Liquid-Liquid Phase Separation of TDP-43 and FUS in Physiology and Pathology of Neurodegenerative Diseases

Abstract

Liquid-liquid phase separation of RNA-binding proteins mediates the formation of numerous membraneless organelles with essential cellular function. However, aberrant phase transition of these proteins leads to the formation of insoluble protein aggregates, which are pathological hallmarks of neurodegenerative diseases including ALS and FTD. TDP-43 and FUS are two such RNA-binding proteins that mislocalize and aggregate in patients of ALS and FTD. They have similar domain structures that provide multivalent interactions driving their phase separation in vitro and in the cellular environment. In this article, we review the factors that mediate and regulate phase separation of TDP-43 and FUS. We also review evidences that connect the phase separation property of TDP-43 and FUS to their functional roles in cells. Aberrant phase transition of TDP-43 and FUS leads to protein aggregation and disrupts their regular cell function. Therefore, restoration of functional protein phase of TDP-43 and FUS could be beneficial for neuronal cells. We discuss possible mechanisms for TDP-43 and FUS aberrant phase transition and aggregation while reviewing the methods that are currently being explored as potential therapeutic strategies to mitigate aberrant phase transition and aggregation of TDP-43 and FUS.

Keywords: ALS; FUS; TDP-43; liquid-liquid phase separation; stress granules.

FIGURE 1

Domain architecture of TDP-43 and FUS. TDP-43 and FUS contain similar structural domains with some notable differences. Both contain a prion-like domain (PrLD), which is characterized by a high concentration of glycine, glutamine, asparagine, and tyrosine amino acids with low sequence complexity; however, the TDP-43 PrLD contains one tyrosine residue whereas the FUS PrLD is comprised of multiple tyrosines that can be phosphorylated. Additionally, the FUS PrLD overlaps with an arginine-glycine-rich (RGG) domain between residues 165 and 267. Notably, FUS contains multiple RGG domains throughout its structure in addition to a zinc-finger motif (ZnF). Both TDP-43 and FUS contain nuclear localization signals (NLS), although they differ in sub-classifications and subsequent nuclear import receptor interactions. Both proteins also contain RNA recognition motifs (RRMs), which are important domains for RNA-binding. Created with BioRender.com.

FIGURE 2

Phase-separated membraneless organelles have distinct components and functions. Schematic of membraneless organelles implicated in TDP-43 and/or FUS biology. Neurons contain nucleoli, which are condensed regions associated with ribosomal RNA synthesis. TDP-43 and FUS have been observed to localize to the nucleolus, which is commonly marked by fibrillarin and rRNA (see Nucleoli). Cajal bodies (CBs) are closely associated with nucleoli and commonly found in neurons. Major CB markers include coilin and the presence of small Cajal body-specific RNA (scaRNA). While not demonstrated to colocalize, TDP-43 is implicated in CB formation and regulation (see Cajal Bodies). Another nuclear membraneless organelle relevant to TDP-43 and FUS biology includes paraspeckles (see Paraspeckles). The long non-coding RNA (lncRNA) NEAT1 is an integral RNA component of paraspeckle organization, and these condensates are commonly marked by NONO. TDP-43 has been demonstrated to be important regulator of paraspeckle formation and it is shown to localize to the shell of paraspeckles, whereas FUS is generally localized to the core (West et al., 2016). Cytoplasmic stress granules are recently implicated in neurodegenerative diseases, and G3BP1 has been demonstrated as a central protein involved in SG formation (Yang et al., 2020). Stress granules stall protein translation and mRNA is a common RNA component of these condensates. Both TDP-43 and FUS demonstrate SG recruitment in neurons (see Stress Response). A final membraneless organelle covered in this review includes transport granules. These granules are observed in dendrites and axons of neurons. TDP-43 and FUS are both implicated in their formation, and these condensates often include mRNA, which is shuttled away from the soma for local translation (see Transport Granules and Local Translation). Created with BioRender.com.

FIGURE 3

Reversing aberrant phase transition of nuclear RNA-binding proteins (RBPs) as a potential therapeutic option for patients with ALS/FTD. In ALS/FTD, nuclear RBPs are first mislocalized to the cytoplasm. In the cytoplasm, RBPs can undergo reversible liquid-liquid phase separation (LLPS) to form liquid condensates. In some cases, RBPs undergo aberrant phase transition into a solid-like phase where the RBPs can assemble into fibrils. Multiple methods of disassembling and disaggregating proteins are proposed, including the use of proteins, RNAs, and small molecules. These biomolecules could potentially reverse aberrant phase transition and allow for subsequent nuclear localization of the RBPs by canonical mechanisms. Created with BioRender.com.