bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2025–12–28
25 papers selected by
Viktor Korolchuk, Newcastle University



  1. Cells. 2025 Dec 12. pii: 1976. [Epub ahead of print]14(24):
      Lysosomes are central effectors of cellular maintenance, integrating the degradation of damaged organelles and protein aggregates with macromolecule recycling and metabolic signaling. In neurons, lysosomes are particularly crucial due to the cells' long lifespan, polarized architecture, and high metabolic demands. Proper regulation of lysosomal function is essential to sustain proteostasis, membrane turnover, and synaptic integrity. Although lysosomal dysfunction has been extensively studied in neurodegenerative diseases, far less is known about how lysosomal capacity and function are maintained-or fail to be maintained-with age in non-diseased neurons. In this review, we summarize current understanding of neuronal lysosomal dynamics, discuss methodological challenges in assessing lysosomal capacity and function, and highlight recent advances that reveal age-associated decline in neuronal lysosomal competence.
    Keywords:  TFEB; aging; autolysosome; autophagy; endolysosome; lysosome; neuron
    DOI:  https://doi.org/10.3390/cells14241976
  2. Autophagy. 2025 Dec 26.
      The conjugation of mammalian Atg8 (ATG8)-family proteins to membrane components is a fundamental process in membrane ATG8ylation (lipidation). While membrane ATG8ylation is well-characterized, protein ATG8ylation, the direct conjugation of ATG8 to cellular proteins, remains enigmatic. In this study, we demonstrate that protein ATG8ylation depends exclusively on ATG4, ATG3, and ATG7. We discovered that the core macroautophagy/autophagy E1 enzyme ATG7 serves a dual role: it is not only the essential E1 enzyme for protein ATG8ylation but also a key substrate. We determined that ATG7 K140 is the modification site and show that protein ATG8ylation of ATG7 forms a mono-LC3B conjugate. We demonstrated that this self-modification creates a negative-feedback loop by hindering the ATG7-ATG3 interaction, thereby attenuating autophagic flux. Our findings redefine ATG7 as a central player and regulator in the protein ATG8ylation cascade, revealing a new mechanism of autophagy regulation.
    Keywords:  ATG7; LC3; post-translational modification; protein ATG8ylation; self-regulation
    DOI:  https://doi.org/10.1080/15548627.2025.2609929
  3. Autophagy. 2025 Dec 26.
      Proteotoxic stress, arising from conditions that cause misfolded protein accumulation, is closely linked to the pathogenesis of multiple diseases. Macroautophagy/autophagy activation is considered a compensatory mechanism to maintain protein homeostasis, but the underlying regulatory mechanisms remain incompletely understood. Here, we show that proteotoxic stress induced by proteasome inhibition, puromycin treatment, or polyglutamine-expanded HTT (huntingtin) expression promotes nuclear accumulation of TFEB and TFE3, key regulators of lysosomal biogenesis and autophagy. Mechanistically, TFEB activation under proteotoxic stress occurs independently of canonical MTORC1 inactivation mediated by TSC2 or ATF4. Instead, it involves non-canonical inhibition of MTORC1 via RRAG GTPases. Proteotoxic stress disrupts the RRAGC-TFEB interaction, preventing TFEB recruitment to lysosomes and subsequent MTORC1 phosphorylation. An activated RRAGC mutant rescues impaired lysosomal localization and nuclear accumulation of TFEB, while co-overexpression of FLCN and FNIP2, a GAP for RRAGC, partially restores stress-induced TFEB dephosphorylation. In addition, proteasome inhibition activates non-canonical autophagy. Deletion of ATG16L1 or ATG5, which blocks Atg8-familyh protein lipidation and sequesters the FLCN-FNIP2 complex, partially abolishes proteotoxic stress-induced TFEB dephosphorylation and nuclear accumulation. Together, these findings demonstrate that proteotoxic stress triggers both non-canonical autophagy and TFEB-mediated canonical autophagy, with Atg8-family protein lipidation contributing to TFEB activation. Our results provide novel insights into how proteotoxic stress engages non-canonical MTORC1 inhibition and TFEB activation, thereby enhancing understanding of cellular adaptation to proteotoxic stress.
    Keywords:  Autophagy; MTORC1; RRAG GTPase; TFEB; lysosomal biogenesis; proteosome
    DOI:  https://doi.org/10.1080/15548627.2025.2608973
  4. Cells. 2025 Dec 15. pii: 1993. [Epub ahead of print]14(24):
      The basic helix-loop-helix (bHLH) transcription factor 'Atoh8' is involved in the regulation of several developmental processes and pathologies. It regulates organogenesis, reprogramming, stem cell fate determination, and cancer development. However, the mechanisms underlying these observations remain unclear. Unlike many tissue-specific bHLH factors, Atoh8 is ubiquitously expressed during development as well as in adult tissues. In this study, we explored whether Atoh8 modulates basic cellular functions, which may reveal a common mechanism that could explain the diverse observations reported in the literature. Our findings demonstrate that the loss of Atoh8 impairs autophagy. In both primary myoblasts and mouse embryonic stem cells lacking Atoh8, we observed differential expression of LC3B-II, TFEB, and accumulation of p62, indicating impairment of autophagy. Furthermore, mass spectrometric analysis performed on C2C12 and Atoh8 overexpressing C2C12 myoblasts revealed significant alterations in the expression of proteins associated with mitochondrial and lysosomal functions. Finally, Cut&Tag sequencing performed in Atoh8 overexpressing C2C12 cells revealed that Atoh8 binds to multiple genes involved in autophagosome assembly. Overall, this study underscores that Atoh8 is a critical regulator of macroautophagy, and its reduction disrupts the autophagic process, whereas its overexpression results in increased autophagic flux.
    Keywords:  Atoh8; Tfeb; autophagy; lysosome; macroautophagy; metabolism; p62
    DOI:  https://doi.org/10.3390/cells14241993
  5. Front Aging. 2025 ;6 1750125
      
    Keywords:  NAD+; ROCK inhibitor; autophagy and mitophagy; lactate; lifestyles including sleep/fasting/exercise; mitochondria and mitochondrial DNA; neurodegeneration; rapamycin
    DOI:  https://doi.org/10.3389/fragi.2025.1750125
  6. Neurotox Res. 2025 Dec 27. 44(1): 2
      The objective of the present review is to discuss the involvement of altered mitochondrial quality control in Mn-induced neurotoxicity. Existing data demonstrate that mitochondrial autophagy (mitophagy) and brain mitochondrial unfolded protein response (mtUPR) are activated in response to Mn exposure to counteract the Mn-induced mitochondrial dysfunction. Both mitophagy and mtUPR have significant overlap and mechanistic intersections with the integrated stress response (ISR). Increased Mn exposures impair mitochondrial dynamics, further aggravating Mn-induced mitochondrial dysfunction. Specifically, Mn suppresses PTEN-induced kinase 1 (PINK1)-Parkin-dependent mitophagy through a variety of mechanisms, including nitric oxide synthase 2 (NOS2)-dependent PINK1 S-nitrosylation, inhibition of transcription factor EB (TFEB) signaling, and mammalian target of rapamycin complex 1 (mTORC1) activation. In addition, Mn promotes mitochondrial fission by up-regulating dynamin-1-like protein (Drp1) expression and phosphorylation via the activation of c-Jun N-terminal kinase (JNK) and inhibition of sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) pathways. Concomitantly, Mn impairs mitochondrial fusion by inhibiting mitofusin (Mfn) 1/2 and dynamin-like 120 kDa protein (Opa1) expression, leading to a reduction in mitochondrial size and disruption of the mitochondrial network. High-dose Mn exposure results in inhibition of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α)/nuclear factor erythroid 2-related factor 2 (NRF2)-dependent mitochondrial biogenesis. The latter may be mediated by inhibition of SIRT1/SIRT3 activity, as well as modulation of PINK1/ zinc finger protein 746 (ZNF746)/PGC-1α axis. Alterations in the mitochondrial quality control system may contribute to Mn-induced neuronal damage and neuroinflammation, indicating that dysregulation of the brain mitochondrial dynamics is an important mechanism by which Mn induces its neurotoxicity.
    DOI:  https://doi.org/10.1007/s12640-025-00776-w
  7. bioRxiv. 2025 Dec 12. pii: 2025.12.09.693285. [Epub ahead of print]
      Cells dynamically regulate chromatin in response to nutrient flux which promotes the transcriptional changes necessary for adaptation. The mechanistic target of rapamycin complex 1 (mTORC1) kinase integrates nutrient signaling with chromatin regulation, yet whether chromatin stability feeds back to mTORC1 activation and stress adaption remains unknown. We previously identified histone H3 at lysine 37 (H3K37) as essential for the response to mTORC1 stress such that mutation of H3K37 to alanine (H3K37A) causes cell death upon mTORC1 inhibition. Herein, we show that H3K37-dependent chromatin stability prevents proteasome-mediated histone degradation, restricts mTORC1 signaling, and safeguards mitochondrial homeostasis during mTORC1 stress. Genetic interaction analyses reveal that H3K37A combined with mutants that destabilize chromatin, including loss of the Set2 H3K36 methyltransferase, Rpd3S histone deacetylase, or multiple histone deposition pathways, causes synthetic lethality when mTORC1 is inhibited. Transcriptome analysis indicates that H3K37A misregulates the mitochondrial transcriptome during mTORC1 stress, which increases mitochondrial reactive oxygen species (ROS) and triggers lethal mitochondrial retrograde signaling. Inactivation of retrograde signaling, or ROS neutralization, rescues viability of H3K37A and chromatin stability mutants during mTORC1 stress. These findings establish chromatin stability as a key safeguard that restrains mTORC1 activity and prevents toxic mitochondrial stress during metabolic adaptation.
    DOI:  https://doi.org/10.64898/2025.12.09.693285
  8. Autophagy. 2025 Dec 24.
      Microautophagy involves the direct uptake of cytoplasmic materials by lysosomes, but its regulation, including substrate specificity, has remained largely unclear in mammalian cells. Microlipophagy, a form of lipid droplet microautophagy, has been suggested in mammalian cells, yet the molecular basis that links lysosomes to lipid droplets and supports their uptake has not been elucidated. In our recent study, we showed that the lysosomal membrane protein LAMP2B mediates this process via its cytoplasmic region, which can bind phosphatidic acid, a lipid present on lipid droplets. We also found that this pathway depends on the ESCRT machinery and proceeds independently of macroautophagy. In this commentary, we summarize these findings and describe how LAMP2B affects lipid droplet degradation in cells. We describe that LAMP2B overexpression protects mice from high-fat-diet-induced obesity and related disorders. We also outline a model of microautophagy and microautophagy-like processes in which LAMP2 isoforms use their cytoplasmic regions to recognize distinct cargos.
    Keywords:  Autophagy; LAMP2; LAMP2B; lipid droplet; microautophagy; microlipophagy
    DOI:  https://doi.org/10.1080/15548627.2025.2609920
  9. Cell Death Discov. 2025 Dec 23.
      Monkeypox virus (MPXV) is a globally reemerging pathogen that poses a significant threat to public health, representing the most impactful Orthopoxvirus infection in humans since the eradication of smallpox. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process essential for maintaining cellular homeostasis, and it can exert either pro-viral or anti-viral effects during infections. Poxviruses interaction with the autophagy machinery remains poorly understood, and the specific interplay between MPXV and autophagy has not been documented. In this study, we infected Calu-3 cells with MPXV and observed that the virus significantly impairs autophagic flux by upregulating Rubicon, a known negative regulator of autophagy. Notably, silencing Rubicon restored autophagic flux and led to a marked reduction in MPXV replication. Overall, our findings reveal a novel mechanism by which MPXV inhibits autophagy through the modulation of Rubicon, suggesting that autophagy activation may be a potential therapeutic strategy for MPXV.
    DOI:  https://doi.org/10.1038/s41420-025-02920-z
  10. Autophagy. 2025 Dec;21(12): 2535-2536
      Although KRAS-driven tumors exhibit elevated macroautophagy/autophagy, the extent to which this process diverges from canonical regulatory pathways has not been well characterized. In a recent study published in Cell Research, Wang et al. unveil a novel form of non-canonical autophagy driven by oncogenic RAS mutations, which they termed RAS-induced non-canonical autophagy via ATG8ylation (RINCAA). This pathway operates through a unique MAPK/p38-ULK1-PI4KB axis, diverging significantly from canonical starvation-induced autophagy. The research not only elucidates a new regulatory mechanism but also identifies a potential, highly specific therapeutic target for RAS-mutant cancers.Abbreviations: PI4KB, phosphatidylinositol 4-kinase beta; PtdIns4P, phosphatidylinositol-4-phosphate; RINCAA, RAS-induced non-canonical autophagy via Atg8ylation; ULK1, unc-51 like autophagy activating kinase 1; WIPI2, WD repeat domain phosphoinositide-interacting protein 2.
    Keywords:  Autophagy; RAS; RINCAA; oncogene; p38-ULK1-PI4KB axis
    DOI:  https://doi.org/10.1080/15548627.2025.2555048
  11. Cells. 2025 Dec 08. pii: 1947. [Epub ahead of print]14(24):
      Autophagy is a cellular catabolic mechanism that facilitates the degradation of cytoplasmic components, thereby restoring energy homeostasis and mitigating cellular damage. This process functions as a housekeeping system, essential for maintaining organismal viability under stressful conditions. Numerous studies have highlighted the role of autophagy in regulating various physiological processes, including metabolic pathways. Notably, certain autophagy-related genes may play a relevant role in metabolic disorders, extending beyond their involvement in the autophagic process, and may offer potential therapeutic avenues for treating energy-related metabolic diseases. This review summarizes the roles of various components of each autophagic complex and the regulators involved in the autophagic process. In particular, it explores the intricate relationship between autophagy and several metabolic diseases, including type 2 diabetes mellitus (T2DM), obesity, and non-alcoholic fatty liver disease (NAFLD).
    Keywords:  autophagy; metabolic diseases; non-alcoholic fatty liver disease; obesity; type 2 diabetes mellitus
    DOI:  https://doi.org/10.3390/cells14241947
  12. Cell Death Discov. 2025 Dec 24.
      Mitochondrial dysfunction is one of the core drivers of aging. It is manifested by reactive oxygen species (ROS) accumulation, mitochondrial DNA (mtDNA) mutations, imbalanced energy metabolism, and abnormal biosynthesis. Mitochondrial autophagy maintains cellular homeostasis by selectively removing damaged mitochondria through mechanisms including the ubiquitin-dependent pathway (PINK1/Parkin pathway) and the ubiquitin-independent pathway (mediated by receptors such as BNIP3/FUNDC1). During aging, the decrease in mitochondrial autophagy efficiency leads to the accumulation of damaged mitochondria, forming a cycle of mitochondrial damage-ROS-aging damage and aggravating aging-related diseases such as neurodegenerative diseases and cardiovascular pathologies. The targeted regulation of mitochondrial autophagy (drug modulation and exercise intervention) can restore mitochondrial function and slow aging. However, autophagy has a double-edged sword effect; moderate activation is anti-aging, but excessive activation or dysfunction accelerates the pathological process. Therefore, targeting mitochondrial autophagy may be an effective anti-aging technique; however, future focus should be on the tissue-specific regulatory threshold and the dynamic balance mechanism to achieve precise intervention.
    DOI:  https://doi.org/10.1038/s41420-025-02913-y
  13. Biochem J. 2025 Dec 23. pii: BCJ20253459. [Epub ahead of print]483(1):
      Loss-of-function mutations in PINK1 and PRKN cause familial forms of Parkinson's disease (PD). In vitro studies have revealed incredible insights into the molecular and cell-biological function of these genes, which have focused predominantly on mitophagy - the autophagic degradation of damaged mitochondria. The mechanisms of PINK1/Parkin function ultimately require investigation in an in vivo context using classic genetic approaches in animal models. In this context, Drosophila models have proven to be remarkably informative, in part due to robust phenotypes arising from null mutations. They have revealed important insights into the function of the Pink1 and parkin orthologues, much of which has proven to be conserved in humans. The simplicity, speed and genetic tractability make Drosophila an excellent in vivo model to interrogate the physiological functions of Pink1 and parkin and to rapidly test emerging hypotheses arising from in vitro work. They also represent a powerful model with which to explore the pathological consequences of Pink1/parkin loss in a whole-organism context. In this regard, several themes have emerged from recent studies that likely have significance for the neurodegenerative process in humans, including aberrant activation of immune signalling and consequent inflammation, disruptions to gut integrity and disturbed mitochondrial calcium handling. In this review, we evaluate the current evidence regarding the mechanism(s) of Pink1/parkin-mediated mitochondrial turnover in Drosophila, and discuss the potential implications of recent developments on the consequences of Pink1/parkin mutations and how these may inform the pathogenesis of PD.
    Keywords:   Drosophila ; PINK1; Parkin; Parkinson’s disease; autophagy; calcium signalling; immune signalling; mitochondria; mitophagy; mtDNA; neurodegeneration
    DOI:  https://doi.org/10.1042/BCJ20253459
  14. Autophagy. 2025 Dec 26.
      Mitochondrial dysfunction is widely recognized as a key driver of aging and neurodegenerative diseases, with mitophagy acting as an essential cellular mechanism for the selective clearance of damaged mitochondria. While pharmacological activation of mitophagy has been reported to exert beneficial effects across multiple neurodegenerative diseases, its functional relevance in amyotrophic lateral sclerosis (ALS) remains poorly characterized. Our recent study published in EMBO Molecular Medicine demonstrates that PINK1-PRKN-dependent mitophagy is markedly impaired in ALS motor neurons. Through high-content drug screening, we identified a potent mitophagy agonist isoginkgetin (ISO), a bioflavonoid from Ginkgo biloba that stabilizes the PINK1-TOMM complex on the outer mitochondrial membrane, enhances PINK1-PRKN-dependent mitophagy, and ameliorates motor neuron degeneration in ALS-like Caenorhabditis elegans, mouse models, and induced pluripotent stem cell-derived motor neurons. Consequently, ISO is able to alleviate ALS-associated phenotypes. In this commentary, we contextualize these findings broadly to discuss whether pharmacologically induced mitophagy can act as an effective therapeutic strategy, distinct from current clinical approaches, for the development of ALS-targeted treatments.
    Keywords:  ALS; PINK1-Parkin; isoginkgetin; mitophagy; motor neurons
    DOI:  https://doi.org/10.1080/15548627.2025.2610450
  15. Autophagy Rep. 2026 ;5(1): 2602971
      Reticulophagy regulator 1 (RETREG1)/Family with sequence similarity 134 member B (FAM134B) is a selective endoplasmic reticulum (ER)-phagy receptor that mediates starvation-induced macro-ER-phagy, but whether it participates in other pathways mediating ER turnover has remained unclear. Here, we unveil a previously unrecognized role for RETREG1 in micro-ER-phagy and show how the murine leukemia virus (MLV) accessory protein glycosylated group-specific antigen (glycoGag) exploits this pathway to antagonize the host restriction factor SERINC5 (serine incorporator 5). GlycoGag binds SERINC5 in the endoplasmic reticulum (ER) and selectively recruits RETREG1 to eliminate SERINC5 through an autophagosome-independent process that bypasses ATG3 (autophagy-related), ATG5, ATG7, BECN1 (Beclin-1), LC3 (microtubule-associated protein 1 light chain 3) lipidation, and PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3)/hVPS34 (vacuolar protein sorting 34). RETREG1 knockout abolishes degradation of ER-retained SERINC5, whereas endolysosomal turnover of surface SERINC5 remains partially intact, demonstrating that glycoGag utilizes dual ER-phagy and endolysosomal routes to suppress SERINC5. These findings expand the functional repertoire of RETREG1 in autophagy, identify that retroviruses repurpose micro-ER-phagy to circumvent SERINC5-mediated restriction, and reveal ER-phagy as an understudied battleground in the ongoing arms race between cellular restriction factors and viral accessory proteins.
    Keywords:  Autophagy; ER-phagy; FAM134B; RETREG1; SERINC5; glycogag; micro-ER-phagy; restriction factor; reticulophagy; retrovirus
    DOI:  https://doi.org/10.1080/27694127.2025.2602971
  16. Autophagy. 2025 Dec 23.
      Members of the mammalian Atg8-protein family (ATG8), including the MAP1LC3/LC3 and GABARAP subfamilies, play essential roles in selective macroautophagy/autophagy. However, their functional distinctions during viral infection remain poorly understood. Here, we show that S-adenosyl-L-methionine (SAM)-binding viral proteins, such as nsp14 from coronavirus and NP868R from African swine fever virus (ASFV), reprogram autophagy by shifting antiviral LC3B activity toward GABARAP-mediated mitophagy in an ATG4A-dependent manner. Mechanistically, the SAM-binding motif allows these viral proteins to stabilize ATG4A mRNA, thereby increasing ATG4A expression and redirecting autophagic flux from LC3B-mediated virophagy to GABARAP-dependent mitophagy. This shift suppresses innate immune responses by targeting both MAVS-dependent interferon signaling and virophagy, ultimately enhancing viral replication. Collectively, our findings uncover a previously unrecognized immune evasion strategy in which SAM-binding viral proteins rewire autophagy from antiviral to proviral pathways.
    Keywords:  ATG4A; ATG8 family; GABARAP; LC3B; mitophagy; virophagy
    DOI:  https://doi.org/10.1080/15548627.2025.2608972
  17. Dev Biol. 2025 Dec 23. pii: S0012-1606(25)00344-6. [Epub ahead of print]
      Autophagy is an evolutionarily conserved catabolic process that is essential for maintaining cellular and developmental homeostasis in eukaryotes. Drosophila oogenesis offers a robust model for investigating the spatial and temporal regulation of autophagy within a complex developmental framework that involves cells from both germline and somatic lineages. This tightly regulated cascade of events enables the differentiation of a germline stem cell into a mature oocyte. Autophagy contributes to cellular quality control, nutrient sensing, and the regulation of developmental cell death, all of which are critical for proper egg development and maturation. Disruption of autophagy influences oogenesis, resulting in defective egg chamber development, altered apoptotic dynamics, abnormally shaped mitochondria and compromised mitophagy. Methodological advances, including immunofluorescence-based detection, live imaging using fluorescent reporters, and ultrastructural analysis via transmission electron microscopy, have significantly enhanced the ability to monitor autophagic activity in the ovary. This review summarizes current evidence that establishes autophagy as a key regulatory mechanism during oogenesis. Additionally, it offers an opportunity to investigate the role of autophagy in various cellular processes, including cell division, gene amplification, endocycling, collective cell migration, and cytoplasmic streaming for embryonic polarity, nurse cell dumping, and programmed cell death during Drosophila oogenesis.
    Keywords:  Drosophila; aging; autophagy; cell death; germarium; germline stem cells; mitophagy; nurse cells; oogenesis; starvation; vitellogenesis
    DOI:  https://doi.org/10.1016/j.ydbio.2025.12.013
  18. Mol Biol Cell. 2025 Dec 24. mbcE25040190
      Activation of the cGAS-STING pathway stimulates innate immune signaling as well as LC3B lipidation and ubiquitylation at Golgi-related vesicles upon STING trafficking. Although ubiquitylation at these subcellular sites has been associated with regulating NF-κB-related innate immune signaling, the mechanisms of Golgi-localized polyubiquitin chain regulation of immune signaling is not well understood. We report here that the ubiquitin- and LC3B-binding proteins, TNIP1 and autophagy receptors p62, NBR1, NDP52, TAX1BP1, and OPTN associate with STING-induced ubiquitin and LC3B-labeled vesicles, and that p62 and NBR1 act redundantly in spatial clustering of the LC3B-labeled vesicles in the perinuclear region. We also find that while TBK1 kinase activity is not required for the recruitment of TNIP1 and the autophagy receptors, it plays a role in sequestration of the LC3B-labeled vesicles. The ubiquitin binding domains, rather than the LC3-interacting regions, of TNIP1 and OPTN are specifically important for their recruitment to Ub/LC3B-associated perinuclear vesicles, and OPTN is also recruited through a TBK1-dependent mechanism. Functionally, we find that TNIP1 plays a role in STING-mediated innate immune signaling, acting as a negative regulator of IRF3-mediated gene expression. Together, these results highlight autophagy-independent mechanisms of autophagy receptors and TNIP1 with unanticipated roles in regulating STING-mediated innate immunity.
    DOI:  https://doi.org/10.1091/mbc.E25-04-0190
  19. bioRxiv. 2025 Dec 12. pii: 2025.12.09.693280. [Epub ahead of print]
      LRRK2 is implicated in lysosomal functions, but the physiological upstream cues that engage endogenous LRRK2 activity are incompletely defined. Here we show that lysosomal swelling serves as a selective and reversible trigger for LRRK2-mediated Rab phosphorylation, without requiring membrane damage. Acute inhibition of PIKfyve, but not the general disruption of phosphoinositide signaling, induces the robust accumulation of phosphorylated Rabs across endolysosomal membranes. Rescue of swelling through pharmacological restoration of lysosomal ionic imbalances from PIKfyve inhibition suppresses LRRK2 activation without restoring lysosomal function. Mechanical lysosomal swelling from indigestible osmolyte uptake causes a dose-dependent increase in LRRK2-mediated Rab phosphorylation on both swollen and non-swollen lysosomes. Together, these findings identify LRRK2 as a sensor of lysosomal volume and mechanical stress, not specifically membrane damage or PIKfyve inhibition. As lysosomal swelling is a shared pathological feature across LRRK2 -linked diseases, these results reframe LRRK2 as part of an endolysosomal surveillance system responsive to lysosomal distension.
    DOI:  https://doi.org/10.64898/2025.12.09.693280
  20. Anal Bioanal Chem. 2025 Dec 23.
      Autophagy is a complex self-degradative process that recycles cytoplasmic components through lysosomal degradation, enabling cells to maintain homeostasis during stress and nutrient deprivation. Despite major advances in understanding the basic mechanisms of autophagy, important gaps remain in translating them to human diseases. This study investigated the metabolic fingerprints and footprints of two mechanistically different autophagy inducers, Torin1 (mTOR-dependent) and Tat-Beclin1 (mTOR-independent), in mouse embryonic fibroblasts (MEF). Multi-platform untargeted metabolomics and lipidomics analyses were performed at 3 and 18 h exposure to elucidate both intracellular and extracellular metabolic changes using liquid chromatography-high-resolution mass spectrometry coupled to drift tube ion mobility, complemented by [13C]-glucose tracing. Torin1 exposure caused downregulation of TCA cycle intermediates, accumulation of purine degradation products, enhanced phospholipid catabolism, and triglycerides' enrichment. In contrast, Tat-Beclin1 preserved central carbon metabolism, promoted recovery of glutathione levels, and redirected diglycerides toward the biosynthesis of polyunsaturated phosphocholines (PC) and C18-containing phosphoethanolamines (PE). Despite these compound-specific responses, several common alterations were observed, including downregulation of ceramides, upregulation of ether-linked PEs, consistent enrichment of PC O-12:0_16:0, lyso-PE 22:6, PC 16:0_20:4, PC 16:0_22:5, and depletion of PE 32:1, PE 34:2, and PE 38:6, along with secretion of unsaturated fatty acids and uptake of sphingomyelin 35:1;O2 and cytosine from the extracellular compartment. Together, these results show that Torin1 and Tat-Beclin1 trigger distinct yet partly overlapping metabolic programs. The metabolic signatures identified here provide reference profiles for future mechanistic studies and highlight candidate biomarkers that may support early functional evaluation of autophagy modulators in disease-relevant settings.
    Keywords:  Metabolic fingerprinting; Stable-isotope tracing; Tat-Beclin1; Torin1; mTOR signaling
    DOI:  https://doi.org/10.1007/s00216-025-06275-3
  21. bioRxiv. 2025 Dec 19. pii: 2025.12.17.694986. [Epub ahead of print]
      Macroautophagy is a cellular process where cytosolic material is captured in double membrane vesicles, termed autophagosomes, which fuse with the vacuole or lysosomes leading to the degradation of the captured contents. In yeast, the biogenesis of autophagosomes is initiated by the fusion of a few small vesicles which contain the integral membrane protein Atg9. Atg9 vesicle trafficking is in part regulated by the peripheral membrane protein Atg23. However, the structure of Atg23 and the mechanism by which Atg23 interacts with Atg9 are currently unknown. Therefore, we determined the crystal structure for a monomeric form of Atg23 and characterized the interaction between Atg23 and Atg9. This work reveals that Atg23 contains a novel fold which is consistent with the AlphaFold 3 prediction except that the helices running towards the dimerization region have a bend giving a more curved global architecture than the prediction. In addition, we demonstrate that conserved sequences in both the N and C-terminal regions of Atg9 bind to a hydrophobic cavity on Atg23.
    DOI:  https://doi.org/10.64898/2025.12.17.694986
  22. bioRxiv. 2025 Dec 18. pii: 2025.12.17.689083. [Epub ahead of print]
      Misfolding of aggregation-prone proteins underpins diseases known as proteinopathies. One of these proteins, alpha-synuclein, is a component of aggregates in neurodegenerative conditions such as Parkinson's disease. The melanosomal protein PMEL, which forms physiologic amyloid scaffold structures on which melanin is organized in melanosomes, similarly ectopically accumulates in the dermis in many forms of cutaneous hyperpigmentation. Here, we demonstrate in a wide range of common clinical pigmentary disorders, as well as in primary melanocyte and mouse models examined by molecular, proteomic, and electron microscopic tools, that melanocytic alpha-synuclein is a prominent component of intracellular protein aggregates bound to similar proteins as in Parkinson's disease, as well as melanized extracellular protein deposits. Using the Real Time Quaking-Induced Conversion Assay (RT-QuIC), we demonstrate that UV induces misfolded melanosomal proteins to self-propagate, augmenting this pathology in prion-like fashion. CUT&RUN chromatin profiling and single-cell RNA-seq demonstrate that melanocytes utilize microphthalmia-associated transcription factor (MITF)-regulated autophagy to counteract protein aggregation, identifying aggregate removal as a core function of tanning. In contrast to extracellular aggregation, impaired intracellular aggregate removal contributes to melanocyte senescence, which conversely exacerbates chronic hypopigmentation and photoaging-related discoloration. These findings identify melanosomal proteinopathy as a common contributor to melanocyte dysfunction and suggest aggregate-focused management approaches.
    DOI:  https://doi.org/10.64898/2025.12.17.689083
  23. bioRxiv. 2025 Dec 19. pii: 2025.12.18.695023. [Epub ahead of print]
      Cell surface receptors such as NOTCH1 must be tightly regulated to ensure developmental fidelity and prevent pathological activation. Although the proteolytic steps culminating in nuclear NOTCH1 signaling are established, how cells prevent excessive or uncontrolled activation has remained unresolved. Here we identify the autophagy-related protein UVRAG as a negative regulator of NOTCH1. Upon receptor activation, UVRAG, acting independently of autophagy, recruits and activates the E3-ligase ITCH to catalyze K27-linked ubiquitination of membrane-tethered NOTCH1, thereby licensing ESCRT-dependent lysosomal degradation. Disruption of the UVRAG-ITCH-ESCRT axis stabilizes activated NOTCH1 intermediates and amplifies oncogenic signaling. In T-cell leukemia models driven by constitutive NOTCH1 activity, restoring UVRAG expression reinstates receptor turnover, suppresses disease progression, and improves therapeutic response. These findings define a ubiquitin-directed safeguard circuit that enforces NOTCH1 signaling homeostasis and reveals a tunable axis for intervention in NOTCH1-driven cancers.
    DOI:  https://doi.org/10.64898/2025.12.18.695023
  24. Brain Sci. 2025 Nov 25. pii: 1260. [Epub ahead of print]15(12):
      Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SN) and the presence of intracellular α-synuclein (αSyn) aggregates known as Lewy bodies (LB). αSyn, a presynaptic protein, is believed to play a crucial role in synaptic function, neurotransmitter release, and neuronal plasticity. However, its misfolding and aggregation are thought to be central to PD pathogenesis. This review provides a comprehensive analysis of αSyn's role in PD, exploring its normal physiological functions, pathological mechanisms, and therapeutic potential. The pathological transformation of αSyn involves structural alterations that promote oligomerization and fibrillization, leading to toxic gain-of-function effects. These aggregates disrupt cellular homeostasis through mechanisms including mitochondrial dysfunction, oxidative stress, lysosomal impairment, and endoplasmic reticulum stress. Furthermore, pathogenic αSyn is thought to exacerbate neurodegeneration via prion-like spread along interconnected neuronal circuits. Emerging evidence highlights the frequent co-occurrence of other proteinopathies, such as tau and amyloid-β, which may synergistically accelerate disease progression. Targeting αSyn has emerged as a potential therapeutic strategy. Approaches such as immunotherapy, small-molecule inhibitors, gene silencing, and modulation of protein degradation pathways (e.g., autophagy and proteasomal systems) are actively being explored. Additionally, lifestyle-based interventions, particularly exercise, have shown neuroprotective effects, potentially mediated by irisin-a myokine implicated in protein clearance and synaptic resilience-underscoring the importance of multimodal strategies in PD management.
    Keywords:  biomarkers; co-pathology; immunotherapy; neurodegeneration; prion-like spread; synucleinopathy
    DOI:  https://doi.org/10.3390/brainsci15121260
  25. Mol Brain. 2025 Dec 25.
      Mitochondrial dysfunction and abnormalities in mitochondrial quality control contribute to the development of neurodegenerative diseases. Parkinson's disease is a neurodegenerative disease that causes motor problems mainly due to the loss of dopaminergic neurons in the substantia nigra pars compacta. Axonal mitochondria in neurons reportedly differ in properties and morphologies from mitochondria in somata or dendrites. However, the function and morphology of axonal mitochondria in human dopaminergic neurons remain poorly understood. To define the function and morphology of axonal mitochondria in human dopaminergic neurons, we newly generated tyrosine hydroxylase (TH) reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines from one control and one PRKN-mutant patient iPSC lines and differentiated these iPSC lines into dopaminergic neurons in two-dimensional monolayer cultures or three-dimensional midbrain organoids. Immunostainings with antibodies against axonal and dendritic markers showed that axons could be better distinguished from dendrites of dopaminergic neurons in the peripheral area of three-dimensional midbrain organoids than in two-dimensional monolayers. Live-cell imaging and correlative light-electron microscopy in peripheral areas of midbrain organoids derived from control TH-GFP iPSCs demonstrated that axonal mitochondria in dopaminergic neurons had lower membrane potential and were shorter in length than those in non-dopaminergic neurons. Although the mitochondrial membrane potential did not significantly differ between dopaminergic and non-dopaminergic neurons derived from PRKN-mutant patient lines, these differences tended to be similar to those in control lines. These results were also largely consistent with those of our previous study on somatic mitochondria. The findings of the present study indicate that midbrain organoids are an effective tool to distinguish axonal from dendritic mitochondria in dopaminergic neurons. This may facilitate the analysis of axonal mitochondria to provide further insights into the mechanisms of dopaminergic neuron degeneration in patients with Parkinson's disease.
    Keywords:  Axonal mitochondria; Dopaminergic neurons; Electron microscopy; Live-cell imaging; Midbrain organoids
    DOI:  https://doi.org/10.1186/s13041-025-01268-w