Corina Ehnert

GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence.

We report that GTP cyclohydrolase (GCH1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, is a key modulator of peripheral neuropathic and inflammatory pain. BH4 is an essential cofactor for catecholamine, serotonin and nitric oxide production. After axonal injury, concentrations of BH4 rose in primary sensory neurons, owing to upregulation of GCH1. After peripheral inflammation, BH4 also increased in dorsal root ganglia (DRGs), owing to enhanced GCH1 enzyme activity. Inhibiting this de novo BH4 synthesis in rats attenuated neuropathic and inflammatory pain and prevented nerve injury–evoked excess nitric oxide production in the DRG, whereas administering BH4 intrathecally exacerbated pain. In humans, a haplotype of the GCH1 gene (population frequency 15.4%) was significantly associated with less pain following diskectomy for persistent radicular low back pain. Healthy individuals homozygous for this haplotype exhibited reduced experimental pain sensitivity, and forskolin-stimulated immortalized leukocytes from haplotype carriers upregulated GCH1 less than did controls. BH4 is therefore an intrinsic regulator of pain sensitivity and chronicity, and the GTP cyclohydrolase haplotype is a marker for these traits.

Bradykinin and peripheral sensitization

Abstract Pain hypersensitivity after tissue injury and inflammation is contributed to by a reduction in the threshold and an increase in the responsiveness of the peripheral terminals of high-threshold nociceptor neurons, the phenomenon of peripheral sensitization. Bradykinin, acting via G-protein-coupled receptors expressed by the sensory neurons, links to multiple intracellular signaling pathways that in turn interact with voltage-gated and ligand-gated ion channels, changing their properties in such a way as to enhance the response to peripheral stimuli.

The calpain inhibitor MDL 28170 prevents inflammation-induced neurofilament light chain breakdown in the spinal cord and reduces thermal hyperalgesia

&NA; Since long‐term hyperexcitability of nociceptive neurons in the spinal cord has been suggested to be caused and maintained by changes of protein expression we assessed protein patterns in lumbar spinal cord during a zymosan induced paw inflammation employing two‐dimensional (2D) gel electrophoresis. 2D PAGE revealed a time‐dependent breakdown of scaffolding proteins one of which was neurofilament light chain (NFL) protein, which has been previously found to be important for axonal architecture and transport. Nociception induced breakdown of NFL in the spinal cord and dorsal root ganglias was prevented by pretreatment of the animals with a single dose of the specific inhibitor of the protease calpain (MDL‐28170) which has been shown to be the primary protease involved in neurofilament degradation in neurodegenerative diseases. Treatment with the calpain inhibitor also provided anti‐inflammatory and anti‐hyperalgesic effects in the zymosan‐induced paw inflammation model irrespective of whether the drug was administered systemically (i.p.) or delivered onto the lumbar spinal cord. This suggests that the activation of calpain is involved in the sensitization of nociceptive neurons what is partly due to neurofilament breakdown but cleavage of other calpain substrates may also be involved. Our results indicate that inhibition of pathological calpain activity may present an interesting novel drug target in the treatment of pain and inflammation.

Protein associated with Myc (PAM) is involved in spinal nociceptive processing

PAM (protein associated with Myc) is a potent inhibitor of adenylyl cyclases (ACs) which is primarily expressed in neurones. Here we describe that PAM is highly expressed in dorsal horn neurones and motoneuron of the spinal cord, as well as in neurones of dorsal root ganglia in adult rats. PAM mRNA expression is differentially regulated during development in both spinal cord and dorsal root ganglia of rats, being strongest during the major respective synaptogenic periods. In adult rats, PAM expression was up‐regulated in the spinal cord after peripheral nociceptive stimulation using zymosan and formalin injection, suggesting a role for PAM in spinal nociceptive processing. Since PAM inhibited Gαs‐stimulated AC activity in dorsal root ganglia as well as spinal cord lysates, we hypothesized that PAM may reduce spinal nociceptive processing by inhibition of cAMP‐dependent signalling. Accordingly, intrathecal treatment with antisense but not sense oligonucleotides against PAM increased basal and Gαs‐stimulated AC activity in the spinal cord and enhanced formalin‐induced nociceptive behaviour in adult rats. Taken together our findings demonstrate that PAM is involved in spinal nociceptive processing.

Essential role of the synaptic vesicle protein synapsin II in formalin-induced hyperalgesia and glutamate release in the spinal cord.

The synaptic vesicle protein synapsin II plays an important role in the regulation of neurotransmitter release and synaptic plasticity. Here, we investigated its involvement in the synaptic transmission of nociceptive signals in the spinal cord and the development of pain hypersensitivity. We show that synapsin II is predominantly expressed in terminals and neuronal fibers in superficial laminae of the dorsal horn (laminae I–II). Formalin injection into a mouse hindpaw normally causes an immediate and strong release of glutamate in the dorsal horn. In synapsin II deficient mice this glutamate release is almost completely missing. This is associated with reduced nociceptive behavior in the formalin test and in the zymosan‐induced paw inflammation model. In addition, the formalin evoked increase in the number of c‐Fos IR neurons is significantly reduced in synapsin II knockout mice. Touch perception and motor coordination, however, are normal indicating that synapsin II deficiency does not generally disrupt sensory and/or motor functions. Antisense‐mediated transient knockdown of synapsin II in the spinal cord of adult animals also reduced the nociceptive behavior. As the antisense effect is independent of a potential role of synapsin II during development we suggest that the hypoalgesia in synapsin II deficient mice does involve a direct ‘pain‐facilitating’ effect of synapsin II and is not essentially dependent on potentially occurring developmental alterations. The distinctive role of synapsin II for pain signaling probably results from its specific localization and possibly from a specific control of glutamate release.

Nerve injury evoked loss of latexin expression in spinal cord neurons contributes to the development of neuropathic pain

Nerve injury leads to sensitization mechanisms in the peripheral and central nervous system which involve transcriptional and post-transcriptional modifications in sensory nerves. To assess protein regulations in the spinal cord after injury of the sciatic nerve in the Spared Nerve Injury model (SNI) we performed a proteomic analysis using 2D-difference gel electrophoresis (DIGE) technology. Among approximately 2300 protein spots separated on each gel we detected 55 significantly regulated proteins after SNI whereof 41 were successfully identified by MALDI-TOF MS. Out of the proteins which were regulated in the DIGE analyses after SNI we focused on the carboxypeptidase A inhibitor latexin because protease dysfunctions contribute to the development of neuropathic pain. Latexin protein expression was reduced after SNI which could be confirmed by Western Blot analysis, quantitative RT-PCR and in-situ hybridisation. The decrease of latexin was associated with an increase of the activity of carboxypeptidase A indicating that the balance between latexin and carboxypeptidase A was impaired in the spinal cord after peripheral nerve injury due to a loss of latexin expression in spinal cord neurons. This may contribute to the development of cold allodynia because normalization of neuronal latexin expression in the spinal cord by AAV-mediated latexin transduction or administration of a small molecule carboxypeptidase A inhibitor significantly reduced acetone-evoked nociceptive behavior after SNI. Our results show the usefulness of proteomics as a screening tool to identify novel mechanisms of nerve injury evoked hypernociception and suggest that carboxypeptidase A inhibition might be useful to reduce cold allodynia.

The impact of CREB and its phosphorylation at Ser142 on inflammatory nociception.

Peripheral noxious stimulation leads to phosphorylation and thereby activation of the transcription factor CREB in the spinal cord. CREB phosphorylation occurs mainly at serine 133, but the phosphorylation site at serine 142 may also be important. We investigated the impact of spinal CREB protein levels and phosphorylation at Ser142 on the nociceptive behaviour in rat and mouse models of inflammatory nociception. Downregulation of total CREB protein in the rat spinal cord by antisense-oligonucleotides resulted in antinociceptive effects. After peripheral noxious stimulation CREB was phosphorylated in the spinal cord at serine 133 and 142 indicating a potential role of both residues in nociceptive processing. However, Ser142 mutant mice developed equal behavioural correlates of hyperalgesia as wild-type mice in different inflammatory models. Thus, our data confirm that CREB is essential for spinal nociceptive processing. However, prevention of phosphorylation only at serine 142 is not sufficient to modulate the nociceptive response.