Phosphorylation Of Neuronal Cytoskeleton

磷酸化过程在神经元细胞骨架的结构组织中起着重要作用。神经丝蛋白的合成、运输和组装在发育和空间上受特定的蛋白激酶的调节，这些蛋白广泛地磷酸化不同的基序，如在NF-M和NF-H的羧基末端尾部区域的Lys-Ser-Pro(KSP)重复序列。这种磷酸化稳定了轴突中的神经营养因子网络，并影响了神经元的轴突运输和传导速度。细胞周期蛋白依赖性激酶-5(CDK5)被认为能磷酸化NF和tau蛋白中的KSP基序。后者的磷酸化仅发生在阿尔茨海默病-S病脑中发现的tau蛋白中的相同位置。神经营养因子的异常磷酸化也与神经退行性疾病有关。我们之前已经报道了CDK5缺失小鼠的产生和特征。为了阐明CDK5在体内的功能，我们检查了这些缺失小鼠中由于CDK5基因中断而出现的神经发育缺陷。这些小鼠表现出一种新的表型，具有围产期致死性。CDK5基因缺失小鼠的大脑皮质分层模式与神经元的出生日期相反，丘脑皮质轴突的轨迹混乱。显然，受影响的是皮质神经元迁移的中间阶段。海马区有异常分层，小脑缺乏典型的叶状结构。脑干和脊髓表现为神经元的膨胀性变化。细胞骨架元素的异常磷酸化发生在CDK5基因缺失小鼠的脊髓和脑干的神经元胞体中。对CDK5缺失的胚胎干细胞来源的嵌合体的研究表明，CDK5对小脑中的神经元迁移具有细胞自主效应。有趣的是，p35基因缺失的小鼠也有倒置的大脑皮层分层模式和异常的轴突轨迹，但这些小鼠是存活的。P35基因缺失小鼠的小脑表现出细微的表型变化，但脑干、脊髓和周围神经系统没有受到影响。我们还开展了研究，以确定CDK5基因缺失小鼠神经系统中CDK5表达的靶向重建是否会导致表型逆转和围产儿死亡。我们还利用组织特异性表达策略确定了CDK5在大脑和外周器官中的功能意义。为了达到这些目标，我们建立并鉴定了p35启动子驱动的高表达CDK5的转基因小鼠(TgCDK5小鼠)。这些小鼠与CDK5杂合子小鼠杂交，获得了只在神经组织中表达CDK5的小鼠(TgKO)。TgKO小鼠出生时是正常的，没有表现出任何发育异常。CDK5表达的恢复逆转了脑干神经元胞体细胞骨架元素的异常磷酸化。这项研究证实了神经系统中CDK5的表达对胚胎发育和存活至关重要。为了阐明CDK5在神经退行性疾病中的作用，特别是在运动神经元中，我们产生了CDK5条件基因敲除，其中CDK5在成人脑中的表达被取消。为此，我们利用loxP系统，通过Cre重组酶的靶向表达，在出生后切除了CDK5基因。这些小鼠表现出异常的姿势和神经肌肉协调。我们的初步分析表明，在这些小鼠的脊髓运动神经元中，核因子-H的积聚与LASK CHAC的外向表达有关。我们仍在对这一表型进行详细的分析。N-甲基-D-天冬氨酸(NMDA)类谷氨酸受体对发育、突触传递、学习和记忆至关重要；它们是中枢神经系统病理性疾病的靶标。NMDA受体被丝氨酸/苏氨酸和酪氨酸激酶磷酸化。在此，我们在体外和完整细胞中证明了细胞周期蛋白依赖性激酶-5(CDK5)与丝氨酸1295处的NR2A亚单位结合并磷酸化。此外，我们发现选择性的CDK5抑制剂罗斯科维汀可以阻断大鼠CA1海马神经元的长时程增强(LTP)诱导和NMDA诱发的电流。这些结果表明，CDK5通过上调NMDA受体在突触传递和可塑性中起关键作用。

Phosphorylation process plays an important role in the structural organization of neuronal cytoskeleton. Synthesis, transport and assembly of neurofilament (NF) proteins are developmentally and spatially regulated by specific kinases that extensively phosphorylate different motifs such as Lys-Ser-Pro (KSP) repeats in the carboxyl-terminal tail domain of NF- M and NF-H. This phosphorylation stabilizes the NF network in the axon, and to affect the axonal transport and conduction velocity in the neurons. Cyclin dependent kinase-5 (Cdk5) is believed to phosphorylate KSP motifs in NF and tau protein. The later phosphorylation occurs exclusively at the same sites found in the tau protein from Alzheimer?s disease brain. Abnormal NF phosphorylation has also been associated with neuro-degenerative diseases. We have previously reported the generation and characterization of Cdk5 null mice. In order to elucidate the function of Cdk5 in vivo, we have examined neuro-developmental deficiencies that arise as a consequence of disruption of the Cdk5 gene in these null mice. These mice exhibit a novel phenotype with perinatal lethality. The cerebral cortical layering pattern of the Cdk5 null mice is inverted with respect to the birthdate of neurons, and the trajectories of the thalamocortical axons were disordered. Evidently it is the intermediate stage of cortical neuronal migration that are affected. There is an abnormal stratification in the hippocampus, and the cerebellum lacks typical foliation. The brain stem and the spinal cord display ballooning change of the neurons. Aberrant phosphorylation of cytoskeletal elements occurs in neuronal cell bodies of Cdk5 null mice in the spinal cord and the brain stem. Studies with Cdk5 null embryonic stem (ES) cell derived chimeras indicated a cell autonomous effect of Cdk5 on neuronal migration in the cerebellum. Interestingly, p35 null mice also have an inverted cerebral cortical layering pattern and aberrant axonal trajection, but these mice are viable. The cerebellum of the p35 null mice shows subtle phenotypic changes, but the brain stem, spinal cord and peripheral nervous system are unaffected. We also carried out studies to determine whether targeted reconstitution of Cdk5 expression in the nervous system of Cdk5 null mice leads to reversal of the phenotype and perinatal mortality. We also determined the functional significance of Cdk5 in the brain and peripheral organs using a tissue specific expression strategy. Towards these goals, we generated and characterized the transgenic mice overexpressing Cdk5 driven by p35 Promoter (TgCdk5 mice). These mice were crossed with Cdk5 heterozygous mice to obtain mice that only express Cdk5 in the nervous tissue (TgKO). The TgKO mice were born normal and did not exhibit any developmental abnormality. The restoration of Cdk5 expression reversed the aberrant phosphorylation of cytoskeletal elements in the soma of brain stem neurons. This investigation confirms that Cdk5 expression in the nervous system is critical for embryonic development and survival. In order to delineate the role of Cdk5 in neurodegenerative disorders and specially in motor neurons, we have generated the Cdk5 conditional knockouts in which Cdk5 expression is abrogated in the adult brain. For this purpose we utilized loxP system to excise Cdk5 gene postnatally by the targeted expression of Cre recombinase enzyme. These mice exhibit abnormal postures and neuromuscular coordination. Our initial analysis indicates accumulation of NF-H in motor neurons of spinal cord in these mice associated with lack ChAc exoression. We are still carrying out detailed analysis of this phenotype. Members of the N-methyl-D-aspartate (NMDA) class of glutamate receptors are critical for development, synaptic transmission, learning and memory; they are targets of pathological disorders in the central nervous system. NMDA receptors are phosphorylated by both serine/threonine and tyrosine kinases . Here, we demonstrate that cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at serine1295 in vitro and in intact cells. Moreover, we show that roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation (LTP) induction and NMDA-evoked currents in rat CA1 hippocampal neurons. These results suggest that Cdk5 plays a key role in synaptic transmission and plasticity through its up-regulation of NMDA receptors.