HSC70-SIM is essential for affinity with other CMA client proteins, such as PKM2. We thus posit that the SIM of HSC70 binds SUMOylated client proteins in a lock-and-key manner to confer substrate selectivity during CMA. To further test our hypothesis, we used label-free quantitative mass spectrometry to study the HSC70-SIM mutant interactome, and generated a proteome-wide SUMO-mediated CMA client pool. We then validated this model by studying YEATS domain containing 2 (YEATS2) from the protein pool, and demonstrated that YEATS2 is SUMOylated at K592, targeting it to CMA. Our work uncovers the SUMO-SIM interaction as a fundamental mechanism governing CMA substrate selectivity and identifies a potential CMA client proteome to deepen our understanding of its pathophysiological relevance.
    Keywords:  Hsc70; O-GlcNAcase; SUMOylation; YEATS2; chaperone-mediated autophagy (CMA)
    DOI:  https://doi.org/10.1016/j.jbc.2025.110314
  18. J Biol Chem. 2025 Jun 03. pii: S0021-9258(25)02184-2. [Epub ahead of print] 110334
      Mitochondrial tRNA processing defects have been associated with some clinical presentations including deafness. Especially, a deafness-linked m.7516delA mutation impaired the 5' end processing of RNA precursors and mitochondrial translation. In this study, we investigated the mechanism by m.7516delA mutation induced-deficiencies mitigate organellular and cellular integrity. The m.7516delA mutation downregulated the expression of nucleus encoding subunits and upregulated assemble factors of complex IV and altered the assembly and activities of oxidative phosphorylation (OXPHOS) complexes. The impairment of OXPHOS alleviated mitochondrial quality control processes, including the imbalanced mitochondrial dynamics via increasing fission with abnormal mitochondrial morphology. The m.7516delA mutation upregulated both ubiquitin-dependent and independent mitophagy pathways, evidenced by increasing levels of Parkin, BNIP3, NIX and MFN2-ubiquitination and altering interaction between MFN2 and MUL1 or Parkin, to facilitate the degradation of severely damaged mitochondria. Strikingly, the m.7516delA mutation activated integrated stress response (ISR) pathway, evidenced by upregulation of GCN2, P-GCN2, p-eIF2α, CHOP, ATF4 and elevating the nucleus-location of ATF5 to minimizes the damages in defective mitochondria. Both activation of ISR and PINK1/Parkin mitophagy pathways ameliorate the cell homeostasis via elevating the autophagy process and upregulating apoptotic pathways. Our findings provide new insights into underlying aberrant RNA processing-induced dysfunctions reprogrammed organelles and cellular integrity.
    DOI:  https://doi.org/10.1016/j.jbc.2025.110334
  19. Clin Transl Med. 2025 Jun;15(6): e70361
       AIMS: Adventitial remodelling in hypertension is characterized by a transformation of adventitial fibroblasts (AFs) into myofibroblasts. Previous studies have highlighted the crucial role of discoidin domain receptor 2 (DDR2) in vascular remodelling. Since DDR2-sustained tyrosine phosphorylation activates PI3K, which may inhibit autophagy through the mTOR signalling pathway, we aimed to investigate whether DDR2 contributes to mTOR-mediated autophagy suppression and subsequently promotes AFs transformation and adventitial remodelling.
    METHODS AND RESULTS: Single-cell RNA sequencing revealed that DDR2 was upregulated in adventitial fibroblasts (AFs) in angiotensin II (Ang II, 1000 ng/min/kg) administrated wild-type (WT) mice. In AFs, rapamycin, an autophagy agonist, significantly attenuated Ang II-induced autophagy suppression and phenotype switching, whereas the autophagy inhibitor chloroquine (CQ) exacerbated these effects. DDR2 inhibition significantly alleviated PI3K/Akt/mTOR pathway-mediated autophagy suppression and subsequently inhibited AFs phenotypic switching. Conversely, DDR2 overexpression aggravated autophagy suppression and AFs phenotypic switching. Consistent with the cellular findings, prophylactic administration of rapamycin (4 mg/kg/d) or conditional knockout of Ddr2 in mice ameliorated autophagy suppression, AFs differentiation and adventitial remodelling in vivo.
    CONCLUSION: DDR2 serves as a critical mediator of autophagy suppression during Ang II-induced phenotypic transformation of AFs and adventitial remodelling. Targeting DDR2 signalling attenuates autophagy dysfunction and inhibits AFs activation, thereby mitigating pathological adventitial remodelling. These findings highlight DDR2 as a potential therapeutic target for preventing conditions driven by aberrant adventitial remodelling.
    Keywords:  adventitial fibroblasts; autophagy; discoidin domain receptor 2; phenotypic switch
    DOI:  https://doi.org/10.1002/ctm2.70361
  20. PLoS Pathog. 2025 Jun;21(6): e1013201
      Severe fever with thrombocytopenia syndrome virus (SFTSV), a tick-borne bunyavirus, causes an emerging viral hemorrhagic fever with a high mortality rate. SFTSV nonstructural protein S (NSs) is a virulence factor that sequesters antiviral proteins into autophagic vesicles for degradation to escape host immune response. SAFA (Nuclear scaffold attachment factor A), an RNA sensor, recognizes viral RNA and is retained in the cytoplasm upon RNA virus SFTSV infection and then activates innate immunity. It is unclear whether NSs mediates the escape of SAFA-mediated antiviral response. Here we showed that SFTSV NSs can inhibit SAFA-dependent antiviral response via autophagy. We used SAFA-NLS (the nuclear localization signal) mutant to transfect SAFA knocked-out MEF cells and found that the cytoplasmic SAFA promoted innate immune response to poly(I:C) stimulating. Importantly, NSs interacted with the AAA+ domain of SAFA and retained SAFA in the cytoplasm thereby suppressing SAFA-mediated antiviral response. Mechanistically, SFTSV NSs degraded cytoplasmic SAFA via SQSTM1/p62-dependent autophagy and sequestered SAFA into autophagic vesicles for degradation through promoting the interaction between SAFA and LC3. In conclusion, our results indicate a novel mechanism of SFTSV NSs to escape host antiviral immune response by recruiting SAFA into autophagic flux for degradation.
    DOI:  https://doi.org/10.1371/journal.ppat.1013201
  21. J Transl Med. 2025 Jun 05. 23(1): 631
       BACKGROUND: Parkinson's disease (PD) is a sporadic neurodegenerative disorder with a rising incidence. Environmental toxins are considered the main etiological factor. The increasing use of polystyrene nanoparticles (PS-NPs) has raised concerns about their potential neurotoxic effects in PD.
    OBJECTIVES: This study aimed to investigate the impact of PS-NPs on the onset and progression of PD and the underlying mechanisms.
    METHODS: The breach of the blood-brain barrier (BBB) by PS-NPs was assessed using bioluminescence imaging, fluorescence observation, Pyrolysis-Gas Chromatography-Mass Spectrometry (Py-GCMs), transmission electron microscope (TEM), and Evans blue staining. To evaluate the potential promotion of PD by PS-NPs, a 30-day repeated oral administration study was conducted in vivo, during which behavioral changes and alterations in dopaminergic neurons in the substantia nigra were assessed. In vitro cytotoxicity assays were performed following PS-NPs intervention. Molecular biology techniques, including Western blotting and immunofluorescence, were employed to analyze proteins related to pyroptosis and autophagy-lysosomal pathway in both in vivo and in vitro settings. Additionally, proteomic sequencing was utilized to identify the upstream regulator of the autophagy-lysosomal pathway (ALP), and the effects of modulating this target protein on the ALP-pyroptosis pathway were analyzed.
    RESULTS: Bioluminescence imaging and Py-GCMs confirmed that PS-NPs entered the brain within 1.5 h. Evans blue staining and TEM showed PS-NPs damaged the BBB. The 30-day oral toxicity revealed that PS-NPs exacerbated behavioral abnormalities and caused dopaminergic neuron loss. Western blotting and immunofluorescence indicated that PS-NPs induced pyroptosis, disrupted autophagic flux, and lowered protein levels involved in autophagosome-lysosome fusion, both in vivo and in vitro. Furthermore, PS-NPs activated the mechanistic target of rapamycin (mTOR) and inhibited the nuclear translocation of Transcription Factor EB (TFEB). Proteomic sequencing identified a deficit of Tuberous Sclerosis Complex (TSC) 2 protein within the mTOR pathway. Immuno-coprecipitation and Coomassie Blue Fast Staining revealed that PS-NPs bound to TSC2 protein, causing disassembly of TSC1-TSC2 complex.
    CONCLUSION: These findings underscore how PS-NPs accelerated PD onset and progression by disrupting autophagosome-lysosome fusion through TSC2-mTOR-TFEB axis, which triggered protein degradation disorders and pyroptosis in dopaminergic neurons. The molecular mechanisms could inform environmental safety regulations concerning nanoplastics and inspire therapeutic strategies for PD.
    Keywords:  Autophagosome-lysosome fusion; Parkinson’s disease (PD); Polystyrene nanoplastics (PS-NPs); Pyroptosis; Tuberous Sclerosis Complex 2 protein (TSC2)
    DOI:  https://doi.org/10.1186/s12967-025-06634-9
  22. Free Radic Biol Med. 2025 Jun 02. pii: S0891-5849(25)00745-2. [Epub ahead of print]237 195-209
      Pancreatic cancer is a highly aggressive malignancy with a significant unmet medical need, as current treatments often yield poor responses. Ferroptosis, a recently recognized form of regulated cell death, has garnered increasing attention for its potential in cancer therapy. However, the molecular links connecting autophagy to ferroptosis remain largely unclear. In this study, we identified that the redox related protein glucose-6-phosphate dehydrogenase (G6PD) is overexpressed in pancreatic cancer and correlates with poor prognosis, promoting cancer cell proliferation and migration. Using PANC-1 and MiaPaCa-2 pancreatic cancer cell lines, we demonstrated that the treatment with ferroptosis-inducing compound RSL3 induced glycolytic dysfunction and significantly downregulated G6PD expression. Moreover, G6PD knockdown in these cell lines impaired the cellular antioxidant defence capability by decreasing the NADPH and GSH contents, leading to increased lipid peroxidation and malondialdehyde (MDA) accumulation. Particularly, G6PD depletion exacerbated RSL3 induced oxidative stress and synergistically promoted autophagy-dependent ferroptosis. Mechanistically, we found that G6PD knockdown disrupted redox homeostasis, triggering the activation of AMPK-mTOR pathway to induce autophagy. Furthermore, pharmacological inhibition of AMPK (with Compound C) rescued ferroptosis induced by G6PD knockdown and RSL3, whereas mTOR inhibition (with Rapamycin) further augmented cell death. Altogether, these findings suggest that G6PD contributes to ferroptosis resistance in pancreatic cancer cells by modulating oxidative balance and autophagy via the AMPK-mTOR pathway, highlighting its potential as a therapeutic target.
    Keywords:  AMPK-mTOR pathway; Autophagy; Ferroptosis; G6PD; Redox balance
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2025.06.002
  23. bioRxiv. 2025 May 22. pii: 2025.05.22.655499. [Epub ahead of print]
      All eukaryotic cells secrete exosomes, a type of extracellular vesicles (EVs) derived from the endocytic compartments known as multivesicular bodies (MVBs), or late endosomes (LEs). Exosomes contain a diverse range of cargo such as nucleic acids, proteins, lipids and small molecules but whether these contents have a biological function remains an area of intense investigation. Over the last decade, numerous studies have described the transcriptome of exosomes but very little is known about the RNA content of the MVBs, the source compartment for exosome biogenesis. Here we determine the small-RNA transcriptome of highly purified MVBs and report that various classes of nuclear small regulatory RNAs such as small-Cajal body associated RNAs (scaRNAs), small-nucleolar RNAs (snoRNAs) and small-nuclear RNAs (snRNAs) traffic to MVBs. We show that this RNA-trafficking requires the function of ESCRT machinery but is independent of canonical LC3 lipidation mediated selective autophagy. Furthermore, blocking the activity of a PI3K Class 3 enzyme, VPS34, required for recruitment of the ESCRT machinery to the endosome, prevents the turnover of these nuclear RNAs in MVBs. Our results provide a mechanism for targeting nuclear ribonucleoprotein complexes (RNPs), such as Cajal bodies, for degradation and turnover by the cytoplasmic endo-lysosomal pathway.
    Significance Statement: Endosomes are cytoplasmic, membrane-bound subcellular organelles that are sites for biogenesis of exosomes, a class of extracellular vesicles, thought to mediate intercellular communication via their packaged cargo such as RNA. Previous studies have focused on the transcriptome of exosomes however very little is known about the identity of RNAs and mechanisms by which they are sorted into endosomes. Here we report a comprehensive endosome transcriptome and provide evidence that several nuclear RNA-protein complexes (RNPs) sort into endosomes, a previously unappreciated phenomenon. We show that this process requires the activity of endosomal sorting complexes and phospholipids characteristic of cellular endocytic compartments. Our study provides a mechanism for recycling and disposal of unwanted nuclear RNPs by the cytoplasmic endolysosomal pathway.
    DOI:  https://doi.org/10.1101/2025.05.22.655499
  24. Neurobiol Dis. 2025 Jun 02. pii: S0969-9961(25)00201-3. [Epub ahead of print] 106985
      DEP-domain containing-5 (DEPDC5) is part of the GATOR1 complex that inhibits the mechanistic target of rapamycin complex-1 (mTORC1). Loss-of-function mutations in human DEPDC5 are the most common cause of lesional or non-lesional focal epilepsies associated with mTOR hyperactivation. Depdc5 silencing in mature neurons leads to excitation/inhibition imbalance and increased excitatory synapse strength. However, no link exists between mTORC1 hyperactivity and the increased activity of glutamatergic synapses. Here, we found that genetic deletion of Depdc5 in a conditional knockout (cKO) mouse recapitulates the excitatory/inhibitory imbalance observed after transient Depdc5 silencing, with increased strength of excitatory transmission and unaffected inhibitory transmission. In Depdc5 cKO neurons, the increased glutamate quantal size and response to exogenous glutamate are attributable to a higher density of GluA1-containing AMPA glutamate receptors due to a shift of the GluA1 subunit from the intracellular pool to the plasma membrane. The DEPDC5 protein interaction network included WDR48, WDR20, and USP46, a ubiquitin-specific protease that regulates GluA1, as key binding partners, along with previously established components of the mTORC1 signaling pathway. In the absence of DEPDC5, USP46 levels increase, and ubiquitination of GluA1 decreases accordingly. Either knockdown of USP46 or rapamycin treatment rescues both the increased glutamate quantal size and USP46 increase caused by Depdc5 deletion, indicating that USP46 overexpression depends on mTORC1 hyperactivity. The data indicate that the DEPDC5/mTORC1 system physiologically controls the excitatory strength by negatively modulating USP46 activity and AMPA receptor deubiquitination, and that failure of this effect can contribute to the development of the Depdc5-linked epileptic phenotype.
    Keywords:  AMPA receptors; DEPDC5 interactome; Depdc5 mutations; Deubiquitination; Excitatory synaptic strength; Primary neurons
    DOI:  https://doi.org/10.1016/j.nbd.2025.106985
  25. Tissue Cell. 2025 May 28. pii: S0040-8166(25)00271-X. [Epub ahead of print]96 102991
      Glioblastoma is one of the most debilitating and extremely aggressive tumors with a median survival of less than a year. Glioblastoma have high metastatic potential and frequently acquire chemoresistance. The current multimodal treatment approaches for glioblastoma include surgical tumor resurrection, radiotherapy, and chemotherapy but these approaches leave the patient with long-term disabilities such as depletion of cognitive abilities, leukoencephalopathy, and recurrence in 6-8 months. Glioma cells are highly dependent on autophagy to survive and proliferate. Autophagy inhibition is proved to be a beneficial strategy for restricting glioma growth. However, due to the lack of specific autophagy inhibitors, the autophagy pathway cannot be efficiently targeted. Understanding the vulnerabilities in autophagy gene expression can help to design better autophagy inhibitors. This study demonstrates the differential expression of GABARAP family members in glioblastoma. Our study highlights the differential expression of GABARAP family members in response to autophagy inhibition and induction. Moreover, the knockdown of specific GABARAP family members enhanced proliferation and reduced temozolomide (TMZ) sensitivity of glial cells by decreasing the p53 expression. The selective expression pattern of GABARAP genes in glioblastoma can be utilized to screen for patients who might respond better to temozolomide treatment. The differential expression of GABARAP family members highlights the subtle regulation of the autophagy pathway in response to environmental cues.
    Keywords:  ATG8; Autophagy; Glioblastoma; P53; TMZ
    DOI:  https://doi.org/10.1016/j.tice.2025.102991
  26. J Hazard Mater. 2025 May 29. pii: S0304-3894(25)01685-1. [Epub ahead of print]494 138769
      Deoxynivalenol (DON) is a chemically stable mycotoxin with a slow natural degradation rate. Consumption of DON-contaminated food and feed poses significant health risks to human and livestock, leading to reduced productivity and substantial economic losses. The functionality of intestinal stem cells (ISCs) are compromised following sustained intracellular deoxynivalenol (DON) stress. Yet, it remains unclear how membrane receptors integrate extracellular DON to impair orderly ISC fate commitments. Here, we found that mechanistic target of rapamycin complex 1 (mTORC1), as well as its upstream signaling pathways such as insulin, mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase-Akt (PI3K/Akt), are involved in DON restraining ISC proliferation and differentiation to disrupt piglet jejunal epithelial structural integrity through single-cell RNA sequencing (scRNA-seq). Using the ex vivo porcine intestinal organoid and in vitro IPEC-J2 cell line, we identified that mTORC1 activation and tuberous sclerosis complex 2 (TSC2) knockout could repair DON-induced ISC injury. Furthermore, DON repressed the TSC2/mTORC1 upstream membrane receptors insulin receptor (IR) and epidermal growth factor receptor (EGFR); conversely, overexpression of IR or EGFR, especially co-overexpression of both, maintained the ISC regeneration in the presence of DON. Importantly, exothermic reactions between DON and the extracellular domains of IR/EGFR monitored by isothermal titration calorimetry (ITC) revealed a composite response consisting of DON recruitment and IR/EGFR conformational dynamics. Therefore, we have ascertained that the extracellular DON regulates intracellular TSC2/mTORC1 activity to restrict ISC function through the interaction with membrane receptors IR and EGFR.
    Keywords:  Deoxynivalenol; Insulin and epidermal growth factor receptors; Intestinal stem cells; Piglets; TSC2/mTOR signaling
    DOI:  https://doi.org/10.1016/j.jhazmat.2025.138769
  27. Cancer Metab. 2025 Jun 05. 13(1): 27
       BACKGROUND: Despite decades of efforts to find successful treatment approaches, cachexia remains a major unmet medical need. This condition, that affects patients with diverse underlying conditions, is characterized by severe muscle loss and is associated with reduced quality of life and limited survival. Search for underlying mechanisms that may guide cachexia treatment has mainly evolved around potential atrophy-inducing roles of inflammatory mediators, and in cancer patients, tumor-derived factors. Recently, a new paradigm emerged as it is becoming evident that specific immune cells inhabit atrophic muscle tissue. Arginase 1 (Arg1) expression is characteristic of these immune cells. Studies of potential contributions of these immune cells to loss of muscle mass and function is in its infancy, and the contribution of ARG1 to these processes remains elusive.
    METHODS: Analyses of RNA sequencing data from murine cachexia models and comprehensive, unbiased open approach proteomics analyses of skeletal myotubes was performed. In vitro techniques were employed to evaluate mitochondrial function and capacity in skeletal muscle cells and cardiomyocytes. Functional bioassays were used to measure autophagy activity. ARG1 level in patients' plasma was evaluated using ELISA, and the association between ARG1 level and patient survival, across multiple types of cancer, was examined using the online database Kaplan-Meier plotter.
    RESULTS: In line with arginine-degrading activity of ARG1, we found signs of arginine restriction in atrophic muscles. In response to arginine restriction, mitochondrial functions and ATP generation was severely compromised in both skeletal muscle cells and in cardiomyocytes. In skeletal muscle cells, arginine restriction enhanced the expression of autophagic proteins, suggesting autophagic degradation of cellular content. Reduction in mitochondria marker TIMM23 supports selective autophagic degradation of mitochondria (mitophagy). In arginine starved cardiomyocytes, mitochondrial dysfunction is accompanied by both increased bulk autophagy and mitophagy. In cancer patients, we found an association between ARG1 expression and accelerated weight loss and reduced survival, further supporting a role of ARG1-producing cells in cachexia pathogenesis.
    CONCLUSION: Together, our findings point to a mechanism for cachexia which depends on expansion of ARG1-expressing myeloid cells, local restriction of arginine, loss of mitochondrial capacity and induced catabolism in skeletal muscle cells and in the heart.
    Keywords:  ARG1; Arginine; Autophagy; Cachexia; Cancer; Mitochondria; Mitophagy; Muscle; Myeloid-derived suppressor cell; Neutrophil
    DOI:  https://doi.org/10.1186/s40170-025-00396-0
  28. Nat Commun. 2025 Jun 05. 16(1): 5209
      The proper function of lysosomes depends on their ability to store and release calcium. While several lysosomal calcium release channels have been described, how lysosomes replenish their calcium stores in placental mammals has not been determined. Using genetic depletion and overexpression techniques combined with electrophysiology and visualization of subcellular ion concentrations and their fluxes across the lysosomal membrane, we show here that TMEM165 imports calcium to the lysosomal lumen and mediates calcium-induced lysosomal proton leakage. Accordingly, TMEM165 accelerates the recovery of cells from cytosolic calcium overload thereby enhancing cell survival while causing a significant acidification of the cytosol. These data indicate that in addition to its previously identified role in the glycosylation of proteins and lipids in the Golgi, a fraction of TMEM165 localizes on the lysosomal limiting membrane, where its putative calcium/proton antiporter activity plays an essential role in the regulation of intracellular ion homeostasis and cell survival.
    DOI:  https://doi.org/10.1038/s41467-025-60349-5
  29. bioRxiv. 2025 May 14. pii: 2025.05.13.653911. [Epub ahead of print]
      Biallelic pathogenic variants in STRADA , an upstream regulator of the mechanistic target of rapamycin (mTOR) pathway, result in megalencephaly, drug-resistant epilepsy, and severe intellectual disability. This study explores how mTOR pathway hyperactivity alters cell fate specification in dorsal and ventral forebrain development using STRADA knock-out human stem cell derived brain organoids. In both dorsal and ventral forebrain STRADA knock-out organoids, neurogenesis is delayed, with a predilection for progenitor renewal and proliferation and an increase in outer radial glia. Ventrally, interneuron subtypes shift to an increase in neuropeptide-Y expressing cells. Inhibition of the mTOR pathway with rapamycin results in rescue for most phenotypes. When mTOR pathway variants are present in all cells of the developing brain, overproduction of interneurons and altered interneuron cell fate may underlie mechanisms of megalencephaly, epilepsy, and cognitive impairment. Our findings suggest mTOR inhibition during fetal brain development as a potential therapeutic strategy in STRADA deficiency.
    DOI:  https://doi.org/10.1101/2025.05.13.653911
  30. Cell Rep. 2025 May 30. pii: S2211-1247(25)00530-3. [Epub ahead of print]44(6): 115759
      Chloroplasts are photosynthetic organelles and one of the major protein-containing organelles in green plants and algae. Although chloroplast contents or entire chloroplasts can be cleared by various vesicular pathways and autophagy, canonical chlorophagy receptors remain unidentified. Also, whether chlorophagy can be enhanced to benefit plants remains unknown. Here, we report the design and validation of a synthetic chlorophagy receptor that promotes plant fitness. The receptor LIR-SNT-BFP contains a fragment spanning the LIR/AIM of NBR1 and the N-terminal amphipathic helix of SFR2. The synthetic receptor localizes to chloroplasts and recruits ATG8a in planta. Induced expression of the synthetic receptor promotes microautophagy of entire chloroplasts, independent of ATG5 or ATG7. Meanwhile, it induces chloroplast fission. Notably, moderate induction of chlorophagy promotes rosette growth, whereas excessive chlorophagy appears detrimental. Induced chlorophagy also partially suppresses herbicide-induced leaf chlorosis. Our study provides proof of concept for controlling chloroplast degradation using a synthetic chlorophagy receptor.
    Keywords:  ATG8; CP: Cell biology; CP: Plants; autophagy; biomass; chloroplast; chloroplast fission; herbicide; microautophagy; receptor; vacuole
    DOI:  https://doi.org/10.1016/j.celrep.2025.115759
  31. J Am Chem Soc. 2025 Jun 06.
      Regulating membrane protein abundance through Lysosome Targeting Chimera (LYTAC) holds significant promise in addressing various diseases. However, the precise structural control of LYTAC molecules and how to improve their treatment efficacy remain elusive. In this study, we develop a multifunctional phototriggered LYTAC platform, named PT-LYTAC, to enhance targeted protein degradation using a photoactive bispecific aptamer chimera (PBAC). PBAC is designed with a precise modular approach that integrates an NIR photosensitive molecule into a bispecific aptamer chimera. Taking advantage of the low molecular weight and easy synthesis of the DNA aptamers, PBAC can efficiently transport the therapeutically relevant membrane protein PTK7 to lysosomes for degradation through the lysosomal pathway. Moreover, our investigation reveals that the multifunctional PT-LYTAC platform, enabled by DNA aptamers, promotes protein degradation by modulating cellular autophagy. By the combination of targeted protein degradation and spatiotemporally controllable regulation of intracellular oxidative stress, the function of tumor cells can be significantly inhibited. Under NIR laser irradiation, PT-LYTAC completely suppresses colorectal cancer growth with just one dose and a single laser treatment, all without any apparent side effects. We anticipate that this novel PT-LYTAC will expand the use of DNA-based LYTAC drugs and provide a new dimension for targeted protein degradation.
    DOI:  https://doi.org/10.1021/jacs.5c05456
  32. Redox Biol. 2025 May 27. pii: S2213-2317(25)00213-7. [Epub ahead of print]84 103700
      Autophagy has crucial roles for ischemia/reperfusion (I/R) injury. To define the role of the autophagy hub protein p62/SQSTM1 in I/R injury, we conducted gain-of-function and loss-of-function experiments in a set of cell types, including two neuron-like cell lines, primary neurons, brain endothelial and astroglial-like cells, which we combined with mouse ischemic stroke studies. p62 levels post-I/R increased alongside intracellular ROS changes. p62 overexpression increased and p62 knockdown or pharmacological deactivation reduced I/R injury. Autophagic flux was p62-dependent, but oxygen-independent. Using p62 domain deletion mutants we identified p62's ZZ domain as key factor mediating autophagy and cell death. Death-promoting effects of p62 involved elevated ROS burden. At the same time, p62 activated a broad network of cytoprotective responses, which included NRF2-associated antioxidant signaling and inhibition of the pro-inflammatory NFκB pathway, which were bidirectionally linked with p62, and downregulation of the ER stress sensor BiP/GRP78 with consecutive activation of the UPR PERK branch. Our study establishes p62 as a master regulator of I/R injury, which offers itself as target for stroke therapies.
    Keywords:  Apoptosis; Middle cerebral artery occlusion; Necroptosis; Nuclear factor erythroid-2-related factor-2; Nuclear factor-kB; Oxygen-glucose deprivation; Protein kinase RNA-Like endoplasmic reticulum kinase; Reactive oxygen species; Reoxygenation; Stress signaling
    DOI:  https://doi.org/10.1016/j.redox.2025.103700
  33. Biotechniques. 2025 May 31. 1-13
      Mitophagy, a crucial mitochondrial quality control system for cellular stress adaptation, is a key focus in pathophysiology and drug discovery. Developing a simple and versatile mitophagy flux assay is vital for advancing our understanding of cellular responses. Addressing a gap in systematic methods, we employ the photoactivatable fluorescent protein mito-Kaede in C2C12 myocytes, demonstrating its remarkable versatility in quantifying mitophagy flux responses under various stimuli, including carbonyl cyanide m-chlorophenyl hydrazone (CCCP), TNF-α, lipopolysaccharide (LPS), and hypoxia. This study underscores the validity and distinctive advantages of the mito-Kaede assay through comparative analysis with conventional assays including Western blotting (WB), potentially providing valuable insights for both mitophagy flux analysis and drug development.
    Keywords:  Autophagy; flux measurement; mito-Kaede; mitochondria; mitophagy; quantitative assay; stimulated response kinetics; time-lapse
    DOI:  https://doi.org/10.1080/07366205.2025.2505357
  34. Front Cell Neurosci. 2025 ;19 1588645
      Neurodegenerative diseases are a diverse group of neurological disorders, in which abnormal mitochondrial function is closely associated with their development and progression. This has generated significant research interest in the field. The proper functioning of mitochondria relies on the dynamic regulation of the mitochondrial quality control system. Key processes such as mitochondrial biogenesis, mitophagy, and mitochondrial dynamics (division/fusion) are essential for maintaining this balance. These processes collectively govern mitochondrial function and homeostasis. Therefore, the mitochondrial quality control system plays a critical role in the onset and progression of neurodegenerative diseases. This article provides a concise overview of the molecular mechanisms involved in mitochondrial biogenesis, mitophagy, and mitochondrial dynamics, explores their interactions, and summarizes current research progress in understanding the mitochondrial quality control system in the context of neurodegenerative diseases.
    Keywords:  Alzheimer’s disease; Huntington’s disease; Parkinson’s disease; amyotrophic lateral sclerosis; mitochondrial quality control
    DOI:  https://doi.org/10.3389/fncel.2025.1588645
  35. Autophagy Rep. 2025 ;4(1): 2507266
      Quiescence is a conserved, reversible state of proliferative arrest, characterized by changes in cell physiology and metabolism. Many cells spend a considerable part of their lifetime in quiescence, including adult stem cells or microorganisms facing unfavorable environmental conditions. Cells can remain quiescent for long periods of time while retaining their viability and reproductive capacity, indicating a need to maintain protein homeostasis. Given the changes in intracellular organization, it has been unclear how protein quality control (PQC) functions in quiescent cells. In our recent study, we examined model misfolded proteins expressed in glucose-depleted quiescent yeast cells and found that quiescent cells maintain an active PQC that relies primarily on selective protein degradation, requiring the ubiquitin-proteasome system, intact nucleus-vacuole junctions and autophagy. Our results highlight the relevance of mitigating misfolded proteins in quiescence.
    Keywords:  Nucleus-vacuole junction; Vac8; proteasome storage granule; protein aggregate; protein quality control; quiescence; yeast
    DOI:  https://doi.org/10.1080/27694127.2025.2507266
  36. Toxicol Appl Pharmacol. 2025 May 29. pii: S0041-008X(25)00197-8. [Epub ahead of print]502 117421
      Resveratrol has confirmed effectiveness in alleviating myocardial ischemia/reperfusion(I/R) injury. However, the underlying mechanisms remain unclear. Mitochondrial dysfunction in injured cardiomyocytes activates autophagy, and excessive autophagy during reperfusion implicates aggravated injury. Considering that resveratrol preserves mitochondrial function by down-regulating VDAC1 expression, we speculated that the cardioprotective effect of resveratrol is achieved by mitochondrial regulation, and we wonder whether it is accomplished by ultimately modulating autophagy. Therefore, this study investigated the mechanism of resveratrol against myocardial I/R injury regarding autophagy regulation and explored the signal pathway. Herein, we established an anoxia/reoxygenation(A/R) model to simulate myocardial I/R injury in vitro. The expressions of VDAC1, Beclin1, LC3-II/I, Parkin, and PINK1 were detected by Western blot; the LDH activity and mPTP opening were measured by spectrophotometry; the ROS levels and mitochondrial membrane potential (ΔΨm) were examined by flow cytometry; the sublocalisation of Parkin and the autophagic vacuoles (AVs) were observed by laser confocal microscopy. Results suggested that resveratrol attenuated A/R injury by inhibiting autophagy, manifested as lower LDH activity, higher cell viability with decreased LC3-II/LC3-I ratio, down-regulated Beclin1 expression, and reduced number of AVs. In addition, stabilised mitochondrial membrane potential, inhibited ROS production and mPTP opening indicated maintained mitochondrial homeostasis. Compared with the A/R group, resveratrol pretreatment down-regulated the PINK1, Parkin, and VDAC1 expressions, accompanied by decreased colocalization of mitochondria with Parkin, suggesting the involved PINK1/Parkin signal pathway. Transfection with pFLAG-VDAC1 reversed resveratrol-induced mitophagy inhibition and cardioprotection. In conclusion, resveratrol protects cardiomyocytes by inhibiting excessive autophagy induced by the VDAC1/PINK1/Parkin pathway during A/R injury.
    Keywords:  Anoxia/reoxygenation; Autophagy; Cardiomyocytes; Resveratrol; Voltage-dependant anion channel 1
    DOI:  https://doi.org/10.1016/j.taap.2025.117421
  37. J Neuroinflammation. 2025 Jun 02. 22(1): 147
       BACKGROUND: The ε4 isoform of apolipoprotein E (ApoE) is the most significant genetic risk factor for Alzheimer's disease. Glial cells are the main source of ApoE in the brain, and in microglia, the ε4 isoform of ApoE has been shown to impair mitochondrial metabolism and the uptake of lipids and Aβ42. However, whether the ε4 isoform alters autophagy or lysosomal activity in microglia in basal and inflammatory conditions is unknown.
    METHODS: Altogether, microglia-like cells (iMGs) from eight APOE3/3 and six APOE4/4 human induced pluripotent stem cell (iPSC) lines were used in this study. The responses of iMGs to Aβ42, LPS and IFNγ were studied by metabolomics, proteomics, and functional assays.
    RESULTS: Here, we demonstrate that iMGs with the APOE4/4 genotype exhibit reduced basal pinocytosis levels compared to APOE3/3 iMGs. Inflammatory stimulation with a combination of LPS and IFNγ or Aβ42 induced PI3K/AKT/mTORC signaling pathway, increased pinocytosis, and blocked autophagic flux, leading to the accumulation of sequestosome 1 (p62) in both APOE4/4 and APOE3/3 iMGs. Exposure to Aβ42 furthermore caused lysosomal membrane permeabilization, which was significantly stronger in APOE4/4 iMGs and positively correlated with the secretion of the proinflammatory chemokine IL-8. Metabolomics analysis indicated a dysregulation in amino acid metabolism, primarily L-glutamine, in APOE4/4 iMGs.
    CONCLUSIONS: Overall, our results suggest that inflammation-induced metabolic reprogramming places lysosomes under substantial stress. Lysosomal stress is more detrimental in APOE4/4 microglia, which exhibit endo-lysosomal defects.
    Keywords:  Alzheimer’s disease; Apolipoprotein E; Lysosomal dysfunction; Microglia; iPSC
    DOI:  https://doi.org/10.1186/s12974-025-03470-y
  38. Autophagy Rep. 2025 ;4(1): 2511724
      Monitoring the delivery of single proteins and protein complexes to the vacuole by autophagy or other processes in yeast Saccharomyces cerevisiae mainly relies on western blot or fluorescence microscopy analyses using endogenous tagging of the protein of interest with GFP. However, these approaches are semi-quantitative and next to impossible with proteins of low abundancy because of the insensitive nature of the methods. Here, we describe the creation of a new PCR-based integration cassette to endogenously tag specific proteins with the truncated version of the vacuolar phosphatase Pho8. The vacuolar activation of Pho8 allows the quantitative measurement of vacuolar delivery using a colorimetric enzymatic assay. This approach has the advantages of a more quantitative interpretation of data and relies on the appearance of a signal rather than its disappearance. As a proof-of-principle, we examined the vacuolar delivery of known cargoes of bulk autophagy and endocytosis. This new system will be of great value to the whole community working within the field of autophagy and other transport pathways to the vacuole.
    Keywords:  Pho8∆60; autophagy; endocytosis; turnover; vacuolar phosphatase; vacuole
    DOI:  https://doi.org/10.1080/27694127.2025.2511724
  39. bioRxiv. 2025 May 21. pii: 2025.05.20.654652. [Epub ahead of print]
      Neurodegenerative diseases affect 1 in 12 people globally and remain incurable. Central to their pathogenesis is a loss of neuronal protein maintenance and the accumulation of protein aggregates with aging. We engineered bioorthogonal tools which allowed us to tag the nascent neuronal proteome and study its turnover with aging, its propensity to aggregate, and its interaction with microglia. We discovered neuronal proteins degraded on average twice as slowly between 4- and 24-month-old mice with individual protein stability differing between brain regions. Further, we describe the aged neuronal 'aggregome' encompassing 574 proteins, nearly 30% of which showed reduced degradation. The aggregome includes well-known proteins linked to disease as well as a trove of proteins previously not associated with neurodegeneration. Unexpectedly, we found 274 neuronal proteins accumulated in microglia with 65% also displaying reduced degradation and/or aggregation with age. Among these proteins, synaptic proteins were highly enriched, suggesting a cascade of events emanating from impaired synaptic protein turnover and aggregation to the disposal of these proteins, possibly by the engulfment of synapses by microglia. These findings reveal the dramatic loss of neuronal proteome maintenance with aging which could be causal for age-related synapse loss and cognitive decline.
    DOI:  https://doi.org/10.1101/2025.05.20.654652
  40. bioRxiv. 2025 May 19. pii: 2025.05.16.654477. [Epub ahead of print]
      Lysosomal damage is an endogenous danger signal to the cell, but its significance for innate immunity and how specific signaling pathways are engaged by this stressor remain unclear. Here, we uncover an immune-inducible pathway that connects lysosomal damage to mitochondrial DNA (mtDNA) efflux and type I IFN production. Lysosomal damage elicits mitochondrial outer membrane permeabilization (MOMP) via BAK/BAX macropores; however, the inner mitochondrial membrane (IMM) prevents wholesale mtDNA release in resting cells. Priming with type II IFN (IFN-γ) induced the antibacterial effector apolipoprotein L-3 (APOL3), which upon transient lysosomal damage, targets mitochondria undergoing MOMP and selectively permeabilizes the IMM to enhance mtDNA release and activate cGAS/STING signaling. Biochemical and cellular reconstitution revealed that analogous to its bactericidal detergent-like mechanism, APOL3 solubilizes cardiolipin to permeabilize the IMM. Our findings illustrate how cells use an antibacterial protein to expedite the breakdown of endosymbiosis and facilitate a heightened response to injury and infection.
    DOI:  https://doi.org/10.1101/2025.05.16.654477
  41. Cell Commun Signal. 2025 May 31. 23(1): 256
       INTRODUCTION: Cardiac mitochondrial function is intricately regulated by various processes, ultimately impacting metabolic performance. Additionally, protein turnover is crucial for sustained metabolic homeostasis in cardiomyocytes.
    OBJECTIVE: Here, we studied the role of mTOR in OPA-1 cleavage and its consequent effects on mitochondrial dynamics and energetics in cardiomyocytes.
    RESULTS: Cultured rat cardiomyocytes treated with rapamycin for 6-24 h showed a significant reduction in phosphorylation of p70S6K, indicative of sustained inhibition of mTOR. Structural and functional analysis revealed increased mitochondrial fragmentation and impaired bioenergetics characterized by decreases in ROS production, oxygen consumption, and cellular ATP. Depletion of either the mitochondrial protease OMA1 or the mTOR regulator TSC2 by siRNA, coupled with an inducible, cardiomyocyte-specific knockout of mTOR in vivo, suggested that inhibition of mTOR promotes mitochondrial fragmentation through a mechanism involving OMA1 processing of OPA-1. Under homeostatic conditions, OMA1 activity is kept under check through an interaction with microdomains in the inner mitochondrial membrane that requires prohibitin proteins (PHB). Loss of these microdomains releases OMA1 to cleave its substrates. We found that rapamycin both increased ubiquitination of PHB1 and decreased its abundance, suggesting proteasomal degradation. Consistent with this, the proteasome inhibitor MG-132 maintained OPA-1 content in rapamycin-treated cardiomyocytes. Using pharmacological activation and inhibition of AMPK our data supports the hypothesis that this mTOR-PHB1-OMA-OPA-1 pathway impacts mitochondrial morphology under stress conditions, where it mediates dynamic changes in metabolic status.
    CONCLUSIONS: These data suggest that mTOR inhibition disrupts mitochondrial integrity in cardiomyocytes by promoting the degradation of prohibitins and OPA-1, leading to mitochondrial fragmentation and metabolic dysfunction, particularly under conditions of metabolic stress.
    Keywords:  AMPK; Mitochondrial fusion; OMA1; OPA-1; Prohibitin; Rapamycin; mTOR
    DOI:  https://doi.org/10.1186/s12964-025-02240-w
  42. Mol Neurobiol. 2025 May 31.
      Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) accumulation, autophagic lysosomal pathway (ALP) dysfunction, mitochondrial abnormalities, and neuroinflammation. Physical exercise (PE) protects against AD, but its molecular mechanisms remain unclear. We hypothesize that PE-mediated upregulation of REV-ERBα and TFEB pathways mitigates AD-related dysfunctions. Acute effects of FK506, a calcineurin inhibitor, were assessed as a TFEB suppressor in mice subjected to aerobic exercise. Chronic treadmill training (8 weeks, 4 sessions/week) was performed in APP/PS1 mice to evaluate hippocampal adaptations through functional tests, imaging, and molecular analyses. Acute FK506 administration inhibited Ppp3ca and Ppp3r1 expression without altering Tfeb levels. Chronic PE improved aerobic capacity, strength, coordination, and memory, promoted neuronal survival, and decreased Aβ levels in APP mice. It also elevated REV-ERBα protein and Nr1 d1 expression in wild-type and APP mice, increased ALP activity, and reduced abnormal mitochondria in the hippocampus of APP mice. A positive correlation between REV-ERBα and Nr1 d1 levels was observed in the 2-min NOR test. Public RNA-seq data revealed lower NR1D1 mRNA in extracellular vesicles from the human frontal cortex of AD patients compared to controls. PE prevents cognitive decline in APP/PS1 mice, enhancing memory, physical performance, and hippocampal health. These benefits are associated with ALP activation, mitochondrial improvements, and reduced neuroinflammation. REV-ERBα may mediate these protective effects, but further studies using pharmacological and genetic models are needed to confirm its role.
    Keywords:  Amyloid-β; Autophagic lysosomal pathway; Calcineurin; Neuroinflammation; REV-ERBα; TFEB; TLR4
    DOI:  https://doi.org/10.1007/s12035-025-05066-2
  43. Ageing Res Rev. 2025 Jun 03. pii: S1568-1637(25)00142-4. [Epub ahead of print]110 102796
      Age-related macular degeneration (AMD) is the most common disease of the elderly that leads to the loss of sight. So far, no satisfactory therapy exists for this complex eye disease. The appearance of extracellular deposits, called drusen, on the outside of the retinal pigment epithelium (RPE) is considered to be the main clinical hallmark of AMD. Whilst the mechanisms of drusen formation are not well known, secreted material from the RPE, during its degeneration, is thought to contribute to the development of AMD. Various unconventional protein secretion (UPS) pathways are considered to be routes for the delivery of material which form the drusen. The two main forms of UPS are secretory autophagy, which is responsible for the cleansing of cellular debris from the RPE cells and endosomal secretion which carries material outside of the cell via exosomes. These pathways are unconventional in the sense that they comprise the delivery of material to the exterior of cells by bypassing the Golgi apparatus. Although secretory autophagy and exosome release are regarded as different routes by which cells exude material, they share similarities, such as common molecular participants and that their routes converge. Therefore, manipulation of these two processes might be useful in a therapy against AMD by diminishing the destructive drusen progression in the vicinity of the RPE.
    Keywords:  Drusen; exosome; extracellular vesicle; retinal pigment epithelium; secretory autophagy; unconventional protein secretion
    DOI:  https://doi.org/10.1016/j.arr.2025.102796
  44. Cell Death Dis. 2025 Jun 05. 16(1): 437
      Satellite cells are muscle-resident stem cells that maintain and repair muscle. Increasing evidence supports the contributing role of satellite cells in Duchenne muscular dystrophy (DMD), a lethal degenerative muscle disease caused by loss of dystrophin. However, whether or not satellite cells exhibit dysfunction due to loss of dystrophin remains unresolved. Here, we used single-cell RNA-sequencing (scRNA-seq) to determine how dystrophin deficiency impacts the satellite cell transcriptome and cellular composition by comparing satellite cells from mdx and the more severe D2-mdx DMD mouse models. DMD satellite cells were disproportionally found within myogenic progenitor clusters and a previously uncharacterized DMD-enriched cluster. Despite exposure to different dystrophic environments, mdx and D2-mdx satellite cells exhibited overlapping dysregulation in gene expression and associated biological pathways. When comparing satellite stem cell versus myogenic progenitor populations, we identified unique dysfunctions between DMD and healthy satellite cells, including apoptotic cell death and senescence, respectively. Pseudotime analyses revealed differences in cell fate trajectories, indicating that DMD satellite cells are stalled in their differentiation capacity. In vivo regeneration assays confirmed that DMD satellite cells exhibit impaired myogenic gene expression and cell fate dynamics during regenerative myogenesis. These defects in differentiation capacity are accompanied by impaired senescence and autophagy dynamics. Finally, we demonstrate that inducing autophagy can rescue the differentiation of DMD progenitors. Our findings provide novel molecular evidence of satellite cell dysfunction in DMD, expanding on our understanding of their role in its pathology and suggesting pathways to target and enhance their regenerative capacity.
    DOI:  https://doi.org/10.1038/s41419-025-07755-1
  45. bioRxiv. 2025 May 23. pii: 2024.05.24.595621. [Epub ahead of print]
      While delivery of supplemental oxygen is a life-saving therapy, exposure to high levels of oxygen, called hyperoxia, is associated with increased mortality in the intensive care unit (ICU). Hyperoxia leads to oxidant-mediated acute lung injury (ALI) and pulmonary cell death, called hyperoxic acute lung injury (HALI). Elucidation of molecular mechanisms in HALI could identify therapeutic targets in ALI. In the current study, we examined in vivo effects of HALI on Beclin-1 (BCN1), a molecule that regulates autophagy and cell death. Effects of HALI on BCN1 and autophagy markers were examined in wildtype mice. Analysis of BCN1 and autophagy was completed via Western blot, RT-qPCR, and immunohistochemistry. In wildtype mice, HALI led to increased BCN1 in the lung and in the alveolar epithelium. HALI resulted in significant alterations in markers of autophagy in the lung, including reduced microtubule-associated protein 1B-light chain (LC3B)-II/-I ratios, suggesting reduced autophagic flux. HALI caused increased LDH release in human alveolar type-II cells derived from induced pluripotent stem cells (AT2s iPSC ), as well as reduced LC3B-II/-I ratios. We previously showed that inhibition of the tyrosine kinase receptor epidermal growth factor receptor (EGFR) is protective in HALI. EGFR Wa5/+ mice, which have genetically reduced EGFR activity and improved survival in HALI, showed increased total BCN1, reduced phosphorylated-(p-)/total BCN1 ratios, and decreased LC3B-II/-I ratios in the lung in HALI compared with wildtype. Administration of wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor which decreases BCN1-mediated autophagy, led to increased mortality in HALI in wildtype mice. These data support that regulation of BCN1 and autophagy by EGFR is a protective mechanism in HALI, a pathway which warrants further study for its therapeutic potential.
    KEY MESSAGES: HALI is associated with increased mortality in the ICU and causes alveolar epithelial cell death, but the role of BCN1 in HALI is not well defined. This study shows that BCN1, a molecule involved in autophagy and cell death, is regulated by EGFR in HALI in vivo . These results are significant because regulation by BCN1 and autophagy by EGFR is a novel pathway in HALI with therapeutic potential that warrants further study.
    DOI:  https://doi.org/10.1101/2024.05.24.595621
  46. FEBS J. 2025 Jun 01.
      PINK1 (PTEN-induced kinase 1) and Parkin (parkin RBR E3 ubiquitin protein) ligase are important regulators for cells to maintain mitochondrial number and functional homeostasis. Here, we established a PINK1/Parkin-based mitophagy drug evaluation method using quantitative Förster resonance energy transfer (FRET) imaging in living cells. A stable model of carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-induced mitophagy was established, verified by increased colocalization of mitochondria with LC3 aggregates, decreased mitochondrial membrane potential (MMP), and increased intracellular reactive oxygen species (ROS) level. Next, by silencing PINK1 and overexpressing LC3 proteins in MCF-7 cells, it was verified that PINK1 and Parkin significantly promoted CCCP-induced mitophagy, in which CCCP promoted the direct interaction of PINK1 and Parkin. Quantitative FRET imaging analysis for the cells coexpressing CFP-PINK1 and YFP-Parkin was used to assess the action of five drugs [3-methyladenine (3-MA), CCCP, doxorubicin hydrochloride (DOX), metformin (Met), resveratrol (RSV)] on the interaction between PINK1 and Parkin. After 6 h of treatment with these drugs, the CCCP, DOX, Met, and RSV groups showed significantly higher maximum donor-centric FRET efficiency (EDmax) than the control group, suggesting that these four drugs promoted the direct interaction between PINK1 and Parkin. While the 3-MA group showed similar EDmax to the control group, suggesting that 3-MA did not promote direct interaction between PINK1 and Parkin. We also performed these experiments in HeLa cells and obtained the same results, further demonstrating that the PINK1/Parkin-based quantitative FRET drug screening method is a potential tool for mitophagy drug screening in living cells.
    Keywords:  PINK1; Parkin; drug screening; mitophagy; quantitative FRET
    DOI:  https://doi.org/10.1111/febs.70146
  47. Mol Neurobiol. 2025 Jun 01.
      To explore the potential protective role and underlying mechanism of the sigma-1 receptor (σ1R) on inner blood-retinal barrier (iBRB) function after retinal ischemia/reperfusion (I/R) injury. A retinal I/R injury model was established in C57BL/6 mice, and the protective effects of σ1R were evaluated using the σ1R agonist SA4503 and the antagonist BD1047. In vitro, an oxygen-glucose deprivation/reperfusion (OGD/R) cell model was used to investigate the regulatory role of σ1R on ER stress, autophagy, and tight junction (TJ) proteins via Western blotting, immunofluorescence, and fluorescently labeled autophagic flux assays. Specific activators and inhibitors of ER stress and autophagy were also employed to elucidate the mechanism of σ1R regulation. Activating σ1R with the agonist SA4503 notably decreased Evans blue leakage in vivo and prevented the disassembly of tight junction proteins both in vivo and in vitro. These advantageous effects disappeared when pretreated with the σ1R antagonist BD1047. σ1R activation mitigated I/R-induced endoplasmic reticulum (ER) stress and autophagy, but these protective benefits were nullified by the administration of tunicamycin (an ER stress activator) or rapamycin (an autophagy activator). Tunicamycin inhibited SA4503's effects on ER stress and autophagy, while rapamycin specifically blocked SA4503's impact on autophagy. Furthermore, an ER stress inhibitor (4-phenylbutyrate) and inhibitors of ER-related signaling pathways (ATF6 inhibitor, IRE1 inhibitor, and PERK inhibitor) effectively blocked I/R-induced autophagy. Activating σ1R had protective effects on iBRB and capillary endothelial function after retinal I/R injury. These effects might involve inhibition of ER stress-dependent autophagy and the degradation of tight junction proteins.
    Keywords:  Autophagy; Endoplasmic reticulum stress; Inner blood-retinal barrier; Retinal ischemia/reperfusion injury; Sigma-1 receptor
    DOI:  https://doi.org/10.1007/s12035-025-05038-6
  48. Sci Adv. 2025 Jun 06. 11(23): eadv4033
      The endolysosomal pathway plays an evolutionarily conserved role in pathogen clearance, and viruses have evolved complex mechanisms to evade this host defense system. Here, we describe a previously unidentified aspect of coronaviral infection, whereby the master transcriptional activator of lysosomal homeostasis-TFEB-is targeted for proteasomal-mediated degradation upon viral infection. Through mass spectrometry analysis and an unbiased small interfering RNA screen, we identify that TFEB protein stability is coordinately regulated by the E3 ubiquitin ligase subunit DCAF7 and the PAK2 kinase. We derive a series of novel small molecules that interfere with the DCAF7-TFEB interaction. These agents inhibit virus-induced TFEB degradation and demonstrate broad antiviral activities including attenuating severe acute respiratory syndrome coronavirus 2 infection in two animal models. Together, these results delineate a virally triggered pathway that impairs lysosomal homeostasis in the host. Small molecule E3 ubiquitin ligase DCAF7 inhibitors that restore lysosomal function represent a novel class of host-directed, antiviral therapies useful for current and potentially future coronaviral variants.
    DOI:  https://doi.org/10.1126/sciadv.adv4033