Understanding essential roles of microtubule regulators during synapse formation and maintenance

神经系统功能的一个关键先决条件是神经元通过称为突触的专门细胞连接与其他细胞通信的能力。突触包含复杂的机制，用于将信号快速传输到伙伴细胞。一旦形成，突触必须保持在可塑状态，突触的过早丧失被认为是衰老和神经退行性疾病中神经元衰退的潜在原因。然而，尽管如此重要，突触维持的机制却知之甚少。该项目的首要目标是提供这样的理解，从而弥补我们对大脑老化和退化过程的知识的重要空白。在神经元的生命过程中，其突触机制不断循环。为此，它的构建模块必须在神经元细胞体和非常远的突触（人类最远可达一米远）之间有效运输，仅通过称为轴突的索状神经元突起连接。突触蛋白沿着轴突的这种精确运动是通过马达蛋白实现的，马达蛋白结合到含有突触蛋白的运输囊泡上，并沿着由平行的微管束（MT）形成的沿着高速公路行进。MT是动态的丝状聚合物，在神经元的整个生命过程中不断构建和降解，并且这些过程必须被调节以维持适当的轴突运输。MT的数量需要得到很好的控制，它们必须具有正确的翻译后修饰（PTM）以促进正确的马达蛋白相互作用，并且它们必须保持其捆绑的组织-所有这些都是为了防止运输的阻塞或减缓。为此，MT通过MT结合蛋白（MTBP）进行调节，MTBP可以控制MT解聚/聚合、稳定、交联和PTM。因此，似乎很明显，MTBP，通过控制MT网络，可以调节轴突运输，因此也突触的维持和神经元的存活，这一致病链可以提供重要的解释，为什么一些MTBP与神经退行性疾病。然而，基于MTBP的突触维持机制仍然知之甚少。例如，MTBP Tau是几十年前发现的。它与阿尔茨海默病和额颞叶痴呆症有关，因此受到了广泛的研究。然而，它在健康和疾病中的功能仍然令人惊讶地知之甚少。这是由于支持MT调节的调节网络的复杂性和鲁棒性，这在实验上难以破译。为了解决这个问题，我使用了一种遗传模式生物，果蝇果蝇，我对这个系统以及其中神经元细胞骨架的作用和调节有着丰富的经验。我已经提供了大量的原则性证据，证明调节机制可以被破译并应用于高等动物。除了实验的巨大的顺从性和速度之外，果蝇细胞骨架研究的根本优势是可以组合地操纵和研究基因的效率。因此，在这个项目中，我利用了我的发现，即当与第二个MTBP（称为Shot）的丢失相结合时，tau的功能变得明显。只有在联合缺失时，才会出现新的表型，即由突触蛋白的轴突运输崩溃引起的突触的急剧丧失。这种表型提供了强大的读数来破译潜在的机制，这将是该项目的一个关键目标。此外，我将研究这些机制的神经元存活的相关性，并评估它们在小鼠神经元中的潜在保护作用。这项工作将解开重要的新机制的理解，将推进对大脑发育，衰老和退化的研究。

A key prerequisite for nervous system function is the capability of neurons to communicate with other cells via specialised cell junctions called synapses. Synapses contain complex machinery for rapid transmission of signals to partner cells. Once formed, synapses have to be maintained in a plastic state, and precocious loss of synapses is considered a potential cause of neuronal decay in ageing and in neurodegenerative diseases. However, in spite of this importance, the mechanisms underlying synaptic maintenance are very little understood. The overarching aim of this project is to deliver such understanding, thus bridging an important gap in our knowledge about processes of ageing and degeneration in the brain.During a neuron's life, its synaptic machinery is constantly recycled. To this end, its building blocks have to be efficiently transported between the neuronal cell body and the very distant synapses (up to a meter away in humans), connected only by a cable-like neuronal protrusion called the axon. Such precise movement of synaptic proteins along the axon is achieved by motor proteins which bind to transport vesicles containing synaptic proteins and trail along highways made out of parallel bundles of microtubules (MTs). MTs are dynamic filamentous polymers which are continuously built and degraded throughout a neuron's life, and these processes have to be regulated to sustain proper axonal transport. The number of MTs needs to be well controlled, they have to bear the right posttranslational modifications (PTMs) to promote the right motor protein interactions, and they have to maintain their bundled organisation - all so that blockage or slowdown of transport is prevented. For this, MTs are regulated through MT-binding proteins (MTBPs) which can control MT de/polymerisation, stabilisation, cross-linkage and PTMs. It seems therefore obvious that MTBPs, through controlling MT networks, can regulate axonal transport and consequently also synaptic maintenance and neuronal survival, and this causative chain could provide important explanations for why a number of MTBPs are associated with neurodegenerative disease. However, MTBP-based mechanisms of synaptic maintenance remain poorly understood. For example, the MTBP Tau was discovered several decades ago. It has been associated with Alzheimer's Disease and Frontotemporal Dementia and has therefore been intensely researched. However, its function in health and disease remains surprisingly poorly understood. This is due to the complexity and robustness of the regulatory networks underpinning MT regulation which are experimentally difficult to decipher. To tackle this problem I am using a genetic model organism, the fruit fly Drosophila, I have extensive experience with this system and the role and regulation of the neuronal cytoskeleton therein. I have provided substantial proof of principle that regulatory mechanisms can be deciphered and applied to higher animals. Apart from the enormous amenability and speed of experimentation, the fundamental advantage for cytoskeletal research in Drosophila is the efficiency with which genes can be manipulated and investigated in combination. Thus, on this project, I capitalise on my finding that functions of tau become apparent when combined with loss of a second MTBP, called Shot. Only upon combined deletion does a new phenotype occur consisting in dramatic loss of synapses caused by collapse of axonal transport of synaptic proteins. This phenotype provides robust readouts to decipher the underlying mechanisms, which will be one key objective of this project. In addition, I will study the relevance of these mechanisms for neuronal survival and assess their potential conservation in mouse neurons. This work will unlock important new mechanistic understanding that will advance research on brain development, ageing and degeneration.