Conditional deletion of Pip5k1c in sensory ganglia and effects on nociception and inflammatory sensitization

Phosphatidylinositol 4-phosphate 5-kinase type 1 gamma (Pip5k1c) generates phosphatidylinositol 4,5-bisphosphate, also known as PI(4,5)P2 or PIP2. Many pronociceptive signaling pathways and receptor tyrosine kinases signal via PIP2 hydrolysis. Previously, we found that pain signaling and pain sensitization were reduced in Pip5k1c+/− global heterozygous knockout mice. Here, we sought to evaluate the extent to which dorsal root ganglia selective deletion of Pip5k1c affected nociception in mice. Initially, we crossed sensory neuron-selective Advillin-Cre mice with a conditional Pip5k1c knockout (cKO) allele (Pip5k1cfl/fl). However, these mice displayed an early onset proprioceptive deficit. To bypass this early onset phenotype, we used two different tamoxifen-inducible Cre lines (Brn3a-Cre-ERT2 and Advillin-Cre-ERT2) to conditionally delete Pip5k1c in adults. Tamoxifen induced high efficiency deletion of PIP5K1C in dorsal root ganglia and slightly reduced PIP5K1C in spinal cord and brain in Brn3a-Cre-ERT2 × Pip5k1cfl/fl (Brn3a cKO) mice while PIP5K1C was selectively deleted in dorsal root ganglia with no changes in spinal cord or brain in Advillin-Cre-ERT2 × Pip5k1cfl/fl (Advil cKO) mice. Acute thermosensation and mechanosensation were not altered in either line relative to wild-type mice. However, thermal hypersensitivity and mechanical allodynia recovered more rapidly in Brn3a cKO mice, but not Advil cKO mice, following hind paw inflammation. These data collectively suggest that PIP5K1C regulates nociceptive sensitization in more regions of the nervous system than dorsal root ganglia alone

Prostatic Acid Phosphatase Is Expressed in Peptidergic and Nonpeptidergic Nociceptive Neurons of Mice and Rats

Thiamine monophosphatase (TMPase, also known as Fluoride-resistant acid phosphatase or FRAP) is a classic histochemical marker of small- to medium-diameter dorsal root ganglia (DRG) neurons and has primarily been studied in the rat. Previously, we found that TMPase was molecularly identical to Prostatic acid phosphatase (PAP) using mice. In addition, PAP was expressed in a majority of nonpeptidergic, isolectin B4-binding (IB4+) nociceptive neurons and a subset of peptidergic, calcitonin gene-related peptide-containing (CGRP+) nociceptive neurons. At the time, we were unable to determine if PAP was present in rat DRG neurons because the antibody we used did not cross-react with PAP in rat tissues. In our present study, we generated a chicken polyclonal antibody against the secretory isoform of mouse PAP. This antibody detects mouse, rat and human PAP protein on western blots. Additionally, this antibody detects PAP in mouse and rat small- to medium-diameter DRG neurons and axon terminals in lamina II of spinal cord. In the rat, 92.5% of all PAP+ cells bind the nonpeptidergic marker IB4 and 31.8% of all PAP+ cells contain the peptidergic marker CGRP. Although PAP is found in peptidergic and nonpeptidergic neurons of mice and rats, the percentage of PAP+ neurons that express these markers differs between species. Moreover, PAP+ axon terminals in the rat partially overlap with Protein kinase Cγ (PKCγ+) interneurons in dorsal spinal cord whereas PAP+ axon terminals in the mouse terminate dorsal to PKCγ+ interneurons. Collectively, our studies highlight similarities and differences in PAP localization within nociceptive neurons of mice and rats

Pain-relieving prospects for adenosine receptors and ectonucleotidases

Adenosine receptor agonists have potent antinociceptive effects in diverse preclinical models of chronic pain. In contrast, the efficacy of adenosine or adenosine receptor agonists at treating pain in humans is unclear. Two ectonucleotidases that generate adenosine in nociceptive neurons were recently identified. When injected spinally, these enzymes have long-lasting adenosine A1 receptor (A1R)-dependent antinociceptive effects in inflammatory and neuropathic pain models. Furthermore, recent findings indicate that spinal adenosine A2A receptor activation can enduringly inhibit neuropathic pain symptoms. Collectively, these studies suggest the possibility of treating chronic pain in humans by targeting specific adenosine receptor subtypes in anatomically defined regions with agonists or with ectonucleotidases that generate adenosine

Topographically Distinct Epidermal Nociceptive Circuits Revealed by Axonal Tracers Targeted to Mrgprd

The brain receives sensory input from diverse peripheral tissues, including the skin, the body's largest sensory organ. Using genetically encoded axonal tracers expressed from the Mrgprd locus, we identify a subpopulation of nonpeptidergic, nociceptive neurons that project exclusively to the skin, and to no other peripheral tissue examined. Surprisingly, Mrgprd+ innervation is restricted to the epidermis and absent from specialized sensory structures. Furthermore, Mrgprd+ fibers terminate in a specific layer of the epidermis, the stratum granulosum. This termination zone is distinct from that innervated by most CGRP+ neurons, revealing that peptidergic and nonpeptidergic epidermal innervation is spatially segregated. The central projections deriving from these distinct epidermal innervation zones terminate in adjacent laminae in the dorsal spinal cord. Thus, afferent input from different layers of the epidermis is conveyed by topographically segregated sensory circuits, suggesting that at least some aspects of sensory information processing may be organized along labeled lines

Orphan G protein-coupled receptors MrgA1 and MrgC11 are distinctively activated by RF-amide-related peptides through the G{alpha}q/11 pathway

MrgA1 and MrgC11 belong to a recently identified family of orphan G-protein coupled receptors, called mrgs (mas-related genes). They are only expressed in a specific subset of sensory neurons that are known to detect painful stimuli. However, the precise physiological function of Mrg receptors and their underlying mechanisms of signal transduction are not known. We therefore have screened a series of neuropeptides against human embryonic kidney (HEK) 293 cells that stably express either MrgA1 or MrgC11 to identify ligands and/or agonists. MrgA1- or MrgC11-specific agonists stimulated dose-dependent increases in intracellular free Ca2+ in a pertussis toxin-insensitive manner, but failed to alter basal or forskolin-stimulated levels of intracellular cAMP. Furthermore, studies using embryonic fibroblasts derived from various G{alpha} protein knockout mice demonstrated that both the MrgA1 and MrgC11 receptors are coupled to the G{alpha}q/11 signaling pathway. Screening of neuropeptides identified surrogate agonists, most of these peptides included a common C-terminal -RF(Y)G or -RF(Y) amide motif. Structure-function studies suggest that endogenous ligands of Mrg receptors are likely to be RF(Y)G and/or RF(Y) amide-related peptides and that postprocessing of these peptides may serve to determine Mrg receptor-ligand specificity. The differences in ligand specificity also suggest functional diversity amongst the Mrg receptors

Recombinant ecto-5'-nucleotidase (CD73) has long lasting antinociceptive effects that are dependent on adenosine A1 receptor activation

<p>Abstract</p> <p>Background</p> <p>Ecto-5'-nucleotidase (NT5E, also known as CD73) hydrolyzes extracellular adenosine 5'-monophosphate (AMP) to adenosine in nociceptive circuits. Since adenosine has antinociceptive effects in rodents and humans, we hypothesized that NT5E, an enzyme that generates adenosine, might also have antinociceptive effects <it>in vivo</it>.</p> <p>Results</p> <p>To test this hypothesis, we purified a soluble version of mouse NT5E (mNT5E) using the baculovirus expression system. Recombinant mNT5E hydrolyzed AMP in biochemical assays and was inhibited by α,β-methylene-adenosine 5'-diphosphate (α,β-me-ADP; IC₅₀= 0.43 μM), a selective inhibitor of NT5E. mNT5E exhibited a dose-dependent thermal antinociceptive effect that lasted for two days when injected intrathecally in wild-type mice. In addition, mNT5E had thermal antihyperalgesic and mechanical antiallodynic effects that lasted for two days in the complete Freund's adjuvant (CFA) model of inflammatory pain and the spared nerve injury (SNI) model of neuropathic pain. In contrast, mNT5E had no antinociceptive effects when injected intrathecally into adenosine A₁receptor (<it>A</it>₁<it>R, Adora1</it>) knockout mice.</p> <p>Conclusion</p> <p>Our data indicate that the long lasting antinociceptive effects of mNT5E are due to hydrolysis of AMP followed by activation of A₁R. Moreover, our data suggest recombinant NT5E could be used to treat chronic pain and to study many other physiological processes that are regulated by NT5E.</p

A High Throughput Assay to Identify Small Molecule Modulators of Prostatic Acid Phosphatase

Prostatic acid phosphatase (PAP) is expressed in nociceptive neurons and functions as an ectonucleotidase. Injection of the secretory isoform of PAP has potent antinociceptive effects in mouse models of chronic pain. These data suggested that a small molecule activator of PAP may have utility as a novel therapeutic for chronic pain, while inhibitors could be used to acutely inhibit PAP in vitro and in vivo. To identify small molecule modulators of PAP activity, we validated a high throughput, fluorescence-based biochemical assay and then used this assay to screen a compound library. We decreased the frequency of false positive activators by subtracting compound fluorescence from the final assay fluorescence. This approach significantly reduced the number of false positive activators found in the screen. While no activators were confirmed, seven novel inhibitors of PAP were identified. Our results suggest this high throughput assay could be used to identify small molecule modulators of PAP activity

Secretion and N-Linked Glycosylation Are Required for Prostatic Acid Phosphatase Catalytic and Antinociceptive Activity

Secretory human prostatic acid phosphatase (hPAP) is glycosylated at three asparagine residues (N62, N188, N301) and has potent antinociceptive effects when administered to mice. Currently, it is unknown if these N-linked residues are required for hPAP protein stability and activity in vitro or in animal models of chronic pain. Here, we expressed wild-type hPAP and a series of Asn to Gln point mutations in the yeast Pichia pastoris X33 then analyzed protein levels and enzyme activity in cell lysates and in conditioned media. Pichia secreted wild-type recombinant (r)-hPAP into the media (6–7 mg protein/L). This protein was as active as native hPAP in biochemical assays and in mouse models of inflammatory pain and neuropathic pain. In contrast, the N62Q and N188Q single mutants and the N62Q, N188Q double mutant were expressed at lower levels and were less active than wild-type r-hPAP. The purified N62Q, N188Q double mutant protein was also 1.9 fold less active in vivo. The N301Q mutant was not expressed, suggesting a critical role for this residue in protein stability. To explicitly test the importance of secretion, a construct lacking the signal peptide of hPAP was expressed in Pichia and assayed. This “cellular” construct was not expressed at levels detectable by western blotting. Taken together, these data indicate that secretion and post-translational carbohydrate modifications are required for PAP protein stability and catalytic activity. Moreover, our findings indicate that recombinant hPAP can be produced in Pichia—a yeast strain that is used to generate biologics for therapeutic purposes

PAPupuncture has localized and long-lasting antinociceptive effects in mouse models of acute and chronic pain

AbstractAcupuncture has been used for millennia to treat pain, although its efficacy and duration of action is limited. Acupuncture also has brief (1–2 h) antinociceptive effects in mice and these effects are dependent on localized adenosine A1 receptor (A1R) activation. Intriguingly, adenosine 5’-monophosphate (AMP) is basally elevated near acupuncture points. This finding suggested that it might be possible to inhibit nociception for a longer period of time by injecting prostatic acid phosphatase (PAP, ACPP) into acupuncture points. PAP is an ectonucleotidase that dephosphorylates extracellular AMP to adenosine, has a long half-life in vivo and is endogenously found in muscle tissue surrounding acupuncture points. Here, we found that injection of PAP into the popliteal fossa—a space behind the knee that encompasses the Weizhong acupuncture point—had dose- and A1R-dependent antinociceptive effects in mouse models of acute and chronic pain. These inhibitory effects lasted up to six days following a single injection, much longer than the hour-long inhibition provided by acupuncture. Antinociception could be transiently boosted with additional substrate (AMP) or transiently blocked with an A1R antagonist or an inhibitor of phospholipase C. This novel therapeutic approach—which we term “PAPupuncture”—locally inhibits pain for an extended period of time (100x acupuncture), exploits a molecular mechanism that is common to acupuncture, yet does not require acupuncture needle stimulation

Mrgprd Enhances Excitability in Specific Populations of Cutaneous Murine Polymodal Nociceptors

The Mas-related G protein-coupled receptor D (Mrgprd) is selectively expressed in nonpeptidergic nociceptors that innervate the outer layers of mammalian skin. The function of Mrgprd in nociceptive neurons and the physiologically relevant somatosensory stimuli that activate Mrgprd^-expressing (Mrgprd^+) neurons are currently unknown. To address these issues, we studied three Mrgprd knock-in mouse lines using an ex vivo somatosensory preparation to examine the role of the Mrgprd receptor and Mrgprd+ afferents in cutaneous somatosensation. In mouse hairy skin, Mrgprd, as marked by expression of green fluorescent protein reporters, was expressed predominantly in the population of nonpeptidergic, TRPV1-negative, C-polymodal nociceptors. In mice lacking Mrgprd, this population of nociceptors exhibited decreased sensitivity to cold, heat, and mechanical stimuli. Additionally, in vitro patch-clamp studies were performed on cultured dorsal root ganglion neurons from Mrgprd^(–/–) and Mrgprd^(+/–) mice. These studies revealed a higher rheobase in neurons from Mrgprd^(–/–) mice than from Mrgprd^(+/–) mice. Furthermore, the application of the Mrgprd ligand β-alanine significantly reduced the rheobase and increased the firing rate in neurons from Mrgprd^(+/–) mice but was without effect in neurons from Mrgprd^(–/–) mice. Our results demonstrate that Mrgprd influences the excitability of polymodal nonpeptidergic nociceptors to mechanical and thermal stimuli