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，以及为什么它们在神经退行性疾病中会断开连接？ 我们已经开发了一个哺乳动物神经退行性疾病的蛋白激酶Cdk5失活的果蝇模型。这是负责将tau磷酸化为神经元缠结中发现的形式的两种主要蛋白质之一的苍蝇同系物，神经元缠结是许多形式的人类神经变性的特征。今年，我们发表的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小鼠中观察到的神经退行性综合征之间的许多表型相似性，从而验证了我们的果蝇突变体作为哺乳动物疾病过程的有价值模型。我们还确定了在我们的果蝇模型中疾病进展早期发生的两个病理过程，这在以前的哺乳动物疾病研究中没有描述过。一个是未能分离神经元的部分，动作电位启动（轴突起始段），另一个是神经突完整性的调节改变，如发育程序性树突重塑的速率和发作缺陷所示。这两个过程都是很好的候选者，可能在哺乳动物疾病中发挥重要作用。因此，对我们来说，一个关键问题是确定它们在神经退行性变的整个过程中的重要性。作为第一步，我们现在已经开始剖析这些表型背后的分子机制。 轴突起始段的一个关键成分是支架蛋白锚蛋白。我们发现，神经元特异性锚蛋白的一个亚型，本地化的AIS和缺乏Cdk5活性的果蝇突变体未能显示这种本地化。我们还发现，锚蛋白是很重要的Cdk5依赖性AIS规范，因为降低或提高Ank2的表达模拟，分别，Cdk5的增益和功能AIS表型的损失。这有力地表明，Cdk 5对AIS形成和维持的作用易于部分地通过其对锚蛋白2的调节来介导。关于树突状细胞的稳定性，一个核心问题是建立在什么样的点在发育重塑过程中Cdk5活性起着调节作用。微管细胞骨架的溶解是已知的最早的，可能也是树突修剪的限速事件。我们的数据现在表明，Cdk5的行为上游的微管拆卸树突修剪。这将使Cdk5活性最早，在树突解体的起始事件。这一假设的一个关键测试是证明微管稳定性是否确实是上位Cdk5活性重塑。为此，我们已经开发并发表了一种新的方法，该方法支持在器官型培养中发育中的果蝇大脑的发育重塑。通过允许实时监测重塑过程和获得大脑的药理学操作，该技术为我们提供了无与伦比的时间分辨率，用于解剖Cdk5相关神经突拆卸之前和期间的事件序列。我们目前正在应用这种方法来验证或证伪的假设，树突的完整性的调节是一个中心，直接功能的Cdk5在体内。

We seek to answer two questions: how do neurons become connected during development, and why do they become disconnected during neurodegenerative disease? We have developed a 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 valuable model of the mammalian disease process. We also identified two pathological processes that occur early in disease progression in our fly model that had not previously been described in studies of the mammalian diseases. One is failure to segregate the portion of the neuron where action potentials initiate (the axon initial segment), the other is altered regulation of neurite integrity, as assayed by defects in the rate and onset of developmentally programmed dendritic remodeling. Both of these are good candidates for processes that could play an important role in mammalian disease. A key question for us, therefore, is to determine their importance in the overall process of neurodegeneration. As a first step we have now begun to dissect the molecular mechanisms underlying each of these phenotypes. A key component of the axon initial segment is the scaffolding protein ankyrin. We find that one isoform of the neuron-specific ankyrin, localizes to the AIS and that Drosophila mutants lacking Cdk5 activity fail to show this localization. We also find that ankyrin is apt to be important for Cdk5-dependent AIS specification, since lowering or raising Ank2 expression mimics, respectively, the Cdk5 gain and loss of function AIS phenotypes. This strongly suggests that the effect of Cdk5 on AIS formation and maintenance is apt to be mediated, in part, through its regulation of Ankyrin 2. Regarding dendritic stability, a central question is to establish at what point in the process of developmental remodeling Cdk5 activity plays a regulatory role. Dissolution of the microtubule cytoskeleton is the earliest known, and probably the rate-limiting, event in dendrite pruning. Our data now show that Cdk5 acts upstream of microtubule disassembly in dendrite pruning. This would make Cdk5 activity the earliest, initiating event in dendrite disassembly. A key test of this hypothesis is to demonstrate whether microtubule stability is indeed epistatic to Cdk5 activity in remodeling. To this end, we have developed and published a novel method that supports developmental remodeling of the developing Drosophila brain in an organotypic culture. By allowing both live monitoring of the remodeling process and access for pharmacological manipulation of the brain, this technique provides us with unparalleled temporal resolution for dissecting the sequence of events before and during Cdk5-associated neurite disassembly. We are currently applying this method to validate or falsify the hypothesis that regulation of dendrite integrity is a central, direct function of Cdk5 in vivo.