Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43

Abstract

The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of frontotemporal lobar degeneration. Among modulators of autophagy, only vacuolar-ATPase inhibitors, such as bafilomycin A1 (Baf), increased the levels of the full-length and cleaved forms of TDP-43 and the autophagosome marker LC3-II (microtubule-associated proteins 1A/1B light chain 3B) in extracellular vesicle fractions prepared from the culture media of HeLa, SH-SY5Y, or NSC-34 cells, whereas vacuolin-1, MG132, chloroquine, rapamycin, and serum starvation did not. The C-terminal fragment of TDP-43 was required for Baf-induced TDP-43 secretion. The Baf treatment induced the translocation of the aggregate-prone GFP-tagged C-terminal fragment of TDP-43 and mCherry-tagged LC3 to the plasma membrane. The Baf-induced secretion of TDP-43 was attenuated in autophagy-deficient ATG16L1 knockout HeLa cells. The knockdown of PGRN induced the secretion of cleaved TDP-43 in an autophagy-dependent manner in HeLa cells. The KO of PGRN in mouse embryonic fibroblasts increased the secretion of the cleaved forms of TDP-43 and LC3-II. The treatment inducing TDP-43 secretion increased the nuclear translocation of GFP-tagged transcription factor EB, a master regulator of the autophagy-lysosomal pathway in SH-SY5Y cells. These results suggest that the secretion of TDP-43 is promoted by dysregulation of the PGRN-driven autophagy-lysosomal pathway.

Keywords: TAR DNA-binding protein 43; amyotrophic lateral sclerosis; autolysosome; autophagosome; autophagy; extracellular vesicles; frontotemporal lobar degeneration; lysosome; progranulin.

Figure 1

Bafilomycin A1 (Baf) increases TDP-43, TSG101, and LC3-II levels in the EV fraction.A–C, HeLa cells expressing mRFP-GFP-LC3 were exposed to vehicle (DMSO), 100 nM Baf, 1 μM MG, or 100 nM Vac for 24 h. A, representative images of these cells are shown. Nuclei were stained with DAPI (blue). Scale bar represents 20 μm. B, autophagosome and (C) autolysosome numbers calculated from three independent experiments including at least 20 cells are shown. D and E, HeLa cells exposed to vehicle (DMSO), 100 nM Baf, 1 μM MG, or 100 nM Vac for 24 h were stained with AcidiFluor ORANGE. D, representative images of live cells stained with AcidiFluor ORANGE (red) in each treatment. Nuclei were stained with Hoechst 33342 (blue). Scale bar represents 20 μm. E, relative signal intensities normalized against cells exposed to vehicle were calculated from three independent experiments including at least 330 cells. F–J, HeLa cells were exposed to DMSO, 100 nM Baf, 1 μM MG, or 100 nM Vac for 24 h. The cell, P1 (20,000g pellet), and P2 (110,000g pellet) fractions were prepared as shown in the Experimental procedures section. F, representative immunoblots (TDP-43 detected by monoclonal, C-terminal, and N-terminal antibodies, TSG101, LC3, and α-tubulin) of each fraction are shown. The dashed square indicates the bands of cleaved TDP-43 used for quantification. G–J, densitometric data on (G) full-length TDP-43, (H) cleaved TDP-43, (I) TSG101, and (J) LC3-II in the cell, P1, and P2 fractions were calculated from immunoblotting results. Relative signal intensities normalized against cells exposed to Baf are shown. In bar graphs, data are presented as means ± SD (N = 3). ∗ indicates p < 0.05 by the two-tailed unpaired t test or Mann–Whitney U test. DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; EV, extracellular vesicle; LC3, microtubule-associated proteins 1A/1B light chain 3B; MG, MG132; mRFP, monomeric red fluorescent protein; TDP-43, TAR DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein; Vac, vacuolin-1.

Figure 2

Inhibition of autophagosome–lysosome fusion by bafilomycin A1 (Baf) is required for TDP-43 secretion via autophagy-associated vesicles. HeLa cells were exposed to indicated treatment for 24 h. A, representative immunoblots (TDP-43 detected by the monoclonal antibody, TSG101, LC3, and α-tubulin) of each fraction are shown. The dashed square indicates bands of cleaved TDP-43 used for quantification. B–E, densitometric data on (B) full-length TDP-43, (C) cleaved TDP-43, (D) TSG101, and (E) LC3-II in the cell, P1, and P2 fractions were calculated from immunoblotting results. Relative signal intensities normalized against cells exposed to Baf are shown. In bar graphs, data are presented as means ± SD (N = 3). ∗ indicates p < 0.05 by the two-tailed unpaired t test. LC3, microtubule-associated proteins 1A/1B light chain 3B; Rapa, rapamaycin; SS, serum starvation; TDP-43, TAR DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Figure 3

Proteomic analyses suggest different intracellular origins of EV in the P1 and P2 fractions derived from HeLa cells exposed to bafilomycin A1 (Baf). HeLa cells were exposed to vehicle (DMSO) or 100 nM Baf for 24 h. Cell, P1, and P2 fractions in each treatment were prepared. A, immuno-EM images of vesicles in the P1 and P2 fractions derived from cells exposed to 100 nM Baf using the rabbit anti-C-terminal TDP-43 antibody. The image surrounded by a red brown square was magnified to compare vesicles between the P1 and P2 fractions. Scale bar represents 500 nm. B, a Venn diagram showing the distribution of proteins in the P1 and P2 fractions identified by LC–MS/MS analyses. C, representative immunoblots (ACTN4, ADAM10, p62, and α-tubulin) of the cell, P1, and P2 fractions are shown. D–F, densitometric data on (D) ACTN4, (E) ADAM10, and (F) p62 in the P1 and P2 fractions were calculated from immunoblotting results. In bar graphs, data are presented as means ± SD (N = 3) normalized against the P1 or P2 fraction derived from cells exposed to Baf. ∗ indicates p < 0.05 by the two-tailed unpaired t test. DMSO, dimethyl sulfoxide; EV, extracellular vesicle; TDP-43, TAR DNA-binding protein 43 kDa.

Figure 4

TARDBP knockdown enhances the extracellular release of LC3-II from bafilomycin A1 (Baf)–treated cells. HeLa cells transfected with control or TARDBP siRNA for 48 h were further cultured in 100 nM Baf-containing medium for 24 h. The cell, P1, and P2 fractions were prepared as shown in the Experimental procedures section. A, representative immunoblots (TDP-43, TSG101, LC3, and α-tubulin) are shown. The dashed square indicates the bands of cleaved TDP-43 used for quantification. B–E, densitometric data on (B) full-length TDP-43, (C) cleaved TDP-43, (D) TSG101, and (E) LC3-II in each fraction were calculated from immunoblotting results. Relative signal intensities normalized against cells transfected with control siRNA are shown. In bar graphs, data are presented as means ± SD (N = 3). ∗ indicates p < 0.05 by the two-tailed unpaired t test. LC3, microtubule-associated proteins 1A/1B light chain 3B; TDP-43, TAR DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Figure 5

The C-terminal region of TDP-43 is required for bafilomycin A1 (Baf)-induced TDP-43 secretion.A and B, HeLa cells stably expressing mCherry-LC3 transfected with GFP-tagged full-length (amino acids 1–414), the N-terminal fragment (amino acids 1–273), or C-terminal fragment (amino acids 162–414) of TDP-43 were exposed to 100 nM Baf for 24 h. A, representative confocal images of live cells in each treatment are shown. Nuclei were stained with Hoechst 33342. Scale bar represents 20 μm. Arrowheads indicate the colocalization of TDP-43 amino acids 162 to 414 and mCherry-LC3. B, densitometric data of fluorescence images were calculated from three independent experiments including at least 20 cells. The percentage of the GFP-TDP-43 signal colocalized with mCherry-LC3 to the total GFP-TDP-43 signal is shown. C–E, HeLa cells transfected with TDP-43 amino acids 1 to 414, TDP-43 amino acids 1 to 273, or TDP-43 amino acids 162 to 414 were exposed to 100 nM Baf for 24 h, and the cell, P1, and P2 fractions were prepared. C, representative immunoblots (TDP-43, GFP, and α-tubulin) of the cell, P1, and P2 fractions are shown. D and E, the ratio of GFP-TDP-43 levels in the (D) P1 and (E) P2 fractions to the cellular fraction calculated from the densitometric data of immunoblotting results are normalized against cells transfected with TDP-43 amino acids 162 to 414. In bar graphs, data are presented as means ± SD (N = 3). Values with a different superscript indicate p < 0.05 by Tukey’s post hoc test. LC3, microtubule-associated proteins 1A/1B light chain 3B; TDP-43, TAR DNA-binding protein 43 kDa.

Figure 6

The bafilomycin A1 (Baf) treatment induces the localization of GFP-tagged C-terminal TDP-43 (amino acids 162–414) and mCherry-LC3 to the plasma membrane. HeLa cells transfected with the TDP-43 amino acids 162 to 414 construct or mCherry-LC3 construct were exposed to vehicle or 100 nM Baf for 24 h. A and B, representative confocal images of live cells transfected with the TDP-43 amino acids 162 to 414 construct (green) exposed to (A) vehicle or (B) 100 nM Baf are shown. The plasma membrane (PM) was stained with PlasMem Bright Red (red). Nuclei were stained with Hoechst 33342. C and D, representative confocal images of live cells transfected with the mCherry-LC3 construct (red) exposed to (C) vehicle or (D) 100 nM Baf are shown. The PM was stained with PlasMem Bright Green (green). The image surrounded by a yellowsquare was magnified. Scale bar represents 5 μm. LC3, microtubule-associated proteins 1A/1B light chain 3B; TDP-43, TAR DNA-binding protein 43 kDa.

Figure 7

ATG16L1 is required for bafilomycin A1 (Baf)-induced TDP-43 secretion.A and B, ATG16L1 WT or KO HeLa cells transfected with the mCherry-LC3 construct were exposed to 100 nM Baf for 24 h. Representative confocal live-cell images of ATG16L1 (A) WT or (B) KO cells transfected with the mCherry-LC3 (red) construct exposed to Baf. The plasma membrane (PM) was stained with PlasMem Bright Green (green). The image surrounded by a yellowsquare was magnified. Scale bar represents 5 μm. C–F, ATG16L1 WT or KO HeLa cells were exposed to 100 nM Baf for 24 h. The cell, P1, and P2 fractions were prepared as shown in the Experimental procedures section. C, representative immunoblots (ATG16L1, TDP-43, TSG101, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates bands of cleaved TDP-43 used for quantification. D–F, densitometric data on (D) full-length TDP-43, (E) cleaved TDP-43, and (F) TSG101 in the cell, P1, and P2 fractions were calculated from immunoblotting results. Relative signal intensities normalized against ATG16L1 WT cells are shown. In bar graphs, data are presented as means ± SD (N = 3). ∗ indicates p < 0.05 by the two-tailed unpaired t test. LC3, microtubule-associated proteins 1A/1B light chain 3B; TDP-43, transactive response DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Figure 8

Chloroquine, an inhibitor of autolysosome formation and lysosomal acidification, does not increase the secretion of TDP-43 or LC3-II.A and B, HeLa cells stained with DALGreen were exposed to vehicle (DMSO), 100 nM Baf, 100 nM Con, or 100 μM CQ for 2 h. A, representative images of live cells stained with DALGreen (green) in each treatment are shown. Nuclei were stained with Hoechst 33342 (blue). Scale bar represents 50 μm. B, relative signal intensities normalized against cells exposed to vehicle were calculated from three independent experiments including at least 74 cells. C and D, HeLa cells exposed to vehicle (DMSO), 100 nM Baf, 100 nM Con, or 100 μM CQ for 24 h were stained with AcidiFluor ORANGE. C, representative images of live cells stained with AcidiFluor ORANGE (red) in each treatment. Nuclei were stained with Hoechst 33342 (blue). Scale bar represents 20 μm. D, relative signal intensities normalized against cells exposed to vehicle were calculated from three independent experiments including at least 332 cells. E–I, HeLa cells were exposed to vehicle (DMSO), 100 nM Baf, 100 nM Con, or 100 μM CQ for 24 h. The cell, P1, and P2 fractions were prepared as shown in the Experimental procedures section. E, representative immunoblots (TDP-43, TSG101, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates the bands of cleaved TDP-43 used for quantification. F–I, densitometric data on (F) full-length TDP-43, (G) cleaved TDP-43, (H) TSG101, and (I) LC3-II were calculated from immunoblotting results. Relative signal intensities normalized against cells exposed to the vehicle are shown. In bar graphs, data are presented as means ± SD (N = 3). Values with a different superscript indicate p < 0.05 by Tukey’s post hoc test. Baf, bafilomycin A1; Con, concanamycin A; CQ, chloroquine; DMSO, dimethyl sulfoxide; LC3-II, microtubule-associated proteins 1A/1B light chain 3B; TDP-43, transactive response DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Figure 9

The knockdown of GRN increases the secretion of TDP-43 and LC3-II in an autophagy-dependent manner.A and B, HeLa cells transfected with control or GRN siRNA for 48 h were stained with DALGreen and incubated for 2 h. A, representative live-cell images stained with DALGreen (green) are shown. Nuclei were stained with Hoechst 33342 (blue). Scale bar represents 50 μm. B, relative signal intensities normalized against cells transfected with control siRNA were calculated from three independent experiments including at least 78 cells. C and D, HeLa cells transfected with control or GRN siRNA for 48 h were stained with AcidiFluor ORANGE, and the acidity of lysosomes was compared. C, representative live-cell images stained with AcidiFluor ORANGE and photographed at a bright field are shown. Scale bar represents 100 μm. D, relative signal intensities normalized against cells transfected with control siRNA were calculated from three independent experiments including at least 627 cells. E–I, HeLa cells transfected with control or GRN siRNA were incubated for 72 h. The cell, P1, and P2 fractions were prepared as shown in the Experimental procedures section. E, representative immunoblots (PGRN, TDP-43, TSG101, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates the bands of cleaved TDP-43 used for quantification. F–I, densitometric data on (F) full-length TDP-43, (G) cleaved TDP-43, (H) TSG101, and (I) LC3-II were calculated from immunoblotting results. Relative signal intensities normalized against cells transfected with GRN siRNA are shown. J–M, ATG16L1 KO HeLa cells transfected with control or GRN siRNA were incubated for 72 h. The cell, P1, and P2 fractions were prepared as shown in the Experimental procedures section. N is a sample from normal HeLa cells. J, representative immunoblots (PGRN, ATG16L1, TDP-43, TSG101, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates the bands of cleaved TDP-43 used for quantification. K–M, densitometric data on (K) full-length TDP-43, (L) cleaved TDP-43, and (M) TSG101 were calculated from immunoblotting results. Relative signal intensities normalized against cells transfected with GRN siRNA are shown. In bar graphs, data are presented as means ± SD (N = 3). ∗ indicates p < 0.05 by the two-tailed unpaired t test or Mann–Whitney U test. LC3, microtubule-associated proteins 1A/1B light chain 3B; PGRN, progranulin; TDP-43, transactive response DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Figure 10

The KO of Grn increases the secretion of TDP-43 and LC3-II from MEFs.A, the base sequences of the genome-edited regions of PGRN in WT and KO mice were elucidated by Sanger sequencing. Arrowheads in the WT alignment show the two bases deleted by CRISPR–Cas9 genome editing. B, representative confocal images of PGRN (green) immunostaining of MEFs derived from PGRN WT and KO mice. Nuclei were stained with DAPI (blue). Arrowheads indicate vesicle-like structures. The image surrounded by a white square in WT MEFs was magnified. Scale bar represents 5 μm. C–H, PGRN WT and KO MEFs were cultured for 72 h. The cell, P1, and P2 fractions were prepared as shown in the Experimental procedures section. C, representative immunoblots (PGRN, TDP-43, TSG101, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates bands of cleaved TDP-43 used for quantification. D, the P1 and P2 fractions treated with or without PNGase F were immunoblotted with PGRN and α-tubulin antibodies. Representative immunoblots of the cell, P1, and P2 fractions are shown (N = 3). E–H, densitometric data on (E) full-length TDP-43, (F) cleaved TDP-43, (G) TSG101, and (H) LC3-II were calculated from immunoblotting results. Relative signal intensities normalized against PGRN KO MEFs are shown. In bar graphs, data are presented as means ± SD (N = 3). ∗ indicates p < 0.05 by the two-tailed unpaired t test. DAPI, 4′,6-diamidino-2-phenylindole; LC3-II, microtubule-associated proteins 1A/1B light chain 3B; MEF, mouse embryonic fibroblast; PGRN, progranulin; TDP-43, transactive response DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Figure 11

Treatments leading to the secretion of TDP-43 promote TFEB-GFP nuclear localization in SH-SY5Y cells.A and B, SH-SY5Y cells stably expressing TFEB-GFP were exposed to DMSO, 100 nM Baf, 100 nM Con, 100 μM CQ, or 100 nM Vac for 24 h. A, representative confocal images are shown. Nuclei were stained with DAPI (blue). Scale bar represents 20 μm. B, the percentage of cells in which TFEB-GFP translocated in the nucleus calculated from three independent experiments including at least 102 cells is shown. C and D, SH-SY5Y cells stably expressing TFEB-GFP were transfected with control, TARDBP #1, TARDBP #2, GRN, or STX17 siRNA for 48 h. C, representative confocal images are shown. Nuclei were stained with DAPI (blue). Scale bar represents 20 μm. D, the percentage of cells in which TFEB-GFP translocated in the nucleus calculated from three independent experiments including at least 120 cells is shown. E–I, SH-SY5Y cells transfected with control or TFEB siRNA were incubated for 72 h. The cell, P1, and P2 fractions were prepared as shown in the Experimental procedures section. E, representative immunoblots (TFEB, TDP-43, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates the bands of cleaved TDP-43 used for quantification. F–I, densitometric data on (F) TFEB, (G) full-length TDP-43, (H) cleaved TDP-43, and (I) LC3-II were calculated from immunoblotting results. Relative signal intensities normalized against cells transfected with control siRNA are shown. In bar graphs, data are presented as means ± SD (N = 3). Values with a different superscript indicate p < 0.05 by Tukey’s post hoc test. ∗ indicates p < 0.05 by the two-tailed unpaired t test. Baf, bafilomycin A1; Con, concanamycin A; CQ, chloroquine; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; TDP-43, transactive response DNA-binding protein 43 kDa; TFEB, transcription factor EB; Vac, vacuolin-1.