Roles of Cdk5 in neurodevelopment and neurodegeneration

在过去一年中，我们在两个新的方向上扩大了我们在这一项目中的努力。 - 在过去的几年里，我们发现果蝇的转录组学分析为识别和解剖涉及神经变性、衰老和死亡的过程提供了非常丰富的静脉。然而，转录组是从生物体的实际生理学中移除的几个步骤。 转录变化必须通过剪接、RNA稳定性、翻译、蛋白质定位、翻译后修饰和其他过程进行过滤，然后才能在动物生理学中读出其后果。因此，我们认为有必要用另一种更接近动物生理学的系统测量来补充我们的转录组学研究。为此，我们与NIH Metabolomics Core合作，在野生型动物和由于Cdk 5/p35活性改变而注定要经历神经变性的动物中，查询果蝇整个生命周期的完整代谢组，并在存在和不存在细菌微生物组的情况下进行。这些实验才刚刚开始，但我们相信，它们将为衰老、疾病和死亡带来的生理变化提供有价值的、意想不到的见解。 - 与其他研究人员的大量证据一致，我们以前的转录组学研究将线粒体功能作为与衰老和神经退行性变相关的关键细胞通路。因此，在过去的一年中，我们已经开始详细分析线粒体的定位和活动在苍蝇的大脑，无论是在野生型和果蝇与改变Cdk 5/p35活性，重点是大脑的区域，我们知道是敏感的Cdk 5相关的变性。初步实验表明，在注定要经历变性的果蝇中，线粒体在轴突的不同部分的差异定位。我们还获得并表达了线粒体代谢和功能的不同方面的生物探针，包括氧化还原状态，ATP/ADP比率和NAD+/NADP比率的不同标志物。初步实验验证了这些生物探针的活性，并表明在大脑退化敏感区域的线粒体功能确实可能发生变化。这些实验需要重复和扩展。如果得到验证，未来的实验将致力于测试我们观察到的线粒体效应是否具有致病性，或者它们是否反映了旨在保护大脑免受损伤的补偿机制。

In the past year, we have expanded our efforts in this project in two new directions. - Over the previous several years, we had found that transcriptomic profiling of Drosophila offered an exceptionally rich vein for identifying and dissecting processes involved in neurodegeneration, aging, and mortality. However, the transcriptome is several steps removed from the actual physiology of the organism. Transcriptional changes have to be filtered through splicing, RNA stability, translation, protein localization, post-translational modification and other processes before their consequences can be read-out in animal physiology. We therefore felt it was essential to complement our transcriptomic studies with another systems measure that is closer to animal physiology. To that end, we have initiated a collaboration with the NIH Metabolomics Core to query the complete metabolome of Drosophila across the lifespan, both in wild type animals and in animals destined to undergo neurodegeneration due to altered activity of Cdk5/p35, and to do so in both the presence and absence of a bacterial microbiome. These experiments are just beginning, but we are confident that they will provide valuable and unexpected insights into the physiological changes that accompany aging, disease, and mortality. - Consistent with a great deal of evidence from other investigators, our previous transcriptomic studies had targeted mitochondrial function as a key cellular pathway associated with aging and neurodegeneration. Therefore, in the past year we have initiated detailed analysis of mitochondrial localization and activity in the fly brain, both in wild type and in flies with altered Cdk5 /p35 activity, focusing on regions of the brain that we know to be sensitive to Cdk5-associated degeneration. Preliminary experiments suggest differential localization of mitochondria to different portions of the axon in flies destined to undergo degeneration. We have also obtained and expressed bioprobes for different aspects of mitochondrial metabolism and function, including different markers of redox state, ATP/ADP ratio, and NAD+/NADP ratio. Preliminary experiments validate the activity of those bioprobes and suggest that there may indeed be changes in mitochondrial function in degeneration-sensitive areas of the brain. These experiments need to be repeated and extended. If validated, future experiments will be directed toward testing whether mitochondrial effects we observe are pathogenic in nature, or whether they reflect compensatory mechanisms meant to protect the brain from damage.