Fusion Of Lysosomes Research Articles

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.

PDZD8-deficient mice manifest behavioral abnormalities related to emotion, cognition, and adaptation due to dyslipidemia in the brain

Although dyslipidemia in the brain has been implicated in neurodegenerative disorders, the molecular mechanisms underlying its pathogenesis have been largely unclear. PDZD8 is a lipid transfer protein and mice deficient in PDZD8 (PDZD8-KO mice) manifest abnormal accumulation of cholesteryl esters (CEs) in the brain due to impaired lipophagy, the degradation system of lipid droplets. Here we show the detailed mechanism of PDZD8-dependent lipophagy. PDZD8 transports cholesterol to lipid droplets (LDs), and eventually promotes fusion of LDs and lysosomes. In addition, PDZD8-KO mice exhibit growth retardation, hyperactivity, reduced anxiety and fear, increased sensorimotor gating, and impaired cued fear conditioned memory and working memory. These results indicate that abnormal CE accumulation in the brain caused by PDZD8 deficiency affects emotion, cognition and adaptive behavior, and that PDZD8 plays an important role in the maintenance of brain function through lipid metabolism.

A BORC-dependent molecular pathway for vesiculation of cell corpse phagolysosomes

Phagocytic clearance is important to provide cells with metabolites and regulate immune responses, but little is known about how phagolysosomes finally resolve their phagocytic cargo of cell corpses, cell debris, and pathogens. While studying the phagocytic clearance of non-apoptotic polar bodies in C.elegans, we previously discovered that phagolysosomes tubulate into small vesicles to facilitate corpse clearance within 1.5 h. Here, we show that phagolysosome vesiculation depends on amino acid export by the solute transporter SLC-36.1 and the activation of TORC1. We demonstrate that downstream of TORC1, BLOC-1-related complex (BORC) is de-repressed by Ragulator through the BORC subunit BLOS-7. In addition, the BORC subunit SAM-4 is needed continuously to recruit the small GTPase ARL-8 to the phagolysosome for tubulation. We find that disrupting the regulated GTP-GDP cycle of ARL-8 reduces tubulation by kinesin-1, delays corpse clearance, and mislocalizes ARL-8 away from lysosomes. We also demonstrate that mammalian phagocytes use BORC to promote phagolysosomal degradation, confirming the conserved importance of TOR and BORC. Finally, we show that HOPS is required after tubulation for the rapid degradation of cargo in small phagolysosomal vesicles, suggesting that additional rounds of lysosome fusion occur. Thus, by observing single phagolysosomes over time, we identified the molecular pathway regulating phagolysosome vesiculation that promotes efficient resolution of phagocytosed cargos.

Myo1b Promotes Premature Endothelial Senescence and Dysfunction via Suppressing Autophagy: Implications for Vascular Aging.

Endothelial cell (EC) senescence characterized by an irreversible growth arrest leading to endothelial dysfunction has been implicated in vascular aging and aging-associated cardiovascular diseases. Autophagy plays a crucial role in the modulation of cellular senescence. Our previous showed that myosin 1b (Myo1b), one family of nonfilamentous class-1 myosin, was reported to be involved in the modulation of human smooth muscle cell senescence. However, the role of Myo1b in the modulation of EC senescence with links to autophagy has yet to be elucidated. In this study, we sought to explore the role of Myo1b in endothelial senescence and further elucidate the underlying mechanisms. Here, we show prominent upregulation of Myo1b in senescent ECs in comparison with nonsenescence ECs in both mRNA and protein expression levels. Silencing Myo1b in senescent cells ameliorates endothelial dysfunctions and reverses endothelial senescence phenotypic changes such as senescence-associated-β-galactosidase activity, cyclin-dependent kinase inhibitor p21WAF1, expression of vascular adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1), and the senescence-associated cytokines. In contrast, in nonsenescent cells, overexpressing Myo1b promotes endothelial senescence and suppresses autophagy through the impairment of autophagosome and lysosome fusion. The interaction between Myo1b and LRRK2 through Myo1b tail domain promotes intracellular calcium elevation, which results in the inhibition of autophagic flux. In vitro and in vivo aging models, Myo1b knockdown in senescent ECs and wild type-aged mice is able to enhance autophagy and ameliorate aging-associated endothelial dysfunction. Taken together, our studies reveal a new function for Myo1b, that is, to couple LRRK2 assembly to promote an increase in intracellular calcium level, which impairs the autophagosome-lysosome fusion, and ultimately the promotion of EC senescence and vascular aging.

Diabetes influences the fusion of autophagosomes with lysosomes in SH-SY5Y cells and induces Aβ deposition and cognitive dysfunction in STZ-induced diabetic rats

Diabetes has been regarded as an independent risk factor for Alzheimer’s disease (AD). Our previous study found that diabetes activated autophagy, but lysosome function was impaired. Autophagy-lysosome dysfunction may be involved in Aβ deposition in diabetic cognitive impairment. In the present study, we used STZ-induced diabetic rats and SH-SY5Y cells to investigate whether diabetes inhibits autophagosome fusion with lysosomes. We found that in the in vivo study, STZ-induced diabetic rats exhibited cognitive dysfunction, and the lysosome function-related factors CTSL, CTSD, and Rab7 were decreased (P < 0.05). In an in vitro study, the mRFP-GFP-LC3 assay showed that the fusion of autophagosomes with lysosomes was partly blocked in SH-SY5Y cells. High glucose treatment downregulated the number of autophagolysosomes, downregulated CTSD, CTSL, and Rab7 expression (P < 0.05), and then influenced the function of ACP2 to partly block the fusion of autophagosomes and lysosomes to inhibit Aβ clearance. These findings indicate that high glucose treatment affected lysosome function, interfered with the fusion of autophagosomes with lysosomes, and partly blocked autophagic flux to influence Aβ clearance.

Physiology of autophagy

The process of intracellular components being broken down by lysosomes is known as autophagy. By destroying intracellular components and giving cells breakdown products, autophagy plays a crucial part in preserving and regulating cell homeostasis [1]. The most recent studies have demonstrated that autophagy serves a variety of physiological and pathological functions, some of which are intricate. There are fewer autophagic vacuoles and poorer lysosome fusion as autophagy decreases with age. The innate immune system is strengthened by activating the autophagic pathway, which has also been shown to be helpful in avoiding different foreign infections. Tau and synuclein protein accumulation result from a decrease in autophagic protein clearance in neurodegenerative diseases. Similar to this, lysosomal lipid accumulation results from autophagic failure. Tumors are first suppressed by autophagy, but they are subsequently immune system-protected. Muscle and cardiac issues are favourably and negatively regulated by autophagy, respectively.

Biochemical and Microscopic Analyses for Sphingolipids and Its Related Molecules in Phagosomes.

Glycosphingolipids (GSLs) form GSL-enriched microdomains, together with sphingomyelin (SM), cholesterol, glycosylphosphatidylinositol (GPI)-anchored proteins, and membrane-associated signaling molecules. GSL-enriched microdomains mediate a variety of physiological functions, including innate immune responses. Innate immune responses are initialized by the binding of host pattern recognition receptors (PRRs) to pathogen-associated molecular patterns (PAMPs) expressed in microorganisms. This binding triggers phagocytosis and leads to the formation of a phagosome-containing microorganism and the subsequent lysosomal fusion with a phagosome. To detect the molecular interaction between GSL-enriched microdomains, sphingolipids, and signaling molecules from the uptake of the microorganism until the phagosome-containing microorganism fuses with lysosomes, biochemical and microscopic approaches are indispensable. Here, we describe the detailed methods for isolating phagosomes and observing the molecular interaction using a superresolution microscope. Our methodology provides a strategy for exploring the molecular interaction between the host and pathogen and for developing new treatment approaches.

Rab26 controls secretory granule maturation and breakdown in Drosophila

At the onset of Drosophila metamorphosis, plenty of secretory glue granules are released from salivary gland cells and the glue is deposited on the ventral side of the forming (pre)pupa to attach it to a dry surface. Prior to this, a poorly understood maturation process takes place during which secretory granules gradually grow via homotypic fusions, and their contents are reorganized. Here we show that the small GTPase Rab26 localizes to immature (smaller, non-acidic) glue granules and its presence prevents vesicle acidification. Rab26 mutation accelerates the maturation, acidification and release of these secretory vesicles as well as the lysosomal breakdown (crinophagy) of residual, non-released glue granules. Strikingly, loss of Mon1, an activator of the late endosomal and lysosomal fusion factor Rab7, results in Rab26 remaining associated even with the large glue granules and a concomitant defect in glue release, similar to the effects of Rab26 overexpression. Our data thus identify Rab26 as a key regulator of secretory vesicle maturation that promotes early steps (vesicle growth) and inhibits later steps (lysosomal transport, acidification, content reorganization, release, and breakdown), which is counteracted by Mon1.

Increased mTOR Signaling and Impaired Autophagic Flux Are Hallmarks of SARS-CoV-2 Infection.

The COVID-19 (Coronavirus Disease 2019), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), severely affects mainly individuals with pre-existing comorbidities. Here our aim was to correlate the mTOR (mammalian/mechanistic Target of Rapamycin) and autophagy pathways with the disease severity. Through western blotting and RNA analysis, we found increased mTOR signaling and suppression of genes related to autophagy, lysosome, and vesicle fusion in Vero E6 cells infected with SARS-CoV-2 as well as in transcriptomic data mining of bronchoalveolar epithelial cells from severe COVID-19 patients. Immunofluorescence co-localization assays also indicated that SARS-CoV-2 colocalizes within autophagosomes but not with a lysosomal marker. Our findings indicate that SARS-CoV-2 can benefit from compromised autophagic flux and inhibited exocytosis in individuals with chronic hyperactivation of mTOR signaling.

Neocryptotanshinone protects against myocardial ischemia-reperfusion injury by promoting autolysosome degradation of protein aggregates via the ERK1/2-Nrf2-LAMP2 pathway

BackgroundAggrephagy is a critical compensatory mechanism for the elimination of misfolded proteins resulting from stress and depends on the autolysosome degradation of protein aggregates. However, there have been few mechanism research related to aggrephagy in myocardial ischemia/reperfusion (I/R) injury. Neocryptotanshinone (NCTS) is a fat-soluble active compound extracted from Salvia miltiorrhiza, and may be cardioprotective against I/R. However, the efficacy and specific mechanism of NCTS on I/R have not been studied. PurposeThe current study aimed to investigate the molecular mechanism of NCTS involved in the therapeutic effect on I/R, with a special emphasis on the up-regulation of the ERK1/2-Nrf2-LAMP2 pathway to increase autolysosomal degradation during aggrephagy. MethodsA rat model of myocardial I/R injury was constructed by left anterior descending (LAD) ligation-reperfusion. To verify cardiac protection, autolysosome clearance of protein aggregates, and their intracellular biological mechanism, an oxygen-glucose deprivation/recovery (OGD/R)-induced H9c2 cardiomyocyte model was created. ResultsNCTS was found to have a significant cardioprotective effect in I/R rats as evidenced by remarkably improved pathological anatomy, decreased myocardial damage indicators, and substantially enhanced cardiac performance. Mechanistically, NCTS might boost the levels of LAMP2 mRNA and protein, total and Ser40 phosphorylated Nrf2, and Thr202/Tyr204p-ERK1/2 protein. Simultaneously, the cytoplasmic Nrf2 level was reduced after NCTS administration, which was contrary to the total Nrf2 content. However, these beneficial changes were reversed by the co-administration with ERK1/2 inhibitor, PD98059. NCTS therapy up-regulated Rab7 protein content, Cathepsin B activity, and lysosomal acidity, while down-regulating autophagosome numbers, Ubiquitin (Ub), and autophagosome marker protein accumulations through the above signaling pathway. This might indicate that NCTS enhanced lysosomal fusion and hydrolytic capacity. It was also found that NCTS intervention limited oxidative stress and cellular apoptosis both in vivo and in vitro. ConclusionsWe reported for the first time that NCTS promoted the autolysosome removal of protein aggregation both in vivo and in vitro, to exert the therapeutic advantages of myocardial I/R injury. This was reliant on the up-regulation of the ERK1/2-Nrf2-LAMP2 signaling pathway.

AP2S1 regulates APP degradation through late endosome-lysosome fusion in cells and APP/PS1 mice.

AP2S1 is the sigma 2 subunit of adaptor protein 2 (AP2) that is essential for endocytosis. In this study, we investigated the potential role of AP2S1 in intracellular processing of amyloid precursor protein (APP), which contributes to the pathogenesis of Alzheimer disease (AD) by generating the toxic β-amyloid peptide (Aβ). We found that knockdown or overexpression of AP2S1 decreased or increased the protein levels of APP and Aβ in cells stably expressing human full-length APP695, respectively. This effect was unrelated to endocytosis but involved lysosomal degradation. Morphological studies revealed that silencing of AP2S1 promoted the translocalization of APP from RAB9-positive late endosomes (LE) to LAMP1-positive lysosomes, which was paralleled by the enhanced LE-lysosome fusion. In support, silencing of vacuolar protein sorting-associated protein 41 (VPS41) that is implicated in LE-lyso fusion prevented AP2S1-mediated regulation of APP degradation and translocalization. In APP/PS1 mice, an animal model of AD, AAV-mediated delivery of AP2S1 shRNA in the hippocampus significantly reduced the protein levels of APP and Aβ, with the concomitant APP translocalization, LE-lyso fusion and the improved cognitive functions. Taken together, these data uncover a LE-lyso fusion mechanism in APP degradation and suggest a novel role for AP2S1 in the pathophysiology of AD.

Toosendanin Induces Hepatocyte Damage by Inhibiting Autophagic Flux via TFEB-Mediated Lysosomal Dysfunction.

Toosendanin (TSN) is a triterpenoid from the fruit or bark of Melia toosendan Sieb et Zucc, which has clear antitumor and insecticidal activities, but it possesses limiting hepatotoxicity in clinical application. Autophagy is a degradation and recycling mechanism to maintain cellular homeostasis, and it also plays an essential role in TSN-induced hepatotoxicity. Nevertheless, the specific mechanism of TSN on autophagy-related hepatotoxicity is still unknown. The hepatotoxicity of TSN in vivo and in vitro was explored in this study. It was found that TSN induced the upregulation of the autophagy-marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) and P62, the accumulation of autolysosomes, and the inhibition of autophagic flux. The middle and late stages of autophagy were mainly studied. The data showed that TSN did not affect the fusion of autophagosomes and lysosomes but significantly inhibited the acidity, the degradation capacity of lysosomes, and the expression of hydrolase cathepsin B (CTSB). The activation of autophagy could alleviate TSN-induced hepatocyte damage. TSN inhibited the expression of transcription factor EB (TFEB), which is a key transcription factor for many genes of autophagy and lysosomes, such as CTSB, and overexpression of TFEB alleviated the autophagic flux blockade caused by TSN. In summary, TSN caused hepatotoxicity by inhibiting TFEB-lysosome-mediated autophagic flux and activating autophagy by rapamycin (Rapa), which could effectively alleviate TSN-induced hepatotoxicity, indicating that targeting autophagy is a new strategy to intervene in the hepatotoxicity of TSN.

Investigating the role of lysosomal exocytosis in Alzheimer’s disease

AbstractBackgroundMany studies have implicated the endosomal‐lysosomal system in the production of beta‐amyloid (Aβ), but the actual compartments involved in Aβ secretion have never been visualized. Although lysosomes are expected to be a site of Aβ clearance, studies have also shown them to be a site of Aβ accumulation. Lysosomal exocytosis is well recognized as a major secretory pathway in non‐neuronal cells but is virtually unstudied in neurons. This project investigates the role and mechanism of lysosomal secretion in neurons. We hypothesize that lysosomal exocytosis is a pathway for Aβ release and could be modulated by Rab27b in iPSC‐derived human neurons.MethodTo study lysosomal exocytosis in a neuronal system, our lab established normal and mutant APP Swedish expressing iPSC‐neurons. The involvement of lysosome‐related secretory proteins (Rab27b, Munc13‐4 and synaptotagmin‐7) were examined using siRNA, dominant‐negative or shRNA constructs. Cells were co‐transduced with a pH‐sensitive dual‐fluorescent lysosomal marker (pHluorin‐LAMP1‐mAppleFP) to identify and track lysosomes. Cells were also loaded with fluorescent exogenous Aβ or a dye to mark endogenous Aβ, and then treated with ionomycin to induce exocytosis. Total internal reflection fluorescence (TIRF) microscopy was performed to capture videos of molecular events occurring within 250nm from the plasma membrane. Imaris software was used to analyze co‐localization of LAMP‐1 with Aβ, and the number and timing of lysosomal secretory events.ResultIn untreated cells, loaded Aβ accumulated in lysosomes. After treatment with ionomycin, a dramatic decrease of lysosomal Aβ was observed. In live cells, TIRF microscopy captured lysosomes docking and fusing with the plasma membrane over 150 seconds after ionomycin treatment. This was visualized by pHluorin‐LAMP1 (which is normally dark in an acidic pH) fluorescing green upon lysosomal fusion with the plasma membrane. Overexpression of Rab27b N133I mutant significantly increased lysosomal exocytosis of Aβ compared to wild‐type, and Rab27b shRNA dramatically reduced lysosomal exocytosis and Aβ secretion.ConclusionOur results demonstrate that lysosomal exocytosis is a novel secretion pathway in human neurons and identify potential pharmacological targets that regulate clearance of toxic Aβ.

RAB7A GTPase Is Involved in Mitophagosome Formation and Autophagosome-Lysosome Fusion in N2a Cells Treated with the Prion Protein Fragment 106-126.

Failed communication between mitochondria and lysosomes causes dysfunctional mitochondria, which may induce mitochondria-related neurodegenerative diseases. Here, we show that RAB7A, a small GTPase of the Rab family, mediates the crosstalk between these two important organelles to maintain homeostasis in N2a cells treated with PrP106-126. Specifically, we demonstrate that mitophagy deficiency in N2a cells caused by PrP106-126 is associated with dysregulated RAB7A localization in mitochondria. Cells lacking RAB7A display decreased mitochondrial colocalization with lysosomes and significantly increased mitochondrial protein expression, resulting in inhibited mitophagy. In contrast, overexpression of GTP-bound RAB7A directly induces lysosome colocalization with mitochondria. Further study revealed that GTP-bound RAB7A protects mitochondrial homeostasis by supporting autophagosome biogenesis. Moreover, wesuggest that depletion of RAB7A leads to gross morphological changes in lysosomes, which prevents autophagosome-lysosome fusion and interferes with the breakdown of autophagic cargo within lysosomes. Overexpression of GTP-bound RAB7A can also alleviate PrP106-126-induced morphological damage and dysfunction of mitochondria, reducing neuronal apoptosis. Collectively, our data demonstrate that RAB7A successfully drives mitochondria to the autophagosomal lumen for degradation, suggesting that the communication of proteotoxic stress from mitochondria to lysosomes requires RAB7A, as a signaling molecule, to establish a link between the disturbed mitochondrial network and its remodeling. These findings indicate that small molecules regulating mitophagy have the potential to modulate cellular homeostasis and the clinical course of neurodegenerative diseases. Proposed model of mitophagy regulated by RAB7A. (1) Accumulating PrP106-126 induced mitophagy. (2) RAB7A is recruited to mitochondria. (3) ATG5-12 and ATG9A (5) vesicles are recruited to the autophagosome formation sites in a RAB7A-dependent manner. The ATG5-12 complex recruits and anchors LC3-I to form active LC3-II (4), accelerating mitophagosomal formation. The ATG9A vesicles are thought to be a source of membranes for autophagosome assembly. The recruitment of proteins and lipids induces membrane expansion and subsequent closure to form the mitophagosome. (6) Maintenance of the normal low lysosomal PH depends on active (GTP-bound) RAB7A. (7) RAB7A recruits effector molecules responsible for tight membrane interactions, and directly or indirectly, the subsequent autophagosome merges with the lysosome, and the cargo is completely degraded.

Metabolomics of V2O5 nanoparticles and V2O5 nanofibers in human airway epithelial BEAS-2B cells

Vanadium is a toxic metal listed by the IARC as possibly carcinogenic to humans. Manufactured nanosize vanadium pentoxide (V2O5) materials are used in a wide range of industrial sectors and recently have been developed as nanomedicine for cancer therapeutics, yet limited information is available to evaluate relevant nanotoxicity. In this study we used high-resolution metabolomics to assess effects of two V2O5 nanomaterials, nanoparticles and nanofibers, at exposure levels (0.01, 0.1, and 1 ppm) that did not cause cell death (i.e., non-cytotoxic) in a human airway epithelial cell line, BEAS-2B. As prepared, V2O5 nanofiber exhibited a fibrous morphology, with a width approximately 63 ± 12 nm and length in average 420 ± 70 nm; whereas, V2O5 nanoparticles showed a typical particle morphology with a size 36 ± 2 nm. Both V2O5 nanoparticles and nanofibers had dose-response effects on aminosugar, amino acid, fatty acid, carnitine, niacin and nucleotide metabolism. Differential effects of the particles and fibers included dibasic acid, glycosphingolipid and glycerophospholipid pathway associations with V2O5 nanoparticles, and cholesterol and sialic acid metabolism associations with V2O5 nanofibers. Examination by transmission electron microscopy provided evidence for mitochondrial stress and increased lysosome fusion by both nanomaterials, and these data were supported by effects on mitochondrial membrane potential and lysosomal activity. The results showed that non-cytotoxic exposures to V2O5 nanomaterials impact major metabolic pathways previously associated with human lung diseases and suggest that toxico-metabolomics may be useful to evaluate health risks from V2O5 nanomaterials.

Intracellular cholesterol transport inhibition Impairs autophagy flux by decreasing autophagosome–lysosome fusion

BackgroundAutophagy is an intracellular degradation process crucial for homeostasis. During autophagy, a double-membrane autophagosome fuses with lysosome through SNARE machinery STX17 to form autolysosome for degradation of damaged organelle. Whereas defective autophagy enhances cholesterol accumulation in the lysosome and impaired autophagic flux that results Niemann-Pick type C1 (NPC1) disease. However, exact interconnection between NPC1 and autophagic flux remain obscure due to the existence of controversial reports.ResultsThis study aimed at a comparison of the effects of three autophagic inhibitor drugs, including chloroquine, U18666A, and bafilomycin A1, on the intracellular cholesterol transport and autophagy flux. Chloroquine, an autophagic flux inhibitor; U1866A, a NPC1 inhibitor, and bafilomycin A, a lysosomotropic agent are well known to inhibit autophagy by different mechanism. Here we showed that treatment with U1866A and bafilomycin A induces lysosomal cholesterol accumulation that prevented autophagic flux by decreasing autophagosome–lysosome fusion. We also demonstrated that accumulation of cholesterol within the lysosome did not affect lysosomal pH. Although the clearance of accumulated cholesterol by cyclodextrin restored the defective autophagosome–lysosome fusion, the autophagy flux restoration was possible only when lysosomal acidification was not altered. In addition, a failure of STX17 trafficking to autophagosomes plays a key role in prevention of autophagy flux caused by intracellular cholesterol transport inhibitors.ConclusionsOur data provide a new insight that the impaired autophagy flux does not necessarily result in lysosomal cholesterol accumulation even though it prevents autophagosome–lysosome fusion.3SNC-nf_k5hppf9--WdwopVideo abstract

A plant virus hijacks phosphatidylinositol-3,5-bisphosphate to escape autophagic degradation in its insect vector

ABSTRACT Hosts can initiate macroautophagy/autophagy as an antiviral defense response, while viruses have developed multiple ways to evade the host autophagic degradation. However, little is known as to whether viruses can target lipids to subvert autophagic degradation. Here, we show that a low abundant signaling lipid, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), is required for rice black-streaked dwarf virus (RBSDV) to evade the autophagic degradation in the insect vector Laodelphax striatellus. RBSDV binds to PtdIns(3,5)P2 and elevates its level through its main capsid protein P10, leading to inhibited autophagy and promoted virus propagation. Furthermore, we show that PtdIns(3,5)P2 inhibits the autophagy pathway by preventing the fusion of autophagosomes and lysosomes through activation of Trpml (transient receptor potential cation channel, mucolipin), an effector of PtdIns(3,5)P2. These findings uncover a strategy whereby a plant virus hijacks PtdIns(3,5)P2 via its viral capsid protein to evade autophagic degradation and promote its survival in insects.

Capsaicin alleviates cisplatin-induced muscle loss and atrophy in vitro and in vivo.

Cisplatin (CP) is a widely used chemotherapeutic drug with subsequent adverse effects on different organs and tissues including skeletal muscle loss and atrophy as the most common clinical symptoms. The molecular mechanism of cisplatin-induced muscle atrophy is not clearly understood. However, recent significant advances indicate that it is related to an imbalance in both the protein status and apoptosis. Capsaicin (CAP) is one of the major ingredients in chilli peppers. It is a valuable pharmacological agent with several therapeutic applications in controlling pain and inflammation with particular therapeutic potential in muscle atrophy. However, the mechanisms underlying its protective effects against cisplatin-induced muscle loss and atrophy remain largely unknown. This study aims to investigate capsaicin's beneficial effects on cisplatin-induced muscle loss and atrophy in vitro and in vivo. The anti-muscle-atrophic effect of capsaicin on cisplatin-induced muscle loss was investigated using in vivo and in vitro studies. By using the pretreatment model, pretreated capsaicin for 24h and treated with cisplatin for 48h, we utilized a C2 C12 myotube formation model where cell viability analysis, immunofluorescence, and protein expression were measured to investigate the effect of capsaicin in hampering cisplatin-induced muscle atrophy. C57BL/6 mice were administered capsaicin (10, 40mg/kg BW) as a pretreatment for 5weeks and cisplatin (3mg/kg BW) for seven consecutively days to assess muscle atrophy in an animal model for protein and oxidative stress examination, and the grip strength was tested to evaluate the muscle strength. Our study results indicated that cisplatin caused lower cell viability and showed a subset of hallmark signs typically recognized during atrophy, including severe reduction in the myotube diameter, repression of Akt, and mTOR protein expression. However, pretreatment with capsaicin could ameliorate cisplatin-induced muscle atrophy by up-regulating the protein synthesis in skeletal muscle as well as down-regulating the markers of protein degradation. Additionally, capsaicin was able to downregulate the protein expression of apoptosis-related markers, activated TRPV1 and autophagy progress modulation and the recovery of lysosome function. In vivo, capsaicin could relieve oxidative stress and cytokine secretion while modulating autophagy-related lysosome fusion, improvinggrip strength, and alleviating cisplatin-induced body weight loss and gastrocnemius atrophy. These findings suggest that capsaicincan restore cisplatin-induced imbalance between protein synthesis and protein degradation pathways and it may have protective effects against cisplatin-induced muscle atrophy.

Combination of polythyleneimine regulating autophagy prodrug and Mdr1 siRNA for tumor multidrug resistance

Multidrug resistance (MDR) has been restricting the efficacy of chemotherapy, which mainly include pump resistance and non-pump resistance. In order to fight overall MDR, a novel targeted gene/drug co-deliver nano system is developed, which can suppress the drug efflux pumps and modulate autophagy to overcoming both pump and non-pump resistance. Here, small interfere RNA (siRNA) is incorporated into polymer-drug conjugates (PEI-PTX, PP) which are composed of polyethyleneimine (PEI) and paclitaxel (PTX) via covalent bonds, and hyaluronic acid (HA) is coated on the surface of PP/siRNA to achieve long blood cycle and CD44-targeted delivery. The RNA interference to mdr1 gene is combined with autophagy inhibition by PP, which efficiently facilitate apoptosis of Taxol-resistant lung cancer cells (A549/T). Further study indicates that PEI in PP may play a significant role to block the autophagosome–lysosome fusion process by means of alkalizing lysosomes. Both in vitro and in vivo studies confirm that the nanoassemblies can successfully deliver PTX and siRNA into tumor cells and significantly inhibited A549/T tumor growth. In summary, the polymeric nanoassemblies provide a potential strategy for combating both pump and non-pump resistance via the synergism of RNAi and autophagy modulation.

Palmitate Inhibits Mouse Macrophage Efferocytosis by Activating an mTORC1-Regulated Rho Kinase 1 Pathway: Therapeutic Implications for the Treatment of Obesity.

Every day, billions of our cells die and get cleared without inducing inflammation. When, clearance is improper, uncleared cells undergo secondary necrosis and trigger inflammation. In addition, proper efferocytosis would be required for inducing resolution of inflammation, thus clearance deficiencies in the long term lead to development of various chronic inflammatory diseases. Increasing evidence indicates that obesity, itself being a low-grade inflammatory disease, predisposes to a variety of other chronic inflammatory diseases. Previous studies indicated that this later might be partially related to an impaired efferocytosis induced by increased uptake of circulating saturated fatty acids by macrophages in obese people. Here, we show that palmitate inhibits efferocytosis by bone marrow-derived macrophages in a dose-dependent manner. Palmitate triggers autophagy but also activates an energy-sensing mTORC1/ROCK1 signaling pathway, which interferes with the autophagosome-lysosome fusion, resulting in accumulation of the cellular membranes in autophagosomes. We propose that lack of sufficient plasma membrane supply attenuates efferocytosis of palmitate-exposed macrophages. AMP-activated protein kinase activators lead to mTORC1 inhibition and, consequently, released the palmitate-induced efferocytosis block in macrophages. Thus, they might be useful in the treatment of obesity not only by affecting metabolism thought so far. ROCK1 inhibitors could also be considered.