The Mechanisms Of Leflunomide-induced Autophagy And Enhancing Degradation Of Mutant SOD1 Protein And Intervention Study Of Leflunomide In ALS Transgenic Mice

Amyotrophic lateral sclerosis(ALS),commonly known as ALS,is a neurodegenerative disease characterized by damage to the anterior horn of the spinal cord,the motor nuclei of the brain stem and motor neurons in the cerebral cortex.At present,a variety of pathogenic genes are known.In Chinese patients with sporadic and familial ALS,the motor neurons often carry the mutated copper/zinc Superoxide dismutase 1(SOD1)gene,and the most common one is the accumulation of the mutated protein SOD1G93A aggregates,which leads to the death of motor neurons.Currently,there is no effective treatment.Autophagy is a general term for the degradation process of intracellular material components peculiar to eukaryotic cells by lysosomes.It is a common mechanism of purification of misfolding,protein or damaged organelles that commonly occurs in the development and aging of eukaryotic cells.Autophagy is regulated by a variety of signaling pathways,including JAK-STAT signaling pathway,MAPK-JNK pathway and PI3K-AKT-mtor pathway.c-Jun amino-terminal kinase(JNK)is the downstream response molecule of the MAPK(mitogen-activated protein kinase)signaling pathway in mammalian cells.The JNK signaling pathway plays an important role in cell differentiation,autophagy,apoptosis and stress.Previous studies have shown that the anti-rheumatoid arthritis drug leflunomide and its active metabolite N-[4-(Trifluoromethyl)phenyl]-2-cyano-3-hydroxy-2-butenamide(A77 1726)can activate TAK1 by inhibiting S6K1 Induces the occurrence of autophagy and further degrades SOD1G93A mutant protein aggregates.We recently discovered that A77 1726 can phosphorylate and activate JNK downstream of TAK1,but the role of JNK in A77 1726-induced autophagy remains unclear.Therefore,we hypothesized that leflunomide and its active metabolite A77 1726 could induce autophagy by phosphorylation and activation of JNK,and JNK played a key role in A77 1726's induction of autophagy to clear SOD1G93A aggregates.To verify the hypothesis,NSC34 cells were first treated with A77 1726 and autophagy-related proteins were analyzed by Western blotting.The results showed that A771726 could phosphorylate and activate MAPK-JNK signaling pathway related proteins such as TAKS412?MKK4S257/T261?JNKT183/Y185?Beclin1s93?Bcl-2S70,and enhance the expression levels of autophagy marker protein LC3II and autophagy substrate marker p62,and the induction effect of A77 1726 was positively correlated with the action time and dose.When the expression of TAK1 was disturbed,the phosphorylation levels of JNK and Beclinl and the expression of autophagy marker substrate p62 were inhibited.The results showed that the upstream signaling molecule TAK1 could regulate the activation and autophagy of JNK mediated by A77 1726.To further verify the role of JNK in the autophagy induced by A771726,we used inhibitor SP600125 and gene knockout to inhibit the expression of JNK gene in NSC34 cells,respectively.Western blot results showed that the phosphorylation levels of Bcl-2 and Beclinl induced by A77 1726 decreased with the inhibition of JNK,while the expression levels of LC3? and p62 decreased,and autophagy was blocked.Confocal analysis also showed that the number of autophagosomes in cytoplasm significantly decreased after JNK inhibition.The above results indicate that JNK plays a crucial role in autophagy induced by A77 1726.Then,we investigated the role of JNK in A77 1726 induced autophagy in clearing SOD1G93A aggregates.Western blot results showed that in the SOD1G93A group,A77 1726 significantly reduced the accumulation of SOD1G93A mutant protein aggregates,but when the JNK inhibitor SP600125 was added or the JNK gene was knocked out,the scavenging effect was significantly inhibited.In order to more intuitively and clearly reflect the key role of JNK in the degradation of SOD1G93A aggregates mediated by A77 1726,the cells were transfected with GFP-SOD1WT or GFP-SOD1G93A plasmids,and then treated with SP600125 and A77 1726.microplate reader and flow cytometry analysis showed that A77 1726 significantly reduced the mean fluorescence intensity of GFP-SOD1G93A transfected cells compared with the untreated group.However,when JNK inhibitor SP600125 was added,the average fluorescence intensity of GFP-SOD1G93A cells significantly increased.The above experimental results showed that A77 1726 can induce autophagy by phosphorylation and activation of JNK,and JNK plays a key role in A77 1726 inducing autophagy to clear SOD1G93A aggregates.Finally,we used amyotrophic lateral sclerosis transgenic SOD1G93A miceas animal models,and gave them different doses of leflunomide(5,10,20,30 mg/kg body weight/day)for short-term intervention One month,through the monitoring of the onset time and course of disease,the effect of leflunomide on delaying the onset of ALS and prolonging the survival of SOD1G93A mice was preliminarily evaluated.The results of the in vivo test showed that the oral administration of Leflunomide(5mg/kg/day,10mg/kg/day)can significantly prolong the survival period of ALS model mice.The above research results will provide important in vivo and in vitro experimental basis for exploring the influence of autophagy regulation on the pathology of ALS.