Microarray data can tip off researchers to new molecular mechanisms, and two recent studies from the Journal of Neuroscience draw on that information to uncover proteins driving nociception and neuropathic pain. In the June 27 issue, a team led by Z. David Luo at the University of California, Irvine, US, shows that thrombospondin-4 (TSP4), an extracellular matrix glycoprotein expressed in astrocytes in the spinal cord dorsal horn, heightens neuropathic pain in rodents, likely by promoting abnormal synapse formation. The researchers also demonstrate that inhibiting TSP4 in the animals diminishes nerve injury-induced pain. In another paper, published June 20, Mark Hoon and colleagues at the National Institutes of Health, Bethesda, Maryland, US, show that neuromedin B (NMB), a neuropeptide expressed in sensory neurons, acts peripherally in mice to cause local swelling and nociceptive sensitization, and may act centrally to selectively mediate sensation of painful heat in the dorsal horn. The new findings reveal unexplored pain pathways, offer new experimental tools for dissecting spinal pain processing, and provide new potential analgesic targets.
A new source of neuropathic pain
A series of discoveries led Luo and colleagues to connect TSP4 to pain. Previously, coauthor Ben Barres at Stanford University, California, US, and other researchers showed that astrocytes secrete TSP family members that promote synapse formation in the developing brain (Christopherson et al., 2005; Xu et al., 2010) by binding the calcium channel alpha2delta-1 (α2δ-1) subunit, the receptor for the anti-seizure and analgesic drug gabapentin (Eroglu et al., 2009). Luo’s group was studying α2δ-1 in neuropathic pain, and Barres struck up a collaboration. Luo dug through his existing microarray data on dorsal root ganglia (DRG) gene expression in rats with nerve injury-induced pain (Valder et al., 2003), and found that TSP4 was upregulated.
In the new study, co-first authors Doo-Sik Kim and Kang-Wu Li and colleagues investigated the expression and function of TSP4 in the spinal nerve ligation (SNL) model of neuropathic pain in rodents. They found TSP4 present in the dorsal horn, mainly in astrocytes, and documented increased TSP4 expression levels after nerve injury, coincident with development of tactile allodynia, and mechanical and thermal hyperalgesia, on the injury side. Meanwhile, blocking TSP4 with intrathecal antibodies or antisense oligodeoxynucleotides decreased SNL-induced pain. Blocking TSP4 not only reversed the behavioral hypersensitivities, but also had prophylactic value: Preemptive treatment with the antibodies prevented the development of allodynia after SNL, and deletion of the TSP4 gene also protected mice from SNL-induced pain. Finally, the authors found that intrathecal injection of TSP4 into healthy rats was sufficient to cause the behavioral hypersensitivities. Studies of spinal cord slices from those animals revealed a mechanistic underpinning of TSP4-induced pain: The protein boosted presynaptic excitatory input to dorsal horn neurons.
Together, the results build a strong case that TSP4 mediates pain after nerve injury, though the mechanism requires further elucidation. Given the previous evidence that thrombospondins promote synapse formation during brain development, Luo and colleagues theorize that a similar process takes place in the spinal cord, to painful effect. “We think that nerve injury causes reactivation of astrocytes in the spinal cord, which causes overproduction and secretion of thrombospondin-4,” Luo said. In turn, the elevated TSP4 “may cause abnormal excitatory synaptogenesis,” such that “normal stimulation from the periphery will induce exaggerated sensory responses in the spinal cord.” Blocking TSP4 or other molecules involved in that process could be a new strategy for pain therapy, he said.
A new nociceptive neuropeptide
Rather than draw on previous microarray results, Hoon made a fresh survey of molecules potentially involved in pain sensation, using a differential expression strategy. He and his colleagues compared whole-genome expression patterns in mouse trigeminal ganglia with those in the geniculate ganglion, which is involved in processing taste sensation, rather than pain. With that approach, Hoon and coauthors Santosh Mishra and Sarah Holzman identified differential expression of the neuropeptide NMB in the trigeminal ganglia, and in the DRG as well. In the trigeminal ganglia, NMB expression partially overlapped that of two well-known nociceptive markers, calcitonin gene-related peptide (CGRP) and the transient receptor potential channel TRPV1, in a subset of sensory neurons. That expression data, consistent with another recent finding of NMB expression in DRG nociceptive neurons in mice (Fleming et al., 2012), suggested that NMB might play a role in pain.
The investigators found that certainly was the case in peripheral tissues: Injection of NMB into the mouse hind paw triggered local swelling and hypersensitivity to painful heat and mechanical stimuli. Likewise, pretreatment with an NMB receptor antagonist decreased mustard oil-induced swelling and hyperalgesia. The same antagonist, in combination with others for CGRP and another nociceptive neuropeptide, substance P, reduced pain and inflammation even further, which suggests that targeting multiple neuropeptides may provide more pain relief than single-target approaches, Hoon said.
Hoon and colleagues also propose that NMB acts as a central neurotransmitter. They found expression of the NMB receptor in a small subset of interneurons located in the superficial layers of the spinal cord dorsal horn, an area that processes nociceptive inputs. They also discovered that selectively ablating those neurons, by conjugating NMB to the ribosome-inactivating protein saporin, diminished animals’ sensitivity to painful heat but had no effects on painful pressure sensitivity or itching, suggesting a selective role for the neuropeptide.
“We thought it was really notable that [the NMB-responsive neurons] were so specifically tuned,” Hoon said. Alongside previous evidence that neurons expressing the receptor for gastrin releasing peptide (GRP), a peptide closely related to NMB, specifically mediate itch (Sun et al., 2009), the new result “points to the idea that there are perhaps specific circuits in the spinal cord that convey very specific types of signal.” (For more discussion on modality-specific circuits, see PRF discussion and interview.) The nature of those circuits is still mysterious, Hoon said, but neuropeptides like NMB could serve as experimental tools to tease them apart.
Comments
Wenqin Luo, University of Pennsylvania
The work from Mishra and colleagues recently published in The Journal of Neuroscience provides many interesting insights into the in vivo function of Neuromedin B (NMB) signaling in the mammalian somatosensory system. Previous work from Sun and colleagues suggested that gastrin releasing peptide (GRP) and its receptor mediate itch sensation (Sun et al., 2007); thus it is a logical step to investigate if the other mammalian bombesin-related peptide, NMB, has similar or different functions. The authors found that NMB is not expressed in the spinal cord, but is expressed in a subset of nociceptive neurons in the trigeminal ganglion (TG). In addition, they found that the receptor for NMB, NMBR, is expressed in the spinal target of nociceptors, superficial dorsal horn spinal cord neurons, by using a GENSAT transgenic mouse line containing a BAC expressing EGFP from the NMBR locus. Though the loss of GFP+ neurons following ablation with NMB-conjugated toxin supports the idea that the neurons expressing GFP endogenously express NMBR, the claim could be much stronger if the colocalization of GFP and endogenous NMBR, either by immunohistochemistry or in-situ hybridization, could have been demonstrated.
To address the function of NMB signaling in somatosensation, the authors ablated NMBR+ neurons using NMB-saporin injection in mice. The authors performed behavioral tests on those animals and concluded that NMBR+ neurons are important for the detection of thermal pain, but not mechanical pain or itch. However, the current experiments performed on NMB-saporin-treated mice are not sufficient to rule out a role for NMB in itch sensation as the authors have only tested histamine-related itch behavior. It has been shown that histaminergic itch and nonhistaminergic itch are mediated through different receptors and sensory pathways (Patel et al., 2011). As shown in our recent paper, NMB is highly expressed in somatosensory neurons that express the G-protein coupled receptor MrgprA3 (Fleming et al., 2012), which is critical for the detection of chloroquine-induced non-histaminergic itch (Liu et al., 2009). Therefore, a more thorough characterization of NMB-saporin- treated mice exposed to both histaminergic and nonhistaminergic compounds would clarify the question of whether NMB is involved in the transmission of itch information.
Nevertheless, the work from Mishra and colleagues is exciting as it provides clear functional evidence for NMB signaling in the mammalian somatosensory system.
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