Chronic neuropathic pain caused by peripheral nerve injury is associated with global changes in gene expression in damaged neurons. To understand the molecular mechanisms underlying neuropathic pain, it is essential to elucidate how nerve injury alters gene expression and how the change contributes to the development and maintenance of chronic pain. MicroRNAs are non-protein-coding RNA molecules that regulate gene expression in a wide variety of biological processes mainly at the level of translation. This study investigated the possible involvement of microRNAs in gene regulation relevant to neuropathic pain. The analyses focused on a sensory organ-specific cluster of microRNAs that includes miR-96, -182, and -183. RT-PCR analyses confirmed that these microRNAs were highly enriched in the dorsal root ganglion (DRG) of adult rats. Using the L5 spinal nerve ligation (SNL) model of chronic neuropathic pain, we observed a significant reduction in expression of these microRNAs in injured DRG neurons compared to controls. In situ hybridization and immunohistochemical analyses revealed that these microRNAs are expressed in both myelinated (N52 positive) and unmyelinated (IB4 positive) primary afferent neurons. They also revealed that the intracellular distributions of the microRNAs in DRG neurons were dramatically altered in animals with mechanical hypersensitivity. Whereas microRNAs were uniformly distributed within the DRG soma of non-allodynic animals, they were preferentially localized to the periphery of neurons in allodynic animals. The redistribution of microRNAs was associated with changes in the distribution of the stress granule protein TIA-1. These data demonstrate that SNL induces changes in expression levels and patterns of miR-96, -182, and -183, implying their possible contribution to chronic neuropathic pain through translational regulation of pain-relevant genes. Moreover, stress granules were suggested to be assembled and associated with microRNAs after SNL, which may play a role in modification of microRNA-mediated gene regulation in DRG neurons.

In the fruit fly Drosophila melanogaster, a network of circadian pacemaker neurons drives daily rhythms in rest and activity. The ion channel NARROW ABDOMEN (NA), orthologous to the mammalian sodium leak channel NALCN, functions downstream of the molecular circadian clock in pacemaker neurons to promote behavioral rhythmicity. To better understand the function and regulation of the NA channel, we have characterized two putative auxiliary channel subunits in Drosophila, unc79 (aka dunc79) and unc80 (aka CG18437). We have generated novel unc79 and unc80 mutations that represent strong or complete loss-of-function alleles. These mutants display severe defects in circadian locomotor rhythmicity that are indistinguishable from na mutant phenotypes. Tissue-specific RNA interference and rescue analyses indicate that UNC79 and UNC80 likely function within pacemaker neurons, with similar anatomical requirements to NA. We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels. Yet despite this relationship, we find that the requirement for unc79 and unc80 in circadian rhythmicity cannot be bypassed by increasing NA protein expression, nor can these putative auxiliary subunits substitute for each other. These data indicate functional requirements for UNC79 and UNC80 beyond promoting channel subunit expression. Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain. Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

The purpose of this study was to devise a strategy for the derivation of corneal endothelial cells (CEnCs) from adult fibroblast-derived induced pluripotent stem cells (iPSCs). IPSCs were generated from an adult human with normal ocular history via expression of OCT4, SOX2, KLF4 and c-MYC. Neural crest cells (NCCs) were differentiated from iPSCs via addition of CHIR99021 and SB4315542. NCCs were driven toward a CEnC fate via addition of B27, PDGF-BB and DKK-2 to CEnC media. Differentiation of NCCs and CEnCs was evaluated via rt-PCR, morphological and immunocytochemical analysis. At 17 days post-NCC induction, there were notable changes in cell morphology and upregulation of the neural crest lineage transcripts PAX3, SOX9, TFAP2A, SOX10 and p75NTR and the proteins p75/NGFR and SOX10. Exposure of NCCs to B27, PDGF-BB and DKK-2 induced a shift in morphology from a spindle-shaped neural phenotype to a tightly-packed hexagonal appearance and increased expression of the transcripts ATP1A1, COL8A1, COL8A2, AQP1 and CDH2 and the proteins ZO-1, N-Cad, AQP-1 and Na+/K+ATPase. Replacement of NCC media with CEnC media on day 3, 5 or 8 reduced the differentiation time needed to yield CEnCs. IPSC-derived CEnCs could be used for evaluation of cornea endothelial disease pathophysiology and for testing of novel therapeutics.