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. 2002 Feb 15;22(4):1363-72.
doi: 10.1523/JNEUROSCI.22-04-01363.2002.

A role of the ubiquitin-proteasome system in neuropathic pain

Andrew Moss  1 , Gordon Blackburn-Munro, Emer M Garry, James A Blakemore, Tracey Dickinson, Roberta Rosie, Rory Mitchell, Susan M Fleetwood-Walker
Affiliations

A role of the ubiquitin-proteasome system in neuropathic pain

Andrew Moss et al. J Neurosci. .

Abstract

Neuropathic pain (characterized by hyperalgesia and allodynia to mechanical and thermal stimuli) causes cellular changes in spinal dorsal horn neurons, some of which parallel those in synaptic plasticity associated with learning. Ubiquitin C-terminal hydrolase (UCH) appears to play a key role in long-term facilitation in Aplysia. The cooperation of UCH with the proteolytic enzyme complex known as the proteasome is required for the degradation of a number of signaling molecules within the cell that may remove normal restraints on synaptic plasticity. We have used electrophysiology, in situ hybridization histochemistry, semiquantitative RT-PCR, Western blotting, and in vivo behavioral reflex analysis to investigate the ubiquitin-proteasome system in a model of neuropathic pain. In neuropathic animals, ionophoretic application of selective proteasome inhibitors attenuated dorsal horn neuron firing evoked by normally innocuous brush or cold stimuli and by noxious mustard oil stimuli. In control animals, only mustard oil-evoked responses were inhibited. Intrathecal administration of proteasome inhibitors attenuated hyperalgesia and allodynia in neuropathic rats. Expression of UCH-L1 (a rat homolog of Aplysia neuronal UCH and of the human UCH-L1, also known as PGP 9.5) and its mRNA were selectively increased within the ipsilateral dorsal horn of neuropathic rats, supporting the idea of a role for the ubiquitin-proteasome system in nociceptive processing. Proteasome inhibitors selectively attenuate allodynic and hyperalgesic responses in neuropathic pain, with some reduction in normal nociceptive, but not non-nociceptive responses, and potentially represent a novel therapeutic strategy for neuropathic pain.

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Figures

Fig. 1.
Fig. 1.
Typical effects of ionophoretic application of the proteasome inhibitor lactacystin on stimulus-evoked sensory responses of single dorsal horn neurons in naive and neuropathic animals. In naive animals, lactacystin had no effect on brush-evoked activation of dorsal horn neuron cell firing rate, measured as action potentials per second (A/s), but it caused a selective attenuation of mustard oil-evoked activation of dorsal horn neuron cell firing (a, b). In contrast, in neuropathic animals, lactacystin reduced dorsal horn neuron firing evoked by application of brush, mustard oil, and cold stimuli to the neuronal receptive field area (c–e). Thebar (bottom right) indicates 1 min duration. Lines above the firing records indicate the duration of the drug application by ionophoresis, close to the recorded cell. This is measured in nanoamperes (nA) of current used to eject the drug and was increased sequentially by 10 nA every 1–2 min.
Fig. 2.
Fig. 2.
The effects of intrathecal administration of the proteasome inhibitors epoxomicin and MG-132 on reflex withdrawal responses to noxious thermal, innocuous mechanical, and innocuous cold stimuli in CCI rats. Data are presented as the mean paw withdrawal latency (in seconds) to noxious thermal stimulation (a,b), paw withdrawal threshold in milliNewtons per millimeters squared (mN/mm2) to mechanical stimulation (c, d), and suspended paw elevation time in seconds [SPET(s)] to cold water (at 4°C) (e, f), before and after intrathecal administration of epoxomicin (0.75 nmol/50 μl) (a–c) and MG-132 (5 nmol/50 μl) (d–f), respectively (atarrow). Injection of vehicle (0.5% dimethylformamide in saline) had no detectable effect on reflexes. Paw withdrawal latency from noxious heat and innocuous cold stimuli ipsilateral to CCI showed significant differences between pre-drug and post-drug administration values (†p < 0.05; one-way ANOVA, followed by a Dunnett's post hoc test). Significant differences between contralateral and ipsilateral paw are indicated (*p < 0.05; Student's paired t test). Paw withdrawal thresholds to mechanical stimulation ipsilateral to CCI showed significant differences between pre-drug and post-drug administration values (†p < 0.05; Kruskal–Wallis one-way ANOVA, followed by a post hocDunn's test). Significant differences between contralateral and ipsilateral paw withdrawal thresholds are indicated (*p< 0.05; Mann–Whitney U test).
Fig. 3.
Fig. 3.
Determination of levels of mRNA for ubiquitin C-terminal hydrolase-L1 (UCH-L1). a, Relative abundance of UCH-L1 mRNA from spinal cord of CCI-treated rats as assessed by semiquantitative RT-PCR normalized to the signal obtained for the cellular housekeeping enzyme GAPDH. Densitometric analysis indicated that the relative UCH-L1 expression was similar in all conditions except in tissue ipsilateral to CCI at 14 d, where expression was significantly greater than that in contralateral samples (*p < 0.05 by paired Student's ttest; n = 3). Further analysis of the changes at 14 d after CCI was made by in situ methods. Seeb and Table 2 for quantification data. b,In situ hybridization histochemistry to show UCH-L1 mRNA expression in lamina I of the rat medial dorsal horn. High-power, light-field photomicrographs showing that expression of mRNA for UCH-L1 was higher ipsilateral to CCI after 14 d, compared with contralateral tissue and naive dorsal horn. Scale bar, 100 μm.
Fig. 4.
Fig. 4.
Western blot analysis of the expression of UCH-L1 protein. Western blots of spinal cord samples from neuropathic, naive, and sham-operated rats. a shows an increase in UCH-L1 protein expression ipsilateral (Ipsi) to CCI in neuropathic animals. No change from naive samples was observed in the sham-operated or contralateral side (Contra) of CCI samples. b represents UCH-L1 expression as a percentage of GAPDH expression in terms of relative gray scale values after quantitative densitometry of ECL films. Data are presented as mean ± SEM (n = 3; *p < 0.05, from control contralateral values; Student's paired ttest).
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