Sphingosine-1-Phosphate and the S1P3 Receptor Initiate Neuronal Retraction via RhoA/ROCK Associated with CRMP2 Phosphorylation.

Serena Quarta,Dusan Matusica,Antonio Ferrer-Montiel,Christine E Bandtlow,Maria Camprubí-Robles,Michaela Kress,Rainer V Haberberger,Rüdiger Schweigreiter,Richard L Proia

doi:10.3389/fnmol.2017.00317

Abstract

The bioactive lipid sphingosine-1-phosphate (S1P) is an important regulator in the nervous system. Here, we explored the role of S1P and its receptors in vitro and in preclinical models of peripheral nerve regeneration. Adult sensory neurons and motor neuron-like cells were exposed to S1P in an in vitro assay, and virtually all neurons responded with a rapid retraction of neurites and growth cone collapse which were associated with RhoA and ROCK activation. The S1P1 receptor agonist SEW2871 neither activated RhoA or neurite retraction, nor was S1P-induced neurite retraction mitigated in S1P1-deficient neurons. Depletion of S1P3 receptors however resulted in a dramatic inhibition of S1P-induced neurite retraction and was on the contrary associated with a significant elongation of neuronal processes in response to S1P. Opposing responses to S1P could be observed in the same neuron population, where S1P could activate S1P1 receptors to stimulate elongation or S1P3 receptors and retraction. S1P was, for the first time in sensory neurons, linked to the phosphorylation of collapsin response-mediated protein-2 (CRMP2), which was inhibited by ROCK inhibition. The improved sensory recovery after crush injury further supported the relevance of a critical role for S1P and receptors in fine-tuning axonal outgrowth in peripheral neurons.

Highlights

MATERIALS AND METHODSNeuritogenesis is a complex event which involves a controlled balance between elongation and retraction of neuronal processes
20% of adult dorsal root ganglia (DRG) neurons had visible processes when neurons were grown in culture for 24 h with 1 μM S1P compared to the 43% of neurons which were treated with vehicle (Figures 1A,C; 42.88 ± 1.82% for ctrl vs. 19.33 ± 4.93% for S1P-treated, p = 0.000391, χ 2-test)
S1P synthesis was previously suggested to act downstream of the NOGO signaling pathway, which has a significant anti-regenerative potential (Kempf et al, 2014) and nerve growth factor (NGF) signaling causes sphingosine kinase type 1 (SphK1) translocation to the plasma membrane with subsequent activation of the S1P1 and S1P2 receptors (Toman et al, 2004)

Summary

MATERIALS AND METHODS

Neuritogenesis is a complex event which involves a controlled balance between elongation and retraction of neuronal processes (da Silva and Dotti, 2002). S1P is an important lipid mediator (Spiegel and Milstien, 2002) and can bind five specific G protein-coupled receptors (GPCRs), S1P1–S1P5. FTY720 stimulates axon regeneration in mice after facial nerve axotomy through a signaling cascade involving S1P receptors, G12/13 G-proteins, RhoA-GTPases and the transcription factors SRF/MRTF (Anastasiadou and Knöll, 2016). Together the data of the present study point towards a critical importance of S1P3 receptor for an overall deceleration of axonal growth, inducing fast neurite retraction through activation of Rho and Rho kinase (ROCK) and consequent phosphorylation of the collapsin response-mediated protein-2 (CRMP2). S1P3 receptor ablation partially improved nerve regeneration in a preclinical model of peripheral nerve injury

Ethics Statement

RESULTS

DISCUSSION