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.

Small RGK GTPases, Rad, Gem, Rem1, and Rem2, are potent inhibitors of high-voltage-activated (HVA) Ca(2+) channels expressed in heterologous expression systems. However, the role of this regulation has never been clearly demonstrated in the nervous system. Using transcriptional analysis, we show that peripheral nerve injury specifically upregulates Gem in mice dorsal root ganglia. Following nerve injury, protein expression was increased in ganglia and peripheral nerve, mostly under its phosphorylated form. This was confirmed in situ and in vitro in dorsal root ganglia sensory neurons. Knockdown of endogenous Gem, using specific small-interfering RNA (siRNA), increased the HVA Ca(2+) current only in the large-somatic-sized neurons. Combining pharmacological analysis of the HVA Ca(2+) currents together with Gem siRNA-transfection of larger sensory neurons, we demonstrate that only the P/Q-type Ca(2+) channels were enhanced. In vitro analysis of Gem affinity to various CaVβx-CaV2.x complexes and immunocytochemical studies of Gem and CaVβ expression in sensory neurons suggest that the specific inhibition of the P/Q channels relies on both the regionalized upregulation of Gem and the higher sensitivity of the endogenous CaV2.1-CaVβ4 pair in a subset of sensory neurons including the proprioceptors. Finally, pharmacological inhibition of P/Q-type Ca(2+) current reduces neurite branching of regenerating axotomized neurons. Taken together, the present results indicate that a Gem-dependent P/Q-type Ca(2+) current inhibition may contribute to general homeostatic mechanisms following a peripheral nerve injury.

Identification of the molecular mechanisms governing sensory neuron subtype excitability is a key requisite for the development of treatments for somatic sensory disorders. Here, we show that the Na,K-ATPase modulator Fxyd2 is specifically required for setting the mechanosensitivity of Aδ-fiber low-threshold mechanoreceptors and sub-populations of C-fiber nociceptors, a role consistent with its restricted expression profile in the spinal somatosensory system. We also establish using the spared nerve injury model of neuropathic pain, that loss of Fxyd2 function, either constitutively in Fxyd2-/- mice or acutely in neuropathic rats, efficiently alleviates mechanical hypersensitivity induced by peripheral nerve lesions. The role of Fxyd2 in modulating Aδ- and C-fibers mechanosensitivity likely accounts for the anti-allodynic effect of Fxyd2 knockdown. Finally, we uncover the evolutionarily conserved restricted expression pattern of FXYD2 in human dorsal root ganglia, thus identifying this molecule as a potentially promising therapeutic target for peripheral neuropathic pain management.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that selectively affects upper and lower motoneurons. Dismantlement of the neuromuscular junction (NMJ) is an early pathological hallmark of the disease whose cellular origin remains still debated. We developed an in vitro NMJ model to investigate the differential contribution of motoneurons and muscle cells expressing ALS-causing mutation in the superoxide dismutase 1 (SOD1) to neuromuscular dysfunction. The primary co-culture system allows the formation of functional NMJs and fosters the expression of the ALS-sensitive fast fatigable type II-b myosin heavy chain (MHC) isoform. Expression of SOD1G93A in myotubes does not prevent the formation of a functional NMJ but leads to decreased contraction frequency and lowers the slow type I MHC isoform transcript levels. Expression of SOD1G93A in both motoneurons and myotubes or in motoneurons alone however alters the formation of a functional NMJ. Our results strongly suggest that motoneurons are a major factor involved in the process of NMJ dismantlement in an experimental model of ALS.

Amyotrophic lateral sclerosis (ALS) is an incurable paralytic disorder primarily typified by the selective and progressive degeneration of motoneurons in the brain and spinal cord. ALS causes muscle wasting and atrophy, resulting eventually in respiratory failure and death within 3-5 years of diagnosis. Vitamin D is a potent secosteroid hormone with diverse biological functions that include protection against neuronal damage. The detrimental consequences of vitamin D dietary deficiency have been documented in other neurodegenerative diseases. However, the protective effect of vitamin D on motoneuron and the influence of its levels on disease course remains elusive. Here we found that the biologically active form of vitamin D significantly potentiated the effect of neurotrophic factors and prevented motoneurons from a Fas-induced death, while electrophysiological properties of motoneurons were not affected. In ALS patients, we report that a severe vitamin D deficiency accelerates by 4 times the rate of decline and were associated with a marked shorter life expectancy. Our findings support a neuroprotective function of vitamin D on motoneurons and propose vitamin D as a reliable prognostic factor of ALS.

Ca2+ influx through voltage-gated calcium channels probably influences neuronal ontogenesis. Many developing neurones transiently express T-type/Cav3 calcium channels that contribute to their electrical activity and potentially to their morphological differentiation. Here we have characterized the electrophysiological properties and the functional role of a large T-type calcium current that is present in mouse developing primary vestibular neurones at embryonic day E17. This T-type current showed fast activation and inactivation, as well as slow deactivation kinetics. The overlap of activation and inactivation parameters produced a window current between -65 and -45 mV. Recovery from short-term inactivation was slow suggesting the presence of the Cav3.2 subunit. This T-type current was blocked by micromolar concentrations of Ni2+ and was inhibited by fast perfusion velocities in a similar fashion to recombinant Cav3.2 T-type channels expressed in HEK-293 cells. More importantly, current clamp experiments have revealed that the T-current could elicit afterdepolarization potentials during the repolarization phase of action potentials, and occasionally generate calcium spikes. Taken together, we demonstrate that the Cav3.2 subunit is likely to be the main T-type calcium channel subunit expressed in embryonic vestibular neurones and should play a key role in the excitability of these neurones during the ontogenesis of vestibular afferentation.

Dorsal root ganglia (DRG) neurons exhibit a wide molecular heterogeneity in relation to the various sensory modalities (mechanoception, thermoception, nociception) that they subserve. Finding markers of subpopulations is an important step in understanding how these neurons convey specific information. We identified fibroblast growth factor homologous factor 1 (FHF1) in a search for markers of subpopulations of DRG neurons. FHFs constitute a family of four factors that share some structural properties with fibroblast growth factors (FGFs) but are functionally distinct. They are expressed in specific subsets of neurons and are involved in the modulation of sodium channel activity. The pattern of expression of FHF1 in the DRG was determined during development, in the adult and after axotomy. We show that in the adult, FHF1 is expressed in two populations, one composed of nociceptors and another in which no neurotrophic factor receptors were detected (panTrk-/c-Ret-). Interestingly, in the nociceptors, FHF1 expression was restricted to a subset of TrkA+/calcitonin gene-related peptide (CGRP)-positive neurons. Neurofilament 200 (NF-200) and peripherin labeling indicates that 70% of the FHF1-expressing neurons contribute to A-fibers and 30% to C-fibers. FHF1 interacts with the Na(v)1.9 sodium channel isoform, which is strongly expressed in cRet+/isolectin-B4 binding neurons, but we show that FHF1 is not expressed in the cRet+/IB4+ subclass and that it does not colocalize with Na(v)1.9. Our results argue strongly against the possibility that FHF1 has a modulatory effect on this channel in cRet+/IB4+ neurons, but FHF1 could play a role in a distinct subset of TrkA+/CGRP+ nociceptors.

Loss-of-function mutations in the potassium-chloride cotransporter KCC3 lead to Andermann syndrome, a severe sensorimotor neuropathy characterized by areflexia, amyotrophy and locomotor abnormalities. The molecular events responsible for axonal loss remain poorly understood. Here, we establish that global or neuron-specific KCC3 loss-of-function in mice leads to early neuromuscular junction (NMJ) abnormalities and muscular atrophy that are consistent with the pre-synaptic neurotransmission defects observed in patients. KCC3 depletion does not modify chloride handling, but promotes an abnormal electrical activity among primary motoneurons and mislocalization of Na+/K+-ATPase α1 in spinal cord motoneurons. Moreover, the activity-targeting drug carbamazepine restores Na+/K+-ATPase α1 localization and reduces NMJ denervation in Slc12a6-/- mice. We here propose that abnormal motoneuron electrical activity contributes to the peripheral neuropathy observed in Andermann syndrome.

Functionally distinct classes of dorsal root ganglia (DRG) somatosensory neurons arise from neural crest cells (NCCs) in two successive phases of differentiation assumed to be respectively and independently controlled by the proneural genes Neurog2 and Neurog1. However, the precise role of Neurog2 during this process remains unclear, notably because no neuronal loss has been reported hitherto in Neurog2-/- mutants. Here, we show that at trunk levels, Neurog2 deficiency impairs the production of subsets of all DRG neuron subtypes. We establish that this phenotype is highly dynamic and reflects multiple defects in NCC-derived progenitors, including somatosensory-to-melanocyte fate switch, apoptosis, and delayed differentiation which alters neuronal identity, all occurring during a narrow time window when Neurog2 temporarily controls onset of Neurog1 expression and neurogenesis. Collectively, these findings uncover a critical period of cell fate plasticity and vulnerability among somatosensory progenitors and establish that Neurog2 function in the developing DRG is broader than initially envisaged.

Matrix Gla protein (Mgp) and bone Gla protein (Bgp) are vitamin-K dependent proteins that bind calcium in their γ-carboxylated versions in mammals. They are recognized as positive (Bgp) or negative (Mgp and Bgp) regulators of biomineralization in a number of tissues, including skeletal tissues of bony vertebrates. The Mgp/Bgp gene family is poorly known in cartilaginous fishes, which precludes the understanding of the evolution of the biomineralization toolkit at the emergence of jawed vertebrates. Here we took advantage of recently released genomic and transcriptomic data in cartilaginous fishes and described the genomic loci and gene expression patterns of the Mgp/Bgp gene family. We identified three genes, Mgp1, Mgp2, and Bgp, in cartilaginous fishes instead of the single previously reported Mgp gene. We describe their genomic loci, resulting in a dynamic evolutionary scenario for this gene family including several events of local (tandem) duplications, but also of translocation events, along jawed vertebrate evolution. We describe the expression patterns of Mgp1, Mgp2, and Bgp in embryonic stages covering organogenesis in the small-spotted catshark Scyliorhinus canicula and present a comparative analysis with Mgp/Bgp family members previously described in bony vertebrates, highlighting ancestral features such as early embryonic, soft tissues, and neuronal expressions, but also derived features of cartilaginous fishes such as expression in fin supporting fibers. Our results support an ancestral function of Mgp in skeletal mineralization and a later derived function of Bgp in skeletal development that may be related to the divergence of bony vertebrates.

Mouse chromaffin cells in acute adrenal slices exhibit two distinct spiking patterns, a repetitive mode and a bursting mode. A sodium background conductance operates at rest as demonstrated by the membrane hyperpolarization evoked by a low Na+ -containing extracellular saline. This sodium background current is insensitive to TTX, is not blocked by Cs+ ions and displays a linear I-V relationship at potentials close to chromaffin cell resting potential. Its properties are reminiscent of those of the sodium leak channel NALCN. In the adrenal gland, Nalcn mRNA is selectively expressed in chromaffin cells. The study fosters our understanding of how the spiking pattern of chromaffin cells is regulated and adds a sodium background conductance to the list of players involved in the stimulus-secretion coupling of the adrenomedullary tissue.

Although cardio-vascular incidents and sudden cardiac death (SCD) are among the leading causes of premature death in the general population, the origins remain unidentified in many cases. Genome-wide association studies have identified Meis1 as a risk factor for SCD. We report that Meis1 inactivation in the mouse neural crest leads to an altered sympatho-vagal regulation of cardiac rhythmicity in adults characterized by a chronotropic incompetence and cardiac conduction defects, thus increasing the susceptibility to SCD. We demonstrated that Meis1 is a major regulator of sympathetic target-field innervation and that Meis1 deficient sympathetic neurons die by apoptosis from early embryonic stages to perinatal stages. In addition, we showed that Meis1 regulates the transcription of key molecules necessary for the endosomal machinery. Accordingly, the traffic of Rab5(+) endosomes is severely altered in Meis1-inactivated sympathetic neurons. These results suggest that Meis1 interacts with various trophic factors signaling pathways during postmitotic neurons differentiation.

The cation-Cl(-) cotransporters participate to neuronal Cl(-) balance and are responsible for the post-natal Cl(-) switch in central neurons. In the adult peripheral nervous system, it is not well established whether a Cl(-) transition occurs during maturation. We investigated the contribution of cation-Cl(-) cotransporters in the Cl(-) handling of sensory neurons derived from the dorsal root ganglia (DRG) of neonatal mice (postnatal days 1-6) and adult mice. Gramicidin-perforated patch-clamp recordings in wild-type neurons revealed that Cl(-) accumulated to very high values in P1-6 sensory neurons and decreased in adulthood. In post-natal sensory neurons, quantitative RT-PCR showed that NKCC1, KCC1 and KCC3 had a higher transcript expression level compared to KCC2 and KCC4. NKCC1 was the main cation-Cl(-) cotransporter controlling Cl(-) accumulation at this developmental stage. In adulthood, the KCC3 transcript was produced in larger amounts than the other cation-Cl(-) cotransporter transcripts and RT-PCR shows larger expression of the shorter KCC3a isoform in adult DRG. Pharmacological inhibitors of cation-Cl(-) cotransporters and the use of KCC3(-/-) mice demonstrated that NKCC1 sustained Cl(-) accumulation in the majority of adult sensory neurons while KCC3 contributed to Cl(-) extrusion in a subset of these neurons. Beta-galactosidase detection in adult KCC3(-/-) DRG showed that KCC3 transcripts were present in all adult sensory neurons suggesting a KCC3 isoform specific regulation of Cl(-) handling. The contribution of KCC3 to Cl(-) extrusion in a subset of sensory neurons indicates that KCC3 could play a major role in GABAergic/glycinergic transmission.

Axonal regeneration is one of the greatest challenges in severe injuries of peripheral nerve. To provide the bridge needed for regeneration, biological or synthetic tubular nerve constructs with aligned architecture have been developed. A key point for improving axonal regeneration is assessing the effects of substrate geometry on neuronal behavior. In the present study, we used an extracellular matrix-micropatterned substrate comprising 3 µm wide lines aimed to physically mimic the in vivo longitudinal axonal growth of mice peripheral sensory and motor neurons. Adult sensory neurons or embryonic motoneurons were seeded and processed for morphological and electrical activity analyses after two days in vitro. We show that micropattern-guided sensory neurons grow one or two axons without secondary branching. Motoneurons polarity was kept on micropattern with a long axon and small dendrites. The micro-patterned substrate maintains the growth promoting effects of conditioning injury and demonstrates, for the first time, that neurite initiation and extension could be differentially regulated by conditioning injury among DRG sensory neuron subpopulations. The micro-patterned substrate impacts the excitability of sensory neurons and promotes the apparition of firing action potentials characteristic for a subclass of mechanosensitive neurons. The line pattern is quite relevant for assessing the regenerative and developmental growth of sensory and motoneurons and offers a unique model for the analysis of the impact of geometry on the expression and the activity of mechanosensitive channels in DRG sensory neurons.

In order to shed light on transcriptional networks involved in adult peripheral nerve repair program, we propose for the first time an organization of the transcriptional dynamics of the mouse dorsal root ganglia (DRG) following a sciatic nerve lesion. This was done by a non-hierarchical bioinformatical clustering of four Serial Analysis of Gene Expression libraries performed on DRG at embryonic day E13, neonatal day P0, adult and adult 3 days post-sciatic nerve section. Grouping genes according to their expression profiles shows that a combination of down-regulation of genes expressed at the adult stages, re-expression of embryonic genes and induction of a set of de novo genes takes place in injured neurons. Focusing on this latter event highlights Ddit3, Timm8b and Oazin as potential new injury-induced molecular actors involved in a stress response pathway. Their association with the traumatic state was confirmed by real-time PCR and in situ hybridization investigations. Clustering analysis allows us to distinguish developmental re-programming events from nerve-injury-induced processes and thus provides a basis for molecular understanding of transcriptional alterations taking place in the DRG after a sciatic nerve lesion.

Neuronal Ca2+ entry elicited by electrical activity contributes to information coding via activation of K+ and Cl- channels. While Ca2+-dependent K+ channels have been extensively studied, the molecular identity and role of Ca2+-activated Cl- channels (CaCCs) remain unclear. Here, we demonstrate that TMEM16F governs a Ca2+-activated Cl- conductance in spinal motoneurons. We show that TMEM16F is expressed in synaptic clusters facing pre-synaptic cholinergic C-boutons in α-motoneurons of the spinal cord. Mice with targeted exon deletion in Tmem16f display decreased motor performance under high-demanding tasks attributable to an increase in the recruitment threshold of fast α-motoneurons. Remarkably, loss of TMEM16F function in a mouse model of amyotrophic lateral sclerosis (ALS) significantly reduces expression of an activity-dependent early stress marker and muscle denervation, delays disease onset, and preserves muscular strength only in male ALS mice. Thus, TMEM16F controls motoneuron excitability and impacts motor resistance as well as motor deterioration in ALS.

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.

A prior peripheral nerve injury in vivo, promotes a rapid elongated mode of sensory neurons neurite regrowth in vitro. This in vitro model of conditioned axotomy allows analysis of the cellular and molecular mechanisms leading to an improved neurite re-growth. Our differential interference contrast microscopy and immunocytochemistry results show that conditioned axotomy, induced by sciatic nerve injury, did not increase somatic size of adult lumbar sensory neurons from mice dorsal root ganglia sensory neurons but promoted the appearance of larger neurites and growth cones. Using atomic force microscopy on live neurons, we investigated whether membrane mechanical properties of growth cones of axotomized neurons were modified following sciatic nerve injury. Our data revealed that neurons having a regenerative growth were characterized by softer growth cones, compared to control neurons. The increase of the growth cone membrane elasticity suggests a modification in the ratio and the inner framework of the main structural proteins.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor system leading to generalized paralysis and death of patients. The understanding of early pathogenic mechanisms will help to define early diagnostics criteria that will eventually provide basis for efficient therapeutics. Early symptoms of ALS usually include muscle weakness or stiffness. Therefore, mechanical response of differentiated myotubes from primary cultures of mice, expressing the ALS-causing SOD1 G93A mutation, was examined by atomic force microscopy. Simultaneous acquisition of topography and cell elasticity of ALS myotubes was performed by force mapping method, compared with healthy myotubes and supplemented with immunofluorescence and qRT-PCR studies. Wild type myotubes reveal a significant difference in elasticity between a narrow and a wide population, consistent with maturation occurring with higher actin expression relative to myosin together with larger myotube width. However, this is not true for SOD1 G93A expressing myotubes, where a significant shift of thin population towards higher elastic modulus values was observed. We provide evidence that SOD1 mutant induces structural changes that occurs very early in muscle development and well before symptomatic stage of the disease. These findings could significantly contribute to the understanding of the role of skeletal muscle in ALS pathogenesis.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable paralytic disorder caused by the progressive death of upper and lower motoneurons. Although numerous strategies have been developed to slow disease progression and improve life quality, to date only a few therapeutic treatments are available with still unsatisfactory therapeutic benefits. The secretome of dental pulp stem cells (DPSCs) contains numerous neurotrophic factors that could promote motoneuron survival. Accordingly, DPSCs confer neuroprotective benefits to the SOD1G93A mouse model of ALS. However, the mode of action of DPSC secretome on motoneurons remains largely unknown. Here, we used conditioned medium of human DPSCs (DPSCs-CM) and assessed its effect on survival, axonal length, and electrical activity of cultured wildtype and SOD1G93A motoneurons. To further understand the role of individual factors secreted by DPSCs and to circumvent the secretome variability bias, we focused on GDF15 and HB-EGF whose neuroprotective properties remain elusive in the ALS pathogenic context. DPSCs-CM rescues motoneurons from trophic factor deprivation-induced death, promotes axon outgrowth of wildtype but not SOD1G93A mutant motoneurons, and has no impact on the spontaneous electrical activity of wildtype or mutant motoneurons. Both GDF15 and HB-EGF protect SOD1G93A motoneurons against nitric oxide-induced death, but not against death induced by trophic factor deprivation. GDF15 and HB-EGF receptors were found to be expressed in the spinal cord, with a two-fold increase in expression for the GDF15 low-affinity receptor in SOD1G93A mice. Therefore, the secretome of DPSCs appears as a new potential therapeutic candidate for ALS.