Lysosomal Stability Research Articles

Up-Regulated Expression of LAMP2 and Autophagy Activity during Neuroendocrine Differentiation of Prostate Cancer LNCaP Cells.

Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and βIII tubulin (βIII tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1μM AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer.

TMEPAI increases lysosome stability and promotes autophagy

Autophagy is emerging as a critical response of normal and cancer cells to environmental changes and plays an important role in cell metabolism and maintenance of damaged organelles. Transmembrane prostate androgen-induced protein (TMEPAI) is a pro-tumorigenic factor with high expression in tumor cells. In this study, we showed that depletion of TMEPAI leads to lysosomal labilization and inhibits autophagy. Further study showed that the inhibition of autophagy induced by the depletion of TMEPAI is involved in regulation of Beclin-1. Depletion of TMEPAI increases the sensitivity of cancer cells to chemotherapeutic drugs. Our study reveals the role of TMEPAI in promoting lysosome stability and autophagy, which might be used as a target for cancer chemotherapeutic treatment.

Effect of L-arginine and L-name on lysosomal cysteine proteases activity and lysosomal membranes permeability in rat aorta

Aim. To study the effect of L-arginine and its analogue N-nitro-L-arginine methyl ester (L-NAME) alone and in combination on lysosomal cysteine proteolysis and lysosomal membranes state in rat aorta.Methods. The study was performed on male Wistar rats kept under standard vivarium conditions and divided into three control and three experimental groups of 8 animals each. The experimental samples included groups with L-arginine and/or L-NAME administration. The indicators were studied in the rat aorta homogenate precipitating and non precipitating fractions. Acid phosphatase activity was determined by a standardized method of «end point», the cathepsins B, L and H activity was studied by spectrofluorimetric method.Results. When simulating the changes of nitric oxide synthesis level using L-arginine, the increase of the total cathepsins activity was detected, acid phosphatase lability coefficient was reduced, what is characterized by general lysosomal membranes stabilization. L-NAME group, in contrast, is characterized by a decrease in the cathepsin B and H activity indicators, differences from arginine group were observed in the cathepsin H in lysosomal and general fractions, lysosomal membrane is labile. Combined drugs administration reduces the total cathepsins activity, while there is an increase of the acid phosphatase total activity, all indicators suggest lysosomal membranes labilization.Conclusion. L-arginine at a dose of 500 mg/kg causes increase in the total cathepsins B, L and H activity in rat aorta due to lysosomal fraction; L-arginine action leads to lysosomal membranes stabilization; L-NAME group in cathepsin H shows a decrease in the cathepsins secretion level with decreased total activity due to both factions; combined administration of arginine + L-NAME group in cathepsin B is characterized by an increase in secretion due to lysosomes membrane labilization.

Correction: Lens Epithelium-Derived Growth Factor Is an Hsp70-2 Regulated Guardian of Lysosomal Stability in Human Cancer.

Correction: Lens Epithelium-Derived Growth Factor Is an Hsp70-2 Regulated Guardian of Lysosomal Stability in Human Cancer.

Oxidative responsiveness to multiple stressors in the key Antarctic species, Adamussium colbecki: Interactions between temperature, acidification and cadmium exposure

High-latitude marine ecosystems are ranked to be among the most sensitive regions to climate change since highly stenothermal and specially adapted organisms might be seriously affected by global warming and ocean acidification. The present investigation was aimed to provide new insights on the sensitivity to such environmental stressors in the key Antarctic species, Adamussium colbecki, focussing also on their synergistic effects with cadmium exposure, naturally abundant in this area for upwelling phenomena. Scallops were exposed for 2 weeks to various combinations of Cd (0 and 40 μgL-1), pH (8.05 and 7.60) and temperature (−1 and +1 °C). Beside Cd bioaccumulation, a wide panel of early warning biomarkers were analysed in digestive glands and gills including levels of metallothioneins, individual antioxidants and total oxyradical scavenging capacity, onset of oxidative cell damage like lipid peroxidation, lysosomal stability, DNA integrity and peroxisomal proliferation. Results indicated reciprocal interactions between multiple stressors and their elaboration by a quantitative hazard model based on the relevance and magnitude of effects, highlighted a different sensitivity of analysed tissues. Due to cellular adaptations to high basal Cd content, digestive gland appeared more tolerant toward other prooxidant stressors, but sensitive to variations of the metal. On the other hand, gills were more affected by various combinations of stressors occurring at higher temperature.

Effect of glutamate on lysosomal membrane permeabilization in primary cultured cortical neurons.

Glutamate is the principal neurotransmitter in the central nervous system. Glutamate-mediated excitotoxicity is the predominant cause of cerebral damage. Recent studies have shown that lysosomal membrane permeabilization (LMP) is involved in ischemia-associated neuronal death in non-human primates. This study was designed to investigate the effect of glutamate on lysosomal stability in primary cultured cortical neurons. Glutamate treatment for 30 min induced the permeabilization of lysosomal membranes as assessed by acridine orange redistribution and immunofluorescence of cathepsin B in the cytoplasm. Inhibition of glutamate excitotoxicity by the NMDA receptor antagonist MK-801 and the calcium chelator ethylene glycolbis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid, rescued lysosomes from permeabilization. The role of calpain and reactive oxygen species (ROS) in inducing LMP was also investigated. Ca2+ overload following glutamate treatment induced the activation of calpain and the production of ROS, which are two major contributors to neuronal death. It has been reported that lysosomal-associated membrane protein 2 (LAMP2) and heat shock protein (HSP)70 are two calpain substrates that promote LMP in cancer cells; however, it was found that calpains were activated by glutamate, but only LAMP2 was subsequently degraded. Furthermore, LMP was not alleviated by treatment with the calpain inhibitors calpeptin and SJA6017, which blocked the cleavage of the calpain substrate α-fodrin. It was demonstrated that LMP was significantly alleviated by treatment with the antioxidant N-Acetyl-L-cysteine, indicating that LMP involvement in early glutamate excitotoxicity may be mediated partly by ROS rather than calpain activation. Overall, these data shed light on the role of ROS-mediated LMP in early glutamate excitotoxicity.

PIG7 promotes leukemia cell chemosensitivity via lysosomal membrane permeabilization.

PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the “verge of apoptosis”. When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways.

In vitro effects of L-arginine on lysosomal cysteine proteases activity in isolated experiment and in the state of oxidative stress

Aim. Assessment of direct influence of arginine on lysosomal cysteine proteases activity in vitro, in isolation as well as the stimulation of oxidative stress.
 Methods. The study was conducted on the 72 female conventional mature Wistar rats 280-320 g divided into 6 series of 12 rats each. Lysosome slurries were isolated from the liver of intact animals with a subsequent in vitro incubation in a sucrose solution, in the presence of L-arginine, as well as in the presence of L-arginine accompanied by the stimulation of oxidative stress. Samples of control groups were exposed in vitro with the addition of isolate and oxidant, respectively. Each batch was reproduced three times, incubation was performed at 37 °C in a water bath for 1, 2 and 4 hours. The activity of cathepsins B, L and H was studied using spectrofluorimetric method in two fractions - intra- and extralysosomal. Acid phosphatase activity was used as the main marker of membrane labialization.
 Results. One hour Incubation with 5 mM arginine in vitro led to inhibition of the cathepsin H activity and lysosomal membrane damage, however, further increase in incubation time led to its stabilization. In vitro exposure to 5 mM H2O2 caused an increase in activity of cathepsines B and L and the drop in the cathepsin H activity without obvious changes in the distribution of enzymes between extra and intralysosomal fractions. In a state of oxidative stress 2-hour in vitro incubation with 5 mM arginine reduced the permeability of lysosomal membranes for cathepsines B, H and L; while 4-hour incubation led to the destabilization of lysosomal membranes.
 Conclusion. The direct effect of arginine at a concentration of 5 mM within the 1,2 and 4-hour time intervals leads to a distinct change as a lysosomal cysteine protease activity and stability of lysosomal membranes.

Effect of L-arginine and carnitine on cathepsin L and H activity and lysosomal membranes permeability in myocardium in expressed hyperhomocysteinemia

Aim. To study the activity of lysosomal cysteine proteases (cathepsins L, H) and acid phosphatase, changing of permeability, stability of myocardial lysosomal membranes in rats in experimental expressed hyperhomocysteinemia model, and while administering L-arginine and carnitine. Methods. The study was performed on male Wistar rats kept on standard vivarium conditions divided into three control and three experimental groups of 8 animals each. Experimental samples were administered methionine, or combination of L-arginine and carnitine with methionine. The level of serum homocysteine was measured by ELISA. Cathepsin L and H activity was detected by spectrofluorimetric method. Acid phosphatase activity was recorded using the «end point» method. Results. In the model of expressed hyperhomocysteinemia the increase of cathepsin H total activity due to both lysosomal and nonlysosomal fractions was found. These changes were observed along with the general increase of lysosomal membranes permeability. When correcting hyperhomocysteinemia with L-arginine and carnitine a decrease of cathepsin L and H levels was noted as well as positive effect on the myocardial lysosomal membranes stability. Conclusion. Expressed hyperhomocysteinemia is accompanied by statistically significant increase of both lysosomal and cytoplasmic fractions of the cathepsin H activity, indicating the lysosomal membranes permeabilisation phenomenon; L-carnitine and arginine correct hyperhomocysteinaemia effects, leading to cathepsin L and H reduced activity and having a stabilizing effect on the lysosomal membranes of cardiomyocytes.

Developing a sentinel mollusc species for toxicity assessment: metal exposure, dose and response – laboratory v. field exposures and resident organisms

Environmental contextMetal contamination in estuarine sediments can affect ecosystem health. Molluscs are commonly used as environmental indicators because they accumulate contaminants that cause adverse health effects. We investigated metal uptake and effects in the Sydney cockle, comparing exposure to contaminated lake sediments in situ and in laboratory aquariums. Although differences were observed between the different exposure types, all approaches were found to be valid for investigating metal health effects in this organism. AbstractRelationships between exposure, tissue dose and biological responses of the benthic marine bivalve Anadara trapezia to an estuarine sediment zinc, copper, lead, cadmium and selenium contamination gradient in Lake Macquarie, NSW, were evaluated using three approaches. Organisms were exposed to sediments in laboratory aquaria, caged in situ in the lake and lake resident organisms collected. Dose included total metal tissue burden and subcellular metal distribution to determine metabolically available metal. Response indices were total antioxidant capacity, lipid peroxidation, lysosomal stability and condition index. Bioaccumulation of total metals was higher in the laboratory and resident organisms than in those transplanted in the field but the contribution of individual metals to the total differed. Laboratory-exposed organisms had increased concentrations of cadmium and lead in their biologically active and detoxified metal fractions but not of the essential elements zinc and copper. Subcellular metal distribution patterns were the same in resident organisms but cadmium and lead burdens were higher in both fractions. Biomarker responses were similar in laboratory, transplanted and resident organisms. Total antioxidant capacity was significantly reduced and lipid peroxidation and lysosomal destabilisation significantly increased in all metal-exposed organisms compared with the reference A. trapezia. Condition index of laboratory-exposed organisms was significantly lower than in situ, resident and reference organisms. Clear metal exposure–dose–response relationships have been demonstrated for A. trapezia in laboratory and in situ experiments. Non-resident organisms, in both exposure scenarios, gave similar responses to resident metal-exposed organisms, showing all approaches are valid when investigating effects in this species.

Para-toluenesulfonamide induces tongue squamous cell carcinoma cell death through disturbing lysosomal stability.

Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors. In the present study, we investigated the inhibitory effects of PTS on tongue squamous cell carcinoma (Tca-8113) and explored the lysosomal and mitochondrial changes after PTS treatment in vitro. High-performance liquid chromatography showed that PTS selectively accumulated in Tca-8113 cells with a relatively low concentration in normal fibroblasts. Next, the effects of PTS on cell viability, invasion, and cell death were determined. PTS significantly inhibited Tca-8113 cells’ viability and invasive ability with increased cancer cell death. Flow cytometric analysis and the lactate dehydrogenase release assay showed that PTS induced cancer cell death by activating apoptosis and necrosis simultaneously. Morphological changes, such as cellular shrinkage, nuclear condensation as well as formation of apoptotic body and secondary lysosomes, were observed, indicating that PTS might induce cell death through disturbing lysosomal stability. Lysosomal integrity assay and western blot showed that PTS increased lysosomal membrane permeabilization associated with activation of lysosomal cathepsin B. Finally, PTS was shown to inhibit ATP biosynthesis and induce the release of mitochondrial cytochrome c. Therefore, our findings provide a novel insight into the use of PTS in cancer therapy.

Further evidences for the mode of action of the larvicidal m-pentadecadienyl-phenol isolated from Myracrodruon urundeuva seeds against Aedes aegypti

Nowadays, dengue fever is considered the most important arbovirosis worldwide and its control is still based upon combating the vector Aedes aegypti. Besides monitoring of mosquito populations resistant to conventional insecticides, the search for new environmentally safe insecticides and conduction of molecular studies focusing on the elucidation of mode of action and possible resistance mechanisms are considered the key for a sustainable management of the mosquito vector. Thus, the present work aimed to assess changes in protein expression of 3rd-instar larvae of Ae. aegypti after exposure to the natural insecticide m-pentadecadienyl-phenol. Bidimensional electrophoresis followed by mass spectrometry resulted in identification of 12 proteins differentially expressed between control and treated groups. Larvae exposed to the toxic compound for 24h showed elevated detoxification response (glutathione-S-transferase), increased levels of stress-related proteins (HSP70) as well as evidence of lysosome stabilization to enable survival. Furthermore, expression of proteins involved in protection of peritrophic membrane and metabolism of lipids indicated systemic effect of toxic effects in treated larvae.

Physiological Indicators of Stress and Morbidity in Commercially Handled AbaloneHaliotis iris

ABSTRACT Abalone are enigmatic, displaying subtle responses to stress, including a delayed onset of morbidity, and death after severe stress. The present study therefore sought to further elucidate the nature of stress responses in the blackfoot abalone Haliotis iris and develop practical indices to quantify stress and forecast survival. Abalone were challenged with air exposure (16°C, 4–120 h). Physiological responses were assessed at three organizational levels: whole organism (heart rate), extracellular (hemolymph metal concentrations), and intracellular (hemocyte lysosome stability). Hemolymph sodium:potassium ratio showed a strong negative correlation with emersion time (R2 = 0.82) and a reasonable capacity to predict subsequent survival (R2 = 0.50); a [Na]:[K] of 23 was representative of an undisturbed abalone, whereas values less than 15 typically indicated morbidity. Hemocytes tended to lyse before lysosome destabilization could be quantified by neutral red loss; the stability assay therefore fail...

Cellular responses of eastern oysters, Crassostrea virginica, to titanium dioxide nanoparticles

Because of the continued development and production of a variety of nanomaterials and nanoparticles, their uptake and effects on the biota of marine ecosystems must be investigated. Filter feeding bivalve molluscs are highly adapted for capturing particles from the external environment and readily internalize nano- and micro-sized particles through endocytosis, so they are commonly used as valuable indicator species for nanoparticle studies. In these studies, adult eastern oysters, Crassostrea virginica, were exposed to a range of titanium dioxide nanoparticle (TiO2-NP) concentrations (5, 50, 500, and 5000 μg/L) in conjunction with natural sunlight. Isolated hepatopancreas tissues were also exposed to the same TiO2-NP concentrations using particles exposed to similar light and dark conditions. Dose-dependent decreases in lysosomal stability were observed in the adult oyster studies as well as in the isolated tissues, at exposures as low as 50 μg/L. Titanium accumulation in isolated hepatopancreas tissue studies was directly correlated to lysosomal destabilization. Based on measurements of lipid peroxidation as an indicator of oxidative stress, TiO2-NPs toxicity was not related to increased ROS production over the short-term course of these exposures. Analysis of particle size using dynamic light scattering (DLS) indicated that concentration had a significant impact on agglomeration rates, and the small agglomerates as well as individual particles are readily processed by oysters. Overall, this study illustrates that low concentrations of TiO2-NPs may cause sublethal toxicity on oysters, which might be enhanced under natural sunlight conditions. In estuarine environments, where these nanomaterials are likely to accumulate, agglomeration rates, interaction with organics, and responses to sunlight are critical in determining the extent of their bioreactivity and biological impacts.

Cytotoxicity of the association of pesticides Roundup Transorb® and Furadan 350 SC® on the zebrafish cell line, ZF-L

The present study aimed to ascertain the cytotoxicity of pesticides commonly used in rice cultivation, through in vitro assays employing the ZF-L cell line. The in vitro analyses investigated three cellular targets (cell membrane integrity, mitochondrial activity and lysosomal stability) in cells exposed to concentrations of Roundup Transorb® (67.7μgL−1, 135.4μgL−1 and 270.8μgL−1), Furadan 350 SC® (0.1μgL−1, 0.05μgL−1 and 0.02μgL−1). We also tested these products in combination. We analyzed the defensive capacity of the cells by measuring the activity of xenobiotic extruder proteins, as well as the expression of these same proteins. Cytotoxic effects of both pesticides were observed individually, as well as with the mixture of both products; including an inhibitory effect on the activity of xenobiotic extrusion. When exposed to the insecticide Furadan, and also the mixture of Furadan and Roundup, there was an increase in the expression of P glycoproteins (P-gps). There was also a negative correlation with cytotoxicity, mainly exhibited by mitochondrial activity and lysosomal integrity, but also with respect to the activity of P-gps. We observed that concentrations below those allowed by law were toxic regarding all parameters tested in this study, with the exception of mitochondrial function. Taken together, our results suggest that toxicity may be due to the surfactants present in the commercial formulations.

Apoptotic cells induce NF-κB and inflammasome negative signaling.

As they undergo phagocytosis, most early apoptotic cells negatively regulate proinflammatory signaling and were suggested as a major mechanism in the resolution of inflammation. The dextran sulfate sodium model is generally viewed as an epithelial damage model suited to investigate innate immune responses. Macrophages primed with LPS and subsequently exposed to DSS secrete high levels of IL-1β in an NLRP3-, ASC-, and caspase-1-dependent manner. The aim of this research was to test the therapeutic effect of a single dose of apoptotic cells in a DSS-colitis model and to explore possible mechanisms. Primary peritoneal macrophages, the DSS mice model, and Nlrp3-deficient mice, were used to assess the effect apoptotic cells on colitis. Immunohistochemistry, flow-cytometer, and western blots helped to explore the effect and mechanisms. Using a variety of NLRP3 triggering mechanisms, we show that apoptotic cells negatively regulate NF-κB and NLRP3 activation in primary peritoneal macrophages, at pre- and post-transcription levels, via inhibition of reactive oxygen species, lysosomal stabilization, and blocking K+ efflux. This property of apoptotic cells is demonstrated in a dramatic clinical, histological, and immunological amelioration of DSS colitis in Balb/c and B6 mice following a single administration of apoptotic cells.

Enhancing the imaging and biosafety of upconversion nanoparticles through phosphonate coating.

Upconversion nanoparticles (UCNPs), which are generated by doping with rare earth (RE) metals, are increasingly used for bioimaging because of the advantages they hold over conventional fluorophores. However, because pristine RE nanoparticles (NPs) are unstable in acidic physiological fluids (e.g., lysosomes), leading to intracellular phosphate complexation with the possibility of lysosomal injury, it is important to ensure that UCNPs are safely designed. In this study, we used commercially available NaYF4:Er/Yb UCNPs to study their stability in lysosomes and simulated lysosomal fluid. We demonstrate that phosphate complexation leads to REPO4 deposition on the particle surfaces and morphological transformation. This leads to a decline in upconversion fluorescence efficiency as well as inducing pro-inflammatory effects at the cellular level and in the intact lung. In order to preserve the imaging properties of the UCNPs as well as improve their safety, we experimented with a series of phosphonate chemical moieties to passivate particle surfaces through the strong coordination of the organophosphates with RE atoms. Particle screening and physicochemical characterization revealed that ethylenediamine tetra(methylenephosphonic acid) (EDTMP) surface coating provides the most stable UCNPs, which maintain their imaging intensity and do not induce pro-inflammatory effects in vitro and in vivo. In summary, phosphonate coating presents a safer design method that preserves and improves the bioimaging properties of UCNPs, thereby enhancing their biological use.

Autophagic behavior in skeletal muscle cells

Autophagy represents a physiological mechanism responsible for cell homeostasis and its deregulation is involved in several conditions related to muscle mass loss such as aging, anorexia, inflammatory diseases, cancer, disuse and immobilization (1). In our previous work, double membrane vesicles, suggestive of autophagy, appeared after chemotherapeutic treatments in C2C12 myotubes (2). Here, to better understand the autophagic behavior, skeletal muscle cells have been exposed to cisplatin, etoposide and staurosporine and their effects have been investigated by means of morphological, cytofluorimetric and functional analyses. Ultrastructural observations evidenced the presence of autophagic vacuoles containing abnormal mitochondria, nuclear materials and membranes. Flow cytometry evaluation of lysosomal compartment stability revealed an autophagic pattern increase, particularly after cisplatin and staurosporine exposure, suggesting the presence of a cell death mechanism. Since autophagic deregulation is involved in different muscle diseases, further studies are in progress to evaluate possible strategies, such as protein ipo/iper supplementation, to prevent the abnormal autophagic activation induced by chemical triggers, which lead to muscle atrophy (3).

Heat Shock Protein 70.1 (Hsp70.1) Affects Neuronal Cell Fate by Regulating Lysosomal Acid Sphingomyelinase

The inducible expression of heat shock protein 70.1 (Hsp70.1) plays cytoprotective roles in its molecular chaperone function. Binding of Hsp70 to an endolysosomal phospholipid, bis(monoacylglycero)phosphate (BMP), has been recently shown to stabilize lysosomal membranes by enhancing acid sphingomyelinase (ASM) activity in cancer cells. Using the monkey experimental paradigm, we have reported that calpain-mediated cleavage of oxidized Hsp70.1 causes neurodegeneration in the hippocampal cornu ammonis 1 (CA1), whereas expression of Hsp70.1 in the motor cortex without calpain activation contributes to neuroprotection. However, the molecular mechanisms of the lysosomal destabilization/stabilization determining neuronal cell fate have not been elucidated. To elucidate whether regulation of lysosomal ASM could affect the neuronal fate, we analyzed Hsp70.1-BMP binding and ASM activity by comparing the motor cortex and the CA1. We show that Hsp70.1 being localized at the lysosomal membrane, lysosomal lipid BMP levels, and the lipid binding domain of Hsp70.1 are crucial for Hsp70.1-BMP binding. In the postischemic motor cortex, Hsp70.1 being localized at the lysosomal membrane could bind to BMP without calpain activation and decreased BMP levels, resulting in increasing ASM activity and lysosomal stability. However, in the postischemic CA1, calpain activation and a concomitant decrease in the lysosomal membrane localization of Hsp70.1 and BMP levels may diminish Hsp70.1-BMP binding, resulting in decreased ASM activity and lysosomal rupture with leakage of cathepsin B into the cytosol. A TUNEL assay revealed the differential neuronal vulnerability between the CA1 and the motor cortex. These results suggest that regulation of ASM activation in vivo by Hsp70.1-BMP affects lysosomal stability and neuronal survival or death after ischemia/reperfusion.

Lysosomotropic agents: impact on lysosomal membrane permeabilization and cell death.

Lysosomes are acidic organelles essential for degradation, signalling and cell homoeostasis. In addition, they play a key role in cell death. Permeabilization of the lysosomal membrane and release of hydrolytic enzymes to the cytosol accompanies apoptosis signalling in several systems. The regulatory mechanism of lysosomal stability is, however, poorly understood. Lipophilic or amphiphilic compounds with a basic moiety will become protonated and trapped within lysosomes, and such lysosomotropic behaviour is also found in many pharmacological drugs. The natural sphingolipid sphingosine exhibits lysosomotropic detergent ability and is an endogenous candidate for controlling lysosomal membrane permeabilization. The lysosomotropic properties of certain detergents might be of use in lysosome-targeting anticancer drugs and drug delivery system in the future. The present review summarizes the current knowledge on the targeting and permeabilizing properties of lysosomotropic detergents from a cellular and physicochemical perspective.