Delineating the role of Matrin-3 in driving aberrant liquid-liquid phase separation that underpins ALS/FTD

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Abstract Protein misfolding underpins several neurodegenerative disorders including frontotemporal dementia (FTD). In FTD the misfolding of several different RNA-binding proteins (RBPs) with prion-like domains, including TDP-43 and FUS, are implicated. Prion-like domains are intrinsically disordered, making these proteins highly aggregation-prone. RBPs are also known to undergo liquid-liquid phase separation (LLPS). LLPS is a physiological response that allows sequestration and protection of essential cellular components, and for RBPs, RNA is a key modulator of this process. However, the localized concentration of proteins can be quite high upon LLPS, further increasing aggregation propensity. Indeed, pathological inclusions of TDP-43 are found in over half of FTD patients. Further, many other proteins are known to co-aggregate with TDP-43, and synergistic corruption of protein homeostasis may underpin FTD. Recently Matrin-3 (MATR3) aggregation was implicated in FTD, and MATR3 has several key similarities and differences as compared to TDP-43 and FUS. However, MATR3 remains poorly understood, and conflicting results have been reported in different studies. We therefore aim to leverage our unique skillset to delineate the properties of MATR3 with respect to LLPS, RNA binding, and interactions with other FTD-linked proteins. Understanding these mechanisms will provide fundamental knowledge to inform new therapeutic development, particularly for new RNA therapeutics that might restore physiological LLPS. We hypothesize that corrupted binding of MATR3 to RNA and other FTD-linked proteins can synergize, driving FTD pathogenesis. In this proposal, we will investigate the specific mechanisms by which MATR3 undergoes LLPS and how its LLPS is perturbed in FTD. Further, we will investigate the specific mechanism by which LLPS leads to quality control malfunction in cells. We will also investigate how interactions of MATR3 with RNA and other FTD-associated proteins synergize in FTD. To test these ideas, my lab has developed a genetically tractable yeast system to probe the drivers of MATR3 toxicity and aberrant LLPS. We have also pioneered purification of full-length MATR3, which will allow us to reconstitute its putative interactions with RNA and other FTD-associated RBPs. Key findings will be validated in mammalian cells. In sum, we will meet the following aims: 1) Define the molecular determinants of Matrin-3 liquid-liquid phase separation, and how liquid-liquid phase separation is perturbed in ALS/FTD and 2) Elucidate the interactions of Matrin-3 with RNA and other ALS/FTD-associated proteins. Successful completion of this project will provide us with new fundamental knowledge of the factors modulating MATR3 LLPS, how MATR3 LLPS is corrupted in FTD, and how LLPS might be therapeutically modulated.