Protein Phosphorylation And Regulation Of Cytoskeleton In Neuronal Systems

在我们以前的研究中，已经证明了神经元特异性中间丝蛋白（如神经丝（NF））的磷酸化在发育和地形学上受到严格调控，并且通常局限于轴突隔室，并选择性地发生在脯氨酸指导的丝氨酸（Ser）和苏氨酸（Thr）残基上。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。包括AD在内的神经退行性疾病是复杂的慢性疾病，涉及人脑中神经元网络的破坏。其中一个主要因素是区室特异性紧密调节激酶和磷酸酶。 AD a的主要病理诊断之一是过度磷酸化的tau和磷酸化-NF-M/H组装成神经元内成对螺旋丝（PHF）和聚集的磷酸化-NFP（pNFP）。有人提出，这种病理抑制轴突运输，并可能导致神经元凋亡。大量的位点特异性蛋白激酶与tau蛋白和神经丝磷酸化有关，而这些磷酸化又通过Ser/Thr蛋白磷酸酶，主要是蛋白磷酸酶2A（PP 2A）的活性来平衡。PP 2A在脑中与tau和NF共定位，并且可能是维持tau和pNF在神经元细胞体中处于正常去磷酸化状态的另一种关键磷酸酶。据报道，AD脑中PP 2A水平的mRNA显着下调。这表明PHF tau和聚集的pNFP在AD和ALS脑（和其他神经退行性疾病）中的积累也可能是由于不能使过度磷酸化的tau和-NFM/H去磷酸化。这刺激了模型系统的研究，其中PP 2A的去磷酸化活性被冈田酸抑制，诱导类似于AD病理的胞体中异常的细胞骨架磷酸化。磷酸酶抑制剂冈田酸，微胱氨酸和fostriecine（特定于PP 2A）诱导大鼠皮层神经元的NF-M/H的强大的核周磷酸化，而PP 2B抑制剂，环孢素，没有影响，表明PP 2A主要参与调节NF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PP 2A的磷酸酶激活剂。 我们已经证明，除了激酶的磷酸酶的活性，特别是蛋白磷酸酶2A的活性在AD和ALS中下调。另一种方法，我们已经提出，以减少病理负担是使用PP 2A激活剂作为治疗试剂。 为了评估PP 2A激活剂FTY 720（芬戈莫德）的作用，我们采用了冈田酸方案，其在原代大鼠皮层神经元中诱导NF-M/H的神经元胞体中的异常过度磷酸化。事实上，FTY 720以剂量和时间依赖性方式降低NF-M/H磷酸化神经丝的水平。在类似的实验中，在FTY 720处理的皮质神经元中Tau的异常过度磷酸化也减少。FTY 720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，其低浓度在激活PP 2A方面非常有效。此外，它成功地从冈田诱导的细胞凋亡中拯救细胞。 我们提出了动物模型，以探讨磷酸酶激活剂对AD和ALS进展的疗效。在一个这样的协议，成年大鼠注入冈田酸到背海马区诱导AD样综合征。我们认为ALS和AD转基因小鼠是研究PP 2A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.