Enniatin B1-induced lysosomal membrane permeabilization in mouse embryonic fibroblasts.

The mycotoxin enniatin B1 (ENN B1) is widely present in grain-based feed and food products. In the present study, we have investigated how this lipophilic and ionophoric molecule can affect the lysosomal stability and chaperone-mediated autophagy (CMA) in wild-type (WT) and in lysosome-associated membrane proteins (LAMP)-1/2 double-deficient (DD) mouse embryonic fibroblasts (MEF). The cell viability and lysosomal pH were assessed using the Neutral Red (NR) cytotoxicity assay and the LysoSensor® Yellow/Blue DND-160, respectively. Changes in the expression of the CMA-related components LAMP-2 and the chaperones heat shock cognate (hsc) 70 and heat shock protein (hsp) 90 were determined in cytosolic extracts by immunoblotting. In the NR assay, LAMP-1/2 DD MEF cells were significantly less sensitive to ENN B1 than WT MEF cells after 24 h exposure to ENN B1 at levels of 2.5-10 µmol/L. Exposure to ENN B1 at concentrations below the half maximal effective concentration (EC50) (1.5-1.7 µmol/L) increased the lysosomal pH in WT MEF, but not in LAMP-1/2 DD cells, suggesting that lysosomal LAMP-2 is an early target of ENN B1-induced lysosomal alkalization and cytotoxicity in MEF cells. Additionally, cytosolic hsp90 and LAMP-2 levels slightly increased after exposure for 4 h, indicating lysosomal membrane permeabilization (LMP). In summary, it appeared that ENN B1 can destabilize the LAMP-2 complex in the lysosomal membrane at concentrations close to the EC50, resulting in the alkalinization of lysosomes, partial LMP, and thereby leakage of CMA-associated components into the cytosol.

Indomethacin suppresses LAMP-2 expression and induces lipophagy and lipoapoptosis in rat enterocytes via the ER stress pathway.

BACKGROUND: Indomethacin enhances small intestinal epithelial cell apoptosis, which may account for mucosal ulceration. However, the involvement of autophagy in indomethacin-induced enterocyte damage is unreported. METHODS: Using light microscopy and electron microscopy techniques, Western blot analysis, and pharmacological inhibition of autophagy, we investigated the autophagic response of cultured rat enterocytes to indomethacin treatment (200 µM) at various time points. Furthermore, autophagy was examined in enterocytes of rats given indomethacin by gavage (10 mg/kg). RESULTS: Our data indicate that indomethacin induced accumulation of cytoplasmic lipid droplets (LDs) in cultured enterocytes, which was associated with time-dependent autophagic responses. Initially (0-6 h), mediated by endoplasmic reticulum stress and suppression of mammalian target of rapamycin, a predominant cytoprotective lipophagy was activated in indomethacin-treated enterocytes, as evidenced by induction and colocalization of LC3-II with LDs, excessive formation of autophagosomes sequestering LDs (autolipophagosomes; ALPs), and decreased viability of enterocytes on blocking autophagy with 3-methyladenine. On prolonged exposure to indomethacin (6-24 h), there was a decrease of LAMP-2 expression in enterocytes coupled with accumulation of ALPs and LDs with fewer autolysosomes in addition to an elevation of lipoapoptosis. These time-dependent autophagic and apoptotic responses to indomethacin treatment were detected in enterocytes of indomethacin-treated rats, confirming in vitro results. CONCLUSIONS: The findings of this study describe a novel mechanism of enterocyte damage by indomethacin mediated by endoplasmic reticulum stress, accumulation of LDs, and subsequent activation of the early phase of cytoprotective lipophagy. This is followed by a late phase characterized by reduced expression of lysosomal autophagic proteins, accumulation of ALPs, and enhanced lipoapoptosis.

Cholesterol supports the retinoic acid-induced synaptic vesicle formation in differentiating human SH-SY5Y neuroblastoma cells.

Synaptic vesicle formation, vesicle activation and exo/endocytosis in the pre-synaptic area are central steps in neuronal communication. The formation and localization of synaptic vesicles in human SH-SY5Y neuroblastoma cells, differentiated with 12-o-tetradecanoyl-phorbol-13-acetate, dibutyryl cyclic AMP, all-trans-retinoic acid (RA) and cholesterol, was studied by fluorescence microscopy and immunocytochemical methods. RA alone or together with cholesterol, produced significant neurite extension and formation of cell-to-cell contacts. Synaptic vesicle formation was followed by anti-synaptophysin (SypI) and AM1-43 staining. SypI was only weakly detected, mainly in cell somata, before 7 days in vitro, after which it was found in neurites. Depolarization of the differentiated cells with high potassium solution increased the number of fluorescent puncta, as well as SypI and AM1-43 co-localization. In addition to increase in the number of synaptic vesicles, RA and cholesterol also increased the number and distribution of lysosome-associated membrane protein 2 labeled lysosomes. RA-induced Golgi apparatus fragmentation was partly avoided by co-treatment with cholesterol. The SH-SY5Y neuroblastoma cell line, differentiated by RA and cholesterol and with good viability in culture, is a valuable tool for basic studies of neuronal metabolism, specifically as a model for dopaminergic neurons.