Poly(ADP-Ribose) Prevents Pathological Phase Separation of TDP-43 by Promoting Liquid Demixing and Stress Granule Localization

Abstract

In amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD), cytoplasmic aggregates of hyperphosphorylated TDP-43 accumulate and colocalize with some stress granule components, but how pathological TDP-43 aggregation is nucleated remains unknown. In Drosophila, we establish that downregulation of tankyrase, a poly(ADP-ribose) (PAR) polymerase, reduces TDP-43 accumulation in the cytoplasm and potently mitigates neurodegeneration. We establish that TDP-43 non-covalently binds to PAR via PAR-binding motifs embedded within its nuclear localization sequence. PAR binding promotes liquid-liquid phase separation of TDP-43 in vitro and is required for TDP-43 accumulation in stress granules in mammalian cells and neurons. Stress granule localization initially protects TDP-43 from disease-associated phosphorylation, but upon long-term stress, stress granules resolve, leaving behind aggregates of phosphorylated TDP-43. Finally, small-molecule inhibition of Tankyrase-1/2 in mammalian cells inhibits formation of cytoplasmic TDP-43 foci without affecting stress granule assembly. Thus, Tankyrase inhibition antagonizes TDP-43-associated pathology and neurodegeneration and could have therapeutic utility for ALS and FTD.

Shortened TDP43 isoforms upregulated by neuronal hyperactivity drive TDP43 pathology in ALS

“…within the TDP43 C-terminus promotes liquid-phase separation and aggregation (90)(91)(92)(93)(94). Even so, our observations and those of others (64,65) show that sTDP43 is relatively insoluble and prone to aggregation, despite lacking the LCD.…”

“…In keeping with previous studies 65 , overexpressed sTDP43 accumulates in the cytoplasm where it often forms large, insoluble inclusions. The low-complexity domain (LCD) within the TDP43 C-terminus is essential for liquid-phase separation [87][88][89][90] , and has been heavily implicated in TDP43 aggregation. Even so, our observations and those of others 64,65 show that sTDP43 is insoluble and prone to aggregation, despite lacking the LCD.…”

Alphavirus nsP3 ADP-ribosylhydrolase Activity Disrupts Stress Granule Formation

“…In humans, ADP-ribosylation is accomplished primarily by a family of 17 ADP-ribosyltransferases, a subset of which is commonly known as poly(ADP-ribose) polymerases (PARPs) [22][23][24]. Polymers of ADP-ribose [i.e., poly(ADP-ribose or PAR], five PARPs and two isoforms of the degradative enzyme PAR glycohydrolase (PARG) have been identified in SGs, where selective SG proteins are ADP-ribosylated [19,25,26]. Overexpression of these PARPs and PARG isoforms induces and suppresses SG formation, respectively, while PARG knockdown delays SG disassembly [19,25].…”

“…Overexpression of these PARPs and PARG isoforms induces and suppresses SG formation, respectively, while PARG knockdown delays SG disassembly [19,25]. PAR, like RNA, has been proposed to seed formation of non-membranous structures by facilitating the high concentration of low complexity region-containing proteins locally through noncovalent binding to the repetitive monomeric units of the polymer [19,[25][26][27][28]. The non-covalent PAR-protein interaction also facilitates the targeting of specific proteins to SGs [25,26,29], such as TDP-43-a key protein involved in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS).…”

“…PAR, like RNA, has been proposed to seed formation of non-membranous structures by facilitating the high concentration of low complexity region-containing proteins locally through noncovalent binding to the repetitive monomeric units of the polymer [19,[25][26][27][28]. The non-covalent PAR-protein interaction also facilitates the targeting of specific proteins to SGs [25,26,29], such as TDP-43-a key protein involved in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Such a targeting mechanism based on PAR-binding to protein is proposed to regulate TDP-43 localization and prevent the formation of pathological aggregates in ALS patients [25,26].Certain PARP inhibitors prevent the neurotoxicity in neuronal cells [26,30], suggesting that pharmacological modulation of PAR-mediated protein targeting could have potential therapeutic benefit.…”