Advertisement

A “Two-hit” Hypothesis for Inclusion Formation by Carboxyl-terminal Fragments of TDP-43 Protein Linked to RNA Depletion and Impaired Microtubule-dependent Transport*

  • G. Scott Pesiridis
    Affiliations
    From the Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, and
    Search for articles by this author
  • Kalyan Tripathy
    Affiliations
    From the Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, and
    Search for articles by this author
  • Selçuk Tanik
    Affiliations
    From the Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, and
    Search for articles by this author
  • John Q. Trojanowski
    Affiliations
    From the Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, and

    Institute on Aging, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
    Search for articles by this author
  • Virginia M.-Y. Lee
    Correspondence
    To whom correspondence should be addressed: Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 3600 Spruce St., 3rd Floor Maloney Bldg., Philadelphia, PA 19104. Tel.: 215-662-6427; Fax: 215-349-5909;
    Affiliations
    From the Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, and

    Institute on Aging, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant AG17586. This work was also supported by the Koller Foundation for ALS Research as well as a gift from the Podolin family.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. 1–6.
Open AccessPublished:March 24, 2011DOI:https://doi.org/10.1074/jbc.M111.231118
      Carboxyl-terminal fragments (CTFs) of TDP-43 aggregate to form the diagnostic signature inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis, but the biological significance of these CTFs and how they are generated remain enigmatic. To address these issues, we engineered mammalian cells with an inducible tobacco etch virus (TEV) protease that cleaves TDP-43 containing a TEV cleavage site. Regions of TDP-43 flanking the second RNA recognition motif (RRM2) are efficiently cleaved by TEV, whereas sites within this domain are more resistant to cleavage. CTFs containing RRM2 generated from de novo cleavage of nuclear TDP-43 are transported to the cytoplasm and efficiently cleared, indicating that cleavage alone is not sufficient to initiate CTF aggregation. However, CTFs rapidly aggregated into stable cytoplasmic inclusions following de novo cleavage when dynein-mediated microtubule transport was disrupted, RNA was depleted, or natively misfolded CTFs were introduced into these cells. Our data support a “two-hit” mechanism of CTF aggregation dependent on TDP-43 cleavage.

      Introduction

      The heterogeneous nuclear ribonucleoprotein (hnRNP)
      The abbreviations used are: hnRNP, heterogeneous nuclear ribonucleoprotein particle; RNP, ribonucleoprotein particle; FTLD-TDP, frontotemporal lobar degeneration; ALS, amyotrophic lateral sclerosis; CTF, C-terminal fragment; NTF, N-terminal fragment; TEV, tobacco etch virus; RRM, RNA recognition motif; MT, microtubule; CC1, coiled-coil 1; EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine; NLS, nuclear localization signal; eGFP, enhanced GFP.
      TDP-43 (TAR DNA-binding protein of 43 kDa) forms pathological inclusions that are diagnostic hallmarks of amyotrophic lateral sclerosis (ALS) and the major form of frontotemporal lobar degeneration (FTLD-TDP) (
      • Neumann 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.
      ). Missense mutations in TDP-43 provide a genetic link between hnRNP functions and motor neuron disease (
      • Lagier-Tourenne C.
      • Cleveland D.W.
      ,
      • Pesiridis G.S.
      • Lee V.M.-Y.
      • Trojanowski J.Q.
      ). TDP-43 is predominantly localized to the nucleus, where it regulates RNA splicing, mRNA stability, microRNA processing, and transcription (
      • Gregory R.I.
      • Yan K.P.
      • Amuthan G.
      • Chendrimada T.
      • Doratotaj B.
      • Cooch N.
      • Shiekhattar R.
      ,
      • Ou S.H.
      • Wu F.
      • Harrich D.
      • García-Martínez L.F.
      • Gaynor R.B.
      ,
      • Abhyankar M.M.
      • Urekar C.
      • Reddi P.P.
      ). However, in neurodegenerative TDP-43 proteinopathies like ALS and FTLD-TDP, a loss of nuclear TDP-43 coincides with cytoplasmic TDP-43 inclusions (
      • Neumann 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.
      ). These inclusions contain full-length TDP-43 and TDP-43 C-terminal fragments (CTFs) that are hyperphosphorylated and ubiquitinated. However, it is unclear how each of these species of TDP-43 contributes to inclusion formation or disease pathogenesis.
      TDP-43 is a typical 2XRRM-gly hnRNP composed of two tandem RNA recognition motifs (RRM1 and RRM2) followed by a glycine-rich carboxyl terminus (Fig. 1) (
      • Burd C.G.
      • Matunis E.L.
      • Dreyfuss G.
      ). Both RRMs retain nucleic acid binding properties, yet only RRM1 appears essential for RNA splicing (
      • Buratti E.
      • Baralle F.E.
      ). The glycine-rich domain is proposed to interact with other hnRNPs to synergistically affect alternative splicing of specific RNA transcripts (
      • Buratti E.
      • Brindisi A.
      • Giombi M.
      • Tisminetzky S.
      • Ayala Y.M.
      • Baralle F.E.
      ,
      • D'Ambrogio A.
      • Buratti E.
      • Stuani C.
      • Guarnaccia C.
      • Romano M.
      • Ayala Y.M.
      • Baralle F.E.
      ). A common feature of shuttling hnRNPs involved in RNA splicing is that their nuclear export is coupled to the export and maturation of mRNA in distinct ribonucleoprotein (RNP) complexes (
      • Piñol-Roma S.
      ,
      • Piñol-Roma S.
      • Dreyfuss G.
      ). In this respect, the nucleocytoplasmic shuttling of TDP-43 is dependent on its bipartite importin-α nuclear localization signal, a chromosome maintenance region 1 (CRM-1) nuclear export signal in RRM2, and undefined aspects of the carboxyl-terminal glycine-rich domain (
      • Ayala Y.M.
      • Zago P.
      • D'Ambrogio A.
      • Xu Y.F.
      • Petrucelli L.
      • Buratti E.
      • Baralle F.E.
      ,
      • Winton M.J.
      • Igaz L.M.
      • Wong M.M.
      • Kwong L.K.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ).
      Figure thumbnail gr1
      FIGURE 1TEV protease cell system. A, schematic of the TDP-43 cleavage system depicting de novo cleavage of human TDP-43. TDP-43 constructs with a TEV cleavage site are transfected into a stable TREx-293 cell line that expresses a tetracycline-inducible TEV protease specifically localized to the nucleus (TEVnuc) or cytoplasm (TEVcyt). The N-terminal FLAG and C-terminal Myc tag serve as a probe to track fragment localization, trafficking, and solubility. B, immunofluorescence of TREx-293 cells expressing HA-tagged (red) TEVnuc or TEVcyt illustrates their predominantly nuclear and cytoplasmic localization, respectively. Endogenous TDP-43 (green) staining confirms the established TDP-43 nuclear localization pattern in TREx-293 cells. Scale bar, 5 μm. C, anti-HA immunoblot of TEVnuc and TEVcyt TREx-293 cell extracts harvested at 0, 6, 24, and 48 h following the addition of 1 μg/ml tetracycline confirms the inducible expression of HA-tagged TEV protease relative to GAPDH protein loading control. D, the activity of TEVnuc and TEVcyt was confirmed using a luciferase reporter system that is sensitive to TEV protease activity. The TEV test substrate is a fusion of the GAL4 DNA binding domain and the VP-16 trans-activating domain separated by a linker containing the TEV protease cleavage site (GAL4-ENLYFQ/G-VP16). Luciferase activity was measured in the absence (black bars) and presence (white bars) of the TEV inducer tetracycline, demonstrating TEV protease activity in cells. Error bars, 1 S.D. from five replicate experiments.
      CTFs are signatures of disease exclusively associated with TDP-43 pathology, and CTFs cleaved in the middle of RRM2 extending from amino acid 208 to the carboxyl terminus of TDP-43 have been recovered from FTLD-TDP brains (
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ). Overexpression of CTFs of varying lengths in cultured cells produces cytoplasmic aggregates that are ubiquitinated and phosphorylated, recapitulating biochemical properties of authentic TDP-43 inclusions (
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ,
      • Dormann D.
      • Capell A.
      • Carlson A.M.
      • Shankaran S.S.
      • Rodde R.
      • Neumann M.
      • Kremmer E.
      • Matsuwaki T.
      • Yamanouchi K.
      • Nishihara M.
      • Haass C.
      ,
      • Nonaka T.
      • Kametani F.
      • Arai T.
      • Akiyama H.
      • Hasegawa M.
      ,
      • Zhang Y.J.
      • Xu Y.F.
      • Cook C.
      • Gendron T.F.
      • Roettges P.
      • Link C.D.
      • Lin W.L.
      • Tong J.
      • Castanedes-Casey M.
      • Ash P.
      • Gass J.
      • Rangachari V.
      • Buratti E.
      • Baralle F.
      • Golde T.E.
      • Dickson D.W.
      • Petrucelli L.
      ). Experiments performed in vitro demonstrate that CTFs readily aggregate and form fibrillar structures (
      • Chen A.K.
      • Lin R.Y.
      • Hsieh E.Z.
      • Tu P.H.
      • Chen R.P.
      • Liao T.Y.
      • Chen W.
      • Wang C.H.
      • Huang J.J.
      ,
      • Johnson B.S.
      • Snead D.
      • Lee J.J.
      • McCaffery J.M.
      • Shorter J.
      • Gitler A.D.
      ). However, CTF expression in transgenic flies is well tolerated without neurotoxicity or motor phenotypes (
      • Li Y.
      • Ray P.
      • Rao E.J.
      • Shi C.
      • Guo W.
      • Chen X.
      • Woodruff 3rd, E.A.
      • Fushimi K.
      • Wu J.Y.
      ,
      • Lu Y.
      • Ferris J.
      • Gao F.B.
      ). This raises the question of whether TDP-43 CTFs initiate the formation of TDP-43 inclusions and play a role in neurotoxicity.
      To elucidate the role of de novo generated TDP-43 CTFs in TDP-43 proteinopathies, we established a mammalian cell system that stably expresses an inducible form of the TEV protease to enable temporally regulatable site-specific cleavage of TDP-43. Cleavage of nuclear TDP-43 produces CTFs that are soluble, transported to the cytoplasm, and efficiently cleared from cells. However, de novo generated CTFs, aggregated to form inclusions in cells when they also expressed insoluble cytoplasmic CTF seeds, were depleted of RNA, or their dynein-mediated MT transport was disrupted. These novel findings suggest a “two-hit” hypothesis for the formation of CTF-rich TDP-43 inclusions in FTLD-TDP and ALS wherein a second deleterious event or “second hit” is required to form inclusions following the generation of CTFs.

      EXPERIMENTAL PROCEDURES

      Generation of TEV Cell Lines and Plasmids

      The TEV protease coding sequence was cloned into pCDNA/5TO plasmid (Invitrogen) with an N-terminal hemagglutinin tag (HA-TEV; TEVcyt) for cytoplasmic localization or a triple SV40 nuclear localization signal (NLS) for nuclear localization (NLSSV40-HA-TEV-NLSSV40-NLSSV40; TEVnuc) similar to Pauli et al. (
      • Pauli A.
      • Althoff F.
      • Oliveira R.A.
      • Heidmann S.
      • Schuldiner O.
      • Lehner C.F.
      • Dickson B.J.
      • Nasmyth K.
      ). TREx-293 (Invitrogen) cells transfected with the TEVcyt and TEVnuc plasmids using Lipofectamine 2000 (Invitrogen) were grown in selection media (DMEM, 10% tetracycline-screened FBS, glutamine, penicillin/streptomycin, 5 μg/ml blasticidin, 200 μg/ml hygromycin) at 37 °C followed by subcloning for individual clones expressing TEVnuc and TEVcyt. Stable clones were screened for HA-tagged TEVnuc and TEVcyt expression following an overnight incubation in the presence of 1 μg/ml tetracycline.
      The wild type coding sequence of TDP-43 was amplified by primer extension from a cDNA clone (
      • Winton M.J.
      • Igaz L.M.
      • Wong M.M.
      • Kwong L.K.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ) using primers that encode an N-terminal FLAG tag and a C-terminal Myc tag, followed by insertion into HindIII/XhoI of pcDNA 3.1(+) (Invitrogen). The TEV protease cleavage site (ENLYFQ↓G) was inserted at amino acid positions Gln182, Glu204, Arg208, Gly215, Lys224, Ala260, and Arg272 of TDP-43 by Exsite mutagenesis (Stratagene) (supplemental Table 1). GFP-mRuby plasmid for mammalian cell expression was generated by primer extension to amplify eGFP cDNA, followed by insertion into NheI and BglII of the mRuby expression vector (
      • Kredel S.
      • Oswald F.
      • Nienhaus K.
      • Deuschle K.
      • Röcker C.
      • Wolff M.
      • Heilker R.
      • Nienhaus G.U.
      • Wiedenmann J.
      ). TDP-43(182tev) was subcloned from the pCDNA 3.1(+) plasmid into GFP-mRuby using HindIII and BamHI.
      The Gal4DBD-TEV-VP16 test substrate was made by primer extension of VP16 from pAct (Promega) and inserted into the NotI and KpnI restriction sites of the pBind vector (Promega) to make pBind-Act. Oligonucleotides encoding the TEV protease site were annealed and inserted into BamHI and MluI restriction sites of the pBind-Act vector. Myc-tagged coiled coil 1 (CC1) in pCDNA 3.1 was a gift from Dr. Holzbaur (University of Pennsylvania).

      De Novo TDP-43 Cleavage Assays

      De novo cleavage of TDP-43 was performed in TEVnuc or TEVcyt cells by transfection of a cell monolayer with the appropriate FLAG-TDP-43(tev)-Myc or GFP-TDP-43(tev)-mRuby plasmids using Fugene HD (Roche Applied Science). After 16 h of TDP-43 maturation, TEVnuc or TEVcyt expression was induced with 1 μg/ml tetracycline for indicated time periods. To determine whether GFP-TDP-43(208–414) inclusions sequester de novo cleaved TDP-43, TEVnuc cells were cotransfected with FLAG-TDP-43(182tev)-Myc and GFP-TDP-43(208–414) (in pcDNA5/TO) overnight, followed by induction of TEVnuc and GFP-TDP-43(208–414) for the simultaneous expression of GFP-TDP-43(208–414) and cleavage of FLAG-TDP-43(182tev)-Myc.

      Biochemical Extractions

      Cell lysates were prepared according to the method of Igaz et al. (
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ) except a less stringent lysis buffer, RSB-150 (20 mm Tris, pH 7.4, 0.15 m NaCl, 2.5 mm MgCl2 with protease inhibitors, 0.5 mm phenylmethylsulfonyl fluoride, leupeptin, N-p-tosyl-l-phenylalanine chloromethyl ketone, Nα-tosyl-l-lysine chloromethyl ketone, trypsin inhibitor, and phosphatase inhibitors 2 mm imidazole, 1 mm NaF, and 1 mm sodium orthovanadate; Sigma), was used, followed by extraction of the insoluble material with urea buffer (
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ). Approximately 25–30 μg of the soluble RSB-150 fraction and ∼75 μg of the insoluble urea fraction were separated on SDS-polyacrylamide gels and transferred to 0.2-μm nitrocellulose for immunoblot with the specified antibodies.
      The relative solubility of CTFs extracted from cells that produce CTFs by TEVnuc cleavage was compared with aggregate-prone CTFs and CTFs extracted from FTLD-TDP brain tissue. For de novo cleaved CTFs, cells were transfected with FLAG-TDP-43(182tev) without a C-terminal Myc tag, followed by TEVnuc cleavage and sequential extraction in RSB-150, followed by urea buffer extraction. Aggregate-prone CTFs were generated by transfection of cDNA coding for amino acids 182–414 (see supplemental Table 1) followed by sequential extraction. CTFs were extracted from FTLD-TDP brain tissue using ∼5 g of brain tissue (temporal lobe) as described previously (
      • Neumann 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.
      ) except the tissue was extracted in RSB-150 (5 ml/g of tissue), followed by urea buffer (0.1 ml/g of tissue).

      Antibodies and Immunofluorescence

      For immunoblot analysis, N-terminal fragments (NTFs) and CTFs along with full-length TDP-43 were visualized using a monoclonal antibody (mAb) anti-FLAG (Sigma), anti-eGFP (Millipore), and previously described polyclonal antibodies that recognize the N terminus (TDP-43n) and C terminus of TDP-43 (TDP-43c) (
      • Igaz 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.-Y.
      ). Phosphorylated TDP-43 was visualized using a rat mAb that recognizes phosphorylated TDP-43 at serines 409 and 410 (1D3) (
      • Neumann 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.-Y.
      ). HA-tagged TEV and GAPDH were visualized with a rat mAb anti-HA (3F10, Roche Applied Science) and mouse anti-GAPDH (Advanced Immunochemical), respectively.
      For immunofluorescence, cells were fixed in 4% paraformaldehyde, followed by lysis in 0.2% Triton X-100 (Sigma) and blocking in BSA buffer as described previously (
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ). To detect FLAG and Myc-tagged TDP-43, cells were incubated overnight at 4 °C in a mixture of 1:2,000 mouse anti-FLAG (Sigma) and 1:3,000 rabbit anti-Myc (Sigma), followed by anti-mouse and anti-rabbit secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 594 (Vector Laboratories). In experiments using GFP-TDP-43(208–414), a 1:200 dilution of an anti-mouse antibody conjugated to aminomethylcoumarin acetate was used to detect the anti-FLAG antibody (Vector Laboratories). Triple fluorescence of GFP and mRuby-tagged TDP-43 with CC1-Myc was detected using 1:2,000 rabbit anti-Myc (Sigma) and 1:250 goat anti-mouse conjugated with aminomethylcoumarin acetate (Vector Laboratories). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted onto glass slides using Fluoromount G (SouthernBiotech).
      Microscopy data were collected on an Olympus BX51 microscope equipped with an Olympus PD71 camera. Where appropriate, the number of cells showing cytoplasmic CTFs, nuclear NTFs, and uncleaved TDP-43(tev) substrates were counted using ImagePro Plus (Media Cybernetics) and expressed as a percentage of the number of transfected cells.

      Pulse-Chase Experiments

      Pulse-chase experiments were performed as described by Jansens and Braakman (
      • Jansens A.
      • Braakman I.
      ). TEVnuc cells transfected with flag-TDP-43(182tev)-Myc were incubated overnight at 37 °C. After a 15-min incubation in depletion medium, cells were pulsed for 10 min in 2 ml of labeling medium (DMEM (−Met −Cys)) + 100 μCi/ml [35S]methionine) followed by different chase periods in full medium containing excess methionine and cysteine (DMEM, 10% tetracycline screened fetal bovine serum, glutamine, penicillin/streptomycin, 5 mm methionine, 5 mm cysteine) supplemented with 1 μg/ml tetracycline for induction of TEVnuc expression. Cells were harvested at each time point by scraping with a rubber policeman in 500 μl of radioimmune precipitation assay buffer followed by sonication and centrifugation at 16,000 × g for 15 min at 4 °C. Soluble lysates were incubated with protein A/G beads conjugated with either 5 μg of anti-FLAG (Sigma) or 5 μg of a mouse mAb that recognizes the C terminus of TDP-43 (
      • Igaz 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.-Y.
      ). Immunoprecipitations were incubated for 2 h at 4 °C, followed by four 1-ml washes in radioimmune precipitation assay buffer. Proteins were eluted with 2× SDS-gel loading buffer, separated by SDS-PAGE, and transferred to a 0.2-μm nitrocellulose membrane. The amount of radioactivity in each protein band was assessed by autoradiography using a high density phosphor screen and a PhosphorImager, followed by quantification using ImageQuant (Amersham Biosciences). The total level of each TDP-43 species was assessed by immunoblot using anti-TDPc and the anti-FLAG. The clearance of each TDP-43 species was fit using a first order decay model of the form [A] = [A]oe−τt, where [A] is the normalized concentration of TDP-43 at time t, and τ is the mean lifetime. The half-life was calculated using the relationship, t½ = τln2.

      Inhibition of the Proteasome- and Dynein-mediated Transport

      To inhibit the proteasome, TEVnuc cells transfected with FLAG-TDP-43(182tev)-Myc were treated with 10 μm clasto-lactacystin-β-lactone (EMD Biosciences) for 5 h before the addition of 1 μg/ml tetracycline for an additional 16 h. The change in CTF level was calculated from the CTF band intensities obtained from immunoblot of five replicate experiments using the Multigauge (Fuji) image analysis software package.
      Dynein-mediated MT transport was inhibited using erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (Sigma) or by coexpression of the CC1 domain from the p150(glued) subunit of dynactin (
      • Zhapparova O.N.
      • Burakov A.V.
      • Nadezhdina E.S.
      ). Dynein function before and after inhibition with EHNA and CC1-Myc was assessed by analysis of Golgi morphology by immunostaining with the Golgi marker golgin (GM-130) (BD Transduction Laboratories). Cells transfected with FLAG-TDP-43(182tev)-Myc overnight were treated with 0.1 mm EHNA for 5 h followed by 16 h of TEVnuc cleavage. The effect of CC1-mediated dynein inhibition on CTF localization was performed by cotransfection of cells with GFP-TDP-43(182tev)-mRuby and Myc-tagged CC1 for 24 h, followed by cleavage with TEVnuc for 16 h.

      RNase Treatment

      RNase treatment of cells was performed as described previously (
      • Prasanth K.V.
      • Sacco-Bubulya P.A.
      • Prasanth S.G.
      • Spector D.L.
      ,
      • Prasanth K.V.
      • Prasanth S.G.
      • Xuan Z.
      • Hearn S.
      • Freier S.M.
      • Bennett C.F.
      • Zhang M.Q.
      • Spector D.L.
      ). Briefly, TEVnuc cells transfected with GFP-TDP-43(182tev)-mRuby were incubated in the presence and absence of 1 μg/ml tetracycline for 16 h of TEVnuc expression to generate CTFs. Cells were washed with cytoskeleton buffer (CSK buffer; 0.1 m NaCl, 0.3 m sucrose, 3 mm MgCl2, 10 mm PIPES, pH 6.8), followed by incubation with CSK buffer plus 0.05% Triton X-100 for 3 min on ice. Cells washed twice with CSK buffer were incubated with 1 mg/ml RNase A (Sigma) in CSK buffer for 15 min followed by fixation in 4% paraformaldehyde and DAPI staining. For sequential extractions, cells were lysed by sonication in RSB-150, 1 mg/ml RNase A, followed by centrifugation and extraction of the soluble and insoluble fractions as described (
      • Igaz 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.-Y.
      ).

      RESULTS

      Generation of TEV Protease Cell Lines

      The protease that cleaves TDP-43 in FTLD-TDP and ALS to produce a CTF of ∼25 kDa that is phosphorylated and aggregated is unknown. Our approach to study TDP-43 cleavage and downstream events from CTF production utilized the highly specific TEV protease. The engineered TDP-43 proteolysis system was designed by inserting the TEV cleavage site (ENLYF(Q↓G)) at specific positions in TDP-43 possessing a FLAG tag at the N terminus and a Myc tag at the C terminus (i.e. FLAG-TDP-43-Myc) (Fig. 1A). Cells expressing a tetracycline-inducible TEV protease generated FLAG-tagged NTFs and Myc-tagged CTFs that were further tracked by immunofluorescence and immunoblot of cell extract (Fig. 1A). These novel tools allowed us to elucidate the cell biology and fate of NTFs and CTFs through de novo cleavage of TDP-43.
      Two stable cell lines of the tetracycline-on TREx-293 cell system were generated that contain a tetracycline-inducible TEV protease that is either targeted to the nucleus (TEVnuc) or remains in the cytoplasm (TEVcyt). To verify if TEVnuc and TEVcyt were properly localized, we examined the distribution of the hemaggluttin-tagged TEV proteases by immunofluorescence. TEVnuc appeared to co-localize with nuclear TDP-43, whereas TEVcyt appeared predominantly cytoplasmic (Fig. 1B). As previously shown (
      • Pauli A.
      • Althoff F.
      • Oliveira R.A.
      • Heidmann S.
      • Schuldiner O.
      • Lehner C.F.
      • Dickson B.J.
      • Nasmyth K.
      ), expression of the TEV protease does not alter the cell morphology, nor is it toxic (data not shown). TEV expression was observed after 6 h of induction with tetracycline and maintained expression throughout the experiment (Fig. 1C).
      To verify that the TEV protease is biologically active in cells, we utilized a UASGAL4 promoter-driven luciferase reporter that is sensitive to a TEV-cleavable GAL4-VP16 transcription factor (Fig. 1D). In the absence of tetracycline, no TEVnuc or TEVcyt was expressed (Fig. 1C), and the intact GAL4-ENLYFQ/G-VP16 activated transcription of the luciferase gene in both cell lines (Fig. 1D). Induction of TEVnuc and TEVcyt with tetracycline activated cleavage of the GAL4-ENLYFQ/G-VP16 test substrate, resulting in the loss of luciferase activity (Fig. 1D). This demonstrates that expression of TEVcyt and TEVnuc is tightly regulated by the tetracycline repressor and is functionally active in mammalian cells.

      TEV Site Scanning in TDP-43 Defines CTF Stability

      To probe protease sites in TDP-43 that are structurally accessible and susceptible to TEV cleavage, we inserted the TEV recognition sequence (ENLYF(Q↓G)) at specific amino acid positions in TDP-43 (Fig. 2A). A TEV recognition site inserted at Gln182 tests cleavage within the unstructured linker between RRM1 and RRM2, generating a CTF with RRM2 and the entire C-terminal domain (Fig. 2, A and B). TEV cleavage within the highly structured RRM2 at Arg208 (Fig. 2B) was generated to mimic a putative cleavage position identified by sequencing CTFs purified from FTLD-TDP brain tissue (
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ). TEV cleavage at Ala260 and Arg272 was tested to probe the fate of CTFs and NTFs generated by cleavage in the glycine-rich C-terminal domain (Fig. 2A). Cells transfected with each construct followed by TEVnuc cleavage were assessed by immunofluorescence and immunoblot to detect localization and levels of full-length TDP-43 and cleavage products (Fig. 2, C and D).
      Figure thumbnail gr2
      FIGURE 2TEV site scanning of TDP-43 defines fragmentation properties. A, schematic of FLAG-TDP-43-Myc with arrows indicating the amino acid position of TEV protease cleavage sites in reference to the RRMs and the glycine-rich domain (gly). B, model of TDP-43 RRM domains (Protein Data Bank entries 2cqg and 3d2w) superimposed onto the backbone of the hnRNP-A1 structure (Protein Data Bank entry 1pgz) illustrates the position of each TEV site insertion (red) in the context of the three-dimensional structure. The loops in RRM2 are numbered in order from the N terminus to the C terminus. C, immunofluorescence of TEVnuc cells transfected with FLAG-TDP-43-Myc in the absence (WT) and presence of TEV cleavage site (ENLYF(Q↓G)). TDP-43 cleavage was assessed after 24 h of TEVnuc cleavage. The localization of N-terminal FLAG (green) and C-terminal Myc (red) depicts specific cells that have the uncut nuclear TDP-43 (merge, yellow), cells with a cytoplasmic CTF pattern (white arrows), and cells with NTF only nuclei (white arrowheads). Scale bar, 5 μm. D, immunoblot of cell extracts from C transfected with FLAG-TDP-43-Myc with the indicated TEV protease cleavage position (top) in the absence (−) and presence (+) of the TEV protease inducer, tetracycline. Each panel illustrates TDP-43 NTFs, CTFs, and TEVnuc expression probed with anti-FLAG, anti-TDPc, and anti-HA, respectively. *, nonspecific degradation of TDP-43.
      Cleavage of TDP-43 at Gln182 produced CTFs with a prominent cytoplasmic localization pattern, following cleavage by TEVnuc (Fig. 2C, arrows). However, some cells showed complete clearance of all CTFs while retaining abundant nuclear NTFs (Fig. 2C, arrowheads). Immunoblots of extracts from cells treated to induce Gln182 cleavage showed NTFs and CTFs that migrate with the expected Mr of each fragment (Fig. 2D). In contrast, there were no TDP-43 cleavage products in the absence of a TEV cleavage site (Fig. 2D). This illustrates that cleavage of TDP-43 at a position that includes RRM2 and the C-terminal domain generates stable CTFs that translocate to the cytoplasm, whereas the corresponding NTFs are retained in the nucleus.
      Cleavage of TDP-43 at Arg208 does not produce significant levels of the expected NTFs and CTFs (Fig. 2D). This is most likely due to the inaccessibility of Arg208 to TEVnuc cleavage (Fig. 2B) (
      • Kuo P.H.
      • Doudeva L.G.
      • Wang Y.T.
      • Shen C.K.
      • Yuan H.S.
      ). We further probed the susceptibility of cleavage within RRM2 by inserting additional cleavage sites in four loops that connect elements of secondary structure in RRM2 (Fig. 2, A and B). Although these loops would be predicted to be more accessible to cleavage than Arg208, these sites (including glutamic acid 204 in loop 1, glycine 215 in loop 2, lysine 224 in loop 3, and glycine 252 in loop 5) were also resistant to TEV cleavage (data not shown). Thus, for reasons that are not entirely clear, cleavage positions in RRM2 are resistant to cleavage.
      De novo cleaved CTFs that lack RRM2 are rapidly cleared from the cell. Cleavage of TDP-43 at Ala260 produces unstable CTFs, with only a small number of cells showing weak cytoplasmic CTF staining and virtually no detectable CTFs by immunoblot (Fig. 2, C and D). This observation is even more pronounced when TDP-43 cleavage occurs in the middle of the most glycine-rich sequence at Arg272. This site is homologous with a physiological cleavage site in hnRNP-A1 that is conserved in TDP-43 (
      • Williams K.R.
      • Stone K.L.
      • LoPresti M.B.
      • Merrill B.M.
      • Planck S.R.
      ). In comparison, NTFs generated by both Ala260 and Arg272 cleavage are not readily cleared and are retained in the nucleus (Fig. 2, C and D).
      The TEV site scanning data demonstrate three distinct types of TDP-43 cleavage: 1) efficient cleavage to generate stable CTFs that translocate to the cytoplasm, 2) inaccessible cleavage sites in RRM2 that are resistant to cleavage, and 3) efficient cleavage sites in the C-terminal domain that generate unstable rapidly degraded CTFs. Because de novo cleavage of TDP-43 at Gln182 efficiently produces stable CTFs that translocate to the cytoplasm, we studied these CTFs in more detail to understand if and how secondary perturbations might induce them to form abnormal CTF aggregates similar to those seen in neurodegenerative TDP-43 proteinopathies, such as ALS and FTLD-TDP.
      Because nuclear cleavage of TDP-43 produces cytoplasmic CTFs without inclusions, we tested whether cytoplasmic cleavage of TDP-43 generates a similar distribution of CTF and NTF localization. Here, TEVcyt cleavage is expected to target the small pool of TDP-43 that shuttles between the nucleus and cytoplasm (
      • Ayala Y.M.
      • Zago P.
      • D'Ambrogio A.
      • Xu Y.F.
      • Petrucelli L.
      • Buratti E.
      • Baralle F.E.
      ,
      • Winton M.J.
      • Igaz L.M.
      • Wong M.M.
      • Kwong L.K.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ). TEVcyt cleavage of TDP-43 at Gln182 produced a similar distribution of cytoplasmic CTFs, whereas NTFs generated in the cytoplasm translocated back into the nucleus without appreciable cytoplasmic localization (supplemental Fig. 1). Therefore, CTFs generated by cytoplasmic cleavage of TDP-43 show similar diffuse cytoplasmic distribution as those generated by nuclear cleavage of TDP-43.

      Nuclear Cleavage of TDP-43 Causes Rapid Nuclear Export and CTF Clearance

      Nuclear cleavage of TDP-43 produced CTFs that shuttled out of the nucleus, where they are redistributed throughout the cytoplasm (Fig. 3A). This phenomenon is reproducible in multiple cell types including the motor neuron-like NSC-34 cells (supplemental Fig. 2). In NSC-34 cells, de novo cleaved CTF localization extends out to the periphery of the cell and throughout the neurite-like projections. This suggests that cellular trafficking may play a role in the cytoplasmic CTF redistribution. However, rather than aggregating as in previous models of CTF expression (
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ,
      • Dormann D.
      • Capell A.
      • Carlson A.M.
      • Shankaran S.S.
      • Rodde R.
      • Neumann M.
      • Kremmer E.
      • Matsuwaki T.
      • Yamanouchi K.
      • Nishihara M.
      • Haass C.
      ,
      • Nonaka T.
      • Kametani F.
      • Arai T.
      • Akiyama H.
      • Hasegawa M.
      ,
      • Zhang Y.J.
      • Xu Y.F.
      • Cook C.
      • Gendron T.F.
      • Roettges P.
      • Link C.D.
      • Lin W.L.
      • Tong J.
      • Castanedes-Casey M.
      • Ash P.
      • Gass J.
      • Rangachari V.
      • Buratti E.
      • Baralle F.
      • Golde T.E.
      • Dickson D.W.
      • Petrucelli L.
      ), de novo cleaved CTFs were rapidly cleared in virtually all cells within 48 h of TEVnuc expression (Fig. 3A). Cells that degrade and clear CTFs are indicated by the nuclear localization of the remaining NTF (Fig. 3A). This illustrates that CTFs and NTFs have significantly different rates of clearance following TDP-43 cleavage.
      Figure thumbnail gr3
      FIGURE 3CTF and NTF localization and clearance rates. A, double immunofluorescence illustrating the localization of N-terminal FLAG (green) and C-terminal Myc (red), following de novo cleavage of FLAG-TDP-43(182tev)-Myc with TEVnuc. Cells fixed at 0, 24, and 48 h after TEVnuc expression depict CTF export and clearance with nuclear retention of the NTF. Scale bar, 5 μm. B, TEVnuc cells transfected with FLAG-TDP-43(182tev)-Myc were labeled with [35S]methionine for 10 min followed by the indicated chase intervals in media containing 1 μg/ml tetracycline to activate TEVnuc expression. Immunoprecipitations of endogenous TDP-43, uncleaved and cleaved FLAG-TDP-43(182tev)-Myc, Gln182-cleaved CTFs, and Gln182-cleaved NTFs were separated on SDS-polyacrylamide gels, followed by autoradiography (top panels). The [35S]methionine intensities of each TDP-43 species were quantified and plotted as a function of time (graph). The first-order decay model is shown with the half-life of each species summarized below. Error bars, 1 S.D. derived from three replicate experiments. Inset, average raw data trace from initiation of the chase and induction of TEVnuc cleavage for the CTF, NTF, and FLAG-TDP-43(182tev)-Myc in the presence of TEVnuc. C, nuclear export of CTFs larger than 55 kDa demonstrated by TEVnuc cleavage in cells transfected with GFP-TDP-43(182tev)-mRuby. Fluorescence microscopy of N-terminal GFP and C-terminal mRuby at different time points demonstrates similar clearance kinetics for CTF-Myc (A) and CTF-mRuby (C).
      To further characterize the relative clearance rates of NTFs and CTFs, we labeled total protein in cells transfected with FLAG-TDP-43(182tev)-Myc with [35S]methionine, followed by different chase periods in media that induce TEVnuc expression (Fig. 3B). The turnover of the full-length protein in cells without TEVnuc expression is consistent with the turnover rate of endogenous TDP-43, with a half-life of ∼34–40 h (Fig. 3B). In the presence of TEVnuc, FLAG-TDP-43(182tev)-Myc rapidly decays with a half-life of 1.7 h, reflecting the kinetics of TEVnuc catalysis in cells (Fig. 3B). Levels of CTFs and NTFs increase with similar kinetics as the loss of the full-length substrate (Fig. 3B, inset). The NTF decay is similar to decay of full-length TDP-43, with a half-life of ∼34 h (Fig. 3B), whereas the CTF half-life is only 4.2 h (Fig. 3B). This reflects the combined rate of efficient nuclear export, cytoplasmic transport, and degradation of CTFs.
      Next, we determined whether the efficient nuclear export of CTFs was a result of passive diffusion through the nuclear pore. It is generally accepted that proteins larger than ∼40 kDa are not efficiently transported across the nuclear pore by diffusion (
      • Bonner W.M.
      ). Because CTFs with a C-terminal Myc tag are only 26 kDa, we attached the 30-kDa mRuby protein to the carboxyl terminus of TDP-43(182tev) to generate a ∼56-kDa CTF following TEVnuc cleavage. Cells transfected with a GFP-TDP-43(182tev)-mRuby showed a typical nuclear localization pattern in the absence of cleavage (Fig. 3C). If CTFs simply diffuse out of the nucleus, the nuclear export of the ∼56-kDa CTF would be expected to be blocked or impaired after TEVnuc cleavage. However, following cleavage with TEVnuc, the expected ∼56-kDa CTFs appeared in the cytoplasm with a similar distribution and frequency compared with Myc-tagged CTFs (Fig. 3C). This suggests that CTFs do not simply diffuse through the nuclear pore. The CTF-mRuby fragment also exhibited similar clearance rates, suggesting that the larger mRuby fusion does not dramatically affect the rate of nuclear export, cytoplasmic distribution, and CTF clearance (Fig. 3C).
      Reports of CTF and full-length TDP-43 degradation suggest that the proteasome is primarily responsible for the degradation of both CTFs and full-length TDP-43 (
      • Winton M.J.
      • Igaz L.M.
      • Wong M.M.
      • Kwong L.K.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ,
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ,
      • Zhang Y.J.
      • Gendron T.F.
      • Xu Y.F.
      • Ko L.W.
      • Yen S.H.
      • Petrucelli L.
      ). To verify that de novo cleaved CTFs are degraded by the proteasome, cells transfected with FLAG-TDP-43(182tev)-Myc were treated with the proteasome inhibitor clasto-lactacystin-β-lactone during CTF production with TEVnuc. Protein extracted from clasto-lactacystin-β-lactone-treated cells show a 3-fold increase in CTF levels compared with untreated controls (supplemental Fig. 3). This confirms that de novo CTFs are degraded by the proteasome. Interestingly, a 3-fold increase in the level of cytoplasmic CTFs did not change the cytoplasmic localization pattern or solubility of the CTF (data not shown). These observations show that CTF clearance includes transport of CTFs to the cytoplasm, followed by efficient degradation by the proteasome.

      Comparison of Aggregate-prone and de Novo Generated CTFs

      The biochemical properties of CTFs generated from de novo cleavage with TEV were compared with CTFs extracted from FTLD-TDP brain (
      • Neumann 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.
      ,
      • Igaz 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.-Y.
      ) and in cells transiently transfected with a cDNA encoding a CTF containing amino acids 182–414. De novo cleavage of TDP-43 at Gln182 resulted in CTFs and NTFs that were extracted in the soluble fraction (Fig. 4A). In contrast, overexpression of the same CTF by transfection shows that it is predominantly extracted in the insoluble fraction and is hyperphosphorylated at serines 409 and 410, a robust marker of TDP-43 pathology (Fig. 4A). The pathological CTFs extracted from FTLD-TDP brain tissue are heavily phosphorylated and present exclusively in the insoluble fraction (
      • Neumann 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.-Y.
      ,
      • Hasegawa 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.
      ) (Fig. 4A). Therefore, CTFs generated by de novo cleavage of TDP-43 possess a distinct set of biochemical properties compared with overexpression of truncated TDP-43 constructs. This indicates that a second event or perturbation of these CTFs may be required to convert CTFs generated by de novo cleavage into insoluble aggregates. Therefore, we tested three plausible conditions that are linked to mechanisms of motor neuron disease to determine if one or more of these perturbations would initiate aggregation of de novo cleaved CTFs: 1) seeding cells with aggregate-prone CTFs, 2) depleting RNA, and 3) disrupting cellular trafficking.
      Figure thumbnail gr4
      FIGURE 4De novo cleaved CTFs colocalize with cytoplasmic GFP-TDP-43(208–414) aggregates. A, the solubility of CTFs generated by de novo cleavage of FLAG-TDP-43(182tev) with TEVnuc was compared with CTFs extracted from cells transfected with a cDNA encoding the same CTF (TDP-43; positions 182–414) and CTFs extracted from FTLD-TDP brain tissue. Immunoblots probed with C terminus-specific anti-TDP-43c and the phosphoserine 409/410-specific TDP-43 antibody (P-409/410) show levels of total and phosphorylated CTFs. NTFs were probed with N terminus-specific anti-TDP-43n. B, immunofluorescence of TEVnuc cells cotransfected with the aggregation-prone GFP-TDP-43(208–414) and the uncleavable FLAG-TDP-43-Myc shows colocalization of the full-length TDP-43 stained with FLAG (blue) and Myc (red) with GFP-TDP-43(208–414). Induction of both the TEVnuc and GFP-TDP-43(208–414) is regulated by the tetracycline repressor. C, cotransfection of GFP-TDP-43(208–414) with the uncleavable FLAG-TDP-43-Myc in the absence of tetracycline shows that full-length TDP-43 is normally localized to the nucleus prior to GFP-TDP-43(208–414) induction. D, cells cotransfected as in C in the presence of 1 μg/ml tetracycline show that GFP-TDP-43(208–414) overexpression generates cytoplasmic inclusions (arrows) that do not colocalize or sequester the nuclear FLAG-TDP-43-Myc. E, cotransfection of GFP-TDP-43(208–414) and the cleavable form of FLAG-TDP-43(182tev)-Myc is localized to the nucleus as in B (data not shown). In the presence of 1 μg/ml tetracycline, CTFs generated by de novo TEVnuc cleavage show formation of inclusions that contain both GFP-TDP-43(208–414) CTFs (green) and de novo cleaved Myc-tagged CTFs (red). The FLAG-tagged NTF remains nuclear (blue). The differential interference contrast (DIC) images show inclusions and cell morphology. Scale bar, 5 μm.
      To determine if aggregate-prone CTF seeds can sequester soluble de novo cleaved CTFs, we performed a TEVnuc cleavage assay in cells cotransfected with FLAG-TDP-43(182tev)-Myc and a GFP-tagged CTF. Here, a tetracycline-inducible form of GFP-TDP-43(208–414) (
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ,
      • Zhang Y.J.
      • Gendron T.F.
      • Xu Y.F.
      • Ko L.W.
      • Yen S.H.
      • Petrucelli L.
      ) serves as the aggregating seed in cells producing CTFs by TEVnuc cleavage. The co-induction of GFP-TDP-43(208–414) and the TEV protease overcomes the problem that coexpression of full-length TDP-43 and CTFs causes co-aggregation (
      • Nonaka T.
      • Kametani F.
      • Arai T.
      • Akiyama H.
      • Hasegawa M.
      ) (Fig. 4B). Immunofluorescence of cells cotransfected with an uncleavable form of FLAG-TDP-43-Myc and GFP-TDP-43(208–414) showed the expected nuclear localization pattern for TDP-43 in the absence of GFP-TDP-43(208–414) inducer tetracycline (Fig. 4C). The addition of tetracycline showed the expected nuclear localization of FLAG-TDP-43-Myc and no colocalization with cytoplasmic GFP-TDP-43(208–414) inclusions (Fig. 4D). Therefore, maturation of full-length TDP-43 before expression of the aggregate-prone GFP-TDP-43(208–414) prevents their colocalization and coaggregation routinely observed by cotransfection (
      • Nonaka T.
      • Kametani F.
      • Arai T.
      • Akiyama H.
      • Hasegawa M.
      ). This also confirms previous observations that overexpression of CTFs in cells leads to the formation of cytoplasmic inclusions but does not sequester nuclear TDP-43 (
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ,
      • Zhang Y.J.
      • Xu Y.F.
      • Cook C.
      • Gendron T.F.
      • Roettges P.
      • Link C.D.
      • Lin W.L.
      • Tong J.
      • Castanedes-Casey M.
      • Ash P.
      • Gass J.
      • Rangachari V.
      • Buratti E.
      • Baralle F.
      • Golde T.E.
      • Dickson D.W.
      • Petrucelli L.
      ). However, when cells were transfected with FLAG-TDP-43(182tev)-Myc and GFP-TDP-43(208–414), followed by TEVnuc cleavage, we observed striking colocalization of the de novo cleaved CTFs with the GFP-TDP-43(208–414) inclusions (Fig. 4E). Although only ∼10% of all transfected cells contained both GFP-TDP-43(208–414) inclusions and de novo cleaved CTFs, nearly all of these cells displayed colocalization of GFP-TDP-43(208–414) CTF and the de novo cleaved CTFs. Therefore, the introduction of aggregate-prone CTF seeds can recruit soluble and otherwise non-aggregate-prone CTFs into inclusions formed by the CTF seeds.

      RNA Dependence of CTF Aggregation

      In contrast to direct expression of CTFs that are not trafficked to the nucleus and localized to the cytoplasm (
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ), the generation of CTFs by de novo cleavage starts with full-length TDP-43 in the nucleus and presumably retains its RNA associated functions. Indeed, cotransfection of cells with the TEV-cleavable FLAG-TDP-43(182tev)-Myc substrate and the CFTR RNA-splicing reporter plasmid illustrates that this protein retains its functional activity for CFTR splicing (supplemental Fig. 4). Therefore, the uncleaved FLAG-TDP-43(182tev)-Myc used in this study associates with nuclear RNA and retains its function in RNA splicing. Because TDP-43 interacts with hnRNPs and RNAs that are efficiently exported to the cytoplasm (
      • Buratti E.
      • Brindisi A.
      • Giombi M.
      • Tisminetzky S.
      • Ayala Y.M.
      • Baralle F.E.
      ,
      • Piñol-Roma S.
      ,
      • Mili S.
      • Shu H.J.
      • Zhao Y.
      • Piñol-Roma S.
      ,
      • Ling S.C.
      • Albuquerque C.P.
      • Han J.S.
      • Lagier-Tourenne C.
      • Tokunaga S.
      • Zhou H.
      • Cleveland D.W.
      ), we determined the effect of RNA on CTF aggregation.
      To determine whether RNA alters CTF solubility or localization, we removed the cellular pool of RNA by treating cells containing cytoplasmic CTFs with RNase. For these experiments, cells transfected with GFP-TDP-43(182tev)-mRuby were incubated overnight in the presence and absence of TEVnuc, followed by a 15-min RNase treatment before fixation. In the presence of RNase, uncleaved TDP-43 displays a speckled nuclear pattern consistent with the RNA binding-deficient localization phenotype for TDP-43 (Fig. 5A) (
      • Buratti E.
      • Baralle F.E.
      ,
      • Ayala Y.M.
      • Zago P.
      • D'Ambrogio A.
      • Xu Y.F.
      • Petrucelli L.
      • Buratti E.
      • Baralle F.E.
      ). RNase-treated cells containing cytoplasmic CTFs showed a remarkable redistribution of CTFs from the diffuse cytoplasmic pattern to inclusion-like foci (Fig. 5B). This striking alteration in the localization of CTFs suggests that the distribution of CTFs can be altered by removal of cellular RNA and that direct or indirect interactions of TDP-43 CTFs with RNA may prevent them from aggregating. Biochemical extraction of cells possessing cytoplasmic CTFs showed a corresponding shift in CTF solubility in cells treated with RNase (Fig. 5C), further supporting the possibility that soluble CTFs are stabilized by interactions with RNA.
      Figure thumbnail gr5
      FIGURE 5RNA dependence of de novo cleaved CTF localization. A and B, RNase pretreatment causes a redistribution of full-length TDP-43 and CTFs. TEVnuc cells transfected with GFP-TDP-43(182tev)-mRuby were incubated overnight in the absence (A) and presence (B) of TEVnuc, followed by a 15-min pretreatment in CSK buffer containing 1 mg/ml RNase A (see “Experimental Procedures”). CTFs appear redistributed as cytoplasmic inclusions (B, arrows). Scale bar, 5 μm. C, sequential extraction of cells transfected with FLAG-TDP-43(182tev)-Myc, followed by cleavage and RNase treatment, shows a shift in de novo cleaved CTF to the insoluble fraction. Symbols mark the migration of FLAG-TDP-43(182tev)-Myc (**) and endogenous TDP-43 (*).

      Cytoplasmic CTF Distribution Is Dependent on Dynein Transport

      Because de novo cleaved nuclear CTFs are trafficked to the cytoplasm, we asked if disruption of cellular transport mechanisms would affect the distribution of CTFs following de novo cleavage. To achieve this, we targeted the MT motor protein dynein using two separate approaches that inhibit dynein function in cells producing CTFs by de novo cleavage of TDP-43: 1) the dynein inhibitor EHNA or 2) the protein inhibitor of dynein CC1. Both approaches effectively disrupt dynein-dependent MT transport because dynein-dependent localization of the Golgi apparatus (
      • Corthésy-Theulaz I.
      • Pauloin A.
      • Pfeffer S.R.
      ) is disrupted in both EHNA- and CC1-treated cells (supplemental Fig. 5).
      EHNA treatment had no effect on the nuclear localization of the FLAG-TDP-43(182tev)-Myc in the absence of TEVnuc cleavage (Fig. 6, A and C). This is consistent with the normal nuclear staining of endogenous TDP-43 after treatment with EHNA (supplemental Fig. 6). However, in the presence of TEVnuc cleavage, cells treated with EHNA showed a greater frequency of cytoplasmic CTFs compared with untreated cells (Fig. 6, compare B with D). Rare cytoplasmic CTF inclusions were seen only in the EHNA-treated cells (Fig. 6D, arrow). Further quantification showed that ∼60% of transfected cells displayed cytoplasmic CTFs after EHNA treatment compared with ∼20% in the control cells (Fig. 6E). Correspondingly, only ∼24% of transfected cells treated with EHNA showed complete CTF clearance compared with ∼54% in control cells (Fig. 6E). Because the integrity of MTs and dynein localization are not dramatically altered in EHNA-treated cells (supplemental Fig. 6), perhaps EHNA-mediated inhibition of dynein impairs the dynamic transport and clearance of CTFs.
      Figure thumbnail gr6
      FIGURE 6Cytoplasmic CTF localization is dependent upon dynein-mediated MT transport. Immunofluorescence of cells transfected with FLAG-TDP-43(182tev)-Myc in the absence (A and B) and presence (C and D) of 0.1 mm EHNA for 16 h. TEVnuc cleavage in the absence of EHNA shows the typical frequency of cytoplasmic CTFs (B), whereas TEVnuc cleavage in the presence of EHNA (D) depicts an increase in the number of transfected cells possessing cytoplasmic CTFs (compare B and D). E, quantification of cells treated with EHNA (n = 312) and controls (n = 292) in A–D displayed as the percentage of transfected cells that contain cytoplasmic CTFs, cleared CTFs represented by nuclear NTF (green), and uncleaved protein. Error bars, 1 S.D. of the mean. F, biochemical extraction of cells cotransfected with GFP-TDP-43(182tev)-mRuby and CC1-Myc or pcDNA 3.1 in the presence and absence of TEVnuc. Note the increase in CTF levels in CC1-Myc-transfected cells. The immunoblot with anti-GFP and anti-TDP-43c shows the respective N terminus- and C terminus-specific bands. G–J, GFP and mRuby fluorescence of cells transfected in F show four distinct phenotypes: normal nuclear localization of uncleaved TDP-43 in the presence of CC1-Myc (G), diffuse cytoplasmic CTFs (red) in cells expressing CC1-Myc (H), cytoplasmic CTF inclusions (red) in cells expressing CC1-Myc (I), and cleavage-dependent full-length inclusions (merge of green and red fluorescence) (J). Scale bar, 5 μm.
      To further demonstrate the role of dynein in CTF transport and clearance, we cotransfected cells with GFP-TDP-43(182tev)-mRuby and CC1-Myc, followed by cleavage with TEVnuc. CC1 is a domain in the p150(glued) subunit of dynactin that interacts with dynein and inhibits dynein motor function (
      • Zhapparova O.N.
      • Burakov A.V.
      • Nadezhdina E.S.
      ). Immunoblots of biochemical extract from cells cotransfected with CC1-Myc and GFP-TDP-43(182tev)-mRuby show an increased level of CTFs in cells containing the dynein inhibitor CC1-Myc. This supports the hypothesis that CTF clearance is dependent on dynein transport (Fig. 6F). Notably, we observed four distinct phenotypes of cytoplasmic CTFs in cells cotransfected with the dynein inhibitor CC1-Myc. Consistent with the EHNA treatment of uncleaved TDP-43, overexpression of CC1-Myc did not affect the nuclear localization of the uncleaved GFP-TDP-43(182tev)-mRuby protein (Fig. 6G). In the presence of TEVnuc, the majority of cotransfected cells displayed cytoplasmic CTFs with a diffuse cytoplasmic localization pattern similar to cells treated with EHNA (Fig. 6, compare D with H). However, ∼15% of cotransfected cells harboring cytoplasmic CTFs showed a clear redistribution of CTFs in the form of discrete CTF inclusions (Fig. 6I). Surprisingly, ∼5% of cells with CTFs also showed cytoplasmic inclusions formed by full-length TDP-43 (Fig. 6J). Because full-length TDP-43 inclusions were not observed in the CC1-Myc transfected cells without cleavage (Fig. 6G), this implies that formation of inclusions formed by full-length TDP-43 is dependent on TDP-43 cleavage. Taken together, these observations define a novel role for dynein-mediated transport in modulating the distribution of CTFs, and they also imply that defects in axonal transport observed in mouse models of motor neuron disease (
      • Zhang B.
      • Tu P.
      • Abtahian F.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ) may contribute to the aggregation of CTFs with full-length TDP-43 in inclusions.

      DISCUSSION

      CTFs are major components of TDP-43 inclusions in TDP-43 proteinopathies. Therefore, understanding TDP-43 proteolytic processing is likely to be as fundamental in providing insights into the mechanism(s) of TDP-43 pathogenesis as elucidating mechanisms of Aβ generation has been for understanding the pathobiology of Alzheimer disease (
      • De Strooper B.
      • Annaert W.
      ). Because the protease that generates TDP-43 CTFs in FTLD-TDP and ALS remains unknown, we established the TEV protease system in mammalian cells as a novel model system to study nuclear TDP-43 cleavage and fate of N-terminal and C-terminal fragments of TDP-43. Our findings show that RRM2 in TDP-43 is relatively resistant to cleavage and that the stability of de novo cleaved CTFs is dependent on RRM2 and the C-terminal glycine-rich domain. Unlike previous studies demonstrating that acute expression of cytoplasmic CTFs results in the formation of cytoplasmic aggregates (
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ,
      • Dormann D.
      • Capell A.
      • Carlson A.M.
      • Shankaran S.S.
      • Rodde R.
      • Neumann M.
      • Kremmer E.
      • Matsuwaki T.
      • Yamanouchi K.
      • Nishihara M.
      • Haass C.
      ,
      • Nonaka T.
      • Kametani F.
      • Arai T.
      • Akiyama H.
      • Hasegawa M.
      ,
      • Zhang Y.J.
      • Xu Y.F.
      • Cook C.
      • Gendron T.F.
      • Roettges P.
      • Link C.D.
      • Lin W.L.
      • Tong J.
      • Castanedes-Casey M.
      • Ash P.
      • Gass J.
      • Rangachari V.
      • Buratti E.
      • Baralle F.
      • Golde T.E.
      • Dickson D.W.
      • Petrucelli L.
      ), CTFs generated from de novo TDP-43 cleavage at Gln182 are efficiently exported from the nucleus and degraded by the proteasome with a relatively short half-life. Thus, other factors must contribute to CTF aggregation. We show that cleavage of TDP-43 coupled with loss of associations with RNA- or MT-dependent dynein transport may initiate the formation of cytoplasmic CTF inclusions.
      Cleavage of TDP-43 at positions in RRM2 by TEV protease does not generate appreciable CTFs or NTFs. This is probably due to the high degree of structural integrity and stability of RRM2 (
      • Kuo P.H.
      • Doudeva L.G.
      • Wang Y.T.
      • Shen C.K.
      • Yuan H.S.
      ). However, because CTFs with Arg208 at the amino terminus have been recovered from FTLD-TDP brains (
      • Igaz L.M.
      • Kwong L.K.
      • Chen-Plotkin A.
      • Winton M.J.
      • Unger T.L.
      • Xu Y.
      • Neumann M.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      ), our data suggest that a structural change must occur before a protease can gain access to these protected cleavage positions. Alternatively, it is possible that smaller fragments, such as Arg208 CTFs, are generated from larger CTFs similar to the Gln182 CTF studied here. Consistent with this possibility, CTFs extracted from FTLD-TDP brains show a range of CTF sizes, including those with an apparent molecular weight similar to that of Gln182 CTF (
      • Neumann 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.
      ,
      • Igaz 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.-Y.
      ).
      It is interesting that de novo cleavage of TDP-43 at Ala260 or Arg272 did not yield any detectable CTFs. Because these CTFs lack RRM2, it is likely that RRM2 is responsible for the increased stability of Gln182 CTFs. Alternatively, it is possible that the loss of the nuclear export sequence in RRM2 prevents export of these shorter CTFs and promotes their nuclear degradation. In contrast, cleavage at Gln182, Ala260, and Arg272 produces NTFs that are retained in the nucleus with a half-life that is similar to the full-length protein. This suggests that NTFs may be degraded in a similar manner as nuclear TDP-43. Because NTFs are generally not observed in post-mortem FTLD-TDP or ALS brains (
      • Igaz 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.-Y.
      ), it is possible that TDP-43 may become modified before cleavage to generate an NTF that is more rapidly cleared in the brain.
      Studies of TDP-43 complexes with protein and RNA are beginning to provide a clearer understanding of TDP-43 as a component of large RNP complexes (
      • Buratti E.
      • Brindisi A.
      • Giombi M.
      • Tisminetzky S.
      • Ayala Y.M.
      • Baralle F.E.
      ,
      • Kim S.H.
      • Shanware N.P.
      • Bowler M.J.
      • Tibbetts R.S.
      ,
      • Sephton C.F.
      • Cenik C.
      • Kucukural A.
      • Dammer E.B.
      • Cenik B.
      • Han Y.
      • Dewey C.M.
      • Roth F.P.
      • Herz J.
      • Peng J.
      • Moore M.J.
      • Yu G.
      ). Our observations that CTF transport are dependent on interactions of TDP-43 with RNA and on dynein-mediated MT transport are consistent with its classification as a shuttling RNP protein (
      • Piñol-Roma S.
      • Dreyfuss G.
      ). However, we speculate that a chain of events following de novo cleavage of TDP-43 must occur to alter CTF solubility and clearance, as outlined in Fig. 7. TDP-43 is a nuclear hnRNP that is likely to exist in both free and RNA-associated states. Model cleavage of TDP-43 by TEV protease at Gln182 generates NTFs and CTFs that stimulate the nuclear export of CTFs and nuclear retention of NTFs. CTFs do not passively diffuse across the nuclear pore, suggesting that nuclear export of the CTFs is associated with a specific transport mechanism (
      • Nishimura A.L.
      • Zupunski V.
      • Troakes C.
      • Kathe C.
      • Fratta P.
      • Howell M.
      • Gallo J.M.
      • Hortobágyi T.
      • Shaw C.E.
      • Rogelj B.
      ). Therefore, we propose that CTFs are bound to or “piggy-backed” onto RNA as they exit the nucleus and undergo transport throughout the cytoplasm. This is consistent with established models of hnRNP nuclear export as a mRNA-RNP complex that is dependent on RRM2 (
      • Piñol-Roma S.
      ,
      • Piñol-Roma S.
      • Dreyfuss G.
      ). However, a second event, such as the loss of direct or indirect interactions with RNA or failed MT transport, could initiate the aggregation process. Once initiated, the aggregated CTFs can serve as a nidus to recruit additional de novo generated CTFs forming large cytoplasmic inclusions, as was seen when the aggregate-prone GFP-TDP-43(208–414) seed was expressed in cells.
      Figure thumbnail gr7
      FIGURE 7Model of CTF cleavage and transport. TDP-43 in the unbound or RNA-associated state is a substrate for TEVnuc-mediated cleavage. Nuclear retention of NTFs may contribute to their long lived stability and may potentially generate alternative RNA targets in the absence of RRM2 and the glycine-rich C terminus. Nuclear CTFs are exported to the cytoplasm potentially “piggy-backed” with RNA as an RNP transport particle. Cytoplasmic RNPs are transported throughout the cell via cytoskeleton networks, including MTs. Loss of RNA or the RNP transport mechanisms alters CTF distribution and clearance and potentially contributes to CTF aggregation.
      In summary, we established a unique cell model to investigate the proteolytic cleavage of TDP-43. This system is adaptable to biological processes that involve proteolysis of a specific protein in cells. For TDP-43, we defined three specific types of cleavage that provide tangible clues to a mechanism of TDP-43 pathogenesis that includes proteolysis. Significantly, our data suggest that physical changes in protein structure must occur before cleavage sites within RRM2 are exposed and that CTF aggregation is a “two-hit” or two-step process that includes TDP-43 cleavage coupled to additional subsequent cellular stresses that may serve as “triggers” for TDP-43 aggregation and the onset of TDP-43 proteinopathies, as exemplified by ALS and FTLD-TDP.

      Acknowledgments

      We thank Dr. Greg Van Duyne for the TEV protease construct, Dr. Erika Holzbaur for the CC1-Myc construct, Andrew Huang and Chi Li for technical assistance, and Drs. Todd Cohen, Linda Kwong and Eddie Lee for critical reading of the manuscript.

      Supplementary Material

      REFERENCES

        • Neumann 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: 130-133
        • Lagier-Tourenne C.
        • Cleveland D.W.
        Cell. 2009; 136: 1001-1004
        • Pesiridis G.S.
        • Lee V.M.-Y.
        • Trojanowski J.Q.
        Hum. Mol. Genet. 2009; 18: R156-R162
        • Gregory R.I.
        • Yan K.P.
        • Amuthan G.
        • Chendrimada T.
        • Doratotaj B.
        • Cooch N.
        • Shiekhattar R.
        Nature. 2004; 432: 235-240
        • Ou S.H.
        • Wu F.
        • Harrich D.
        • García-Martínez L.F.
        • Gaynor R.B.
        J. Virol. 1995; 69: 3584-3596
        • Abhyankar M.M.
        • Urekar C.
        • Reddi P.P.
        J. Biol. Chem. 2007; 282: 36143-36154
        • Burd C.G.
        • Matunis E.L.
        • Dreyfuss G.
        Mol. Cell. Biol. 1991; 11: 3419-3424
        • Buratti E.
        • Baralle F.E.
        J. Biol. Chem. 2001; 276: 36337-36343
        • Buratti E.
        • Brindisi A.
        • Giombi M.
        • Tisminetzky S.
        • Ayala Y.M.
        • Baralle F.E.
        J. Biol. Chem. 2005; 280: 37572-37584
        • D'Ambrogio A.
        • Buratti E.
        • Stuani C.
        • Guarnaccia C.
        • Romano M.
        • Ayala Y.M.
        • Baralle F.E.
        Nucleic Acids Res. 2009; 37: 4116-4126
        • Piñol-Roma S.
        Semin. Cell Dev. Biol. 1997; 8: 57-63
        • Piñol-Roma S.
        • Dreyfuss G.
        Nature. 1992; 355: 730-732
        • Ayala Y.M.
        • Zago P.
        • D'Ambrogio A.
        • Xu Y.F.
        • Petrucelli L.
        • Buratti E.
        • Baralle F.E.
        J. Cell Sci. 2008; 121: 3778-3785
        • Winton M.J.
        • Igaz L.M.
        • Wong M.M.
        • Kwong L.K.
        • Trojanowski J.Q.
        • Lee V.M.-Y.
        J. Biol. Chem. 2008; 283: 13302-13309
        • Igaz L.M.
        • Kwong L.K.
        • Chen-Plotkin A.
        • Winton M.J.
        • Unger T.L.
        • Xu Y.
        • Neumann M.
        • Trojanowski J.Q.
        • Lee V.M.-Y.
        J. Biol. Chem. 2009; 284: 8516-8524
        • Dormann D.
        • Capell A.
        • Carlson A.M.
        • Shankaran S.S.
        • Rodde R.
        • Neumann M.
        • Kremmer E.
        • Matsuwaki T.
        • Yamanouchi K.
        • Nishihara M.
        • Haass C.
        J. Neurochem. 2009; 110: 1082-1094
        • Nonaka T.
        • Kametani F.
        • Arai T.
        • Akiyama H.
        • Hasegawa M.
        Hum. Mol. Genet. 2009; 18: 3353-3364
        • Zhang Y.J.
        • Xu Y.F.
        • Cook C.
        • Gendron T.F.
        • Roettges P.
        • Link C.D.
        • Lin W.L.
        • Tong J.
        • Castanedes-Casey M.
        • Ash P.
        • Gass J.
        • Rangachari V.
        • Buratti E.
        • Baralle F.
        • Golde T.E.
        • Dickson D.W.
        • Petrucelli L.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7607-7612
        • Chen A.K.
        • Lin R.Y.
        • Hsieh E.Z.
        • Tu P.H.
        • Chen R.P.
        • Liao T.Y.
        • Chen W.
        • Wang C.H.
        • Huang J.J.
        J. Am. Chem. Soc. 2010; 132: 1186-1187
        • Johnson B.S.
        • Snead D.
        • Lee J.J.
        • McCaffery J.M.
        • Shorter J.
        • Gitler A.D.
        J. Biol. Chem. 2009; 284: 20329-20339
        • Li Y.
        • Ray P.
        • Rao E.J.
        • Shi C.
        • Guo W.
        • Chen X.
        • Woodruff 3rd, E.A.
        • Fushimi K.
        • Wu J.Y.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 3169-3174
        • Lu Y.
        • Ferris J.
        • Gao F.B.
        Mol. Brain. 2009; 2: 30
        • Pauli A.
        • Althoff F.
        • Oliveira R.A.
        • Heidmann S.
        • Schuldiner O.
        • Lehner C.F.
        • Dickson B.J.
        • Nasmyth K.
        Dev. Cell. 2008; 14: 239-251
        • Kredel S.
        • Oswald F.
        • Nienhaus K.
        • Deuschle K.
        • Röcker C.
        • Wolff M.
        • Heilker R.
        • Nienhaus G.U.
        • Wiedenmann J.
        PLoS One. 2009; 4: e4391
        • Igaz 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.-Y.
        Am. J. Pathol. 2008; 173: 182-194
        • Neumann 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.-Y.
        Acta Neuropathol. 2009; 117: 137-149
        • Jansens A.
        • Braakman I.
        Methods Mol. Biol. 2003; 232: 133-145
        • Zhapparova O.N.
        • Burakov A.V.
        • Nadezhdina E.S.
        Biochemistry. 2007; 72: 1233-1240
        • Prasanth K.V.
        • Sacco-Bubulya P.A.
        • Prasanth S.G.
        • Spector D.L.
        Mol. Biol. Cell. 2003; 14: 1043-1057
        • Prasanth K.V.
        • Prasanth S.G.
        • Xuan Z.
        • Hearn S.
        • Freier S.M.
        • Bennett C.F.
        • Zhang M.Q.
        • Spector D.L.
        Cell. 2005; 123: 249-263
        • Kuo P.H.
        • Doudeva L.G.
        • Wang Y.T.
        • Shen C.K.
        • Yuan H.S.
        Nucleic Acids Res. 2009; 37: 1799-1808
        • Williams K.R.
        • Stone K.L.
        • LoPresti M.B.
        • Merrill B.M.
        • Planck S.R.
        Proc. Natl. Acad. Sci. U.S.A. 1985; 82: 5666-5670
        • Bonner W.M.
        J. Cell Biol. 1975; 64: 421-430
        • Zhang Y.J.
        • Gendron T.F.
        • Xu Y.F.
        • Ko L.W.
        • Yen S.H.
        • Petrucelli L.
        Mol. Neurodegener. 2010; 5: 33
        • Hasegawa 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: 60-70
        • Mili S.
        • Shu H.J.
        • Zhao Y.
        • Piñol-Roma S.
        Mol. Cell. Biol. 2001; 21: 7307-7319
        • Ling S.C.
        • Albuquerque C.P.
        • Han J.S.
        • Lagier-Tourenne C.
        • Tokunaga S.
        • Zhou H.
        • Cleveland D.W.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 13318-13323
        • Corthésy-Theulaz I.
        • Pauloin A.
        • Pfeffer S.R.
        J. Cell Biol. 1992; 118: 1333-1345
        • Zhang B.
        • Tu P.
        • Abtahian F.
        • Trojanowski J.Q.
        • Lee V.M.-Y.
        J. Cell Biol. 1997; 139: 1307-1315
        • De Strooper B.
        • Annaert W.
        Annu. Rev. Cell Dev. Biol. 2010; 26: 235-260
        • Kim S.H.
        • Shanware N.P.
        • Bowler M.J.
        • Tibbetts R.S.
        J. Biol. Chem. 2010; 285: 34097-34105
        • Sephton C.F.
        • Cenik C.
        • Kucukural A.
        • Dammer E.B.
        • Cenik B.
        • Han Y.
        • Dewey C.M.
        • Roth F.P.
        • Herz J.
        • Peng J.
        • Moore M.J.
        • Yu G.
        J. Biol. Chem. 2011; 286: 1204-1215
        • Nishimura A.L.
        • Zupunski V.
        • Troakes C.
        • Kathe C.
        • Fratta P.
        • Howell M.
        • Gallo J.M.
        • Hortobágyi T.
        • Shaw C.E.
        • Rogelj B.
        Brain. 2010; 133: 1763-1771