细胞周期蛋白依赖性激酶5(CDK5)是参与神经元稳态和对神经元存活至关重要的发育的蛋白激酶。此外,它的失调与神经退行性病变如阿尔茨海默病和帕金森病有关。出于这个原因,我们的目的是使用CRISPR/Cas9技术,在人诱导多能干细胞(hiPSCs)来源的神经元中建立CDK5缺陷遗传模型.我们获得了FN2.1hiPSC系的杂合CDK5+/-克隆,该克隆保留了hiPSC的干性和多能潜能。然后,神经干细胞(NSC)和其他神经元来自CDK5+/-KOFN2.1hiPSCs,使用识别谱系特异性标记物(NSC的SOX-1、SOX-2和NESTIN,神经元的TUJ-1、MAP-5和MAP-2)的抗体,通过免疫荧光染色验证其表型。我们发现,CDK5/-KOhiPSC衍生的神经元的增殖率增加,同时NEUN和P35表达水平降低。然而,形态分析显示,CDK5缺乏导致主体长度增加,小学,和继发性神经突和神经元体细胞区。作为一个整体,我们发现,CDK5的缺陷并不损害hiPSC神经元分化,而是去调节增殖和神经突生长,有利于伸长。特定激酶的活性失调导致异常,例如神经退行性疾病中轴突连接受损。因此,旨在使激酶活性正常化的治疗方法,例如CDK5,可能有助于防止脆弱神经元的变性。
Cyclin-dependent kinase 5 (
CDK5) is a protein kinase involved in neuronal homeostasis and development critical for neuronal survival. Besides, its deregulation is linked to neurodegenerative pathologies such as Alzheimer\'s and Parkinson\'s diseases. For that reason, we aimed to generate a deficient
CDK5 genetic model in neurons derived from human-induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 technology. We obtained a heterozygous CDK5+/- clone for the FN2.1 hiPSC line that retained hiPSC stemness and pluripotent potential. Then, neural stem cells (NSCs) and further neurons were derived from the
CDK5+/- KO FN2.1 hiPSCs, and their phenotype was validated by immunofluorescence staining using antibodies that recognize lineage-specific markers (SOX-1, SOX-2, and NESTIN for NSCs and TUJ-1, MAP-5, and MAP-2 for neurons). We found that the proliferation rate increased in
CDK5+/- KO hiPSC-derived neurons concomitantly with a reduction in NEUN and P35 expression levels. However, the morphometric analysis revealed that
CDK5 deficiency caused an increase in the length of the main, primary, and secondary neurites and the neuronal soma area. As a whole, we found that a deficit in CDK5 does not impair hiPSC neuronal differentiation but deregulates proliferation and neurite outgrowth, favoring elongation. The misregulated activity of specific kinases leads to abnormalities such as impaired axonal connectivity in neurodegenerative diseases. Thus, therapeutic approaches aimed at normalizing the activity of kinases, such as CDK5, may help prevent the degeneration of vulnerable neurons.