Acute central nervous system injuries, including ischemic stroke, intracerebral hemorrhage, subarachnoid hemorrhage, traumatic brain injury, and spinal cord injury, are a major global health challenge. Identifying optimal therapies and improving the long-term neurological functions of patients with acute central nervous system injuries are urgent priorities. Mitochondria are susceptible to damage after acute central nervous system injury, and this leads to the release of toxic levels of reactive oxygen species, which induce cell death. Mitophagy, a selective form of autophagy, is crucial in eliminating redundant or damaged mitochondria during these events. Recent evidence has highlighted the significant role of mitophagy in acute central nervous system injuries. In this review, we provide a comprehensive overview of the process, classification, and related mechanisms of mitophagy. We also highlight the recent developments in research into the role of mitophagy in various acute central nervous system injuries and drug therapies that regulate mitophagy. In the final section of this review, we emphasize the potential for treating these disorders by focusing on mitophagy and suggest future research paths in this area.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with limited therapeutic options. Previously, we have shown that a combination of multiple probiotic strains can regulate intestinal flora, increase serum short-chain fatty acids (SCFAs), reduce abnormal protein accumulation in the spinal cord, and protect neurons. It is necessary to explore the mechanism to provide therapeutic targets for ALS.

This study utilizes live cell imaging, mouse behavioral research, immunofluorescence, Electron microscopy, Western Blot, and polymerase chain reaction to explore the impact of various SCFAs on ALS animal and cell models, as well as their underlying mechanisms.

We found SCFAs, including butyrate and propionate can increase the levels of acetylated histones, enhance the expression of autophagy-related genes and regulate autophagy, leading to a decrease in abnormal SOD1 aggregation, reduction of cell damage, and enhancement of cell proliferation in NSC34-SOD1G93A cells. Furthermore, systemic administration of butyrate and propionate can regulate autophagy, reduce SOD1 aggregation, and protect spinal cord neurons in SOD1G93A mice. However, these favorable effects of butyrate and propionate are greatly decreased at later stages of the disease process in SOD1G93A mice.

Our study revealed that the positive impact of SCFAs in autophagy could be a promising focus for ALS therapy. However, this effect might have different impacts in different stages of ALS.

Endolysosomes, considered the cellular recycling compartments, receive and degrade materials from multiple pathways. However, whether endolysosomes can acquire cargo through alternative mechanisms remains unclear. Here, we identify a previously unrecognized endolysosomal pathway for material uptake. In this process, endolysosomes extend two membrane protrusions that envelop and ultimately engulf autophagosomes, independently of autophagosome-endolysosome fusion and the endosomal sorting complex required for transport complex (ESCRT)-mediated microautophagy. The endolysosomes containing internalized autophagosomes, acquire additional autophagosomes through homotypic fusion. A subset of autophagosomes is marked by F-actin on their membranes and the majority of them contain the ER protein Sec61β and the peroxisomal protein Pex16 within their lumens, whereas mitochondria remain excluded. Our discovery of this endolysosomal process unveils a previously uncharacterized pathway for cargo acquisition by endolysosomes.

The transmembrane autophagy protein ATG9 has multiple functions essential for autophagosome formation. Here, we uncovered a novel function of ATG-9 in regulating lysosome biogenesis and integrity in Caenorhabditis elegans. Through a genetic screen, we identified that mutations attenuating the lipid scrambling activity of ATG-9 suppress the autophagy defect in epg-5 mutants, in which non-degradative autolysosomes accumulate. The scramblase-attenuated ATG-9 mutants promote lysosome biogenesis and delivery of lysosome-localized hydrolases and also facilitate the maintenance of lysosome integrity. Through manipulation of phospholipid levels, we found that a reduction in phosphatidylethanolamine (PE) also suppresses the autophagy defects and lysosome damage associated with impaired lysosomal degradation. Our results reveal that modulation of phospholipid composition and distribution, e.g., by attenuating the scramblase activity of ATG-9 or reducing the PE level, regulates lysosome function and integrity.

The multi-step macroautophagy/autophagy process ends with the cargo-laden autophagosome fusing with the lysosome to deliver the materials to be degraded. The metazoan-specific autophagy factor EPG5 plays a crucial role in this step by enforcing fusion specificity and preventing mistargeting. How EPG5 exerts its critical function and how its deficiency leads to diverse phenotypes of the rare multi-system disorder Vici syndrome are not fully understood. Here, we report the first structure of human EPG5 (HsEPG5) determined by cryo-EM and AlphaFold2 modeling. Our structure revealed that HsEPG5 is constructed from helical bundles analogous to tethering factors in membrane trafficking pathways but contains a unique protruding thumb domain positioned adjacent to the atypical tandem LIR motifs involved in interaction with the GABARAP subfamily of Atg8-family proteins. Our NMR spectroscopic, molecular dynamics simulations and AlphaFold modeling studies showed that the HsEPG5 tandem LIR motifs only bind the canonical LIR docking site (LDS) on GABARAP without engaging in multivalent interaction. Our co-immunoprecipitation analysis further indicated that full-length HsEPG5-GABARAP interaction is mediated primarily by LIR1. Finally, our biochemical affinity isolation, X-ray crystallographic analysis, affinity measurement, and AlphaFold modeling demonstrated that this mode of binding is observed between Caenorhabditis elegans EPG-5 and its Atg8-family proteins LGG-1 and LGG-2. Collectively our work generated novel insights into the structural properties of EPG5 and how it potentially engages with the autophagosome to confer fusion specificity.ABBREVIATIONS: ATG: autophagy related; CSP: chemical shift perturbation; eGFP: enhanced green fluoresent protein; EM: electron microscopy; EPG5: ectopic P-granules 5 autophagy tethering factor; GST: glutathione S-transferase; HP: hydrophobic pocket; HSQC: heteronuclear single-quantum correlation; ITC: isothermal titration calorimetry; LDS: LC3 docking site; LIR: LC3-interacting region; MD: molecular dynamics; NMR: nuclear magnetic resonance; TEV: tobacco etch virus.

Host-directed therapies (HDTs) have been investigated as a potential solution to combat intracellular and drug-resistant bacteria. HDTs stem from extensive research on the intricate interactions between the host and intracellular bacteria, leading to a treatment approach that relies on immunoregulation. To improve the bioavailability and safety of HDTs, researchers have utilized diverse drug delivery systems (DDS) to encapsulate and transport therapeutic agents to target cells. In this review, we first introduce the three mechanisms of bactericidal action and intracellular bacterial evasion: autophagy, reactive oxygen species (ROS), and inflammatory cytokines, with a particular focus on autophagy. Special attention is given to the detailed mechanism of xenophagy in clearing intracellular bacteria, a crucial selective autophagy process that specifically targets and degrades intracellular pathogens. Following this, we present the application of DDS to modulate these regulatory methods for intracellular bacteria elimination. By integrating insights from immunology and nanomedicine, this review highlights the emerging role of DDS in advancing HDTs for intracellular bacterial infections and paving the way for innovative therapeutic interventions.

Multiple environmental factors contribute to digestive system damage caused by food contamination in both humans and animals. Mycotoxins, such as deoxynivalenol (DON) and T-2 toxin, have emerged as the most significant factors due to their extensive contamination and difficulty in removal. Transcription factor EB (TFEB) serves as a crucial transcriptional regulator governing lysosomal biogenesis and autophagy, a lysosomal-driven degradation system that safeguards cells against harmful stressors. However, little is known about whether the post-translational modification of TFEB affects autophagy activity, which could explain the toxicity disparity between DON and T-2 toxin. Here, we discovered that T-2 toxin induces excessive autophagy by significantly reducing TFEB acetylation, whereas DON surprisingly inhibits autophagy activity via maintaining high TFEB acetylation, which impairs lysosomal biogenesis, thereby boosting their respective toxicity. Mechanically, the T-2 toxin decreases TFEB acetylation via enhanced SIRT1-TFEB interaction and SIRT1 deacetylase activity, while DON maintains high TFEB acetylation by reversing the process. Together, our study revealed that the acetylation state of TFEB mediated by SIRT1 alters autophagy phenotypes in intestinal cells, shedding light on the various toxicological mechanisms and an important target of DON and T-2 toxin.

Autophagy-tethering compounds (ATTECs) as a new targeted protein degradation technology could directly bind targets to LC3 (a key autophagosome-associated protein) and subsequently result in the degradation of targets via the autophagolysosomal pathway. Herein, we developed a new LC3 ligand screening strategy using an NIR fluorescent probe with both pH-sensitive and LC3-targeted features. The presence of both the probe and a potential LC3 ligand leads to competitive binding to LC3 in cells and hinders the probe from entering and being activated in acidic lysosomes via the autophagy pathway. Notably, LD5, an in-house compound of our lab, was screened out as a potential LC3 ligand by the strategy, and its capacity of binding to LC3 was further verified by SPR technology. By using LD5 as the LC3 binding moiety, two ATTECs were synthesized, which exhibited significant activities in degrading PCSK9 and lipid droplets, respectively, and further validated the feasibility of our LC3 ligand screening strategy.

Dehydroepiandrosterone (DHEA), a precursor of sex hormones, has been implicated in the pathogenesis of non-alcoholic steatohepatitis (NASH) or metabolic dysfunction-associated steatohepatitis (MASH), with studies suggesting a strong correlation between DHEA levels and disease severity. In this study, we demonstrated that DHEA alleviated lipotoxicity-induced hepatic damage by promoting autophagy. Our findings demonstrate that DHEA-induced autophagy is mediated by estrogen receptor alpha (ER-α) and androgen receptor (AR) activation and protects hepatic cells against palmitate-induced apoptosis, steatosis, and inflammasome activation. DHEA treatment in a murine NASH model induced significant autophagy in the liver, further supporting the hepatoprotective role of DHEA. Collectively, our results identified DHEA as a pro-autophagic hormone with therapeutic potential for the treatment of lipotoxicity in NASH.

Increasing evidence has shown that programmed cell death (PCD) plays a crucial role in tumorigenesis and cancer progression. The components of PCD are complex and include various mechanisms such as apoptosis, necroptosis, alkaliptosis, oxeiptosis, and anoikis, all of which are interrelated in their functions and regulatory pathways. Given the significance of these processes, it is essential to conduct a comprehensive study on PCD to elucidate its multifaceted nature. Key signaling pathways, particularly the caspase signaling pathway, the RIPK1/RIPK3/MLKL pathway, and the mTOR signaling pathway, are pivotal in regulating PCD and influencing tumor progression. In this review, we briefly describe the generation mechanisms of different PCD components and focus on the regulatory mechanisms of these three major signaling pathways within the context of global PCD. Furthermore, we discuss various tumor therapeutic compounds that target different signaling axes of these pathways, which may provide novel strategies for effective tumor therapy and help improve patient outcomes in cancer treatment.

Autophagy is a "self-eating" biological process that degrades cytoplasmic contents to ensure cellular homeostasis. Its response to stimuli occurs in two stages: Within a few to several hours of exposure to a stress condition, autophagic flow rapidly increases, which is mediated by post-translational modification (PTM). Subsequently, the transcriptional program is activated and mediates the persistent autophagic response. O-linked β-N-acetylglucosamine (O-GlcNAc) modification is an inducible and dynamically cycling PTM; mounting evidence suggests that O-GlcNAc modification participates in the total autophagic process, including autophagy initiation, autophagosome formation, autophagosome-lysosome fusion, and transcriptional process. In this review, we summarize the current knowledge on the emerging role of O-GlcNAc modification in regulating autophagy-associated proteins and explain the different regulatory effects on autophagy exerted by O-GlcNAc modification.

Triple-negative breast cancer (TNBC) is a subtype with the worst prognosis and there is still a lack of effective treatment. Exosomes (Exos) secreted by cancer cells to tumor microenvironment play an important role in cancer progression. We have demonstrated that the function of Rasal2 in the modulation of breast cancer progression is exos-mediated, but the relationship between Rasal2 and exosome secretion remains elusive.

Rasal2 knock-out (KO) MDA-MB-231 cells were conducted by crispr-cas9 technique and Rab27a knock-down (KD) in Rasal2 KO MDA-MB-231 cells (KO + KD) were further established by siRNA-mate plus transfection Reagent. Control (CT)/KO MDA-MB-231 cells stably overexpressing GFP-LC3 were generated by using GFP-LC3 plamisd. Transmission electron microscope (TEM), nanoparticle tracking analysis (NTA) and western blot analysis (WB) were used to identify exos derived from TNBC. Confocal microscopy was used to observe the autophagic flux and the colocalization of autophagosomes and multivesicular bodies (MVBs). Co-immunoprecipitation analysis was performed to determine the interaction between Rasal2 and Rab27a. Immunohistochemical analysis were used to detect the expression levels of autophagy-related proteins in tumor tissues of xenograft mice inoculated with CT/KO/KO + KD MDA-MB-231 cells.

In this paper, we found that Rasal2 KO disrupts autophagic flux and induces secretory autophagy to promote autophagic-exos secretion in TNBC. Moreover, Rasal2 inhibits the activity of Rab27a which regulates vesicles transport and fusion, and Rab27a mediates Rasal2 KO-induced autophagic-exos secretion. Additionally, Rab27a KD inhibits Rasal2 KO-induced secretory autophagy, thereby promoting TNBC progression both in vivo and in vitro.

Collectively, these findings delineated the role of Rab27a in TNBC progression modulated by Rasal2 through autophagy-exos pathway and suggested that it is of great significance for the early diagnosis, targeted therapy and prognosis judgement of TNBC from the perspective of tumor microenvironment.

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract, primarily driven by KIT or PDGFRA mutations. Programmed cell death (PCD), including apoptosis, autophagy, and ferroptosis, plays a crucial role in GIST pathogenesis, progression, and treatment response. Non-coding RNAs (ncRNAs) have emerged as key regulators of PCD pathways, influencing GIST proliferation, metastasis, and drug resistance, particularly in response to tyrosine kinase inhibitors (TKIs) such as imatinib. Apoptosis suppression is strongly associated with poor prognosis, while autophagy contributes to tumor dormancy and TKI resistance. Ferroptosis, a novel iron-dependent cell death pathway, represents a promising therapeutic target. Recent evidence suggests that ncRNAs modulate these PCD pathways through interactions with key molecular regulators such as miR-494, miR-30a, and lncRNAs, which affect signaling networks including PI3K/AKT, MAPK, and mTOR. Furthermore, ncRNAs have mediated secondary resistance to imatinib by promoting autophagic flux and altering ferroptosis sensitivity. Understanding the molecular interplay between ncRNAs and PCD in GIST provides novel insights into disease mechanisms and offers potential therapeutic strategies to overcome drug resistance. Targeting ncRNA-mediated regulation of apoptosis, autophagy, and ferroptosis may enhance treatment efficacy and improve patient outcomes. Future research should focus on elucidating the mechanistic roles of ncRNAs in PCD pathways to develop innovative diagnostic and therapeutic approaches for GIST.

Necrosis at the ischemic distal end of flap transplants increases patients' pain and economic burden. Reactive oxygen species (ROS) and mitochondrial damage are crucial in regulating parthanatos, but the mechanisms linking disrupted macroautophagic/autophagic flux to parthanatos in ischemic flaps remain unclear. The results of western blotting, immunofluorescence staining, and a proteomic analysis revealed that the autophagic protein SNAP29 was deficient in ischemic flaps, resulting in disrupted autophagic flux, increased ROS-induced parthanatos, and aggravated ischemic flap necrosis. The use of AAV vector to restore SNAP29 in vivo mitigated the disruption of autophagic flux and parthanatos. Additionally, quantification of the total m6A level and RIP-qPCR, MeRIP-qPCR, and RNA stability assessments were performed to determine differential Snap29 mRNA m6A methylation levels and mRNA stability in ischemic flaps. Various in vitro and in vivo tests were conducted to verify the ability of METTL3-mediated m6A methylation to promote SNAP29 depletion and disrupt autophagic flux. Finally, we concluded that restoring SNAP29 by inhibiting METTL3 and YTHDF2 reversed the "autophagy-mitochondrial crisis", defined for the first time as disrupted autophagic flux, mitochondrial damage, mitochondrial protein leakage, and the occurrence of parthanatos. The reversal of this crisis ultimately promoted the survival of ischemic flaps.Abbreviations: AAV = adeno-associated virus; ACTA2/α-SMA = actin alpha 2, smooth muscle, aorta; AIFM/AIF = apoptosis-inducing factor, mitochondrion-associated; ALKBH5 = alkB homolog, RNA demythelase; Baf A1 = bafilomycin A1; CQ = chloroquine; DHE = dihydroethidium; ECs = endothelial cells; F-CHP = 5-FAM-conjugated collagen-hybridizing peptide; GO = gene ontology; HUVECs = human umbilical vein endothelial cells; KEGG = Kyoto Encyclopedia of Genes and Genomes; LC-MS/MS = liquid chromatography-tandem mass spectrometry; LDBF = laser doppler blood flow; m6A = N6-methyladenosine; MAP1LC3/LC3 = microtubule-associated protein 1 light chain 3; MeRIP = methylated RNA immunoprecipitation; METTL3 = methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit; NAC = N-acetylcysteine; OGD = oxygen glucose deprivation; PAR = poly (ADP-ribose); PARP1 = poly (ADP-ribose) polymerase family, member 1; PECAM1/CD31 = platelet/endothelial cell adhesion molecule 1; ROS = reactive oxygen species; RT-qPCR = reverse transcription quantitative polymerase chain reaction; RIP = RNA immunoprecipitation; SNAP29 = synaptosomal-associated protein 29; SNARE = soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1 = sequestosome 1; SRAMP = sequence-based RNA adenosine methylation site predicting; STX17 = syntaxin 17; TMT = tandem mass tag; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling; VAMP8 = vesicle-associated membrane protein 8; WTAP = WT1 associating protein; YTHDF2 = YTH N6-methyladenosine RNA binding protein 2; 3' UTR = 3'-untranslated region.

Bacillus thuringiensis produces insecticidal crystal (Cry) proteins that target and destroy insect pest midgut epithelial cells, ultimately leading to larval death. However, exposure to sublethal concentrations of Cry proteins can activate defense responses to counteract toxicity. Here, we revealed a moderate autophagy response to a sublethal dose of Cry9Aa in the larval midgut of Chilo suppressalis through autophagosome detection by electron microscopy, Western blot analysis of the Atg8-PE/Atg8 ratio, and visualization of Atg8-PE puncta. Additionally, differential gene expression analysis showed significant upregulation of the autophagy receptor genes sequestosome 1 (sqstm1) and atg12, supporting a potential role for autophagy in the response to Cry9Aa intoxication. Knocking down sqstm1 increased larval susceptibility to Cry9Aa by 30%, highlighting its role in defense, whereas atg12 knockdown increased susceptibility by only 8%. Our findings suggest that sqstm1 contributes to C. suppressalis defense against Cry9Aa intoxication through a mechanism response that may not be strictly dependent on canonical autophagy.

Due to their biocompatibility, biodegradability, and suitable mechanical properties, magnesium-based biodegradable implants are emerging as a promising alternative to traditional metal implants. The Mg-4Y-3RE (WE43) biodegradable alloy is among the most extensively studied and widely utilized magnesium alloys in clinical applications. As an absorbable and degradable metallic material, magnesium alloys undergo gradual degradation, wear, and fracture within the body. These alloys reduce the long-term risks associated with permanent implants but generate insoluble byproducts that accumulate in surrounding tissues. Following the implantation of magnesium alloys, granulation tissue and fibrous encapsulation typically form around the material. However, limited research has addressed the interaction between insoluble byproducts of magnesium alloys and macrophages. This study focused on the biological effects of macrophages during the second stage of the host inflammatory response in the degradation process of magnesium alloy. Using subcutaneous implantation of WE43 magnesium alloy sheets, observations were made regarding the degradation components, morphological changes in surrounding tissues, and the biological effects of macrophages upon phagocytosis of insoluble byproducts. The primary degradation products of WE43 in vivo were identified as Ca3 (PO4)2, Mg3(PO4)2, Na3PO4, NaCa (PO4), MgSO4, MgCO3, NaCl, Mg24Y5, and Mg12YNd. Postimplantation, levels of IL-1β and IL-18 in adjacent tissues significantly increased (p < 0.05). By 8 weeks, compared to nitinol alloy, significant thickening of the fibrous capsule (p < 0.05) was observed, accompanied by substantial inflammatory cell infiltration, vascularization, and the presence of macrophages and multinucleated giant cells. Macrophages were observed extending pseudopodia to enclose and phagocytose particles, forming phagosomes and creating a relatively isolated microenvironment around the engulfed substances, where further particle degradation occurred. Following the phagocytosis of degradation products, macrophages exhibited increased lysosome numbers, mitochondrial swelling and damage, phagolysosome formation, and autophagosome development. Furthermore, the degradation products were observed to induce elevated reactive oxygen species (ROS) production in macrophages, activation of P2X7 receptors, enhanced IL-6 secretion, endoplasmic reticulum stress, autophagy, and activation of the NLRP3 inflammasome pathway. This study provides novel insights and contributes a theoretical foundation for a more comprehensive understanding of magnesium alloy degradation in vivo.

Selective autophagy targets specific cellular cargo for degradation. In this issue, Zhao et al. (https://doi.org/10.1083/jcb.202410150) uncovered that Rab GTPases serve as pivotal "autophagy cues" for recruitment of cargo receptors to facilitate mitophagy, lipophagy, and xenophagy, contributing to the precise spatiotemporal regulation of selective autophagy.

Mitochondria-derived vesicles (MDVs) participate in early cellular defence mechanisms initiated in response to mitochondrial damage. They maintain mitochondrial quality control (MQC) by clearing damaged mitochondrial components, thereby ensuring the normal functioning of cellular processes. This process is crucial for cell survival and health, as mitochondria are the energy factories of cells, and their damage can cause cellular dysfunction and even death. Recent studies have shown that MDVs not only maintain mitochondrial health but also have a significant impact on tumour progression. MDVs selectively encapsulate and transport damaged mitochondrial proteins under oxidative stress and reduce the adverse effects of mitochondrial damage on cells, which may promote the survival and proliferation of tumour cells. Furthermore, it has been indicated that after cells experience mild stress, the number of MDVs significantly increases within 2-6 h, whereas mitophagy, a process of clearing damaged mitochondria, occurs 12-24 h later. This suggests that MDVs play a critical role in the early stress response of cells. Moreover, MDVs also have a significant role in intercellular communication, specifically in the tumour microenvironment. They can carry and transmit various bioactive molecules, such as proteins, nucleic acids, and lipids, which regulate tumour cell's growth, invasion, and metastasis. This intercellular communication may facilitate tumour spread and metastasis, making MDVs a potential therapeutic target. Advances in MDV research have identified novel biomarkers, clarified regulatory mechanisms, and provided evidence for clinical use. These breakthroughs pave the way for novel MDV-targeted therapies, offering improved treatment alternatives for cancer patients. Further research can identify MDVs' role in tumour development and elucidate future cancer treatment horizons.

Neurodegenerative diseases (NDs), such as Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis, and frontotemporal dementia, are well known to pose formidable challenges for their treatment due to their intricate pathogenesis and substantial variability among patients, including differences in environmental exposures and genetic predispositions. One of the defining characteristics of NDs is widely reported to be the buildup of misfolded proteins. For example, Alzheimer disease is marked by amyloid beta and hyperphosphorylated Tau aggregates, whereas Parkinson disease exhibits α-synuclein aggregates. Amyotrophic lateral sclerosis and frontotemporal dementia exhibit TAR DNA-binding protein 43, superoxide dismutase 1, and fused-in sarcoma protein aggregates, and Huntington disease involves mutant huntingtin and polyglutamine aggregates. These misfolded proteins are the key biomarkers of NDs and also serve as potential therapeutic targets, as they can be addressed through autophagy, a process that removes excess cellular inclusions to maintain homeostasis. Various forms of autophagy, including macroautophagy, chaperone-mediated autophagy, and microautophagy, hold a promise in eliminating toxic proteins implicated in NDs. In this review, we focus on elucidating the regulatory connections between autophagy and toxic proteins in NDs, summarizing the cause of the aggregates, exploring their impact on autophagy mechanisms, and discussing how autophagy can regulate toxic protein aggregation. Moreover, we underscore the activation of autophagy as a potential therapeutic strategy across different NDs and small molecules capable of activating autophagy pathways, such as rapamycin targeting the mTOR pathway to clear α-synuclein and Sertraline targeting the AMPK/mTOR/RPS6KB1 pathway to clear Tau, to further illustrate their potential in NDs' therapeutic intervention. Together, these findings would provide new insights into current research trends and propose small-molecule drugs targeting autophagy as promising potential strategies for the future ND therapies. SIGNIFICANCE STATEMENT: This review provides an in-depth overview of the potential of activating autophagy to eliminate toxic protein aggregates in the treatment of neurodegenerative diseases. It also elucidates the fascinating interrelationships between toxic proteins and the process of autophagy of "chasing and escaping" phenomenon. Moreover, the review further discusses the progress utilizing small molecules to activate autophagy to improve the efficacy of therapies for neurodegenerative diseases by removing toxic protein aggregates.

Xenophagy plays a crucial role in restraining the growth of intracellular bacteria in macrophages. However, the machinery governing autophagosome‒lysosome fusion during bacterial infection remains incompletely understood. Here, we utilize leprosy, an ideal model for exploring the interactions between host defense mechanisms and bacterial infection. We highlight the glycoprotein nonmetastatic melanoma protein B (GPNMB), which is highly expressed in macrophages from lepromatous leprosy (L-Lep) patients and interferes with xenophagy during bacterial infection. Upon infection, GPNMB interacts with autophagosomal-localized STX17, leading to a reduced N-glycosylation level at N296 of GPNMB. This modification promotes the degradation of SNAP29, thus preventing the assembly of the STX17-SNAP29-VAMP8 SNARE complex. Consequently, the fusion of autophagosomes with lysosomes is disrupted, resulting in inhibited cellular autophagic flux. In addition to Mycobacterium leprae, GPNMB deficiency impairs the proliferation of various intracellular bacteria in human macrophages, suggesting a universal role of GPNMB in intracellular bacterial infection. Furthermore, compared with their counterparts, Gpnmbfl/fl Lyz2-Cre mice presented decreased Mycobacterium marinum amplification. Overall, our study reveals a previously unrecognized role of GPNMB in host antibacterial defense and provides insights into its regulatory mechanism in SNARE complex assembly.

The heavy metal cadmium (Cd) is an important environmental factor that induces liver injury and contributes to liver disease. Ongoing research aims to refine our understanding of the pathogenesis of cadmium-induced liver injury and the interactions between the various mechanisms. Oxidative stress, described as a pathophysiological basis of liver injury, is a process in which reactive oxygen species are generated, causing the destruction of hepatocyte structure and cellular dysfunction. Additionally, the activation of oxidative stress downstream signals regulates several forms of cell death, such as apoptosis, necroptosis, autophagy, ferroptosis, and pyroptosis, which significantly contributes to liver damage. Furthermore, the interplay between different types of programmed cell death highlights the complexity of liver injury mechanisms. This review summarizes the role of programmed cell death in Cd-induced liver injury and explores the relationships between different programmed cell death pathways, which is expected to provide new insights into the mechanisms of Cd-induced liver injury.

Autophagy, an evolutionarily conserved process, plays an important role in cellular homeostasis and human diseases. Cardiovascular dysfunction, which presents during cancer treatment or in cancer-free individuals years after treatment, is a growing clinical challenge. Millions of cancer survivors and patients face an unpredictable risk of developing cardiotoxicity. Cardiotoxicity due to cancer treatment, as well as cancer progression, has been linked to autophagy dysregulation. Modulating autophagy has been further proposed as a therapeutic treatment for both cancer and cardiovascular disorders. The safe and effective use of autophagy modulation as a cardioprotective strategy during cancer treatment especially requires careful consideration and experimentation to minimize the impact on cancer treatment. We focus here on recent advances in targeted autophagy modulation strategies that utilize interdisciplinary approaches in biomedical sciences and are potentially translatable to treat cardiotoxicity and improve cancer treatment outcomes. This review highlights non-small molecule autophagy modulators to enhance targeted therapy, nanomedicine for autophagy modulation and monitoring, and in vitro models and future experiments needed to bring novel autophagy discoveries from basic research to clinical translation.

Subcellular inter-organellar crosstalk among lysosome, endoplasmic reticulum (ER), and mitochondrion is crucial for cancer cell survival and is a promising target in cancer treatment; however, efficiently disrupting these interactive networks is challenging. Herein, a communication interception strategy is presented, which specifically disrupts inter-organellar crosstalk by lysosomal contents leakage along with their trajectory and pre-activates autophagic flux to augment the lysosome-associated autophagy blocking for preventing the self-repair of this subcellular disorder. Briefly, fullerenols containing multiple hydroxyl groups (MF) tear the lysosomal phospholipid membrane through direct interaction, which causes lysosomal contents (calcium ions and cathepsins) to leak into the cytoplasm, subsequently leading to endoplasmic reticulum stress and mitochondrial dysfunction with redox imbalance and metabolic reprogramming. mTOR inhibitors activate and amplify autophagy, then impaired lysosomes prevent their fusion with autophagosome, and thus autophagy is paralyzed along with autolysosome accumulation. Consequently, the cellular homeostasis is compromised by destroyed inter-organellar networks without self-repair by autophagy, thereby triggering PANoptotic processes and leading to a remarkable anti-tumor therapeutic efficacy in vitro and in vivo. This strategy demonstrates the selective cytotoxicity of non-toxic nanomaterials that interfere with subcellular inter-organellar crosstalk, offering a novel method for designing tumor therapies.

Ischemic heart disease remains a major global health challenge, with myocardial ischemia-reperfusion injury (MIRI) being one of its most common and severe pathophysiological complications. The pathogenesis of MIRI is multifaceted, involving oxidative stress, inflammatory responses, apoptotic pathways, and autophagic regulation. Notably, autophagy exerts a dual regulatory effect, where maintaining optimal autophagic flux is essential for cardiac homeostasis. Emerging evidence underscores the crucial role of non-coding RNAs (ncRNAs) in modulating these pathological processes. In particular, long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs) have been identified as key regulators of autophagy-mediated MIRI progression through complex molecular networks. This review provides a systematic analysis of the molecular pathways through which ncRNAs influence MIRI pathogenesis, with a specific focus on their autophagy-regulatory mechanisms. These insights may enhance our understanding of MIRI pathobiology and facilitate the development of novel therapeutic strategies.

Pituitary tumors, arising from the pituitary gland, can be classified as functioning or non-functioning based on their hormone production. Previous studies demonstrated that impairment of cellular processes, such as autophagy, a crucial cellular recycling mechanism, has been implicated in pituitary tumorigenesis and hormone dysregulation. This review comprehensively examines the intricate relationship between autophagy and pituitary tumors. We explore the multifaceted role of autophagy in cancer, highlighting its dual nature as both a tumor suppressor and a promoter depending on the context. We also discuss the specific mechanisms of autophagy, including macroautophagy, mitophagy, crinophagy, and their relevance to pituitary tumorigenesis and hormone regulation. Furthermore, we analyze the current literature regarding the impact of various therapeutic interventions in pituitary tumor cells, with both autophagy-promoting and autophagy-inhibiting strategies. We address the challenges in interpreting autophagy activity and its complex interplay with hormone production. Current evidence suggests the potential of targeting autophagy as a therapeutic approach for pituitary tumors, emphasizing further research and clinical trials to determine the optimal strategy for individual patients and improve long-term outcomes.

The rising prevalence of diabetes mellitus (DM) is undermining global efforts to eliminate tuberculosis (TB). Most studies found that patients with pulmonary TB and DM have more cavitary lung lesions, higher mycobacterial burden on the lungs, longer periods of infectiousness, and worse outcomes. Both human and animal studies indicate that TB-DM is associated with impaired innate and adaptive immune responses, resulting in delayed bacterial clearance. Similar observations have been noted in other infections, such as those caused by Klebsiella pneumoniae, where DM contributes to increased susceptibility and worse outcomes due to compromised immune functions including defective phagocytosis and impaired early immune cell recruitment. This review delves into the mechanisms of immune dysfunction in TB-DM, exploring how DM increases TB susceptibility and severity. By elucidating these complex interactions, this review aims to offer insights into more effective strategies for managing and improving outcomes for patients with this challenging comorbidity.

Macroautophagy involves the sequestration of cytoplasmic contents in a double-membrane autophagosome and its subsequent delivery to lysosomes for degradation and recycling. In Caenorhabditis elegans, autophagy participates in diverse processes such as stress resistance, cell fate specification, tissue remodeling, aging, and adaptive immunity. Genetic screens in C. elegans have identified a set of metazoan-specific autophagy genes that form the basis for our molecular understanding of steps unique to the autophagy pathway in multicellular organisms. Suppressor screens have uncovered multiple mechanisms that modulate autophagy activity under physiological conditions. C. elegans also provides a model to investigate how autophagy activity is coordinately controlled at an organismal level. In this chapter, we will discuss the molecular machinery, regulation, and physiological functions of autophagy, and also methods utilized for monitoring autophagy during C. elegans development.

Lysosomes, as crucial organelles within cells, carry out diverse biological functions such as waste degradation, regulation of the cellular environment, and precise control of cell signaling. This paper reviews the core functions and structural characteristics of lysosomes, and delves into the current research status of lysosomes damage repair mechanisms. Subsequently, we explore in depth the close association between lysosomes and various diseases, including but not limited to age-related chronic diseases, neuro-degenerative diseases, tumors, inflammation, and immune imbalance. Additionally, we also provide a detailed discussion of the application of lysosome-targeted substances in the field of regenerative medicine, especially the enormous potential demonstrated in key areas such as stem cell regulation and therapy, and myocardial cell repair. Though the integration of multidisciplinary research efforts, we believe that lysosomes damage repair mechanisms will demonstrate even greater application value in disease treatment and regenerative medicine.

Neonatal necrotizing enterocolitis (NEC) is a devastating intestinal disease that primarily affects preterm infants. Unfortunately, no specific treatment for NEC is currently available, making it crucial to further investigate its underlying mechanisms. In this study, we aimed to identify the key target gene, CHI3L1, which was significantly upregulated in the intestinal tissues of both affected children and model mice from the GEO database. CHI3L1 is known to play important roles in inflammatory and immune responses, as well as in tissue damage and repair, all of which are closely associated with the development of NEC. We conducted validations at both the cellular and animal levels, demonstrating that the inhibition or knockdown of CHI3L1 significantly reduced the severity of NEC. Mechanistic investigations revealed that the knockdown of CHI3L1 inhibited the PI3K-Akt-FoxO1 signalling pathway, alleviating excessive autophagy in intestinal epithelial cells and subsequently reducing injury and inflammatory responses. Clinical studies have revealed that elevated serum CHI3L1 expression in paediatric patients is associated with both the occurrence and severity of necrotising enterocolitis NEC, demonstrating positive correlations with the Duke Abdominal Assessment Scale (DAAS), C-reactive protein (CRP), procalcitonin (PCT), red cell distribution width (RDW), and lactate dehydrogenase (LDH) levels. In conclusion, our findings confirmed a close relationship between CHI3L1 and the occurrence and severity of NEC, suggesting that it may mitigate inflammatory responses and tissue damage by alleviating excessive autophagy in intestinal epithelial cells. Therefore, targeting CHI3L1 may be an effective strategy to combat NEC.

The Vici syndrome protein EPG5 acts as a tethering factor determining the fusion specificity of autophagosomes with late endosomes/lysosomes. Here we demonstrated that during C. elegans development, EPG-5 modulates SMA and MAB TGFB/TGF-β signaling in controlling body size and also WNT signaling in regulating cell migration. EPG-5 is required for retrograde trafficking of the TGFB receptor SMA-6 and WLS/Wntless homolog MIG-14. In epg-5 mutants, SMA-6 and MIG-14 are trapped within hybrid endosomal structures, which colocalize with SNX-1- and SNX-3-labeled vesicles, respectively. Basolateral recycling processes of transmembrane cargos H.s.TFR/hTfR and H.s.IL2RA/hTAC are also defective in epg-5 mutants. Depletion of EPG-5 causes defective RAB-5 and RAB-7, and RAB-5 and RAB-10 conversion, leading to the formation of these hybrid vesicles. The defects in endocytic trafficking and autophagy in epg-5 mutants are ameliorated by knocking down components of the HOPS complex. Our study demonstrates the intersection between the autophagy pathway and the endocytic pathway, providing insights into the pathogenesis of amyotrophic lateral sclerosis (ALS) and Vici syndrome.Abbreviations: ALM: anterior lateral microtubule; ATG: autophagy related; AVM: anterior ventral microtubule; CORVET: class C core vacuole/endosome tethering; DAF-4: abnormal dauer formation 4; DIC: differential interference contrast; EPG: ectopic PGL granules; EPG-5: ectopic P granules 5; GAP: GTPase activating protein; GFP: green fluorescent protein; HOPS: homotypic fusion and vacuole protein sorting; H.s.IL2RA/hTAC: human interleukin 2 receptor subunit alpha; H.s.TFR/hTfR: human transferrin receptor; L1/L4: the first/fourth larval; mCh: mCherry; MIG-14: abnormal cell migration 14; PLM: posterior lateral microtubule; PVM: posterior ventral microtubule; RAB: ras-related protein; RFP: red fluorescent protein; RME-1: receptor mediated endocytosis 1; SMA-6: small 6; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SNX: sorting nexin; TBC-2: TBC1 (Tre-2/Bub2/Cdc16) domain family 2; TGFB/TGF-β: transforming growth factor beta; TGN: trans-Golgi network; VPS: related to yeast vacuolar protein sorting factor; WT: wild type.

Transcriptional regulation of genes involved in the macroautophagy/autophagy-lysosome pathway acts as an important mechanism for controlling autophagy activity. The factors that globally regulate autophagy activity at the transcriptional level during C. elegans development remain unknown. Here we showed that the THAP domain-containing transcription factor LIN-15B modulates autophagy activity during C. elegans development. Loss of function of lin-15B suppresses the autophagy defect caused by impaired autophagosome maturation and promotes lysosome biogenesis and function. LIN-15B maintains the repressed state of genes involved in the autophagy pathway. Accordingly, loss of function of lin-15B upregulates a plethora of genes involved in autophagosome formation and maturation as well as lysosome biogenesis and function. The cytoplasm-to-nucleus translocation of LIN-15B is mediated by the T16G12.6/IMPORTIN 13/IPO-13 receptor and modulated by nutrient status. Our study uncovers that LIN-15B integrates environmental cues into transcriptional control of a network of genes involved in autophagy in C. elegans.Abbreviations: ATG: autophagy related; DIC: differential interference contrast; EPG: ectopic PGL granules; ER: endoplasmic reticulum; FOXO: forkhead box O; GFP: green fluorescent protein; SQST-1: SeQueSTosome related 1; SynMuv: synthetic multivulva; IPO-13: importin 13; TFEB: transcription factor EB.

Periodontitis, a prevalent and chronic inflammatory disease, is intricately linked with macroautophagy/autophagy, which has a dual role in maintaining periodontal homeostasis. Despite its importance, the precise interplay between autophagy and periodontitis pathogenesis remains to be fully elucidated. In this study, our investigation revealed that the ubiquitination of RAB7A, mediated by reduced levels of the deubiquitinating enzyme USP4 (ubiquitin specific peptidase 4), disrupts normal lysosomal trafficking and autophagosome-lysosome fusion, thereby contributing significantly to periodontitis progression. Specifically, through genomic and histological analysis of clinical gingival samples, we observed a decreased RAB7A expression and impaired autophagic activity in periodontitis. This was further substantiated through experimental periodontitis mice, where RAB7A inactivation was shown to directly affect autophagy efficiency and drive periodontitis progression. Next, we explored the function of active RAB7A to promote lysosomal trafficking dynamics and autophagosome-lysosome fusion, which was inhibited by RAB7A ubiquitination in macrophages stimulated by Porphyromonas gingivalis (P. g.), one of the keystone pathogens of periodontitis. Last, by proteomics analysis, we revealed that the ubiquitination of RAB7A was mediated by USP4 and validated that upregulation of USP4 could attenuate periodontitis in vivo. In conclusion, these findings highlight the interaction between USP4 and RAB7A as a promising target for therapeutic intervention in managing periodontal diseases.Abbreviation: 3-MA: 3-methyladenine; Baf A1:bafilomycin A1; BECN1: beclin 1, autophagy related; CEJ-ABC: cementoenamel junctionto alveolar bone crest; IL1B/IL-1β: interleukin 1 beta; KD:knockdown; LPS: lipopolysaccharide; MOI: multiplicity of infection;OE: overexpression; P.g.: Porphyromonasgingivalis; RILP: Rabinteracting lysosomal protein; ScRNA-seq: single-cell RNA sequencing; SQSTM1/p62: sequestosome 1; S.s.: Streptococcus sanguinis; USP4:ubiquitin specific peptidase 4.

Ferroptosis is an iron-dependent programmed death caused by the imbalance of lipid peroxides in cells. Unlike apoptosis, autophagy and necrosis, ferroptosis is mainly induced by the small molecule compound erastin. The main characteristics of ferroptosis were glutathione (GSH) depletion, inactivation of glutathione peroxidase 4 (GPX4) and reactive oxygen species (ROS) promoting lipid peroxidation. Eventually, the imbalance of lipid peroxidation regulation in cells leads to ferroptosis. The lipid metabolic pathway ultimately contributes to ferroptosis through the production of lipid peroxides. In addition, other cellular metabolic pathways can also regulate ferroptosis, such as the antioxidant metabolic pathway, which inhibits ferroptosis by clearing lipid peroxides and reducing cell membrane damage. Long non-coding RNAs (lncRNAs) are non-coding transcripts more than 200 nucleotides in length and are a less classified group of RNA transcripts that are associated with tumorigenesis and metastasis and are more tissue or cell type specific than protein-coding genes. Studies on the molecular profile of lncRNAs in plasma samples from liver cancer patients show that differentially expressed lncRNAs are mainly concentrated in biological functions related to tumorigenesis, such as cell metastasis, immune response and metabolic regulation. With different biological functions in physiological and pathological environments, the specific expression patterns of lncRNAs coordinate cell state, development, differentiation, and disease.

The macroautophagy/autophagy proteins ATG2A and ATG2B transfer lipids for phagophore membrane growth. They also form stable complexes with WDR45 and WDR45B. Our previous study demonstrated that WDR45 and WDR45B mediate autophagosome-lysosome fusion in neural cells. Given the defective autophagosome formation in cells lacking both ATG2s, their role in later autophagy stages is hard to explore. Here, we report that in neuroblastoma-derived Neuro-2a (N2a) cells, knocking down (KD) Atg2a, but not Atg2b, results in significant accumulation of SQSTM1/p62 and MAP1LC3-II/LC3-II, indicating impaired autophagy. Atg2a deficiency does not affect autophagosome formation, but reduces colocalization of autophagosomal LC3 with late endosomal/lysosomal RFP-RAB7, suggesting impaired autophagosome-lysosome fusion. ATG2A interacts with the SNARE proteins STX17, SNAP29, and VAMP8, facilitating their assembly. Overexpression of ATG2A partially rescues the autophagosome-lysosome fusion defects in Wdr45- and Wdr45b-deficient cells. ATG2 and another tether protein, EPG5, function partially redundantly in mediating autophagosome-lysosome fusion. Thus, ATG2A plays a key role in neural autophagy by tethering autophagosomes with lysosomes for fusion.Abbreviations: AAV: adeno-associated virus; ATG2Ar: RNAi-resistant ATG2A; Baf: bafilomycin A1; co-IP: co-immunoprecipitation; CQ: chloroquine; DKD: double knockdown; DKO: double knockout; ER: endoplasmic reticulum; KD: knockdown; KO: knockout; MIL: membrane-impermeable Halo ligand; MPL: membrane-permeable Halo ligand; N2a: Neuro-2a; NC negative control; PG: phagophore; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; TEM: Transmission electron microscopy; TM: transmembrane domain; WT: wild-type.

Autophagy is a natural process whereby damaged or dying parts of a cell are eliminated and recycled. The term autophagy usually refers to macroautophagy, which is one of three types of autophagy, alongside microautophagy and chaperone-mediated autophagy. Autophagy is activated by adenosine monophosphate-activated protein kinase (AMPK) and inhibited by mammalian target of rapamycin (mTOR) through their interference with Unc-51-like kinase 1 (ULK1). Dysregulated autophagy is deeply involved in autoimmune glomerular diseases. Upregulated autophagy can induce inflammation and activate innate and adaptive immunity. However, autophagy may also exert a protective role on podocytes, enhance endothelial cell function, and preserve proximal tubular epithelial cells during ischemic or endotoxic acute kidney injury (AKI). Hydroxychloroquine (HCQ) can downregulate increased autophagy and is widely used in lupus nephritis. HCQ causes alkalinization, which results in vacuolization of lysosomes and inhibition of their functions. By inhibiting autophagic activity, HCQ may reduce inflammation and innate immunity, inhibit the activation of T cells, restore the T helper 17/T regulator balance, restrict the production of pro-inflammatory cytokines, and modulate co-stimulatory molecules. This reduces the risk of flares, spares the dosage of glucocorticoids, improves lupus activity, and prevents the thrombotic effects of anti-phospholipid antibodies. Recent studies showed that HCQ can also reduce proteinuria in IgA nephropathy (IgAN) and membranous nephropathy (MN). Drugs that improve mitochondrial function or enhance autophagy, such as metformin, sodium-glucose co-transporter 2 (SGLT2) inhibitors or mTOR inhibitors, may exert protective effects on podocytes and reduce proteinuria in MN or focal segmental glomerulosclerosis (FSGS).

The mechanisms of adipose tissue activation and inactivation have been a hot topic of research in the last decade, from which countermeasures have been attempted to be found against obesity as well as other lipid metabolism-related diseases, such as type 2 diabetes mellitus and non-alcoholic fatty liver disease. Autophagy has been shown to be closely related to the regulation of adipocyte activity, which is involved in the whole process including white adipocyte differentiation/maturation and brown or beige adipocyte generation/activation. Dysregulation of autophagy in adipose tissue has been demonstrated to be associated with obesity. On this basis, we summarize the pathways and mechanisms of autophagy involved in the regulation of lipid metabolism and present a review of its pathophysiological roles in lipid metabolism-related diseases, in the hope of providing ideas for the treatment of these diseases.

Liquid storage of semen is a widely used technology for promoting genetic improvement in goat breeding. The short shelf life of spermatozoa greatly limits the application of liquid storage, which urgently needs to explore the underlying regulatory factors. Autophagy as a cellular catabolic process plays critical roles in eliminating damaged material, that thus protects the function and fertilizing ability of spermatozoa. Nevertheless, the regulatory mechanisms of autophagy in goat spermatozoa under liquid storage remain unclear. In this study, the typical morphologic abnormalities and ultrastructural changes in goat spermatozoa, such as plasma membrane swollen and shrunken, acrosome exfoliation, and axoneme exposure, were observed after liquid storage at 4°C. Moreover, assessment of the formation of autophagy in liquid-stored goat spermatozoa was performed by a morphological "gold standard" of electron microscopy. Notably, a large number of vesicles with double-membrane structure indicating autophagosome were found to surround the aberrant spermatozoa, suggesting the activation of autophagy. Several proteins, such as LC3, ATG5, and p62, exhibited differential expression after liquid storage, which further validated the occurrence of autophagy in liquid-stored goat spermatozoa. Furthermore, chloroquine treatment was used to inhibit the autophagy of spermatozoa, which caused a significantly decrease in the quality of liquid-stored spermatozoa, including motility, viability, plasma membrane integrity, and acrosome integrity. Significant increase in ROS and MDA levels of spermatozoa and significant decrease in Ca2+ influx and protein tyrosine phosphorylation of spermatozoa were also detected after chloroquine-induced autophagy inhibition. The ultrastructural observation of double-membrane autophagosome provides strong evidences for the activation of autophagy in goat spermatozoa under liquid storage. The inhibition of autophagy mediated by chloroquine indicated that autophagy plays vital roles in the survival of spermatozoa. These results facilitate understanding the activation of autophagy in spermatozoa and provide valuable references for uncovering the underlying regulatory mechanisms of liquid storage of goat spermatozoa.

Autophagy impairments have been implicated in various aging conditions. Previous studies in cervical motor neurons show an age-dependent increase in the key autophagy proteins LC3 and p62, reflecting autophagy impairment and autophagosome accumulation. Chloroquine is commonly used to inhibit autophagy by preventing autophagosome-lysosome fusion and may thus emulate the effects of aging on the neuromuscular system. Indeed, acute chloroquine administration in old mice decreases maximal transdiaphragmatic pressure generation, consistent with aging effects. We hypothesized that chloroquine alters diaphragm muscle neuromuscular junction (NMJ) morphology and increases denervation. Adult male and female C57BL/6 × 129J mice between 5 and 8 months of age were used to examine diaphragm muscle NMJ morphology and denervation following daily intraperitoneal injections of chloroquine (10 mg/kg/d) or vehicle for 7 days. The motor end-plates and pre-synaptic terminals were fluorescently labeled with α-bungarotoxin and anti-synaptophysin, respectively. Confocal microscopy was used to assess pre- and post-synaptic morphology and denervation. At diaphragm NMJs, chloroquine treatment decreased pre-synaptic volume by 12% compared to the vehicle (p < 0.05), with no change in post-synaptic volume. Chloroquine treatment increased the proportion of partially denervated NMJs by 2.7-fold compared to vehicle treatment (p < 0.05). The morphological changes observed were similar to those previously reported in the diaphragm muscles of 18-month-old mice. These findings highlight the importance of autophagy in the maintenance of the structural properties at adult NMJs in vivo.