Protein Phosphorylation And Regulation Of Cytoskeleton In Neuronal Systems

我们已经证明，磷酸化的许多受体位点上的蛋白质，如Tau和神经丝（NF）的拓扑结构受到严格监管，一般局限于轴突室，并选择性地发生在脯氨酸指导的丝氨酸（Ser）和苏氨酸（Thr）残基。人们认识到，在神经退行性疾病，如阿尔茨海默病（AD）和肌萎缩性侧索硬化症（ALS），病理学的特征是异常磷酸化的细胞骨架蛋白在这些残基上的胞体中的积累，表明地形调节已受到损害。脯氨酸指导的Ser/Thr残基在神经系统中稳定磷酸化。最近，我们实验室的研究表明，肽基脯氨酰异构酶1（Pin 1）稳定了正常和应激神经元中的神经丝磷酸化。Pin 1选择性结合磷酸化的Ser/Thr-Pro残基，并将顺式异构体转化为更稳定的反式异构体。NF-M/H的多个SP重复以磷酸化特异性方式被Pin 1稳定。Pin 1调节兴奋毒性和氧化应激诱导的核周NF-M/H磷酸化 调节神经元细胞骨架蛋白的局部磷酸化的其他因子是磷酸酶。我们发现蛋白磷酸酶2A（PP 2A）在神经元胞体中高表达，冈田酸（OA）、微囊藻毒素LR和fostriecin抑制PP 2A活性可导致神经丝核周过度磷酸化，Pin 1 siRNA和显性（DN）Pin 1抑制Pin 1可抑制冈田酸（OA）诱导的神经丝核周异常磷酸化。为了确定OA处理的神经元中响应Pin 1抑制的NF异常过度磷酸化是否是NF激酶活化的结果，或者是PP 2A介导的NF去磷酸化的直接作用，我们在加入PP 2A抑制剂之前用激酶抑制剂（JNK、ERK和cdk 5）测试NF磷酸化。在加入OA之前，用JNK抑制剂（SP 600125）、ERK抑制剂（PD 98059）或Cdk 5抑制剂roscovitine（OA）对皮质神经元进行未处理和激酶抑制剂处理，没有改变磷酸化NF积累的水平，表明由PP 2A介导的NF的异常磷酸化不是由于JNK/ERK/Cdk 5激酶途径的激活。因此，Pin 1直接调节PP 2A介导的NF过度磷酸化。长期以来，磷酸化被认为是调节神经丝（NF）相互作用和轴突运输，进而影响轴突的稳定性和轴突的成熟。我们在原代皮层神经元中转染GFP-NF-H，并通过定量远端与近端轴突中GFP-NF-H的总水平以及通过实时监测GFP标记结构的运动来监测GFP-NF-H的转运，在OA治疗前后。转染的GFP NF-H很好地易位到正常神经元的轴突隔室中。当皮质神经元用OA或fostriecin（Fos）处理2小时时，NF易位被破坏，如仅在细胞体和近端轴突中通过GFP-NF-H观察到的。在OA/Fos处理的神经元中，由于细胞体中NF的过度磷酸化，NF转位被破坏。Pin 1的敲低挽救了OA/Fos处理的神经元中的NF轴突易位。这些结果表明Pin 1在NF动力学中可能发挥作用。这项研究强调了PP 2A通过Pin 1的一种新的信号传导作用，并暗示Pin 1作为一种治疗靶点，可以减少AD、PD和ALS等神经退行性疾病中神经丝蛋白的异常磷酸化。

We have demonstrated that phosphorylation of the numerous acceptor sites on such proteins as Tau and neurofilaments (NFs) were tightly regulated topographically and generally confined to the axonal compartment and selectively occurs on proline directed serine (Ser) and threonine (Thr) residues. It was recognized that in neurodegenerative disorders such as Alzheimers disease (AD) and Amyotrophic lateral sclerosis (ALS), the pathology was characterized by an accumulation of aberrantly phosphorylated cytoskeletal proteins in perikarya on these residues, suggesting that topographic regulation had been compromised. The proline directed Ser/Thr residues are stably phosphorylated in the nervous system. The mechanism of this stable phosphorylation is not understood.Recently, studies from our laboratory have shown that peptidyl prolyl isomerase 1 (Pin1) stabilizes the neurofilament phosphorylation in normal and stressed neurons. Pin1 selectively binds to the phosphorylated Ser/Thr-Pro residues and converts the cis isomers to the more stable trans isomers. The multiple SP repeats of NF-M/H are stabilized by Pin1 in a phosphorylation specific manner. Pin1 modulates the excitotoxic and oxidative stress induced perikaryal phosphorylation of NF-M/H. The other factors regulating the topographic phosphorylation of neuronal cytoskeletal proteins are phosphatases. We found that protein phosphatase 2A (PP2A) expression is high in neuronal cell bodies and inhibition of PP2A activity by okadaic acid (OA), microcystin LR and fostriecin leads to perikaryal hyperphosphorylation of neurofilaments.Inhibition of Pin1 by Pin1 siRNA and dominant (DN) Pin1 inhibits okadaic acid (OA) induced aberrant perikaryal phosphorylation of neurofilaments. In order to determine if aberrant hyperphosphorylation of NF in OA-treated neurons in response to Pin1 inhibition is a consequence of NF kinase activation, or a direct effect of PP2A mediated dephosphorylation of NF, we tested the NF phosphorylation with kinase inhibitiors (JNK, ERK and cdk5) prior to the addition of PP2A inhibitor. Non-treated and kinase inhibitor treatment of cortical neurons with JNK inhibitor (SP600125),ERK inhibitor (PD98059) or Cdk5 inhibitor, roscovitine (OA) prior to the addition of OA did not change the levels of phospho-NF accumulation, suggesting that the aberrant phosphorylation of NF mediated by PP2A is not due to the activation of JNK/ERK/Cdk5 kinase pathways. Thus, Pin1 directly modulates PP2A mediated hyperphosphorylation of NF. Phosphorylation has long been considered to regulate neurofilament (NF) interaction and axonal transport, and, in turn, influence axonal stability and their maturation of axons. We transfected GFP-NF-H in primary cortical neurons and monitored transport of GFP-NF-H by quantifying the total levels of GFP-NF-H in distal versus the proximal axons, as well as by real-time monitoring the movement of GFP-tagged structures, before and after treatment with OA. Transfected GFP NF-H is well translocated in to the axonal compartment in normal neurons. When cortical neurons are treated with OA or fostriecin (Fos) for 2 h, NF translocation is disrupted as observed by the GFP-NF-H in the cell bodies and proximal axons only. The NF translocation is disrupted in OA/Fos treated neurons due to the hyperphosphorylation of NF in the cell bodies. Knockdown of Pin1 rescues the NF axonal translocation in OA/Fos treated neurons. These results indicate a possible role of Pin1 in NF dynamics. This study highlights a novel signaling role of PP2A by Pin1 and implicates Pin1 as a therapeutic target to reduce aberrant phosphorylation of neurofilament proteins in neurodegenerative disorders such as AD, PD and ALS.