A seeding reaction recapitulates intracellular formation of Sarkosyl-insoluble transactivation response element (TAR) DNA-binding protein-43 inclusions

Abstract

The transactivation response element (TAR) DNA-binding protein-43 (TDP-43) is a nuclear protein that normally regulates transcription and splicing. Abnormal accumulation of insoluble inclusions containing TDP-43 has been recently reported in the affected tissues of amyotrophic lateral sclerosis (ALS) patients. Here, we show that intracellular aggregation of TDP-43 can be triggered by transduction of fibrillar aggregates prepared from in vitro functional TDP-43. Sarkosyl is found to be incapable of solubilizing those intracellularly seeded aggregates of TDP-43, which is consistent with the observation that TDP-43 inclusions in ALS patients are sarkosyl-insoluble. In addition, intracellular seeding in our cell models reproduces ubiquitination of TDP-43 aggregates, which is another prominent feature of TDP-43 inclusions in ALS patients. Although it has been so far difficult to initiate disease-associated changes of TDP-43 using cultured cell models, we propose that a seeding reaction is a key to construct a model to monitor TDP-43 pathologies.

FIGURE 1.

An assay for DNA binding of in vitro refolded TDP-43 proteins. A, a schematic representation of our filter binding assay using biotinylated single-stranded DNA (sDNA) as a substrate for TDP-43 proteins. A nitrocellulose membrane traps proteins including DNA-bound and unbound TDP-43, whereas unbound DNAs are recovered on a nylon membrane. B, 0.1 μm of either biotinylated (TG)₁₂ (left) or biotinylated (AC)₁₂ (right) was mixed with TDP-43^FL (wild type as well as ALS mutant) at indicated concentrations. These mixtures were filtered through a nitrocellulose membrane (upper) overlaid on a nylon membrane (lower), and DNAs trapped on each of membranes were probed with Streptavidin-HRP.

FIGURE 2.

Formation of sarkosyl-insoluble fibrillar aggregates of TDP-43^FL proteins. A, aggregation kinetics of 1 μm refolded TDP-43^FL proteins monitored by the increase of solution turbidity at 405 nm: open circles, WT; filled circles, M337V; open squares, A382T. Three independent experiments were performed to estimate errors. Error bars indicate S.E. B, formation of TDP-43^FL aggregates with limited solubility in a buffer containing 1% sarkosyl. After the turbidity changes reached plateau (∼1,600 min in A), ultracentrifugation separated the soluble supernatant (lane 1, soluble fraction) from insoluble pellets, which were resuspended and sonicated in PBS containing 1% sarkosyl with the same volume as that of the corresponding supernatant. Ultracentrifugation again separated supernatant (lane 2, sarkosyl-soluble fraction) from the insoluble pellets, which were resolubilized in PBS containing 2% SDS with the same volume as that of the corresponding supernatant (lane 3, sarkosyl-insoluble fraction). Equal volumes of the samples were boiled in the presence of β-mercaptoethanol, loaded on a 12.5% SDS-PAGE gel, and stained with Coomassie Brilliant Blue. C, 1 μm TDP-43 before (open bars) and after (∼1,600 min in A, filled bars) aggregation was mixed with 25 μm thioflavin T. Fluorescence intensity was measured as described under “Experimental Procedures.” Three independent experiments were done to estimate errors. Error bars indicate S.E. D–F, electron micrograms of TDP-43^FL aggregates: WT (D), M337V (E), and A382T (F). The bar in each panel represents 50 nm.

FIGURE 3.

Identification of a core structure of TDP-43^FL fibrillar aggregates. A, peptides corresponding to the mass peaks detected (see supplemental Fig. S1) are mapped on the primary sequence of TDP-43. Details are also summarized in supplemental Table S1. Numbers indicated above the primary sequence of TDP-43 (shown as open bars) represent the amino acid numbers. Four TDP-43 truncates examined here (TDP-43^N, TDP-43^N-1, TDP-43^C, and TDP-43^2-C) are also indicated in the figure. B, an SDS-PAGE analysis of reduced solubility of TDP-43 truncates after agitation with 1,200 rpm at 37 °C for 21 h. Samples were ultracentrifuged at 110,000 × g for 30 min to separate soluble supernatant (s) and insoluble pellets (p). Insoluble pellets were redissolved in PBS containing 2% SDS before loading an SDS-PAGE gel.

FIGURE 4.

A seeded fibrillation of TDP-43 proteins in vitro and in vivo. A, a seeded fibrillation of refolded TDP-43^2-C monitored by the increase of ThT fluorescence intensity. In the presence or absence of 0.3 μm (monomer base) sonicated TDP-43^2-C aggregates as seeds, 3 μm TDP-43^2-C was incubated at 37 °C without constant agitation (see “Experimental Procedures”). In this experimental condition, ThT fluorescence exhibited no changes until at least 4,000 min in the absence of added seeds but increased after ∼1,000 min of lag time in the presence of seeds. A red curve represents a sigmoidal fit to the data. As shown in the inset, an SDS-PAGE analysis has shown that all TDP-43^2-C molecules remain soluble without added seeds but become completely insoluble by seeding after incubation for 4,000 min. Soluble supernatant (s) and insoluble pellets (p) after ultracentrifugation are shown. B–G, intracellular seeded aggregation of TDP-43^2-C-HA (B–D) and TDP-43^FL-HA (E–G) overexpressed in HEK293T cells by transduction of exogenous TDP-43^2-C aggregates modified with Alexa Fluor 555 (red in C and F). Cells were fixed, stained with an anti-HA-fluorescein antibody (green in B and E), and observed using a confocal microscope. Merged images are shown in D and G, and clear representative co-localization of intracellular TDP-43 proteins (green) with transduced TDP-43^2-C aggregates (red) is shown by white arrows. Nuclei were counterstained by Hoechst 33342 (blue in D and G). H and I, a seeding reaction by transduction of His-TDP-43^2-C aggregates reduces solubility of intracellular TDP-43^2-C-HA (H) and TDP-43^FL-HA (I) in the presence of 1% sarkosyl. HEK293T cells expressing either TDP-43^2-C-HA (H) or TDP-43^FL-HA (I) were transduced with exogenous His-TDP-43^2-C aggregates, lysed in the presence of 1% sarkosyl, and ultracentrifuged to prepare soluble supernatant (s) and insoluble pellets (p). The sample volume of insoluble fractions that are loaded on a gel is 10 times more than that of soluble fractions (see “Experimental Procedures”). Western blotting using an anti-HA antibody (left panels) has shown increased amounts of TDP-43^FL/2-C proteins in the insoluble pellets after transduction of aggregates. Transduced TDP-43^2-C aggregates were also probed by using an anti-His antibody (right panels).

FIGURE 5.

Intracellularly seeded TDP-43 aggregates with limited sarkosyl solubility are polyubiquitinated. A–D, immunocytochemistry of HEK293T cells was performed by double-staining with rat anti-HA-fluorescein antibodies (green, upper panels) and rabbit anti-Ub antibodies followed with Alexa Fluor 555-anti-rabbit antibodies (red, middle panels). Nuclei were counterstained with Hoechst 33342 (blue, lower panels). HEK293T cells transiently expressing TDP-43^2-C-HA (A and B) and TDP-43^FL-HA (C and D) were examined with (B and D) or without (A and C) transduction of exogenous His-TDP-43^2-C aggregates. Representative clear co-localization of seeded TDP-43 aggregates (green) with ubiquitin (red) is shown by white arrows (see merged images in lower panels). E and F, sarkosyl-insoluble TDP-43 proteins (TDP-43^2-C-HA (E) and TDP-43^FL-HA (F)) seeded by exogenous His-TDP-43^2-C aggregates were immunoprecipitated with anti-HA antibodies. A Western blotting analysis of immunoprecipitants by using anti-HA antibodies (E and F, left panels) confirms successful precipitation of sarkosyl-insoluble TDP-43 in the samples transduced with TDP-43^2-C aggregates. When these Western blots were probed with anti-Ub antibodies (E and F, right panels), smears in the high molecular weight region were observed in the seeded samples, suggesting polyubiquitination of seeded TDP-43^FL/2-C aggregates.