Accumulation Of Autophagosomes Research Articles

Adipose deficiency and aberrant autophagy in a Drosophila model of MPS VII is corrected by pharmacological stimulators of mTOR

Mucopolysaccharidosis type VII (MPS VII) is a recessively inherited lysosomal storage disorder caused due to β-glucuronidase (β-GUS) enzyme deficiency. Prominent clinical symptoms include hydrops fetalis, musculoskeletal deformities, neurodegeneration and hepatosplenomegaly leading to premature death in most cases. Apart from these, MPS VII is also characterized as adipose storage deficiency disorder although the underlying mechanism of this lean phenotype in the patients or β-GUS-deficient mice still remains a mystery. We addressed this issue using our recently developed Drosophila model of MPS VII (the CG2135-/- fly), which also exhibited a significant loss of body fat. We report here that the lean phenotype of the CG2135−/− larvae is due to fewer number of adipocytes, smaller lipid droplets and reduced adipogenesis. Our data further revealed that there is an abnormal accumulation of autophagosomes in the CG2135−/− larvae due to autophagosome-lysosome fusion defect. Decreased lysosome-mediated turnover also led to attenuated mTOR activity in the CG2135−/− larvae. Interestingly, treatment of the CG2135−/− larvae with mTOR stimulators, 3BDO or glucose, led to the restoration of mTOR activity with simultaneous correction of the autophagy defect and adipose storage deficiency. Our finding thus established a hitherto unknown mechanistic link between autophagy dysfunction, mTOR downregulation and reduced adiposity in MPS VII.

Regulation of basal autophagy by calmodulin availability.

Macroautophagy (hereafter autophagy) is a process that degrades cellular components to maintain homeostasis. The Ca2+ sensor calmodulin (CaM) regulates numerous cell functions but is a limiting factor due to its insufficient availability for all target proteins. However, evidence that CaM availability regulates basal autophagy is lacking. Here, we have tested this hypothesis. CaM antagonists W-7, trifluoperazine and CGS9343b cause autophagosome accumulation and inhibit basal autophagic flux in the same manner as does chloroquine. These reagents promote the activity of AMP-activated protein kinase (AMPK) but not that of the mechanistic target of rapamycin (mTOR). Competitive binding assays using CaM sensors with different Ca2+ dependencies showed that chloroquine directly binds CaM in a Ca2+ -dependent fashion. The CaM antagonists have disparate effects on cytoplasmic Ca2+ , triggering from none to robust signals, indicating that their consistent inhibition of autophagy is due to inhibition of CaM and not Ca2+ . Chelating intracellular Ca2+ reduces the effect of the CaM antagonists to accumulate LC3-II, indicating that they do so by inhibiting CaM-dependent activities at basal Ca2+ level. The CaM antagonists cause lysosomal alkalinisation. Consistently, buffering CaM with a high-affinity CaM-binding protein that binds CaM at resting Ca2+ level increases lysosomal pH. Enhanced CaM buffering using a chimeric protein that contains two high-affinity CaM-binding sites that can collectively bind CaM at a large range of Ca2+ further increases lysosomal pH and increases LC3-II accumulation and AMPK activity, but not that of mTOR. These data demonstrate that CaM availability is required for basal autophagy.

Activation of Autophagy by Low-Dose Silica Nanoparticles Enhances Testosterone Secretion in Leydig Cells.

Silica nanoparticles (SNPs) can cause abnormal spermatogenesis in male reproductive toxicity. However, the toxicity and toxicological mechanisms of SNPs in testosterone synthesis and secretion in Leydig cells are not well known. Therefore, this study aimed to determine the effect and molecular mechanism of low doses of SNPs in testosterone production in Leydig cells. For this, mouse primary Leydig cells (PLCs) were exposed to 100 nm Stöber nonporous spherical SNPs. We observed significant accumulation of SNPs in the cytoplasm of PLCs via transmission electron microscopy (TEM). CCK-8 and flow cytometry assays confirmed that low doses (50 and 100 μg/mL) of SNPs had no significant effect on cell viability and apoptosis, whereas high doses (more than 200 μg/mL) decreased cell viability and increased cell apoptosis in PLCs. Monodansylcadaverine (MDC) staining showed that SNPs caused the significant accumulation of autophagosomes in the cytoplasm of PLCs. SNPs activated autophagy by upregulating microtubule-associated protein light chain 3 (LC3-II) and BCL-2-interacting protein (BECLIN-1) levels, in addition to downregulating sequestosome 1 (SQSTM1/P62) level at low doses. In addition, low doses of SNPs enhanced testosterone secretion and increased steroidogenic acute regulatory protein (StAR) expression. SNPs combined with rapamycin (RAP), an autophagy activator, enhanced testosterone production and increased StAR expression, whereas SNPs combined with 3-methyladenine (3-MA) and chloroquine (CQ), autophagy inhibitors, had an opposite effect. Furthermore, BECLIN-1 depletion inhibited testosterone production and StAR expression. Altogether, our results demonstrate that low doses of SNPs enhanced testosterone secretion via the activation of autophagy in PLCs.

Repurposing econazole as a pharmacological autophagy inhibitor to treat pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the highest mortality among carcinomas. The pathogenesis of PDAC requires elevated autophagy, inhibition of which using hydroxychloroquine has shown promise. However, current realization is impeded by its suboptimal use and unpredictable toxicity. Attempts to identify novel autophagy-modulating agents from already approved drugs offer a rapid and accessible approach. Here, using a patient-derived organoid model, we performed a comparative analysis of therapeutic responses among various antimalarial/fungal/parasitic/viral agents, through which econazole (ECON), an antifungal compound, emerged as the top candidate. Further testing in cell-line and xenograft models of PDAC validated this activity, which occurred as a direct consequence of dysfunctional autophagy. More specifically, ECON boosted autophagy initiation but blocked lysosome biogenesis. RNA sequencing analysis revealed that this autophagic induction was largely attributed to the altered expression of activation transcription factor 3 (ATF3). Increased nuclear import of ATF3 and its transcriptional repression of inhibitor of differentiation-1 (ID-1) led to inactivation of the AKT/mammalian target of rapamycin (mTOR) pathway, thus giving rise to autophagosome accumulation in PDAC cells. The magnitude of the increase in autophagosomes was sufficient to elicit ER stress-mediated apoptosis. Furthermore, ECON, as an autophagy inhibitor, exhibited synergistic effects with trametinib on PDAC. This study provides direct preclinical and experimental evidence for the therapeutic efficacy of ECON in PDAC treatment and reveals a mechanism whereby ECON inhibits PDAC growth.

Autophagosome-lysosome fusion is facilitated by plectin-stabilized actin and keratin 8 during macroautophagic process.

Autophagy is a lysosome-mediated degradative process that removes damaged proteins and organelles, during which autophagosome-lysosome fusion is a key step of the autophagic flux. Based on our observation that intermediate cytofilament keratin 8 (KRT8) enhances autophagic clearance in cells under oxidative stress condition, we investigated whether KRT8 supports the cytoplasmic architectural networks to facilitate the vesicular fusion entailing trafficking onto filamentous tracks. We found that KRT8 interacts with actin filaments via the cytolinker, plectin (PLEC) during trafficking of autophagosome. When PLEC was knocked down or KRT8 structure was collapsed by phosphorylation, autophagosome-lysosome fusion was attenuated. Inhibition of actin polymerization resulted in accumulation of autophagosomes owing to a decrease in autophagosome and lysosome fusion. Furthermore, myosin motor protein was found to be responsible for vesicular trafficking along the actin filaments to entail autolysosome formation. Thus, the autophagosome-lysosome fusion is aided by PLEC-stabilized actin filaments as well as intermediate cytofilament KRT8 that supports the structural integrity of actin filaments during macroautophagic process under oxidative stress condition.

Ethanol potentiates mirtazapine-induced cardiotoxicity by inducing dysfunctional autophagy via HMGB1-dependent Akt/mTOR signaling pathway

Previous surveys have revealed that mirtazapine (MIR), one of the most commonly prescribed antidepressants, is associated with a higher risk of adverse cardiac events compared with other newer antidepressants. Chronic ethanol (EtOH) abuse could also lead to myocardial injuries. Concerning the common comorbidity of major depression and alcohol dependence, combined consumption of MIR and EtOH might be prevalent in patients with depression, resulting in an additive or synergistic cardiotoxic effect. To this end, the present study evaluated cardiotoxicity induced by MIR-plus-EtOH in vivo (male C57BL/6J mice) and in vitro (H9c2 cardiomyoblasts), Further research on the role of autophagy and underlying signaling pathway were carried out in H9c2 cells. We found that EtOH exacerbated MIR-induced cardiotoxicity both in vivo and in vitro. Furthermore, EtOH significantly potentiated MIR-induced dysfunctional autophagy as reflected by upregulated protein levels of LC3-II, p62, Beclin1 and LAMP-1. Pharmacological inhibition of autophagy by 3-methyladenine alleviated MIR-plus-EtOH-induced myocardial injury. High mobility group box 1 (HMGB1) is a positive regulator of autophagy. In our work, HMGB1 knockdown decreased autophagosome accumulation and boosted viability in H9c2 cells. Additionally, HMGB1 blockage markedly upregulated p-Akt/Akt and p-mTOR/mTOR levels which were suppressed in MIR-plus-EtOH treated cells. In general, the present study demonstrates that EtOH potentiates MIR-induced cardiotoxicity which might be attributed to dysfunctional autophagy via inhibiting Akt/mTOR signaling pathway, while HMGB1 knockdown might contribute to improve autophagy flux.

Selective elimination of pluripotent stem cells by PIKfyve specific inhibitors.

SummaryInhibition of PIKfyve phosphoinositide kinase selectively kills autophagy-dependent cancer cells by disrupting lysosome homeostasis. Here, we show that PIKfyve inhibitors can also selectively eliminate pluripotent embryonal carcinoma cells (ECCs), embryonic stem cells, and induced pluripotent stem cells under conditions where differentiated cells remain viable. PIKfyve inhibitors prevented lysosome fission, induced autophagosome accumulation, and reduced cell proliferation in both pluripotent and differentiated cells, but they induced death only in pluripotent cells. The ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells was confirmed with xenografts derived from ECCs. Pretreatment of ECCs with the PIKfyve specific inhibitor WX8 suppressed their ability to form teratocarcinomas in mice, and intraperitoneal injections of WX8 into mice harboring teratocarcinoma xenografts selectively eliminated pluripotent cells. Differentiated cells continued to proliferate, but at a reduced rate. These results provide a proof of principle that PIKfyve specific inhibitors can selectively eliminate pluripotent stem cells in vivo as well as in vitro.

PM2.5 induces inflammatory responses via oxidative stress-mediated mitophagy in human bronchial epithelial cells.

Fine particulate matter (PM2.5) is a ubiquitous air pollutant, and it has been reported to be closely associated with lung inflammatory injury. In this study, the potential molecular mechanisms underlying PM2.5-induced cellular inflammation in human bronchial epithelial (BEAS-2B) cells were investigated. Ambient PM2.5 particulates from Suzhou, China, were collected and re-suspended in ultrapure water. Cellular damages, characterized by oxidative stress, mitochondrial injury, and inflammatory cytokine production, were determined in 24h PM2.5-treated BEAS-2B cells with or without 3-methyladenine (3-MA; autophagy inhibitor) pretreatment. Biomarkers related to oxidative damage, inflammatory injury and autophagy signaling pathways were also measured. Uptake of PM2.5 in BEAS-2B cells induced cellular oxidative damage, mitochondrial injury, and inflammatory responses as indicated by a significant decrease in GSH/GSSG ratio, increased MDA content, dilated mitochondria with loss and rupture of crista, and production of inflammatory cytokines. Activation of Nrf-2/TXNIP-mediated NF-κB and Bnip3L/NIX-dependent mitophagy signaling pathways, as well as accumulation of autophagosomes and autolysosomes, were also observed. A 6 h pretreatment of 3-MA increased PM2.5-induced oxidative damage and cellular inflammation as indicated by increasing protein levels of HO-1, TXNIP, Bnip3L/NIX and IL-8 gene expression. PM2.5 induced cellular inflammatory injury by oxidative stress, mitochondrial dysfunction, and mitophagy initiation. Although induction of Bnip3L/NIX-mediated mitophagy in BEAS-2B cells appeared to confer protection in response to PM2.5, dysfunction of autophagic flux may be a critical contributor to defective mitophagy and cellular inflammatory response.

Tfeb-Mediated Transcriptional Regulation of Autophagy Induces Autosis during Ischemia/Reperfusion in the Heart.

Autosis is a unique form of cell death with characteristic morphological and biochemical features caused by dysregulated autophagy. Autosis is observed in the heart during the late phase of ischemia/reperfusion (I/R), when marked accumulation of autophagosomes is induced. We previously showed that the excessive accumulation of autophagosomes promotes autosis in cardiomyocytes. Although the inhibition of autophagic flux via the upregulation of Rubicon induces the accumulation of autophagosomes during I/R, it appears that additional mechanisms exacerbating autophagosome accumulation are required for the induction of autosis. Here, we show that Tfeb contributes to the induction of autosis during the late phase of I/R in the heart. During myocardial reperfusion, Tfeb is activated and translocated into the nucleus, which in turn upregulates genes involved in autophagy and lysosomal function. The overexpression of Tfeb enhanced cardiomyocyte death induced by a high dose of TAT-Beclin 1, an effect that was inhibited by the downregulation of Atg7. Conversely, the knockdown of Tfeb attenuated high-dose TAT-Beclin1-induced death in cardiomyocytes. Although the downregulation of Tfeb in the heart significantly decreased the number of autophagic vacuoles and inhibited autosis during I/R, the activation of Tfeb activity via 3,4-dimethoxychalcone, an activator of Tfeb, aggravated myocardial injury during I/R. These findings suggest that Tfeb promotes cardiomyocyte autosis during the late phase of reperfusion in the heart.

Belimumab Decreases Autophagy and Citrullination in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus.

Belimumab (BLM) is a B lymphocyte stimulator (BLyS) inhibitor approved for the treatment of systemic lupus erythematosus (SLE). Autophagy is a cell survival mechanism involved in the pathogenesis of SLE. Citrullination is a post-translational modification catalyzed by peptidylarginine deiminase (PAD) enzymes. Autophagy and citrullination may generate neoepitopes, evoking an autoimmune response. No previous studies have investigated the connection of these processes, and how BLM could affect them, in SLE. Ex vivo autophagy and protein citrullination were analyzed by western blot in lysates from 26 SLE patients’ PBMCs at baseline and after 2, 4, and 12 weeks of BLM administration, and from 16 healthy donors’ PBMCs. Autophagic PBMCs were identified by the immunofluorescent detection of the autophagy-associated proteins LC3B (LC3 puncta) and LAMP-1. Autophagosome accumulation was evaluated in CD14− (PBLs) and CD14+ (monocytes) SLE cells. The presence of the BLyS receptors BAFF-R, BCMA, and TACI on SLE CD4+, CD8+ T cells and monocytes, as well as serum IL-18 levels, was also assessed. Following BLM administration, we observed a decrease in autophagy and citrullination, with a lowering of LC3-II, citrullinated vimentin, and PAD4 expression levels in PBMCs from SLE patients. LC3-II levels showed a correlation with the SLE Disease Activity Index 2000 (SLEDAI-2K) after 12 weeks of therapy. The LC3B/LAMP-1 analysis confirmed the reduction in autophagy. A lesser autophagosome accumulation occurred in PBLs and monocytes which, in turn, seemed to be the main cellular populations contributing to autophagy. A reduction in patients’ serum IL-18 concentrations occurred. CD4+ and CD8+ cells weakly expressed BAFF receptors; monocytes expressed only BAFF-R. BLM could impact on autophagy and citrullination, offering an opportunity for a deeper understanding of these mechanisms in SLE, and a possible tool for the clinical management of SLE.

Tributyltin inhibits autophagy by decreasing lysosomal acidity in SH-SY5Y cells

Tributyltin (TBT) is an environmental pollutant that remains in marine sediments and is toxic to mammals. For example, TBT elicits neurotoxic and immunosuppressive effects on rats. However, it is not entirely understood how TBT causes toxicity. Autophagy plays a pivotal role in protein quality control and eliminates aggregated proteins and damaged organelles. We previously reported that TBT dephosphorylates mammalian target of rapamycin (mTOR), which may be involved in enhancement of autophagosome synthesis, in primary cultures of cortical neurons. Autophagosomes can accumulate due to enhancement of autophagosome synthesis or inhibition of autophagic degradation, and we did not clarify whether TBT alters autophagic flux. Here, we investigated the mechanism by which TBT causes accumulation of autophagosomes in SH-SY5Y cells. TBT inhibited autophagy without affecting autophagosome–lysosome fusion before it caused cell death. TBT dramatically decreased the acidity of lysosomes without affecting lysosomal membrane integrity. TBT decreased the mature protein level of cathepsin B, and this may be related to the decrease in lysosomal acidity. These results suggest that TBT inhibits autophagic degradation by decreasing lysosomal acidity. Autophagy impairment may be involved in the mechanism underlying neuronal death and/or T-cell-dependent thymus atrophy induced by TBT.

Autophagy inducers lead to transient accumulation of autophagosomes in Arabidopsis roots.

This study reveals that plant roots show a rapid termination of autophagy induction, offering a plant model for studying how excessive autophagy is deterred. In eukaryotes, autophagy is an intracellular mechanism that is important for recycling nutrients by degrading various macromolecules and organelles in vacuoles and lysosomes. Autophagy is induced when the nutrient supply to plant cells is limited. The protein kinase target of rapamycin (TOR) complex negatively regulates autophagy when nutrients are present in adequate amounts. The TOR inhibitor AZD8055 is an autophagy inducer that is useful for studying starvation-induced autophagy in plant cells. The mechanism by which AZD8055 increases the autophagic flux in plant cells has not been studied in detail. Here, we show that AZD8055-induced autophagy requires phosphatidylinositol 3-kinase activity and canonical AUTOPHAGY-RELATED (ATG) genes in Arabidopsis thaliana. Autophagic flux rapidly increased in seedlings treated with AZD8055. Unexpectedly, autophagy induction was transient in root cells and terminated earlier than in cotyledon cells. Transient induction is partly caused by a temporary effect of AZD8055 on phagophore initiation. These findings indicate a TOR-independent mechanism for terminating autophagy induction, thereby paving the way for elucidating how excess autophagy is prevented in plant roots.

Cornel Iridoid Glycoside and Its Effective Component Regulate ATPase Vps4A/JNK to Alleviate Autophagy Deficit with Autophagosome Accumulation.

Improving autophagy-lysosome fusion has been considered a key method in the treatment of Alzheimer's disease (AD). Cornel iridoid glycoside (CIG) is extracted from Cornus officinalis and has been shown to promote the clearance of tau oligomers via the autophagy pathway. However, the mechanisms of CIG on autophagy deficits are not understood. Here, we found autophagy deficit and tau aggregation in the brains of P301S tau transgenic mice and MAPT cells edited using CRISPR-Cas9 technology. CIG decreased tau aggregation and alleviated autophagic markers involving the JNK/Beclin-1 signaling pathway which demonstrated CIG that might enhance lysosome formation by upregulating ATPase Vps4A expression. Knocking down VPS4A increased autophagosome accumulation and attenuated the effect of CIG on p62. In addition, CIG had no effect on tau oligomers but still inhibited the level of tau monomer in VPS4A knockout cells. The effective component (Sweroside, SWE) of CIG attenuated tau oligomers accumulation and increased Vps4A level but not CHMP2B. SWE could not change the level of tau oligomers in VPS4A knockout cells. In conclusion, CIG suppressed autophagosome accumulation by regulating the ATPase Vps4A/JNK. SWE is a core of active factors of CIG in Vps4A regulation. These findings suggest CIG may be a potential drug in AD treatment.

Neuroprotection of Triptolide against Amyloid-Beta1-42-induced toxicity via the Akt/mTOR/p70S6K-mediated Autophagy Pathway.

Triptolide is a natural active compound that has significant neuroprotective properties and shows promising effects in the treatment of Alzheimer's disease (AD). Recent studies have shown that autophagy occurs in AD. In this study, we determined whether autophagy regulated by triptolide ameliorates neuronal death caused by amyloid-Beta1-42 (Aβ1-42). We examined the effects of triptolide on cell viability, autophagy, apoptosis, and the protein kinase B/mammalian target of the rapamysin/70 kDa ribosomal protein S6 kinase (Akt/mTOR/p70S6K) signaling pathway in PC12 cells. The results indicated that triptolide treatment exhibited a cytoprotective effect against cell injury induced by Aβ1-42. Triptolide also reduced apoptosis and enhanced cell survival by decreasing autophagosome accumulation and inducing autophagic degradation. Furthermore, our results also showed that activating the Akt/mTOR/p70S6K mechanism was one reason for the protection of triptolide. Triptolide treatment protected against Aβ1-42-induced cytotoxicity by decreasing autophagosome accumulation, and inducing autophagic degradation in PC12 cells. These findings also suggest that the reduction of autophagosome accumulation observed in triptolide-treated cells was Akt/mTOR/p70S6K pathway dependent. Overall, triptolide exhibits a neuron protective effect and this study provides new insight into AD prevention and treatment.

Emerging investigator series: long-term exposure of amorphous silica nanoparticles disrupts the lysosomal and cholesterol homeostasis in macrophages

Long-term ASiNP exposure led to lysosomal density and size increments as well as protein changes, and autophagosome and cholesterol accumulation in cells.

Repurposing Oxiconazole against Colorectal Cancer via PRDX2-mediated Autophagy Arrest

Colorectal cancer (CRC) is one of the most common malignancies worldwide, yet successful treatment still remains a challenge. In this study, we found that oxiconazole (OXI), a broad-spectrum antifungal agent, exhibits certain anti-tumor effect against CRC. Autophagy arrest and subsequent apoptosis are characterized as pivotal events involving OXI-induced growth suppression of CRC cells. Mechanistically, OXI downregulates the protein levels of peroxiredoxin-2 (PRDX2), an antioxidant enzyme, for reactive oxygen species (ROS) detoxication, to initiate autophagy by inactivating the Akt/mTOR pathway and inhibiting RAB7A-mediated fusion of autophagosome and lysosome, which lead to extreme accumulation of autophagosomes and subsequent growth suppression of CRC cells. Consistently, interfering with autophagy or overexpressing PRDX2 significantly impedes OXI-induced growth suppression of CRC cells. Moreover, OXI plus oxaliplatin, a mainstay drug for CRC treatment, achieves an improved anti-tumor effect. Taken together, our findings bring novel mechanistic insights into OXI-induced autophagy arrest and the growth inhibitory effect on CRC cells, and suggest a promisingly therapeutic role of OXI for CRC treatment.

High Glucose Accelerates Tumor Progression by Regulating MEDAG-Mediated Autophagy Levels in Breast Cancer.

Recent studies have shown that diabetes is a major risk factor for breast cancer (BC), but the mechanism is incompletely understood. Mesenteric estrogen-dependent adipogenesis (MEDAG) plays a significant role in both glucose uptake and BC development. However, the relationship between MEDAG and BC under high glucose (HG) conditions remains unclear. In our study, MEDAG expression was higher in BC tissue from diabetic patients than in BC tissue from nondiabetic patients. HG promoted BC progression in vitro and in vivo by upregulating MEDAG expression. Furthermore, MEDAG deficiency increased the autophagosome number and autophagic flux. Moreover, inhibition of autophagy partially reversed MEDAG knockdown (MEDAGKD)-induced suppression of tumorigenic biological behaviors and epithelial-mesenchymal transition (EMT) progression. Finally, MEDAG significantly suppressed AMPK phosphorylation. Additionally, the AMPK inhibitor Compound C markedly reduced autophagosome accumulation and antitumor effects in MEDAGKD cells. Treatment with the AMPK activator AICAR exhibited similar effects in MEDAG-overexpressing (MEDAGOE) cells. In conclusion, the MEDAG-AMPK-autophagy axis is vital to BC progression in diabetic patients. Our findings provide a novel treatment target for BC in patients with diabetes.

Hgs Deficiency Caused Restrictive Cardiomyopathy via Disrupting Proteostasis.

The molecular mechanisms underlying restrictive cardiomyopathy (RCM) are not fully understood. Hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) is a vital element of Endosomal sorting required for transport (ESCRT), which mediates protein sorting for degradation and is crucial for protein homeostasis (proteostasis) maintenance. However, the physiological function and underlying mechanisms of HGS in RCM are unexplored. We hypothesized that HGS may play vital roles in cardiac homeostasis. Cardiomyocyte-specific Hgs gene knockout mice were generated and developed a phenotype similar to human RCM. Proteomic analysis revealed that Hgs deficiency impaired lysosomal homeostasis in cardiomyocytes. Loss of Hgs disrupted cholesterol transport and lysosomal integrity, resulting in lysosomal storage disorder (LSD) with aberrant autophagosome accumulation and protein aggregation. Suppression of protein aggregation by doxycycline treatment attenuated cardiac fibrosis, and diastolic dysfunction in Hgs-knockout mice. These findings uncovered a novel physiological role of HGS in regulating cardiac lysosomal homeostasis and proteostasis, suggesting that the deficient HGS contributes to LSD-associated RCM-like cardiomyopathy.

Weakened interaction of ATG14 and the SNARE complex blocks autophagosome-lysosome fusion contributes to fluoride-induced developmental neurotoxicity

Fluoride is capable of inducing developmental neurotoxicity, but the mechanisms involved remain unclear. We aimed to explore the role of autophagosome-lysosome fusion in developmental fluoride neurotoxicity, particularly focusing on the interaction between ATG14 and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. We developed in vivo models of Sprague–Dawley rats exposed to sodium fluoride (NaF) from the pregnancy of parental rats until the offspring were two months old and in vitro models of NaF and/or Ad-ATG14-treated SH-SY5Y cells. We assessed neurobehavioral changes in offspring and further investigated the effects of NaF exposure on autophagic flux, apoptosis, autophagosome-lysosome fusion, and the interaction between ATG14 and the SNARE complex. NaF exposure impaired offspring learning and memory capabilities and induced the accumulation of autophagosomes and autophagic flux blockage and apoptosis, as indicated by increased LC3-II, p62, and cleaved-caspase-3 expression in vivo and in vitro. In addition, NaF treatment downregulated the protein expression of ATG14 and the SNARE complex and induced autophagosome-lysosome fusion blockage as evidenced by decreased ATG14, STX17, SNAP29, and VAMP8 expression and diminished colocalization of autophagosomes and lysosomes in vivo and in vitro. Furthermore, ATG14 upregulation enhanced the interaction of ATG14 and the SNARE complex to facilitate autophagosome-lysosome fusion, thereby restoring autophagic flux and alleviating NaF-induced apoptosis. In conclusion, NaF exhibited developmental neurotoxicity by restraining the interaction of ATG14 with the SNARE complex and hindering autophagosome-lysosome fusion, thereby participating in the occurrence and development of fluoride neurotoxicity. Notably, ATG14 upregulation protects against developmental fluoride neurotoxicity, and ATG14 may serve as a promising biomarker for further epidemiological investigation.

LCN2 deficiency ameliorates doxorubicin-induced cardiomyopathy in mice

Doxorubicin (DOX) is an effective anticancer drug with the side effect of irreparable cardiomyopathy. Lipocalin-2 (LCN2) has been identified as an important regulator of oxidative stress and inflammation in cardiovascular disease pathophysiology. Here, we demonstrate that LCN2 deletion increases autophagic flux in the DOX-treated hearts. Mice were injected intraperitoneally six times with 30 mg/kg DOX. Echocardiography showed that DOX-treated wild-type (WT) mice had markedly weaker cardiac function compared to saline-treated WT mice. In DOX-treated LCN2 knockout (KO) mice, cardiac function was partially restored. Histological analysis showed a reduction in cardiomyocyte diameter in DOX-treated WT mice that was ameliorated in DOX-treated LCN2KO mice. Cardiac levels of phosphorylated signal transducer and activator of transcription 3, LCN2, heme oxygenase-1, and NAD (P) H dehydrogenase were markedly greater in DOX-treated WT mice than in DOX-treated LCN2KO mice. Light chain 3B (LC3B)II expression was higher in DOX-treated WT mice, but lower in DOX-treated LCN2KO mice when compared to saline-treated WT mice. Less co-localization of LC3B and lysosomal-associated membrane protein 1 was observed in DOX-treated WT mice than in DOX-treated LCN2KO mice. LCN2 co-localized with LC3B-stained cells in the DOX-treated WT mouse heart, but not in the DOX-treated LCN2KO mouse heart. These findings indicate that the cardiotoxic effect of DOX is due to autophagosome accumulation mediated by LCN2 upregulation and that LCN2 may inhibit autophagic flux, leading to DOX-induced cardiomyopathy.