Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Na v s) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na v s remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Na v s, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Na v 1.7-specific inhibitor, ProTxII, allowed the isolation of Na v 1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na v 1.7 and Na v 1.8 currents. The redistribution of Na v 1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L -/-). SNS-Nedd4L -/-mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na v 1.7 and Na v 1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na v s and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

SummaryGene targeting in embryonic stem (ES) cells remains best practice for introducing complex mutations into the mouse germline. One aspect in this multistep process that has not been streamlined with regard to the logistics and ethics of mouse breeding is the efficiency of germline transmission: the transmission of the ES cell‐derived genome through the germline of chimeras to their offspring. A method whereby male chimeras transmit exclusively the genome of the injected ES cells to their offspring has been developed. The new technology, referred to as goGermline, entails injecting ES cells into blastocysts produced by superovulated homozygous Tsc22d3 floxed females mated with homozygous ROSA26‐Cre males. This cross produces males that are sterile due to a complete cell‐autonomous defect in spermatogenesis. The resulting male chimeras can be sterile but when fertile, they transmit the ES cell‐derived genome to 100% of their offspring. The method was validated extensively and in two laboratories for gene‐targeted ES clones that were derived from the commonly used parental ES cell lines Bruce4, E14, and JM8A3. The complete elimination of the collateral birth of undesired, non‐ES cell‐derived offspring in goGermline technology fulfills the reduction imperative of the 3R principle of humane experimental technique with animals. genesis 54:326–333, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

Prenatal exposure to mercury causes neurodevelopmental disorders and neurological pathologies in infants, such as microcephaly and mental retardation. Despite critical importance, the molecular interactions leading to mercury toxicity are yet to be elucidated. We first used a cell-free assay to investigate mercury effects on purified γ-secretase activity. Next, we treated adult Drosophila melanogaster with mercury and collected control and mercury-treated embryos, which were subjected to mild hypotonic protein extraction, or immunostained to reveal nervous system morphology. Embryos left to develop into adults were examined for wing phenotypes. Relative to control metals, we found that mercury strongly inhibits in vitro γ-secretase processing of both amyloid-β precursor protein (APP) and Notch. Mercury inhibited APP and Notch cleavage in a dose-dependent manner, with IC(50) values of 50-125 nM, and is therefore comparable in potency to benchmark γ-secretase inhibitors. Immunoblot analysis of embryonic protein extracts showed that mercury inhibits Notch cleavage by γ-secretase in vivo. This is accompanied by severe neurodevelopmental abnormalities in embryos and adult wing-notching phenotypes. Our findings provide first evidence that mercury is a direct and potent γ-secretase inhibitor and suggest that inhibition of γ-secretase and disruption of the Notch developmental pathway potentially contribute to mercury-induced toxicity in the nervous system.

Homeodomain proteins are encoded by homeobox genes and regulate development and differentiation in many neuronal systems. The mouse vomeronasal organ (VNO) generates in situ mature chemosensory neurons from stem cells. The roles of homeodomain proteins in neuronal differentiation in the VNO are poorly understood. Here we have characterized the expression patterns of 28 homeobox genes in the VNO of C57BL/6 mice at postnatal stages using multicolor fluorescent in situ hybridization. We identified 11 homeobox genes (Dlx3, Dlx4, Emx2, Lhx2, Meis1, Pbx3, Pknox2, Pou6f1, Tshz2, Zhx1, Zhx3) that were expressed exclusively in neurons; 4 homeobox genes (Pax6, Six1, Tgif1, Zfhx3) that were expressed in all non-neuronal cell populations, with Pax6, Six1 and Tgif1 also expressed in some neuronal progenitors and precursors; 12 homeobox genes (Adnp, Cux1, Dlx5, Dlx6, Meis2, Pbx2, Pknox1, Pou2f1, Satb1, Tshz1, Tshz3, Zhx2) with expression in both neuronal and non-neuronal cell populations; and one homeobox gene (Hopx) that was exclusively expressed in the non-sensory epithelium. We studied further in detail the expression of Emx2, Lhx2, Meis1, and Meis2. We found that expression of Emx2 and Lhx2 initiated between neuronal progenitor and neuronal precursor stages. As far as the sensory neurons of the VNO are concerned, Meis1 and Meis2 were only expressed in the apical layer, together with Gnai2, but not in the basal layer.

Homeobox genes constitute a large family of genes widely studied because of their role in the establishment of the body pattern. However, they are also involved in many other events during development and adulthood. The main olfactory epithelium (MOE) is an excellent model to study neurogenesis in the adult nervous system. Analyses of homeobox genes during development show that some of these genes are involved in the formation and establishment of cell diversity in the MOE. Moreover, the mechanisms of expression of odorant receptors (ORs) constitute one of the biggest enigmas in the field. Analyses of OR promoters revealed the presence of homeodomain binding sites in their sequences. Here we characterize the expression patterns of a set of 49 homeobox genes in the MOE with in situ hybridization. We found that seven of them (Dlx3, Dlx5, Dlx6, Msx1, Meis1, Isl1, and Pitx1) are zonally expressed. The homeobox gene Emx1 is expressed in three guanylate cyclase(+) populations, two located in the MOE and the third one in an olfactory subsystem known as Grüneberg ganglion located at the entrance of the nasal cavity. The homeobox gene Tshz1 is expressed in a unique patchy pattern across the MOE. Our findings provide new insights to guide functional studies that aim to understand the complexity of transcription factor expression and gene regulation in the MOE. J. Comp. Neurol. 524:2713-2739, 2016. © 2016 Wiley Periodicals, Inc.