Exploring the cell biology of neuronal ageing and the underlying mechanisms

随着预期寿命的增加，人们的寿命足够长，可以承受老龄化的负面影响。影响老年人并对生活质量产生深远影响的最重要的健康问题是由于脑组织衰退而导致的精神或认知能力下降。关于正常衰老过程中大脑功能退化的一些最重要但尚未解决的问题是：这些与年龄相关的神经元变化的性质是什么？什么（病理）机制导致改变？它们可以预防或治疗吗？这些问题的答案将为正常衰老过程中大脑功能的下降提供解释，并成为改善认知能力下降策略的起点。老年脑神经元的各种亚细胞变化包括蛋白质聚集、细胞骨架衰变、轴突萎缩和突触退化，最终导致神经元通讯缺陷。我们假设这些变化是由细胞内降解系统的缺陷引发的，即泛素-蛋白酶体和自体吞噬体-溶酶体系统，这两个系统都是清除细胞中不需要的和受损的蛋白质和细胞器所必需的。此外，TOR和IIS信号通路是已知的各种细胞过程的协调，并且在生物体水平上与寿命有关。我们假设这些通路通过调节细胞内降解系统来影响神经元衰老，验证这一假设需要神经元衰老模型，其中候选机制的操作可以与亚细胞过程的详细读数相结合。在哺乳动物物种中进行这样的研究是非常漫长的（由于年龄尺度），费力和昂贵的，它引起了伦理问题。在这里，我们介绍了一个神经元模型的衰老在大脑中的果蝇。果蝇表现出衰老的行为特征，如学习能力下降。与这种现象相关，我们的模型神经元显示出逐渐积累的形态特征，模仿灵长类动物大脑中突出的衰老标志。其中包括：1）Tau蛋白的缠结状聚集体，使人想起受阿尔茨海默氏症或衰老影响的大脑，2）轴突微管束的不稳定，3）营养不良轴突的出现，以及4）突触的衰退。在这个项目中，我们将通过结合细胞器动力学、轴突运输、超微结构分析和神经元活动的实时成像来进一步开发这个读出库。更重要的是，我们将把我们的读数作为一个独特的机会来测试我们的工作假设。为此，我们将利用果蝇的高效组合遗传学，并使用现成的遗传工具来操纵泛素-蛋白酶体和自噬体-溶酶体系统，以及TOR和IIS途径。我们将通过我们模型中与年龄相关的读数提供的鲁棒量化来评估这些操作是否以及如何影响神经元，从而将有关衰老机制的系统知识带到神经元的亚细胞水平。这些研究将巩固我们的模型，并建立其用于未来神经元老化研究的用途-具有高效的实验手段和独特的能力，快速和成本效益地进行这种研究。另一方面，我们将提供从我们的假设中得出的机械内部。由于我们的模型显示了从衰老的人类大脑中已知的特征，这种方法具有很大的潜力来推进我们的知识，然后可以将其输入翻译管道。

With the increase in life expectancy people live long enough to suffer from the negative effects of ageing. The single most important health problem affecting elderly people and profoundly impacting on the quality of life is mental or cognitive decline due to the decay of brain tissues. Some of the most important but unresolved questions about the deterioration of brain functions during normal aging are: What is the nature of these age-related alterations in neurons? What (patho-) mechanisms cause the alterations? Can they be prevented or treated? Answers to these questions will provide explanations for the decline in brain function during normal ageing, and be a starting point for the development of strategies to ameliorate cognitive decline. Various subcellular changes reported for neurons of aged brains include protein aggregation, cytoskeletal decay, axonal atrophy and deterioration of synapses culminating in neuronal communication deficits. We hypothesise that these changes are triggered by deficiencies in intracellular degradation systems, i.e. the ubiquitin-proteasome and auto-phagosome-lysosome systems, which are both necessary to clean cells from unwanted and damaged proteins and organelles. In addition, the TOR and IIS signalling pathways are known orchestrators of various cellular processes and have been linked to longevity at the organism level. We hypothesise that these pathways influence neuronal ageing through regulating the intracellular degradation system.Testing this hypothesis requires neuron models of ageing in which manipulations of candidate mechanisms can be combined with detailed readouts for subcellular processes. Performing such studies in mammalian species is extremely lengthy (due to age scales), laborious and expensive, and it raises ethical concerns. Here we introduce a neuron model of ageing in the brain of the fruit fly Drosophila. Drosophila shows behavioural features of ageing, such as a decline of learning performance. Correlating with this phenomenon, our model neurons display gradual build-up of morphological features that mimic prominent hallmarks of ageing in the primate brain. These include: 1) tangle-like aggregates of Tau protein reminiscent of brains affected by Alzheimer's or ageing, 2) destabilisation of axonal microtubule bundles, 3) the occurrence of dystrophic axons, and 4) the decay of synapses. On this project we will develop this repertoire of readouts further by incorporating live imaging of organelle dynamics, axonal transport, ultrastructural analysis and neuronal activity. More importantly, we will apply our readouts as a unique opportunity to test our working hypothesis. For this, we will capitalise on the highly efficient combinatorial genetics of Drosophila and use readily available genetic tools to manipulate the ubiquitin-proteasome and autophagosome-lysosome systems, as well as the TOR and IIS pathways. We will assess whether and how these manipulations affect neurons through robust quantification provided by the age-related readouts in our model, thus taking systemic knowledge about ageing mechanisms to the subcellular level of neurons.On the one hand, these studies will consolidate our model and establish its use for future studies of neuronal ageing - with highly efficient experimental means and the unique ability to perform such research fast and cost-effectively. On the other hand, we will provide mechanistic insides derived from our hypothesis. Since our model shows characteristic hallmarks known from the ageing human brain, this approach has a great potential to advance our knowledge which can then be fed into the translational pipeline.