Autophagic Vesicles Research Articles

The autophagic protein FYCO1 controls TNFRSF10/TRAIL receptor induced apoptosis and is inactivated by CASP8 (caspase 8)

ABSTRACT Apoptosis is a tightly controlled cell death program executed by proteases, the so-called caspases. It plays an important role in tissue homeostasis and is often dysregulated in cancer. Here, we identified FYCO1, a protein that promotes microtubule plus end-directed transport of autophagic and endosomal vesicles as a molecular interaction partner of activated CASP8 (caspase 8). The absence of FYCO1 sensitized cells to basal and TNFSF10/TRAIL-induced apoptosis by receptor accumulation and stabilization of the Death Inducing Signaling Complex (DISC). Loss of FYCO1 resulted in impaired transport of TNFRSF10B/TRAIL-R2/DR5 (TNF receptor superfamily member 10b) to the lysosomes in TNFSF10/TRAIL-stimulated cells. More in detail, we show that FYCO1 interacted via its C-terminal GOLD domain with the CCZ1-MON1A complex, which is necessary for RAB7A activation and for the fusion of autophagosomal/endosomal vesicles with lysosomes. We demonstrated that FYCO1 is a novel and specific CASP8 substrate. The cleavage at aspartate 1306 resulted in the release of the C-terminal GOLD domain, inactivating FYCO1 function, and allowing for the progression of apoptosis. Furthermore, the lack of FYCO1 resulted in a stronger and prolonged formation of the TNFRSF1A/TNF-R1 signaling complex. Thus, FYCO1 limits the ligand-induced and steady-state signaling of TNFR-superfamily members, providing a control mechanism that fine-tunes both apoptotic and inflammatory answers. Abbreviations: AP: affinity purification; CHX: cycloheximide; co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; DISC: death-inducing signaling complex; DR: death receptors; doxy: doxycycline; GEF: guanine nucleotide exchange factor; ind: inducible; KD: knockdown; KO: knockout; MS: mass spectrometry; shRNA: short hairpin RNA; siRNA: small interfering RNA; TIP: two-step co-immunoprecipitation; WB: western blot.

Selenomethionine Inhibits NF-κB-mediated Inflammatory Responses of Bovine Mammary Epithelial Cells Caused by Klebsiella pneumoniae by Increasing Autophagic Flux.

Klebsiella pneumoniae (K. pneumoniae) is one of the major pathogens causing bovine clinical mastitis. Autophagy maintains cellular homeostasis and resists excessive inflammation in eukaryotic organisms. Selenomethionine (Se-Met) is commonly used as a source of selenium supplementation for dairy cows. This study aimed to investigate the effects of Se-Met on inflammatory responses mediated by nuclear factor-kappa B (NF-κB) through autophagy. We infected bovine mammary epithelial cell line (MAC-T) with K. pneumoniae and examined the expression of autophagy-related proteins and changes in autophagic vesicles, LC3 puncta, and autophagic flux at various intervals. The results showed that K. pneumoniae activated the early-stage autophagy of MAC-T cells. The levels of LC3-II, Beclin1, and ATG5, as well as the number of LC3 puncta and autophagic vesicles, increased after 2h post-treatment. However, the late-stage autophagic flux was blocked. Furthermore, the effect of autophagy on NF-κB-mediated inflammation was investigated with different autophagy levels. The findings showed that enhanced autophagy inhibited the K. pneumoniae-induced inflammatory responses of MAC-T cells. The opposite results were found with the inhibition of autophagy. Finally, we examined the effect of Se-Met on NF-κB-mediated inflammation based on autophagy. The results indicated that Se-Met alleviated K. pneumoniae-induced autophagic flux blockage, inhibited NF-κB-mediated inflammation, and decreased the adhesion of K. pneumoniae to MAC-T cells. The inhibitory effect of Se-Met on NF-κB-mediated inflammation could be partially blocked by the autophagy inhibitor chloroquine (CQ). Overall, Se-Met attenuated K. pneumoniae-induced NF-κB-mediated inflammatory responses by enhancing autophagic flux.

#3794 AUTOPHAGY PATHWAY ACTIVATION IN IMMUNE-INFLAMMATORY CELLS OF PERITONEAL DIALYSIS PATIENTS

Abstract Background and Aims The better understanding of the biological pathways related to chronic kidney disease (CKD) may be useful for the identification of new therapeutic targets to improve the management of CKD patients. High-throughput technologies with innovative bioinformatics approaches for data analysis are very useful to achieve this objective. Method We reinterrogated our previous gene expression microarray data obtained from peripheral mononuclear cells (PBMCs) of 5 healthy subjects (HS), 9 chronic kidney disease patients with KDOQI stage II-III (CKD) and 10 CKD patients undergoing peritoneal dialysis treatment (PD). Advanced statistical and machine learning methods, including support vector machine learning and partial least squares discriminant analysis, were used for bioinformatics. Western blot and flow cytometry, then, was used to validate these results in an independent study group (10 HS, 10 CKD and 15 PD). Results Statistical analysis revealed that the transcriptomic profile of PD was significantly different from HS and CKD. Instead, no significant differences were observed between HS and CKD. A total of 348 genes (p<0.0001, FDR<5%) were able to differentiate PD patients from the other two study groups. Autophagy resulted one of the most up-regulated pathways in PD and the autophagy related 5 (ATG5), a gene encoding for a key protein involved in autophagic vesicle formation, was the top discriminative transcript (as demonstrated also by the VIP score). Western blot for ATG5 and LC3BII, main phagosome proteins, performed on the validation cohort, confirmed the activation of autophagy pathway only in PBMC of PD patients. Conclusion Our high-throughput data demonstrated, for the first time, that the autophagy pathway is activated in immune-cells of PD patients. This condition may significantly impact their immune-response and -homeostasis and, probably, it represents the last attempt of these circulating cells to avoid apoptosis.

Pooled analysis of frontal lobe transcriptomic data identifies key mitophagy gene changes in Alzheimer's disease brain.

The growing prevalence of Alzheimer's disease (AD) is becoming a global health challenge without effective treatments. Defective mitochondrial function and mitophagy have recently been suggested as etiological factors in AD, in association with abnormalities in components of the autophagic machinery like lysosomes and phagosomes. Several large transcriptomic studies have been performed on different brain regions from AD and healthy patients, and their data represent a vast source of important information that can be utilized to understand this condition. However, large integration analyses of these publicly available data, such as AD RNA-Seq data, are still missing. In addition, large-scale focused analysis on mitophagy, which seems to be relevant for the aetiology of the disease, has not yet been performed. In this study, publicly available raw RNA-Seq data generated from healthy control and sporadic AD post-mortem human samples of the brain frontal lobe were collected and integrated. Sex-specific differential expression analysis was performed on the combined data set after batch effect correction. From the resulting set of differentially expressed genes, candidate mitophagy-related genes were identified based on their known functional roles in mitophagy, the lysosome, or the phagosome, followed by Protein-Protein Interaction (PPI) and microRNA-mRNA network analysis. The expression changes of candidate genes were further validated in human skin fibroblast and induced pluripotent stem cells (iPSCs)-derived cortical neurons from AD patients and matching healthy controls. From a large dataset (AD: 589; control: 246) based on three different datasets (i.e., ROSMAP, MSBB, & GSE110731), we identified 299 candidate mitophagy-related differentially expressed genes (DEG) in sporadic AD patients (male: 195, female: 188). Among these, the AAA ATPase VCP, the GTPase ARF1, the autophagic vesicle forming protein GABARAPL1 and the cytoskeleton protein actin beta ACTB were selected based on network degrees and existing literature. Changes in their expression were further validated in AD-relevant human in vitro models, which confirmed their down-regulation in AD conditions. Through the joint analysis of multiple publicly available data sets, we identify four differentially expressed key mitophagy-related genes potentially relevant for the pathogenesis of sporadic AD. Changes in expression of these four genes were validated using two AD-relevant human in vitro models, primary human fibroblasts and iPSC-derived neurons. Our results provide foundation for further investigation of these genes as potential biomarkers or disease-modifying pharmacological targets.

Cinchonine exerts anti-tumor and immunotherapy sensitizing effects in lung cancer by impairing autophagic-lysosomal degradation

Currently, there are several treatments approaches available for lung cancer; however, patients who develop drug resistance or have poor survival rates urgently require new therapeutic strategies for lung cancer. In autophagy, damaged proteins or organelles are enclosed within autophagic vesicles with a bilayer membrane structure and transported to the lysosomes for degradation and recirculation. Autophagy is a crucial pathway involved in the clearance of reactive oxygen species (ROS) and damaged mitochondria. Meanwhile, inhibiting autophagy is a promising strategy for cancer treatment. In this study, we found for the first time that Cinchonine (Cin) can act as an autophagy suppressor and exert anti-tumor effects. Cin significantly inhibited the proliferation, migration, and invasion of cancer cells in vitro and the tumor growth and metastasis in vivo, without obvious toxic effects. We found that Cin suppressed the autophagic process by blocking autophagosome degradation through the inhibition of the maturation of lysosomal hydrolases. Cin-mediated autophagy inhibition resulted in the elevated ROS level and the accumulation of damaged mitochondria, which in turn promoted apoptosis. N-acetylcysteine, a potential ROS scavenger, significantly suppressed Cin-induced apoptosis. Additionally, Cin upregulated programmed death-ligand 1 (PD-L1) expression in lung cancer cells by inhibiting autophagy. Compared with monotherapy and control group, the combined administration of anti-PD-L1 antibody and Cin significantly reduced tumor growth. These results suggest that Cin exerts anti-tumor effects by inhibiting autophagy, and that the combination of Cin and PD-L1 blockade has synergistic anti-tumor effects. The data demonstrates the significant clinical potential of Cin in lung cancer treatment.

Profiling of purified autophagic vesicle degradome in the maturing and aging brain

Autophagy disorders prominently affect the brain, entailing neurodevelopmental and neurodegenerative phenotypes in adolescence or aging, respectively. Synaptic and behavioral deficits are largely recapitulated in mouse models with ablation of autophagy genes in brain cells. Yet, the nature and temporal dynamics of brain autophagic substrates remain insufficiently characterized. Here, we immunopurified LC3-positive autophagic vesicles (LC3-pAVs) from the mouse brain and proteomically profiled their content. Moreover, we characterized the LC3-pAV content that accumulates after macroautophagy impairment, validating a brain autophagic degradome. We reveal selective pathways for aggrephagy, mitophagy, and ER-phagy via selective autophagy receptors, and the turnover of numerous synaptic substrates, under basal conditions. To gain insight into the temporal dynamics of autophagic protein turnover, we quantitatively compared adolescent, adult, and aged brains, revealing critical periods of enhanced mitophagy or degradation of synaptic substrates. Overall, this resource unbiasedly characterizes the contribution of autophagy to proteostasis in the maturing, adult, and aged brain.

Ambient NO2 hinders neutrophil extracellular trap formation in rats: Assessment of the role of neutrophil autophagy

NO2 has been known to impair immunity and exacerbate susceptibility to infectious diseases. However, scant notice has been taken of the effect of NO2 on neutrophils. Neutrophil extracellular traps (NETs) formation is necessary for NETosis development by neutrophils as an immune system against pathogens. By analyzing the morphology and signature components of NETs, we focused for the first time on finding that 10 ppm of NO2 exposure for 15 consecutive days can hinder the formation of NETs. Next, we used NO2 in vivo derivatives to probe the mechanism for NETs formation in vitro. Our findings showed that NO2 suppression of respiratory burst levels and mitogen-activated protein kinase (MAPK)/Phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling was related to NO2 reduction in NETs formation. Inhibition of phorbol myristate acetate (PMA)-induced NETs formation by NO2 hindered autophagy, as evidenced by increased mTOR protein expression, decreased LC3 protein expression, and reduced autophagic vesicles. By activating mTOR-mediated autophagy, rapamycin (Rapa) reduced the inhibition of PMA-induced NETs by NO2. This study will provide valuable insights into the mechanisms of immunotoxicity of NO2, new insights into the etiology of diseases linked to NETs formation, and a theoretical basis for protection against such illnesses.

Peroxisome proliferator-activated receptor γ activation ameliorates liver fibrosis-differential action of transcription factor EB and autophagy on hepatocytes and stellate cells.

Peroxisome proliferator-activated receptor γ (PPARγ) activation suppresses HSC activation and liver fibrosis. Moreover, autophagy is implicated in hepatic lipid metabolism. Here, we determined whether PPARγ activation ameliorates HSC activation by downregulating transcription factor EB (TFEB)-mediated autophagy. Atg7 or Tfeb knockdown in human HSC line LX-2 cells downregulated the expression of fibrogenic markers including α smooth muscle actin, glial fibrillary acidic protein, and collagen type 1. Conversely, Atg7 or Tfeb overexpression upregulated fibrogenic marker expression. Rosiglitazone (RGZ)-mediated PPARγ activation and/or overexpression in LX-2 cells and primary HSCs decreased autophagy, as indicated by LC3B conversion, total and nuclear-TFEB contents, mRFP-LC3 and BODIPY 493/503 colocalization, and GFP-LC3 and LysoTracker colocalization. RGZ treatment decreased liver fat content, liver enzyme levels, and fibrogenic marker expression in high-fat high-cholesterol diet-fed mice. Electron microscopy showed that RGZ treatment restored the high-fat high-cholesterol diet-mediated lipid droplet decrease and autophagic vesicle induction in primary HSCs and liver tissues. However, TFEB overexpression in LX-2 cells offset the aforementioned effects of RGZ on autophagic flux, lipid droplets, and fibrogenic marker expression. Activation of PPARγ with RGZ ameliorated liver fibrosis and downregulation of TFEB and autophagy in HSCs may be important for the antifibrotic effects of PPARγ activation.

Raising awareness of TBC1 domain‐containing kinase (TBCK) epileptic encephalopathy among Puerto Rican children

AbstractBackgroundTBC1 domain‐containing kinase (TBCK) syndrome is a rare autosomal recessive genetic disorder that presents with infantile hypotonia, intellectual disability, motor impairment, and drug‐resistant epilepsy. The abnormal TBCK protein alters the mammalian target of rapamycin complex 1 (mTORC1), leading to accumulation of autophagic vesicles within fibroblasts. Thirty‐five previously reported individuals with TBCK were identified.MethodsWe present four children with TBCK due to the p.R126X (p.ARG126*) mutation, a variant predominating among children of Puerto Rican descent. This mutation has been associated with TBCK‐encephaloneuropathy or Boricua syndrome.DiscussionThese four of our patients were diagnosed within a span of 6 months, suggesting that the prevalence of the syndrome could be higher than expected among individuals from Puerto Rico. We believe this finding was largely due to the increasing availability and affordability of modern genetic testing in Puerto Rico, illustrating the importance of equitable access to genetic testing to all pediatric patients with similar symptomatology. Prompt diagnosis facilitates appropriate medical management and prognosis counseling.

Overexpression of DAPK1 and Beclin1 under oxygen and glucose deprivation conditions promotes excessive autophagy and apoptosis in A549 cells

AbstractIn this study, we aimed to determine the specific roles of death‐associated protein kinase 1 (DAPK1) and Beclin1 in non‐small cell lung cancer (NSCLC) under oxygen and glucose deprivation (OGD). We found that OGD caused most cells to shrink, aggregate, and produce many vacuoles in the cytoplasm. Transmission electron microscopy revealed the presence of autophagic vesicles in the OGD group but not in the Control group. Moreover, the cell counting kit‐8 assay showed that cell proliferation was reduced in the OGD group. Quantitative reverse transcription‐polymerase chain reaction, western blot, and cell function assays showed that DAPK1 overexpression under OGD promoted apoptosis and autophagy in A549 cells. The coimmunoprecipitation assay confirmed the interaction between DAPK1 and Beclin1 protein. Moreover, knockdown of Beclin1 inhibited autophagy, but its overexpression promoted apoptosis in A549 cells. In vivo tumorigenesis experiment revealed that overexpression of DAPK1 promoted A549 cell apoptosis. Collectively, overexpression of DAPK1 and Beclin1 under OGD promoted excessive autophagy and apoptosis in A549 cells. Our study may provide a novel therapeutic target and theoretical basis for NSCLC treatment.

Echovirus induces autophagy to promote viral replication via regulating mTOR/ULK1 signaling pathway.

Among enteroviruses, echovirus can cause severe illnesses in neonates or infants, with high morbidity and mortality. Autophagy, a central component of host defense mechanisms, can function against diverse infections. In the present study, we investigated the interplay between echovirus and autophagy. We demonstrated that echovirus infection increases LC3-II expression dose-dependently, accompanied by an increased intracellular LC3 puncta level. In addition, echovirus infection induces the formation of autophagosome. These results suggest that echovirus infection induces autophagy machinery. Furthermore, phosphorylated mTOR and ULK1 were both decreased upon echovirus infection. In contrast, both levels of the vacuolar protein sorting 34 (VPS34) and Beclin-1, the downstream molecules which play essential roles in promoting the formation of autophagic vesicles, increased upon virus infection. These results imply that the signaling pathways involved in autophagosome formation were activated by echovirus infection. Moreover, induction of autophagy promotes echovirus replication and viral protein VP1 expression, while inhibition of autophagy impairs VP1 expression. Our findings suggest that autophagy can be induced by echovirus infection via regulating mTOR/ULK1 signaling pathway and exhibits a proviral function, revealing the potential role of autophagy in echovirus infection.

G-protein signaling modulator 1 promotes colorectal cancer metastasis by PI3K/AKT/mTOR signaling and autophagy.

Colorectal cancer is the second most common malignant tumor worldwide. A deeper insight into the mechanisms underlying colorectal cancer metastasis is urgently needed. G-protein signaling modulator 1 and autophagy play critical roles in tumor migration and invasion. However, the biological functions and regulatory networks of G-protein signaling modulator 1 and autophagy have not yet been fully studied. We performed immunohistochemistry and clinic-pathological characteristic analysis in 328 human colorectal cancer specimens to identify the clinical role of G-protein signaling modulator 1 in colorectal cancer. An in vitro coculture system and a tumor metastasis mouse model were used to explore the biological function of G-protein signaling modulator 1 on tumor metastasis. Autophagic flux detection like GFP-LC3B signal immunofluorescence and electron microscope observation of autophagic vesicles and confocal microscope detection were used to gain insights into the underlying role of G-protein signaling modulator 1 in autophagy. We found that G-protein signaling modulator 1 was abundantly expressed in colorectal cancer tissues and was associated with lymph node metastasis and poor prognosis. Furthermore, our bioinformatic and functional studies demonstrated that G-protein signaling modulator 1 significantly promoted cell migration and invasion, both in vitro and in vivo. Mechanistically, we demonstrated that G-protein signaling modulator 1 could promote colorectal cancer cell migration and invasion and inhibit autophagy and by activating the PI3K/AKT/mTOR pathway. We proposed that G-protein signaling modulator 1 promotes colorectal cancer metastasis by modulating autophagy through the PI3K/AKT/mTOR pathway.

PPARα Inhibits Astrocyte Inflammation Activation by Restoring Autophagic Flux after Transient Brain Ischemia

Astrocyte inflammation activation is an important cause that hinders the recovery of motor function after cerebral ischemia. However, its molecular mechanism has not yet been clearly clarified. The peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated nuclear transcriptional factor. This study aims to further clarify the role of PPARα in astrocyte inflammation activation after cerebral ischemia and to explore the underlying mechanism. Astrocyte activation was induced in an in vivo model by transient middle cerebral artery occlusion (tMCAO) in mice. The in vitro model was induced by an oxygen-glucose deprivation/reoxygenation (OGD/R) in a primary culture of mouse astrocyte. PPARα-deficient mice were used to observe the effects of PPARα on astrocyte activation and autophagic flux. Our results showed that PPARα was mainly expressed in activated astrocytes during the chronic phase of brain ischemia and PPARα dysfunction promoted astrocyte inflammatory activation. After cerebral ischemia, the expressions of LC3-II/I and p62 both increased. Autophagic vesicle accumulation was observed by electron microscopy in astrocytes, and the block of autophagic flux was indicated by an mRFP-GFP-LC3 adenovirus infection assay. A PPARα deficit aggravated the autophagic flux block, while PPARα activation preserved the lysosome function and restored autophagic flux in astrocytes after OGD/R. The autophagic flux blocker bafilomycin A1 and chloroquine antagonized the effect of the PPARα agonist on astrocyte activation inhibition. This study identifies a potentially novel function of PPARα in astrocyte autophagic flux and suggests a therapeutic target for the prevention and treatment of chronic brain ischemic injury.

The synthetic TRPML1 agonist ML-SA1 rescues Alzheimer-related alterations of the endosomal-autophagic-lysosomal system.

Abnormalities in the endosomal-autophagic-lysosomal (EAL) system are an early event in Alzheimer's disease (AD) pathogenesis. However, the mechanisms underlying these abnormalities are unclear. The transient receptor potential channel mucolipin 1(TRPML1, also known as MCOLN1), a vital endosomal-lysosomal Ca2+ channel whose loss of function leads to neurodegeneration, has not been investigated with respect to EAL pathogenesis in late-onset AD (LOAD). Here, we identify pathological hallmarks of TRPML1 dysregulation in LOAD neurons, including increased perinuclear clustering and vacuolation of endolysosomes. We reveal that induced pluripotent stem cell (iPSC)-derived human cortical neurons expressing APOE ε4, the strongest genetic risk factor for LOAD, have significantly diminished TRPML1-induced endolysosomal Ca2+ release. Furthermore, we found that blocking TRPML1 function in primary neurons by depleting the TRPML1 agonist PI(3,5)P2 via PIKfyve inhibition, recreated multiple features of EAL neuropathology evident in LOAD. This included increased endolysosomal Ca2+ content, enlargement and perinuclear clustering of endolysosomes, autophagic vesicle accumulation and early endosomal enlargement. Strikingly, these AD-like neuronal EAL defects were rescued by TRPML1 reactivation using its synthetic agonist ML-SA1. These findings implicate defects in TRPML1 in LOAD EAL pathogenesis and present TRPML1 as a potential therapeutic target.

Role of Autophagy Mediated by AMPK/DDiT4/mTOR Axis in HT22 Cells Under Oxygen and Glucose Deprivation/Reoxygenation.

Background: cerebral ischemia/reperfusion (I/R) injury is an important complication of ischemic stroke, and autophagy is one of the mechanisms of it. In this study, we aimed to determine the role and mechanism of autophagy in cerebral I/R injury. Methods: the oxygen and glucose deprivation/reoxygenation (OGD/R) method was used to model cerebral I/R injury in HT22 cells. CCK-8 and LDH were conducted to detect viability and damage of the cells, respectively. Apoptosis was measured by flow cytometry and Tunel staining. Autophagic vesicles of HT22 cells were assessed by transmission electron microscopy. Western blotting analysis was used to examine the protein expression involving AMPK/DDiT4/mTOR axis and autophagy-related proteins. 3-Methyladenine and rapamycin were, respectively, used to inhibit and activate autophagy, compound C and AICAR acted as AMPK inhibitor and activator, respectively, and were used to control the starting link of AMPK/DDiT4/mTOR axis. Results: autophagy was activated in HT22 cells after OGD/R was characterized by an increased number of autophagic vesicles, the expression of Beclin1 and LC3II/LC3I, and a decrease in the expression of P62. Rapamycin could increase the viability, reduce LDH leakage rate, and alleviate cell apoptosis in OGD/R cells by activating autophagy. 3-Methyladenine played an opposite role to rapamycin in OGD/R cells. The expression of DDiT4 and the ratio of p-AMPK/AMPK were increased after OGD/R in HT22 cells. While the ratio of p-mTOR/mTOR was reduced by OGD/R, AICAR effectively increased the number of autophagic vesicles, improved viability, reduced LDH leakage rate, and alleviated apoptosis in HT22 cells which suffered OGD/R. However, the effects of compound C in OGD/R HT22 cells were opposite to that of AICAR. Conclusions: autophagy is activated after OGD/R; autophagy activator rapamycin significantly enhanced the protective effect of autophagy on cells of OGD/R. AMPK/DDiT4/mTOR axis is an important pathway to activate autophagy, and AMPK/DDiT4/mTOR-mediated autophagy significantly alleviates cell damage caused by OGD/R.

Lysosomal dysfunction in Schwann cells is involved in bortezomib-induced peripheral neurotoxicity.

Bortezomib (BTZ) is a proteasome inhibitor serves as a first-line drug for multiple myeloma treatment. BTZ-induced peripheral neuropathy (BIPN) is the most common adverse effect of BTZ with an incidence as high as 40-60%. However, the pathological mechanisms underlying BIPN remain largely unclear. BTZ leads to dramatic Schwann cell demyelination in sciatic nerves. Previous studies implied that myelin debris was predominantly degraded via autophagy-lysosome pathway in Schwann cells. However, the association of autophagy with BIPN has not been made. Mice were treated with BTZ (2mg/kg, i.v.) on Day1 and Day4 each week for continuous 4weeks. BTZ-treated mice showed enhanced mechanical hyperalgesia, decreased tail nerve conduction and sciatic nerve demyelination. Unexpectedly, BTZ led to the accumulation of autophagic vesicles, LC3-II and p62 in the sciatic nerve. Moreover, BTZ blocked autophagic flux in RSC96 Schwann cells as determined by mcherry-GFP-LC3 assay, suggesting BTZ may impair lysosomal function rather than inducing autophagy in Schwann cells. BTZ significantly reduced the lysosomal activity in Schwann cells as determined by reduced LysoTracker Red and DQ-Red-BSA staining and increased the level of immature Cathepsin B (CTSB). Remarkably, lysosomal activators PP242 and Torin1, significantly reversed the blockage of autophagic flux by BTZ. We further verified that Torin1 rescued the demyelination, nerve conduction and reduced the mechanical hyperalgesia in BIPN mice. Additionally, Torin1 did not compromise the efficacy of BTZ in suppressing multiple myeloma RPMI8226 cell. Taken together, we identified that lysosomal dysfunction in Schwann cells caused by BTZ is involved in the BIPN pathology. Improved lysosomal function in Schwann cells can be a promising strategy for BIPN treatment.

Metformin Inhibits Autophagy, Mitophagy and Antagonizes Doxorubicin-Induced Cardiomyocyte Death.

The antidiabetic drug metformin has been shown to reduce cardiac injury under various pathological conditions, including anticancer drug doxorubicin (DOX)-induced cardiotoxicity, which makes metformin a prime candidate for repurposing. However, the mechanisms that mediate the cardioprotective effects of metformin remain highly controversial. In this study, we tested a prevailing hypothesis that metformin activates autophagy/mitophagy to reduce DOX cardiotoxicity. FVB/N mice and H9C2 cardiac myoblasts were treated with metformin, respectively. Autophagy/mitophagy was determined by Western blot analysis of microtubule-associated protein light chain 3, form-II (LC3-II), a well-established marker of autophagic vesicles. Although metformin had minimal effects on basal LC3-II levels, it significantly inhibited the accumulation of LC3-II levels by the lysosomal protease inhibitors pepstatin A and E64d in both total cell lysates and mitochondrial fractions. Also, dual fluorescent autophagy/mitophagy reporters demonstrated that metformin slowed the degradation rate of autophagic cargos or mitochondrial fragments in the lysosomes. These surprising results suggest that metformin inhibits rather than stimulates autophagy/mitophagy, sharply contrasting the popular belief. In addition, metformin diminished DOX-induced autophagy/mitophagy as well as cardiomyocyte death. Together, these results suggest that the cardioprotective effects of metformin against DOX cardiotoxicity may be mediated by its ability to inhibit autophagy and mitophagy, although the underlying molecular mechanisms remain to be determined.

Estrogen receptor alpha mediates 17β-estradiol, up-regulates autophagy and alleviates hydrogen peroxide-induced vascular senescence.

Atherosclerosis threatens human health by developing cardiovascular diseases, the deadliest disease world widely. The major mechanism contributing to the formation of atherosclerosis is mainly due to vascular endothelial cell (VECs) senescence. We have shown that 17β-estradiol (17β-E2) may protect VECs from senescence by upregulating autophagy. However, little is known about how 17β-E2 activates the autophagy pathway to alleviate cellular senescence. Therefore, the aim of this study is to determine the role of estrogen receptor (ER) α and β in the effects of 17β-E2 on vascular autophagy and aging through in vitro and in vivo models. Hydrogen peroxide (H2O2) was used to establish Human Umbilical Vein Endothelial Cells (HUVECs) senescence. Autophagy activity was measured through immunofluorescence and immunohistochemistry staining of light chain 3 (LC3) expression. Inhibition of ER activity was established using shRNA gene silencing and ER antagonist. Compared with ER-β knockdown, we found that knockdown of ER-α resulted in a significant increase in the extent of HUVEC senescence and senescence-associated secretory phenotype (SASP) secretion. ER-α-specific shRNA was found to reduce 17β-E2-induced autophagy, promote HUVEC senescence, disrupt the morphology of HUVECs, and increase the expression of Rb dephosphorylation and SASP. These in vitro findings were found consistent with the in vivo results. In conclusion, our data suggest that 17β-E2 activates the activity of ER-α and then increases the formation of autophagosomes (LC3 high expression) and decreases the fusion of lysosomes with autophagic vesicles (P62 low expression), which in turn serves to decrease the secretion of SASP caused by H2O2 and consequently inhibit H2O2-induced senescence in HUVEC cells.

Abstract A031: Combination of hedgehog targeting and autophagy inhibitor potentiate cytotoxicity in 3D spheroids of cervical cancer: A promising therapeutic strategy in metastatic disease

Abstract Cervical cancer is the second most common malignancy in women and a major cause of morbidity and mortality worldwide. Statistics show that present biologic therapy has had little impact on survival in recurrent and metastatic disease. Consequently, there is an urgent need to explore the best treatment options for advanced cervical cancer. Hedgehog (Hh) signaling pathway has been shown to play an important role in metastasis, recurrence and drug resistance of cervical cancer. Therefore, inhibition of hedgehog signaling pathway is emerging as a new strategy for the treatment of advanced cervical cancer. Reports from various solid tumors show that efficacy of targeted therapy can be compromised due to the cytoprotective effect of autophagy upregulation in cancer cells. Therefore, the present study was designed to elucidate the effects of targeting Hh pathway on autophagic flux in cervical cancer spheroids (cervicospheroids). We developed 3D cervicospheroids as an in vitro model to study the Hh signaling and role of autophagy in cervical cancer. Characterisation of the cervicospheroids showed high expression of stemness genes like oct4, sox2 , nanog and CD 133 as well as epithelial to mesenchymal trasition genes like vimentin, snail and twist indicating that spheroids were enriched in cancer stem cell population. Acridine orange staining of 3D spheroids showed that autophagy was also enhanced in the cervicospheroids compared to monolayer culture. We observed a significant positive correlation between the expression of hedgehog mediator, Gli1 and autophagy protein, LC31 in immunohistochemistry data of archival sections of 45 patients of advanced cervical cancer. A higher autophagy flux, as indicated by presence of larger number autophagic vesicle compared to normal cervix, was also observed in these sections. These results further validated our in vitro observations in the 3D model of cervicospheres. MTT data, showed that autophagy help to evade the cytotoxicity of the chemotherapeutic drug, cisplatin in the cervicospheres. We further found an aberrantly activated Hh pathway in the cervicospheroids and observed that the effect of cisplatin and Hh inhibitor, GANT61 was potentiated when the autophagy inhibitor , chloroquine, was added to the 3D spheroid culture. These results led us to conclude that that 3-D spheroid grown in nonadherent conditions can be used as surrogate model to study molecular pathways involved in metastasis. Based on our present findings, we suggest a combinatorial approach targeting both cytoprotective autophagy and Hh pathway as a new therapeutic approach for targeting cancer stem cell population in cervical cancer . Citation Format: Shalmoli Bhattacharyya, Renaissa De, Radhika Srinivasan. Combination of hedgehog targeting and autophagy inhibitor potentiate cytotoxicity in 3D spheroids of cervical cancer: A promising therapeutic strategy in metastatic disease [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A031.

EGFR-dependent suppression of synaptic autophagy is required for neuronal circuit development

The development of neuronal connectivity requires stabilization of dynamic axonal branches at sites of synapse formation. Models that explain how axonal branching is coupled to synaptogenesis postulate molecular regulators acting in a spatiotemporally restricted fashion to ensure branching toward future synaptic partners while also stabilizing the emerging synaptic contacts between such partners. We investigated this question using neuronal circuit development in the Drosophila brain as a model system. We report that epidermal growth factor receptor (EGFR) activity is required in presynaptic axonal branches during two distinct temporal intervals to regulate circuit wiring in the developing Drosophila visual system. EGFR is required early to regulate primary axonal branching. EGFR activity is then independently required at a later stage to prevent degradation of the synaptic active zone protein Bruchpilot (Brp). Inactivation of EGFR results in a local increase of autophagy in presynaptic branches and the translocation of active zone proteins into autophagic vesicles. The protection of synaptic material during this later interval of wiring ensures the stabilization of terminal branches, circuit connectivity, and appropriate visual behavior. Phenotypes of EGFR inactivation can be rescued by increasing Brp levels or downregulating autophagy. In summary, we identify a temporally restricted molecular mechanism required for coupling axonal branching and synaptic stabilization that contributes to the emergence of neuronal wiring specificity.