Autophagy最新文献

The liver orchestrates systemic metabolism, and its dysfunction drives diseases including metabolic dysfunction-associated steatotic liver disease (MASLD) and hepatocellular carcinoma (HCC). ATG9A, an autophagy-related transmembrane protein and lipid scramblase, regulates lipid dynamics, yet its role in hepatic pathogenesis remains unclear. Using multi-model approaches, we demonstrate that liver-specific ATG9A overexpression in mice enhanced autophagic flux but impaired autophagosome degradation. ATG9A disrupted hepatic lipid metabolism, reduced lipid droplet accumulation and exacerbated inflammation and fibrosis. Furthermore, we identified PLA2G6 as an ATG9A binding protein. ATG9A-PLA2G6 interaction accelerated phosphatidylcholine degradation, perturbing fatty acid metabolism and causing mitochondrial dysfunction. Besides, ATG9A promoted tumor growth in vivo, independent of canonical macroautophagy/autophagy. Our findings redefine ATG9A as a dual metabolic effector, driving liver disease progression through lipid remodeling and organelle stress. The ATG9A-PLA2G6 axis presents a therapeutic target for metabolic liver disorders and HCC.

Macroautophagy/autophagy was previously shown to play a critical role in the hippocampus for memory formation, with age-related autophagy deficits being further linked to cognitive decline. However, the neuronal subtypes where autophagy is required to form new memories remain unknown. Given the well-established role of PVALB (parvalbumin) interneurons in hippocampus-dependent memory formation and consolidation, we examined whether autophagy in these cells is required for such complex behaviors. We show that contrary to other neuronal subtypes, the vast majority of PVALB neurons, with the exception of cerebellar Purkinje cells, survive and are maintained long-term independently of autophagy. However, autophagy controls the homeostasis of mitochondria, endoplasmic reticulum, and synaptic proteins within PVALB interneurons, ultimately regulating their synaptic excitation, neuronal excitability and excitation-inhibition balance in the hippocampus. Consequently, mice with conditional impairment of autophagy in PVALB-expressing neurons exhibit impaired inhibitory neurotransmission and deficits in hippocampus-dependent memory. Taken together, these findings identify PVALB interneurons as key cellular substrates of autophagy in the context of learning and memory.Abbreviation: ATG5: autophagy related 5; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CA1: cornu ammonis 1; CALCOCO1: calcium binding and coiled coil domain 1; ER: endoplasmic reticulum; GABA: gamma-aminobutyric acid; GRIA/AMPAR: glutamate receptor, ionotropic, AMPA; GRIN2A/NR2A/GluN2A: glutamate receptor, ionotropic, NMDA2A (epsilon 1); PRKN: parkin RBR E3 ubiquitin protein ligase; PC: pyramidal cells; PJ: Purkinje; PVALB: parvalbumin; RTN3: reticulon 3; SQSTM1/p62: sequestosome 1.

Skeletal muscle is a heterogeneous tissue consisting of fibers with distinct contractile speeds, metabolic profiles, and cellular signaling. This heterogeneity may extend to mitochondrial quality control processes such as mitophagy. Using mt-Keima mice, we found that mitophagic activity was greater in the fast-twitch, glycolytic extensor digitorum longus (EDL) compared to the slow-twitch, oxidative soleus (SOL) muscle. Live imaging of quadriceps (QUAD) muscle revealed two distinct fiber populations: those with high total mt-Keima signal but low mitophagic activity, and others with low signal but higher mitophagic activity. Additionally, we observed skeletal muscle type and regional differences in autophagic and mitophagic protein content. Further, select mitophagic proteins strongly correlated with mitochondrial proteins across different regions of the gastrocnemius, while others did not. These findings highlight the complexity of mitophagy regulation in skeletal muscle and emphasize the importance of considering muscle phenotype, including fiber type, region, and mitochondrial content when studying mitophagy.

Macroautophagy/autophagy enables macrophages to degrade intracellular Mycobacterium tuberculosis (Mtb), and this defense depends on E3 ubiquitin ligases such as PRKN/PARKIN/PARK2 and SMURF1, which tag Mtb-associated structures for lysosomal clearance. Deubiquitinases (DUBs) counter ubiquitin ligases by removing ubiquitin from molecular targets. We hypothesized that DUBs might offset ubiquitin ligase activity and negatively regulate host immunity to Mtb. Here, we identify USP15 (ubiquitin specific peptidase 15) as a negative regulator of MAP1LC3/LC3-dependent targeting pathways (consistent with xenophagy or CASM/LAP-related ATG8ylation) that mediate macrophage immunity to Mtb. Using a targeted knockdown screen in mouse macrophages, we found that Usp15 loss increased K63-linked ubiquitination and LC3 recruitment to Mtb-associated structures, leading to reduced bacterial replication. These effects required USP15's catalytic activity and were reversed by knockdown of PRKN or inhibition of autophagy initiation. In primary human macrophages, USP15 knockdown similarly enhanced LC3 targeting and restricted Mtb growth. Importantly, pharmacological inhibition of USP15 with a selective small molecule decreased Mtb burden in human macrophages. Our findings identify USP15 as a suppressor of macrophage immunity and suggest that targeting deubiquitinases may represent a promising host-directed therapeutic strategy against tuberculosis.Abbreviations: CFU: colony-forming unit; DUBs: deubiquitinases; K48-Ub: K48-linked ubiquitin; K63-Ub: K63-linked ubiquitin; Mtb-pLux: luminescent Mtb strain Mtb; Mycobacterium tuberculosis; MOI: multiplicity of infection; NTC: non-targeting control; TB: tuberculosis.

Macroautophagy (hereafter referred to as autophagy) requires the coordinated action of approximately 20 ATG (autophagy related) genes. Duplication of ATG genes has had a major impact on the evolution of the autophagy pathway among major lineages. One duplication hotspot is in vertebrates. However, the exact duplication timing, post-duplication evolutionary divergence patterns, and its relation to functional differences among paralogs have not been investigated in detail. Here, we demonstrate that most ATG genes were likely duplicated by whole-genome duplication events near the root of vertebrates. We compared the sequence and gene expression divergence between paralogs and categorized the evolutionary fates (i.e., how ancestral function is divided between paralogs). Within the paralog pairs that evolved most asymmetrically, namely BECN, WIPI (WIPI1 and WIPI2), and ATG16, one paralog likely retained the ancestral function, allowing the other to evolve under less constraint. While no obvious asymmetry was observed between ATG9A and ATG9B in non-mammalian vertebrates, ATG9B experienced marked sequence divergence and expression level reduction in mammals, suggesting a shift in balance. Expression patterns among the ULK-1 (ULK1 and ULK2), GABARAP (GABARAP and GABARAPL1), and LC3 (LC3A and LC3B) pairs were more consistent with hypofunctionalization/dosage sharing, such that ancestral function depends on both paralogs. We also demonstrate that both ULK1 and ULK2 can support autophagy, whereas only BECN1, but not BECN2, has autophagic function and discuss the relationship between autophagic function and evolutionary divergence. The present detailed analysis of ATG gene duplication in vertebrates provides a critical timeline for interpreting functional differentiation between homologs.Abbreviations: ATG: autophagy related; BLAST: Basic Local Alignment Search Tool; DKO: double knockout; GFP: green fluorescent protein; GLMM: generalized linear mixed model; KO: knockout; LC3: MAP1LC3; MEF: mouse embryonic fibroblast; ns: non-significant; PAML: Phylogenetic Analysis by Maximum Likelihood; RPKM: reads per kilobase per million mapped reads; SVA: surrogate variable analysis; TMM: trimmed mean of M values; TMR: tetramethylrhodamine; WT: wild type.

Micronuclei are formed during cell division when acentric fragments or lagging chromosomes cannot be incorporated into the primary nucleus. Macroautophagy/autophagy may reduce chromosomal instability (CIN) by clearing isolated, atypical micronuclei. Other studies implicate that the loss of autophagy disrupts DNA repair pathways. However, whether aberrant mitosis contributing to CIN occurs when autophagy is inhibited has yet to be evaluated. We found impaired autophagy initiation contributes to CIN and facilitates the formation of micronuclei and other abnormal nuclear phenotypes either by genetic or pharmacological manipulation in multiple cell lines. We also found that loss of the integral autophagy protein ATG9A resulted in various types of mitotic errors that can contribute to the formation of micronuclei. ATG9A also localizes to centrosomes and midbody during cell division. Autophagy inhibition causes the overactivation and mislocalization of TBK1 (TANK binding kinase 1) into cytoplasmic, punctate structures that colocalize with SQSTM1/p62. This overactivation interferes with its function in cell division as a mitotic kinase and its role at the centrosome. These results indicate that loss of autophagy contributes to genomic instability from multiple angles, one of which being aberrant cell division.

Macroautophagy/autophagy plays a crucial role in maintaining cellular homeostasis and protecting against osteoarthritis (OA). Its dysregulation contributes to OA progression by promoting chondrocyte senescence, inflammation, and cartilage degradation. Enhancing autophagic activity thus represents a promising therapeutic strategy for OA. In this study, we identified lactucopicrin (LCP) as an effective autophagy activator that alleviates OA progression in a mouse model induced by the destabilization of the medial meniscus, by reducing cartilage degeneration and preserving matrix integrity. Mechanistically, LCP enhances ZDHHC4-catalyzed palmitoylation of the cargo receptor CCDC50, facilitating the selective autophagic degradation of MAP2K4/MKK4, leading to the suppression of MAPK/JNK signaling and the attenuation of chondrocyte senescence. Structural analysis reveals that LCP directly binds to His72 of ZDHHC4 via its p-hydroxybenzoic acid moiety, boosting enzymatic activity and promoting selective autophagy. These findings establish a novel ZDHHC4-CCDC50-MAP2K4/MKK4-MAPK/JNK regulatory axis linking palmitoylation, autophagy, and senescence, and identify LCP as a promising agent for targeting this pathway to inhibit OA progression. Furthermore, this study provides mechanistic insights into the crosstalk between autophagy, protein palmitoylation, and cellular senescence in degenerative joint disease.Abbreviation: ABE: acyl-biotin exchange; ADAMTS5: ADAM metallopeptidase with thrombospondin type 1 motif 5; CCDC50: coiled-coil domain containing 50; COL2A1: collagen, type II, alpha 1; COL10A1: collagen, type X, alpha 1; DARTS: drug affinity responsive target stability; DHHC: Asp-His-His-Cys catalytic motif; GOT1/AST: glutamic-oxaloacetic transaminase 1, soluble; GPT/ALT: glutamic pyruvic transaminase, soluble; H₂O_2: hydrogen peroxide; LCP: lactucopicrin; IL6: interleukin 6; MAPK/JNK: mitogen-activated protein kinase; MAP2K4/MKK4: mitogen-activated protein kinase kinase 4; MMP13: matrix metallopeptidase 13; OA: osteoarthritis; p-MAPK/JNK: phosphorylated mitogen-activated protein kinase; SASP: senescence-associated secretory phenotype; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; ZDHHC: zinc finger, DHHC domain containing.

Apoptotic bodies (ABs) are a type of extracellular vesicles (EVs) that could contribute to the paracrine effect of stem cells. However, their potential in treating cardiovascular diseases is largely unexplored. This study investigated the therapeutic effects of ABs derived from human umbilical cord mesenchymal stem cells (MSCs) on cardiac recovery in a porcine model of myocardial infarction (MI). In vitro, ABs reduced apoptosis and cytotoxicity in cardiomyocytes under oxygen and glucose deprivation (OGD) conditions and enhanced the capacity of migration and tube formation in endothelial cells. In vivo, akin to MSCs, administration of ABs improved contractile function, reduced infarct size, and mitigated adverse remodeling in pig hearts with MI, concomitantly with increased cardiomyocyte survival and angiogenesis. These cardioprotective effects were mediated through the regulation of autophagy by activating the adenosine monophosphate - activated protein kinase (AMPK) and transcription factor EB (TFEB) signaling pathways. microRNAs contained in ABs were sequenced, revealing that let-7f-5p was the most abundant. let-7f-5p promoted AMPK phosphorylation by targeting protein phosphatase 2 regulatory subunit B alpha (PPP2R2A) and decreased TFEB phosphorylation by targeting MAP4K3 to regulate autophagy, thereby contributing to the effects of ABs. Overall, these findings indicate that MSC-derived ABs have the potential to be a promising and effective acellular therapeutic option for treating MI.

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.Abbreviations: Aβ: β-amyloid; ACM: apoptotic conditioned medium; AD: Alzheimer disease; AIF1/IBA1: allograft inflammatory factor 1; CNS: central nervous system; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; ELISA: enzyme linked immunosorbent assay; GFAP: glial fibrillary acidic protein; GSDMD: gasdermin D; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PANX1: pannexin 1; PBS: phosphate-buffered saline; SQSTM1/p62: sequestosome 1; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; RT-PCR: reverse transcription quantitative real-time polymerase chain reaction; SMOX: spermidine oxidase; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; UV: ultraviolet; WT: wild-type.