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.

细胞周期蛋白依赖性激酶 5 (Cdk5) 是 cdks 家族的成员。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 或其亚型 p39）的关联对于激酶活性至关重要。我们使用传统的 Cdk5 敲除小鼠 (Cdk5-/-) 分析了 Cdk5 的作用。 Cdk5-/- 小鼠表现出与由于神经元迁移缺陷导致的皮质层状结构破坏相关的胚胎致死性。此外，在 Cdk5-/- 小鼠的神经元中观察到染色质变化，例如具有偏心核的气球状细胞体。这伴随着磷酸化神经丝-H (pNF-H) 免疫反应性的积累。 pNF-H 的这种积累常见于颅神经核和脊髓腹侧神经元运动神经元的细胞体中。由于传统 Cdk5-/- 小鼠的胚胎致死性，尚未对这种神经元病理学进行进一步分析，以阐明成年 CNS 中的磷酸化异常。除了Cdk5在发育阶段的重要作用外，还表明Cdk5在成熟的CNS中发挥多种功能，例如神经细胞骨架的磷酸化、突触传递和多巴胺能信号传导。为了确定 Cdk5 在成人 CNS 中的作用，我们使用条件基因靶向策略（称为 Cre-loxP 系统）培育了用于破坏 Cdk5 基因的小鼠，该策略控制空间和时间基因破坏。我们使用鼠类 NF-H 启动子生成了 Cre 转基因小鼠（mNFH-cre-12 系），仅在神经元中空间表达 Cre 重组酶，并在出生前后暂时表达，以避免 cdk5 基因破坏造成的胚胎致死。我们通过将 Cdk5 floxed 小鼠与 mNF-H-cre-12 转基因小鼠杂交，成功创建了条件 KO 小鼠 (Cdk5D/D)。这些 Cdk5D/D 小鼠能够存活并且具有生育能力。这些小鼠还表现出缓慢的运动异常进展，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，导致其一生运动功能异常。 Cdk5-/- 小鼠表现出与由于神经元迁移缺陷导致的皮质层状结构破坏相关的胚胎致死性。而由于 Cdk5 激活剂亚型的冗余，p35-/- 小鼠表现出比 Cdk5-/- 小鼠更温和的表型。此外，具有与 Cdk5-/- 小鼠相同表型的 p35-/-p39-/- 小鼠证实了这些亚型中的冗余。 BrdU 标记的神经元出生日期揭示了 Cdk5-/- 小鼠大脑皮层的倒层结构。大脑皮层层结构的倒置模式是卷轴器和扰码器/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，表明这些突变体中分别突变的基因产物 Reelin 和 Dab1 在皮质发育过程中在共同的信号通路中发挥作用。虽然 Cdk5-/- 和 p35-/- 与 reeler 和 scrambler/yotari 小鼠有一些相似之处，但 Cdk5-/- 和 p35-/- 小鼠胚胎大脑皮层的发育也显示出与 reeler 和 scrambler/yotari 小鼠的显着差异，例如作为预板的分裂。在野生型小鼠中，连续波的迁移神经元以由内而外的方式形成皮质板，将前板分成边缘区和亚板。在 reeler 和 scrambler/yotari 突变体中，迁移的皮质神经元似乎无法分裂前板，并且皮质板神经元在前板下方以相反的顺序堆叠。在 Cdk5-/- 和 p35-/- 小鼠中，较早出生的神经元成功地分裂了前板，然而，较晚出生的神经元堆积在亚板下方的倒置层中。发育中的神经系统中神经元迁移的两种一般模式已被描述：核（或体）易位（也称为核运动）和运动。最近使用活体切片培养观察到大脑皮层中这两种径向迁移模式的存在。基于我们对 Cdk5-/- 小鼠大脑皮层的观察，我们提出，较早出生的神经元可能使用不依赖于 Cdk5 的核易位模式，而较晚出生的神经元在大脑皮层中的迁移模式则依赖于 Cdk5。最近，报道了 Cdk5-/- 和 p35-/-p39-/- 小鼠的脑干结构发育缺陷，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-/- 小鼠后脑的神经元迁移。在面核和下橄榄核中发现神经元迁移的选择性缺陷，其余脑干核团正常形成，其中脑桥核被认为是通过核易位迁移方式形成的。由于在 reeler 小鼠中已描述了面核和下橄榄神经元迁移缺陷，因此使用 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，从而允许轴突生长的微管重排。 oCdk5 的活性受到与其神经元激活剂 p35 和 p39 相关的严格调节。尽管 tau 在体外可以被 Cdk5 磷酸化，但其在体内的作用仍不清楚。在这里，我们发现 tau 在大脑发育过程中被 Cdk5/p39 磷酸化，导致其与微管的亲和力降低。 p39在胚胎后脑和脊髓以及出生后大脑皮层中的表达水平较高，而p35在整个大脑发育过程中的大脑皮层中最为突出。 Cdk5 tau 磷酸化的能力在与 p39 相关时比与 p35 相关时更高。 Cdk5-/- 小鼠大脑中 Ser-202 和 Thr-205 的 Tau 磷酸化减少，但 p35-/- 小鼠大脑中没有减少，这表明 Cdk5/p39 负责 tau 的体内磷酸化。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动态的特性，这对于大脑的正常发育是必需的。