Regulation of the Na,K-ATPase Gamma-Subunit FXYD2 by Runx1 and Ret Signaling in Normal and Injured Non-Peptidergic Nociceptive Sensory Neurons

Dorsal root ganglia (DRGs) contain the cell bodies of sensory neurons which relay nociceptive, thermoceptive, mechanoceptive and proprioceptive information from peripheral tissues toward the central nervous system. These neurons establish constant communication with their targets which insures correct maturation and functioning of the somato-sensory nervous system. Interfering with this two-way communication leads to cellular, electrophysiological and molecular modifications that can eventually cause neuropathic conditions. In this study we reveal that FXYD2, which encodes the gamma-subunit of the Na,K-ATPase reported so far to be mainly expressed in the kidney, is induced in the mouse DRGs at postnatal stages where it is restricted specifically to the TrkB-expressing mechanoceptive and Ret-positive/IB4-binding non-peptidergic nociceptive neurons. In non-peptidergic nociceptors, we show that the transcription factor Runx1 controls FXYD2 expression during the maturation of the somato-sensory system, partly through regulation of the tyrosine kinase receptor Ret. Moreover, Ret signaling maintains FXYD2 expression in adults as demonstrated by the axotomy-induced down-regulation of the gene that can be reverted by in vivo delivery of GDNF family ligands. Altogether, these results establish FXYD2 as a specific marker of defined sensory neuron subtypes and a new target of the Ret signaling pathway during normal maturation of the non-peptidergic nociceptive neurons and after sciatic nerve injury

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

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

<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

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