Phosphorylation Of Neuronal Cytoskeleton In Neurodegener

Neurodegene 中神经元细胞骨架的磷酸化

• 批准号: 6673990
• 负责人: Ashok B. KULKARNI
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6673990
• 关键词: Alzheimer's disease; NMDA receptors; amyotrophic lateral sclerosis; cell cycle proteins; cell migration; cyclin dependent kinase; cytoskeleton; developmental neurobiology; enzyme activity; enzyme induction /repression; gene targeting; laboratory mouse; motor neurons; neurofilament proteins; neurogenesis; phosphoproteins; phosphorylation

Cyclin-dependent kinase 5 (Cdk5) is a member of the family of cdks. Unlike other cdks, Cdk5 activity is detected mainly in postmitotic neurons. Association of Cdk5 with a neuron-specific regulatory subunit, either p35 or its isoforms p39, is critical for kinase activity. We have analyzed Cdk5 roles using conventional knockout mice of Cdk5 (Cdk5-/-). Cdk5-/- mice exhibit embryonic lethality associated with disruption of the cortical laminar structures due to defective neuronal migration. Additionally, chromatolytic changes such as a ballooned cell soma with eccentric nucleus were observed in neurons of Cdk5-/- mice. This is accompanied by an accumulation of phosphorylated neurofilament-H (pNF-H) immunoreactivity. This accumulation of pNF-H was typical seen in the cell soma of the motor neurons in the cranial nerve nuclei and spinal cord ventral neurons. Because of embryonic lethality of conventional Cdk5-/- mice, further analysis of this neuronal pathology has not been progressed to clarify this phosphorylation abnormality in adult CNS. Addition to the important roles of Cdk5 in developmental stage, it is also implicated that Cdk5 plays multiple functions in mature CNS such as phosphorylation of neurocytoskeletons, synaptic transmission and dopaminergic signaling . To determine the role of Cdk5 in adult CNS, we have generated the mice for disruption of Cdk5 gene using conditional gene targeting strategy, called Cre-loxP system, with controlling spatial and temporal gene disruption. We have generated the Cre transgenic mouse (mNFH-cre-12 line) using murine NF-H promoter for expressing the Cre recombinase spatially just only in neuron and temporarily from around birth to escape the embryonic lethality from cdk5 gene disruption. We have succeeded in creating conditional KO mice (Cdk5D/D) after crossing Cdk5 floxed mice and mNF-H-cre-12 transgenic mice. These Cdk5D/D mice were viable and fertile. These mice also showed slow progression of locomotor abnormalities including hunched back posture. We have also found that neuron-specific cdk5 gene disruption causes abnormal accumulation of pNF-H with decline of choline acetyltransferase (ChAT) immunoreactivity in motor neuron of spinal cord and the abnormal motor function in its life. Cdk5-/- mice exhibit embryonic lethality associated with disruption of the cortical laminar structures due to defective neuronal migrations. Whereas p35-/- mice showed milder phenotypes than Cdk5-/- mice due to the redundancy of Cdk5 activator isoforms. Moreover p35-/-p39-/- mice with the phenotype identical to Cdk5-/- mice confirm redundancy in in these isofroms. Neuronal birthdate labeling by BrdU revealed an inverted layer structure in cerebral cortex of Cdk5-/- mice. An inverted pattern of layer structure in the cerebral cortex is a well-known characteristic of the reeler and scrambler/yotari. These mutant mice exhibit nearly identical phenotypes suggesting that the gene products mutated in these mutants, Reelin and Dab1 respectively, act in a common signaling pathway during cortical development. While Cdk5-/- and p35-/- demonstrate some similarities with reeler and scrambler/yotari mice, the development of the embryonic cerebral cortex in Cdk5-/- and p35-/- mice also shows significant differences from reeler and scrambler/yotari such as the split of the preplate. In the wild-type mice, successive waves of migrating neurons form the cortical plate in an inside-out fashion, splitting the preplate into the marginal zone and subplate. In reeler and scrambler/yotari mutants, the migrating cortical neurons appear incapable of splitting the preplate, and cortical plate neurons stack up in the inverted order beneath the preplate. In Cdk5-/- and p35-/- mice, earlier-born neurons successfully split the preplate, however, late-born neurons stack up in an inverted layer under the subplate. Two general modes of neuronal migration have been described in the developing nervous system: nuclear (or somal) translocation (also called nucleokinesis) and locomotion. Presence of these two modes of radial migration in the cerebral cortex is recently observed using living slice culture. Based on our observation in the cerebral cortex of the Cdk5-/- mice, we proposed that earlier-born neurons might use nuclear translocation mode which is Cdk5-independent whereas migration mode of late-born neurons is Cdk5-dependent in the cerebral cortex . Recently, developmental defects of brain stem structures have been reported in Cdk5-/- and p35-/-p39-/- mice including the lack of inferior olive, however, characterization of migration defects of these abnormalities remains to be elucidated. In order to characerizeCdk5-dependent and independent modes of neuronal migrations, we analyzed neuronal migrations in the hindbrain of Cdk5-/- mice in detail. Selective defects of neuronal migration were identified in facial nucleus and inferior olive, and rest of other brainstem nuclei formed normally including pontine nucleus which is considered to be formed by nuclear translocation mode of migration. Since neuronal migration defects of facial nucleus and inferior olive have been described in reeler mice, a possible relation of Cdk5/p35 with Reelin signaling and its effect on neuronal migration were further studied using double mutant mice for p35 and Dab1 as well as Dab1 mutant mice. The microtubule-associated protein tau is a developmentally regulated family of neuronal phosphoproteins. The increase in tau phosphorylation reduces its ability to bind and stabilize axonal microtubules, allowing microtubule rearrangements underlying axonal growth. The activity oCdk5 is tightly regulated by association with its neuronal activators, p35 and p39. Although tau can be phosphorylated by Cdk5 in vitro, the in vivo roles remained to be unclear. Here, we show that tau is phosphorylated by Cdk5/p39 during brain development, resulting in a reduction of its affinity for microtubules. The p39 expression level was higher in embryonic hindbrain and spinal cord and in postnatal cerebral cortex while that of p35 was most prominent in cerebral cortex throughout brain development. The ability of Cdk5 for tau phosphorylation was higher in association with p39 rather than in association with p35. Tau phosphorylation at Ser-202 and Thr-205 was decreased in Cdk5-/- mice brains but not in p35-/- mice brains, suggesting that Cdk5/p39 is responsible for in vivo phosphorylation of tau. This suggests that Cdk5 may provide the microtubules with more dynamic property in a region-specific and developmentally regulated manner through the tau phosphorylation which would be necessary for a proper brain development.

周期蛋白依赖性激酶5 （Cdk5）是cdks家族的一员。与其他cdks不同，Cdk5活性主要在有丝分裂后神经元中检测到。Cdk5与神经元特异性调节亚基（p35或其同工异构体p39）的关联对激酶活性至关重要。我们使用传统的Cdk5敲除小鼠（Cdk5-/-）分析了Cdk5的作用。Cdk5-/-小鼠表现出胚胎致命性，与神经元迁移缺陷引起的皮质层状结构破坏有关。此外，在Cdk5-/-小鼠的神经元中观察到染色质的溶解变化，如球状细胞胞体和偏心核。这伴随着磷酸化神经丝- h （pNF-H）免疫反应性的积累。这种pNF-H的积累在脑神经核和脊髓腹侧神经元的运动神经元细胞体中是典型的。由于常规Cdk5-/-小鼠的胚胎致死性，对这种神经病理的进一步分析尚未取得进展，以阐明成年中枢神经系统中的这种磷酸化异常。除了在发育阶段发挥重要作用外，Cdk5在成熟中枢神经系统中还发挥多种功能，如神经细胞骨架磷酸化、突触传递和多巴胺能信号传导。为了确定Cdk5在成人中枢神经系统中的作用，我们使用条件基因靶向策略（称为Cre-loxP系统）生成Cdk5基因破坏小鼠，并控制空间和时间基因破坏。我们利用小鼠NF-H启动子培育了Cre转基因小鼠（mNFH-cre-12系），该启动子仅在神经元中空间表达Cre重组酶，并在出生前后暂时表达Cre重组酶，以避免cdk5基因破坏的胚胎致死性。我们通过将Cdk5粘接小鼠与mNF-H-cre-12转基因小鼠杂交，成功培育出条件型KO小鼠（Cdk5D/D）。这些Cdk5D/D小鼠是有活力和可生育的。这些小鼠还表现出运动异常的缓慢进展，包括驼背姿势。我们还发现神经元特异性cdk5基因破坏导致pNF-H异常积累，脊髓运动神经元胆碱乙酰转移酶（ChAT）免疫反应性下降，运动功能异常。