Molecular Roles of Cdk5 in Neuronal Functions and Pain Signaling

Cdk5 在神经元功能和疼痛信号传导中的分子作用

• 批准号: 7733915
• 负责人: Ashok B. KULKARNI
• 金额: $ 68.8万
• 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/7733915
• 关键词: Afferent Neurons; Agonist; Analgesics; Antibodies; Apoptosis; Automobile Driving; Biochemical Pathway; Brain; Calcium; Calpain; Capsaicin; Carrageenan; Cell Cycle; Cell physiology; Collaborations; Cyclin-Dependent Kinase 5; Defect; Development; Diabetes Mellitus; Disease; Dose; Drug Industry; ERG gene; Enzymes; Evaluation; Exhibits; Face; Future; Homeostasis; Human Genome; Hybrids; Hypersensitivity; Inflammation; Injection of therapeutic agent; Knockout Mice; Knowledge; Laboratories; Link; Luciferases; MAP Kinase Gene; MAPK11 gene; MAPK14 gene; MAPK8 gene; MEKs; Malignant Neoplasms; Mediating; Messenger RNA; Molecular; Mus; NF-kappa B; NFKB Signaling Pathway; Nervous system structure; Neuraxis; Neurobiology; Neurodegenerative Disorders; Neurons; Nociception; Nociceptors; Numbers; Oral; PC12 Cells; Pain; Pathway interactions; Peripheral; Peripheral Nervous System; Phosphorylation; Phosphotransferases; Play; Process; Proline; Protein Kinase; Protein Kinase Inhibitors; Protein Overexpression; Protein-Serine-Threonine Kinases; Proteins; Regulation; Role; Screening procedure; Signal Transduction; Site; Stimulus; TRPV1 gene; Techniques; Testing; Therapeutic; Threonine; Time; Tooth structure; Tumor Necrosis Factor-alpha; Two-Dimensional Gel Electrophoresis; Validation; Western Blotting; Work; Yeasts; attenuation; base; cytokine; enzyme activity; heat stimulus; human TNF protein; inhibitor/antagonist; interest; mouse model; novel; programs; promoter; protein kinase inhibitor; receptor; research and development; research study; response; transcription factor; vector

Cdk5 regulates pain signaling: Our present studies are primarily focused on delineating the roles of Cdk5 in the central and peripheral nervous system with a special emphasis on nociception and pain. We have earlier identified expression of Cdk5 and p35 in nociceptive neurons, and discovered that this expression is modulated during peripheral inflammation. We also discovered that induced inflammation results in increased calpain activity in sensory neurons, which activates cleavage of p35 to p25 and subsequently increases Cdk5 kinase activity. The p35-/- mice, which exhibit significantly decreased Cdk5 activity, showed delayed responses towards painful thermal stimulation compared to their wild-type controls. In contrast, mice overexpressing p35 with elevated levels of Cdk5 activity were more sensitive to painful thermal stimuli than controls. We analyzed TRPV1 for potential phosphorylation by Cdk5 and found that Cdk5 can directly phosphorylate TRPV1 at threonine 407, and this in turn modulates agonist-induced calcium influx. We also 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 Cdk5-/- mice die perinatally, we generated primary nociceptor-specific Cdk5 conditional knockout (COKO) mice to identify the precise role of Cdk5 in primary afferent pain signaling. In the basal state, the conditional knockout mice had significant hypoalgesia, confirming the direct role of normal Cdk5 activity in primary afferents. Collectively, our findings show a novel molecular mechanism for the functional regulation of TRPV1 by Cdk5 and suggest that Cdk5/p35 may be a target for development of analgesic drugs. The aim of our current study in FY08 was to identify the proinflammatory molecules that regulate Cdk5/p35 activity in response to inflammation. We constructed a vector that contains the mouse p35 promoter driving luciferase expression. We transiently transfected this vector in PC12 cells to test the effect of several cytokines on p35 transcriptional activity and Cdk5 activity. Our results indicate that tumor necrosis factor-alpha (TNF-alpha) activates p35 promoter activity in a dose- and time-dependent manner and concomitantly upregulates Cdk5 activity. Because TNF-alpha is known to activate ERK1/2, p38 MAPK, JNK, and NF-kB signaling pathways, we examined their involvement in the activation of p35 promoter activity. MEK inhibitor, which inhibits ERK activation, decreased p35 promoter activity, while the inhibitors of p38 MAPK, JNK, and NF-kB increased p35 promoter activity, indicating that these pathways regulate p35 expression differently. The mRNA and protein levels of early gene response 1 (Egr-1), a transcription factor, were increased by TNF-alpha treatment, and this increase was dependent on ERK signaling. In a mouse model of inflammation-induced pain in which carrageenan injection into the hind paw causes hypersensitivity to heat stimuli, TNF-alpha mRNA was increased at the site of injection. These findings suggest that TNF-alpha-mediated regulation of Cdk5 activity plays an important role in inflammation-induced pain signaling. Phosphoproteomic analysis of Cdk5 targets: The human genome encodes over 500 different protein kinases, the key regulatory enzymes that catalyze the phosphorylation of proteins at about 100,000 different sites to reversibly control their functional activities. Defects in specific protein kinases have been linked to over 400 diseases, and about 25% of all pharmaceutical industry research and development is now focused on the discovery and evaluation of protein kinase inhibitors for therapeutic applications. Cdk5 has become a target of high interest to the drug industry because of its key role in neuronal homeostasis. So far more than 40 different Cdk5 substrates have been identified, and abnormal Cdk5 activity has been implicated in several disease processes, including neurodegenerative disorders, cancer, and diabetes. However, a global profiling of protein phosphorylation mediated by Cdk5 is still not available. Our current knowledge about such profiling comes from experiments performed in different laboratories mainly based on 2-dimensional gel electrophoresis or yeast 2-hybrid screening. Because of the limitations of these techniques at the point of validation of targeted proteins, we took a different approach to resolve this issue. We compared phosphorylation status and total protein levels of 258 different proteins by simple Western blotting analyses of Cdk5-/- and WT wild-type brains. The antibodies used in this analysis are already proven to be highly specific for their targeted sites in different biochemical pathways. We based our selection of these proteins on some known and predicted functions of Cdk5 and further categorized them into 6 different groups. The first group contained 25 different proteins involved in apoptosis, the second consisted of 77 different other kinases, the third consisted of 39 different substrates for these kinases, the fourth consisted of 43 different proteins involved in the cell cycle, the fifth contained 37 different proteins involved in numerous neurobiological functions, and the sixth consisted of 37 different proteins controlling different kinase pathways. This phosphoproteomic screening gave us a broad overview of Cdk5 targets involved in these biochemical pathways and cellular processes. We will develop strategies to help us identify the role of Cdk5 in tooth pain and develop potential analgesics. Work on other potentially important questions involving Cdk5 have been shifted to ongoing major collaborations with a number of leading laboratories in neurobiology. This shift will benefit us in our increasingly predominant program on Cdk5 and pain signaling.

Cdk5调节疼痛信号：我们目前的研究主要集中在描述Cdk5在中枢和周围神经系统中的作用，特别强调伤害和疼痛。我们早前已经确定了Cdk5和p35在伤害性神经元中的表达，并发现这种表达在外周炎症中受到调节。我们还发现，诱导炎症导致感觉神经元钙蛋白酶活性增加，从而激活p35到p25的裂解，随后增加Cdk5激酶活性。p35-/-小鼠Cdk5活性显著降低，与野生型对照相比，对疼痛热刺激的反应延迟。相比之下，过度表达p35且Cdk5活性水平升高的小鼠对疼痛的热刺激比对照组更敏感。我们分析了TRPV1被Cdk5磷酸化的可能性，发现Cdk5可以直接磷酸化TRPV1的苏氨酸407，这反过来调节激动剂诱导的钙内流。我们还发现，抑制Cdk5活性导致辣椒素诱导的DRG神经元钙内流的衰减，并且这种衰减是可逆的。这些观察结果表明，cdk5介导的TRPV1磷酸化对于辣椒素介导的钙通过该受体内流是重要的。由于Cdk5-/-小鼠会在围产期死亡，我们构建了初级伤害感受器特异性Cdk5条件敲除（COKO）小鼠，以确定Cdk5在初级传入疼痛信号中的确切作用。在基础状态下，条件敲除小鼠有明显的痛觉减退，证实了正常Cdk5活性在原发事件中的直接作用。总之，我们的研究结果揭示了Cdk5调控TRPV1功能的一种新的分子机制，并提示Cdk5/p35可能是开发镇痛药物的靶点。

项目成果

Cyclin-dependent kinase 5 is required for control of neuroblast migration in the postnatal subventricular zone

• DOI: 10.1523/jneurosci.1014-07.2007
• 发表时间: 2007-11-21
• 期刊: JOURNAL OF NEUROSCIENCE
• 影响因子: 5.3
• 作者: Hirota, Yuki;Ohshima, Toshio;Sawamoto, Kazunobu
• 通讯作者: Sawamoto, Kazunobu

Cdk5 is required for multipolar-to-bipolar transition during radial neuronal migration and proper dendrite development of pyramidal neurons in the cerebral cortex

• DOI: 10.1242/dev.02854
• 发表时间: 2007-06-15
• 期刊: DEVELOPMENT
• 影响因子: 4.6
• 作者: Ohshima, Toshio;Hirasawa, Motoyuki;Mikoshiba, Katsuhiko
• 通讯作者: Mikoshiba, Katsuhiko

Long-term enzyme correction and lipid reduction in multiple organs of primary and secondary transplanted Fabry mice receiving transduced bone marrow cells.

对接受转导骨髓细胞的初次和二次移植法布里小鼠的多个器官进行长期酶校正和脂质减少。

• DOI: 10.1073/pnas.120177997
• 发表时间: 2000
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Takenaka,T;Murray,GJ;Qin,G;Quirk,JM;Ohshima,T;Qasba,P;Clark,K;Kulkarni,AB;Brady,RO;Medin,JA
• 通讯作者: Medin,JA

Mutant superoxide dismutase 1 causes motor neuron degeneration independent of cyclin-dependent kinase 5 activation by p35 or p25.

突变的超氧化物歧化酶 1 会导致运动神经元变性，与 p35 或 p25 激活细胞周期蛋白依赖性激酶 5 无关。

• DOI: 10.1046/j.1471-4159.2003.02256.x
• 发表时间: 2004
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: Takahashi,Satoru;Kulkarni,AshokB
• 通讯作者: Kulkarni,AshokB

Control of cyclin‐dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability

• DOI: 10.1111/j.1471-4159.2005.03058.x
• 发表时间: 2005-04
• 期刊: Journal of Neurochemistry
• 影响因子: 4.7
• 作者: Fan-Yan Wei;K. Tomizawa;T. Ohshima;A. Asada;Taro Saito;C. Nguyen;J. Bibb;K. Ishiguro;Ashok B Kulkarni;H. Pant;K. Mikoshiba;H. Matsui;S. Hisanaga