Autophagy最新文献

Duchenne muscular dystrophy (DMD) is caused by the loss of DMD (dystrophin), leading to sarcolemmal fragility and progressive muscle degeneration. Although adeno-associated viral (AAV) microdystrophin (µDMD) therapies have advanced clinically, their benefits remain partial, highlighting the need to identify secondary cellular defects that limit therapeutic efficacy. In our recent study, we demonstrated that lysosomal dysfunction is a conserved, intrinsic, and persistent feature of DMD pathology. Using mouse, canine, and human dystrophic muscle, we show marked lysosomal membrane permeabilization (LMP), impaired acidification, defective proteolysis, and inefficient membrane repair, all hallmarks of compromised lysosomal integrity. Cholesterol accumulation within dystrophic myofibers further exacerbates these defects, linking lipid dysregulation to lysosomal injury and accelerated muscle degeneration. We find macroautophagy/autophagy impairment in DMD stems in part from reduced autophagosome-lysosome fusion, reframing autophagy failure as a downstream consequence of lysosomal damage. µDMD gene therapy only partially corrects these abnormalities and does not fully restore lysosomal stability. In contrast, combining µDMD with the lysosome-activating disaccharide trehalose produces synergistic benefits, improving muscle strength, architecture, and molecular signatures beyond either treatment alone. These findings position lysosomal dysfunction as a central driver of DMD pathophysiology and support therapeutic strategies that pair gene restoration with lysosomal enhancement.Abbreviation: AAV: adeno-associated virus; DAGC: DMD-associated glycoprotein complex; DMD: Duchenne muscular dystrophy; FDA: Food and Drug Administration; LMP: lysosome membrane permeabilization; MTOR: mechanistic target of rapamycin kinase; µDMD: microdystrophin.

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 known blocks Atg8-family 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.Abbreviations: ALP, autophagy-lysosomal pathway; ATF4, activating transcription factor 4; Baf A1, bafilomycin A₁; CHX, cycloheximide; BTZ, bortezomib; CFZ, carfilzomib; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; DQ-BSA, dequenched-bovine serum albumin; EIF4EBP1/4EBP1, eukaryotic translation initiation factor 4E binding protein 1; ER, endoplasmic reticulum; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MG132, carbobenzoxy-Leu-Leu-leucinal; MTORC1, mechanistic target of rapamycin kinase complex 1; RPS6KB1/p70, ribosomal protein S6 kinase B1; RRAG, Ras related GTP binding; SQSTM1/p62, sequestosome 1; TFE3, transcription factor E3; TFEB, transcription factor EB; TSC2, TSC complex subunit 2; tfLC3, tandem fluorescent LC3; UPS, ubiquitin-proteasome system.

Apoptosis, a programmed cell death process activated in Alzheimer disease (AD), is not limited to neurons but extends to all cell types within the central nervous system (CNS). However, how apoptotic cells mediate their impact on surrounding cells and contribute to the pathological progression of AD remains largely unclear. Here, we report that in 5×FAD mice, cells surrounding amyloid-β (Aβ) plaques undergo apoptosis, which occurs concurrently with elevated macroautophagy/autophagy. The autophagic flux, nevertheless, is impaired in AD, as evidenced by the simultaneous accumulation of MAP1LC3/LC3 and SQSTM1/p62. As a result, although there is an increased formation of autophagosomes, misfolded proteins fail to undergo proper degradation in the subsequent process. By profiling the "metabolomic secretome" of primary neurons and glial cells under different apoptotic stimuli, we identified spermidine as a conserved apoptotic metabolite messenger in the CNS. Spermidine is actively released from apoptotic neurons or glia cells and functions in a paracrine manner to induce autophagy activation in neighboring cells. Such an effect of enhancing autophagic flux promotes both the cargo encapsulation within autophagosomes and degradation in autolysosomes in nearby cells. Conversely, the blockade of spermidine release impairs autophagic flux, thereby exacerbating cognitive impairment and pathological progression in AD. These findings reveal a link between cell apoptosis and autophagy in AD, suggesting that spermidine supplementation could serve as a promising therapeutic strategy.

Radiotherapy is a fundamental step in the combined treatment of glioblastoma (GBM), while radioresistance of GBM causes limitation of therapeutic efficacy. Natural killer (NK) cells, a potential target of immunotherapy, have attracted considerable attention due to the robust cancer cell-targeted cytotoxicity in combined treatment with radiotherapy, suggesting NK cell regulation might be a radiosensitization strategy. Here we show that a cytotoxic subset of NK cells could be stimulated by ionizing radiation (IR) and accumulate in the GBM tumor microenvironment (TME). Co-culturing with NK cells significantly enhances the GBM cell response to IR, and pharmaceutically depleting NK cells in mice elevates IR-induced tumor growth delay. Specifically, GZMB should be the radiosensitization effector secreted by NK cells. Suppressing GZMB activity remarkably impairs NK-mediated GBM radiosensitization. Meanwhile, administrating exogenous GZMB improves irradiation dose-survival response in vitro or in a xenograft model. Mechanically, GZMB blocks autophagosome-lysosome fusion in GBM cells by directly recognizing and cleaving SDC1, a key regulator of autophagosome maturation, at the valine 225 and aspartate 228 sites. Uncleavable mutation of SDC1 reverses GZMB-mediated radiosensitization in GBM. Further studies demonstrate that cleavage of SDC1 obstructs the localization of TGM2, a key MAP1LC3/LC3 recognizer, on the lysosome surface. Clinical data reveal GBM patients with an SDC1 valine 225 or aspartate 228 mutation display lower response to radiotherapy. In this study, we disclose the critical role of NK cells in tumor radiotherapy through secreting GZMB and impeding autophagosome maturation, as well as propose a potential strategy combining radiotherapy and NK-based immunotherapy against radioresistant GBM.Abbreviations: DEGs: differentially expressed genes; GBM: glioblastoma; GZMB: granzyme B; IL: interleukin; IR: ionizing radiation; IRS: immunoreactive score; LAMP: lysosomal associated membrane protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; mSDC1: mutant SDC1; NK: natural killer; PRF1: perforin 1; SDC1: syndecan 1; SNAP29: synaptosome associated protein 29; SQSTM1: sequestosome 1; STX17: syntaxin 17; TGM2: transglutaminase 2; TME: tumor microenvironment; TGD: tumor growth delay; VAMP8: vesicle associated membrane protein 8; WT: wild type.

Macroautophagy/autophagy exerts multilayered protective functions in intestinal epithelial cells (IECs) while a loss-of-function genetic variant in ATG16L1 (autophagy related 16 like 1) is associated with risk for developing Crohn disease (CD). Westernization of diet, partly characterized by excess of long-chain fatty acids, contributes to CD, and a metabolic control of intestinal inflammation is emerging. Here, we report an unexpected inflammatory function for ATG16L1-mediated autophagy in Crohn-like metabolic enteritis of mice induced by polyunsaturated fatty acid (PUFA) excess in a western diet. Dietary PUFAs induce ATG16L1-mediated conventional autophagy in IECs, which is required for PUFA-induced chemokine production and metabolic enteritis. By transcriptomic and lipidomic profiling of IECs, we demonstrate that ATG16L1 is required for PUFA-induced inflammatory stress signaling specifically mediated by TLR2 (toll-like receptor 2) and the production of arachidonic acid metabolites. Our study identifies ATG16L1-mediated autophagy in IECs as an inflammatory hub driving metabolic enteritis, which challenges the perception of protective autophagy in the context of diet westernization.Abbreviations: AA: arachidonic acid; ATG16L1: autophagy related 16 like 1; CD: Crohn disease; CXCL1: C-X-C motif chemokine ligand 1; ER: endoplasmic reticulum; GFP: green fluorescent protein; GPX4: glutathione peroxidase 4; IBD: inflammatory bowel disease; IECs: intestinal epithelial cells; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; PUFA: polyunsaturated fatty acid; SDA: stearidonic acid; TLR2: toll-like receptor 2; WT: wild-type.

Haploinsufficiency of TBK1 causes familial ALS and frontotemporal dementia (FTD), yet the mechanisms by which TBK1 loss leads to neurodegeneration remain unclear. Using deep proteomics and phospho-proteomics, we demonstrate that TBK1 regulates select macroautophagy/autophagy factors, targeting cargo receptors and autophagy initiation factors, and also sustains the phosphorylation of the late endosomal marker RAB7A in stem cells and stem cell-derived excitatory neurons. We further uncovered novel TBK1-dependent phosphorylation sites in the key autophagy protein SQSTM1/p62. Loss of TBK1 function results in a cell-autonomous neurodegenerative phenotype characterized by impaired neurite outgrowth and lysosomal dysfunction.

The vacuolar-type H⁺-translocating ATPase (V-ATPase) plays a pivotal role in cellular homeostasis by acidifying endosomes and lysosomes, regulating key processes such as autophagy and membrane trafficking. While the importance of V-ATPase in these functions is well-established, the methodologies for studying its assembly and function remain varied and under-characterized. In this study, we systematically validated and compared methodologies for assessing V-ATPase assembly and endo/lysosomal acidification under physiological and high-fat conditions, both in vitro and in vivo. Various techniques, including fractionation, immunoprecipitation, immunofluorescence microscopy, and proximity ligation assays, were evaluated using cardiomyocyte cell lines, rat models of lipid overload, and two heart-specific V-ATPase-knockout mouse models (V-ATPase subunits ATP6V1G1 and ATP6V0D2). High palmitate (HP) and bafilomycin A₁ (BafA) were used to manipulate v-ATPase function, while a colorimetric assay assessed proton-pumping activity. Results consistently showed that HP and BafA induced V-ATPase disassembly and inhibited proton-pumping activity, leading to impaired endo/lysosomal acidification and autophagy inhibition upon fusion of autophagosomes with lysosomes. Similar findings were observed in vivo, where a high-fat diet (HFD) reproduced the effects of HP on cardiac tissue. The methodologies were further validated in two heart-specific V-ATPase-knockout mouse models, demonstrating consistent outcomes across different experimental approaches. This study establishes a robust framework for evaluating V-ATPase assembly and function. The validated methodologies reveal that lipid overload inhibits autophagy and contributes to insulin resistance by inducing V-ATPase disassembly and subsequent lysosomal dysfunction. These findings offer insights into the molecular mechanisms underlying metabolic diseases and provide valuable tools for further research.

Lysosome homeostasis is vital for cellular fitness due to the essential roles of this organelle in various pathways. Given their extensive workload, lysosomes are prone to damage, which can stimulate lysosomal quality control mechanisms such as biogenesis, repair, or autophagic removal - a process termed lysophagy. Despite recent advances highlighting lysophagy as a critical mechanism for lysosome maintenance, the extent of lysosome integrity perturbation and the magnitude of lysophagy in vivo remain largely unexplored. Additionally, the pathophysiological relevance of lysophagy is poorly understood. To address these gaps, it is necessary to develop quantifiable methods for evaluating lysosome damage and lysophagy flux in vivo. To this end, we created two transgenic mouse lines expressing a tandem fluorescent LGALS3/galectin 3 probe (tfGAL3), either constitutively or conditionally under Cre recombinase control, utilizing the property of LGALS3 to recognize damaged lysosomes. This tool enables spatiotemporal visualization of lysosome damage and lysophagy activity at single-cell resolution in vivo. Systemic analysis across various organs, tissues, and primary cultures from these lysophagy reporter mice revealed significant variations in basal lysophagy, both in vivo and in vitro. Additionally, this study identified substantial changes in lysosome integrity and lysophagy flux in different tissues under stress conditions such as starvation, acute kidney injury and diabetic modeling. In conclusion, these complementary lysophagy reporter models are valuable resources for both basic and translational research.Abbreviation: AAV: adeno-associated virus; ATG7: autophagy related 7; CA-tfGAL3: cre-recombinase-activated tandem fluorescent LGALS3; DAPI: 4',6-diamidino-2-phenylindole; DM: diabetes mellitus; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HFD: high-fat diet; Igs2/H11/Hipp11: intergenic site 2; IST1: IST1 factor associated with ESCRT-III; KI: knock-in; LAMP1: lysosomal-associated membrane protein 1; LGALS3: lectin, galactoside-binding, soluble, 3; LLOMe: L-leucyl-L-leucine methyl ester hydrobromide; MEFs: mouse embryonic fibroblasts; NaOx: sodium oxalate; PDCD6IP: programmed cell death 6 interacting protein; PTECs: proximal tubular epithelial cells; RFP: red fluorescent protein; STZ: streptozotocin; TAM: tamoxifen; tfGAL3: tandem fluorescent LGALS3; TMEM192: transmembrane protein 192.

Macroautophagy/autophagy has long been viewed as being strictly dependent on vacuolar or lysosomal acidity, with the vacuolar-type H⁺-translocating ATPase (V-ATPase) functioning mainly as a proton pump that sustains degradation. Our recent paper overturns this paradigm, revealing that loss of V-ATPase activity paradoxically induces a selective autophagy program in nutrient-replete Saccharomyces cerevisiae. Vacuolar deacidification triggers a signaling cascade through the Gcn2-Gcn4/ATF4 integrated stress response, which drives Atg11-dependent ribophagy even when TORC1 remains active. This "V-ATPase-dependent autophagy" operates as a self-corrective feedback loop: when the vacuole's degradative capacity falters, it signals its own dysfunction to restore homeostasis. Tryptophan and NAD⁺ metabolism modulate this response, linking metabolic balance to autophagy induction. This discovery reframes the vacuole/lysosome from a passive endpoint to an active sensor of cellular integrity. It also challenges the use of V-ATPase inhibitors such as bafilomycin A₁ as neutral autophagy flux blockers, because inhibition itself can stimulate autophagy induction. Collectively, these findings position the V-ATPase as a bidirectional regulator - both gatekeeper and sentinel - governing how cells translate organelle stress into adaptive autophagy.Abbreviation: ATG: autophagy related; FL: follicular lymphoma; TORC1: TOR complex 1; V-ATPase: vacuolar-type H⁺-translocating ATPase.

Intrahepatic triglyceride breakdown and recycling occur through lipolysis and lipid droplet (LD) macroautophagy/autophagy to regulate systemic fat partitioning. We recently demonstrated that MC3R is important for hepatic autophagy and peripheral metabolism, beyond its established functions in the CNS, where it affects energy homeostasis, feeding regulation, and puberty. MC3R agonists activate hepatocyte autophagy through LC3-II activation, TFEB cytoplasmic-to-nuclear translocation, and subsequent downstream autophagy gene activation. Global mc3r knockout mice develop obesity with increased hepatic triglyceride accumulation and blunted hepatocellular autophagosome-lysosome docking, leading to defective lipid droplet clearance. Hepatic Mc3r reactivation in global knockouts improves hepatocellular autophagy, lipid metabolism, mitochondrial respiration, energy expenditure, body fat, and body weight. These results reveal an autonomous role for hepatic MC3R in regulating lipid droplet autophagy, liver steatosis, and systemic adiposity.Abbreviation: AP:autophagosome; CNS:central nervous system; EIF4EBP1: eukaryotic translation initiationfactor 4E binding protein 1; EM: electron microscopy; LD: lipiddroplets; GFP: green fluorescent protein; MAFLD: metabolic-associatedfatty liver disease; MAP1LC3/LC3-II: microtubule-associated protein 1light chain 3-II; MC3R: melanocortin 3 receptor; MTORC1: mechanistictarget of rapamycin kinase complex 1; NDP-42:norleucine D-phenylalanine compound-42; TFEB: transcriptionfactor EB.