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Although the neurotoxicity of ZnO nanoparticles (NPs) has been evaluated in animal and nerve cell culture models, these models cannot accurately mimic human brains. Three-dimensional (3D) brain organoids based on human-induced pluripotent stem cells have been developed to study the human brains, but this model has rarely been used to evaluate NP neurotoxicity. We used 3D brain organoids that express cortical layer proteins to investigate the mechanisms of ZnO NP-induced neurotoxicity. Cytotoxicity caused by high levels of ZnO NPs (64 μg/mL) correlated with high intracellular Zn ion levels but not superoxide levels. Exposure to a non-cytotoxic concentration of ZnO NPs (16 μg/mL) increased the autophagy-marker proteins LC3B-II/I but decreased p62 accumulation, whereas a cytotoxic concentration of ZnO NPs (64 μg/mL) decreased LC3B-II/I proteins but did not affect p62 accumulation. Fluorescence micro-optical sectioning tomography revealed that 64 μg/mL ZnO NPs led to decreases in LC3B proteins that were more obvious at the outer layers of the organoids, which were directly exposed to the ZnO NPs. In addition to reducing LC3B proteins in the outer layers, ZnO NPs increased the number of micronuclei in the outer layers but not the inner layers (where LC3B proteins were still expressed). Adding the autophagy flux inhibitor bafilomycin A1 to ZnO NPs increased cytotoxicity and intracellular Zn ion levels, but adding the autophagy inducer rapamycin only slightly decreased cellular Zn ion levels. We conclude that high concentrations of ZnO NPs are cytotoxic to 3D brain organoids via defective autophagy and intracellular accumulation of Zn ions.

Quinacrine, a fluorescent amphipathic amine, has been used as a vital fluorescent probe to visualize vesicular storage of ATP in the field of purinergic signaling. However, the mechanism(s) by which quinacrine represents vesicular ATP storage remains to be clarified. The present study investigated the validity of the use of quinacrine as a vial fluorescent probe for ATP-storing organelles. Vesicular nucleotide transporter (VNUT), an essential component for vesicular storage and ATP release, is present in very low density lipoprotein (VLDL)-containing secretory vesicles in hepatocytes. VNUT gene knockout (Vnut(−/−)) or clodronate treatment, a VNUT inhibitor, disappeared vesicular ATP release (Tatsushima et al., Biochim Biophys Acta Molecular Basis of Disease 2021, e166013). Upon incubation of mice’s primary hepatocytes, quinacrine accumulates in a granular pattern into the cytoplasm, sensitive to 0.1-μM bafilomycin A(1), a vacuolar ATPase (V-ATPase) inhibitor. Neither Vnut(−/−) nor treatment of clodronate affected quinacrine granular accumulation. In vitro, quinacrine is accumulated into liposomes upon imposing inside acidic transmembranous pH gradient (∆pH) irrespective of the presence or absence of ATP. Neither ATP binding on VNUT nor VNUT-mediated uptake of ATP was affected by quinacrine. Consistently, VNUT-mediated uptake of quinacrine was negligible or under the detection limit. From these results, it is concluded that vesicular quinacrine accumulation is not due to a consequence of its interaction with ATP but due to ∆pH-driven concentration across the membranes as an amphipathic amine. Thus, quinacrine is not a vital fluorescent probe for vesicular ATP storage.

Metal copper oxide nanoparticles (nano-CuO) are under mass production and have been widely utilized in many fields including catalysis, gas sensors, semiconductor materials, etc. The broad applications of nano-CuO have increased the possibility of risk to incidental exposure to the environment, and therefore, an in-depth investigation of their effects on live cells is required. This study investigated the impact of the nano-CuO on SH-SY5Y cells, and findings showed that the ratio of LC3-II/LC3-I was significantly increased in SH-SY5Y cells when the cells were treated with nano-CuO. However, if the autophagy inhibitor Bafilomycin A1 (Baf A1) was co-treated, the ratio of LC3-II/LC3-I was further improved. These outcomes might indicate that autophagy flux was permanently elevated by adding nano-CuO. Further results found highly activated levels of long noncoding RNAs (lncRNAs) under nano-CuO treatment. The data illustrate a mechanism that nano-CuO can promote autophagy and activate lncCyt b-AS/ND5-AS/ND6-AS in SH-SY5Y cells and have critical implications for nanoparticle biomedical applications.

Natural diphyllin glycosides were identified as potent vacuolar H+ -ATPase (V-ATPase) inhibitors. A series of diphyllin β-hydroxyl amino derivatives were designed and synthesized as novel diphyllin derivatives. Most of these derivatives displayed potent cytotoxicity against six cancer cell lines with IC50 values in the submicromolar to nanomolar concentration range. Compounds 2b, 2c, 2l, 2m, and 2n showed similar V-ATPase inhibitory potency to Bafilomycin A1. Compound 2l exhibited potent activity of modulation of lysosomal pH and cytoplasmic pH.

Lysosomes are important sites for macromolecular degradation, defined by an acidic lumenal pH of ∼4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein (FP)-based pH biosensor called Fluorescence Indicator REporting pH in Lysosomes (FIRE-pHLy). This biosensor was targeted to lysosomes with lysosomal-associated membrane protein 1 (LAMP1) and reported lumenal pH between 3.5 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan pH-sensitive FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content quantitative imaging. We expressed FIRE-pHLy in several cellular models and quantified the alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust, and versatile lysosomal pH biosensor, that has broad applications for investigating pH dynamics in aging- and lysosome-related diseases, as well as in lysosome-based drug discovery.

Background Bovine mammary epithelial cells after calving undergo serious metabolic challenges and oxidative stress both of which could compromise autophagy. Transcription factor EB (TFEB)-mediated autophagy is an important cytoprotective mechanism against oxidative stress. However, effects of TFEB-mediated autophagy on the oxidative stress of bovine mammary epithelial cells remain unknown. Therefore, the main aim of the study was to investigate the role of TFEB-mediated autophagy in bovine mammary epithelial cells experiencing oxidative stress. Results H2O2 challenge of the bovine mammary epithelial cell MAC-T increased protein abundance of LC3-II, increased number of autophagosomes and autolysosomes while decreased protein abundance of p62. Inhibition of autophagy via bafilomycin A1 aggravated H2O2-induced reactive oxygen species (ROS) accumulation and apoptosis in MAC-T cells. Furthermore, H2O2 treatment triggered the translocation of TFEB into the nucleus. Knockdown of TFEB by siRNA reversed the effect of H2O2 on protein abundance of LC3-II and p62 as well as the number of autophagosomes and autolysosomes. Overexpression of TFEB activated autophagy and attenuated H2O2-induced ROS accumulation. Furthermore, TFEB overexpression attenuated H2O2-induced apoptosis by downregulating the caspase apoptotic pathway. Conclusions Our results indicate that activation of TFEB mediated autophagy alleviates H2O2-induced oxidative damage by reducing ROS accumulation and inhibiting caspase-dependent apoptosis.

Pharmacological inhibition of vacuolar-type H+-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-Å. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7’-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c., Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Here authors report the cryo-EM structure of bafilomycin A1-bound V-ATPase with six bafilomycin A1 molecules bound to the c-ring and reveal the molecular basis for Bafilomycin A1 inhibition of the V-ATPase.

Glutamate is the major excitatory neurotransmitter in the brain and is involved in many brain functions. In this study, we investigated whether typhaneoside, a flavonoid from Typhae angustifolia pollen, affects endogenous glutamate release from rat cortical synaptosomes. Using a one-line enzyme-coupled fluorometric assay, glutamate release stimulated by the K+ channel blocker 4-aminopyridine was monitored to explore the possible underlying mechanisms. The vesicular transporter inhibitor bafilomycin A1 and chelation of extracellular Ca2+ ions with EGTA suppressed the effect of typhaneoside on the induced glutamate release. Nevertheless, the typhaneoside activity has not been affected by the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate. The synaptosomal plasma membrane potential was assayed using a membrane potential-sensitive dye DiSC3(5), and cytosolic Ca2+ concentrations ([Ca2+]C) was monitored by a Ca2+ indicator Fura-2. Results showed that typhaneoside did not alter the synaptosomal membrane potential but lowered 4-aminopyridine-induced increases in [Ca2+]C. Furthermore, the Cav2.2 (N-type) channel blocker ω-conotoxin GVIA blocked Ca2+ entry and inhibited the effect of typhaneoside on 4-aminopyridine-induced glutamate release. However, the inhibitor of intracellular Ca2+ release dantrolene and the mitochondrial Na+/Ca2+ exchanger blocker 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one have no effect on the suppression of glutamate release mediated by typhaneoside. Moreover, inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) prevented the inhibitory effect of typhaneoside on induced glutamate release. Typhaneoside reduced 4-aminopyridine-induced phosphorylation of ERK1/2 and the major presynaptic ERK target synapsin I, which is a synaptic vesicle-associated protein. In conclusion, these findings suggest a role for typhaneoside in modulating glutamate release by suppressing voltage-dependent Ca2+ channel mediated presynaptic Ca2+ influx and the MAPK/ERK/synapsin I signaling cascade.

A new bafilomycin derivative (1) and another seven known bafilomycins (2-8) were isolated from feces-derived Streptomyces sp. HTL16. The structure of 1 was elucidated by 1D and 2D NMR spectroscopic analysis. Biological testing demonstrated that these bafilomycins exhibited potent antiviral activities against the influenza A and SARS-CoV-2 viruses, with IC50 values in the nanomolar range, by inhibiting the activity of endosomal ATP-driven proton pumps.

Background Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and broke out as a global pandemic in late 2019. The acidic pH environment of endosomes is believed to be essential for SARS-CoV-2 to be able to enter cells and begin replication. However, the clinical use of endosomal acidification inhibitors, typically chloroquine, has been controversial with this respect. Methods In this study, RT-qPCR method was used to detect the SARS-CoV-2N gene to evaluate viral replication. The CCK-8 assay was also used to evaluate the cytotoxic effect of SARS-CoV-2. In situ hybridization was used to examine the distribution of the SARS-CoV-2 gene in lung tissues. Hematoxylin and eosin staining was also used to evaluate virus-associated pathological changes in lung tissues. Results In this study, analysis showed that endosomal acidification inhibitors, including chloroquine, bafilomycin A1 and NH4CL, significantly reduced the viral yields of SARS-CoV-2 in Vero E6, Huh-7 and 293T-ACE2 cells. Chloroquine and bafilomycin A1 also improved the viability and proliferation of Vero E6 cells after SARS-CoV-2 infection. Moreover, in the hACE2 transgenic mice model of SARS-CoV-2 infection, chloroquine and bafilomycin A1 reduced viral replication in lung tissues and alleviated viral pneumonia with reduced inflammatory exudation and infiltration in peribronchiolar and perivascular tissues, as well as improved structures of alveolar septum and pulmonary alveoli. Conclusions Our research investigated the antiviral effects of endosomal acidification inhibitors against SARS-CoV-2 in several infection models and provides an experimental basis for further mechanistic studies and drug development.

Vacuolar ATPase (v-ATPase) is the main proton pump that acidifies vesicles such as lysosomes. Disruption in the lysosomal localization of v-ATPase leads to lysosomal dysfunction, thus contributing to the pathogenesis of lysosomal storage disorders and neurodegenerative diseases such as Alzheimer's disease. Recent studies showed that increases in cyclic AMP (cAMP) levels acidify lysosomes and consequently enhance autophagy flux. Although the upregulation of v-ATPase function may be the key mechanism underlying the cAMP-mediated lysosomal acidification, it is unknown whether a mechanism independent of v-ATPase may be contributing to this phenomenon. In the present study, we modeled v-ATPase dysfunction in brain cells by blocking lysosomal acidification in cortical astrocytes through treatment with bafilomycin A1, a selective v-ATPase inhibitor. We observed that cAMP reversed the pH changes via the activation of protein kinase A; interestingly, cAMP also increased autophagy flux even in the presence of bafilomycin A1, suggesting the presence of an alternative route of proton entry. Notably, pharmacological inhibitors and siRNAs of H+ /K+ -ATPase markedly shifted the lysosomal pH toward more alkaline values in bafilomycin A1/cAMP-treated astrocytes, suggesting that H+ /K+ -ATPase may be the alternative route of proton entry for lysosomal acidification. Furthermore, the cAMP-mediated reversal of lysosomal pH was nullified in the absence of ZnT3 that interacts with H+ /K+ -ATPase. Our results suggest that the H+ /K+ -ATPase/ZnT3 complex is recruited to lysosomes in a cAMP-dependent manner and functions as an alternative proton pump for lysosomes when the v-ATPase function is downregulated, thus providing insight into the potential development of a new class of lysosome-targeted therapeutics in neurodegenerative diseases.

Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron-derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.

Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembrane ion fluxes. Endolysosomal CLC Cl-/H+ exchangers function as electric shunts for proton pumping and in luminal Cl- accumulation. We now report three unrelated children with severe neurodegenerative disease, who carry the same de novo c.1658A>G (p.Tyr553Cys) mutation in CLCN6, encoding the late endosomal Cl-/H+-exchanger ClC-6. Whereas Clcn6-/- mice have only mild neuronal lysosomal storage abnormalities, the affected individuals displayed severe developmental delay with pronounced generalized hypotonia, respiratory insufficiency, and variable neurodegeneration and diffusion restriction in cerebral peduncles, midbrain, and/or brainstem in MRI scans. The p.Tyr553Cys amino acid substitution strongly slowed ClC-6 gating and increased current amplitudes, particularly at the acidic pH of late endosomes. Transfection of ClC-6Tyr553Cys, but not ClC-6WT, generated giant LAMP1-positive vacuoles that were poorly acidified. Their generation strictly required ClC-6 ion transport, as shown by transport-deficient double mutants, and depended on Cl-/H+ exchange, as revealed by combination with the uncoupling p.Glu200Ala substitution. Transfection of either ClC-6Tyr553Cys/Glu200Ala or ClC-6Glu200Ala generated slightly enlarged vesicles, suggesting that p.Glu200Ala, previously associated with infantile spasms and microcephaly, is also pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H+-driven Cl- accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectrum of clinical features depends on the differential impact of the allele on ClC-6 function.

Bafilomycin A1 (BAF A1) is a macrolide antibiotic that, in addition to its antibacterial activity, can induce apoptosis of cancer cells. Glycosylation plays an important role in the modification of antibiotics as it can improve their bioactivity. However, glycosylation of BAF A1 has not been previously reported. Bacillus licheniformis glycosyltransferase (GTs) Bl-YjiC and Bacillus subtilis GTs Bs-YjiC were expressed successfully in a heterologous manner. The glycosylation of BAF A1 with UDP-glucose, UDP-galactose or UDP-N-acetylglucosamine was catalyzed using the enzymes Bl-YjiC and Bs-YjiC. Our results demonstrated that Bl-YjiC can only utilize UDP-glucose as the donor, while Bs-YjiC can utilize all three glycosyl donors. The glycosylation site was demonstrated by MS/MS to be the hydroxyl group at the C21 position of BAF A1. The anti-proliferative effects of glucosyl BAF A1 (BAF-Glc) on HeLa cells indicate that this novel antibiotic is superior to BAF A1. The IC50 for BAF-Glc was determined to be 5.47 μM. Here, we report the production of glycosylated BAF A1 for the first time, and we show that the produced BAF-Glc exhibited better anticancer activity than BAF A1. This work provides theoretical and experimental support for the development of novel anticancer bafilomycins.

Cisplatin plays a key role in treating small cell lung cancer (SCLC); however, the rapid development of cisplatin resistance limits its treatment effect. The detailed mechanisms of cisplatin-resistance, particularly in SCLC, remain unclear. We analyzed the differentially expressed genes (DEGs) between cisplatin-resistant small cell lung cancer cell line H446/CDDP and its parental cell line H446, using the transcriptome sequencing technique. Gene ontology (GO) analysis and the subsequent tests demonstrated that the functions of protein ubiquitination and autophagy are more active in the H446/CDDP cells. Autophagy plays a protective role in the H446/CDDP cells by using the autophagy inhibitors, 3-methyladenine and bafilomycin A1. Moreover, antimalarial drugs that inhibit autophagy by increasing the pH of lysosomes can also enhance cisplatin-induced cell death.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, caused by mutation in the gene encoding lamin A/C, which produces a truncated protein called progerin. In cells from HGPS patients, progerin accumulates at the nuclear membrane (NM), where it causes NM deformations. In this study, we investigated whether progerin-induced NM deformation involved ESCRT-III, a protein complex that remodels nuclear and cytoplasmic membranes. The ESCRT-III protein CHMP4B was recruited to sites of aberrant NM proliferation in human cells ectopically expressing progerin and in patient-derived HGPS fibroblasts. Derepression of NM deformation in these cells was observed following depletion of CHMP4B or an ESCRT-III adaptor, ALIX. Treatment with rapamycin (which induce autophagic clearance of progerin and reverse progerin-induced cellular phenotypes) down-regulated progerin-induced NM deformation, whereas treatment with bafilomycin A1 (an inhibitor of autophagy and lysosome-based degradation) or CHMP4B depletion antagonized the effects of rapamycin. These results indicate that the ALIX-mediated ESCRT-III pathway plays a suppressive role in progerin-induced NM deformation and suggest that autophagy down-regulates progerin-induced NM deformation in a manner dependent on ESCRT-III machinery.

Melanosomes are specialized membrane-bound organelles that are involved in melanin synthesis. Unlike melanosome biogenesis, the melanosome degradation pathway is poorly understood. Among the cellular processes, autophagy controls degradation of intracellular components by cooperating with lysosomes. In this study, we showed that ursolic acid inhibits skin pigmentation by promoting melanosomal autophagy, or melanophagy, in melanocytes. We found that B16F1 cells treated with ursolic acid suppressed alpha-melanocyte stimulating hormone (α-MSH) stimulated increase in melanin content and activated autophagy. In addition, we found that treatment with ursolic acid promotes melanosomal degradation, and bafilomycin A1 inhibition of autophagosome-lysosome fusion blocked the removal of melanosomes in α-MSH-stimulated B16F1 cells. Furthermore, depletion of the autophagy-related gene 5 (ATG5) resulted in significant suppression of ursolic acid-mediated anti-pigmentation activity and autophagy in α-MSH-treated B16F1 cells. Taken together, our results suggest that ursolic acid inhibits skin pigmentation by increasing melanosomal degradation in melanocytes.

Background The proper topography of implant surface can induce macrophages polarization, whereas the regulation mechanism has not been fully deciphered. The study aimed to examine the regulation mechanism of macrophages M2 polarization by titanium (Ti) implant surface micro/nano topography. Results Firstly, the titanium implant micropits-nanotubular surface with ~30 nm diameters (MNT) can induce the M2 polarization of RAW264.7 spontaneously, as indicated by the spindle-like cell morphological alteration and specific molecular marker arginase-1 (Arg1) expression. Next, the autophagic vacuoles (AVs) number is significantly increased on MNT surface, as confirmed by the monodansylcadaverine (MDC) and CYTO-ID staining as well as the transmission electron microscope (TEM) observation. In addition, increasing or decreasing the autophagosomes number by rapamycin or 3-methyladenine (3-MA) will result in augmentation or attenuation of Arg1. Furthermore, blocking the fusion between autophagosomes and lysosomes by bafilomycin also significantly reduces Arg1, even in the presence of rapamycin. Finally, the ERK phosphorylation is selectively upregulated on MNT surface and the AVs number and Arg1 expression are significantly suppressed by U0126 treatment. Conclusion Our findings suggest that the ERK-Beclin-1-autophagy axis may play a pivotal role in the regulation of M2 polarization induced by nanotopography.

Metal nanoparticles-based sensors invoked much research attention in the biomedical field, especially in applications involving live cell imaging and monitoring. Here, a simple cost-effective method is adopted to synthesize glutathione coated copper nanoclusters (Cu-GSH NCs) with strong bright red fluorescence (625 nm). The clusters were found to be containing five Cu(0) atoms complexed with one molecule of glutathione (GSH) as evidenced by MALDI-TOF MS analysis. The synthesized Cu-GSH NCs system responds linearly to the pH in the acidic and alkaline ranges with a high degree of in vitro pH reversibility, projecting its potential as a real time pH sensor. Higher intensity emission observed in acidic conditions can be exploited for its employability as cellular organelle markers. The imaging and sensing potential of Cu-GSH NCs in the live human adenocarcinoma cell line, the HeLa cells, was tested. The treatment of HeLa cells for 48 h imparted deep red fluorescence, owing to the lower level of intracellular pH in cancer cells. In contrast, the imaging using normal cell lines (L-132, lung epithelial cell line) showed significantly lower fluorescence intensity as compared to that of HeLa cells. The subcellular pH-dependent fluorescence emission of Cu-GSH NCs was further assessed by treating HeLa cells with proton pump (V-ATPase) inhibitor Bafilomycin A1, which increases the vesicular pH. Interestingly, the fluorescent intensity of HeLa cells decreases with increasing concentration of Bafilomycin A1 in the presence of Cu-GSH NCs, as evidenced by the fluorescence microscopic images and quantitative fluorescent output. Accordingly, the developed Cu-GSH NCs system can be employed as an efficient pH-based bioimaging probe for the detection of cancer cells with an implied potential for the label free subcellular organelle tracking and marking. Importantly, the Cu-GSH NCs can be used for live cell pH imaging owing to their high degree of reversibility in sensing of pH variation.

Poly(ethylene imine)–poly(l-lysine)–poly(l-glutamic acid) (PKE) polymers with various glutamic acid portions were synthesized by ring opening polymerization of l-lysine N-carboxyanhydride (NCA) and l-glutamic acid NCA from poly(ethylene imine) 1.8 kDa (PEI1.8k) as a macroinitiator. It was found that their glutamic acid residues could buffer endosomal pH. PK5E9 polymer could form nanoparticles by self-assembly and nanosized polyplexes, possessing pH-responsive charge-conversion properties. PK5E9 or its polyplex nanoparticles showed polyhedral structures with bumpy surfaces. Its cytotoxicity was marginal at both pH 7.4 and 6.0, and its transfection efficiency was highly increased at pH 6.0. The improved transfection efficiency in acidic conditions was thought to be induced by elevated cellular uptake of the polyplexes via charge-conversion from negative to positive charges. Its transfection was also found to be mediated by endosomal escape through endosome buffering by bafilomycin A1-treated transfection. I...

Die Autophagie ist ein evolution��r konservierter zellul��rer Wiederaufbereitungs-mechanismus, der zelleigene Makromolek��le und Organellen in seine Grundbausteine zerlegt und dem Zellmetabolismus zur Wahrung der Zellhom��ostase wieder zur Verf��gung stellt. Dies geschieht durch Einschluss der Zielstrukturen in eine Doppelmembranstruktur, das Autophagosom, und mittels konsekutiver Fusion des Autophagosoms mit einem degradativen Lysosom. Nervenzellen geh��ren aufgrund ihres postmitotischen Zustandes sowie hoher metabolischer Aktivit��t zu jenen Zelltypen, die besonders auf die Autophagie angewiesen sind. Deshalb l��sst sich eine Neurodegeneration durch eine Ausschaltung der basalen neuronalen Autophagie ausl��sen. Aus diesem Grund ger��t die Autophagie im Kontext akuter und chronischer Stressreize zunehmend in den wissenschaftlichen Fokus. Gleichwohl sind die quantitativen Variationen der Autophagiekapazit��t verschiedener Nervenzelltypen bisher ein unbearbeitetes Gebiet in der Autophagieforschung. Diese Variationen sind von besonderem Interesse, weil viele Erkrankungen des Nervensystems zumindest initial einen selektiven Befall bestimmter Nervenzelltypen zeigen und Variationen in der Autophagiekapazit��t eine Grundlage f��r eine differenzielle Vulnerabilit��t verschiedener Neuronentypen darstellen k��nnen. Die hirnorganotypische Schnittkultur ist zur Untersuchung dieser Frage vorteilhaft, weil sie das organotypische Mikromilieu mit einer leichten Zug��nglichkeit und einer Manipulierbarkeit der ��u��eren Umst��nde kombiniert. Im Rahmen der vorliegenden Untersuchung wurde die cerebell��re organotypische Schnittkultur als experimentelles System f��r den zelltypspezifischen quantitativen Vergleich neuronaler Autophagie etabliert und anhand eines Teils des Autophagieapparates getestet, der Autophagosomenformation. Hierzu wurden [1] ein Vergleich zwischen cerebell��ren Schnittkulturen und nativem Kleinhirngewebe aus altersentsprechenden M��usen, [2] eine Untersuchung der Entwicklung des Autophagiestatus ��ber die Kulturdauer hinweg sowie [3] eine Verifizierung der allgemeinen Responsivit��t der Schnittkulturen auf in der Autophagieforschung g��ngige Methoden der Manipulation des Autophagiestatus in orientierenden Western Blot-Untersuchungen durchgef��hrt. Anschlie��end erfolgte ein zellspezifischer immunhistochemischer Vergleich zwischen Purkinje-Zellen und Parvalbumin-positiven Interneuronen jeweils nach Behandlungen mit dem etablierten Pharmakon Bafilomycin, das die Degradation von Autophagosomen inhibiert. Die so entstandene Akkumulation von LC3B-positiven Autophagosomen wurde als Ausdruck der Autophagosomenformation gewertet und zwischen den Zelltypen verglichen. Die Ergebnisse zeigen dabei einen deutlichen absolut Unterschied in der basalen Autophagosomenbildung zwischen den Zelltypen, der ma��geblich mit der Somagr����e korreliert, sowie eine zelltyp��bergreifende relative Kinetik der Autophagosomenbildung der untersuchten Neuronen im gleichen organotypischen Mikromilieu., Autophagy is an evolutionarily highly conserved cellular recycling mechanism which degrades macromolecules and organelles in order to supply cell metabolism with nutrients and maintain cellular homeostasis. This is achieved by enclosing target structures in autophagosomes and their consecutive fusion with degradative lysosomes. Postmitotic neurons are particularly dependent on autophagy due to their high metabolic activity. Additionally, neurons are not able to dilute damaged organelles or aggregated macromolecules by mitosis. Consequently, experimentally induced impairment of autophagy leads to neurodegeneration. The basal role of autophagy in cellular homeostasis focuses scientific attention on autophagy as either risk factor or compensating mechanism for both acute and chronic cellular stress. However, knowledge about quantitative variations in autophagic capacities among cell types largely remain a ���blind spot���. Such variations may lead to a selective vulnerability of specific cell types and provide an explanation for the fact that a wide range of diseases of the central nervous system initially manifest in a distinct pattern of cell affliction. The brain organotypic slice culture assay provides a convenient model for investigation of cell type specific differences because it combines an organotypic micro-milieu with an easy accessability and manipulability of external circumstances. This study establishes a cerebellar organotypic slice assay as experimental model for quantitative cell type specific analysis of neuronal autophagy and applies this assay to the quantitative investigation of autophagosome formation. The following basic parameters were established using Western Blot assays: firstly, a comparison of cerebellar slices with samples directly obtained from mice was carried out; secondly, the influence of duration of cultivation was investigated; thirdly, the responsivity of cerebellar slices to commonly used methods for manipulation of autophagy was confirmed. Afterwards, an immunohistochemical comparison of purkinje cells and parvalbumin-positive interneurons after treatment with bafilomycin, which inhibits autophagosome degradation, was conducted. The consecutive accumulation of LC3B-positive Autophagosomes was analyzed as measure for autophagosome formation and compared between cell types. Results showed a marked absolute difference in autophagosome formation between cell types as well as analogous kinetics in the same organotypic micro-milieu.

Cells in the tumor microenvironment experience mechanical stresses, such as compression generated by uncontrolled cell growth within a tissue, increased substrate stiffness due to tumor cell extracellular matrix (ECM) remodeling, and leaky angiogenic vessels which involve low fluid shear stress. With our hypothesis that shear stress increases V–H $$^+$$ -ATPase number density in prostate cancer cells via activation of the mTORC1 and mTORC2 pathways, we demonstrated and quantified such a mechanism in prostate cancer cells. Moderately metastatic DU145 and highly metastatic PC3 prostate cancer cells were subjected to 0.05 dynes $$\hbox {cm}^{-2}$$ wall shear stress for 24 h, followed by immunocytochemistry and fluorescence measurements of $$\beta$$ 1 integrin, endosome, lysosome, V–H $$^{+}$$ -ATPase proton pump, mTORC1, and p-mTORC2 antibodies. Post shear stress migration assays, and the effects of vacuolar proton pump inhibitor Bafilomycin A1 (60 nM, 24 h) as well as shear stress on the ICC fluorescence intensity of the proteins of interest were conducted with DU145 cells. Low fluid shear stress increases the fluorescence intensity of $$\beta$$ 1 integrin, endosome, lysosome, V–H $$^{+}$$ -ATPase, mTORC1, and p-mTORC2 antibodies in PC3 and DU145 cells, and also increased cell migration. However, Bafilomycin A1 decreased fluorescence intensity of all of these proteins in DU145 cells exposed to shear stress, revealing that V–H $$^+$$ -ATPase controls the expression of these proteins. Prostate cancer cell mechanotransduction increases endosomes, lysosomes, and proton pumps—where increases have been associated with enhanced cancer aggressiveness. We also show that the prostate cancer cell’s response to force promotes the cancer drivers mTORC1 and mTORC2.

Oxidative stress-induced DNA damage, the senescence-associated secretory phenotype (SASP), and impaired autophagy all are general features of senescent cells. However, the cross-talk among these events and processes is not fully understood. Here, using NIH3T3 cells exposed to hydrogen peroxide stress, we show that stress-induced DNA damage provokes the SASP largely via cytosolic chromatin fragment (CCF) formation, which activates a cascade comprising cGMP-AMP synthase (cGAS), stimulator of interferon genes protein (STING), NF-κB, and SASP, and that autolysosomal function inhibits this cascade. We found that CCFs accumulate in senescent cells with activated cGAS-STING-NF-κB signaling, promoting SASP and cellular senescence. We also present evidence that the persistent accumulation of CCFs in prematurely senescent cells is partially associated with a defect in DNA-degrading activity in autolysosomes and reduced abundance of activated DNase 2α. Intriguingly, we found that metformin- or rapamycin-induced activation of autophagy significantly lessened the size and levels of CCFs and repressed the activation of the cGAS-STING-NF-κB-SASP cascade and cellular senescence. These effects of autophagy activators indicated that autolysosomal function contributes to CCF clearance and SASP suppression, further supported by the fact that the lysosome inhibitor bafilomycin A1 blocked the role of autophagy-mediated CCF clearance and senescence repression.

The efficient application of smart drug-delivery systems requires further improvement of their cellular uptake and in particular their release from endolysosomal compartments into the cytoplasm of target cells. The usage of virus proteins allows for such developments, as viruses have evolved efficient entry mechanisms into the cell, mediated by their fusion proteins. In our investigations, the transferability of the glycoprotein G which is a fusion protein of the vesicular stomatitis virus (VSV-G) onto the surface of a layer-by-layer (LbL) designed microcarrier was investigated. The assembly of VSV-G as a reversible viral fusion protein onto LbL microcarriers indeed induced an enhanced uptake rate on Vero cells as well as a fast and efficient release of the intact carriers from endolysosomes into the cytoplasm. Additionally, neither virus-associated effects on cellular viability nor activation of an interferon response were detected. Our study emphasizes the suitability of VSV-G as an efficient surface functionalization of drug-delivery systems.

ATP6L, the C subunit of the V‐ATPase V0 domain, is involved in regulating the acidic tumor micro‐environment and may promote tumor progression. However, the expression and functional role of ATP6L in tumors have not yet been well explored. In this study, we found that ATP6L protein overexpression was related to colorectal cancer histological differentiation (P, ATP6L is markedly overexpressed in the poorly differentiated colorectal cancer tissues evidently located in the invasive front and metastatic foci. More importantly, in vitro and in vivo data demonstrated that ATP6L can induce the EMT program in CRC cells. Ectopic ATP6L expression can promote microvessel density in animal xenograft tissue.

Grass carp reovirus (GCRV), the genus Aquareovirus in family Reoviridae, is viewed as the most pathogenic aquareovirus. To understand the molecular mechanism of how aquareovirus initiates productive infection, the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots (QDs)-tracking in combination with biochemical approaches. We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH(4)Cl, chloroquine and bafilomycin A1 (Bafi). Confocal images indicated that GCRV particles could colocalize with Rab5, Rab7 and lysosomes in host cells. Further ultrastructural examination validated that viral particle was found in late endosomes. Moreover, disruption of microtubules with nocodazole clearly blocked GCRV entry, while no inhibitory effects were observed with cytochalasin D treated cells in viral infection, hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament. Notably, viral particles were observed to transport along microtubules by using QD-labeled GCRV. Altogether, our results suggest that GCRV can use endosomes and microtubules to initiate productive infection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-019-00178-1) contains supplementary material, which is available to authorized users.

BMC developmental biology 19, 22 (2019). doi:10.1186/s12861-019-0203-y, Published by BioMed Central, London

Objective:Bafilomycin-A1 and ML9 are lysosomotropic agents, irrespective of cell types. However, the mechanisms of lysosome targeting either bafilomycin-A1 or ML9 are unclear.Methods:The present research has been carried out by different molecular and biochemical analyses like western blot, confocal imaging and FACS studies, as well as molecular docking.Results:Our data shows that pre-incubation of neonatal cardiomyocytes with ML9 for 4h induced cell death, whereas a longer period of time (24h) with bafilomycin-A1 was required to induce an equivalent effect. Neither changes in ROS nor ATP production is associated with such death mechanisms. Flow cytometry, LC3-II expression levels, and LC3-GFP puncta formation revealed a similar lysosomotropic effect for both compounds. We used a molecular docking approach, that predicts a stronger inhibitory activity against V-ATPase-C1 and C2 domains for bafilomycin-A1 in comparison to ML9.Conclusion:Bafilomycin-A1 and ML9 are lysosomotropic agents, involved in cell death events. But such death events are not associated with ATP and ROS production. Furthermore, both the drugs target lysosomes through different mechanisms. For the latter, cell death is likely due to lysosomal membrane permeabilization and release of lysosomal proteases into the cytosol.