Peripheral and orofacial pain sensation is unaffected by the loss of p39

Abstract

Cdk5 is a key neuronal kinase necessary for proper brain development, which has recently been implicated in modulating nociception. Conditional deletion of Cdk5 in pain-sensing neurons attenuates pain responses to heat in both the periphery and orofacial regions. Cdk5 activity is regulated by binding to the activators p35 and p39, both of which possess a cyclin box. Our previous examination of the nociceptive role of the well-characterized Cdk5 activator p35 using mice that either lack or overexpress this regulatory subunit demonstrated that Cdk5/p35 activity affects mechanical, chemical, and thermal nociception. In contrast, the nociceptive role of Cdk5’s other less-studied activator p39 is unknown. Here, we report that the knockout of p39 in mice did not affect orofacial and peripheral nociception. The lack of any algesic response to nociceptive stimuli in the p39 knockout mice contrasts with the hypoalgesic effects that result from the deletion of p35. Our data demonstrate different and nonoverlapping roles of Cdk5 activators in the regulation of orofacial as well as peripheral nociception with a crucial role for Cdk5/p35 in pain signaling.

Figure 1.

Levels of Cdk5 and its regulatory subunit p35, and Cdk5 kinase activity in trigeminal ganglia and brain of wild-type and p39^−/− mice. Real-time PCR analysis revealed significantly reduced levels of p39 mRNA in trigeminal ganglia (a) and brain (b) of p39^−/− mice. p35 and Cdk5 expression levels remained unchanged. Results obtained from six different mice are expressed as mean ± SEM and analyzed by unpaired t test (***p < 0.001). Representative Western blots showing p39, p35, and Cdk5 levels from wild-type and p39^−/− mice (c). Cdk5 activity in trigeminal ganglia and brain of wild-type and p39^−/− mice (d). Data obtained from 4 different mice are presented as mean ± SEM and analyzed by unpaired t test. BR: brain; CPM: counts per minute; Cdk5: cyclin-dependent kinase 5; mRNA: messenger RNA; TG: trigeminal ganglia; WT: wild type.

Figure 2.

Expression and activity profile of p39, p35, and Cdk5 in dorsal root ganglia and spinal cord of p39^−/− mice. qPCR analysis revealed significantly decreased levels of p39 mRNA in the DRG (a) and spinal cord (b) of p39^−/− mice but no changes in p35 or Cdk5 mRNA levels. All the data were normalized to the expression level seen in control animals. Results obtained from 6 to 10 animals are expressed as mean ± SEM and analyzed by an unpaired t test (*p < 0.05, ***p < 0.001). Representative Western blots showing p39, p35, and Cdk5 protein levels in DRG and spinal cord of control and p39^−/− animals (c). Cdk5 activity in DRG (n = 6) and spinal cord (n = 5) of WT and p39^−/− mice (d). Data are presented as mean ± SEM. Cdk5: cyclin-dependent kinase 5; CPM: counts per minute; DRG: dorsal root ganglion; mRNA: messenger RNA; SC: spinal cord; WT: wild type.

Figure 3.

Normal activity and anxiety levels in p39^−/− mice. Total distance travelled and resting time (a), stereotypy and time control and p39^−/− mice spent displaying stereotypic behavior (b). Values represent the mean from four mice during 10-min interval sessions. Data are presented as mean ± SEM and analyzed by unpaired t test. WT: wild type.

Figure 4.

Behavioral responses of wild-type and p39^−/− mice during orofacial operant assays. Responses of wild type and p39^−/− mice to TRPV1 and TRPA1 agonists. Water-deprived mice were tested using a lickometer. Initially, mean licking responses were measured using water. After five training sessions, water was replaced with 5 or 25 μM capsaicin, and aversion to the agonist was measured during 1 h (a). Data are presented as mean ± SEM from four mice during five differenct measurements. Effect of activation of the TRPA1 receptor. Mean licking responses of wild-type p39^−/− mice during lickometer testing using water and two different concentrations of the TRPA1 agonist mustard oil-1 and 100 μM (b). During mechanical testing, mice were able to access a 30% sucrose reward by inserting their snout through the drinking window. Licking behaviour and the time spent licking the reward were measured with no wires (c) or using a plate of 6 + 6 wires (d). Data are presented as mean ± SEM from four mice measured five times in case of the baseline and three times after the induction of nociception. AITC: allyl isothiocyanate; CAP: capsaicin; WT: wild type.

Figure 5.

Behavioral responses of control, p35^−/− and p39^−/− mice to mechanical and thermal stimulation at the periphery. Reaction time and force intensity needed for paw withdrawal was unaffected in the control as well as the p39^−/− mice (a). Results are expressed as mean ± SEM of time and force required to induce paw withdrawal in eight animals per group. p35^−/− mice exhibited a delayed basal response to mechanical stimulation and experienced hypoalgesia compared to control animals (t test, p < 0.001) (b). Data are presented as a mean ± SEM (n = 8). Basal thermal responses were unaffected in p39^−/− mice. Mean paw withdrawal latencies during low (IR30) and high (IR 60) heat stimulation (c). Data are presented as a mean ± SEM (n = 8). WT: wild type.