Lipopolysaccharide induces neuroinflammation in microglia by activating the MTOR pathway and downregulating Vps34 to inhibit autophagosome formation.

BACKGROUND: Microglial activation is a prominent feature of neuroinflammation, which is present in almost all neurodegenerative diseases. While an initial inflammatory response mediated by microglia is considered to be protective, excessive pro-inflammatory response of microglia contributes to the pathogenesis of neurodegeneration. Although autophagy is involved in the suppression of inflammation, its role and mechanism in microglia are unclear. METHODS: In the present study, we studied the mechanism by which lipopolysaccharide (LPS) affects microglial autophagy and the effects of autophagy on the production of pro-inflammatory factors in microglial cells by western blotting, immunocytochemistry, transfection, transmission electron microscopy (TEM), and real-time PCR. In a mouse model of neuroinflammation, generated by intraventricular injection of LPS (5 µg/animal), we induced autophagy by rapamycin injection and investigated the effects of enhanced autophagy on microglial activation by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. RESULTS: We found that autophagic flux was suppressed in LPS-stimulated N9 microglial cells, as evidenced by decreased expression of the autophagy marker LC3-II (lipidated form of MAP1LC3), as well as increased levels of the autophagy adaptor protein SQSTM1. LPS significantly decreased Vps34 expression in N9 microglial cells by activating the PI3KI/AKT/MTOR pathway without affecting the levels of lysosome-associated proteins and enzymes. More importantly, overexpression of Vps34 significantly enhanced the autophagic flux and decreased the accumulation of SQSTM1 in LPS-stimulated N9 microglial cells. Moreover, our results revealed that an LPS-induced reduction in the level of Vps34 prevented the maturation of omegasomes to phagophores. Furthermore, LPS-induced neuroinflammation was significantly ameliorated by treatment with the autophagy inducer rapamycin both in vitro and in vivo. CONCLUSIONS: These data reveal that LPS-induced neuroinflammation in N9 microglial cells is associated with the inhibition of autophagic flux through the activation of the PI3KI/AKT/MTOR pathway, while enhanced microglial autophagy downregulates LPS-induced neuroinflammation. Thus, this study suggests that promoting the early stages of autophagy might be a potential therapeutic approach for neuroinflammation-associated diseases.

Mechanistic target of rapamycin-mediated autophagy is involved in the alleviation of lipopolysaccharide-induced acute lung injury in rats.

Acute lung injury (ALI) is a complex clinical syndrome with high morbidity and mortality rates. Autophagy is an adaptive process that plays a complex role in ALI. The aim of this study was to investigate the effects of autophagy on lipopolysaccharide (LPS)-induced lung injury by establishing a rat ALI model and to further explore the possible mechanisms involved. Rats were pretreated with the autophagy inhibitor 3-methyladenine (3-MA) or the autophagy activator rapamycin before they were challenged with the intratracheal instillation of LPS (5 mg/kg). The level of autophagy in the lung tissue was detected. Lung injury and vascular permeability were assessed. The role of the mechanistic target of rapamycin (mTOR)-mediated Unc-51-like kinase 1 (ULK1) and the class III PI3 kinase VPS34 in autophagy regulation was examined. LPS challenge induced autophagy and rapamycin pretreatment enhanced autophagy activity in LPS-induced ALI rats. LPS caused severe lung injury and high pulmonary vascular permeability, which could be alleviated by enhancing autophagy. In addition, the inhibition of mTOR upregulated the expression of ULK1 and VPS34 and thus increased LPS-induced autophagy. Autophagy plays a protective role in LPS-induced ALI, and enhancing autophagy via the inhibition of mTOR alleviates lung injury and pulmonary barrier function. Moreover, mTOR negatively mediates ULK1 and VPS34 to regulate LPS-induced autophagy in rats.

Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia.

B-cell acute lymphoblastic leukemia is the most common type of pediatric leukemia. Despite improved remission rates, current treatment regimens for pediatric B-cell acute lymphoblastic leukemia are often associated with adverse effects and central nervous system relapse, necessitating more effective and safer agents. Bafilomycin A1 is an inhibitor of vacuolar H(+)-ATPase that is frequently used at high concentration to block late-phase autophagy. Here, we show that bafilomycin A1 at a low concentration (1 nM) effectively and specifically inhibited and killed pediatric B-cell acute lymphoblastic leukemia cells. It targeted both early and late stages of the autophagy pathway by activating mammalian target of rapamycin signaling and by disassociating the Beclin 1-Vps34 complex, as well as by inhibiting the formation of autolysosomes, all of which attenuated functional autophagy. Bafilomycin A1 also targeted mitochondria and induced caspase-independent apoptosis by inducing the translocation of apoptosis-inducing factor from mitochondria to the nucleus. Moreover, bafilomycin A1 induced the binding of Beclin 1 to Bcl-2, which further inhibited autophagy and promoted apoptotic cell death. In primary cells from pediatric patients with B-cell acute lymphoblastic leukemia and a xenograft model, bafilomycin A1 specifically targeted leukemia cells while sparing normal cells. An in vivo mouse toxicity assay confirmed that bafilomycin A1 is safe. Our data thus suggest that bafilomycin A1 is a promising candidate drug for the treatment of pediatric B-cell acute lymphoblastic leukemia.