Nociception-dependent CCL21 induce dorsal root ganglia axonal growth via CCR7-ERK activation

While chemokines were originally described for their ability to induce cell migration, many studies show how chemokines also take part in a variety of other cell functions, acting as adaptable messengers in the communication between a diversity of cell types. In the nervous system, chemokines participate both in physiological and pathological processes, and while their expression is often described on glial and immune cells, growing evidence describe the expression of chemokines and their receptors in neurons, highlighting, their potential in auto- and paracrine signalling. In this study we analysed the role of nociception in the neuronal chemokinome, and their role in axonal growth. We found that stimulating TRPV1+ nociceptors induces a transient increase in CCL21. Interestingly we found that, this CCL21 increases neurite growth of large diameter proprioceptors in vitro. Consistent with this, we show that proprioceptors express the CCL21 receptor CCR7, and a CCR7 neutralizing antibody dose-dependently attenuates CCL21-induced neurite outgrowth. Mechanistically, we found that CCL21 binds locally to its receptor CCR7 at the growth cone, activating the downstream MEK-ERK pathway, that in turn activates N-WASP, triggering actin filament ramification in the growth cone, resulting in increased axonal growth.


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Introduction 30 Classically, chemokines have been associated with leukocyte migration (1). Nevertheless, growing 31 evidence shows they can signal to a great variety of cell types and tissues (2,3). In addition, as 32 conventional chemokine receptors are G-protein coupled receptors (GPCRs), chemokines can initiate 33 a broad variety of intracellular signaling pathways (1). 34 Neurons alter their secretome when exposed to different stimuli and according to their physiological 48 state. In that direction, neuronal activity has shown to modulate neuronal communication, including 49 with microglia or with other neurons beyond classical neurotransmission (15-17). Nociceptor activity 50 after axonal injury is normally associated with pathological neuropathic pain (18), despite that, some 51 studies have started to uncover how nociception participates in the healing process, such as 52 promoting skin or adipose tissue regeneration, as well as neovascularization (19-21). These findings 53 indicate that nociception might function as a key component of the healing machinery, and it is 54 therefore important to study its precise roles in healing and regeneration in different tissues, 55 including the nervous system. 56 In that sense, injured nociceptors have been described to release CCL21, however, whether this 57 expression affected axonal regeneration has not been previously assessed (13). CCL21 has a 58 primordial role in immune cell homing, via its canonical receptor CCR7 (22), but other functions for 59 this chemokine in distinct tissues are also emerging, such as cartilage regeneration (23) and 60 neuropathic pain induction in the CNS (13). Interestingly, in accordance with its function as a 61 migration cue, the CCL21-CCR7 interaction activates intracellular pathways related to chemotaxis, 62 via ERK signaling, this includes actin cytoskeleton remodeling via RhoA (24). Since cellular 63 migration and growth cone dynamics are analogous mechanisms (25,26), we hypothesized that 64 CCL21 could exert a growth promoting effect on neurons. 65 In the present study, we investigated the impact of nociceptor activation in the neuronal 66 chemokinome which led us to find an undescribed mechanism of neuronal communication between 67 two different neuronal types, nociceptors and proprioceptors. Specifically, we found that activation of 68 TRPV1 + nociceptors induce an increase in CCL21 expression. Moreover, we revealed a novel role for 69 this CCL21 in proprioceptors, promoting neurite outgrowth. We then found that the receptor CCR7, 70 expressed in proprioceptors, was required for this effect, which was also dependent on the MEK-71 ERK pathway. Finally, our results disclose a local mechanism in the growth cone, where CCR7 72 expression is concentrated, affecting the actin cytoskeleton, ultimately leading to enhanced neurite 73 outgrowth. 74

CCL21 expression is upregulated upon nociceptor activation 157
While most defined neuronal chemokines are induced after traumatic injury or inflammatory 158 signalling (30), we analysed whether stimulating neuronal activity in the DRG would have an impact 159 in chemokine expression and secretion. To this aim we used DRG neuronal cultures expressing ChR2 160 (from Thy1-ChR2 animals) and subjected them to optical stimulation. We recovered the media 24h 161 later and measured chemokine secretion using a Mouse Chemokine Array (Raybiotech). 162 Interestingly, we found a remarkable increase in CCL21 levels compared to non-stimulated controls 163 (Data not shown). ChR2 expression in DRGs is ubiquitous among all sensory subtypes (Fig 1A-B), 164 however, previous studies had reported CCL21 expression specifically in small diameter TRPV1 + 165 nociceptors after peripheral nerve injury (31,32), so we hypothesized that this CCL21 increase after 166 optogenetic stimulation could be specific of this neuronal subtype. Accordingly, CCL21 expression 167 was increased in TRPV1 + nociceptors 2h after i.pl capsaicin (TRPV1 agonist) injection as compared 168 to vehicle administered animals (Student's T test p= 0.0347) ( Fig. 1C-E). 169

CCL21 promotes neurite outgrowth 170
To test whether this chemokine could also influence axonal growth, we administered CCL21 into the 171 sciatic nerve and cultured disaggregated DRG neurons 24h after. This led to an increment in the 172 neurite outgrowth of ex vivo CCL21-treated DRG neurons when compared to vehicle treated ones 173 (Student's t-test p= 0.0046) ( Fig. 2A Figure 1), 188 suggesting a CCL21 biased CCR7 activation is responsible for this particular mechanism. 189

CCL21-CCR7 activates MEK-ERK pathway 190
We then targeted the two main known downstream actuators of CCR7 activation, the MEK pathway 191 and the Gi/o protein previously known to be involved in axon growth(34-36)). Pharmacological 192 inhibition of MEK with U0126 blocks the neurite outgrowth induced by CCL21 ( Fig. 4A- Ptx: p= 0.7458). 200

CCL21-CCR7-MEK pathway modulates actin cytoskeleton to promote neurite outgrowth 201
Local assessment of the axonal tips revealed larger growth cones after CCL21 treatment (Fig. 5A), 202 this is in consonance with the especially abundant CCR7 expression found on these structures (Fig.  203 5B). We then sought to check the local effects that CCL21 could have in cytoskeletal dynamics of the 204 growth cones. Consequently, we inhibited actin branching by combining CCL21 administration with 205 wiskostatin, an inhibitor of the neural Wiskott-Aldrich syndrome protein (N-WASP), that acts as an 206 Arp2/3 complex activator. Wiskostatin co-administration greatly reduced the CCL21-induced 207 growth, supporting a local effect of CCL21 in the growth cone (two-way ANOVA followed by 208 Bonferroni test; interaction p= 0.0825; one-way ANOVA followed by Bonferroni test; DMSO-Veh 209 vs DMSO-CCL21 p= 0.0207; DMSO-CCL21 vs Wiskostatin-Veh p= 0.0202). 210

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Discussion 211 There are plenty of evidence that neuronal activity can alter neuronal signalling. In that sense, we 212 found that the expression and release of the chemokine CCL21 is enhanced upon neuronal 213 stimulation. Interestingly, we found that specific TRPV1 + nociceptor stimulation is responsible for 214 CCL21 production, similarly to what has already been described upon axonal injury (31). 215 After axonal injury, release of neuronal CCL21 is mainly linked to neuropathic pain (13,37), 216 however, its role in axon growth and regeneration had never been described before. Interestingly, our 217 results revealed that CCL21 induces growth in proprioceptive neurons trough activation of the 218 CCR7-MEK-ERK pathway, exerting an effect on actin dynamics at the growth cone level. 219 CCL21 was first designated as a recruiting cue for leucocytes, specifically stimulating the migration 220 of T cell subpopulations and dendritic cells (22,38-40). More recently, it has also been described to 221 induce migration in other cells such as tumorigenic or mesenchymal stem cells (41-45). 222 Fundamentally, cell migration and axonal growth are events that share similar cellular and molecular 223 machinery (46,47). Thus, molecules that orchestrate one process will most likely be implicated in the 224 other, and vice versa, as for instance what occurs with CXCL12 (48-51). 225 We also describe that CCL21 executes its growth-inducing function through its canonical receptor 226 CCR7, in accordance, we found abundant expression of this receptor on proprioceptive (PV + ) 227 neurons, similarly to the findings of other studies, where neuronal CCR7 is abundantly found in 228 peripheral nerves and hippocampal neurons (52,53 This also suggest caution in the indiscriminate use of analgesic drugs and treatments after injury, as 282 these may hinder nociceptive regenerative signalling, limiting the healing process, similarly to the 283 effects observed by broad immunosuppressive drugs (89) or antioxidants (90) after SCIs. Therefore, 284 appropriate timing and level of analgesic inhibition after injury will likely need to be tailored to 285 provide pain relief while avoiding unwanted effects in hindering the tissue regeneration. An 286 additional intriguing implication is the possibility to modulate nociceptive signalling to achieve tissue 287 regeneration. While therapeutically, inducing nociception is not a reasonable approach, further 288 investigation and characterization of signalling elicited by nociceptor stimulation may increase our 289 understanding of the molecular mechanisms underlying the healing process, and may enable the 290 future design of therapeutic targets and strategies to foster tissue regeneration. 291

Conflict of Interest 292
The authors declare that the research was conducted in the absence of any commercial or financial 293 relationships that could be construed as a potential conflict of interest. 294