TY - JOUR
T1 - Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation
AU - French, Rachel L.
AU - Grese, Zachary R.
AU - Aligireddy, Himani
AU - Dhavale, Dhruva D.
AU - Reeb, Ashley N.
AU - Kedia, Niraja
AU - Kotzbauer, Paul T.
AU - Bieschke, Jan
AU - Ayala, Yuna M.
N1 - Funding Information:
This work was supported by the President’s Research Fund of Saint Louis University Grant 230230, by Amyotrophic Lateral Sclerosis Association and ALS Finding a Cure, and by NINDS, National Institutes of Health Grant K01 NS082391. This work study was also supported in part by Alzheimer’s Dis-ease Core Center Grant P30AG013854 from NIA, National Institutes of Health to Northwestern University (Chicago, IL). The authors declare that they have no conflicts of interest with the contents of this article. The con-tent is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
This work was supported by the President's Research Fund of Saint Louis University Grant 230230, by Amyotrophic Lateral Sclerosis Association and ALS Finding a Cure, and by NINDS, National Institutes of Health Grant K01 NS082391. This work study was also supported in part by Alzheimer's Disease Core Center Grant P30AG013854 from NIA, National Institutes of Health to Northwestern University (Chicago, IL). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2019 French et al.
PY - 2019/4/26
Y1 - 2019/4/26
N2 - Aggregates of the RNA-binding protein TDP-43 (TAR DNA-binding protein) are a hallmark of the overlapping neurode-generative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The process of TDP-43 aggregation remains poorly understood, and whether it includes formation of intermediate complexes is unknown. Here, we analyzed aggregates derived from purified TDP-43 under semidenaturing conditions, identifying distinct oligomeric complexes at the initial time points before the formation of large aggregates. We found that this early oligomerization stageisprimarily driven by TDP-43's RNA-binding region. Specific binding to GU-rich RNA strongly inhibited both TDP-43 oligomerization and aggregation, suggesting that RNA interactions are critical for maintaining TDP-43 solubility. Moreover, we analyzed TDP-43 liquid-liquid phase separation and detected similar detergent-resistant oligomers upon maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, the ALS-linked TDP-43 mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that aggregates generated from purified TDP-43 seed intracellular aggregation detected by established TDP-43 pathology markers. Remarkably, cytoplasmic aggregate seeding was detected earlier for the A315T and M337V variants and was 50% more widespread than for WT TDP-43 aggregates. We provide evidence for an initial step of TDP-43 self-assembly into intermediate oligomeric complexes, whereby these complexes may provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.
AB - Aggregates of the RNA-binding protein TDP-43 (TAR DNA-binding protein) are a hallmark of the overlapping neurode-generative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The process of TDP-43 aggregation remains poorly understood, and whether it includes formation of intermediate complexes is unknown. Here, we analyzed aggregates derived from purified TDP-43 under semidenaturing conditions, identifying distinct oligomeric complexes at the initial time points before the formation of large aggregates. We found that this early oligomerization stageisprimarily driven by TDP-43's RNA-binding region. Specific binding to GU-rich RNA strongly inhibited both TDP-43 oligomerization and aggregation, suggesting that RNA interactions are critical for maintaining TDP-43 solubility. Moreover, we analyzed TDP-43 liquid-liquid phase separation and detected similar detergent-resistant oligomers upon maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, the ALS-linked TDP-43 mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that aggregates generated from purified TDP-43 seed intracellular aggregation detected by established TDP-43 pathology markers. Remarkably, cytoplasmic aggregate seeding was detected earlier for the A315T and M337V variants and was 50% more widespread than for WT TDP-43 aggregates. We provide evidence for an initial step of TDP-43 self-assembly into intermediate oligomeric complexes, whereby these complexes may provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.
UR - http://www.scopus.com/inward/record.url?scp=85065098459&partnerID=8YFLogxK
U2 - 10.1074/jbc.RA118.005889
DO - 10.1074/jbc.RA118.005889
M3 - Article
C2 - 30824544
AN - SCOPUS:85065098459
VL - 294
SP - 6696
EP - 6709
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 17
ER -