The effect of FUS phase transitions on local protein synthesis in axon termini and the implications for neurodegeneration

背景：肌萎缩侧索硬化症(ALS)是一种致命的神经退行性疾病，以运动神经元死亡为特征，导致肌肉无力，并随着时间的推移而恶化。尽管肌萎缩侧索硬化症的病因尚不清楚，但已有几个基因的突变与肌萎缩侧索硬化症有关。其中一个基因是编码在肉瘤中融合的蛋白质(FUS)的基因，这种蛋白质存在于神经元中。FUS是这个项目的重点。FUS的一个有趣的特性是它可以在不同的阶段之间转换。细胞内部是一个水环境，这意味着它由许多分子和离子在水中的溶液组成，FUS与水分子相互作用的方式可能会改变。它不仅可以在溶液中以单一蛋白质的形式存在，也可以以固体或聚集体的形式存在，它还可以进行相分离。这意味着FUS分子有时优先与水分子相互作用，因此在细胞内形成离散的液滴，就像水中的油一样。在某些疾病中，可溶的、固体的和相分离的状态之间的平衡会被破坏，但这种情况发生的确切细胞内机制尚不清楚。众所周知，神经元中FUS不同相状态之间的平衡失调可以影响其信号发送尖端-轴突终末中某些其他蛋白质的产生。一些蛋白质是在这些轴突末端使用特定于单一蛋白质的信使RNA(MRNA)分子在局部合成(产生)的，这一过程被称为翻译。FUS可以结合这些mRNA，这可能会影响它们的翻译，这种方式被认为取决于FUS的相位状态。据预测，与FUS相关的一种信使核糖核酸是烟酰胺/烟酸单核苷酸腺基转移酶2(NMNA2)。由于局部NMNA2的产生有助于维持轴突的健康，这可能在神经退行性疾病中发挥作用。方法：在这个项目中，我们将表征轴突末端的FUS相变，并将它们与局部蛋白质合成(LPS)的变化相关联，然后与细胞整体状态(其表型)的变化相关联。我们将测定nmnat2mRNA在表达正常(野生型)或突变的FUS的轴突中的定位和翻译率，以了解NMNA2蛋白水平的失调是否会导致轴突疾病。我们将建立多个模型系统来研究不同复杂程度的FUS介导的过程，并部署和开发适合分析这些系统的技术。我们将使用非洲爪哇视网膜神经节细胞作为适合于高分辨率成像的相关的内毒素神经元模型系统。我们的目标是使用原子力显微镜和单分子平移成像的组合来关联通过细胞粘度测量的内毒素和FUS相态，这是以前没有尝试过的。我们还将开发分析这些数据集所需的软件。在该项目的后期，我们的目标是通过使用单面照明显微镜在青蛙或鱼的早期胚胎中成像轴突，以成像它们在自然环境中的图像。与EPSRC的研究领域保持一致：这项研究属于EPSRC的生物物理学和软物质物理的研究领域。我们将研究蛋白质相变的生物物理现象，并在长度尺度的层次上追踪其影响。为此，我们将开发新的成像方法来定量研究生物系统。

Context: Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease, characterised by the death of motor neurons, which results in muscle weakness that worsens over time. Though the causes of ALS are unknown, mutations in several genes have been linked to ALS. One of these genes is the one encoding the protein 'Fused in sarcoma' (FUS), which is found in neurons. FUS is the focus of this project. One of FUS's interesting properties is that it can transition between different phases. The inside of the cell is an aqueous environment, which means it consists of many molecules and ions in solution in water, and the way FUS interacts with water molecules can change. Not only can it be found both as a single protein in solution and as a solid or even aggregate, it can also undergo phase separation. This means that FUS molecules sometimes preferentially associate with each other over the water molecules, and so form discrete droplets within the cell, like oil in water. The balance between the soluble, solid, and phase-separated states is disturbed in some diseases, but the exact intracellular mechanisms through which this occurs are unknown.It is known that a disturbance of the balance between FUS's different phase states in neurons can influence the production of certain other proteins in their signal-sending tips, the axon termini. Some proteins are locally synthesised (produced) in these axon termini, using messenger RNA (mRNA) molecules that are specific to a single protein, in a process known as translation. FUS can bind these mRNAs, which can influence their translation, in a way that is thought to depend on FUS's phase state. One mRNA that FUS is predicted to associate with is that specific to the protein nicotinamide/nicotinic acid mononucleotide adenylyltransferase 2 (NMNAT2). As local NMNAT2 production helps maintain axonal health, this may play a role in neurodegenerative diseases.Methodology: In this project, we will characterise FUS phase transitions in axon termini and correlate them with changes in local protein synthesis (LPS) initially, and later with changes in the cell's overall state (its phenotype). Localisation and translation rates of nmnat2 mRNA in axons expressing normal (wild-type) or mutant FUS will be determined, to understand whether dysregulation of NMNAT2 protein levels can cause axons to become diseased.We will set up multiple model systems to investigate FUS-mediated processes at different scales of complexity, and deploy and develop techniques suitable to analyse these systems. We will use Xenopus retinal ganglion cells as a relevant neuronal model system of LPS that is suitable for high-resolution imaging. We aim to use a combination of atomic force microscopy with single molecule translation imaging to correlate LPS with FUS phase state as measured through cellular viscosity, which has not been attempted beforehand. We will also develop the required software to analyse these datasets. Later on in the project, we aim to image axons in their native environment, by imaging them within early frog or fish embryos using single-plane illumination microscopy.Alignment with EPSRC research areas: This research falls under the EPSRC 'Biophysics and Soft Matter Physics' research area. We will study the biophysical phenomenon of protein phase transitions, and trace its effects across a hierarchy of length scales. To this purpose, we will develop new imaging methods to study biological systems in a quantitative manner.