Liquid-Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation

Donya Pakravan; Emiel Michiels; Anna Bratek-Skicki; Mathias De Decker; Joris Van Lindt; David Alsteens; Sylvie Derclaye; Philip Van Damme; Joost Schymkowitz; Frederic Rousseau; Peter Tompa; Ludo Van Den Bosch

doi:10.3390/biom11040548

Liquid-Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation

Donya Pakravan, Emiel Michiels, Anna Bratek-Skicki, Mathias De Decker, Joris Van Lindt, David Alsteens, Sylvie Derclaye, Philip Van Damme, Joost Schymkowitz, Frederic Rousseau, Peter Tompa, Ludo Van Den Bosch

Research output: Contribution to journal › Article › peer-review

32 Citations (Scopus)

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.

Original language	English
Article number	548
Number of pages	21
Journal	Biomolecules
Volume	11
Issue number	4
DOIs	https://doi.org/10.3390/biom11040548
Publication status	Published - 8 Apr 2021

Bibliographical note

Funding Information:
Funding: This research was supported by VIB, the KU Leuven (C1 and “Opening the Future” Fund), the “Fund for Scientific Research Flanders” (FWO-Vlaanderen) and the ALS Liga België (A Cure for ALS). D.P. is doctoral fellow of the VIB international PhD program. PVD holds a senior clinical investigatorship of FWO-Vlaanderen and is supported by the E. von Behring Chair for Neuromuscular and Neurodegenerative Disorders and the KU Leuven funds “Een Hart voor ALS”, “Laeversfonds voor ALS Onderzoek” and the “Valéry Perrier Race against ALS Fund”.

Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Copyright:
Copyright 2021 Elsevier B.V., All rights reserved.

Keywords

Amyotrophic Lateral Sclerosis/metabolism
DNA-Binding Proteins/chemistry
Fluorescence Recovery After Photobleaching
Humans
Hydrophobic and Hydrophilic Interactions
Liquid-Liquid Extraction
Peptides/chemical synthesis
Protein Aggregates
Protein Domains
RNA/chemistry
Recombinant Proteins/biosynthesis
Static Electricity

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10.3390/biom11040548

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title = "Liquid-Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation",

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.",

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author = "Donya Pakravan and Emiel Michiels and Anna Bratek-Skicki and {De Decker}, Mathias and {Van Lindt}, Joris and David Alsteens and Sylvie Derclaye and {Van Damme}, Philip and Joost Schymkowitz and Frederic Rousseau and Peter Tompa and {Van Den Bosch}, Ludo",

note = "Funding Information: Funding: This research was supported by VIB, the KU Leuven (C1 and “Opening the Future” Fund), the “Fund for Scientific Research Flanders” (FWO-Vlaanderen) and the ALS Liga Belgi{\"e} (A Cure for ALS). D.P. is doctoral fellow of the VIB international PhD program. PVD holds a senior clinical investigatorship of FWO-Vlaanderen and is supported by the E. von Behring Chair for Neuromuscular and Neurodegenerative Disorders and the KU Leuven funds “Een Hart voor ALS”, “Laeversfonds voor ALS Onderzoek” and the “Val{\'e}ry Perrier Race against ALS Fund”. Publisher Copyright: {\textcopyright} 2021 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",

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doi = "10.3390/biom11040548",

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AU - Pakravan, Donya

AU - Michiels, Emiel

AU - Bratek-Skicki, Anna

AU - De Decker, Mathias

AU - Van Lindt, Joris

AU - Alsteens, David

AU - Derclaye, Sylvie

AU - Van Damme, Philip

AU - Schymkowitz, Joost

AU - Rousseau, Frederic

AU - Tompa, Peter

AU - Van Den Bosch, Ludo

N1 - Funding Information: Funding: This research was supported by VIB, the KU Leuven (C1 and “Opening the Future” Fund), the “Fund for Scientific Research Flanders” (FWO-Vlaanderen) and the ALS Liga België (A Cure for ALS). D.P. is doctoral fellow of the VIB international PhD program. PVD holds a senior clinical investigatorship of FWO-Vlaanderen and is supported by the E. von Behring Chair for Neuromuscular and Neurodegenerative Disorders and the KU Leuven funds “Een Hart voor ALS”, “Laeversfonds voor ALS Onderzoek” and the “Valéry Perrier Race against ALS Fund”. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

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Y1 - 2021/4/8

N2 - 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.

AB - 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.

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KW - Fluorescence Recovery After Photobleaching

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KW - Hydrophobic and Hydrophilic Interactions

KW - Liquid-Liquid Extraction

KW - Peptides/chemical synthesis

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KW - Protein Domains

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KW - Recombinant Proteins/biosynthesis

KW - Static Electricity

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U2 - 10.3390/biom11040548

DO - 10.3390/biom11040548

M3 - Article

C2 - 33917983

SN - 2218-273X

VL - 11

JO - Biomolecules

JF - Biomolecules

IS - 4

M1 - 548

ER -

Liquid-Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation

Abstract

Bibliographical note

Keywords

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