The mTOR Substrate S6 Kinase 1 (S6K1) Is a Negative Regulator of Axon Regeneration and a Potential Drug Target for Central Nervous System Injury

Abstract

The mammalian target of rapamycin (mTOR) positively regulates axon growth in the mammalian central nervous system (CNS). Although axon regeneration and functional recovery from CNS injuries are typically limited, knockdown or deletion of PTEN, a negative regulator of mTOR, increases mTOR activity and induces robust axon growth and regeneration. It has been suggested that inhibition of S6 kinase 1 (S6K1, gene symbol: RPS6KB1), a prominent mTOR target, would blunt mTOR's positive effect on axon growth. In contrast to this expectation, we demonstrate that inhibition of S6K1 in CNS neurons promotes neurite outgrowth in vitro by twofold to threefold. Biochemical analysis revealed that an mTOR-dependent induction of PI3K signaling is involved in mediating this effect of S6K1 inhibition. Importantly, treating female mice in vivo with PF-4708671, a selective S6K1 inhibitor, stimulated corticospinal tract regeneration across a dorsal spinal hemisection between the cervical 5 and 6 cord segments (C5/C6), increasing axon counts for at least 3 mm beyond the injury site at 8 weeks after injury. Concomitantly, treatment with PF-4708671 produced significant locomotor recovery. Pharmacological targeting of S6K1 may therefore constitute an attractive strategy for promoting axon regeneration following CNS injury, especially given that S6K1 inhibitors are being assessed in clinical trials for nononcological indications.SIGNIFICANCE STATEMENT Despite mTOR's well-established function in promoting axon regeneration, the role of its downstream target, S6 kinase 1 (S6K1), has been unclear. We used cellular assays with primary neurons to demonstrate that S6K1 is a negative regulator of neurite outgrowth, and a spinal cord injury model to show that it is a viable pharmacological target for inducing axon regeneration. We provide mechanistic evidence that S6K1's negative feedback to PI3K signaling is involved in axon growth inhibition, and show that phosphorylation of S6K1 is a more appropriate regeneration indicator than is S6 phosphorylation.