Molecular Roles of Cdk5 in Neuronal Functions and Pain Signaling

Cdk 5和口面机械性疼痛：慢性口面疼痛是一个重要的公共卫生问题。患有口面疼痛病症的患者经常经历机械和热异常性疼痛或痛觉过敏。然而，这种情况的动物模型很少。为了研究orofacial hypo/hyperalgesia和确定是否涉及Cdk 5活性，我们利用特殊的设备来量化小鼠疼痛的机械orofacial刺激的反应。 使用这些设备，我们正在探索Cdk 5和口面疼痛之间的联系，表现为机械性痛觉减退或痛觉过敏。 Cdk 5是否参与口面部机械性疼痛感觉？为了回答这个基本问题，我们正在使用一种改良的口面刺激试验（OST）对野生型，p35-/-和Tgp 35小鼠研究不同的机械刺激对其行为的影响。 这种新的行为测试方法使用了一种冲突范式，允许动物在接受奖励（30%蔗糖）或逃避厌恶刺激之间做出选择，因此动物可以控制伤害性刺激的数量，并可以修改自己的行为。此外，该技术使用自动记录的观察动物的行为提供了独立于干扰器的测试;它们承受的压力较小，并且它们的行为可以以无偏见的方式重复测量。通过使用具有不同数量的镍钛合金丝的板（疼痛水平1：6+6丝，水平2：9+9丝，和水平3：13+13丝）干扰它们获得奖励（30%蔗糖）来实现三种不同水平的疼痛状况。我们目前的研究结果揭示了Tgp 35小鼠（具有增加的Cdk 5活性）对机械刺激的厌恶行为，这可以通过缩短小鼠的总舔舔时间和尝试获得奖励的次数来证明。 在这些小鼠中，奖励舔/面部接触事件的数量大幅减少，机械性疼痛强度增加。相比之下，缺乏p35的小鼠（Cdk 5活性降低）表现出机械性痛觉减退。据我们所知，我们是第一个报告使用口面机械刺激试验在小鼠中，以证明Cdk 5在口面机械伤害性感受中起着重要作用。 新型Cdk 5底物的磷酸化蛋白质组学分析：由于Cdk 5在疼痛信号传导、神经递质释放和神经变性中的重要作用，我们研究了进行磷酸化蛋白质组学筛选是否可行，最初是比较Cdk 5 +/+和Cdk 5-/-小鼠中的磷酸化蛋白。 p35-/-和p39-/-小鼠仍然具有残留的Cdk 5活性，因此我们使用Cdk 5-/-小鼠;因为这些小鼠显示围产期致死性，我们使用从E18.5 Cdk 5 +/+和Cdk 5-/-小鼠胚胎获得的全脑蛋白提取物。 磷酸化蛋白质组学分析是与智利圣地亚哥的智利大学的Christian Gonzalez合作进行的。 从我们的全脑蛋白提取物中分离出磷蛋白，并用胰蛋白酶消化，并对所得肽进行同位素标记，以进行相对和绝对定量。 我们能够识别含有一个或多个Cdk 5磷酸化位点的40种磷蛋白水平的变化。 我们使用基因本体论软件对这40种磷蛋白进行了功能分类。 例如，通过基因本体分析，我们能够将11种磷蛋白分类为参与神经元形态发生，而13种磷蛋白被归类为信号通路。 总之，已经表明Cdk 5调节口面机械性疼痛，我们目前的研究重点是进一步证实这些发现与其他Cdk 5小鼠模型。 此外，我们将大力追求分子研究，以确定新的Cdk 5基板参与疼痛信号。

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.