Exploring the cell biology of neuronal ageing and the underlying mechanisms

随着预期寿命的增加，人们活得足够长，足以承受老龄化的负面影响。影响老年人并深刻影响生活质量的一个最重要的健康问题是由于脑组织腐烂导致的智力或认知能力下降。关于正常衰老过程中大脑功能的退化，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本质是什么？是什么（病理）机制导致了这些改变？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，并成为改善认知衰退策略发展的起点。据报道，老年大脑神经元的各种亚细胞变化包括蛋白质聚集、细胞骨架衰退、轴突萎缩和突触退化，最终导致神经元通信缺陷。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-蛋白酶体和自吞噬体-溶酶体系统，它们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者，并且在生物体水平上与长寿有关。我们假设这些途径通过调节细胞内降解系统影响神经元老化。要验证这一假设，需要老化的神经元模型，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（由于年龄的差异），费力和昂贵的，并且它引起了伦理问题。在这里，我们介绍果蝇大脑中衰老的神经元模型。果蝇表现出衰老的行为特征，比如学习能力下降。与这一现象相关联的是，我们的神经元模型显示出逐渐形成的形态特征，模仿了灵长类动物大脑衰老的显著特征。这些包括：1)Tau蛋白的缠结状聚集体使人联想到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)轴突营养不良的发生，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，进一步发展这些读数。更重要的是，我们将把我们的读数作为一个独特的机会来检验我们的工作假设。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。我们将通过模型中与年龄相关的读数提供的稳健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入神经元的亚细胞水平。一方面，这些研究将巩固我们的模型，并利用高效的实验手段和独特的能力来快速、经济地进行这类研究，为未来的神经元衰老研究奠定基础。另一方面，我们将提供从我们的假设中导出的机械内部。由于我们的模型显示了人类大脑衰老的特征，这种方法有很大的潜力来推进我们的知识，然后将其输入到翻译管道中。

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.