The Mechanisms Of Leflunomide-induced Autophagy And Its Role In Autophagic Degradation Of ALS-linked Mutant SOD1 Protein Aggregates

Amyotrophic lateral sclerosis(ALS)is a fatal neurodegenerative disease and can't be cure at present.90%of diagnosed cases were sporadic ALS,and the remaining 10%were familial ALS.Many pathogenic genes have been found,of which 20%of familial ALS patients carry the mutated Cu/Zn superoxide dismutase(SOD1)gene.There is usually large number of deposits of mutant SOD1G93A protein aggregates found in their motor neurons,which leads to the death of motor neurons.Autophagy is the process by which cells phagocytose,degrade,and recycle their redundant proteins and damaged organelles.In the process of autophagy,Mammalian target of rapamycin(mTOR)and Adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)play an important role.mTOR is a serine/threonine kinase,it phosphorylates ULK1 at S757 site and inhibits autophagosome assembly.On the other hand,AMPK phosphorylates and activates ULK1S555,which induces autophagy.Leflunomide is an autophagy inhibitor that is widely used in the treatment of rheumatoid arthritis,autoimmune diseases,and organ transplantation.Recently we found that Leflunomide and its active metabolite A77 1726 can inhibit the activity of S6K1.S6K1 is a serine/threonine kinase downstream of mTOR,which can be activated and phosphorylated by mTOR.Studies have shown that AMPK activity is up-regulated in S6K1-deficient mice and S6K1-/-myotubes.However,whether S6K1 regulates autophagy in mammalian cells remains to be found.We supposed that leflunomide can activate AMPK by inhibiting S6K1 activation and induce autophagy,which result to the reduction of SOD1G93A aggregates in nervous system and delay of ALS.To test our hypothesis,we first treated NSC34 cells with A77 1726,and autophagy-related proteins were tested through Western blot analysis.The results showed that A77 1726 was capable of feedback activation of PI3K pathway and significantly increased the expression of autophagy marker LC3II in a dose-and time-dependent manner.Confocal analysis showed that compared with the control group,there is a significantly increase of the number of intracytoplasmic autophagosomes in the A77 1726-treated group in NSC34 cells.These results show that A77 1726 indeed induces autophagy.To further verify whether S6K1 inhibition induce autophagy,NSC34 cells were treated with siRNA and S6K1 inhibitor PF-4708671.Western blot analysis showed that LC3II expression was significantly up-regulated after S6K1 inhibition.Confocal analysis also showed that the number of autophagosomes in the cytoplasm significantly increased after S6K1 inhibition,indicating that inhibition of S6K1 induces autophagy.To investigate the potential mechanism of A77 1726-induced autophagy,NSC34 cells were treated with A77 1726,S6K1 siRNA or PF-4708671.Western blot analysis showed that inhibition of S6K1 could up-regulate the phosphorylation of AMPKT172,ACCS79 and ULKlS555,and feedback activate ULK1S757.To investigate the role TAK1 plays in A77 1726-mediated autophagy,NSC34 cells were treated with TAK1 inhibitor 5Z-7-oxozeaenol or TAK1 siRNA followed by A77 1726 treatment.Western blot analysis showed that A77 1726 could phosphorylate and activate TAK1T84/187,and significantly reduce phosphorylation levels of AMPKT172 and ULK1S5555 as well as the expression level of LC3? while its activity was inhibited,suggesting that TAK1 may mediated A77 1726-induced autophagy through affecting AMPK phosphorylation.Finally,we researched the effect of A77 1726 on the degradation of SOD1 mutant protein aggregates.NSC34 cells were transfected with GFP-SOD1WT and GFP-SOD1G93A plasmids and then treated with A77 1726.Confocal microscopy analysis showed that A77 1726 significantly reduced the number of GFP-SOD1 positive cells.Microplate reader and flow cytometry analysis showed that A77 1726 significantly reduced the average fluorescence intensity of GFP-SOD1WT and GFP-SOD1G93A transfected cells.Western blot results showed that A77 1726 significantly reduced the expression of SOD1G93A aggregates.To further find out whether the reduction of GFP-SOD1G93A mutant protein aggregates was degraded by autophagy pathway,autophagy-related gene ATG7 was knocked down and the cells were treated with A77 1726.Western blot analysis showed that knock down of ATG7 could inhibit the expression of LC3?induced by A77 1726 and block the reduction effect of SOD1G93A aggregates.All these results show that A77 1726 can activate autophagy by inhibiting the expression of S6K1 and accelerate the autophagic degradation of SOD1G93A mutant protein aggregates.