Expression of TDP-43 C-terminal Fragments in Vitro Recapitulates Pathological Features of TDP-43 Proteinopathies
2009; Elsevier BV; Volume: 284; Issue: 13 Linguagem: Inglês
10.1074/jbc.m809462200
ISSN1083-351X
AutoresLionel M. Igaz, Linda K. Kwong, Alice Chen‐Plotkin, Matthew J. Winton, Travis L. Unger, Yan Xu, Manuela Neumann, John Q. Trojanowski, Virginia M.‐Y. Lee,
Tópico(s)Parkinson's Disease Mechanisms and Treatments
ResumoThe disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) was identified recently as the TDP-43 (TAR DNA-binding protein 43), thereby providing a molecular link between these two disorders. In FTLD-U and ALS, TDP-43 is redistributed from its normal nuclear localization to form cytoplasmic insoluble aggregates. Moreover, pathological TDP-43 is abnormally ubiquitinated, hyperphosphorylated, and N-terminally cleaved to generate C-terminal fragments (CTFs). However, the specific cleavage site(s) and the biochemical properties as well as the functional consequences of pathological TDP-43 CTFs remained unknown. Here we have identified the specific cleavage site, Arg208, of a pathological TDP-43 CTF purified from FTLD-U brains and show that the expression of this and other TDP-43 CTFs in cultured cells recapitulates key features of TDP-43 proteinopathy. These include the formation of cytoplasmic aggregates that are ubiquitinated and abnormally phosphorylated at sites found in FTLD-U and ALS brain and spinal cord samples. Furthermore, we observed splicing abnormalities in a cell culture system expressing TDP-43 CTFs, and this is significant because the regulation of exon splicing is a known function of TDP-43. Thus, our results show that TDP-43 CTF expression recapitulates key biochemical features of pathological TDP-43 and support the hypothesis that the generation of TDP-43 CTFs is an important step in the pathogenesis of FTLD-U and ALS. The disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) was identified recently as the TDP-43 (TAR DNA-binding protein 43), thereby providing a molecular link between these two disorders. In FTLD-U and ALS, TDP-43 is redistributed from its normal nuclear localization to form cytoplasmic insoluble aggregates. Moreover, pathological TDP-43 is abnormally ubiquitinated, hyperphosphorylated, and N-terminally cleaved to generate C-terminal fragments (CTFs). However, the specific cleavage site(s) and the biochemical properties as well as the functional consequences of pathological TDP-43 CTFs remained unknown. Here we have identified the specific cleavage site, Arg208, of a pathological TDP-43 CTF purified from FTLD-U brains and show that the expression of this and other TDP-43 CTFs in cultured cells recapitulates key features of TDP-43 proteinopathy. These include the formation of cytoplasmic aggregates that are ubiquitinated and abnormally phosphorylated at sites found in FTLD-U and ALS brain and spinal cord samples. Furthermore, we observed splicing abnormalities in a cell culture system expressing TDP-43 CTFs, and this is significant because the regulation of exon splicing is a known function of TDP-43. Thus, our results show that TDP-43 CTF expression recapitulates key biochemical features of pathological TDP-43 and support the hypothesis that the generation of TDP-43 CTFs is an important step in the pathogenesis of FTLD-U and ALS. TDP-43 (TAR DNA-binding protein 43) is the major disease protein of sporadic and familial frontotemporal lobar degeneration (FTLD) 4The abbreviations used are: FTLD-U, frontotemporal lobar degeneration with ubiquitin-positive inclusions; ALS, amyotrophic lateral sclerosis; CFTR, cystic fibrosis transmembrane conductance regulator; CTF, C-terminal fragment; hnRNP, heterogeneous nuclear ribonucleoprotein; IP, immunoprecipitation; mAb, monoclonal antibody; NES, nuclear export signal; NLS, nuclear localization signal; pAb, polyclonal antibody; RRM, RNA recognition motifs; RIPA, radioimmunoprecipitation assay; CFTR, cystic fibrosis transmembrane conductance regulator; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid; RT, reverse transcription; C-t, C terminus; CNS, central nervous system. with ubiquitin-positive, tau-negative inclusions (FTLD-U) with or without motor neuron disease as well as sporadic and the majority of familial amyotrophic lateral sclerosis (ALS) cases (1Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. Feiden W. Kretzschmar H.A. Trojanowski J.Q. Lee V.M.Y. Science. 2006; 314: 130-133Crossref PubMed Scopus (4417) Google Scholar, 2Forman M.S. Trojanowski J.Q. Lee V.M.Y. Curr. Opin. Neurobiol. 2007; 17: 548-555Crossref PubMed Scopus (102) Google Scholar). Human TDP-43 is encoded by the TARDBP gene on chromosome 1. It is a 414-amino acid nuclear protein with two highly conserved RNA recognition motifs (RRM1 and RRM2) and a C-terminal tail with a typical glycine-rich region that mediates protein-protein interactions, including interactions with other heterogeneous ribonucleoprotein (hnRNP) family members such as hnRNP A1, A2/B1, and A3 (3Buratti E. Brindisi A. Giombi M. Tisminetzky S. Ayala Y.M. Baralle F.E. J. Biol. Chem. 2005; 280: 37572-37584Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Thus, TDP-43 is a ubiquitously expressed RNA/DNA-binding protein that also interacts with other nuclear proteins such as splicing factors. As such, TDP-43 is implicated in repression of gene transcription, regulation of exon splicing, and the functions of nuclear bodies (4Ou S.H. Wu F. Harrich D. Garcia-Martinez L.F. Gaynor R.B. J. Virol. 1995; 69: 3584-3596Crossref PubMed Google Scholar, 5Abhyankar M.M. Urekar C. Reddi P.P. J. Biol. Chem. 2007; 282: 36143-36154Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 6Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (495) Google Scholar, 7Mercado P.A. Ayala Y.M. Romano M. Buratti E. Baralle F.E. Nucleic Acids Res. 2005; 33: 6000-6010Crossref PubMed Scopus (194) Google Scholar, 8Bose J.K. Wang I.F. Hung L. Tarn W.Y. Shen C.K. J. Biol. Chem. 2008; 283: 28852-28859Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 9Wang I.F. Reddy N.M. Shen C.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13583-13588Crossref PubMed Scopus (164) Google Scholar). Pathological TDP-43 accumulates as insoluble aggregates in the central nervous system neurons and glia of patients with FTLD-U and ALS (1Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. Feiden W. Kretzschmar H.A. Trojanowski J.Q. Lee V.M.Y. Science. 2006; 314: 130-133Crossref PubMed Scopus (4417) Google Scholar). Moreover, FTLD-U patients can develop ALS, and ALS patients often suffer from a dementia consistent with FTLD-U (10Murphy J.M. Henry R.G. Langmore S. Kramer J.H. Miller B.L. Lomen-Hoerth C. Arch Neurol. 2007; 64: 530-534Crossref PubMed Scopus (181) Google Scholar). We therefore proposed that these diseases are part of a clinicopathological spectrum of the same neurodegenerative process collectively referred to as TDP-43 proteinopathy (1Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. Feiden W. Kretzschmar H.A. Trojanowski J.Q. Lee V.M.Y. Science. 2006; 314: 130-133Crossref PubMed Scopus (4417) Google Scholar, 2Forman M.S. Trojanowski J.Q. Lee V.M.Y. Curr. Opin. Neurobiol. 2007; 17: 548-555Crossref PubMed Scopus (102) Google Scholar). TDP-43 inclusions are present as cytoplasmic, neuritic, or nuclear inclusions, and affected neurons show a dramatic depletion of normal nuclear TDP-43 (1Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. Feiden W. Kretzschmar H.A. Trojanowski J.Q. Lee V.M.Y. Science. 2006; 314: 130-133Crossref PubMed Scopus (4417) Google Scholar, 11Arai T. Hasegawa M. Akiyama H. Ikeda K. Nonaka T. Mori H. Mann D. Tsuchiya K. Yoshida M. Hashizume Y. Oda T. Biochem. Biophys. Res. Commun. 2006; 351: 602-611Crossref PubMed Scopus (1849) Google Scholar, 12Cairns N.J. Neumann M. Bigio E.H. Holm I.E. Troost D. Hatanpaa K.J. Foong C. White 3rd, C.L. Schneider J.A. Kretzschmar H.A. Carter D. Taylor-Reinwald L. Paulsmeyer K. Strider J. Gitcho M. Goate A.M. Morris J.C. Mishra M. Kwong L.K. Stieber A. Xu Y. Forman M.S. Trojanowski J.Q. Lee V.M.Y. Mackenzie I.R. Am. J. Pathol. 2007; 171: 227-240Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). To mimic this nuclear clearance and to model the sequestration of endogenous TDP-43 into cytoplasmic aggregates, we overexpressed TDP-43 with mutated nuclear localization signals (ΔNLS-TDP-43) in cultured cells that showed a reduction in endogenous nuclear TDP-43 and accumulations of insoluble cytoplasmic aggregates (13Winton M.J. Igaz L.M. Wong M.M. Kwong L.K. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2008; 283: 13302-13309Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). Moreover, overexpression of TDP-43 with a mutated nuclear export signal (ΔNES-TDP-43) resulted in the formation of insoluble nuclear TDP-43 aggregates (13Winton M.J. Igaz L.M. Wong M.M. Kwong L.K. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2008; 283: 13302-13309Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). Pathological TDP-43 is hyperphosphorylated, ubiquitinated, and abnormally cleaved so that C-terminal fragments (CTFs) of TDP-43 accumulate in cells of affected CNS areas (1Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. Feiden W. Kretzschmar H.A. Trojanowski J.Q. Lee V.M.Y. Science. 2006; 314: 130-133Crossref PubMed Scopus (4417) Google Scholar). Indeed, we recently showed that insoluble TDP-43 CTFs are selectively enriched in affected cortical regions compared with the spinal cord of both FTLD-U and ALS cases (14Igaz L.M. Kwong L.K. Xu Y. Truax A.C. Uryu K. Neumann M. Clark C.M. Elman L.B. Miller B.L. Grossman M. McCluskey L.F. Trojanowski J.Q. Lee V.M. Am. J. Pathol. 2008; 173: 182-194Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). These observations suggest that TDP-43 is differentially processed in brain versus spinal cord and that TDP-43 CTFs might seed inclusion formation and aggregation in cortical neurons. Because CTFs contain the TDP-43 glycine-rich region, the accumulation of CTF-rich aggregates may result in abnormal interactions with proteins involved in the splicing machinery of affected cells. However, little is known about how pathological TDP-43 is cleaved to generate TDP-43 CTFs or the biochemical properties of disease-related TDP-43 CTFs. Here we address these questions by identifying the cleavage site of an endogenous TDP-43 CTF purified from FTLD-U brains. We also model the aggregation of this and several other TDP-43 CTFs in the cytoplasm of cultured cells and show that expression of TDP-43 CTFs is sufficient to generate cytoplasmic aggregates. Moreover, these insoluble CTFs are ubiquitinated and abnormally phosphorylated at sites similar to those in human FTLD-U and ALS CNS samples. Finally, we demonstrate that cells expressing TDP-43 CTFs display abnormal splicing of the cystic fibrosis transmembrane conductance regulator (CFTR), which is relevant to TDP-43 function because alternative splicing of this gene is known to be regulated by TDP-43 (6Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (495) Google Scholar). Our results recapitulate unique features of pathological TDP-43 that are hallmarks of FTLD-U and ALS and implicate the generation of TDP-43 CTFs as a key event in the pathogenesis of TDP-43 proteinopathies. Constructs-N-terminal truncations (see below) were generated by PCR of human TDP-43 using the following primers: 177-TDP-43, 5′GGATCCATGCTTCCTAATTCTAAGCAAAGCC-3′; 187-TDP-43, 5′-GGATCCATGCCTTTGAGAAGCAGAAAAGTGT-3′; 197-TDP-43, 5′-GGATCCATGCGCTGTACAGAGGACATGACTG-3′; and 208-TDP-43, 5′-GGATCCCGGGGTTCTTCTCTCAGTACGGGGAT-3′ and 5′-TCTAGAGCTACATTCCCCAGCCAGAAGACTTAGA-3′. The addition of a Myc epitope tag to the 5′-end of the TDP-43 N-terminal truncations was achieved by PCR, using the following primers: 5-Myc-177-TDP-43, 5′-GGATCCATGGAACAAAAACTCATCTCGGAAGAGGATCTGCTTCCTAATTCTAAGCAAAGCC-3′; Myc-187-TDP-43, 5′-GGATCCATGGAACAAAAACTCATCTCGGAAGAGGATCTGCCTTTGAGAAGCAGAAAAGTGT-3′; Myc-197-TDP-43, 5′-GGATCCATGGAACAAAAACTCATCTCGGAAGAGGATCTGCGCTGTACAGAGGACATGACTG-3′; and Myc-208-TDP-43, 5′-GGATCCATGGAACAAAAACTCATCTCGGAAGAGGATCTGCGGGAGTTCTTCTCTCAGTACGGGGAT-3′ and 5′-TCTAGAGCTACATTCCCCAGCCAGAAGACTTAGA-3′. All of the PCR products were cloned into the pGEM-T vector (Promega, Madison, WI). Following sequence analysis, the PCR products were subcloned into pcDNA 5/To plasmid (Invitrogen) using restriction sites BamHI and XbaI. A diagram of each TDP-43 CTF is shown in Fig. 1B. Myc-WT-TDP-43, Myc-ΔNLS-TDP-43, and Myc-ΔNES-TDP-43 were previously described (13Winton M.J. Igaz L.M. Wong M.M. Kwong L.K. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2008; 283: 13302-13309Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). Human TDP-43 short hairpin RNA (targeted to the 3′-untranslated region) was obtained from OriGene (Rockville, MD; catalog number TR308946). pcDNA 3.1 α-synuclein was previously described (15Paxinou E. Chen Q. Weisse M. Giasson B.I. Norris E.H. Rueter S.M. Trojanowski J.Q. Lee V.M. Ischiropoulos H. J. Neurosci. 2001; 21: 8053-8061Crossref PubMed Google Scholar). Antibodies-Commercial antibodies used in this study were: rabbit anti-TDP-43 polyclonal antibody (pAb) raised to amino acids 1–260 (Protein Tech Group, Chicago, IL), a human specific mouse monoclonal (mAb) raised to the same TDP-43 sequence (2E2-D3) (Abnova, Taipei, Taiwan), anti-Myc mAb (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), anti-HA (12CA5 mouse mAb; Roche Applied Science), and anti-α-glyceraldehyde-3-phosphate dehydrogenase (6C5 mouse mAb; Advanced ImmunoChemical Inc, Long Beach, CA). A rabbit anti-TDP-43 pAb raised to amino acids 394–414 (C-t TDP-43 pAb) was described previously (14Igaz L.M. Kwong L.K. Xu Y. Truax A.C. Uryu K. Neumann M. Clark C.M. Elman L.B. Miller B.L. Grossman M. McCluskey L.F. Trojanowski J.Q. Lee V.M. Am. J. Pathol. 2008; 173: 182-194Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). A rat phospho-specific mAb that recognizes TDP-43 phosphorylated at Ser409/Ser410 (p409/410 TDP-43) was developed and characterized elsewhere (16Neumann M. Kwong L.K. Lee E.B. Kremmer E. Flatley A. Xu Y. Forman M.S. Troost D. Kretzschmar H.A. Trojanowski J.Q. Lee V.M. Acta Neuropathol. 2009; 117: 137-149Crossref PubMed Scopus (365) Google Scholar). Immunoprecipitation (IP) and N-terminal Cleavage Site Analysis-Sarkosyl-insoluble urea soluble extracts from FTLD-U brains with abundant TDP-43 CTFs were used for IP. Because N-terminal cleaved TDP-43 fragments migrate similarly to IgG light chains, anti-TDP-43 specific mAbs were cross-linked to protein A/G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) with the homobifunctional imidoester cross-linker dimethyl pimelimidate for IP use. Dialyzed urea fractions in RIPA buffer (0.1% SDS, 1% Nonidet P-40, 0.5% sodium dexoycholate, 5 mm EDTA, 150 mm NaCl, 50 mm Tris-HCl, pH 8.0) or diluted SDS fractions from CNS were precleared with protein A/G-agarose and subjected to IP with antibody-protein A/G-agarose beads. Bound proteins and fragments were then eluted from the beads with SDS sample buffer without dithiothreitol (10 mm Tris-HCl, pH 6.8, 1 mm EDTA, 1% SDS, 10% sucrose) at 80 °C to minimize the dissociation of IgG light chain from the bead complex. Eluted proteins were reduced with dithiothreitol at elevated temperature before resolution by 12% Bis-Tris NuPage® (Invitrogen) SDS-PAGE using MOPS buffer system and transferred to sequence grade polyvinylidene difluoride membranes (Bio-Rad). Typically, two gels were run: one for immunoblotting and the other for N-terminal sequencing. For N-terminal sequencing, the membrane was stained with 0.1% Amido Black, and protein bands that correspond to the TDP-43 immuno-positive bands (∼20–25 kDa) on the companion immunoblots were excised. Cleavage sites of TDP-43 fragments were determined by N-terminal automated Edman sequencing on an Applied Biosystems 494 protein sequencer at the Wistar Institute Proteomics Facility. At minimum, eight cycles of sequencing are conducted for amino acid sequence. Cell Culture and Transfection-QBI-293, Neuro2a, and COS-7 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% l-glutamate. The cells were transfected using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. In some experiments, transfected cells were treated with 10 μm MG132 (Sigma-Aldrich) for 16 h. Immunofluorescence Studies-Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100 (Sigma) in PBS for 10 min, blocked with 5% powdered milk in PBS for 2 h, and incubated overnight with primary antibody at 4 °C. Primary antibodies were visualized with secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 594 (Vector Laboratories, Burlingame, CA), and the nuclei were detected using 4′,6′-diamino-2-phenylindole. All of the cells were analyzed using a Nikon TE-2000-E (Nikon, Tokyo, Japan), and images were captured using a CoolSnap-HQ camera (Photometrics, Tuscon, AZ). All of the micrographs show images representative of the total cell population. For quantification of aggregates, several random fields/sample were analyzed, and the percentage of transfected cells that displayed TDP-43 positive accumulations was calculated. Solubility and Biochemical Analysis-To examine the solubility profile of TDP-43, sequential extractions were performed. The cells were washed twice with PBS, lysed in cold RIPA buffer containing 1 mm phenylmethylsulfonyl fluoride, a mixture of protease inhibitors (1 mg/ml pepstatin, leuptin, N-p-Tosyl-l-phenylalanine chloromethyl ketone, Nα-Tosyl-l-lysine chloromethyl ketone hydrochloride, trypsin inhibitor; Sigma), and a mixture of phosphatase inhibitors (2 mm imidazole, 1 mm NaF, 1 mm sodium orthovanadate; Sigma). The cell lysates were sonicated and then cleared by centrifugation at 100,000 × g for 30 min at 4 °C to generate the RIPA soluble samples. To prevent carry-overs, the resulting pellets were washed with RIPA buffer (i.e. resonicated and recentrifuged). Only the supernatants from the first centrifugation were analyzed. RIPA insoluble pellets were then extracted with urea buffer (7 m urea, 2 m thiourea, 4% CHAPS, 30 mm Tris, pH 8.5), sonicated, and centrifuged at 70,000 × g for 30 min at 22 °C. Protease and phosphatase inhibitors were added to all buffers prior to use (1 mm phenylmethylsulfonyl fluoride and a mixture of protease and phosphatase inhibitors). Protein concentration was determined by bicinchoninic acid method (Pierce), and proteins were resolved by 10 or 15% SDS-PAGE and transferred to nitrocellulose membranes. Following transfer, nitrocellulose membranes were blocked in 5% powdered milk and incubated in the primary antibody overnight at 4 °C. Primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, Wegate, PA), and the blots were developed with Renaissance Enhanced Luminal Reagents (PerkinElmer Life Sciences). The digital images were acquired using a Fuji Film Intelligent Darkbox II (Fuji Systems, Stamford, CT). For quantification of TDP-43 CTFs insolubility, densitometric analysis of RIPA-soluble and insoluble fractions of at least three different experiments was performed using Image Quant 5.0 software (Molecular Dynamics Inc, Sunnyvale, CA). Where indicated, cell lysates or postmortem brain tissue from FTLD-U cases sequentially extracted as previously described (1Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. Feiden W. Kretzschmar H.A. Trojanowski J.Q. Lee V.M.Y. Science. 2006; 314: 130-133Crossref PubMed Scopus (4417) Google Scholar) were dephosphorylated by dialysis (20 mmol/liter Tris and 0.2 mmol/liter EDTA, pH 8.0) and treated with Escherichia coli alkaline phosphatase (Sigma) for 2 h at 56 °C. Splicing Analysis-TDP-43 functional activity was assayed through evaluation of CFTR splicing. First, various TDP-43 constructs were transiently transfected into QBI-293 cells using Lipofectamine 2000 reagent (Invitrogen) following standard manufacturer protocols. Forty-eight hours later, a hybrid minigene construct (a generous gift from Dr. F. Baralle, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy) designed to evaluate CFTR exon 9 splicing was transiently transfected into the same cells (6Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (495) Google Scholar, 17Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Supplemental Fig. S3 depicts the structure of the minigene construct, which consists of CFTR exon 9 with portions of the CFTR flanking introns inserted between exons from a hybrid fibronectin-α-globin gene (18Muro A.F. Iaconcig A. Baralle F.E. FEBS Lett. 1998; 437: 137-141Crossref PubMed Scopus (43) Google Scholar). The relative exclusion of exon 9 in the presence of various TDP-43 constructs was then evaluated by primer extension from the flanking exons of exon 9. Total RNA was prepared from cells 72 h after transfection of TDP-43 constructs and 24 h after transfection of the TG(13)T(5) CFTR minigene reporter construct, and RT-PCR was performed using 3 μg of total RNA and 2 μl of the resulting cDNA as described previously (17Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The primers used were: Bra2, TAGGATCCGGTCACCAGGAAGTTGGTTAAATCA; a2-3, CAACTTCAAGCTCCTAAGCCACTGC. PCR conditions were as follows: 95 °C for 10 min (hot start), followed by 30 cycles of denaturing at 95 °C for 30 s, annealing at 57 °C for 30 s, and elongation at 72 °C for 60 s. The PCR products were visualized on a 1.5% agarose gel; relative amounts of different splice products were quantified and visualized using the Agilent 2100 Bioanalyzer on a DNA 1000 chip. The experiments were performed in duplicate and repeated at least three times. N-terminally Cleaved Sites of TDP-43 CTFs-Previously, we have shown that cortical TDP-43 inclusions in FTLD-U and ALS brains are composed predominantly of CTFs (14Igaz L.M. Kwong L.K. Xu Y. Truax A.C. Uryu K. Neumann M. Clark C.M. Elman L.B. Miller B.L. Grossman M. McCluskey L.F. Trojanowski J.Q. Lee V.M. Am. J. Pathol. 2008; 173: 182-194Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). To better understand the biological significance of the CTFs, we determined their cleavage sites by N-terminal sequencing. Cortical urea extracts of FTLD-U brains containing high levels of CTFs were immunoprecipitated with anti-TDP-43 mAb, and the resultant proteins were resolved on SDS-PAGE gels and immunoblotted with a pAb raised to the extreme C terminus (C-t) of TDP-43. Two protein bands with apparent molecular masses of ∼24 and ∼22 kDa were recognized by the anti-C-t TDP-43 pAb (Fig. 1A). The same bands were identified on Amido Black-stained duplicate polyvinylidene difluoride blots and were excised for N-terminal sequencing (Fig. 1A, arrows). Results from the ∼22-kDa band gave a primary sequence beginning at Arg208 in TDP-43 (Fig. 1A, arrow with asterisk), but no sequence was obtained from the ∼24-kDa band. Similar results were obtained from four separate experiments using two different FTLD-U cases. The identification of the N terminus of a CTF together with our previous liquid chromatography/tandem mass spectrometry studies on TDP-43 CTFs showing the presence of residues at the extreme C terminus (1Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. Feiden W. Kretzschmar H.A. Trojanowski J.Q. Lee V.M.Y. Science. 2006; 314: 130-133Crossref PubMed Scopus (4417) Google Scholar) allow us to conclude that we have identified a pathological TDP-43 fragment spanning amino acid residues 208–414 (designated as 208 TDP-43). To study the biochemical properties of TDP-43 CTFs and to determine whether the expression of these fragments recapitulated pathological features of authentic CTFs isolated from FTLD-U and ALS brains, we developed a series of vectors for expression in cultured cells. Plasmids containing the 208 TDP-43 CTF as well as slightly longer CTFs containing residues 177–414, 187–414, or 197–414 of TDP-43 (designated as 177 TDP-43, 187 TDP-43, and 197 TDP-43, respectively) were generated (Fig. 1B). Each TDP-43 CTF cDNA was expressed in QBI-293 cells, a human embryonic kidney cell line, as well as N2a, a mouse neuroblastoma cell line, and the electrophoretic mobility of the fragments migrated between 20 and 25 kDa (Fig. 1C). TDP-43 CTFs Expressed in Cultured Cells Are Insoluble and Hyperphosphorylated-Previous studies have shown that TDP-43 CTFs isolated from FTLD-U and ALS brains are insoluble and hyperphosphorylated (1Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. Feiden W. Kretzschmar H.A. Trojanowski J.Q. Lee V.M.Y. Science. 2006; 314: 130-133Crossref PubMed Scopus (4417) Google Scholar). To determine whether these pathological properties can be recapitulated in cultured cells, we expressed all four TDP-43 CTFs constructs (i.e. 177 TDP-43, 187 TDP-43, 197 TDP-43, and 208 TDP-43) in N2a (Fig. 2) and QBI-293 (supplemental Fig. S1) cells. Sequential extraction of cells overexpressing each of the four CTFs with RIPA and urea buffers showed a progressive increase in the degree of RIPA insolubility going from the largest to the smallest CTF with 177 TDP-43 CTFs being the most soluble and 197 and 208 TDP-43 CTFs being the most insoluble (Fig. 2, A and B, and supplemental Fig. S1). Quantitative immunoblotting showed that almost 100% of the two smaller TDP-43 CTFs are insoluble in RIPA and can only be extracted by urea (Fig. 2B). However, expression of TDP-43 CTFs tagged with the Myc epitope in QBI-293 cells (supplemental Fig. S2) increased their solubility when compared with their untagged counterparts (compare supplemental Figs. S1 and S2). We also detected multiple immunobands (particularly those recovered in the urea fractions) upon expression of TDP-43 CTFs in cultured cells (Fig. 2A and supplemental Fig. S1). Because pathological TDP-43 CTFs recovered from FTLD-U and ALS brains are hyperphosphorylated at multiple sites including hyperphosphorylation at Ser409 and Ser410 (p409/410), we asked whether the CTFs expressed in transfected cells are also hyperphosphorylated (19Hasegawa M. Arai T. Nonaka T. Kametani F. Yoshida M. Hashizume Y. Beach T.G. Buratti E. Baralle F. Morita M. Nakano I. Oda T. Tsuchiya K. Akiyama H. Ann. Neurol. 2008; 64: 60-70Crossref PubMed Scopus (503) Google Scholar). Using a rat mAb specific for p409/410 (16Neumann M. Kwong L.K. Lee E.B. Kremmer E. Flatley A. Xu Y. Forman M.S. Troost D. Kretzschmar H.A. Trojanowski J.Q. Lee V.M. Acta Neuropathol. 2009; 117: 137-149Crossref PubMed Scopus (365) Google Scholar), we found that all four TDP-43 CTFs displayed robust phospho-specific signals and that labeled phospho-immunobands showed slower apparent electrophoretic mobility than the main protein band recognized by the anti-C-t TDP-43 pAb (Fig. 2A and supplemental Fig. S1, red asterisks highlight the same immunobands detected by C-t TDP-43 and p409/410 antibodies, and the black asterisk identifies the main protein band recognized only by the anti-C-t TDP-43 pAb). Significantly, although the electrophoretic mobility of nonphosphorylated 208 TDP-43 CTFs was close to 20 kDa, the phosphorylated counterpart migrated at ∼22 kDa. Moreover, phosphorylated TDP-43 CTFs were detected only in the urea fraction, and phosphorylation at Ser409 and Ser410 was not seen in endogenous TDP-43 recovered from RIPA extractions (Fig. 2A and supplemental Fig. S1). Thus, like pathological TDP-43 CTFs recovered from diseased brains, TDP-43 CTFs in our in vitro models are phosphorylated, and the phosphorylated TDP-43 CTFs are insoluble. To characterize the morphology of these phosphorylated, insoluble TDP-43 CTFs, we conducted double label immunofluorescence analysis of transfected cells using anti-C-t TDP-43 pAb and the rat anti-p409/410 mAb. Phosphorylated Ser409/Ser410 was detected within C-t TDP-43-positive aggregates when each of the four TDP-43 CTFs was expressed (Fig. 2C and supplemental Fig. S1). Phosphorylated endogenous nuclear TDP-43 was not seen in control or untransfected cells, and the foci of aggregated phosphorylated TDP-43 CTFs were observed within diffuse cytoplasmic TDP-43 immunoreactivity when longer, more soluble CTFs (e.g. 177 TDP-43) were expressed (Fig. 2C and supplemental Fig. S1). Occasionally, we observed CTFs in the nucleus, and we attribute this to the overexpression and small size of the CTFs. Finally, we also observed a direct correlation bet
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