The multiple PDZ domain protein Mpdz/MUPP1 regulates opioid tolerance and opioid-induced hyperalgesia

Abstract

Background: Opioids are a mainstay for the treatment of chronic pain. Unfortunately, therapy-limiting maladaptations such as loss of treatment effect (tolerance), and paradoxical opioid-induced hyperalgesia (OIH) can occur. The objective of this study was to identify genes responsible for opioid tolerance and OIH.

Results: These studies used a well-established model of ascending morphine administration to induce tolerance, OIH and other opioid maladaptations in 23 strains of inbred mice. Genome-wide computational genetic mapping was then applied to the data in combination with a false discovery rate filter. Transgenic mice, gene expression experiments and immunoprecipitation assays were used to confirm the functional roles of the most strongly linked gene. The behavioral data processed using computational genetic mapping and false discovery rate filtering provided several strongly linked biologically plausible gene associations. The strongest of these was the highly polymorphic Mpdz gene coding for the post-synaptic scaffolding protein Mpdz/MUPP1. Heterozygous Mpdz +/- mice displayed reduced opioid tolerance and OIH. Mpdz gene expression and Mpdz/MUPP1 protein levels were lower in the spinal cords of low-adapting 129S1/Svlm mice than in high-adapting C57BL/6 mice. Morphine did not alter Mpdz expression levels. In addition, association of Mpdz/MUPP1 with its known binding partner CaMKII did not differ between these high- and low-adapting strains.

Conclusions: The degrees of maladaptive changes in response to repeated administration of morphine vary greatly across inbred strains of mice. Variants of the multiple PDZ domain gene Mpdz may contribute to the observed inter-strain variability in tolerance and OIH by virtue of changes in the level of their expression.

Keywords: Drug tolerance; Gene mapping; Opioid analgesics; Synaptic plasticity.

Fig. 1

Opioid-induced changes in nociceptive thresholds for 23 strains of mice. These data were reproduced from our earlier report [18]. In panel (a) the reduction in mechanical nociceptive thresholds are displayed as a fraction of the baseline thresholds. The strains are displayed from the most to least robustly changed. In panel (b) thermal withdrawal thresholds measured using a Hargreaves’ apparatus are displayed as a fraction of baseline threshold. In panel (c) changes in thermal withdrawal threshold of the tail (tail flick) response are provided. For each trait the mean value is displayed +/− S.E.M., n = 8 mice per strain

Fig. 2

Morphine tolerance, dependence and weight change for 23 strains of mice. Some data were reproduced from our earlier report [18]. In panel (a) the fold change in the ED₅₀ values for morphine dose–response curves are provided. In panel (b) the number of jumps exhibited by the mice in the 15 min period following the administration of naloxone are displayed. In panel (c) the change in weight as a fraction of baseline for each of the 23 strains used in these experiments are provided. For each trait the mean value is displayed +/− S.E.M., n = 8 mice per strain

Fig. 3

Mpdz expression and MUPP1 levels in mouse spinal cord tissue. In these experiments the effects of both genotype and morphine treatment were evaluated. Panel (a) provides the results of qPCR experiments in which the index C57BL/6 and low-adapting 129S1/SvIm strains were used. Baseline Mpdz levels were lower in the 129S1/SvIm strain, but the levels of Mpdz mRNA were unchanged by morphine treatment in both of the strains tested. In panel (b) the results of Western analysis are provided demonstrating that 129S1/SvIm mice have lower spinal levels of Mpdz/MUPP1 protein. For each trait the mean value is displayed +/− S.E.M., n = 6 mice per group. *p < 0.05, ***p < 0.001

Fig. 4

Opioid adaptations in C57BL/6 wild-type and Mpdz ^+/− mice. In panel (a) the results of experiments measuring opioid-induced hyperalgesia using changes in mechanical withdrawal thresholds are displayed. In panel (b) the anangesic effect of cumulitive doses of morphine are shown for wild-type and Mpdz ^+/− mice both before and after 4 days of escalating dose morphine treatment. In panel (c) naloxone-induced jumping behavior for wild-type and Mpdz ^+/− mice is displayed. Finally, in panel (d) data for morphine-induced weight loss are provided. For each trait the mean value is displayed +/− S.E.M., n = 8 mice per strain. ##, **p < 0.01, ###, ****p < 0.001

Fig. 5

Co-immunoprecipitation of MUPP1 with CaMKII in mouse spinal cord tissue. Spinal cord synaptosomes were first incubated with anti-MUPP1 (or IgG as a negative control). After column purification, the immune complexes were eluted and separated on acrylamide gels. Blots were probed with anti-CaMKII to quantify MUPP1-CaMKII association. Panel (a) provides the results of co-immunoprecipitation experiments demonstrating that MUPP1-CaMKII association is unchanged by morphine treatment in C57BL/6 mice. In panel (b) the results suggest MUPP1 protein from 129S1/Svlm and C57BL/6 mice bind CaMKII similarly. For each measurement the mean value is displayed +/− S.E.M., n = 6 mice per group