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The RSK2-RPS6 Axis Promotes Axonal Regeneration in the Peripheral and Central Nervous Systems

Charlotte Decourt, Julia Schaeffe, Beatrice Blot, Antoine Paccard, Blandine Excoffier, Mario Pende, Homaira Nawabi, Stephane Belin 

Abstract

Unlike immature neurons and the ones from the peripheral nervous system (PNS), mature neurons from the central nervous system (CNS) cannot regenerate after injury. In the past 15 years, tremendous progress has been made to identify molecules and pathways necessary for neuroprotection and/or axon regeneration after CNS injury. In most regenerative models, phosphorylated ribosomal protein S6 (p-RPS6) is up-regulated in neurons, which is often associated with an activation of the mTOR (mammalian target of rapamycin) pathway. However, the exact contribution of posttranslational modifications of this ribosomal protein in CNS regeneration remains elusive. In this study, we demonstrate that RPS6 phosphorylation is essential for PNS and CNS regeneration in mice. We show that this phosphorylation is induced during the preconditioning effect in dorsal root ganglion (DRG) neurons and that it is controlled by the p90S6 kinase RSK2. Our results reveal that RSK2 controls the preconditioning effect and that the RSK2-RPS6 axis is key for this process, as well as for PNS regeneration. Finally, we demonstrate that RSK2 promotes CNS regeneration in the dorsal column, spinal cord synaptic plasticity, and target innervation leading to functional recovery. Our data establish the critical role of RPS6 phosphorylation controlled by RSK2 in CNS regeneration and give new insights into the mechanisms related to axon growth and circuit formation after traumatic lesion

Introduction

In contrast to developing neurons or the ones from the peripheral nervous system (PNS), mature neurons from the central nervous system (CNS) fail to regenerate their axons after an insult (neurodegenerative diseases or traumatic lesions). Patients must bear irreversible and permanent motor, cognitive and/or sensory disabilities. The continuous increase of such nervous system disorders worldwide, along with the lack of efficient therapies, makes axon regeneration and functional recovery major challenges of public health.

CNS regenerative failure has both extrinsic and neuronal intrinsic components [1,2]. The mTOR (mammalian target of rapamycin) pathway is one of the key neuronal signaling pathway controlling axon regeneration. Indeed, it has been shown that the activation of mTOR pathway via PTEN (Phosphatase and TENsin homolog) deletion in neurons, triggers robust axon regeneration in the visual system and in the corticospinal tract [3–7]. Subsequently, combinatorial/synergistic approaches, which often include mTOR pathway activation, have led to long-distance regeneration [8,9]. Additionally, the analysis of specific retinal ganglion cells (RGC) subpopulations regenerative capacity revealed osteopontin and IGF as regulators of axon regeneration through mTOR activation [10]. In the PNS, mTOR has also been shown to regulate axon regeneration. However, its exact contribution to this process remains unclear. Indeed, one target of mTOR, S6 kinase 1 (S6K1), inhibits axon regeneration through negative feedback on mTOR [11,12]. In contrast, TSC2 genetic deletion or PTEN inhibition (negative regulators of mTOR pathway) leads to a modest increase of axon regeneration after sciatic nerve lesion [13–15]. Moreover, pharmacological inhibition of mTOR, in cultured DRG (dorsal root ganglia) neurons induces only a mild effect [16,17].

Material and methods

Surgical procedures

Animal care and procedures were performed according to the Grenoble Institute Neurosciences, French (French Ministry of research guidelines) and European guidelines (directive 86/609 and 2010/63) (APAFIS #38155–202205021448189 v5).
For intrathecal injections and dorsal column crush, mice were anesthetized with a mix of ketamine (100 mg/kg) and xylazine (10 mg/kg). Sciatic nerve crush procedure was performed under 3% induction, 2% maintenance isoflurane. For analgesia, paracetamol was given in the drinking water (4 mg/ml) 1 day before and 2 days after surgery. Buprenorphine (0.05 mg/kg) was administered by subcutaneous injection 6 h before dorsal column injury and every 6 h for 3 days after surgery.

Results

RPS6 phosphorylation on Ser 235–236 controls the preconditioning effect and contributes to sciatic nerve regeneration

The phosphorylation of ribosomal protein RPS6 is often used as a readout of mTOR activation [18]. However, the exact contribution of RPS6 during regeneration has never been addressed. Indeed, RPS6 is a core ribosomal protein that carries 5 serine residues that can be phosphorylated (Ser235, Ser236, Ser240, Ser244, and Ser 247) [19,21] (Fig 1A). To understand the role of RPS6 during axon regeneration, we analyzed its dynamics of phosphorylation upon sciatic nerve injury (Fig 1B). We collected 6-week-old wild-type mice lumbar dorsal root ganglia (DRG-L3 to L5) from intact (naive) condition and 1, 3, and 7 days post-sciatic nerve injury (dpi). Western blot analysis using specific anti-p-S6Ser235-236 and anti-p-S6Ser240-244 antibodies revealed that RPS6 phosphorylation on Ser235-236 is up-regulated at 1 dpi and reaches a peak at 3 dpi, before decreasing at 7 dpi (Fig 1C and 1D). On the other hand, RPS6 phosphorylation on Ser240-244 remains overall stable, despite a slight increase only at 3 dpi (Fig 1E). The total level of RPS6 remains stable after injury (Fig 1F). In parallel, we analyzed the levels of phosphorylated RPS6 in DRG sections using immunofluorescence. Consistently with the western blot analysis, we observed an increase of p-S6Ser235-236 expression from 1 dpi, with a peak at 3 dpi. At 7 dpi, the level of p-S6Ser235-236 was back to the control (uninjured) condition (Fig 1G and 1H). Conversely, the level of p-S6Ser240-244 did not display any significant change over time (Fig 1I and 1J). Together, these results show that RSP6 phosphorylation on Ser235-236 is up-regulated in DRG upon sciatic nerve injury.

Discussion

The current lack of efficient therapies for CNS regeneration remains a major challenge. Despite outstanding advances in the modulation of intrinsic regrowth capabilities in the past 15 years [1,8,9,35], full functional recovery has not been achieved yet. Rapidly, mTOR became a key pathway to trigger CNS regeneration [3,4]. However, not only the precise mechanisms by which mTOR leads to axon regeneration remain elusive, but also the exact contribution of one of its main effectors, the phosphorylated RPS6, is unknown. Moreover, during PNS regeneration and the preconditioning effect, mTOR has a modest contribution that can also depend on the experimental design used to assess its function [13,16,17]. These observations suggest that other pathways may be involved in these processes.

In our study, we demonstrate that RPS6 phosphorylation, in particular, on the Serine 235–236 is critical for PNS and CNS regeneration. We show that RPS6 phosphorylation is induced by sciatic nerve injury and is required for the preconditioning effect. In addition, we demonstrate that this phosphorylation is not controlled by mTOR but by the p90S6 kinase, RSK2. Moreover, RSK2 promotes regeneration of the central branch of DRG axons in the spinal cord, skin innervation, synaptic plasticity, and associated functional recovery. Altogether, our work sheds light on the critical role of RPS6 phosphorylation and on the importance of this posttranslational regulation by RSK2.

Acknowledgments

We would like to acknowledge E.Plissonier, T.-N.Nguyen, N. Fayad, and A. Lapierre for laboratory help and discussions. We thank S. Carnicella, M. Bartolomucci, and the GIN behavioral facility that is supported by the Grenoble Center of Excellence in Neurodegeneration (GREEN). This work was supported by the Photonic Imaging Center of Grenoble Institute Neuroscience (Univ Grenoble Alpes–Inserm U1216) which is part of the ISdV core facility and certified by the IBiSA label.

Citation: Decourt C, Schaeffer J, Blot B, Paccard A, Excoffier B, Pende M, et al. (2023) The RSK2-RPS6 axis promotes axonal regeneration in the peripheral and central nervous systems. PLoS Biol 21(4): e3002044. https://doi.org/10.1371/journal.pbio.3002044

Academic Editor: Cody J. Smith, University of Notre Dame, Center for Stem Cells and Regenerative Medicine, UNITED STATES

Received: August 16, 2022; Accepted: February 21, 2023; Published: April 17, 2023

Copyright: © 2023 Decourt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its supporting information files.

Funding: This work was supported by a grant from ANR to SB (ANR-18-CE16-0007). HN is supported by the NRJ Foundation and the European Research Council (ERC-St17-759089). This work was supported by the French National Research Agency under the “Investissements d’avenir” programme (ANR-17-EURE-0003 to CD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.

Abbreviations: CNS, central nervous system; CTB, cholera toxin B; dpi, days post-injury; DRG, dorsal root ganglion; mTOR, mammalian target of rapamycin; PC, precontionned; PNS, peripheral nervous system; p-RPS6, phosphorylated ribosomal protein S6; RGC, retina ganglion cell; RPS6, ribosomal protein S6; vGAT, vesicular gamma aminobutyric acid transporter; Vglut1, vesicular glutamate transporter 1

https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002044#ack

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