Abstract
Aggregates of TAR DNA-binding protein (TDP-43) are a hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although TDP-43 aggregates are an undisputed pathological species at the end stage of these diseases, the molecular changes underlying the initiation of aggregation are not fully understood. The aim of this study was to investigate how phase separation affects self-aggregation and aggregation seeded by pre-formed aggregates of either the low-complexity domain (LCD) or its short aggregation-promoting regions (APRs). By systematically varying the physicochemical conditions, we observed that liquid–liquid phase separation (LLPS) promotes spontaneous aggregation. However, we noticed less efficient seeded aggregation in phase separating conditions. By analyzing a broad range of conditions using the Hofmeister series of buffers, we confirmed that stabilizing hydrophobic interactions prevail over destabilizing electrostatic forces. RNA affected the cooperativity between LLPS and aggregation in a “reentrant” fashion, having the strongest positive effect at intermediate concentrations. Altogether, we conclude that conditions which favor LLPS enhance the subsequent aggregation of the TDP-43 LCD with complex dependence, but also negatively affect seeding kinetics.
Highlights
Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein implicated in transcriptional and post-transcriptional regulation of gene expression
low-complexity domain (LCD) of multiple proteins have been shown to undergo spontaneous liquid–liquid phase separation (LLPS) in the test tube, and this process is affected by conditions such as pH, salt concentration, molecular crowding and temperature [24,36]
We investigated the effect of total yeast RNA on the formation of TDP-43 LCD droplets in phosphate buffer (LLPS+ condition) using Differential interference contrast (DIC)
Summary
Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein implicated in transcriptional and post-transcriptional regulation of gene expression. TDP-43 regulates RNA metabolism, RNA transport and RNA splicing [1,2]. TDP-43 and neurodegenerative diseases was established when TDP-43 was found to be the major component of pathological ubiquitin-positive protein inclusions in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) [3,4]. Heterozygous missense mutations in the TAR DNA-binding protein (TARDBP). Gene encoding TDP-43 are a genetic cause of ALS [5,6]. TDP-43 aggregates have been observed in other neurodegenerative diseases [7,8,9].
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