Phosphorylation Of Neuronal Cytoskeleton In Neurodegener

Neurodegene 中神经元细胞骨架的磷酸化

• 批准号: 6814506
• 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/6814506
• 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; neural degeneration; 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. 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 begun to analyze the mice for conditional disruption of Cdk5 gene using Cre-loxP system, with controlling spatial and temporal gene disruption (Cdk5D/D). These Cdk5D/D mice were viable and fertile and showed slow progression of locomotor abnormalities including hunched back posture. 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 characerize Cdk5-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. We have demonstrated 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. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective loss of motor neurons in the brain and spinal cord. Neurotoxicity mediated by glutamate is thought to play a role in t he neuronal death through intrcellular calcium-dependent signaling cascades. Cdk5 has been proposed as one of the calcium-dependent mediators that mmay caiuse neuronal death observed in this death. Cdk5 is activated in neurons by the association with its activators, p35 or p39. The calcium activated protease calpain cleaves p35 to its truncated product, p25, which eventually causes the cellular mislocalization and prolonged activation of Cdk5. This deregualted Cdk5 induces cytoskeletal disruption and apopotosis. To examine whether inhibition of the calapin-medaited conversion of p35 to p25 can delay the disease progression of ALS, we generate a double transgenic mice in which ALS-linked SOD1 mutant was expressed in a Cdk5 deficient background. The phenotype of these mice provide a direct evidence that the pathogenesis of motor neuron disease in the mutant SOD1 mice is independent of the Cdk5 activation by p35 or p25.

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。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，我们已经开始使用Cre-loxP系统分析小鼠Cdk5基因的条件破坏，控制空间和时间基因破坏（Cdk5D/D）。这些Cdk5D/D小鼠是活的和可生育的，并且表现出包括驼背姿势在内的运动异常的缓慢进展。Cdk5-/-小鼠表现出胚胎致死性，与神经元迁移缺陷引起的皮质层状结构破坏有关。而p35-/-小鼠表现出比Cdk5-/-小鼠更温和的表型，这是由于Cdk5激活子亚型的冗余性。此外，表型与Cdk5-/-小鼠相同的p35-/-p39-/-小鼠证实了这些同工异构体中的冗余。BrdU标记的神经元出生日期揭示了Cdk5-/-小鼠大脑皮层的倒置层结构。在大脑皮层层结构的倒置模式是众所周知的特点，卷轴和扰频/yotari。这些突变小鼠表现出几乎相同的表型，这表明在这些突变体中分别发生突变的基因产物Reelin和Dab1在皮质发育过程中通过共同的信号通路起作用。虽然Cdk5-/-和p35-/-与reeler和scrambler/yotari小鼠表现出一些相似之处，但Cdk5-/-和p35-/-小鼠的胚胎大脑皮层发育也与reeler和scrambler/yotari小鼠表现出显著差异，如预板的分裂。在野生型小鼠中，连续的神经元迁移波以一种由内向外的方式形成皮质板，将预板分成边缘区和亚板。在reeler和扰频者/yotari突变体中，迁移的皮质神经元似乎无法分裂预板，皮质板神经元以相反的顺序堆积在预板下面。在Cdk5-/-和p35-/-小鼠中，较早出生的神经元成功地分裂了预板，然而，较晚出生的神经元在亚板下反向堆叠。在发育中的神经系统中，已经描述了两种一般的神经元迁移模式：核（或染色体）易位（也称为核分裂）和运动。这两种径向迁移模式在大脑皮层的存在是最近用活切片培养观察到的。根据我们对Cdk5-/-小鼠大脑皮层的观察，我们提出早出生的神经元可能使用不依赖Cdk5的核易位模式，而晚出生的神经元在大脑皮层的迁移模式是依赖Cdk5的。最近，在Cdk5-/-和p35-/-p39-/-小鼠中报道了脑干结构的发育缺陷，包括缺乏下橄榄，然而，这些异常的迁移缺陷的特征仍有待阐明。为了表征Cdk5依赖和独立的神经元迁移模式，我们详细分析了Cdk5-/-小鼠后脑中的神经元迁移。发现面核和下橄榄核存在神经元迁移的选择性缺陷，其余脑干核正常形成，包括脑桥核，认为脑桥核是通过核易位迁移方式形成的。由于在reeler小鼠中已经发现了面核和下橄榄树的神经元迁移缺陷，因此我们利用p35和Dab1双突变小鼠以及Dab1突变小鼠进一步研究了Cdk5/p35与Reelin信号传导的可能关系及其对神经元迁移的影响。微管相关蛋白tau是一个受发育调控的神经元磷酸化蛋白家族。tau磷酸化的增加降低了其结合和稳定轴突微管的能力，使微管重排成为轴突生长的基础。oCdk5的活性受其神经元激活剂p35和p39的密切调控。虽然Cdk5在体外可使tau磷酸化，但其在体内的作用尚不清楚。我们已经证明tau蛋白在大脑发育过程中被Cdk5/p39磷酸化，导致其对微管的亲和力降低。p39在胚胎后脑、脊髓和出生后大脑皮层的表达水平较高，而p35在整个大脑发育过程中在大脑皮层的表达水平最为显著。Cdk5磷酸化tau蛋白的能力与p39的相关性高于与p35的相关性。在Cdk5-/-小鼠大脑中，Ser-202和Thr-205位点的Tau磷酸化减少，而在p35-/-小鼠大脑中则没有，这表明Cdk5/p39是体内Tau磷酸化的原因。这表明Cdk5可能通过tau磷酸化以区域特异性和发育调节的方式为微管提供更多的动态特性，这对于大脑的正常发育是必要的。肌萎缩性侧索硬化症（ALS）是一种神经退行性疾病，其特征是大脑和脊髓中运动神经元的选择性丧失。谷氨酸介导的神经毒性被认为通过细胞内钙依赖性信号级联反应在神经元死亡中起作用。Cdk5已被认为是钙依赖性介质之一，可能导致在这种死亡中观察到的神经元死亡。Cdk5在神经元中通过与其激活因子p35或p39的关联而被激活。钙活化蛋白酶calpain将p35切割成其截短的产物p25，最终导致细胞错定位和Cdk5的延长活化。这种失调的Cdk5诱导细胞骨架破坏和细胞凋亡。为了研究抑制calapin介导的p35向p25的转化是否可以延缓ALS的疾病进展，我们产生了双转基因小鼠，其中ALS相关的SOD1突变体在Cdk5缺陷背景下表达。这些小鼠的表型提供了直接证据，表明SOD1突变小鼠运动神经元疾病的发病机制与p35或p25激活Cdk5无关。