ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS

Abstract

Dominant mutations in two functionally related DNA/RNA-binding proteins, trans-activating response region (TAR) DNA-binding protein with a molecular mass of 43 KDa (TDP-43) and fused in sarcoma/translocation in liposarcoma (FUS/TLS), cause an inherited form of ALS that is accompanied by nuclear and cytoplasmic aggregates containing TDP-43 or FUS/TLS. Using isogenic cell lines expressing wild-type or ALS-linked TDP-43 mutants and fibroblasts from a human patient, pulse-chase radiolabeling of newly synthesized proteins is used to determine, surprisingly, that ALS-linked TDP-43 mutant polypeptides are more stable than wild-type TDP-43. Tandem-affinity purification and quantitative mass spectrometry are used to identify TDP-43 complexes not only with heterogeneous nuclear ribonucleoproteins family proteins, as expected, but also with components of Drosha microprocessor complexes, consistent with roles for TDP-43 in both mRNA processing and microRNA biogenesis. A fraction of TDP-43 is shown to be complexed with FUS/TLS, an interaction substantially enhanced by TDP-43 mutants. Taken together, abnormal stability of mutant TDP-43 and its enhanced binding to normal FUS/TLS imply a convergence of pathogenic pathways from mutant TDP-43 and FUS/TLS in ALS.

Fig. 1.

Characterization of isogenic cell lines expressing a single copy of wild-type and ALS-linked TDP-43 mutations. (A) Schematic representation of site-directed recombinase-based system to generate isogenic stable cell lines, in which CMV promoter was used to drive the expression of tetracycline (Tet)-inducible wild-type and mutant genes that were integrated at a common locus [Flp Recognition Target (FRT) site]. Normally, Tet repressor (TetR) binds to Tet operator (TetO), repressing transcription. On addition, binding of Tet to TetR induces a conformation change and releases TetR from TetO, allowing transcription to start. Lower shows the LAP tag of TDP-43. The LAP tag is composed of GFP followed by PreScission protease cleavage sequences and 6× histidine tag. TDP-43 is tagged at the N terminus with myc peptide (EQKLISSEEDL) and at the C terminus with HA peptide (YPYDVPDYA). Three different mutations, G298S, Q331K, and M337V, were used in this study. (B) Expression of transgene in isogenic stable cell lines. The transgenes are under TetR control. The transgenes express on incubating with tetracycline (−, without tetracycline; +, with tetracycline); 20 μg total cell extract was loaded for each lane. Recombinant TDP-43 of known amount is loaded for the quantification, and tubulin is used as loading control. Exposure shown was taken on the same blot for quantification. (C) Immunofluorescence images of TDP-43 in HeLa cells. Both rabbit polyclonal antibody (ProteinTech) and mouse monoclonal antibody (FL4) showed similar staining pattern for nuclear TDP bodies. (D) Fluorescent images of isogenic cell lines. Upper is GFP signal, and Lower is DAPI-stained to mark the nucleus. All LAP-tagged TDP-43 form nuclear speckles that are similar to immunofluorescence images of endogenous TDP-43 (C). (Scale bar, 10 μm.)

Fig. 2.

ALS-linked TDP-43 mutations exhibit longer half-lives. (A) Representative autoradiogram and immunoblots of the pulse-chase assay using isogenic cell lines expressing wild type (wt), G298S, Q331K, and M337V mutations in TDP-43. LAP-tagged wt–TDP-43 and its mutants were immunoprecipitated and run on SDS-PAGE for autoradiography and immunoblotting. Serial dilutions of 0-h point (start of chase) were used to monitor the linearity of the autoradiograph signal. The autoradiograph signals were normalized to the immunoblotting signals and plotted using 0 h as 100%. (B) Half-lives of LAP-tagged TDP-43 and its ALS-linked mutations (n = 5). Error bar represents SEM. (C) Half-lives of TDP-43 in primary human fibroblasts. Wild-type TDP-43 exhibits 4-h half-life, whereas TDP-43 in heterozygote harboring a copy of G298S mutant exhibits 11-h half-life (n = 5). Error bar represents SEM.

Fig. 3.

Quantitative proteomic analysis of TDP-43 using SILAC coupled with TAP. (A) Schematic representation of TAP purification and quantitative analysis using SILAC. (B) Samples from key steps (clarified lysate, immunoprecipitation for GFP (GFP-IP), PreScission elution, and final elution) in purification were probed with TDP-43 antibody, showing that tagged TDP-43 associates and copurifies with endogenous TDP-43. (C) Silver stain of the samples from the final elution of TAP step. Arrow indicates tagged TDP-43. (D) Representative mass spectrum for TDP-43 peptide. The corresponding light isotope-containing peptide is below detection limit, whereas the heavy Lys/Arg-containing peptide is detected. Asterisks indicate the natural occurrences of ¹³C/¹⁵N in the peptide, as revealed by high resolution mass spectrometry. (E) Confirmation of putative TDP-43–associated proteins by immunoblotting, including hnRNP Q, hnRNP A2/B1, hnRNP K, hnRNP H, and interleukin-enhancer binding factor 3/nuclear factor 90 KDa (ILF3/NF90). (F) Summary for putative TDP-43–associated proteins. TDP-associated proteins were identified with peptides containing only heavy Lys/Arg and by multiple different peptides from at least two independent runs. The associated proteins were grouped according to the known assigned functions for the proteins.

Fig. 4.

ALS-linked TDP-43 mutations associate with FUS/TLS. (A) Reciprocal immunoprecipitation shows that TDP-43 interacts with FUS/TLS using HeLa cells. A monoclonal antibody against splicing factor proline/glutamine-rich or polypyrimidine tract binding protein-associated splicing factor (SFPQ/PSF) was used for control. (B) PreScission elution fractions from GFP pull-down samples showed that FUS/TLS associates more prominently with Q331K and M337V mutations in TDP-43. (C) Schematic representation of in situ proximity ligation assay. (D) Quantification of in situ proximity ligation assay (PLA) results (n = 3; cell numbers > 100 per experiment). Error bar represents SEM. A combination of anti-myc antibody and rabbit IgG molecules was used as negative control, whereas a combination of anti-myc and anti–TDP-43 antibodies was used as positive control. The monitored signal used a combination of anti-myc and anti-TLS/FUS antibody. (E) Representative results for in situ proximity ligation assay. (Scale bar, 15 μm.)