Molecular Roles of Cdk5 in Neuronal Functions and Pain Signaling

CDK5和口腔面部机械性疼痛：慢性口腔面部疼痛是一个重大的公共卫生问题。口腔面部疼痛的患者通常会经历机械性和热敏性痛觉或痛觉过敏。然而，针对这种情况的动物模型寥寥无几。为了研究口腔面部痛觉减退/痛敏，并确定是否与CDK5活性有关，我们利用特殊装置量化了小鼠对疼痛机械口腔刺激的反应。使用这些设备，我们正在探索CDK5和表现为机械性痛觉减退或痛觉过敏的口腔面部疼痛之间的联系。 CDK5是否参与口腔面部机械性痛觉？为了回答这个基本问题，我们在野生型、p35-/-和Tgp35小鼠身上使用了一种改进的口腔面部刺激测试(OST)来研究不同机械刺激对它们行为的影响。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)和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，并可以改变自己的行为。此外，这项技术使用自动记录的被观察动物的行为提供了独立于研究人员的测试；它们遭受的压力较小，并且可以以无偏见的方式重复测量它们的行为。通过干扰他们获得奖励(30%蔗糖)来实现三种不同程度的疼痛情况，使用具有不同数量的镍钛合金钢丝的板材(疼痛级别1：6+6钢丝，级别2：9+9钢丝，以及级别3：13+13钢丝)。我们目前的发现揭示了Tgp35小鼠对口面部机械过敏的机械刺激的厌恶行为(这增加了CDK5的活性)，证据是小鼠的总舔时间和尝试获得奖励的次数缩短。在这些机械性疼痛强度增加的小鼠中，奖励舔舔/面部接触事件的数量显著减少。相反，缺乏p35(CDK5活性降低)的小鼠表现出机械性痛觉减退。据我们所知，我们是第一个使用小鼠口面部机械刺激试验来证明CDK5在口面部机械痛觉中起重要作用的报道。 新的CDK5底物的磷酸化蛋白质组学分析：由于CDK5在疼痛信号、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筛选是否可行，最初是比较CDK5+/+和CDK5-/-小鼠的磷化蛋白。P35-/-和p39-/-小鼠仍然有残留的CDK5活性，所以我们使用了CDK5-/-小鼠；因为这些小鼠表现出围产期的致死性，我们使用了从E18.5CDK5+/+和CDK5-/-小鼠胚胎中提取的全脑蛋白提取物。磷酸蛋白质组分析是与智利圣地亚哥智利大学的克里斯蒂安·冈萨雷斯合作进行的。从我们的全脑蛋白提取液中分离出磷蛋白，并用胰酶消化，得到的多肽被同位素标记，以进行相对和绝对定量。我们能够确定包含一个或多个CDK5磷酸化位点的40个磷酸蛋白的水平变化(S)。我们利用基因本体论软件对这40个磷蛋白进行了功能分类。例如，通过基因本体论分析，我们能够将11个磷酸蛋白归类为参与神经元形态发生的蛋白，而13个属于信号通路的类别。 综上所述，在证明了CDK5调节口腔面部机械性疼痛后，我们目前的研究重点是通过其他CDK5小鼠模型进一步证实这些发现。此外，我们将积极开展分子研究，寻找与疼痛信号有关的新的CDK5底物。

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.