Abstract：
Deregulated cyclin-dependent kinase 5 (Cdk5) activity closely correlates with hyperphosphorylated tau, a common pathology found in neurodegenerative diseases. Previous postmortem studies had revealed increased Cdk5 immunoreactivity in amyotrophic lateral sclerosis (ALS); hence, we investigated the effects of Cdk5 inhibition on ALS model mice and neurons in this study. For the in vitro study, motor neuron cell lines with wild-type superoxide dismutase 1 (SOD1) or SOD1G93A and primary neuronal cultures from SOD1G93A transgenic (TG) mice or non-TG mice were compared for the expression of proteins involved in tau pathology, neuroinflammation, apoptosis, and neuritic outgrowth by applying Cdk5-small interfering RNA or Cdk5-short hairpin RNA (shRNA). For the in vivo study, SOD1G93A mice and non-TG mice were intrathecally injected with adeno-associated virus 9 (AAV9)-scramble (SCR)-shRNA or AAV9-Cdk5-shRNA at the age of 5 weeks. Weight and motor function were measured three times per week from 60 days of age, longevity was evaluated, and the tissues were collected from 90-day-old or 120-day-old mice. Neurons with SOD1G93A showed increased phosphorylated tau, attenuated neuritic growth, mislocalization of SOD1, and enhanced neuroinflammation and apoptosis, all of which were reversed by Cdk5 inhibition. Weights did not show significant differences among non-TG and SOD1G93A mice with or without Cdk5 silencing. SOD1G93A mice treated with AAV9-Cdk5-shRNA showed significantly delayed disease onset, delayed rotarod failure, and prolonged survival compared with those treated with AAV9-SCR-shRNA. The brain and spinal cord of SOD1G93A mice intrathecally injected with AAV9-Cdk5-shRNA exhibited suppressed tau pathology, neuroinflammation, apoptosis, and an increased number of motor neurons compared to those of SOD1G93A mice injected with AAV9-SCR-shRNA. Cdk5 inhibition could be an important mechanism in the development of a new therapeutic strategy for ALS.
摘要：
去调节细胞周期蛋白依赖性激酶5(Cdk5)活性与tau过度磷酸化密切相关,在神经退行性疾病中发现的一种常见病理。先前的验尸研究表明，肌萎缩侧索硬化症（ALS）中的Cdk5免疫反应性增加；因此，在本研究中，我们研究了Cdk5抑制对ALS模型小鼠和神经元的影响。对于体外研究，具有野生型超氧化物歧化酶1（SOD1）或SOD1G93A的运动神经元细胞系和来自SOD1G93A转基因（TG）小鼠或非TG小鼠的原代神经元培养物比较了与tau病理学有关的蛋白质的表达，神经炎症，凋亡，和通过应用Cdk5小干扰RNA或Cdk5短发夹RNA（shRNA）的神经质生长。对于体内研究，SOD1G93A小鼠和非TG小鼠在5周龄鞘内注射腺相关病毒9(AAV9)-scramble(SCR)-shRNA或AAV9-Cdk5-shRNA。从60日龄开始，每周测量三次体重和运动功能，寿命进行了评估，并从90天大或120天大的小鼠中收集组织。具有SOD1G93A的神经元显示磷酸化tau增加，神经生长减弱，SOD1的错误定位，并增强神经炎症和细胞凋亡，所有这些都被Cdk5抑制逆转。在有或没有Cdk5沉默的非TG和SOD1G93A小鼠之间，体重没有显着差异。用AAV9-Cdk5-shRNA治疗的SOD1G93A小鼠表现出明显延迟的疾病发作，延迟旋转杆失效,与使用AAV9-SCR-shRNA治疗的患者相比，生存期延长。鞘内注射AAV9-Cdk5-shRNA的SOD1G93A小鼠的脑和脊髓表现出抑制的tau病理，神经炎症，凋亡，与注射AAV9-SCR-shRNA的SOD1G93A小鼠相比，运动神经元数量增加。Cdk5抑制可能是开发ALS新治疗策略的重要机制。
