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特殊细胞连接与其他细胞进行交流的能力。突触包含复杂的机制，用于向伙伴细胞快速传递信号。突触一旦形成，就必须保持在可塑性状态，而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。该项目的总体目标是提供这样的理解，从而弥合我们对大脑衰老和退化过程的知识的重要差距。在神经元的生命中，它的突触机制不断地循环。为此，它的组成部分必须在神经元细胞体和非常遥远的突触（在人类中可达一米远）之间有效地运输，这些突触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由运动蛋白实现的，运动蛋白与含有突触蛋白的运输囊泡结合，沿着由平行微管束（MTs）组成的高速公路行进。mt是动态的丝状聚合物，在神经元的整个生命周期中不断构建和降解，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，它们必须承受正确的翻译后修饰（PTMs）来促进正确的运动蛋白相互作用，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的去聚合、稳定、交联和PTMs。因此，mtbp通过控制MT网络，可以调节轴突运输，从而调节突触维持和神经元存活，这一致病链可以为为什么许多mtbp与神经退行性疾病相关提供重要解释。然而，基于mbp的突触维持机制仍然知之甚少。例如，MTBP Tau是在几十年前被发现的。它与阿尔茨海默病和额颞叶痴呆有关，因此得到了深入的研究。然而，它在健康和疾病中的作用仍然知之甚少。这是由于支持MT调节的调节网络的复杂性和鲁棒性，这在实验上很难破译。为了解决这个问题，我使用了一种遗传模型生物，果蝇，我对这个系统和其中的神经元细胞骨架的作用和调节有丰富的经验。我已经提供了大量的原理证据，证明调节机制可以被破译并应用于高等动物。除了实验的巨大适应性和速度外，果蝇细胞骨架研究的基本优势在于基因可以被操纵和组合研究的效率。因此，在这个项目中，我利用了我的发现，当第二个MTBP（称为Shot）缺失时，tau的功能变得明显。只有在联合缺失的情况下，新的表型才会发生，这种表型由突触蛋白轴突运输的崩溃引起突触的急剧损失。这种表型提供了可靠的读数来破译潜在的机制，这将是这个项目的一个关键目标。此外，我将研究这些机制与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。这项工作将开启重要的新机制理解，将推动对大脑发育、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.