Stress induces dynamic, cytotoxicity-antagonizing TDP-43 nuclear bodies via paraspeckle lncRNA NEAT1-mediated liquid-liquid phase separation

Abstract

Short title (<50 characters): Phase-separated TDP-43 NBs mitigate stress 20 Keywords: TDP-43, nuclear body, RNP granule, phase separation, lncRNA NEAT1 21 Word count: ~6500 words (excluding the title page, abstract, references and figure legends)Recent studies indicate that liquid-liquid phase separation (LLPS) of RNA-binding protein 72 (RBPs) drives the assembly of liquid droplet (LD)-like, membraneless RNP granules in the 73 cytoplasm and nucleoplasm (25)(26)(27)(28)(29). Several ALS-related RBPs including TDP-43, FUS, hnRNP 74 A1, hnRNP A2 and TIA1 are shown to phase separate in vitro (14,(30)(31)(32)(33)(34)(35). The intrinsically 75 disordered LCD domains of the RBPs are thought to mediate the LLPS (28, 31,(36)(37)(38) and 76 posttranslational protein modifications play an important role in determining the biophysical and 77 biological properties of the phase behavior of the RBPs (34,(39)(40)(41)(42)(43)(44)(45)(46). In addition to the LCD, 78 recent studies reveal that RNA is of vital importance in regulating the LLPS (47,48), which can 79 either suppress or promote phase separation depending on the contents and concentrations of 80 the RNAs (25,(49)(50)(51). 81In this study, we find that various cellular stresses induce TDP-43 to form distinct, highly 82 dynamic and reversible NBs, which not only attenuate the cytotoxicity of TDP-43 in mammalian 83 cells but also ameliorate neurodegeneration and behavioral deficits in a Drosophila model of ALS. 84Further investigation with super-resolution microscopy reveals the unexpected opposing 85 functions of the two RRMs in maintaining the core-shell architecture of TDP-43 NBs. 86Furthermore, we demonstrate a crucial role of the paraspeckle scaffolding RNA nuclear-enriched 87 abundant transcript 1 (NEAT1) in promoting TDP-43 LLPS in vitro, which is compromised by the 88 ALS-causing mutation D169G, leading to a defect in forming TDP-43 NBs. Together, our findings 89 demonstrate how stress induces the assembly of TDP-43 NBs in the generally "suppressive" 90 nucleoplasm environment and suggest the involvement of dysfunctional TDP-43 NBs in ALS 91 pathogenesis. 92Phase-separated TDP-43 NBs mitigate stress_CWang Page 6 of 44 RESULTS 93 Arsenic stress induces dynamic and reversible TDP-43 NBs 94TDP-43 protein is predominantly localized to the nucleus but can shuttle between the nucleus 95 and the cytoplasm. In response to cellular stress, TDP-43 is recruited to cytoplasmic SGs (11). 96Interestingly, in an earlier related work of our group (46), we noticed that although arsenite, a 97 commonly used experimental reagent to raise cellular stress (52), induced TDP-43 + cytoplasmic 98SGs, the majority of TDP-43 signal remained in the nucleus. Furthermore, in a great portion of 99 the cells, the nuclear TDP-43 lost the normal diffused pattern and instead exhibited a distinct 100 granular appearance (Movie S1 and Fig. 1, A and B). 101 A membraneless nuclear structure fulfills the requirements of NBs if it is: (1) microscopically 102 visible, (2) enriched with specific nuclear fac...

Multi-phaseted problems of TDP-43 in selective neuronal vulnerability in ALS

“…RNA binding of TDP-43 is mediated by the two RNA recognition motif (RRM) domains (RRM1 and RRM2) that reside in the middle of the primary protein structure [11]. RRM1 and RRM2 have differential affinities with different types of RNAs and, under stress conditions, have distinct functions in the assembly and maintenance of nuclear TDP-43 granules [45]. The RRM2 contains a putative nuclear export signal (NES) predicted via bioinformatics [40,46,47].…”

“…The RRM2 contains a putative nuclear export signal (NES) predicted via bioinformatics [40,46,47]. Mutations in the putative NES or inhibition of the nuclear export receptor exportin-1 (XPO1) by leptomycin B treatment lead to nuclear TDP-43 granule formation [40,45]. However, independent studies have failed to establish TDP-43 as a direct substrate of XPO1 [47][48][49], and exact mechanisms for the nuclear TDP-43 granule formation caused either by the mutation of the putative NES or leptomycin B treatment remined to be clarified.…”

“…These observations elucidate the close linking of the intranuclear phase transition of TDP-43 to its RNA-regulatory roles. The granular appearance of nuclear TDP-43 is enhanced when cells are under stress, assembling dynamic and reversible TDP-43-containing NBs [45,51,71]. Upon arsenite treatment, TDP-43 associates with distinct RNA species, such as long non-coding RNA NEAT1 or short tRNAs, for NB assembly via its two RRM domains [45].…”

Enhanced detection of expanded repeat mRNA foci with hybridization chain reaction

“…These TDP-43 positive structures are thought to contribute to ALS disease pathogenesis [ 78 ]. Given the greater nuclear distribution of G 4 C 2 repeat RNA after stress induction, we evaluated whether there was any significant overlap between endogenous G 4 C 2 repeat RNA and TDP-43, a critical factor in C9orf72 ALS/FTD pathology and ALS pathogenesis as well as a robust marker for nuclear bodies [ 16 , 78 ]. Our untreated C9 fibroblasts appeared to have small TDP-43 nuclear bodies, indicative of them being inherently stressed.…”

Enhanced detection of nucleotide repeat mRNA with hybridization chain reaction

“…These TDP-43 positive structures are thought to contribute to ALS disease pathogenesis (Wang et al 2020). Given the greater nuclear distribution of G4C2 repeat RNA after stress induction, we evaluated whether there was any significant overlap between endogenous G4C2 repeat RNA and TDP43, a critical factor in C9orf72 pathology and ALS pathogenesis as well as a robust marker for nuclear bodies (Duan et al 2019;Wang et al 2020).…”