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. 2007 Sep 26;27(39):10530-4.
doi: 10.1523/JNEUROSCI.3421-07.2007.

Progranulin mediates caspase-dependent cleavage of TAR DNA binding protein-43

Yong-Jie Zhang  1 , Ya-fei Xu, Chad A Dickey, Emanuele Buratti, Francisco Baralle, Rachel Bailey, Stuart Pickering-Brown, Dennis Dickson, Leonard Petrucelli
Affiliations

Progranulin mediates caspase-dependent cleavage of TAR DNA binding protein-43

Yong-Jie Zhang et al. J Neurosci. .

Abstract

TAR DNA binding protein-43 (TDP-43) is the pathologic substrate of neuronal and glial inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTDL-U) and in amyotrophic lateral sclerosis (ALS). Mutations in the progranulin gene (PGRN) have been shown to cause familial FTLD-U. The relationship between progranulin and TDP-43 and their respective roles in neurodegeneration is unknown. We report that progranulin mediates proteolytic cleavage of TDP-43 to generate approximately 35 and approximately 25 kDa species. Suppression of PGRN expression with small interfering RNA leads to caspase-dependent accumulation of TDP-43 fragments that can be inhibited with caspase inhibitor treatment. Cells treated with staurosporine also induced caspase-dependent cleavage and redistribution of TDP-43 from its nuclear localization to cytoplasm. Altered cleavage and redistribution of TDP-43 in cell culture models are similar to findings in FTLD-U and ALS. The results suggest that abnormal metabolism of TDP-43 mediated by progranulin may play a pivotal role in neurodegeneration.

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Figures

Figure 1.
Figure 1.
Proteolytic processing of TDP-43. A, Progranulin knockdown in H4 neuroglioma cell line using siRNA. B, Schematic representation of the amino acid sequence of TDP-43 (Entrez accession number NP_031401) depicting the three caspase-3 recognition motifs, DXXD (bold and underlined), cleavage of which would be predicted to generate fragments with approximate molecular weights of 42 kDa (red), 35 kDa (green), and 25 kDa (blue). C, In vitro cleavage of recombinant TDP-43 protein. GST-tagged TDP-43 protein (2 μg) was incubated with purified cleaved caspase-3, caspase-7, or caspase-8 (2 U; Chemicon) for 4 h. Samples were separated on 10% SDS-PAGE and immunoblotted with rabbit anti-TDP-43 antibody. Similar results were obtained in duplicate experiments. D, In vitro generation of pathologic TDP-43 by cells treated with PGRN siRNA but not by cells treated with control siRNA. Progranulin deficiency leads to an increase in cleaved caspase-3 activity. Treatment with a pan-caspase inhibitor suppresses progranulin-mediated TDP-43 cleavage and caspase-3 activity. Similar results were obtained in triplicate experiments. E, Biochemical analyses of TDP-43 in sporadic and familial (PGRN mutant; Gly333ValfsX28) FTLD-U (Gass et al., 2006) and AD brains. Immunoblots of urea fractions from temporal cortex of FTLD-U patients and PGRN siRNA total cell extract with rabbit anti-TDP-43 antibody showed a pathological profile of TDP-43 similar to that in progranulin knockdown experiments but not in Alzheimer's disease or vascular dementia brains (data not shown). F.L., Full-length.
Figure 2.
Figure 2.
Cleavage, solubility, and cellular localization of TDP-43. A, H4 cells treated with staurosporine (0.2 and 1 μm, 3 h) and PGRN siRNA revealed reduction in full-length TDP-43, proteolytic cleavage of TDP-43, and increases in caspase-3 activity. Similar results were obtained in triplicate experiments. B, Biochemical analyses of TDP-43 in HeLa cells. Immunoblots of cell extracts treated with PRGN siRNA or staurosporine with rabbit anti-TDP-43 antibody revealed the pathologic ∼25 and ∼35 kDa bands in Triton X-100-insoluble fractions. Similar results were obtained in triplicate experiments. C, Subcellular redistribution of TDP-43. Immunofluorescent staining for endogenous TDP-43 in H4 cells treated with control siRNA, PGRN siRNA, or staurosporine (0.2 and 1 μm) with rabbit anti-TDP-43 (green) or mouse monoclonal anti-β-actin (red) and examined by confocal microscopy. The nucleus is stained with 4′,6′-diamidino-2-phenylindole (DAPI; blue). Representative images of a field (n = 5) of one of three independent experiments from each culture. Scale bar, 10 μm. D, TDP-43 immunohistochemistry of motor neurons in ALS shows normal nuclear localization but no cytoplasmic labeling (top), early pathologic immunoreactivity characterized by diffuse granular cytoplasmic with no nuclear labeling (middle), and fully formed fibrillar cytoplasmic skein-like inclusions with no nuclear labeling (bottom). Pathologic redistribution of TDP-43 in cultured cells is similar to that in human disease. Magnification, 40×. Ctrl, Control; Ins, insoluble; Sol, soluble; Stspn, staurosporine.
Figure 3.
Figure 3.
Cellular localization of caspase-resistant TDP-43. A, H4 cells transfected with either myc-tagged wild-type TDP-43 or myc-tagged double caspaseTDP-43 mutant (D89E, 35 kDa fragment; D219E, 25 kDa fragment) in the presence or absence of staurosporine (6 h, 1 μm). Immunoblots of cell extracts probed with mouse anti-myc antibody revealed resistance of the caspase mutant TDP-43 to proteolytic cleavage in cells treated with staurosporine. Similar results were obtained in duplicate experiments. B, Subcellular redistribution of wild-type and caspase mutant TDP-43. Immunofluorescent staining for myc-tagged wild-type and caspase mutant TDP-43 in H4 cells treated with staurosporine (6 h, 1 μm) with mouse anti-TDP-43 (green) and examined by confocal microscopy. The nucleus is stained with 4′,6′-diamidino-2-phenylindole (DAPI; blue). Caspase mutant TDP-43 is retained in the nucleus in the presence of staurosporine. Scale bar, 20 μm. Representative images of one of three independent experiments from each culture. Stspn, Staurosporine; WT, wild type.
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References

    1. Baker M, Mackenzie IR, Pickering-Brown SM, Gass J, Rademakers R, Lindholm C, Snowden J, Adamson J, Sadovnick AD, Rollinson S, Cannon A, Dwosh E, Neary D, Melquist S, Richardson A, Dickson D, Berger Z, Eriksen J, Robinson T, Zehr C, et al. Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. Nature. 2006;442:916–919. - PubMed
    1. Buratti E, Baralle FE. Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9. J Biol Chem. 2001;276:36337–36343. - PubMed
    1. Buratti E, Dork T, Zuccato E, Pagani F, Romano M, Baralle FE. Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping. EMBO J. 2001;20:1774–1784. - PMC - PubMed
    1. Buratti E, Brindisi A, Giombi M, Tisminetzky S, Ayala YM, Baralle FE. TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail: an important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing. J Biol Chem. 2005;280:37572–37584. - PubMed
    1. Cruts M, Gijselinck I, van der Zee J, Engelborghs S, Wils H, Pirici D, Rademakers R, Vandenberghe R, Dermaut B, Martin JJ, van Duijn C, Peeters K, Sciot R, Santens P, De Pooter T, Mattheijssens M, Van den Broeck M, Cuijt I, Vennekens K, De Deyn PP, et al. Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. Nature. 2006;442:920–924. - PubMed

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