GDNF family ligands influence <i>FXYD2</i> expression in adult DRG neurons in vitro and in vivo.

<p>(A) Quantitative analysis of FXYD2-expressing neurons in DRG cultures in the presence or absence of GDNF/NRTN. The picture is representative of a neuronal culture stained with the anti-FXYD2 antibody revealed with DAB as a substrate. On the graph is reported the proportion of FXYD2+ neurons after 3 h in culture, 3 days in culture without added factors, or 3 days in culture with GDNF/NRTN (10 ng/ml each). <i>FXYD2</i> expression was efficiently maintained by addition of factors. (B) QRT-PCR for FXYD2 on L4/5 DRGs dissected from control animals or mice axotomized and injected intrathecally either with saline, GDNF or NRTN solutions. (C–E″) Combined <i>FXYD2</i> in situ hybridization and FluoroGold staining on adult DRG sections from mice axotomized and injected either with saline (C–C″), GDNF (D–D″) or NRTN (E–E″) solutions during 3 days. Double-labeled neurons are virtually absent with saline injection, while they are numerous after GDNF and NRTN treatments. Insets in C″, D″ and E″ show higher magnifications. Insets in C, D and E represent injection quality controls showing IB4 staining on hemisections of the dorsal spinal cord (brackets) ipsilateral to the axotomy, that is normally lost after axotomy and saline injection, but rescued with GDNF or NRTN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029852#pone.0029852-Bennett1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029852#pone.0029852-Bennett2" target="_blank">[12]</a>. (F) Quantification of FluoroGold+/FXYD2+ neurons in the indicated conditions, showing that GDNF family ligands efficiently maintain <i>FXYD2</i> in injured neurons. (G) Quantification of the proportion of FXYD2+ neurons per DRG section in naïve animals (Ctrl) or in axotomized mice injected either with saline, GDNF or NRTN solutions. <i>FXYD2</i> is normally expressed in 57% of the DRG neurons and in 16% after axotomy and saline injection. In GDNF and NRTN injected mice, this proportion reaches 32% and 44%, respectively. (H) Triple-labeling for FXYD2, IB4 and FluoroGold (FG) on adult DRG sections from axotomized mice treated with NRTN. Presence of triple-labeled cells (white arrows) shows that FluoroGold+/FXYD2+ neurons are IB4+ nociceptors. Inset show higher magnification.</p

Loss of <i>FXYD2</i> expression in L4/L5 DRG neurons after sciatic nerve axotomy.

<p>(A, B) <i>FXYD2</i> in situ hybridization on naïve DRGs (A) and injured DRGs 3 days post-axotomy (dpa) (B). (C) Quantification of the percentage of <i>FXYD2</i>+ neurons in naïve and axotomized DRGs 3 dpa, showing a reduction from 57% to 16% after lesion of the sciatic nerve. (D, E) Time course analysis of <i>FXYD2</i> expression in the DRGs from 6 hpa to 7 dpa (D). Quantification reveals a major decrease between 2 and 3 dpa that remains stable at 7 dpa (E). (F) Scheme illustrating retrograde labeling of axotomized DRG neurons with Fluorogold. (G–G″) Combined <i>FXYD2</i> in situ hybridization and FluoroGold staining on DRG sections 3 dpa. Virtually no double-positive cells are found. Arrows and arrowheads point to FluoroGold-negative/FXYD2+ and FluoroGold+/FXYD2-negative neurons, respectively.</p

Restricted expression of FXYD2 in TrkB+ mechanoceptive and Ret+/IB4+ non-peptidergic noniceptive neurons within the DRGs.

<p>(A–J) Double-labeling for <i>FXYD2</i> and <i>TrkA</i>, <i>TrkB</i>, <i>TrkC</i>, <i>Ret</i> or IB4 on adult DRG sections. No co-localization is observed between FXYD2 and TrkA or TrkC. Double-positive neurons are detected with TrkB+ mechanoceptors (arrows in E,F) and Ret+/IB4+ non-peptidergic nociceptors (arrows in G–J). Arrowheads in G,H point to large Ret+ mechanoceptive neurons that are <i>FXYD2</i>-negative. (K,L) Percentages of TrkB+ (K) or IB4+ (L) neurons expressing FXYD2 showing that virtually all the TrkB+ mechanoceptors and the non-peptidergic nociceptors are FXYD2+. (M) Distribution of FXYD2+ neurons in two main neuronal types: the TrkB+ (representing 13%) and the Ret+/IB4+ (representing 85%) populations.</p

<i>FXYD2</i> expression depends on Runx1 and Ret signaling in non-peptidergic nociceptors.

<p>(A–D) <i>FXYD2</i> in situ hybridization on adult DRG sections from control (<i>Runx1^F/F</i>; A,C) and mutant (<i>Runx1^F/F;Wnt1Cre</i>; B,D) animals at P15 (A,B) and P90 (C,D). Insets show higher magnification. In control (A,C), small and larger (respectively, red and green brackets in insets) diameter neurons are detected, while in <i>Runx1</i> mutants at both stages (B,D) only the large diameter population expresses <i>FXYD2</i> (green brackets in insets). (E) Quantification of the proportions of FXYD2+ neurons at P90 showing a reduction of 69% in <i>Runx1</i> mutants. (F,G) Double-labeling for FXYD2 and IB4 on adult DRG sections from control and Runx1 mutant animals at P90, showing a loss of FXYD2 specifically in the IB4+ population in the mutants. Insets show higher magnifications. (H,I) <i>Ret</i> in situ hybridization on DRG sections from control (H) and <i>Runx1</i> mutant (I) animals at P90. Insets show higher magnification. <i>Ret</i> expression is lost in small diameter nociceptors (red brackets in insets) and persists only in large diameter mechanoceptive neurons (green brackets in insets) in <i>Runx1</i> mutants. (J–M) <i>FXYD2</i> in situ hybridizations (J,K) and immunochemistry (L,M) on DRG sections at P15 from control (<i>Ret^F/F</i>; J,L) and <i>Ret</i> mutants (<i>Ret^F/F;Wnt1-Cre</i>; K,M) showing a reduced number of FXYD2+ neurons and expression intensity in the mutants. (N) Quantification of the relative number of <i>FXYD2+</i> neurons showing a reduction of 30% in <i>Ret</i> mutants. (O) Epistatic relationships between Runx1, Ret and FXYD2 in non-peptidergic nociceptors. Runx1 controls (directly or indirectly) the onset of FXYD2 expression partly through Ret regulation. Ret signaling seems involved in ensuring proper levels of <i>FXYD2</i> and in its maintenance at subsequent stages (dashed arrows; see text).</p

Expression profile of <i>FXYD2</i> mRNA and protein during DRG neuron development.

<p>(A) Quantitative RT-PCR analysis of <i>FXYD2</i> expression in the developing DRGs and after axotomy. SAGE tag frequencies for FXYD2 at equivalent stages or conditions are indicated below. (B) Western blot using a FXYD2 antibody shows the presence of the FXYD2 isoforms gamma-a and gamma-b in the adult DRG. Kidney extract is a positive control. (C–F) <i>FXYD2</i> in situ hybridization on mouse DRG sections at E13, P0, P15 and adult. Arrow and arrowhead in F point to FXYD2-positive and FXYD2-negative neurons, respectively. (G) FXYD2 immunochemistry on adult DRG sections. Arrows and Arrowheads point respectively to positive cell bodies and nerve fibers.</p

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 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

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 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