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？ 我们已经建立了一种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一个有价值的模型。我们还确定了在我们的苍蝇模型中疾病进展早期发生的两个病理过程，这两个过程以前在哺乳动物疾病的研究中没有描述过。一个是未能分离动作电位起始的神经元部分(轴突初始段)，另一个是轴突完整性的调节改变，通过发育程序性树突重塑的速度和起始时间的缺陷来衡量。这两个都是在哺乳动物疾病中发挥重要作用的过程的很好候选者。因此，我们面临的一个关键问题是确定它们在整个神经退化过程中的重要性。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ankyrin。我们发现神经元特异性锚蛋白的一种异构体定位于AIS，而缺乏CDK5活性的果蝇突变体没有表现出这种定位。我们还发现，Ankyrin对于依赖CDK5的AIS规范往往是重要的，因为降低或提高Ank2的表达分别模拟了CDK5功能AIS的获得和丢失表型。这有力地表明，CDK5在AIS形成和维持中的作用可能部分是通过其对Ankyrin 2的调节来实现的。关于树突状细胞的稳定性，一个中心问题是确定在发育重塑过程中，CDK5活性在什么时候发挥调节作用。在树突修剪中，微管细胞骨架的溶解是已知的最早的，也可能是速度限制的事件。我们的数据现在表明，在树突修剪过程中，CDK5作用于微管分解的上游。这将使CDK5活性成为树突分解过程中最早的启动事件。对这一假说的一个关键检验是证明微管稳定性是否确实与重构中的CDK5活性上位相关。为此，我们开发并发表了一种新的方法，支持在器官型培养中对发育中的果蝇大脑进行发育重建。通过实时监测重塑过程和对大脑进行药物操作，这项技术为我们提供了无与伦比的时间分辨率，可以解剖CDK5相关轴突分解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这一假设，即树突完整性的调节是体内CDK5的中心、直接功能。

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.