Cyclin-dependent kinase 5 (Cdk5 physiology and pathology)

细胞周期蛋白依赖性激酶 5（Cdk5 生理学和病理学）

• 批准号: 9157547
• 负责人: HARISH C PANT
• 金额: $ 144.46万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 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/9157547
• 关键词: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 1-Methyl-4-phenylpyridinium; Accounting; Affect; Aging; Alzheimer' s Disease; Alzheimer' s disease model; Amino Acids; Amyloid beta-Protein Precursor; Animal Model; Animals; Apoptosis; Autopsy; Behavior; Behavioral; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Assay; Blood - brain barrier anatomy; Brain; Cell Cycle; Cells; Chronic; Complex; Corpus striatum structure; Cyclin-Dependent Kinase 5; Data; Defect; Dementia; Development; Disease; Disease model; Dopamine; Etiology; Exhibits; Fluorescein-5-isothiocyanate; Freezing; Functional disorder; Gait; Gene Mutation; Genetic Transcription; Glucose; Human; Hyperactive behavior; Immunohistochemistry; In Situ; In Vitro; Inflammation; Injection of therapeutic agent; Intraperitoneal Injections; Kinetics; Literature; Memory impairment; Mitochondria; Motor; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Parkinson Disease; Pathology; Pathway interactions; Peptides; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Physiology; Process; Production; Proteins; Reactive Oxygen Species; Reagent; Reporting; Role; Saline; Senile Plaques; Short-Term Memory; Structure; Substantia nigra structure; Testing; Therapeutic; Therapeutic Agents; Toxic effect; Trans-Activators; Transgenic Mice; Transgenic Organisms; age group; age related; amyloid pathology; base; disease phenotype; dopaminergic neuron; extracellular; hyperphosphorylated tau; improved; in vivo; inhibitor/antagonist; motor deficit; motor disorder; mouse model; mutant; neurodegenerative phenotype; neurofilament; neuroinflammation; neuropathology; neurotoxicity; prevent; response; roscovitine; secretase; tau Proteins; therapeutic target

We have began to use TFP5 to study its therapeutic role in PD. Parkinsons disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopamine neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. Recent evidence indicates that cyclin-dependent kinase 5 (Cdk5) is inappropriately activated in several neurodegenerative conditions including PD. To date, strategies to specifically inhibit Cdk5 hyperactivity have not been successful without affecting normal Cdk5 activity. Previously, we reported that TFP5 peptide has neuroprotective effects in animal models of Alzheimer's disease (AD). Here, we show that TFP5/TP5 selective inhibition of Cdk5/p25 hyperactivation in vivo and in vitro, rescues nigrostriatal dopaminergic neurodegeneration induced by 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+),in a mouse model of PD. TP5 peptide treatment also blocked dopamine depletion in the striatum and improved gait dysfunction after MPTP administration. The neuroprotective effect of TFP5/TP5 peptide is also associated with marked reduction in neuroinflamation and apoptosis. Here, we show selective inhibition of Cdk5/p25 hyperactivation by TFP5/TP5 peptide, which identifies the kinase as a potential therapeutic target to reduce neurodegeneration in Parkinson's disease. Because of its involvement in PD, Cdk5/p25 has been identified as a prime therapeutic target for PD. Accordingly, compounds like roscovitine, an inhibitor targeting the ATP binding site in Cdk5 and other cell cycle kinases, have been studied as potential therapeutic agents, however, these drugs are toxic. Our approach to this problem, based on structure and kinetics of the Cdk5/p25complex, resulted in the production of several small truncated peptides of p35, which competed with p25 binding and inhibited Cdk5 hyperactivation in vitro. A small peptide, P5, comprising 24 aa, specifically inhibited Cdk5/p25 activity in cultured cortical neurons, reduced hyperphosphorylated tau and apoptosis, without affecting the normal endogenous Cdk5/p35 activity, nor the activity of several cell cycle kinases (Zheng et al., 2005, Zheng et al., 2010a).The P5 peptide was modified as TFP5 with a transactivator of transcription (Tat) peptide conjugated at the C terminus to facilitate passage through the blood-brain barrier, and fluoresceinisothiocyanate (FITC; a green fluorescent tag) attached at the N terminus as a marker. When injected intraperitoneally into 5XFAD AD model mice, significantly reduced Cdk5/p25 hyperactivity, hyperphosphorylated tau and rescued behavior deficits of spatial working memory and motor deficits (Shukla et al., 2013). Moreover, TFP5 also reduced toxicity in cortical neurons exposed to high glucose (Binukumar et al., 2014). We now find the intraperitoneal injection of TP5 (TFP5 without FITC) into MPTP-induced mice effectively blocks degeneration of dopamine neurons in the SNpc, and almost completely prevents the loss of striatal dopamine and its metabolites. The peptide treatment also ameliorates the MPTP-induced behavioral deficits, inhibits neuroinflamation in vivo and protects MPP+ neurotoxicity in vitro.These results suggest that TFP5/TP5 may be effective in the treatment of Parkinsons disease. We believe TFP5 acts on multiple pathways inhibiting hyper activation of Cdk5 and other kinases involved in inducing neuropathologies to reduce / prevent neurodegenerative phenotypes.

我们已经开始使用TFP 5来研究其在PD中的治疗作用。帕金森病（PD）是一种慢性神经退行性疾病，其特征在于黑质多巴胺神经元的丢失，纹状体多巴胺水平降低，以及随之而来的锥体外系运动功能障碍。最近的证据表明，细胞周期蛋白依赖性激酶5（Cdk 5）在包括PD在内的几种神经退行性疾病中被不适当地激活。迄今为止，特异性抑制Cdk 5活性亢进的策略在不影响正常Cdk 5活性的情况下尚未成功。以前，我们报道了TFP 5肽在阿尔茨海默病（AD）动物模型中具有神经保护作用。在这里，我们表明，TFP 5/TP 5在体内和体外选择性抑制Cdk 5/p25超活化，挽救黑质纹状体多巴胺能神经变性诱导的1-甲基-4-苯基-1，2，3，6-四氢吡啶（MPTP/MPP+），在PD小鼠模型。TP 5肽治疗还阻断纹状体中的多巴胺耗竭，并改善MPTP给药后的步态功能障碍。TFP 5/TP 5肽的神经保护作用还与神经炎症和细胞凋亡的显著减少相关。在这里，我们显示了TFP 5/TP 5肽对Cdk 5/p25超活化的选择性抑制，这将激酶鉴定为减少帕金森病神经变性的潜在治疗靶点。 由于其参与PD，Cdk 5/p25已被确定为PD的主要治疗靶点。因此，化合物如roscovitine（靶向Cdk 5和其他细胞周期激酶中的ATP结合位点的抑制剂）已被研究为潜在的治疗剂，然而，这些药物是有毒的。基于Cdk 5/p25复合物的结构和动力学，我们对这个问题的解决方法导致产生了几个小的截短的p35肽，其与p25结合竞争并在体外抑制Cdk 5超活化。由24个aa组成的小肽P5特异性抑制培养的皮质神经元中的Cdk 5/p25活性，减少过度磷酸化的tau和细胞凋亡，而不影响正常的内源性Cdk 5/p35活性，也不影响几种细胞周期激酶的活性（Zheng等人，2005，Zheng等人，将P5肽修饰为TFP 5，其中在C末端缀合有转录的反式激活因子（达特）肽以促进通过血脑屏障，并且在N末端连接有异硫氰酸荧光素（FITC;绿色荧光标签）作为标记物。当腹膜内注射到5XFAD AD模型小鼠中时，显著降低Cdk 5/p25活动过度、过度磷酸化tau并挽救空间工作记忆和运动缺陷的行为缺陷（Shukla et al.，2013年）。此外，TFP 5还降低了暴露于高葡萄糖的皮质神经元中的毒性（Binukumar等人，2014年）。我们现在发现腹膜内注射TP 5（不含FITC的TFP 5）到MPTP诱导的小鼠中有效地阻断了SNpc中多巴胺神经元的变性，并且几乎完全防止了纹状体多巴胺及其代谢物的损失。TFP 5/TP 5在体内可减轻MPTP诱导的行为障碍，抑制神经炎症，在体外可保护MPP+的神经毒性，提示TFP 5/TP 5可能对帕金森病有治疗作用。 我们认为TFP 5作用于多种途径，抑制Cdk 5和其他参与诱导神经病理学的激酶的过度活化，以减少/预防神经退行性表型。