SUMMARYAlthough microglia are implicated in nerve injury-induced neuropathic pain, how injured sensory neurons engage microglia is unclear. Here we demonstrate that peripheral nerve injury induces de novo expression of colony-stimulating factor 1 (CSF1) in injured sensory neurons. The CSF1 is transported to the spinal cord where it targets the microglial CSF1 receptor (CSF1R). Cre-mediated sensory neuron deletion of Csf1 completely prevented nerve injury-induced mechanical hypersensitivity and reduced microglia activation and proliferation. In contrast, intrathecal injection of CSF1 induces mechanical hypersensitivity and microglial proliferation. Nerve injury also upregulated CSF1 in motoneurons, where it is required for ventral horn microglial activation and proliferation. Downstream of CSF1R, we found that the microglial membrane adapter protein DAP12 is required for both nerve injury- and intrathecal CSF1-induced upregulation of pain-related microglial genes and the ensuing pain, but not for microglia proliferation. Thus, both CSF1 and DAP12 are potential targets for the pharmacotherapy of neuropathic pain.

BackgroundTRPV4 and the cellular cytoskeleton have each been reported to influence cellular mechanosensitive processes as well as the development of mechanical hyperalgesia. If and how TRPV4 interacts with the microtubule and actin cytoskeleton at a molecular and functional level is not known.Methodology and Principal FindingsWe investigated the interaction of TRPV4 with cytoskeletal components biochemically, cell biologically by observing morphological changes of DRG-neurons and DRG-neuron-derived F-11 cells, as well as functionally with calcium imaging. We find that TRPV4 physically interacts with tubulin, actin and neurofilament proteins as well as the nociceptive molecules PKCε and CamKII. The C-terminus of TRPV4 is sufficient for the direct interaction with tubulin and actin, both with their soluble and their polymeric forms. Actin and tubulin compete for binding. The interaction with TRPV4 stabilizes microtubules even under depolymerizing conditions in vitro. Accordingly, in cellular systems TRPV4 colocalizes with actin and microtubules enriched structures at submembranous regions. Both expression and activation of TRPV4 induces striking morphological changes affecting lamellipodial, filopodial, growth cone, and neurite structures in non-neuronal cells, in DRG-neuron derived F11 cells, and also in IB4-positive DRG neurons. The functional interaction of TRPV4 and the cytoskeleton is mutual as Taxol, a microtubule stabilizer, reduces the Ca2+-influx via TRPV4.Conclusions and SignificanceTRPV4 acts as a regulator for both, the microtubule and the actin. In turn, we describe that microtubule dynamics are an important regulator of TRPV4 activity. TRPV4 forms a supra-molecular complex containing cytoskeletal proteins and regulatory kinases. Thereby it can integrate signaling of various intracellular second messengers and signaling cascades, as well as cytoskeletal dynamics. This study points out the existence of cross-talks between non-selective cation channels and cytoskeleton at multiple levels. These cross talks may help us to understand the molecular basis of the Taxol-induced neuropathic pain development commonly observed in cancer patients.

We evaluated the signalling pathway by which estrogen acts in peripheral tissue to produce protein kinase Cepsilon (PKCepsilon)-dependent mechanical hyperalgesia. Specific agonists for the classical estrogen receptors (ER), ERalpha and ERbeta, did not result in activation of PKCepsilon in neurons of dissociated rat dorsal root ganglia. In contrast, G-1, a specific agonist of the recently identified G-protein-coupled estrogen receptor, GPR30, induced PKCepsilon translocation. Involvement of GPR30 and independence of ERalpha and ERbeta was confirmed using the GPR30 agonist and simultaneous ERalpha and ERbeta antagonist ICI 182,780 (fulvestrant). The GPR30 transcript could be amplified from dorsal root ganglia tissue. We found estrogen-induced as well as GPR30-agonist-induced PKCepsilon translocation to be restricted to the subgroup of nociceptive neurons positive for isolectin IB4 from Bandeiraea simplicifolia. Corroborating the cellular results, both GPR30 agonists, G-1 as well as ICI 182,780, resulted in the onset of PKCepsilon-dependent mechanical hyperalgesia if injected into paws of adult rats. We therefore suggest that estrogen acts acutely at GPR30 in nociceptors to produce mechanical hyperalgesia.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

Protein kinase C epsilon (PKCepsilon) is an important intracellular signaling molecule in primary afferent nociceptors, implicated in acute and chronic inflammatory as well as neuropathic pain. In behavioral experiments inflammatory mediators produce PKCepsilon-dependent hyperalgesia only in male rats. The mechanism underlying this sexual dimorphism is unknown. We show that the hormone environment of female rats changes the nociceptive signaling in the peripheral sensory neuron. This change is maintained in culture also in the absence of a gender-simulating environment. Stimulation of beta(2)-adrenergic receptors (beta(2)-AR) leads to PKCepsilon activation in cultured dorsal root ganglia (DRG) neurons derived from male but not from female rats. Addition of estrogen to male DRG neurons produces a switch to the female phenotype, namely abrogation of beta(2)-AR-initiated activation of PKCepsilon. Estrogen interferes downstream of the beta(2)-AR with the signaling pathway leading from exchange protein activated by cAMP (Epac) to PKCepsilon. The interfering action is fast indicating a transcriptional-independent mechanism. Estrogen has a dual effect on PKCepsilon. If applied before beta(2)-AR or Epac stimulation, estrogen abrogates the activation of PKCepsilon. In contrast, estrogen applied alone leads to a brief translocation of PKCepsilon. Also in vivo the activity of estrogen depends on the stimulation context. In male rats, intradermal injection of an Epac activator or estrogen alone induces mechanical hyperalgesia through a PKCepsilon-dependent mechanism. In contrast, injection of estrogen preceding the activation of Epac completely abrogates the Epac-induced mechanical hyperalgesia. Our results suggest that gender differences in nociception do not reflect the use of generally different mechanisms. Instead, a common set of signaling pathways can be modulated by hormones.

Recently, we described estrogen and agonists of the Gprotein coupled estrogen receptor GPR30 to induce protein kinase C (PKC)e-dependent pain sensitization. PKCe phosphorylates the ion channel transient receptor potential, vanilloid subclass I (TRPV1) close to a novel microtubule-TRPV1 binding site. We now modeled the binding of tubulin to the TRPV1 C-terminus. The model suggests PKCe phosphorylation of TRPV1-S800 to abolish the tubulin-TRPV1 interaction. Indeed, in vitro PKCe phosphorylation of TRPV1 hindered tubulin-binding to TRPV1. In vivo, treatment of sensory neurons and F-11 cells with estrogen and the GPR30 agonist, G-1, resulted in microtubule destabilization and retraction of microtubules from filopodial structures. We found estrogen and G-1 to regulate the stability of the microtubular network via PKC phosphorylation of the PKCephosphorylation site TRPV1-S800. Microtubule disassembly was not, however, dependent on TRPV1 ion conductivity. TRPV1 knock-down in rats inverted the effect of the microtubule-modulating drugs, Taxol and Nocodazole, on estrogen-induced and PKCe-dependent mechanical pain sensitization. Thus, we suggest the C-terminus of TRPV1 to be a signaling intermediate downstream of estrogen and PKCe, regulating microtubule-stability and microtubuledependent pain sensitization.

PKC-epsilon is an important intracellular signaling molecule in primary afferent nociceptors, implicated in acute and chronic inflammatory as well as neuropathic pain. In behavioral experiments the inflammatory mediator epinephrine produces PKC-epsilon-dependent hyperalgesia only in male rats. The mechanism underlying this sexual dimorphism is unknown. We show that the hormone environment of female rats changes the nociceptive signaling in the peripheral sensory neuron. This change is maintained in culture also in the absence of a gender-simulating environment. Addition of estrogen to malederived DRG neurons produces a switch to the female phenotype, namely abrogation of beta 2-AR-initiated activation of PKC-epsilon. Estrogen interferes downstream of the beta 2-AR with the signaling pathway leading from Epac to PKC-epsilon. The interfering action is fast indicating a transcription-independent mechanism.As in other systems, estrogen has a dual effect. If applied minutes before beta 2-AR or Epac stimulation, estrogen abrogates the activation of PKC-epsilon. In contrast, estrogen applied alone leads to a brief translocation of PKCepsilon. Also in vivo the activity of estrogen depends on the stimulation context. Intradermal injection of an Epac activator as well as estrogen alone induces mechanical hyperalgesia through a PKC-epsilon-dependent mechanism. In contrast, injection of estrogen preceeding the activation of Epac completely abrogates the Epac-induced mechanical hyperalgesia.Our results indicate that gender differences in nociception do not reflect the use of generally different mechanisms.Instead, the contribution of a common set of signaling pathways can be modulated by hormones.

Spinal cord transplants of embryonic cortical GABAergic progenitor cells derived from the medial ganglionic eminence (MGE) can reverse mechanical hypersensitivity in the mouse models of peripheral nerve injury-and paclitaxel-induced neuropathic pain. Here, we used electrophysiology, immunohistochemistry, and electron microscopy to examine the extent to which MGE cells integrate into host circuitry and recapitulate endogenous inhibitory circuits. Whether the transplants were performed before or after nerve injury, the MGE cells developed into mature neurons and exhibited firing patterns characteristic of subpopulations of cortical and spinal cord inhibitory interneurons. Conversely, the transplanted cells preserved cortical morphological and neurochemical properties. We also observed a robust anatomical and functional synaptic integration of the transplanted cells into host circuitry in both injured and uninjured animals. The MGE cells were activated by primary afferents, including TRPV1-expressing nociceptors, and formed GABAergic, bicucullinesensitive, synapses onto host neurons. Unexpectedly, MGE cells transplanted before injury prevented the development of mechanical hypersensitivity. Together, our findings provide direct confirmation of an extensive, functional synaptic integration of MGE cells into host spinal cord circuits. This integration underlies normalization of the dorsal horn inhibitory tone after injury and may be responsible for the prophylactic effect of preinjury transplants.