期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2017
卷号：114
期号：12
页码：E2466-E2475
DOI：10.1073/pnas.1614462114
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceMutations in TAR-DNA binding protein 43 (TDP-43), an RNA-binding protein (RBP) with multiple functions in RNA metabolism, cause amyotrophic lateral sclerosis (ALS), but it is uncertain how defects in RNA biology cause disease. Purified RNA-binding protein FUS and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) form liquid droplets in vitro through liquid-liquid phase separation. However, the biophysical properties of ribonucleoprotein (RNP) granules composed of wild-type (WT) or ALS-linked TDP-43 have not been studied in primary neurons. We show that TDP-43 WT RNP granules exhibit distinct biophysical properties depending on their axonal location, whereas granules formed by ALS-linked mutant TDP-43 are more viscous and show disrupted axonal transport dynamics. We propose the distinct biophysical properties of these neuronal RNP granules may reflect different maturational states and differential propensity for pathological transformation. Ribonucleoprotein (RNP) granules are enriched in specific RNAs and RNA-binding proteins (RBPs) and mediate critical cellular processes. Purified RBPs form liquid droplets in vitro through liquid-liquid phase separation and liquid-like non-membrane-bound structures in cells. Mutations in the human RBPs TAR-DNA binding protein 43 (TDP-43) and RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), but the biophysical properties of these proteins have not yet been studied in neurons. Here, we show that TDP-43 RNP granules in axons of rodent primary cortical neurons display liquid-like properties, including fusion with rapid relaxation to circular shape, shear stress-induced deformation, and rapid fluorescence recovery after photobleaching. RNP granules formed from wild-type TDP-43 show distinct biophysical properties depending on axonal location, suggesting maturation to a more stabilized structure is dependent on subcellular context, including local density and aging. Superresolution microscopy demonstrates that the stabilized population of TDP-43 RNP granules in the proximal axon is less circular and shows spiculated edges, whereas more distal granules are both more spherical and more dynamic. RNP granules formed by ALS-linked mutant TDP-43 are more viscous and exhibit disrupted transport dynamics. We propose these altered properties may confer toxic gain of function and reflect differential propensity for pathological transformation.
