"Feeling the Distance: Investigating the molecular mechanisms of intrinsic cell size sensing in neurons using stem cells, bioengineering and imaging"

神经元是一种特殊的细胞，它们相互连接，并与我们全身的其他细胞类型相连，并依赖于从我们的神经系统到其他器官的信息和命令。像任何其他细胞一样，它们的形状与它们的功能密切相关，而且由于一些靶器官有时可能距离大脑相当远，神经元本身必须跨越很远的距离。例如，脊髓运动神经元需要将脊髓与我们身体中的每一根肌肉纤维连接起来，有时还可能有轴突--神经元使用一根“电缆”连接到长度超过1米的其他细胞的长突起。这种极端的形状对神经元构成了挑战：资源(蛋白质和RNA)的主要生产中心在细胞体，但高需求和高消耗的主要部位在一个1米长的轴突的末端，这两个部位由一系列转运蛋白分子链连接。此外，轴突末端突触的需求需要与胞体成分的生产相匹配，但对于很长的轴突需求可能会迅速变化，细胞末端之间的巨大距离使得不可能运行一条“及时”生产线。出于这些原因，神经元采用了各种机制来创造局部生产和调节资源，使之远离细胞体进入轴突，使后者更加独立，能够缓冲需求的快速变化。其中一个关键问题是，神经元如何感知自己的长度，并决定实施这些依赖长度的适应，以及是否有一类特定的信号负责感知轴突长度，或者这一过程是否更受多个方面的供需之间复杂的相互作用的指导。这是基础神经生物学的一个基本问题，但对于理解几种人类疾病的基础也是非常相关的，因为在几种神经退行性疾病中，轴突远端的能量供需失衡似乎是一系列问题中较早观察到的事件，最终导致某些神经元的死亡，如肌萎缩侧索硬化症。到目前为止，系统地研究这一机制是复杂的，因为在动物模型中，不可能以简单的方式系统地改变轴突的长度，而细胞培养系统通常有非常短的神经元。因此，大多数提出的神经元感知能力的机制都集中在相对较短的轴突周围，通常远低于1 mm。我们开发了一种新颖的平台，结合生物工程、人类干细胞和先进的成像技术来创建长度可控的人类运动神经元的有序阵列，我们已经使用它成功地证明了当达到一定长度(即“阈值长度”)时，轴突中的几个重要机制发生了根本改变，因此我们完全准备系统地研究神经元大小感知背后的机制，以了解是什么决定了这种“阈值长度”。为此，我们建议使用我们的平台，系统地改变我们观察到的所有随着轴突长度变化的不同路径，以确定它们中是否有任何一个直接负责确定“阈值”长度，如果是，那么导致神经元实施这些适应的事件序列是什么。我们的假设是，将是ATP(细胞能量的货币单位和所有其他功能的基础)的动态将成为早期-如果不是第一个-反馈系统之一，它决定了能量水平在多长时间内不再可持续，需要实施更多的本地生产和维护流程。

Neurons are specialised cells that connect to each other and to other cells types across our bodies and rely information and commands from our nervous system to the other organs. Like any other cell, their shape is intimately linked to their function, and as some of the target organs can be sometimes at considerable distance from the brain, neurons themselves have to span vast distances. For example, spinal Motor Neurons need to connect the spinal cord with every single muscle fibre in our body and can sometime have axons -the long protrusions that neurons use a "cable" to connect to other cells" exceeding 1m in length. This extreme shape poses a challenge for the neurons: the main centre of production for resources (proteins and RNA) is in the cell bodies, but the main site of high demand and consumption is at the end of a 1m long axon, with the two sites connected by a chain of transporter molecules. Moreover, demands in the synapses at the end of the axon need to be matched by production of component in the soma, but for very long axons demands might change swiftly and the vast distance separating the end of the cell make it impossible to run a "just-in-time" production line.For these reasons, neurons have adopted mechanisms to create local production and regulation of resources away from the cell body and into the axon, rendering the latter more independent and capable of buffering fast changes in demand.One key question in all this, is how do neurons sense their own length and decide to enact these length-dependent adaptation, and whether there is one specific class of signals responsible for sensing axonal length or if the process is more guided by a complex interplay between supply and demand across multiple fronts. This is a fundamental question for basic neurobiology, but it is also very relevant for understanding the basis of several human diseases, as in several neurodegenerative disorders imbalances in supply and demand of energy at the far end of the axon seems to be some of the earlier observable events in the chain of problems that ends with the death of certain neurons, like in Amyotrophic Lateral Sclerosis.Up until now it has been complex to study this mechanism systematically, as in animal models it is not possible to systematically change the length of axons in a simple way, and cell culture systems generally have very short neurons. As a result, most of the proposed mechanisms for this sensing capacity of neurons is centred around relatively short axons, usually well below 1mm.We have developed a novel platform that combines bioengineering, human stem cells and advanced imaging to create ordered arrays of human motor neurons with controllable length up to and exceeding 1cm, which we have used to successfully demonstrate that several important mechanisms are fundamentally altered in the axons when a certain length is reached (i.e. "threshold length") and we therefore perfectly poised to systematically study the mechanisms behind neuronal size sensing, to understand what determines this "threshold length". To do so, we propose to use our platform and systematically alter all the different pathways we observed changing with the axonal length, to determine if any of them is directly responsible for determining the "threshold" length, and if so what is the sequence of events that leads the neurons to enact these adaptations. Our hypothesis is that it will be the dynamics of ATP (the cell's unit of currency for energy and basis of all other function) that will be one of the early -if not the first- feedback system, which determines at which length energy levels are no longer sustainable and more local processes for production and upkeep need to be implemented.