Cdk5-mediated phosphorylation and neuronal functions

Cdk5 介导的磷酸化和神经元功能

• 批准号: 7593372
• 负责人: Ashok B. KULKARNI
• 金额: $ 84.9万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593372
• 关键词: Address; Afferent Neurons; Agonist; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Asthma; Ataxic Gait; Attenuated; Behavior Control; Bladder Diseases; Body Weight; Brain; C Fiber; Calcium; Caliber; Capsaicin; Cell Cycle Progression; Cell Cycle Regulation; Cell Nucleus; Cells; Cerebellum; Cloning; Condition; Consensus Sequence; Crohn' s disease; Cyclin-Dependent Kinase 5; Cystitis; Defect; Degenerative polyarthritis; Development; Diagnosis; Disease; Drug Addiction; Family; Future; Gastroesophageal reflux disease; Gated Ion Channel; Gene Targeting; Goals; Heating; Inflammation; Inflammatory Bowel Diseases; Knock-out; Knockout Mice; Learning; Leukotrienes; Ligands; Malignant Bone Neoplasm; Mediating; Memory; Midbrain structure; Modeling; Molecular; Mus; Nervous system structure; Neurodegenerative Disorders; Neurons; Nociception; Nociceptive Stimulus; Nociceptors; Pain; Pancreatitis; Parkinson Disease; Pathogenesis; Pathway interactions; Perception; Perinatal; Peripheral; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Play; Positioning Attribute; Process; Proline; Protein Overexpression; Protein-Serine-Threonine Kinases; Proteins; Protons; Purkinje Cells; Range; Regulation; Reporting; Role; Sensory; Serine; Signal Transduction; Specificity; Spinal Cord; Spinal Ganglia; Structure of trigeminal ganglion; TRPV1 gene; Techniques; Thermal Hyperalgesias; Threonine; Tissues; Vulvodynia; Weight maintenance regimen; allodynia; anandamide; attenuation; base; behavior test; cancer pain; capsaicin receptor; chronic pain; functional genomics; granule cell; hindbrain; improved; inflammatory pain; insight; member; migration; nervous system disorder; novel; postnatal; preference; protein protein interaction; receptor; relating to nervous system; response; superior colliculus Corpora quadrigemina; vanilloid receptor subtype 1

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.

Cdk 5与伤害感受：一般来说，伤害性（疼痛）神经信号源自外周组织中的感觉神经元，并被传递到脊髓中的二级神经元，然后二级神经元将信息传递到大脑中的特定核团以感知疼痛。 伤害感受是由受损组织、感觉神经元和脊髓中分子和细胞机制的激活引起的。 许多细胞通路参与了伤害性信号传导，但其确切的分子机制尚未明确。 伤害感受过程的分子通路的阻断可能导致慢性疼痛状况。 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。 最近的进展，特别是辣椒素受体（也称为vanilloid receptor 1，VR 1，TRPV 1）的克隆和表征，显着提高了我们对伤害性神经元信号转导的理解。 TRPV 1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、花生四烯酸和白三烯激活。 TRPV 1基因敲除小鼠在骨癌疼痛模型中表现出炎症后热痛觉过敏降低和伤害性反应降低。 TRPV 1与广泛的生理状况和疾病有关，例如热痛觉过敏、异常性疼痛、炎性肠病、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TRPV 1受到多个相互作用的控制水平。 TRPV 1的磷酸化对于其响应伤害性刺激的功能至关重要。 我们以前描述的表达和特异性活性的Cdk 5/p35的TRPV 1阳性细胞的背根神经节（DRG）和三叉神经节（TG）和调制的Cdk 5活性在外周炎症疼痛。 我们还发现，p35基因敲除小鼠，这已显着降低Cdk 5活性，行为模仿TRPV 1敲除疼痛表型。 Cdk 5是一种神经元特异性的脯氨酸导向的丝氨酸/苏氨酸激酶，由两种非细胞周期蛋白激活剂p35和/或p39激活。 cdk 5磷酸化丝氨酸和苏氨酸紧邻脯氨酸残基的上游。 除了在+1位上绝对需要脯氨酸外，Cdk 5还显示出对+3位上碱性残基的明显偏好，并使共有序列（S/T）PX（K/H/R）磷酸化。 几个神经元和非神经元底物被Cdk 5磷酸化，新底物的名单正在增加。早期的研究表明，Cdk 5基因敲除和p35/p39双敲除小鼠是胚胎致死的神经元迁移缺陷，而p35基因敲除小鼠显示倒置的神经元分层，同时减弱对有害热的反应;相反，过表达p35的小鼠痛觉过敏。 随后，我们分析了TRPV 1可能被Cdk 5磷酸化。我们报告说，Cdk 5可以直接磷酸化TRPV 1在苏氨酸407，这反过来又调节激动剂诱导的钙内流。 我们最初发现，抑制Cdk 5活性导致辣椒素诱导的培养DRG神经元钙内流衰减，这种衰减是可逆的。 这些观察结果表明，Cdk 5介导的磷酸化的TRPV 1是辣椒素介导的钙离子内流通过该受体的重要。 由于生殖系Cdk 5基因敲除小鼠是胚胎致死性的，p35基因敲除小鼠存在各种神经元疾病，我们产生了初级伤害感受器特异性Cdk 5条件性敲除（Cdk 5-CoKO）小鼠，以确定Cdk 5在初级传入疼痛信号传导中的确切作用。 在基础状态下，条件性基因敲除小鼠表现出显着的痛觉减退，证实了初级传入中正常Cdk 5活性的直接作用。总的来说，我们的研究结果描述了一种新的分子机制，通过Cdk 5的TRPV 1的功能调节，并提供了进一步的见解Cdk 5在疼痛通路中的作用。 CDK 5与大脑发育：由于Cdk 5-/-小鼠的围产期致死率和p35-/-小鼠的代偿表型，Cdk 5在出生后脑发育中的功能作用的分析受到阻碍。 为了研究Cdk 5在出生后中脑-后脑（MHB）发育中的作用，我们通过将Cdk 5 floxed小鼠与Wnt 1-Cre小鼠杂交来产生MHB特异性Cdk 5条件性敲除小鼠。 Wnt 1-Cre介导的Cdk 5条件性敲除（WCOKO）小鼠的体重低于对照组，步态共济失调，出生后早期致死。 组织学分析表明小脑较小，浦肯野细胞迁移缺陷。 对WCOKO小鼠的详细分析表明，颗粒细胞完全缺乏向内迁移。 此外，我们还确定了一个完全缺乏的上级丘在Cdk 5-/-小鼠和它的异常发育在WCOKO小鼠。 这些结果表明Cdk 5在小鼠MHB的发育中起重要作用。