Heat shock-induced phosphorylation of TAR DNA-binding protein 43 (TDP-43) by MAPK/ERK kinase regulates TDP-43 function

TAR DNA-binding protein (TDP-43) is a highly conserved and essential DNA- and RNA-binding protein that controls gene expression through RNA processing, in particular, regulation of splicing. Intracellular aggregation of TDP-43 is a hallmark of amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. This TDP-43 pathology is also present in other types of neurodegeneration including Alzheimer's disease. We report here that TDP-43 is a substrate of MEK, a central kinase in the MAPK/ERK signaling pathway. TDP-43 dual phosphorylation by MEK, at threonine 153 and tyrosine 155 (p-T153/Y155), was dramatically increased by the heat shock response (HSR) in human cells. HSR promotes cell survival under proteotoxic conditions by maintaining protein homeostasis and preventing protein misfolding. MEK is activated by HSR and contributes to the regulation of proteome stability. Phosphorylated TDP-43 was not associated with TDP-43 aggregation, and p-T153/Y155 remained soluble under conditions that promote protein misfolding. We found that active MEK significantly alters TDP-43-regulated splicing and that phosphomimetic substitutions at these two residues reduce binding to GU-rich RNA. Cellular imaging using a phospho-specific p-T153/Y155 antibody showed that phosphorylated TDP-43 was specifically recruited to the nucleoli, suggesting that p-T153/Y155 regulates a previously unappreciated function of TDP-43 in the processing of nucleolar-associated RNA. These findings highlight a new mechanism that regulates TDP-43 function and homeostasis through phosphorylation and, therefore, may contribute to the development of strategies to prevent TDP-43 aggregation and to uncover previously unexplored roles of TDP-43 in cell metabolism

Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation

Aggregates of the RNA-binding protein TDP-43 (TAR DNAbinding protein) are a hallmark of the overlapping neurodegenerative 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 stage is primarily 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 detergentresistant 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 forWTTDP-43 aggregates.We provide evidence for aninitial 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 perturbationsin TDP-43 homeostasisin protein aggregation and ALS-FTD pathogenesis

Inhibition of phase separation disrupts TDP-43 nuclear retention.

(A) Relative nuclear to cytoplasmic signal ratio (N/C) of endogenous TDP-43 in HEK293 cells treated with 1,6 hexanediol (HD, 5% for 5 min) quantified from immunoblotting (S2A Fig), expressed relative to non-treated control. Mean ± SD is shown for 6 independent replicates. (B) Immunofluorescence of control and 1,6-HD-treated HEK293 cells, as seen by confocal microscopy. Polyclonal TDP-43 antibody was used to detect endogenous TDP-43 distribution. Scale bar, 10 μm. (C) HEK293HA-TDP-43 cells expressing HA-tagged WT and A326P, M337P, and M337V mutants located in the conserved α-helical region (CR). Quantification of N/C of HA-TDP-43 relative to WT in each replicate from immunoblotting (S2B Fig). Mean ± SD is shown for 6 independent replicates. ***p p S2 Fig and in S1 Data, respectively. CR, conserved region; HA, hemagglutinin; WT, wild type.</p

TDP-43 nuclear localization depends on the assembly of RNP complexes mediated via multivalent interactions.

RNP TDP-43 complexes in the nucleus contribute to TDP-43 nuclear localization through the formation of higher-order macromolecular assemblies unable to passively diffuse to the cytoplasm. Under aberrant conditions, disruption of RNA binding or self-assembly increases levels of smaller TDP-43 species inducing greater cytoplasmic localization. Chronic defects in RNP assembly may increase TDP-43 misfolding and aggregation in the cytoplasm. RNP, ribonucleoprotein.</p

Supporting data.

TDP-43 is an essential RNA-binding protein strongly implicated in the pathogenesis of neurodegenerative disorders characterized by cytoplasmic aggregates and loss of nuclear TDP-43. The protein shuttles between nucleus and cytoplasm, yet maintaining predominantly nuclear TDP-43 localization is important for TDP-43 function and for inhibiting cytoplasmic aggregation. We previously demonstrated that specific RNA binding mediates TDP-43 self-assembly and biomolecular condensation, requiring multivalent interactions via N- and C-terminal domains. Here, we show that these complexes play a key role in TDP-43 nuclear retention. TDP-43 forms macromolecular complexes with a wide range of size distribution in cells and we find that defects in RNA binding or inter-domain interactions, including phase separation, impair the assembly of the largest species. Our findings suggest that recruitment into these macromolecular complexes prevents cytoplasmic egress of TDP-43 in a size-dependent manner. Our observations uncover fundamental mechanisms controlling TDP-43 cellular homeostasis, whereby regulation of RNA-mediated self-assembly modulates TDP-43 nucleocytoplasmic distribution. Moreover, these findings highlight pathways that may be implicated in TDP-43 proteinopathies and identify potential therapeutic targets.</div

Defects in TDP-43 self-assembly and RNA binding decrease nuclear localization.

Defects in TDP-43 self-assembly and RNA binding decrease nuclear localization.</p

Defects in RNA-mediated TDP-43 self-assembly decrease nuclear localization.

(A) TDP-43 domain organization and model of RNA-mediated TDP-43 self-assembly involving multivalent interactions through the NTD oligomerization and C-terminal assembly. Mutations in the NTD, RRMs, and deletion of the conserved α-helical region (ΔCR) in the C-tail (a.a. 316–343) are highlighted. The basic residues (red) in the NLS were substituted to generate NLSmut, used as control. (B) Immunoblot of total, cytoplasmic and nuclear fractions of HEK293HA-TDP-43 cells. Lamin A/C and tubulin were used as nuclear and cytoplasmic controls, respectively. Source images can be found in S1 Raw Images. (C) HA-TDP-43 nuclear to cytoplasmic signal ratio (N/C) quantified from immunoblotting, normalized to WT in each independent experiment. Mean ± SD is shown for 6 independent replicates. ns, not significant, ***p p S1B Fig and S1 Data, respectively. NLS, nuclear localization signal; NTD, N-terminal domain; RRM, RNA recognition motif.</p

Cellular distribution of WT and mutant TDP-43 as seen by microscopy.

Cellular distribution of WT and mutant TDP-43 as seen by microscopy.</p

TDP-43 macromolecular complex distribution in total cell lysate.

TDP-43 macromolecular complex distribution in total cell lysate.</p

RNA binding and TDP-43 self-assembly exert independent and additive effects on TDP-43 nuclear localization.

(A) HEK293HA-TDP-43 cells expressing HA-tagged WT and mutant TDP-43 probed with HA antibody (top: immunofluorescence, bottom: pseudo-color look-up table). The intensity histogram for each image was independently maximized across the full range. Scale bar, 20 μm. Graph shows automated quantification of the TDP-43 nuclear/cytoplasmic ratio (N/C) at steady state, normalized to WT. Mean ± SD is shown for approximately 1,600 cells/well in 7 independent replicates. ns, not significant, ****p p p 6 or (CA)6 oligonucleotides. Mean ± SD is shown for approximately 1,700 cells/well in 5 independent replicates. Source data can be found in S1 Data. HA, hemagglutinin; WT, wild type.</p