Abstract: In Drosophila , pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). Here, we show that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, β3-octopamine receptor (Octβ3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octβ3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulates ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. We propose that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant.

Abstract: Hypersensitivity to mechanical stimulation is a well documented symptom of neuropathic pain, for which there is currently no effective therapy. Src-family kinases (SFKs) are involved in proliferation and differentiation and in neuronal plasticity, including long-term potentiation, learning, and memory. Here we show that activation of SFKs induced in spinal cord microglia is crucial for mechanical hypersensitivity after peripheral nerve injury. Nerve injury induced a striking increase in SFK phosphorylation in the ipsilateral dorsal horn, and SFKs were activated in hyperactive microglia but not in neurons or astrocytes. Intrathecal administration of the Src-family tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) suppressed nerve injury-induced mechanical hypersensitivity but not heat and cold hypersensitivity. Furthermore, PP2 reversed the activation of extracellular signal-regulated protein kinase (ERK), but not p38 mitogen-activated protein kinase, in spinal microglia. In contrast, there was no change in SFK phosphorylation in primary sensory neurons, and PP2 did not decrease the induction of transient receptor potential ion channel TRPV1 and TRPA1 in sensory neurons. Together, these results demonstrate that SFK activation in spinal microglia contributes to the development of mechanical hypersensitivity through the ERK pathway. Therefore, preventing the activation of the Src/ERK signaling cascade in microglia might provide a fruitful strategy for treating neuropathic pain.

Abstract: To investigate whether activation of mitogen-activated protein kinase (MAPK) in damaged and/or undamaged primary afferents participates in neuropathic pain after partial nerve injury, we examined the phosphorylation of extracellular signal-regulated protein kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK) in the L4 and L5 dorsal root ganglion (DRG) in the L5 spinal nerve ligation (SNL) model. We first confirmed, using activating transcription factor 3 and neuropeptide Y immunoreactivity, that virtually all L4 DRG neurons are spared from axotomy in this model. In the injured L5 DRG, the L5 SNL induced the activation of ERK, p38, and JNK in different populations of DRG neurons. In contrast, in the uninjured L4 DRG, the L5 SNL induced only p38 activation in tyrosine kinase A-expressing small- to medium-diameter neurons. Intrathecal ERK, p38, and JNK inhibitor infusions reversed SNL-induced mechanical allodynia, whereas only p38 inhibitor application attenuated SNL-induced thermal hyperalgesia. Furthermore, the L5 dorsal rhizotomy did not prevent SNL-induced thermal hyperalgesia. We therefore hypothesized that p38 activation in the uninjured L4 DRG might be involved in the development of heat hypersensitivity in the L5 SNL model. In fact, the treatment of the p38 inhibitor and also anti-nerve growth factor reduced SNL-induced upregulation of brain-derived neurotrophic factor and transient receptor potential vanilloid type 1 expression in the L4 DRG. Together, our results demonstrate that the L5 SNL induces differential activation of MAPK in injured and uninjured DRG neurons and, furthermore, that MAPK activation in the primary afferents may participate in generating pain hypersensitivity after partial nerve injury.

Abstract: The p75 neurotrophin receptor (p75NTR) has been implicated in diverse neuronal responses, including survival, cell death, myelination, and inhibition of regeneration. However, the role of p75NTR in neuropathic pain, for which there is currently no effective therapy, has not been explored. Here, we report that the pharmacological blockade of p75NTR in primary sensory neurons reversed neuropathic pain after nerve injury. Nerve injury increased the expression and axonal transport of p75NTR and phosphorylation of TrkA in the uninjured primary afferents. Functional inhibition of p75NTR suppressed injury-induced neuropathic pain and decreased the phosphorylation of TrkA and p38 mitogen-activated protein kinase, and the induction of transient receptor potential channels in dorsal root ganglion (DRG) neurons. Our results show that p75NTR induced in undamaged DRG neurons facilitates TrkA signaling and contributes to heat and cold hyperalgesia.

Abstract: In line with our findings, ABC transporter genes have also been linked to toxin resistance in other species. For example, an orthologue of the gene mapped in this study was recently implicated in Cry1Ac resistance in H. virescens ( 4 ) and two other lepidopteran pests ( 5 ); nucleotide deletions in ABC transporter genes were reported in two of these species. It is interesting to note that binding of Cry1Ac to a preparative form of midgut membrane was abolished in homozygous mutants of H. virescens ( 4 ); however, we found no difference in Cry1Ab binding to the midgut membrane between susceptible and resistant silkworms, suggesting differences in the mechanisms underlying the response to the two classes of toxin or in B. mori . An ABC transporter (subfamily C) homologous to the Bombyx gene is known to function in human multidrug resistance, suggesting that the ABC transporter in B. mori might confer resistance via a detoxification mechanism. Detoxification is expected to act like a dominant trait, in that a heterozygote bearing only a single copy of the functioning gene should still be able to inactivate or excrete some toxin. However, in four lepidopteran species, Bt resistance is a recessive trait that requires two nonfunctioning gene copies. A more plausible mechanism for its mode of action is that the ABC transporter acts in the midgut in conjunction with a toxin receptor, such as a cadherin-like protein or aminopeptidase N for binding to the midgut membrane or insertion of the toxin into the cell. Involvement of a single amino acid difference in this gene for Bt resistance will provide tools for critical functional studies of the transporter in the mechanism of Bt action. Primary importance of the mutation of the ABC transporter for Bt resistance can also facilitate field monitoring for Bt resistance level of lepidopteran pests. To verify the role of BGIBMGA007792-93 in the Bt toxin response, we introduced the ABC transporter gene from the susceptible strain Rin into resistant strain w1-pnd, which is routinely used in silkworm transgenesis. Silkworm transformation was performed based on the GAL4-UAS (upstream activation sequence) system in the silkworm, which is routinely used in the fruit fly. The transgenic silkworms are expected to possess endogenous resistant ABC transporter genes in both sister chromosomes, together with transformed susceptible gene(s) from Rin. They (second- and fourth-instar larvae) were highly susceptible to Bt toxin, consistent with the dominant trait of the susceptible gene. We confirmed expression of the transgene and endogenous genes by real-time reverse transcriptase-PCR. Therefore, this study demonstrates that the germline introduction of a functional form of a gene associated with susceptibility to Bt toxin can alter the resistance phenotype of an insect. We then performed map-based cloning of the Bt resistance gene on chromosome 15 by using BC 1 progeny from crosses between C2 females and F 1 males (C2 female × Rin male). We conducted three rounds of chromosome mapping on 44, 32, and 15 larvae selected after Bt toxin exposure, followed by SNP marker screening on more than 1,000 BC 1 progeny. This allowed us to refine further the region harboring the Bt resistance gene to an 82-kb segment of linkage group 15, in which we identified six candidate genes by using KAIKObase ( http://sgp.dna.affrc.go.jp/KAIKObase/ ); two of these were subsequently removed as a result of incorrect assignments in the database. Of the four remaining candidate genes, two genes were not expressed in the midgut. Given that resistance is presumed to involve proteins with active functions in the gut, we focused on the two remaining genes expressed in this tissue, and examined their complete coding sequences in C2 and Rin strains. One of these genes was identical between the two strains, excluding it from further study; however, many nucleotides were different between C2 and Rin strains in the second gene, BGIBMGA007792-93 , which we annotated as an ATP-binding cassette (ABC) family C transporter. Upon examining six additional resistant and nine susceptible strains for the sequence polymorphisms of this gene, we found that only a single, common amino acid (tyrosine) insertion/deletion located in the second outer loop of a predicted 12-transmembrane structure ( Fig. P1 ) was able to distinguish resistant from susceptible strains, including C2 and Rin. Based on the known function of ABC transporter proteins, we determined that BGIBMGA007792-93 was a plausible candidate for Bt resistance. Inbred strains of the domesticated silkworm Bombyx mori , in which this bacterial pathogen was first reported, show various levels of susceptibility to Bt toxin. Taking advantage of recent advances in genome databases and high-density genetic maps for map-based (i.e., positional) cloning ( 3 ), together with transgenic techniques for the study of gene function, we initiated cloning of a silkworm gene conferring resistance to the Bt toxin Cry1Ab. In these studies, we used two strains differing substantially in their response to Cry1Ab, C2 (resistant) and Rin (susceptible); C2 strain required 300-fold higher toxin dose to be killed than Rin strain. First-generation (F 1 ) hybrids were susceptible, indicating that resistance was a recessive trait. We used SNP-based PCR ( 3 ) to determine the linkage group carrying Bt resistance. Backcross (i.e., BC 1 ) progeny from a cross between an F 1 female (C2 female × Rin male) and a C2 male that survived Bt toxin screening were expected to carry homozygous alleles for Bt resistance. Among 28 linkage groups (chromosomes), we observed homozygous genotypes in all tested progeny only at SNP markers on linkage group 15, indicating that the resistance locus was on this chromosome; all other chromosomes exhibited some heterozygotes. We also examined linkage of other genes previously reported to be associated with Bt resistance, including genes encoding cadherin-like protein, aminopeptidase Ns, alkaline phosphatase, and glycosyltransferases; none of these genes were located on chromosome 15, indicating that they represented different forms of resistance. Additionally, we tested another possible resistance factor, enzyme digestion of protoxin (130 kDa) in digestive tract (i.e., midgut), which creates active form of toxin (60 kDa) in insect midgut. We detected no marked difference in digestion between the two strains, indicating that the resistance gene was unlikely to encode a proteolytic enzyme required for the initial step of toxin activation. Toxins produced by Bacillus thuringiensis (Bt) are widely used for controlling insect pests as insecticidal constituents in agricultural chemicals and transgenic crops. The increasing use of Bt insecticides and widespread cultivation of Bt crops have raised concerns for the potential of accelerated development of Bt resistance in field populations ( 1 ). Despite the broad use of Bt toxin and the discovery of molecules involved in Bt resistance in agricultural pests, such as the tobacco budworm Heliothis virescens , the diamondback moth Plutella xylostella , and the pink bollworm Pectinophora gossypiella , its mode of action is not fully understood ( 2 ).

Abstract: Bombyx mori densovirus type 2 (BmDNV-2), a parvo-like virus, replicates only in midgut columnar cells and causes fatal disease. The resistance expressed in some silkworm strains against the virus is determined by a single gene, nsd-2 , which is characterized as nonsusceptibility irrespective of the viral dose. However, the responsible gene has been unknown. We isolated the nsd-2 gene by positional cloning. The virus resistance is caused by a 6-kb deletion in the ORF of a gene encoding a 12-pass transmembrane protein, a member of an amino acid transporter family, and expressed only in midgut. Germ-line transformation with a wild-type transgene expressed in the midgut restores susceptibility, showing that the defective membrane protein is responsible for resistance. Cumulatively, our data show that the membrane protein is a functional receptor for BmDNV-2. This is a previously undescribed report of positional cloning of a mutant gene in Bombyx and isolation of an absolute virus resistance gene in insects.

Abstract: The capsaicin receptor transient receptor potential V1 (TRPV1; also known as vanilloid receptor 1) is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been reported that extracellular ATP potentiates the TRPV1 currents evoked by capsaicin or protons and reduces the temperature threshold for its activation through metabotropic P2Y receptors in a PKC-dependent pathway, suggesting that TRPV1 activation could trigger the sensation of pain at normal body temperature in the presence of ATP. Here, we show that ATP-induced thermal hyperalgesia was abolished in mice lacking TRPV1, suggesting the functional interaction between ATP and TRPV1 at a behavioral level. However, thermal hyperalgesia was preserved in P2Y 1 receptor-deficient mice. Patch-clamp analyses using mouse dorsal root ganglion neurons indicated the involvement of P2Y 2 rather than P2Y 1 receptors. Coexpression of TRPV1 mRNA with P2Y 2 mRNA, but not P2Y 1 mRNA, was determined in the rat lumbar DRG using in situ hybridization histochemistry. These data indicate the importance of metabotropic P2Y 2 receptors in nociception through TRPV1.

Abstract: Proteinase-activated receptor (PAR) 2 is expressed on a subset of primary afferent neurons and involved in inflammatory nociception. Transient receptor potential vanilloid subfamily 1 (TRPV1) is a sensory neuron-specific cation channel that responds to capsaicin, protons, or heat stimulus. Here, we show that TRPV1 is coexpressed with PAR2 but not with PAR1 or PAR3, and that TRPV1 can functionally interact with PAR2. In human embryonic kidney 293 cells expressing TRPV1 and PAR2, PAR2 agonists increased capsaicin- or proton-evoked TRPV1 currents through a PKC-dependent pathway. After application of PAR2 agonists, temperature threshold for TRPV1 activation was reduced from 42°C to well below the body temperature. PAR2-mediated Fos expression in spinal cord was decreased in TRPV1-deficient mice. The functional interaction was also observed in mouse DRG neurons and proved at a behavioral level. These represent a novel mechanism through which trypsin or tryptase released in response to tissue inflammation might trigger the sensation of pain by PAR2 activation.

Abstract: Microglia in the spinal cord may play an important role in the development and maintenance of neuropathic pain. A metabotropic ATP receptor, P2Y 12 , has been shown to be expressed in spinal microglia constitutively and be involved in chemotaxis. Activation of p38 mitogen-activated protein kinase (MAPK) occurs in spinal microglia after nerve injury and may be related to the production of cytokines and other mediators, resulting in neuropathic pain. However, it remains unknown whether any type of P2Y receptor in microglia is involved in the activation of p38 MAPK and the pain behaviors after nerve injury. Using the partial sciatic nerve ligation (PSNL) model in the rat, we found that P2Y 12 mRNA and protein increased in the spinal cord and peaked at 3 d after PSNL. Double labeling studies revealed that cells expressing increased P2Y 12 mRNA and protein after nerve injury were exclusively microglia. Both pharmacological blockades by intrathecal administration of P2Y 12 antagonist and antisense knockdown of P2Y 12 expression suppressed the development of pain behaviors and the phosphorylation of p38 MAPK in spinal microglia after PSNL. The intrathecal infusion of the P2Y 12 agonist 2-(methythio) adenosine 5′-diphosphate trisodium salt into naive rats mimicked the nerve injury-induced activation of p38 in microglia and elevated pain behaviors. These data suggest a new mechanism of neuropathic pain, in which the increased P2Y 12 works as a gateway of the following events in microglia after nerve injury. Activation of this receptor by released ATP or the hydrolyzed products activate p38 MAPK pathway and may play a crucial role in the generation of neuropathic pain.