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.

依赖细胞周期蛋白的激酶5（CDK5）是CDK家族的成员。与其他CDK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的关联，无论是p35还是其同工型p39，对于激酶活性至关重要。我们使用CDK5的常规基因敲除小鼠（CDK5 - / - ）分析了CDK5角色。 CDK5 - / - 小鼠由于有缺陷的神经元迁移而表现出与破坏皮质层状结构相关的胚胎致死性。此外，在CDK5 - / - 小鼠的神经元中观察到色液变化，例如带有偏心核的气球细胞体。这伴随着磷酸化的神经丝-H（PNF-H）免疫反应性的积累。 PNF-H的这种积累在颅神经核和脊髓腹侧神经元的运动神经元的细胞体内典型。除了CDK5在发育阶段的重要作用外，CDK5在成熟的CN中起多种功能，例如神经细胞骨架的磷酸化，突触传播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系统分析小鼠是否有条件地破坏CDK5基因，并通过控制空间和时间基因破坏（CDK5D/D）。这些CDK5D/d小鼠是可行且肥沃的，表现出运动异常的缓慢进展，包括弯曲的姿势。 CDK5 - / - 小鼠由于有缺陷的神经元迁移而表现出与破坏皮质层状结构相关的胚胎致死性。而p35 - / - 小鼠由于CDK5激活剂同工型的冗余而显示出比CDK5 - / - 小鼠较温和的表型。此外，p35 - / - p39 - / - 具有与CDK5 - / - 小鼠相同的表型的小鼠证实了这些异形体中的冗余。 BRDU的神经元出生日期标记显示CDK5 - / - 小鼠的脑皮质中的倒层结构。大脑皮层中层结构的倒置模式是卷轴和crambler/yotari的众所周知的特征。这些突变小鼠表现出几乎相同的表型，表明在这些突变体，reelin和dab1中突变的基因产物在皮质发育过程中起作用在公共信号传导途径中起作用。虽然CDK5 - / - 和p35 - / - 表现出与reeler和crambler/Yotari小鼠的某些相似之处，而CDK5 - / - / - 和P35 - / - 小鼠在胚胎大脑皮层的发展中也显示出与Reeler和Reeler和Scrambler/Yotari这样的显着差异作为预拆分的分裂。在野生型小鼠中，连续的迁移神经元的波动以内而外的方式形成皮质板，将预元素分为边缘区域和子板。在reeler和scrambler/yotari突变体中，迁移的皮质神经元似乎无法分裂预元素，而皮质板神经元在预序列下方的倒数顺序中堆叠起来。在CDK5 - / - 和p35 - / - 小鼠中，早期出生的神经元成功将预元素分开，但是，后期神经元在子板下方的倒立层中堆叠。在发育中的神经系统中已经描述了神经元迁移的两种一般模式：核（或索尔）易位（也称为核子运动）和运动。最近，使用Living Slice培养观察到了这两种径向迁移模式。基于我们在CDK5 - / - 小鼠的大脑皮层中的观察结果，我们提出，早期出生的神经元可能使用核易位模式，而核易位模式是与CDK5无关的，而后期神经元的迁移模式在大脑皮层中依赖于CDK5。最近，在CDK5 - / - 和p35 - / - p39 - / - 小鼠（包括缺乏下橄榄）中，已经报道了脑干结构的发育缺陷，但是，这些异常的迁移缺陷的表征尚待阐明。为了使CDK5依赖性和独立的神经元迁移模式表征，我们详细分析了CDK5 - / - 小鼠后脑中的神经元迁移。神经元迁移的选择性缺陷在面部核和下橄榄中鉴定出来，其余的其他脑干核正常形成，包括丘脑核，被认为是由核易位迁移模式形成的。由于已经在卷轴小鼠中描述了面部核和下橄榄的神经元迁移缺陷，因此使用双突变体小鼠P35和DAB1和DAB1突变小鼠，CDK5/p35与reelin信号的可能关系及其对神经元迁移的影响。 。微管相关蛋白TAU是一种发育调节的神经元磷蛋白家族。 TAU磷酸化的增加降低了其结合和稳定轴突微管的能力，从而使轴突生长的基础重排。活性OCDK5与其神经元激活剂p35和p39紧密地调节。尽管Tau可以在体外用CDK5磷酸化，但体内角色仍不清楚。我们已经证明，在大脑发育过程中，tau被CDK5/p39磷酸化，从而减少了其对微管的亲和力。 p39表达水平在胚胎后脑和脊髓以及产后脑皮质中较高，而p35的表达水平在整个大脑发育过程中最突出。 CDK5与p39相关的tau磷酸化的能力较高，而不是与p35相关。在CDK5 - / - 小鼠的大脑中，Ser-202和THR-205处的Tau磷酸化降低，但在p35 - / - 小鼠的大脑中却没有磷酸化，这表明CDK5/p39负责Tau的体内磷酸化。这表明CDK5可以通过TAU磷酸化以特定区域和发育调节的方式为微管提供更具动态的性能，这对于适当的大脑发育是必不可少的。肌萎缩性外侧硬化症（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被认为是依赖钙的介质之一，该介质在此死亡中观察到了神经元死亡。 CDK5与其激活剂p35或p39在神经元中激活。钙激活的蛋白酶钙蛋白酶将p35裂解至其截短的产物p25，最终导致细胞错误定位并延长CDK5激活。这种放置的CDK5诱导细胞骨架破坏和凋亡。为了检查抑制加拉蛋白在p35向p25的抑制p25是否会延迟ALS的疾病进展，我们产生了双转基因小鼠，其中在CDK5缺乏背景中表达了ALS连接的SOD1突变体。这些小鼠的表型提供了直接证据，表明突变体SOD1小鼠中运动神经元疾病的发病机理独立于p35或p25的CDK5激活。