Cdk5-mediated phosphorylation and neuronal functions

Cdk5和痛觉：一般来说，痛觉（疼痛）神经信号来自外周组织的感觉神经元，并被传递到脊髓的二级神经元，然后将信息传递到大脑中的特定核，以感知疼痛。伤害感觉是由受损组织、感觉神经元和脊髓的分子和细胞机制的激活引起的。许多细胞通路与痛觉信号有关，但其精确的分子机制尚未明确定义。伤害性过程的分子通路异常可能导致慢性疼痛。因此，这些途径的详细表征是必要的，以制定有效的策略来治疗疼痛。由伤害性刺激激活的信号转导机制尚未完全表征。近年来的研究进展，特别是辣椒素受体（也称为香草酸受体1，VR1, TRPV1）的克隆和鉴定，极大地提高了我们对伤害性神经元信号转导的认识。TRPV1是瞬时受体电位（TRP）家族的成员。它是一种多模态配体门控离子通道，在小直径感觉神经元（c -纤维）中表达，并被热、质子、苯胺和白三烯激活。在骨癌疼痛模型中，TRPV1基因敲除小鼠表现出炎症后热痛觉过敏减轻和伤害性反应降低。TRPV1与广泛的生理状况和疾病有关，如热痛觉过敏、异位性疼痛、炎症性肠病、克罗恩病、外阴痛、骨关节炎、胰腺炎、胃食管反流病、膀胱疾病、膀胱炎和哮喘。越来越多的证据表明，TRPV1受到多个相互作用水平的控制。TRPV1的磷酸化对其响应伤害性刺激的功能至关重要。我们之前描述了Cdk5/p35在trpv1阳性的背根神经节（DRG）和三叉神经节（TG）细胞中的表达和特异性活性，以及Cdk5活性在外周炎性疼痛反应中的调节。我们还发现p35基因敲除小鼠，其Cdk5活性显著降低，行为上模仿TRPV1基因敲除的疼痛表型。

Cdk5 and nociception: In general nociceptive (painful) neural signals originate from sensory neurons in peripheral tissues and are transmitted to second-order neurons in the spinal cord, which then convey the message to specific nuclei in the brain for perception of pain. Nociception results from the activation of molecular and cellular mechanisms in damaged tissue, sensory neurons, and the spinal cord. Many cellular pathways have been implicated in nociceptive signaling, but their precise molecular mechanisms have not been clearly defined. Abnormalities in molecular pathways underlying nociceptive processes may result in chronic pain conditions. It follows that a detailed characterization of these pathways is necessary for developing effective strategies to treat pain. Signal transduction mechanisms activated by nociceptive stimuli have not been fully characterized. Recent advances, especially the cloning and characterization of the capsaicin receptor (also known as vanilloid receptor 1, VR1, TRPV1) remarkably improved our understanding of signal transduction in nociceptive neurons. TRPV1 is a member of the transient receptor potential (TRP) family. It is a polymodal ligand-gated ion channel that is expressed in small-diameter sensory neurons (C-fibers) and activated by heat, protons, anandamide, and leukotrienes. TRPV1 knockout mice show reduced thermal hyperalgesia following inflammation and reduced nociceptive responsiveness in a model of bone cancer pain. TRPV1 has been connected to a broad spectrum of physiological conditions and diseases such as thermal hyperalgesia, allodynia, inflammatory bowel disease, Crohn's disease, vulvodynia, osteoarthritis, pancreatitis, gastroesophageal reflux disease, bladder disease, cystitis, and asthma. There is mounting evidence that TRPV1 is subject to multiple interacting levels of control. Phosphorylation of TRPV1 is critical for its function in response to nociceptive stimuli. We previously described the expression and specific activity of Cdk5/p35 in TRPV1-positive cells of dorsal root ganglia (DRG) and trigeminal ganglia (TG) and the modulation of Cdk5 activity in response to peripheral inflammatory pain. We also found that p35 knockout mice, which have significantly reduced Cdk5 activity, behaviorally mimic the TRPV1 knockout pain phenotype. Cdk5, a neuron-specific, proline-directed serine/threonine kinase is activated by 2 noncyclin activators, p35 and/or p39. Cdk5 phosphorylates serine and threonine immediately upstream of a proline residue. In addition to an absolute requirement for proline in the +1 position, Cdk5 shows a marked preference for a basic residue in the +3 position and phosphorylates the consensus sequence (S/T)PX(K/H/R). Several neuronal and nonneuronal substrates are phosphorylated by Cdk5, and the list of new substrates is increasing. Earlier studies showed that Cdk5 knockout and p35/p39 double-knockout mice are embryonically lethal with neuronal migration defects, whereas p35 knockout mice show inverted neuronal layering with a concurrent attenuated response to noxious heat; conversely mice overexpressing p35 were hyperalgesic. Subsequently, we analyzed TRPV1 for potential phosphorylation by Cdk5. We report that Cdk5 can directly phosphorylate TRPV1 at threonine 407, and this in turn modulates agonist-induced calcium influx. We initially found that inhibiting Cdk5 activity resulted in attenuation of capsaicin-induced calcium influx in cultured DRG neurons, and this attenuation was reversible. These observations suggest that Cdk5-mediated phosphorylation of TRPV1 is important for capsaicin-mediated calcium influx through this receptor. Since germline Cdk5 knockout mice are embryonically lethal and p35 knockout mice present various neuronal disorders, we generated primary nociceptor-specific Cdk5 conditional knockout (Cdk5-CoKO) mice to identify the precise role of Cdk5 in primary afferent pain signaling. In the basal state, the conditional knockout mice showed significant hypoalgesia, confirming the direct role of normal Cdk5 activity in primary afferent. Collectively, our findings describe a novel molecular mechanism for the functional regulation of TRPV1 by Cdk5 and provide further insights into the role of Cdk5 in the pain pathway. Cdk5 and brain development: Analysis of functional roles of Cdk5 in postnatal brain development has been hampered because of perinatal lethality of Cdk5-/- mice and the compensated phenotype of p35-/- mice. To study the role of Cdk5 in postnatal development of the midbrain-hindbrain (MHB), we generated MHB-specific Cdk5 conditional knockout mice by crossing Cdk5 floxed mice with Wnt1-Cre mice. Wnt1-Cre-mediated Cdk5 conditional knockout (WCOKO) mice have lower body weight than controls, an ataxic gait, and early postnatal lethality. Histological analysis indicated a smaller cerebellum with defective migration of the Purkinje cells. A detailed analysis of WCOKO mice showed a complete lack of inward migration of the granule cells. In addition, we also identified a complete absence of the superior colliculus in Cdk5-/- mice and its abnormal development in WCOKO mice. These results indicate that Cdk5 plays important roles in mouse MHB development.