Inhibition of p70 S6 kinase activity by A77 1726 induces autophagy and enhances the degradation of superoxide dismutase 1 (SOD1) protein aggregates

Jing Sun,Xiulong Xu,Jun Sun,Richard A Prinz,Jianxin Yi,Yarong Mu,Ruilong Song,Xinan Jiao,Yuanyuan Jiang,Jingsong Zhou,Yi Li

doi:10.1038/s41419-018-0441-0

Abstract

Autophagy plays a central role in degrading misfolded proteins such as mutated superoxide dismutase 1 (SOD1), which forms aggregates in motor neurons and is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Autophagy is activated when UNC-51-like kinase 1 (ULK1) is phosphorylated at S555 and activated by AMP-activated protein kinase (AMPK). Autophagy is suppressed when ULK1 is phosphorylated at S757 by the mechanistic target of rapamycin (mTOR). Whether p70 S6 kinase 1 (S6K1), a serine/threonine kinase downstream of mTOR, can also regulate autophagy remains uncertain. Here we report that inhibition of S6K1 by A77 1726, the active metabolite of an anti-inflammatory drug leflunomide, induced mTOR feedback activation and ULK1S757 phosphorylation in NSC34 cells, a hybrid mouse motoneuron cell line. Unexpectedly, A77 1726 did not suppress but rather induced autophagy by increasing AMPKT172 and ULK1S555 phosphorylation. Similar observations were made with PF-4708671, a specific S6K1 inhibitor, or with S6K1 siRNA. Further studies showed that A77 1726 induced AMPK phosphorylation by activating the TGF-β-activated kinase 1 (TAK1). Functional studies revealed that A77 1726 induced co-localization of mutant SOD1G93A protein aggregates with autophagosomes and accelerated SOD1G93A protein degradation, which was blocked by inhibition of autophagy through autophagy-related protein 7 (ATG7) siRNA. Our study suggests that S6K1 inhibition induces autophagy through TAK1-mediated AMPK activation in NSC34 cells, and that blocking S6K1 activity by a small molecule inhibitor such as leflunomide may offer a new strategy for ALS treatment.

Highlights

Amyotrophic lateral sclerosis (ALS) is the most common form of adult-onset motoneuron degenerative disease characterized by the selective loss of motoneurons in the ventral horn of the spinal cord, the cerebral cortex, and brainstem nuclei[1, 2]
Confocal microscopic fluorescence analysis revealed that LC3 formed autophagosomes in NSC34 cells in the presence of A77 1726 or rapamycin (Fig. 1k)
Rapamycin targets mechanistic target of rapamycin (mTOR), leading to decreased S6 kinase 1 (S6K1) phosphorylation higher in NSC34 cells treated with A77 1726 or rapamycin than that in the untreated controls (Fig. 1l)

Summary

Introduction

Amyotrophic lateral sclerosis (ALS) is the most common form of adult-onset motoneuron degenerative disease characterized by the selective loss of motoneurons in the ventral horn of the spinal cord, the cerebral cortex, and brainstem nuclei[1, 2]. 90% of ALS is sporadic and does not have an apparent genetic linkage. The remaining 10% is familial and these patients carry a mutant gene[3]. Superoxide dismutase 1 (SOD1) was the first mutated gene to be discovered in familial ALS about two decades ago[4,5,6]. Mutant SOD1 proteins are prone to misfolding and forming aggregates in motoneurons. Several other genes, including TAR DNA-binding protein 43

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Conclusion