Abstract
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Stress induces phase-separated TDP-43 NBs to alleviate cytotoxicity
The two RRMs interact with different RNAs and act distinctly in the assembly of TDP-43 NBs
LncRNA NEAT1 promotes TDP-43 LLPS and is upregulated in stressed neurons
The ALS-causing D169G mutation is NB-defective and forms pTDP-43 cytoplasmic foci
Summary Despite the prominent role of TDP-43 in neurodegeneration, its physiological and pathological functions are not fully understood. Here, we report an unexpected function of TDP-43 in the formation of dynamic, reversible, liquid droplet-like nuclear bodies (NBs) in response to stress. Formation of NBs alleviates TDP-43-mediated cytotoxicity in mammalian cells and fly neurons. Super-resolution microscopy reveals a “core-shell” organization of TDP-43 NBs, antagonistically maintained by the two RRMs. TDP-43 NBs are partially colocalized with nuclear paraspeckles, whose scaffolding lncRNA NEAT1 is dramatically upregulated in stressed neurons. Moreover, increase of NEAT1 promotes TDP-43 liquid-liquid phase separation (LLPS) in vitro. Finally, we uncover that the ALS-associated mutation D169G impairs the NEAT1-mediated TDP-43 LLPS and NB assembly, causing excessive cytoplasmic translocation of TDP-43 to form stress granules that become phosphorylated TDP-43 cytoplasmic foci upon prolonged stress. Together, our findings suggest a stress-mitigating role and mechanism of TDP-43 NBs, whose dysfunction may be involved in ALS pathogenesis.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
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In the revision, we show that the interesting NB-forming defective TDP-43 D169G mutation causes more nuclear export and translocation to stress granules than WT TDP-43 upon prolonged stress, which accumulates cytoplasmic TDP-43 foci that are marked with the phosphorylation disease hallmark pS409/410. And, the cells expressing the D169G mutant are more prone to stress-induced cell death. Moreover, we show that knockdown of NEAT1 dramatically reduced the assembly of TDP-43 NBs in stressed cells, indicating that NEAT1 is required for the nucleation of TDP-43 droplets. Furthermore, by the dot-blot and EMSA assays, we demonstrate that TDP-43-D169G shows a decreased binding affinity to NEAT1 but not total RNAs, which may underlie the impairment of NEAT1-mediated nucleation of TDP-43 droplets in vitro and in the nucleus by the D169G mutation. Finally, we have generated the long-awaited UAS-hTDP-43-D169G transgenic flies, which exhibit not only age-dependent degeneration of the fly eye but also accelerated decline of the locomotor capability during aging compared to the flies expressing WT TDP-43. In addition, we've done many other confirmation assays in vitro, in cells and using primary mouse neurons. Together, these new data strengthen and further support the major argument that the lncRNA NEAT1 plays a pivotal role in the assembly of stress-induced TDP-43 NBs, whose dysfunction may contribute to ALS pathogenesis.