These results indicate that 17 AAG promotes the clearance of the small asynuclein accumulations via lysosomal pathways. Hence we probed Deforolimus AP23573 for LC3, a specific marker for autophagosomes, to test if autophagic activity was induced by 17 AAG. During autophagosome formation endogenous LC3 is processed to LC3 I, an 18 kDa cytosolic isoform, which is converted to LC3 II. The latter is a membrane bound 16 kDa isoform which associates with the autophagosomal membranes and its amount as compared to tubulin or actin correlates with the number of autophagosomes. Cells were incubated for 24 h with increasing concentrations of 17 AAG or with 50 nM 17 AAG for 3 24 h, cell lysates were prepared and subjected to immunoblot analysis. Fig. 4A demonstrates that 17 AAG in a time and concentration dependent manner markedly increased the level of LC3 II. Quantitative evaluation indicates that this effect is maximal after 18 24 h at a concentration of 50 nM.
Since LC3 II itself is degraded by autophagy, we compared LC3 II levels in the absence and presence of the lysosomal inhibitor NH4Cl. Immunoblot analysis revealed that when cells were incubated with 17 AAG in combination with NH4Cl for 24, the level of LC3 II was further augmented in comparison to the treatment with 17 AAG alone, pointing to an enhancement of the autophagic flux by 17 AAG. Neither NH4Cl nor chloroquine alone caused the upregulation of HSP70 or of any other HSPs tested. Quantitative evaluation of the immunoblots indicated that in the presence of NH4Cl the amount of a synuclein was enhanced supporting the notion that the lysosomal pathway is involved in its degradation.
To assess if the stimulatory effects of 17 AAG on macroautophagy is not restricted to the oligodendroglial clonal cell line used in this study, primary cultures of rat brain oligodendrocytes were prepared and subjected to 17 AAG. Oligodendrocytes treated with 17 AAG remained morphologically intact and displayed an arborized morphology. Immunoblot analysis further indicated that 17 AAG in oligodendrocytes increased the levels of LC3 II. Also rapamycin caused an increase in LC3 II. Indirect immunofluorescence corroborated this finding, demonstrating the accumulation of LC3 positive puncta in the cell somata similarly as observed after treatment with the macroautophagy inducer rapamycin. Similarly to primary cultures of oligodendrocytes, 17 AAG caused a marked increase in the level of LC3 II in OLN cells stably expressing asynuclein in the absence of tau.
However, both cell culture systems do not express prefibrillary a synuclein aggregates under normal growth conditions, hence, OLN A53T 3 Methyladenine Inhibits 17 AAG Induced Autophagy and LC3 Puncta Formation The contribution of macroautophagy to the degradation of asynuclein in OLN A53T cells was further confirmed by using the selective inhibitor of macroautophagy, 3 methyladenine. In cells incubated in the presence of 17 AAG and 3 MA simultaneously for 24 h, a synuclein positive aggregates remained to be present throughout the cytoplasm, and thus the aggregate clearing effect of 17 AAG was abolished. Immunoblot analysis of cell extracts depicted that application of 3 MA alone or in combination with 17 AAG prevented or reduced the formation of LC3 II, while the induction of HSP70 was not affected.