FXYD2 antisense oligonucleotide provides an efficient approach for long-lasting relief of chronic peripheral pain.

Chronic pain, whether of inflammatory or neuropathic origin, affects about 18% of the population of developed countries, and most current treatments are only moderately effective and/or cause serious side effects. Therefore, the development of novel therapeutic approaches still represents a major challenge. The Na,K-ATPase modulator FXYD2 is critically required for the maintenance of neuropathic pain in rodents. Here, we set up a therapeutic protocol based on the use of chemically modified antisense oligonucleotides (ASOs) to inhibit FXYD2 expression and treat chronic pain. We identified an ASO targeting a 20-nucleotide stretch in the FXYD2 mRNA that is evolutionarily conserved between rats and humans and is a potent inhibitor of FXYD2 expression. We used this sequence to synthesize lipid-modified forms of ASO (FXYD2-LASO) to facilitate their entry into dorsal root ganglia neurons. We established that intrathecal or intravenous injections of FXYD2-LASO in rat models of neuropathic or inflammatory pain led to a virtually complete alleviation of their pain symptoms, without causing obvious side effects. Remarkably, by using 2'-O-2-methoxyethyl chemical stabilization of the ASO (FXYD2-LASO-Gapmer), we could significantly prolong the therapeutic action of a single treatment up to 10 days. This study establishes FXYD2-LASO-Gapmer administration as a promising and efficient therapeutic strategy for long-lasting relief of chronic pain conditions in human patients.

Neurog2 Deficiency Uncovers a Critical Period of Cell Fate Plasticity and Vulnerability among Neural-Crest-Derived Somatosensory Progenitors.

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.

PRDM12 Is Required for Initiation of the Nociceptive Neuron Lineage during Neurogenesis.

The sensation of pain is essential for the preservation of the functional integrity of the body. However, the key molecular regulators necessary for the initiation of the development of pain-sensing neurons have remained largely unknown. Here, we report that, in mice, inactivation of the transcriptional regulator PRDM12, which is essential for pain perception in humans, results in a complete absence of the nociceptive lineage, while proprioceptive and touch-sensitive neurons remain. Mechanistically, our data reveal that PRDM12 is required for initiation of neurogenesis and activation of a cascade of downstream pro-neuronal transcription factors, including NEUROD1, BRN3A, and ISL1, in the nociceptive lineage while it represses alternative fates other than nociceptors in progenitor cells. Our results thus demonstrate that PRDM12 is necessary for the generation of the entire lineage of pain-initiating neurons.

Inhibition of neuronal FLT3 receptor tyrosine kinase alleviates peripheral neuropathic pain in mice.

Peripheral neuropathic pain (PNP) is a debilitating and intractable chronic disease, for which sensitization of somatosensory neurons present in dorsal root ganglia that project to the dorsal spinal cord is a key physiopathological process. Here, we show that hematopoietic cells present at the nerve injury site express the cytokine FL, the ligand of fms-like tyrosine kinase 3 receptor (FLT3). FLT3 activation by intra-sciatic nerve injection of FL is sufficient to produce pain hypersensitivity, activate PNP-associated gene expression and generate short-term and long-term sensitization of sensory neurons. Nerve injury-induced PNP symptoms and associated-molecular changes were strongly altered in Flt3-deficient mice or reversed after neuronal FLT3 downregulation in wild-type mice. A first-in-class FLT3 negative allosteric modulator, discovered by structure-based in silico screening, strongly reduced nerve injury-induced sensory hypersensitivity, but had no effect on nociception in non-injured animals. Collectively, our data suggest a new and specific therapeutic approach for PNP.

Fxyd2 regulates Aδ- and C-fiber mechanosensitivity and is required for the maintenance of neuropathic pain.

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.

Onset of Spinal Cord Astrocyte Precursor Emigration from the Ventricular Zone Involves the Zeb1 Transcription Factor.

During spinal cord development, astrocyte precursors arise from neuroepithelial progenitors, delaminate from the ventricular zone, and migrate toward their final locations where they differentiate. Although the mechanisms underlying their early specification and late differentiation are being deciphered, less is known about the temporal control of their migration. Here, we show that the epithelial-mesenchymal transition regulator Zeb1 is expressed in glial precursors and report that loss of Zeb1 function specifically delays the onset of astrocyte precursor delamination from the ventricular zone, correlating with transient deregulation of the adhesion protein Cadherin-1. Consequently, astrocyte precursor invasion into the Zeb1-/- mutant white matter is delayed, and induction of their differentiation is postponed. These findings illustrate how fine regulation of adhesive properties influences the onset of neural precursor migration and further support the notion that duration of exposure of migrating astrocyte precursors to environmental cues and/or their correct positioning influence the timing of their differentiation.

Zeb family members and boundary cap cells underlie developmental plasticity of sensory nociceptive neurons.

Dorsal root ganglia (DRG) sensory neurons arise from heterogeneous precursors that differentiate in two neurogenic waves, respectively controlled by Neurog2 and Neurog1. We show here that transgenic mice expressing a Zeb1/2 dominant-negative form (DBZEB) exhibit reduced numbers of nociceptors and altered pain sensitivity. This reflects an early impairment of Neurog1-dependent neurogenesis due to the depletion of specific sensory precursor pools, which is slightly later partially compensated by the contribution of boundary cap cells (BCCs). Indeed, combined DBZEB expression and genetic BCCs ablation entirely deplete second wave precursors and, in turn, nociceptors, thus recapitulating the Neurog1(-/-) neuronal phenotype. Altogether, our results uncover roles for Zeb family members in the developing DRGs; they show that the Neurog1-dependent sensory neurogenesis can be functionally partitioned in two successive phases; and finally, they illustrate plasticity in the developing peripheral somatosensory system supported by the BCCs, thereby providing a rationale for sensory precursor diversity.

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.

The transcription factor c-Maf controls touch receptor development and function.

The sense of touch relies on detection of mechanical stimuli by specialized mechanosensory neurons. The scarcity of molecular data has made it difficult to analyze development of mechanoreceptors and to define the basis of their diversity and function. We show that the transcription factor c-Maf/c-MAF is crucial for mechanosensory function in mice and humans. The development and function of several rapidly adapting mechanoreceptor types are disrupted in c-Maf mutant mice. In particular, Pacinian corpuscles, a type of mechanoreceptor specialized to detect high-frequency vibrations, are severely atrophied. In line with this, sensitivity to high-frequency vibration is reduced in humans carrying a dominant mutation in the c-MAF gene. Thus, our work identifies a key transcription factor specifying development and function of mechanoreceptors and their end organs.

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.

The transcription factor Cux2 marks development of an A-delta sublineage of TrkA sensory neurons.

The developmental process and unique molecular identity between the many different types of dorsal root ganglion (DRG) sensory neurons generated during embryogenesis provide the cellular basis for the distinct perceptual modalities of somatosensation. The mechanisms leading to the generation of different types of nociceptive sensory neurons remain only partly understood. Here, we show that the transcription factor Cux2 is a novel marker of sensory neuron subpopulations of three main sublineages as defined by the expression of neurotrophic factor receptors TrkA, TrkB and TrkC. In particular, it is expressed in a subpopulation of early TrkA(+) neurons that arise during the early, Ngn1-independent initiated neurogenesis in the DRG. Postnatally, Cux2 marks a specific subtype of A-delta nociceptors as seen by expression of TrkA and NF200 but absence of TrpV1. Analysis of Cux2 mutant mice shows that Cux2 is not required for specification of Trk(+) neuronal subpopulations. However, Cux2 mutant mice are hypersensitive to mechanical, but not to heat or cold stimuli, consistent with a requirement in the process of specification of the mechanoreceptive neuron circuit. Hence, our results show that Cux2 is expressed and may participate in development of a specific subtype of myelinated TrkA(+) nociceptors.

OPA1 links human mitochondrial genome maintenance to mtDNA replication and distribution.

Eukaryotic cells harbor a small multiploid mitochondrial genome, organized in nucleoids spread within the mitochondrial network. Maintenance and distribution of mitochondrial DNA (mtDNA) are essential for energy metabolism, mitochondrial lineage in primordial germ cells, and to prevent mtDNA instability, which leads to many debilitating human diseases. Mounting evidence suggests that the actors of the mitochondrial network dynamics, among which is the intramitochondrial dynamin OPA1, might be involved in these processes. Here, using siRNAs specific to OPA1 alternate spliced exons, we evidenced that silencing of the OPA1 variants including exon 4b leads to mtDNA depletion, secondary to inhibition of mtDNA replication, and to marked alteration of mtDNA distribution in nucleoid and nucleoid distribution throughout the mitochondrial network. We demonstrate that a small hydrophobic 10-kDa peptide generated by cleavage of the OPA1-exon4b isoform is responsible for this process and show that this peptide is embedded in the inner membrane and colocalizes and coimmunoprecipitates with nucleoid components. We propose a novel synthetic model in which a peptide, including two trans-membrane domains derived from the N terminus of the OPA1-exon4b isoform in vertebrates or from its ortholog in lower eukaryotes, might contribute to nucleoid attachment to the inner mitochondrial membrane and promotes mtDNA replication and distribution. Thus, this study places OPA1 as a direct actor in the maintenance of mitochondrial genome integrity.

Defective respiratory rhythmogenesis and loss of central chemosensitivity in Phox2b mutants targeting retrotrapezoid nucleus neurons.

The retrotrapezoid nucleus (RTN) is a group of neurons in the rostral medulla, defined here as Phox2b-, Vglut2-, neurokinin1 receptor-, and Atoh1-expressing cells in the parafacial region, which have been proposed to function both as generators of respiratory rhythm and as central respiratory chemoreceptors. The present study was undertaken to assess these two putative functions using genetic tools. We generated two conditional Phox2b mutations, which target different subsets of Phox2b-expressing cells, but have in common a massive depletion of RTN neurons. In both conditional mutants as well as in the previously described Phox2b(27Ala) mutants, in which the RTN is also compromised, the respiratory-like rhythmic activity normally seen in the parafacial region of fetal brainstem preparations was completely abrogated. Rhythmic motor bursts were recorded from the phrenic nerve roots in the mutants, but their frequency was markedly reduced. Both the rhythmic activity in the RTN region and the phrenic nerve discharges responded to a low pH challenge in control, but not in the mutant embryos. Together, our results provide genetic evidence for the essential role of the Phox2b-expressing RTN neurons both in establishing a normal respiratory rhythm before birth and in providing chemosensory drive.

A mesenchymal-like ZEB1(+) niche harbors dorsal radial glial fibrillary acidic protein-positive stem cells in the spinal cord.

In humans and rodents the adult spinal cord harbors neural stem cells located around the central canal. Their identity, precise location, and specific signaling are still ill-defined and controversial. We report here on a detailed analysis of this niche. Using microdissection and glial fibrillary acidic protein (GFAP)-green fluorescent protein (GFP) transgenic mice, we demonstrate that neural stem cells are mostly dorsally located GFAP(+) cells lying ependymally and subependymally that extend radial processes toward the pial surface. The niche also harbors doublecortin protein (Dcx)(+) Nkx6.1(+) neurons sending processes into the lumen. Cervical and lumbar spinal cord neural stem cells maintain expression of specific rostro-caudal Hox gene combinations and the niche shows high levels of signaling proteins (CD15, Jagged1, Hes1, differential screening-selected gene aberrative in neuroblastoma [DAN]). More surprisingly, the niche displays mesenchymal traits such as expression of epithelial-mesenchymal-transition zinc finger E-box-binding protein 1 (ZEB1) transcription factor and smooth muscle actin. We found ZEB1 to be essential for neural stem cell survival in vitro. Proliferation within the niche progressively ceases around 13 weeks when the spinal cord reaches its final size, suggesting an active role in postnatal development. In addition to hippocampus and subventricular zone niches, adult spinal cord constitutes a third central nervous system stem cell niche with specific signaling, cellular, and structural characteristics that could possibly be manipulated to alleviate spinal cord traumatic and degenerative diseases.

Zfh1 promotes survival of a peripheral glia subtype by antagonizing a Jun N-terminal kinase-dependent apoptotic pathway.

In Drosophila subperineurial glia (SPG) ensheath and insulate the nerve. SPG is under strict cell cycle and survival control because cell division or death of such a cell type would compromise the integrity of the blood-nerve barrier. The mechanisms underlying the survival of SPG remain unknown. Here, we show that the embryonic peripheral glia expresses the Zfh1 transcription factor, and in zfh1 mutants a particular SPG subtype, ePG10, undergoes apoptosis. Our findings show that in ePG10, Zfh1 represses the pro-apoptotic RHG-motif gene reaper in a cell-autonomous manner. Zfh1 also blocks the activation of the Jun N-terminal kinase (JNK) pathway, and reducing or enhancing JNK signalling in zfh1 mutants prevents or promotes ePG10 apoptosis. Our study shows a novel function for Zfh1 as an anti-apoptotic molecule and uncovers a cryptic JNK-dependent apoptotic programme in ePG10, which is normally blocked by Zfh1. We propose that, in cells such as SPG that do not undergo self-renewal and survive long periods, transcriptional control of RHG-motif gene expression together with fine tuning of JNK signalling is crucial for cell survival.

Low-threshold mechanoreceptor subtypes selectively express MafA and are specified by Ret signaling.

Low-threshold mechanoreceptor neurons (LTMs) of the dorsal root ganglia (DRG) are essential for touch sensation. They form highly specialized terminations in the skin and display stereotyped projections in the spinal cord. Functionally defined LTMs depend on neurotrophin signaling for their postnatal survival and functioning, but how these neurons arise during development is unknown. Here, we show that specific types of LTMs can be identified shortly after DRG genesis by unique expression of the MafA transcription factor, the Ret receptor and coreceptor GFRalpha2, and find that their specification is Ngn2 dependent. In mice lacking Ret, these LTMs display early differentiation defects, as revealed by reduced MafA expression, and at later stages their central and peripheral projections are compromised. Moreover, in MafA mutants, a discrete subset of LTMs display altered expression of neurotrophic factor receptors. Our results provide evidence that genetic interactions involving Ret and MafA progressively promote the differentiation and diversification of LTMs.

Delays in neuronal differentiation in Mash1/Ascl1 mutants.

The inactivation of a developmental transcription factor may lead to the complete absence of a specific cell type. More commonly, though, it only partially impairs its generation. The modalities of this partial effect have rarely been documented in any detail. Here, we report a novel function for the bHLH transcription factor Ascl1/Mash1 in the generation of the nucleus of the solitary tract (nTS). In Mash1(-/-) late embryos, the nTS is markedly atrophic. Tracing back the origin of this atrophy, we show that nTS precursors appear in the mutants 1 day later than in the wild type and then accumulate at a slower pace. We also show that the previously reported atrophy of the sympathetic chain in Mash1 mutants is similarly preceded by a delay of 1 to 2 days in the appearance of differentiated ganglionic cells. Finally, we provide evidence that the acceleration imposed by Mash1, regardless of the production of post-mitotic cells, affects differentiation itself, both generic and type-specific.

Reciprocal gene replacements reveal unique functions for Phox2 genes during neural differentiation.

The paralogous paired-like homeobox genes Phox2a and Phox2b are involved in the development of specific neural subtypes in the central and peripheral nervous systems. The different phenotypes of Phox2 knockout mutants, together with their asynchronous onset of expression, prompted us to generate two knock-in mutant mice, in which Phox2a is replaced by the Phox2b coding sequence, and vice versa. Our results indicate that Phox2a and Phox2b are not functionally equivalent, as only Phox2b can fulfill the role of Phox2a in the structures that depend on both genes. Furthermore, we demonstrate unique roles of Phox2 genes in the differentiation of specific motor neurons. Whereas the oculomotor and the trochlear neurons require Phox2a for their proper development, the migration of the facial branchiomotor neurons depends on Phox2b. Therefore, our analysis strongly indicates that biochemical differences between the proteins rather than temporal regulation of their expression account for the specific function of each paralogue.

Generation and characterization of Rgs4 mutant mice.

RGS proteins are negative regulators of signaling through heterotrimeric G protein-coupled receptors and, as such, are in a position to regulate a plethora of biological phenomena. However, those have just begun to be explored in vivo. Here, we describe a mouse line deficient for Rgs4, a gene normally expressed early on in discrete populations of differentiating neurons and later on at multiple sites of the central nervous system, the cortex in particular, where it is one of the most highly transcribed Rgs genes. Rgs4(lacZ/lacZ) mice had normal neural development and were viable and fertile. Behavioral testing on mutant adults revealed subtle sensorimotor deficits but, so far, supported neither the proposed status of Rgs4 as a schizophrenia susceptibility gene (by showing intact prepulse inhibition in the mutants) nor (unlike another member of the Rgs family, Rgs9) a role of Rgs4 in the acute or chronic response to opioids.

Sleep-disordered breathing in newborn mice heterozygous for the transcription factor Phox2b.

RATIONALE: Central congenital hypoventilation syndrome (CCHS) is a rare autosomal dominant syndrome present from birth, and characterized by depressed ventilation during sleep. Heterozygous mutations of the homeobox gene Phox2b were recently found in a very high proportion of patients. OBJECTIVES: To determine whether newborn mice with heterozygous targeted deletion of the transcription factor Phox2b would display sleep-disordered breathing. METHODS: We measured breathing pattern using whole-body plethysmography in wild-type and mutant 5-day-old mice, and we classified sleep-wake states using nuchal EMG and behavioral scores. RESULTS: We found that sleep apnea total time was approximately six times longer (8.9 +/- 12 vs. 1.5 +/- 2.2 seconds, p < 0.0015), and ventilation during active sleep was 21% lower (18.4 +/- 5.1 vs. 23.3 +/- 5.5 ml/g/second, p < 0.006) in mutant than in wild-type pups. During wakefulness, apnea time and ventilation were not significantly different between mutant and wild-type pups. Mutant and wild-type pups showed highly similar sleep-wake states. CONCLUSION: Although their respiratory phenotype was much less severe than CCHS, the Phox2b(+/-) mutant mice showed sleep-disordered breathing, which partially modeled the key feature of CCHS.