betadex and 3-methyladenine

A disturbed autophagic pathway leads to chronically increased levels of autophagic vacuoles in Niemann Pick Type-C 1 (NPC1) deficient neurons. Since these accumulations potentially contribute to neuronal cell death associated with the disease, we investigated two pharmacological strategies which potentially reduce the number of autophagic structures under following aspects: efficiency, sustainability and effect on neuronal cell viability. The strategies comprised (i) an interruption of the autophagic flux by the class III PI3K inhibitor 3-methyladenine (3-MA) and (ii) an acceleration of the autophagic execution by 2-hydroxypropyl-β-cyclodextrin (pCD). Our data show that the inhibition of autophagy with 3-MA only initially reduced the number of autophagic vacuoles in cultured neurons. Prolonged treatments with the PI3K-inhibitor reversed this lowering effect. The re-increase in the number of autophagic vacuoles was combined with a defect in the integrity of lysosomes which endangered further survival of cells. The treatment with pCD evoked a slow but sustained reduction of autophagic structures and had no negative effects on neuronal survival.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Adenine; Animals; Autophagy; beta-Cyclodextrins; Cells, Cultured; Cerebral Cortex; Cholesterol; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation; Intracellular Signaling Peptides and Proteins; Lysosomal-Associated Membrane Protein 2; Lysosomes; Mice; Mice, Inbred BALB C; Mice, Transgenic; Microtubule-Associated Proteins; Neurons; Niemann-Pick C1 Protein; Proteins; Time Factors; Vacuoles

Autophagy, evoked by diverse stresses including myocardial ischemia/reperfusion (I/R), profoundly affects the development of heart failure. However, the specific molecular basis of autophagy remains to be elucidated. Here we report that sphingosylphosphorylcholine (SPC), a bioactive sphingolipid, significantly suppressed apoptosis and induced autophagy in cardiomyocytes. Blocking this SPC evoked autophagy by 3-methyladenine (3MA)-sensitized cardiomyocytes to serum deprivation-induced apoptosis. Subsequent studies revealed that SPC downregulated the phosphorylation of p70S6K and 4EBP1 (two substrates of mTOR) but enhanced that of JNK when inducing autophagy. We identified SPC as a switch for the activity of Akt1, a supposed upstream modulator of both mTOR and JNK. Furthermore, β-cyclodextrin, which destroys membrane cholesterol, abolished the SPC-reduced phosphorylation of both Akt and PTEN, thus inhibiting SPC-induced autophagy. In conclusion, SPC is a novel molecule protecting cardiomyocytes against apoptosis by promoting autophagy. The lipid raft/PTEN/Akt1/mTOR signal pathway is the underlying mechanism and might provide novel targets for cardiac failure therapy.

Topics: Adenine; Animals; Animals, Newborn; Apoptosis; Autophagy; beta-Cyclodextrins; Carrier Proteins; Gene Expression Regulation; Intracellular Signaling Peptides and Proteins; MAP Kinase Kinase 4; Membrane Microdomains; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinase 8; Myocytes, Cardiac; Phosphoproteins; Phosphorylcholine; Primary Cell Culture; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sphingosine; TOR Serine-Threonine Kinases