Roles of Cdk5 in neurodevelopment and neurodegeneration

我们试图回答两个问题：神经元在发育过程中是如何连接的，为什么在神经退行性疾病期间它们会断开连接？ 我们之前开发了一个潜在的哺乳动物神经退行性疾病的果蝇模型，方法是使蛋白激酶CDK5失活。这是两种主要蛋白质之一的果蝇同源物，这两种蛋白质负责将tau磷酸化成在神经原纤维缠结中发现的形式，这是许多形式的人类神经退化的特征。今年，我们发表了一些数据，记录了果蝇这种内源性过程中的神经退行性综合征与在人类和老鼠身上观察到的神经退行性综合征之间的许多表型相似之处，从而验证了我们的果蝇突变体是哺乳动物疾病过程的模型。我们还扩展了我们对苍蝇模型中一种新的病理过程的分析，这种过程发生在疾病进展的早期，以前在哺乳动物疾病的研究中没有描述过。去年，我们发现，缺乏CDK5活性会阻止神经元分离动作电位启动的细胞部分(轴突起始部分)。这很可能与神经退行性变的机制有关，因为我们发现，随着果蝇年龄的增长，有缺陷的初始节段区域变得容易发展成粗大的轴突肿胀，后来成为组织学异常的焦点，如组织中的大空泡孔。我们现在发现，这不仅仅是CDK5的一种发育功能。今年，我们证明了CDK5是维持初始片段所必需的，因为引入CDK5的药物抑制剂会导致初始片段的急性丢失，即使它已经形成。 我们还启动了对CDK5失活后果的系统的、全基因组的研究。我们已经使用微阵列对具有或缺乏CDK5活性的成年果蝇进行了基因表达谱分析。我们通过分离幼蝇的RNA，在突变体出现明显的细胞或器官缺陷之前，将我们的努力集中在退化的早期事件上。初步分析发现，在缺乏CDK5活性的突变体中，存在两类基因表达变化。我们观察到对神经功能直接重要的基因的变化，包括感觉转导、突触传递和离子稳态。根据我们的表型分析，我们还观察到了与我们先前假设与CDK5功能相关的各种生物生理过程相关的基因表达的变化。这些包括蛋白质平衡、氧化还原状态、线粒体结构和功能以及应激敏感性。因此，微阵列分析作为表型分析的强大独立验证。此外，由于这种无偏见的全基因组阵列分析未能识别早期研究没有预测到的主要基因类别，这些数据表明我们已经确定了CDK5下游负责其病理后果的主要功能通路。

We seek to answer two questions: how do neurons become connected during development, and why do they become disconnected during neurodegenerative disease? We previously developed a potential Drosophila model of mammalian neurodegenerative disease by inactivating the protein kinase Cdk5. This is the fly homolog of one of the two main proteins responsible for phosphorylating tau into the form found in the neurofibrillary tangles that are characteristic of many forms of human neurodegeneration. This year, we published data documenting many phenotypic similarities between the neurodegenerative syndrome in this endogenous process of flies and that observed in humans and mice, thus validating our Drosophila mutant as a model of the mammalian disease process. We also extended our analysis of a novel pathological process that occurs early in disease progression in our fly model that had not previously been described in studies of the mammalian diseases. Last year, we found that lack of Cdk5 activity prevents a neuron from segregating the portion of the cell where action potentials initiate (the axon initial segment). This is apt to be relevant to the mechanism of neurodegeneration, since we find that the defective initial segment domain becomes prone to development of gross axonal swellings as the fly ages, and later is the focus of histological abnormalities such as large, vacuolated holes in the tissue. We now find that this is not just a developmental function of Cdk5. This year, we showed that Cdk5 is required chronically for maintenance of the initial segment, as introduction of pharmacologic inhibitors of Cdk5 cause acute loss of the initial segment even after it has formed. We also initiated systematic, genome-wide investigations into the consequences of Cdk5 inactivation. We have used microarrays to perform gene expression profiling of adult flies having, or lacking, Cdk5 activity. We have focused our efforts on early events in degeneration by isolating RNA from young flies, at a time before the onset of overt cellular or organismal defects in the mutants. Preliminary analysis reveals two classes of gene expression changes in mutants lacking Cdk5 activity. We observe alterations in genes directly important for neural function, including sensory transduction, synaptic transmission and ion homeostasis. We also observe changes in the expression of genes associated with a variety of organismal physiological processes we had previously hypothesized to be associated with Cdk5 function, based on our phenotypic analysis. These include proteostasis, redox state, mitochondrial structure and function and stress-sensitivity. The microarray analysis, therefore, serves as a powerful independent validation of the phenotypic analysis. Moreover, as this unbiased, genome-wide array analysis failed to identify major classes of genes not predicted by the earlier studies, these data suggest that we have identified the major functional pathways downstream of Cdk5 that are responsible for its pathological consequences.