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

New Insights in the Contribution of Voltage-Gated Nav Channels to Rat Aorta Contraction

BACKGROUND: Despite increasing evidence for the presence of voltage-gated Na(+) channels (Na(v)) isoforms and measurements of Na(v) channel currents with the patch-clamp technique in arterial myocytes, no information is available to date as to whether or not Na(v) channels play a functional role in arteries. The aim of the present work was to look for a physiological role of Na(v) channels in the control of rat aortic contraction. METHODOLOGY/PRINCIPAL FINDINGS: Na(v) channels were detected in the aortic media by Western blot analysis and double immunofluorescence labeling for Na(v) channels and smooth muscle alpha-actin using specific antibodies. In parallel, using real time RT-PCR, we identified three Na(v) transcripts: Na(v)1.2, Na(v)1.3, and Na(v)1.5. Only the Na(v)1.2 isoform was found in the intact media and in freshly isolated myocytes excluding contamination by other cell types. Using the specific Na(v) channel agonist veratridine and antagonist tetrodotoxin (TTX), we unmasked a contribution of these channels in the response to the depolarizing agent KCl on rat aortic isometric tension recorded from endothelium-denuded aortic rings. Experimental conditions excluded a contribution of Na(v) channels from the perivascular sympathetic nerve terminals. Addition of low concentrations of KCl (2-10 mM), which induced moderate membrane depolarization (e.g., from -55.9+/-1.4 mV to -45.9+/-1.2 mV at 10 mmol/L as measured with microelectrodes), triggered a contraction potentiated by veratridine (100 microM) and blocked by TTX (1 microM). KB-R7943, an inhibitor of the reverse mode of the Na(+)/Ca(2+) exchanger, mimicked the effect of TTX and had no additive effect in presence of TTX. CONCLUSIONS/SIGNIFICANCE: These results define a new role for Na(v) channels in arterial physiology, and suggest that the TTX-sensitive Na(v)1.2 isoform, together with the Na(+)/Ca(2+) exchanger, contributes to the contractile response of aortic myocytes at physiological range of membrane depolarization

Collagen XVI is a neural component of the developing and regenerating dorsal root ganglia extracellular matrix.

International audienceCollagen XVI is a homotrimeric molecule harbouring similarities to the FACIT (fibril-associated collagens with interrupted triple helices) family of collagens (Grassel et al., 1996). Collagen XVI is expressed in skin and cartilage where it is integrated into tissue specific aggregates (Grassel et al., 1999; Kassner et al., 2003). In the nervous system, collagen XVI has been detected at lowlevel in the brain and a strong expression was also reported in spinal root fibers during development (Lai and Chu, 1996). In dorsal root ganglia (DRG), analysis of SAGE banks performed by our group during development and after nerve injury (Mechaly et al., 2006) shows a fluctuation of collagen XVI expression between the different conditions and prompted us to study it further. DRGs contain the cell bodies of neurons, the axons of which transmit sensory information from the periphery to the central nervous system. While it is well known that during development and regeneration, neurites require extracellular matrix molecules for growth and guidance (Hari et al., 2004), the composition and the role of the matrix surrounding neurons in the ganglia itself have solicited little interest. Here, we show that collagen XVI is a component of the developingDRG extracellular matrix, that following nerve injury, its expression is increased around neuronal cell bodies and that neurons express collagen XVI in the peripheral nervous system

Oxidative Stress Plays an Important Role in Glutamatergic Excitotoxicity-Induced Cochlear Synaptopathy: Implication for Therapeutic Molecules Screening

International audienceThe disruption of the synaptic connection between the sensory inner hair cells (IHCs) and the auditory nerve fiber terminals of the type I spiral ganglion neurons (SGN) has been observed early in several auditory pathologies (e.g., noise-induced or ototoxic drug-induced or age-related hearing loss). It has been suggested that glutamate excitotoxicity may be an inciting element in the degenerative cascade observed in these pathological cochlear conditions. Moreover, oxidative damage induced by free hydroxyl radicals and nitric oxide may dramatically enhance cochlear damage induced by glutamate excitotoxicity. To investigate the underlying molecular mechanisms involved in cochlear excitotoxicity, we examined the molecular basis responsible for kainic acid (KA, a full agonist of AMPA/KA-preferring glutamate receptors)-induced IHC synapse loss and degeneration of the terminals of the type I spiral ganglion afferent neurons using a cochlear explant culture from P3 mouse pups. Our results demonstrated that disruption of the synaptic connection between IHCs and SGNs induced increased levels of oxidative stress, as well as altered both mitochondrial function and neurotrophin signaling pathways. Additionally, the application of exogenous antioxidants and neurotrophins (NT3, BDNF, and small molecule TrkB agonists) clearly increases synaptogenesis. These results suggest that understanding the molecular pathways involved in cochlear excitotoxicity is of crucial importance for the future clinical trials of drug interventions for auditory synaptopathies

Oxidative Stress Plays an Important Role in Glutamatergic Excitotoxicity-Induced Cochlear Synaptopathy: Implication for Therapeutic Molecules Screening

The disruption of the synaptic connection between the sensory inner hair cells (IHCs) and the auditory nerve fiber terminals of the type I spiral ganglion neurons (SGN) has been observed early in several auditory pathologies (e.g., noise-induced or ototoxic drug-induced or age-related hearing loss). It has been suggested that glutamate excitotoxicity may be an inciting element in the degenerative cascade observed in these pathological cochlear conditions. Moreover, oxidative damage induced by free hydroxyl radicals and nitric oxide may dramatically enhance cochlear damage induced by glutamate excitotoxicity. To investigate the underlying molecular mechanisms involved in cochlear excitotoxicity, we examined the molecular basis responsible for kainic acid (KA, a full agonist of AMPA/KA-preferring glutamate receptors)-induced IHC synapse loss and degeneration of the terminals of the type I spiral ganglion afferent neurons using a cochlear explant culture from P3 mouse pups. Our results demonstrated that disruption of the synaptic connection between IHCs and SGNs induced increased levels of oxidative stress, as well as altered both mitochondrial function and neurotrophin signaling pathways. Additionally, the application of exogenous antioxidants and neurotrophins (NT3, BDNF, and small molecule TrkB agonists) clearly increases synaptogenesis. These results suggest that understanding the molecular pathways involved in cochlear excitotoxicity is of crucial importance for the future clinical trials of drug interventions for auditory synaptopathies

Dependence on KCl of contractile response to veratridine and TTX in the presence of prazosin.

<p>The absence of endothelium was confirmed as described above. Representative recordings of isometric responses to veratridine (vera; 100 µmol/L) (A) or tetrodotoxin (TTX; 1 µmol/L) (B) after contraction induction by KCl (6 and 10 mmol/L) in the presence of 10 µmol/L prazosin (Pz). (C) Summary of results of the experiments described in A and B: the effects of veratridine (100 µmol/L) or TTX (1 µmol/L) on the contraction induced by KCl were expressed as a percentage of maximal contraction induced by PE (1 µmol/L). (D) Effects of TTX expressed as a percentage of KCl-induced contraction. Values represent the mean±s.e.m. of 6 animals, with experiments performed in duplicate. *p<0.05; **p<0.01; ***p<0.001, paired <i>t-test</i>.</p

Membrane potential determination.

<p>Membrane potentials were recorded in the SMCs of aortic rings under basal conditions and after addition of low KCl concentrations, using glass microelectrodes. (A) Representative recordings illustrating membrane potential stability in control experiments (upper panel) and depolarizations induced by the cumulative addition of 5 mmol/L KCl (lower panel). (B) Averaged membrane potentials under basal conditions (CTL) and after addition of 5 mmol/L and 10 mmol/L KCl (as described). *p<0.05; ***p<0.001; two-way ANOVA followed by a Bonferroni post-hoc analysis. Values represent means ±s.e.m. of 4 animals, with experiments performed in duplicate.</p

Characteristics of the contractile responses of endothelium-denuded aortic rings to various agonists.

<p>The agonists used are KCl, phenylephrine (PE), arginine-vasopressin (AVP), and endothelin-1 (ET-1). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007360#s3" target="_blank">Results</a> are presented as E_max, corresponding to the maximal effect elicited by each agonist expressed in terms of isometric tension (g), and EC₅₀ values (in mmol/L or nmol/L), indicating the concentration at which 50% of the maximal effect is induced. Values represent the mean±s.e.m. of 5 animals, with experiments performed in duplicate.</p

Nucleotide sequences of the specific primers used to detect Na_v channel α and β isoforms by RT-PCR.

<p>Each set of primers was designed from mRNA sequences with the Genbank accession numbers indicated above, using the primer design software Light Cycler Probe Design (Roche). They generated PCR products of the predicted length in base pairs.</p

Involvement of aortic SMC Na_v channels in the contractile response to KCl.

<p>The absence of endothelium was confirmed as described above. After washing and 15 min of incubation with prazosin (Pz; 10 µmol/L), suramin (300 µmol/L) and PPADS (30 µmol/L), increasing concentrations of KCl (1 to 100 mmol/L) were added in the absence or presence of TTX (1 µmol/L). (A) Representative recording of the isometric response. (B) Dose-responses to KCl in the absence (▒) or in the presence of 1 µmol/L TTX (•). Data are expressed as a percentage of the maximal KCl-induced contraction and were analyzed with a non-linear fit function to determine EC₅₀ values. Values represent means±s.e.m. of 5 animals, with experiments performed in quadruplicate.</p