Protein Phosphorylation And Regulation Of Cytoskeleton In Neuronal Systems

我们以前的研究已经证明，神经元特异的中间丝蛋白，如神经丝蛋白(NFs)的磷酸化受到发育和地形图的严格调控，一般局限于轴突间隔室，选择性地发生在脯氨酸导向的丝氨酸(Ser)和苏氨酸(Thr)残基上。人们认识到，在阿尔茨海默病(AD)和肌萎缩侧索硬化症(ALS)等神经退行性疾病中，病理特征是核周异常磷酸化的细胞骨架蛋白在这些残基上积聚，这表明地形调节受到了损害。包括阿尔茨海默病在内的神经退行性疾病是一种复杂的慢性疾病，涉及到人脑中神经网络的破坏。其中一个主要因素是隔室特异性的严密调节激酶和磷酸酶。 阿尔茨海默病的主要病理诊断之一是过度磷酸化的tau和磷酸化的核因子-M/H组装成神经元内的成对螺旋细丝(PHF)和聚集性的磷酸化NFP(PNFP)。据认为，这种病理抑制了轴突的运输，并可能导致神经元凋亡。在tau和神经丝的磷酸化过程中，广泛的位点特异性蛋白激酶被涉及，而这些磷酸化又被丝氨酸/苏氨酸蛋白磷酸酶，主要是蛋白磷酸酶2A(PP2A)的活性所平衡。PP2A在大脑中与tau和nfs共定位，可能是另一种关键的磷酸酶，在神经元胞体中维持tau和pNf处于正常的去磷酸化状态。已有研究表明，阿尔茨海默病患者脑组织中PP2A基因的表达水平显著下调。这表明PHF tau和聚集的pNFP在AD和ALS(以及其他神经退行性疾病)中的积累，也可能是由于未能对过度磷酸化的tau和-NFM/H进行去磷酸化所致。这刺激了对模型系统的研究，在该模型系统中，PP2A的去磷酸化活性被冈田酸抑制，诱导类似AD病理的核周细胞骨架异常磷酸化。磷酸酶抑制剂冈田酸、微胱氨酸和Fostriecine(PP2A特异性)可诱导大鼠皮质神经元核周核因子-M/H的强阳性磷酸化，而PP2B抑制剂环孢菌素则无此作用，提示PP2A主要参与调节核因子的磷酸化。很明显，AD治疗的另一种合理方法是研究磷酸酶激活剂的疗效，特别是那些特异性激活PP2A的药物。 我们已经证明，在AD和ALS中，除了蛋白激酶外，磷酸酶的活性，特别是蛋白磷酸酶2A的活性被下调。我们提出的另一种减轻病理负担的方法是使用PP2A激活剂作为治疗试剂。 为了评价PP2A激动剂FTY720对Fingolimod的作用，我们采用冈田酸的方法诱导原代大鼠皮质神经元核周的NF-M/H异常过度磷酸化，FTY720确实以剂量和时间依赖的方式降低了NF-M/H磷酸化神经丝的水平。在一个类似的实验中，FTY720处理的皮质神经元中Tau的异常过度磷酸化也被减少。FTY720是一种免疫抑制剂，广泛用于预防器官移植排斥反应。值得注意的是，它的低浓度在激活PP2A方面非常有效。此外，它还成功地将细胞从冈田克诱导的凋亡中解救出来。 我们建立动物模型来探讨磷酸酶激活剂在AD和ALS进展中的作用。在一个这样的方案中，成年大鼠将冈田酸注入背侧海马区，诱导了一种类似AD的综合症。我们认为ALS和AD转基因小鼠将是研究PP2A激活剂治疗作用的理想模型。

In our previous studies it has been demonstrated that phosphorylation of the neuron specific intermediate filament proteins, such as neurofilaments (NFs) were tightly regulated developmentally and 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. Neurodegenerative diseases including AD are complex and chronic disorders that involve the disruption of the neuronal network in the human brain. One of the major factors is the compartment specific tightly regulate kinases and phosphatases. One of the principle pathologies diagnostic of AD a is hyperphosphorylated tau and phospho-NF-M/H assembled into intraneuronal paired helical filaments (PHF) and aggregated phospho-NFPs (pNFPs). It is proposed that this pathology inhibits axonal transport and may lead to neuronal apoptosis. An extensive repertoire of site-specific protein kinases have been implicated in tau and neurofilament phosphorylation which are balanced, in turn, by the activity of Ser/Thr protein phosphatases, principally protein phosphatase 2A (PP2A). PP2A is colocalized with tau and NFs in the brain and is probably a key another phosphatase that maintains tau and pNFs in a normal dephosphorylated state in the neuronal cell bodies. It has been reported that the mRNA of PP2A level is significantly down regulated in AD brains. This suggests that accumulation of PHF tau and aggregated pNFPs in AD and ALS brains (and other neurodegenerative disorders), may also result from the failure to dephosphorylate hyperphosphorylated tau and -NFM/H. This has stimulated the study of model systems in which dephosphorylating activity of PP2A, inhibited by okadaic acid, induces aberrant cytoskeletal phosphorylation in perikarya resembling AD pathology. Phosphatase inhibitors okadaic acid, microcystine and fostriecine (specific to PP2A) induce robust perikaryal phosphorylation of NF-M/H in rat cortical neurons, whereas, the PP2B inhibitor, cyclosporine, has no effect, suggesting that PP2A is principally involved in modulating NF phosphorylation. It has become evident that another rational approach to AD therapy is a study of the efficacy of phosphatase activators, particularly those that specifically activate PP2A. We have demonstrated that in addition of kinases the activity of phosphatase, specifically , protein phosphatase 2A activity is down regulated in AD and ALS. The other approach, we have proposed to reduce the pathological burden is to use PP2A activators as therapeutic reagents. To evaluate the effects of the PP2A activator FTY720, fingolimod, we employed an okadaic acid protocol that induces an aberrant hyperphosphorylation in neuronal perikarya of NF-M/H in primary rat cortical neurons, Indeed FTY720 reduced the level of NF-M/H phosphorylated neurofilaments in a dose and time dependent manner. In a similar experiment the aberrant hyperphosphorylation of Tau was also reduced in FTY720 treated cortical neurons. FTY720, is an immunosuppressant, extensively used to prevent organ transplant rejection. It is important to note that its low concentration is highly effective in activating PP2A. Furthermore, it succeeds in rescuing cells from okadaic-induced apoptosis. We propose animal models to explore the efficacy of phosphatase activators on the progress of AD and ALS. In one such protocol, adult rats infused with okadaic acid into the dorsal hippocampus region induced an AD-like syndrome. We propose the ALS and AD transgenic mice will be an ideal model to study therapeutic effects of PP2A activators.