Expression of FSHD-related DUX4-FL alters proteostasis and induces TDP-43 aggregation

Abstract

Objective: Pathogenesis in facioscapulohumeral muscular dystrophy (FSHD) appears to be due to aberrant expression, particularly in skeletal muscle nuclei, of the full-length isoform of DUX4 (DUX4-FL). Expression of DUX4-FL is known to alter gene expression and to be cytotoxic, but cell responses to DUX4-FL are not fully understood. Our study was designed to identify cellular mechanisms of pathogenesis caused by DUX4-FL expression.

Methods: We used human myogenic cell cultures to analyze the effects of DUX4-FL when it was expressed either from its endogenous promoter in FSHD cells or by exogenous expression using BacMam vectors. We focused on determining the effects of DUX4-FL on protein ubiquitination and turnover and on aggregation of TDP-43.

Results: Human FSHD myotubes with endogenous DUX4-FL expression showed both altered nuclear and cytoplasmic distributions of ubiquitinated proteins and aggregation of TDP-43 in DUX4-FL-expressing nuclei. Similar changes were found upon exogenous expression of DUX4-FL, but were not seen upon expression of the non-toxic short isoform DUX4-S. DUX4-FL expression also inhibited protein turnover in a model system and increased the amounts of insoluble ubiquitinated proteins and insoluble TDP-43. Finally, inhibition of the ubiquitin-proteasome system with MG132 produced TDP-43 aggregation similar to DUX4-FL expression.

Interpretations: Our results identify DUX4-FL-induced inhibition of protein turnover and aggregation of TDP-43, which are pathological changes also found in diseases such as amyotrophic lateral sclerosis and inclusion body myopathy, as potential pathological mechanisms in FSHD.

Figure 1

Exogenous DUX4-FL increased caspase activation. (A–H) DUX4-FL (red) was expressed in proliferating human myoblasts (A–D) at 24 h after plasmid transfection and in differentiated myotubes (E–H) at 48 h after BacMam vector addition. Cultures were established with myoblasts from a healthy donor. Immunostaining for activated caspase-3 (green) was found in a fraction of myoblasts and myotubes that expressed exogenous DUX4-FL (arrows). Yellow arrowhead in (A–D) indicates a DUX4-FL-positive myoblast without caspase-3 staining. (I) Caspase 3/7 enzymatic activity was increased by BacMam-mediated expression of DUX4-FL, but not DUX4-S, in both proliferating (myoblast) and differentiated (myotube) cultures of human myogenic cells. Error bars = SE, n = 4. *P < 0.05; **P < 0.01.

Figure 2

Exogenous DUX4-FL altered ubquitinated protein distribution in myoblasts. (A–C) After plasmid transfection, DUX4-FL (red) was expressed in a subset of myoblasts; and confocal images of immunostaining for ubiquitinated proteins (Ub-protein, green) showed decreased staining in the nuclei of DUX4-FL-positive (arrows) compared to DUX4-FL-negative (arrowheads) myoblasts. Numbered cells give examples of the classifications of Ub-protein staining patterns that are graphed in (D); with the (DUX4-FL-positive) myoblast labeled “1” classified as low nuclear and low cytoplasmic; and the (DUX4-FL-negative) myoblast labeled “2” classified as mid nuclear and mid cytoplasmic. Additional classification examples are in Figure4. (D) Nuclear and cytoplasmic Ub-protein staining patterns were independently classified as low, mid, or high, as described under (A) and in the text, and the number of myoblasts with each pattern was counted. The predominant effect of exogenous DUX4-FL expression on Ub-protein distribution in myoblasts was to decrease nuclear staining with little effect on cytoplasmic staining.

Figure 3

Exogenous DUX4-FL induced aggregation of TDP-43 in myonuclei. (A–C) DUX4-FL (red) was expressed in proliferating human myoblasts using plasmid transfection. Arrows indicate DUX4-FL-positive myoblasts and arrowheads indicate DUX4-FL-negative myoblasts. (D–H) DUX4-FL was expressed in differentiated myotubes using a BacMam vector. Cultures were established with myoblasts from a healthy donor and examined at 32–48 h after addition of plasmids or BacMam vector. TDP-43 immunostaining (green) in DUX4-FL-negative myoblasts (A–C) and myotubes (I and J) was diffusely distributed in nuclei and absent from the cytoplasm, whereas DUX4-FL-positive myoblasts (A–C) and myotubes (D–H) showed a different, punctate pattern of TDP-43 staining indicative of aggregation. (K and L) TDP-43 immunostaining also showed a punctate pattern after a short heat shock (1 h at 42°C).

Figure 4

Exogenous and endogenous DUX4-FL altered ubiquitinated protein distribution in myotubes. (A–C) A BacMam vector was used to express exogenous DUX4-FL (red) in myotubes. When compared to the low cytoplasmic and uniform nuclear staining for Ub-proteins in DUX4-FL-negative cells (e.g. Fig.2A and arrowhead in D), DUX4-FL-positive myotubes show increased immunostaining for Ub-proteins (green) in the cytoplasm and variable staining, often with aggregates, in nuclei. (D–F) Expression of DUX4-FL from its endogenous promoter in myotubes formed from FSHD myoblasts (17Abic) also led to increased cytoplasmic staining and variable, often punctate, staining for Ub-proteins. Arrows in all panels indicate cells with DUX4-FL-positive nuclei and the arrowheads in (D–F) indicate a cell with a DUX4-FL-negative nucleus. Additional examples of Ub-protein staining in DUX4-FL-negative cells are shown in Figure7E and F. Numbered cells give examples of the classifications of Ub-protein staining patterns graphed in (G); with the (DUX4-FL-positive) myotube labeled “1” in (A) classified as mid nuclear and high cytoplasmic; the (DUX4-FL-negative) myocyte labeled “2” in (D) classified as mid nuclear and low cytoplasmic; and the (DUX4-FL-positive) myotube labeled “3” in (D) classified as high nuclear and mid cytoplasmic. Note that mid and high-nuclear staining can be punctate, rather than uniform across the nuclei. Additional examples are in Figure2. (G) Nuclear and cytoplasmic Ub-protein staining patterns were independently classified as low, mid, or high, and the number of cells with each pattern was counted. The results showed that endogenous DUX4-FL expression in myotubes increased cytoplasmic staining and produced variable changes in nuclear staining for Ub-proteins.

Figure 5

Endogenous DUX4-FL-induced aggregation of TDP-43 in myotube nuclei. Double immunostaining of differentiated cultures for expression of endogenous DUX4-FL (red) and TDP-43 (green) showed that TDP-43 staining was punctate, indicative of aggregation, in nuclei that expressed endogenous DUX4-FL (arrows), whereas nearby nuclei that did not express DUX4-FL (arrowheads) showed a more uniform distribution of TDP-43 immunostaining. Each row shows a different set of nuclei. In the nuclei shown here, DUX4-FL staining was also punctate, however, merged confocal images showed little or no overlap of the distinctly punctate areas of staining for DUX4-FL and Ub-proteins (e.g. H).

Figure 6

Exogenous DUX4-FL slowed protein turnover. (A) GFP protein made unstable by addition of the 16 amino acid CL1 degron sequence (GFP-degron) was expressed by plasmid transfection in HEK293 cells with or without co-expression of exogenous DUX4-FL and with or without MG132 treatment to inhibit protein turnover through the ubiquitin–proteasome system. Immunoblots showed that more of the GFP-degron protein accumulated in cultures that co-expressed DUX4-FL (compare GFP in lanes 1 and 3) indicating that DUX4-FL expression led to stabilization and slowed turnover of the GFP-degron protein. As a positive control, MG132 treatment also led to accumulation of GFP-degron protein (lanes 2 and 4). GAPDH was used a loading control. (B–D) When co-expressed by plasmid transfection in human myoblasts, GFP-degron protein (green) accumulated when co-expressed with DUX4-FL (red).

Figure 7

MG132 treatment changed the distributions of ubiquitinated proteins and TDP-43 in human myoblasts and myotubes. (A–D) In untreated myoblasts, staining for Ub-proteins was diffuse in nuclei and low in the cytoplasm (A), whereas, after MG132 treatment, cytoplasmic staining was high and partially punctate in the cytoplasm and nuclear staining was very low (B). Expression of exogenous DUX4-FL by plasmid did not further change the Ub-staining pattern in myoblasts (C and D). (E–H) Staining for Ub-proteins was uniformly diffuse in nuclei and low in the cytoplasm of untreated myotubes (E and F), whereas MG132 treatment led to decreased nuclear staining and increased, often punctate, cytoplasmic staining (G and H). (I–L) Staining for TDP-43 was uniformly diffuse in nuclei and low in the cytoplasm of untreated myotubes (I and J), whereas MG132 treatment led to a punctate, aggregated pattern of TDP-43 staining in nuclei (arrowheads; K and L).

Figure 8

The punctate immunostaining patterns for ubiquitinated proteins, TDP-43, and DUX4-FL in nuclei showed little co-localization. (A–C) Confocal microscope images for ubiquitinated proteins (green, A) and DUX4-FL (red, C) were merged in (B) to show the relatively small regions of overlap (yellow). (B) Includes blue DNA stain for the nucleus. (D–F) Standard microscope images for ubiquitinated proteins (green, D) and TDP-43 (red, F) were merged in (E) and showed little overlap of the punctate areas of immunostaining in the nucleus. (G–I) Standard microscope images for TDP-43 (green, G) and DUX4-FL (red, I) were merged in (H) and showed little overlap of the punctate areas immunostaining seen in the lower DUX4-FL-positive nucleus. Additional examples are in Figure5. The upper DUX4-FL-negative nucleus in (G–I) shows the nearly uniform nuclear immunostaining for TDP-43 that is seen in the absence of DUX4-FL.

Figure 9

Exogenous expression of DUX4-FL, but not DUX4-S, was followed by increased amounts of insoluble ubiquitinated proteins, TDP-43, and DUX4-FL itself. BacMam DUX4-FL or DUX4-S vectors were added to myogenic cell cultures that had been in differentiation medium for 4 days, and 48 h later the cultures were harvested and separated into a RIPA-soluble fraction and a RIPA-insoluble/urea soluble fraction. Fractions were separated by SDS-PAGE and immunoblotted to identify, as indicated, ubiquitinated proteins (Ub proteins); TDP-43; DUX4-FL and DUX4-S (by V5 epitope tag); and GAPDH (as a marker for the RIPA-soluble fraction). The amounts of ubiquitinated proteins and TDP-43 in the RIPA-soluble fraction was not affected by expression of either DUX4-FL or DUX4-S. In contrast, the RIPA-insoluble/urea soluble fraction had increased amounts of both ubiquitinated proteins and TDP-43 (both the 43 kDa form and the ∽35 kDa form) upon expression of DUX4-FL, but DUX4-S. In addition, much of the DUX4-FL, but little of the DUX4-S, was found in the RIPA-insoluble/urea soluble fraction. All samples were analyzed on the same immunoblot with some lanes re-arranged for presentation as denoted by the vertical dotted lines. The experiment was repeated twice with similar results.