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

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.