Cdk5 and orofacial mechanical pain: Chronic orofacial pain is a significant public health concern. Patients with orofacial pain conditions often experience mechanical and thermal allodynia or hyperalgesia. Nevertheless, there are few animal models for such conditions. In order to study orofacial hypo/hyperalgesia and determine whether Cdk5 activity is involved, we have utilized special devices to quantify the responses of mice to painful mechanical orofacial stimulation. Using these devices, we are exploring the link between Cdk5 and orofacial pain as manifested by mechanical hypoalgesia or hyperalgesia. Is Cdk5 involved in orofacial mechanical pain sensation? To answer this fundamental question, we are using a modified orofacial stimulation test (OST) on wild-type, p35-/-, and Tgp35 mice to study the effect of different mechanical stimuli on their behavior. This new behavioral testing method uses a conflict paradigm that allows animals to make a choice between receiving a reward (30% sucrose) or escaping aversive stimuli, so the animals have control over the amount of nociceptive stimulation and can modify their own behavior. Additionally, this technique provides investigator-independent testing using automatically recorded behavior of the observed animals;they incur less stress, and their behavior can be measured repeatedly in a non-biased fashion. Three different levels of painful conditions were achieved by interfering with their access to a reward (30% sucrose) using plates with different numbers of Nitinol wires (pain level 1: 6+6 wires, level 2: 9+9 wires, and level 3: 13+13 wires). Our current findings reveal aversive behavior to mechanical stimulation with orofacial mechanical hypersensitivity in Tgp35 mice (which have increased Cdk5 activity), as evidenced by shortening of the total licking time and number of attempts the mice make to access the reward. The number of reward licking/facial contact events decreased substantially in these mice with increased mechanical pain intensity. In contrast, mice lacking p35 (with decreased Cdk5 activity) displayed mechanical hypoalgesia. To the best of our knowledge, we are the first to report using the orofacial mechanical stimulation test in mice to demonstrate that Cdk5 plays an important role in orofacial mechanonociception. Phosphoproteomic analysis for novel Cdk5 substrates: Because of the important role of Cdk5 in pain signaling, neurotransmitter release, and neurodegeneration, we examined whether it was practical to conduct phosphoproteomics screening, initially to compare phosphoproteins in Cdk5+/+ and Cdk5-/- mice. The p35-/- and p39-/- mice still have residual Cdk5 activity, so we used Cdk5-/- mice;because these mice show perinatal lethality, we used whole-brain protein extracts obtained from E18.5 Cdk5+/+ and Cdk5-/- mouse embryos. Phosphoproteomic analysis was performed in collaboration with Christian Gonzalez of the University of Chile, Santiago, Chile. Phosphoproteins were isolated from our whole-brain protein extracts and digested with trypsin, and the resulting peptides were isotopically labeled for their relative and absolute quantification. We were able to identify changes in the levels of 40 phosphoproteins containing one or more Cdk5 phosphorylation site(s). We classified these 40 phosphoproteins according to their functions using gene ontology software. With the gene ontology analysis, we were, for example, able to classify 11 of the phosphoproteins as involved in neuronal morphogenesis while 13 were grouped in the category of signaling pathways. In summary, having shown that Cdk5 modulates orofacial mechanical pain, our current research is focused on further confirming these findings with additional Cdk5 mouse models. Furthermore, we will vigorously pursue molecular investigations into identifying novel Cdk5 substrates involved in pain signaling.
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