CaMKK-CaMK1a, a new post-traumatic signalling pathway induced in mouse somatosensory neurons.

Neurons innervating peripheral tissues display complex responses to peripheral nerve injury. These include the activation and suppression of a variety of signalling pathways that together influence regenerative growth and result in more or less successful functional recovery. However, these responses can be offset by pathological consequences including neuropathic pain. Calcium signalling plays a major role in the different steps occurring after nerve damage. As part of our studies to unravel the roles of injury-induced molecular changes in dorsal root ganglia (DRG) neurons during their regeneration, we show that the calcium calmodulin kinase CaMK1a is markedly induced in mouse DRG neurons in several models of mechanical peripheral nerve injury, but not by inflammation. Intrathecal injection of NRTN or GDNF significantly prevents the post-traumatic induction of CaMK1a suggesting that interruption of target derived factors might be a starter signal in this de novo induction. Inhibition of CaMK signalling in injured DRG neurons by pharmacological means or treatment with CaMK1a siRNA resulted in decreased velocity of neurite growth in vitro. Altogether, the results suggest that CaMK1a induction is part of the intrinsic regenerative response of DRG neurons to peripheral nerve injury, and is thus a potential target for therapeutic intervention to improve peripheral nerve regeneration

Perturbation of the CaMKK-CaMK1a pathway reduces neurite growth velocity of injured DRG neurons <i>in vitro</i>.

<p>(<b>A</b>). Phase-contrast illustrative images at 0, 8 and 24 hours after plating of sensory neurons dissected from naïve animals (upper panels) or from mice having undergone a sciatic nerve axotomy 3 days before (lower panels). Scale bar  = 100 µm. Note that after 24 h, sensory neurons from controls exhibit an arborized growth while many axotomized neurons exhibit an elongated growth. The graph on the right illustrates the growth speed of neurons in both conditions. In controls, the arborized neurons extend neurites at a velocity of 24.92 µm/h+/−1.96, while axotomized elongated neurons extend neurites at a velocity of 54.7+/−2.2 µm/h, confirming previous published studies. (<b>B</b>). Quantification of the neurite growth velocity of axotomized elongated neurons put in culture 3 days after a sciatic nerve section during 24 hours without (dark grey column) or with (light grey column) treatment with the CaMKK inhibitor STO-609. Untreated axotomized elongated neurons normally extend neurites at a velocity of 54.7+/−2.2 µm/h. With STO-609 (0.5 µg/µl) treatment, we observed a 25% reduction of the growth speed which drops to 40.8+/−2,6 µm/h. (<b>C</b>). Quantification of the effect of Control or CaMK1a siRNA on the velocity of neurite outgrowth of axotomized elongated neurons. Mice were given intrathecal injections of CaMK1a siRNA or control non-targeting siRNA in transfection agent containing dextran- tetramethylrhodamine as an indicator of transfection. The graph on the left show QRT-PCRs revealing a 46% reduction of CamK1a expression specifically in neurons injected with CamK1a siRNA compared to control siRNA. The neurite growth velocities of axotomized dextran+ and dextran- neurons were evaluated and reported on the graph on the right. CaMK1a siRNA transfection reduced DRG neurite outgrowth from 55+/−2,48 to 30+/−2,47 µm/h while control siRNA had no effect.</p

CaMK1a is induced in DRG neurons by nerve injury and not by inflammation.

<p>(<b>A–C</b>). QRT- PCR analysis of the expression of <i>CaMK1a</i> mRNA in DRG three days (3d) after crush, chronic constriction injury (CCI) or CFA induced inflammation, showing that <i>CaMK1a</i> mRNA is induced by crush and CCI but not by CFA injection. (<b>D–I</b>). Corresponding immunohistochemical staining for CaMK1a protein in the three experimental models showing sections of DRGs ipsi- (<b>D–F</b>) and contralateral (<b>G–I</b>) to the injury site. Note the presence of numerous strongly-labeled neurons in the ipsilateral DRGs from mice after crush (<b>D</b>) or CCI (<b>E</b>) but not CFA (<b>F</b>). Controlateral DRGs were CamK1a-negative for all conditions (<b>G–I</b>).</p

GDNF and NRTN delivery partially normalizes the <i>de novo</i> expression of <i>CaMK1a</i> in DRG neurons.

<p>(<b>A–B</b>). QRT-PCR analysis of the expression of <i>CaMK1a</i> and <i>Beta Actin</i> (<b>A</b>) or <i>ATF3</i>, <i>Sprr1a</i> and <i>NPY</i> (<b>B</b>) mRNA in naive (<b>WT</b>) or axotomized (<b>Axo</b>) DRGs compared to axotomized DRG after GDNF or NRTN intrathecal delivery. Statistically relevant differences (p<0.05) are signified with an “*”in the graphs. (<b>C</b>). Red columns on the graph (left scale) show that the percentage of Fluorogold(FG)+Ret+ neurons over the total number of FG+ neurons remains stable - around 60% - in axotomized DRG after intrathecal injections of either a saline solution, Neurturin (NRTN) or GDNF, thus establishing the Ret+FG+ neurons as a reliable reference population. Dark grey columns on the graph (right scale) show the percentage of CaMK1a+ FG+Ret+ neurons over the total number of FG+Ret+ neurons in the three conditions described above. In saline treated DRGs close to 81% of all Ret+/FG+ neurons are also CaMK1a+. This percentage significantly drops to 38% after NRTN delivery. In GDNF-treated DRGs there a is a slight reduction to 74% which is however below the threshold of significance. (<b>D,E</b>). Double labeling of ATF3 and IB4 on adult spinal cord transverse sections from mice having undergone a sciatic nerve axotomy and injections of either a saline solution (D) or NRTN (E). Only the ipsilateral axotomized side, revealed by the motoneuronal expression of ATF3 (arrows) is shown. The IB4 staining in the dorsal horn of the spinal cord is greatly reduced in animals injected with a saline solution (bracket in D) while it remains strong in animals injected with NRTN (bracket in E). (<b>F,G</b>). Illustration of a double-immunofluroscence experiment used for the quantification, using CaMK1a and Ret antibodies combined with FG detection on transverse sections of axotomized DRGs from animals injected either with a saline solution (F) or NRTN (G). On the right panels are shown close-ups corresponding to the white frames in the enlarged images, showing individual labeling (Fluorogold in red, Ret in green and CaMK1a in blue) and the merged image. Note the presence of many Ret+FG+CaMK1a+ yellow cells in the NRTN injected animals which are rare in the controls.</p

CaMK1a is preferentially induced in large diameter Ret+ neurons after axotomy.

<p>(<b>A–D</b>). Combined CaMK1a immunohistochemistry and retrograde labelling with Fluorogold (FG) on L4–L5 DRG sections three days post-axotomy of the sciatic nerve. FG was applied at the cut nerve stump and specifically labels axotomized neurons. (<b>E</b>). Counts on DRG sections show that 74+/−2% of CaMK1a+ neurons are FG+. (<b>F</b>). Cell soma size distribution of CaMK1a+ neurons in DRG after sciatic nerve axotomy. (<b>G–J</b>). Double-immunofluorescent staining for CaMK1a and NF-200 on sections of L4–L5 DRG three days post-axotomy. (<b>K–N</b>). Double-immunofluorescent staining for CaMK1a and Ret on DRG sections three days post-axotomy shows numerous co-labelled neurons for both proteins. (<b>O</b>). Counts of CamK1a+NF200+ double-labeled cell reveals that about 50% of CaMK1a-positive neurons are NF200+. (<b>P</b>). Size repartition of CamK1a+Ret+ double-labeled cells showing that the vast majority of CaMK1a-positive neurons with medium-large cell soma diameter also express Ret.</p

Primer sequences used for PCR products intended to generate In Situ Hybridization Probes (1) or for Real-Time Polymerase Chain Reaction (2).

<p>Primer sequences used for PCR products intended to generate In Situ Hybridization Probes (1) or for Real-Time Polymerase Chain Reaction (2).</p

Inhibition of neuronal FLT3 receptor tyrosine kinase alleviates peripheral neuropathic pain in mice

Sensitisation of dorsal root ganglia neurons contributes to neuropathic pain. Here the authors demonstrate the cytokine FL contributes to sensitisation of DRGs via its receptor FLT3 expressed on neurons, and identify a novel FLT3 inhibitor that attenuates neuropathic pain in mice