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

Expression of glutamate transporters in the medial and lateral vestibular nuclei during rat postnatal development.

The postnatal developmental expression and the distribution of the glutamate transporters (GLAST, GLT-1 and EAAC1) were analyzed in rat vestibular nuclei (VN), at birth and during the following 4 weeks. Analyses were performed using reverse transcriptase-polymerase chain reaction and immunoblotting of GLAST, GLT-1 and EAAC1 mRNA and protein during the postnatal development of the VN neurons and their afferent connections. We also studied the distribution of each glutamate transporter in the medial and lateral VN by use of immunocytochemistry and confocal microscopy. GLAST, GLT-1 and EAAC1 mRNA and protein were present in the VN at each developmental stage. GLAST was highly expressed mainly in glia from birth to the adult stage, its distribution pattern was heterogeneous depending on the region of the medial and lateral VN. GLT-1 expression increased dramatically during the second and third postnatal weeks. At least during the first postnatal week, GLT-1 was expressed in the soma of neurons. EAAC1 was detected in neurons and decreased from the third week. These temporal and regional patterns of GLAST, GLT-1 and EAAC1 suggest that they play different roles in the maturation of glutamatergic synaptic transmission in the medial and lateral VN during postnatal development

Vestibular Schwann cells are a distinct subpopulation of peripheral glia with specific sensitivity to growth factors and extracellular matrix components

International audienceVestibular nerve Schwann cells are predisposed to develop schwannoma. While knowledge concerning this condition has greatly improved, little is known about properties of normal vestibular Schwann cells. In an attempt to understand this predisposition, we evaluated cell density regulation and proliferative features of these cells taken from 6-day-old rats. Data were compared to those obtained with sciatic Schwann cells. In both vestibular and sciatic 7-day-old cultures, Schwann cells appear as bipolar or flattened cells. However , sciatic and vestibular cells greatly differ in other aspects: on poly-L-lysine coating, sciatic cells specifically synthesize myelin basic protein, while expression of P0 mRNAs is restricted to some vestibular cells. Laminin increases sciatic cell density but not that of vestibular cells. Fibronectin selectively enhances the proliferation of vestibular Schwann cells and lacks an effect on sciatic ones. Comparison of cell density changes between sciatic and vestibular cells shows that they are sensitive to two different sets of growth factors. Progesterone and FGF-2 combined with forskolin selectively enhance the cell density of sciatic glia, while IGF-1 and GDNF specifically increase vestibular cell density. Furthermore, BrdU incorporation assays indicate that GDNF is also a mitogen for vestibular cells. Altogether, vestibular Schwann cells display phenotypic features and responsiveness to exog-enous signals that are significantly different from sciatic Schwann cells, suggesting that vestibular glia form a subpopulation of Schwann cells

Evolution of dental tissue mineralization: an analysis of the jawed vertebrate SPARC and SPARC-L families

Abstract Background The molecular bases explaining the diversity of dental tissue mineralization across gnathostomes are still poorly understood. Odontodes, such as teeth and body denticles, are serial structures that develop through deployment of a gene regulatory network shared between all gnathostomes. Dentin, the inner odontode mineralized tissue, is produced by odontoblasts and appears well-conserved through evolution. In contrast, the odontode hypermineralized external layer (enamel or enameloid) produced by ameloblasts of epithelial origin, shows extensive structural variations. As EMP (Enamel Matrix Protein) genes are as yet only found in osteichthyans where they play a major role in the mineralization of teeth and others skeletal organs, our understanding of the molecular mechanisms leading to the mineralized odontode matrices in chondrichthyans remains virtually unknown. Results We undertook a phylogenetic analysis of the SPARC/SPARC-L gene family, from which the EMPs are supposed to have arisen, and examined the expression patterns of its members and of major fibrillar collagens in the spotted catshark Scyliorhinus canicula, the thornback ray Raja clavata, and the clawed frog Xenopus tropicalis. Our phylogenetic analyses reveal that the single chondrichthyan SPARC-L gene is co-orthologous to the osteichthyan SPARC-L1 and SPARC-L2 paralogues. In all three species, odontoblasts co-express SPARC and collagens. In contrast, ameloblasts do not strongly express collagen genes but exhibit strikingly similar SPARC-L and EMP expression patterns at their maturation stage, in the examined chondrichthyan and osteichthyan species, respectively. Conclusions A well-conserved odontoblastic collagen/SPARC module across gnathostomes further confirms dentin homology. Members of the SPARC-L clade evolved faster than their SPARC paralogues, both in terms of protein sequence and gene duplication. We uncover an osteichthyan-specific duplication that produced SPARC-L1 (subsequently lost in pipidae frogs) and SPARC-L2 (independently lost in teleosts and tetrapods).Our results suggest the ameloblastic expression of the single chondrichthyan SPARC-L gene at the maturation stage reflects the ancestral gnathostome situation, and provide new evidence in favor of the homology of enamel and enameloids in all gnathostomes

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

Molecular footprinting of skeletal tissues in the catshark Scyliorhinus canicula and the clawed frog Xenopus tropicalis identifies conserved and derived features of vertebrate calcification

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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