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• W2020159880 startingPage "20329" @default.
• W2020159880 abstract "Non-amyloid, ubiquitinated cytoplasmic inclusions containing TDP-43 and its C-terminal fragments are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder, and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Importantly, TDP-43 mutations are linked to sporadic and non-SOD1 familial ALS. However, TDP-43 is not the only protein in disease-associated inclusions, and whether TDP-43 misfolds or is merely sequestered by other aggregated components is unclear. Here, we report that, in the absence of other components, TDP-43 spontaneously forms aggregates bearing remarkable ultrastructural similarities to TDP-43 deposits in degenerating neurons of ALS FTLD-U patients. The C-terminal domain of TDP-43 is critical for spontaneous aggregation. Several ALS-linked TDP-43 mutations within this domain (Q331K, M337V, Q343R, N345K, R361S, and N390D) increase the number of TDP-43 aggregates and promote toxicity in vivo. Importantly, mutations that promote toxicity in vivo accelerate aggregation of pure TDP-43 in vitro. Thus, TDP-43 is intrinsically aggregation-prone, and its propensity for toxic misfolding trajectories is accentuated by specific ALS-linked mutations. Non-amyloid, ubiquitinated cytoplasmic inclusions containing TDP-43 and its C-terminal fragments are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder, and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Importantly, TDP-43 mutations are linked to sporadic and non-SOD1 familial ALS. However, TDP-43 is not the only protein in disease-associated inclusions, and whether TDP-43 misfolds or is merely sequestered by other aggregated components is unclear. Here, we report that, in the absence of other components, TDP-43 spontaneously forms aggregates bearing remarkable ultrastructural similarities to TDP-43 deposits in degenerating neurons of ALS FTLD-U patients. The C-terminal domain of TDP-43 is critical for spontaneous aggregation. Several ALS-linked TDP-43 mutations within this domain (Q331K, M337V, Q343R, N345K, R361S, and N390D) increase the number of TDP-43 aggregates and promote toxicity in vivo. Importantly, mutations that promote toxicity in vivo accelerate aggregation of pure TDP-43 in vitro. Thus, TDP-43 is intrinsically aggregation-prone, and its propensity for toxic misfolding trajectories is accentuated by specific ALS-linked mutations. TDP-43 is intrinsically aggregation-prone, and amyotrophic lateral sclerosis-linked mutations accelerate aggregation and increase toxicity.Journal of Biological ChemistryVol. 284Issue 37PreviewVOLUME 284 (2009) PAGES 20329–20339 Full-Text PDF Open Access TDP-43 is a ubiquitously expressed and highly conserved metazoan nuclear protein (1Ayala Y.M. Pantano S. D'Ambrogio A. Buratti E. Brindisi A. Marchetti C. Romano M. Baralle F.E. J. Mol. Biol. 2005; 348: 575-588Crossref PubMed Scopus (278) Google Scholar), which contains two RNA recognition motifs (RRMs) 3The abbreviations used are: RRMRNA recognition motifALSamyotrophic lateral sclerosisFTLD-Ufrontotemporal lobar degeneration with ubiquitin-positive inclusionsYFPyellow fluorescent proteinPBSphosphate-buffered salineHRPhorseradish peroxidaseGFPgreen fluorescent proteinBSAbovine serum albuminCFPcyan fluorescent proteinGSTglutathione S-transferaseEMelectron microscopyPDParkinson diseaseWTwild typePIpropidium iodideTEVtobacco etch virus. and a glycine-rich region in its C-terminal domain (see Fig. 1A). TDP-43 function is uncertain, but it likely plays important roles in pre-mRNA splicing and transcriptional repression (2Ayala Y.M. Misteli T. Baralle F.E. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3785-3789Crossref PubMed Scopus (196) Google Scholar, 3Buratti E. Baralle F.E. J. Biol. Chem. 2001; 276: 36337-36343Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). In ALS and FTLD-U, TDP-43 is depleted from the nucleus and accumulates in ubiquitinated cytoplasmic inclusions (4Neumann 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. Science. 2006; 314: 130-133Crossref PubMed Scopus (4595) Google Scholar). These and other situations of TDP-43 pathology, including some forms of Alzheimer and Parkinson diseases, are now known as TDP-43 proteinopathies (5Forman M.S. Trojanowski J.Q. Lee V.M. Curr. Opin. Neurobiol. 2007; 17: 548-555Crossref PubMed Scopus (102) Google Scholar). Importantly, mutations in the TDP-43 gene (TARDBP) are linked to sporadic and non-SOD1 familial ALS, implying that TDP-43 abnormalities are likely one cause of disease (6Kabashi E. Valdmanis P.N. Dion P. Spiegelman D. McConkey B.J. Vande Velde C. Bouchard J.P. Lacomblez L. Pochigaeva K. Salachas F. Pradat P.F. Camu W. Meininger V. Dupre N. Rouleau G.A. Nat. Genet. 2008; 40: 572-574Crossref PubMed Scopus (1242) Google Scholar, 7Sreedharan J. Blair I.P. Tripathi V.B. Hu X. Vance C. Rogelj B. Ackerley S. Durnall J.C. Williams K.L. Buratti E. Baralle F. de Belleroche J. Mitchell J.D. Leigh P.N. Al-Chalabi A. Miller C.C. Nicholson G. Shaw C.E. Science. 2008; 319: 1668-1672Crossref PubMed Scopus (1992) Google Scholar, 8Van Deerlin V.M. Leverenz J.B. Bekris L.M. Bird T.D. Yuan W. Elman L.B. Clay D. Wood E.M. Chen-Plotkin A.S. Martinez-Lage M. Steinbart E. McCluskey L. Grossman M. Neumann M. Wu I.L. Yang W.S. Kalb R. Galasko D.R. Montine T.J. Trojanowski J.Q. Lee V.M. Schellenberg G.D. Yu C.E. Lancet Neurol. 2008; 7: 409-416Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 9Rutherford N.J. Zhang Y.J. Baker M. Gass J.M. Finch N.A. Xu Y.F. Stewart H. Kelley B.J. Kuntz K. Crook R.J. Sreedharan J. Vance C. Sorenson E. Lippa C. Bigio E.H. Geschwind D.H. Knopman D.S. Mitsumoto H. Petersen R.C. Cashman N.R. Hutton M. Shaw C.E. Boylan K.B. Boeve B. Graff-Radford N.R. Wszolek Z.K. Caselli R.J. Dickson D.W. Mackenzie I.R. Petrucelli L. Rademakers R. PLoS Genet. 2008; 4: e1000193Crossref PubMed Scopus (371) Google Scholar, 10Gitcho M.A. Baloh R.H. Chakraverty S. Mayo K. Norton J.B. Levitch D. Hatanpaa K.J. White 3rd, C.L. Bigio E.H. Caselli R. Baker M. Al-Lozi M.T. Morris J.C. Pestronk A. Rademakers R. Goate A.M. Cairns N.J. Ann. Neurol. 2008; 63: 535-538Crossref PubMed Scopus (528) Google Scholar, 11Yokoseki A. Shiga A. Tan C.F. Tagawa A. Kaneko H. Koyama A. Eguchi H. Tsujino A. Ikeuchi T. Kakita A. Okamoto K. Nishizawa M. Takahashi H. Onodera O. Ann. Neurol. 2008; 63: 538-542Crossref PubMed Scopus (329) Google Scholar). However, despite this synthesis of pathology and genetics, the mechanisms by which TDP-43 might contribute to disease remain unknown and controversial (12Banks G.T. Kuta A. Isaacs A.M. Fisher E.M. Mamm. Genome. 2008; 19: 299-305Crossref PubMed Scopus (62) Google Scholar, 13Rothstein J.D. Ann. Neurol. 2007; 61: 382-384Crossref PubMed Scopus (25) Google Scholar). RNA recognition motif amyotrophic lateral sclerosis frontotemporal lobar degeneration with ubiquitin-positive inclusions yellow fluorescent protein phosphate-buffered saline horseradish peroxidase green fluorescent protein bovine serum albumin cyan fluorescent protein glutathione S-transferase electron microscopy Parkinson disease wild type propidium iodide tobacco etch virus. A key unresolved question is whether TDP-43 is inherently aggregation-prone or whether TDP-43 is sequestered by other aggregated components and is merely a marker of disease (13Rothstein J.D. Ann. Neurol. 2007; 61: 382-384Crossref PubMed Scopus (25) Google Scholar, 14Arai 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 (1929) Google Scholar, 15Sanelli T. Xiao S. Horne P. Bilbao J. Zinman L. Robertson J. J. Neuropathol. Exp. Neurol. 2007; 66: 1147-1153Crossref PubMed Scopus (45) Google Scholar, 16Xiao S. Tjostheim S. Sanelli T. McLean J.R. Horne P. Fan Y. Ravits J. Strong M.J. Robertson J. J. Neurosci. 2008; 28: 1833-1840Crossref PubMed Scopus (56) Google Scholar). Indeed, multiple proteins aside from TDP-43 are found in Sarkosyl-insoluble fractions from FTLD-U patients (14Arai 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 (1929) Google Scholar). Moreover, deconvolution imaging reveals that TDP-43 appears to be excluded from some regions of the ubiquitinated inclusions in ALS (15Sanelli T. Xiao S. Horne P. Bilbao J. Zinman L. Robertson J. J. Neuropathol. Exp. Neurol. 2007; 66: 1147-1153Crossref PubMed Scopus (45) Google Scholar). Here, we assess TDP-43 aggregation in the absence of other components. We then define which domains of TDP-43 are important for this process and determine the direct effects of several ALS-linked TDP-43 mutations on TDP-43 misfolding and toxicity. Our findings bring to light several intrinsic properties of TDP-43 and ALS-linked TDP-43 mutants that likely play important roles in the aberrant TDP-43 proteostasis (17Balch W.E. Morimoto R.I. Dillin A. Kelly J.W. Science. 2008; 319: 916-919Crossref PubMed Scopus (1787) Google Scholar) that contributes to the pathogenesis of ALS, FTLD-U, and other TDP-43 proteinopathies. Yeast cells were grown in rich medium (YPD; yeast/peptone/dextrose) or in synthetic media lacking uracil and containing 2% glucose (SD/-Ura), raffinose (SRaf/-Ura), or galactose (SGal/-Ura). A TDP-43 Gateway entry clone was obtained from Invitrogen, containing full-length human TDP-43 in the vector pDONR221. To generate C-terminally YFP-tagged TDP-43 constructs, we used PCR to amplify TDP-43 without a stop codon and incorporate SpeI and HindIII restriction sites along with a Kozak consensus sequence. The resulting PCR product was cloned into SpeI/HindIII-digested pRS416GAL-YFP to generate the CEN TDP-43YFP fusion construct. Each ALS-linked TDP-43 mutant construct was generated by using the QuikChange® site-directed mutagenesis system (Stratagene) with pRS416GAL-TDP-43-YFP as template. All constructs were verified by DNA sequencing. CEN plasmid constructs (e.g. pRS416GAL-TDP-43-YFP) were transformed into BY4741 (MATa his3 leu2 met15 ura3). Yeast procedures were performed according to standard protocols. We used the polyethylene glycol/lithium acetate method to transform yeast with plasmid DNA. For spotting assays, yeast cells were grown overnight at 30 °C in liquid media containing SRaf/-Ura until they reached log or mid-long phase. Cultures were then normalized for A600 nm, serially diluted and spotted with a Frogger (V&P Scientific) onto synthetic solid media containing glucose (SD/-Ura) or galactose (SGal/-Ura) lacking uracil and were grown at 30 °C for 2–3 days. We performed survivorship assays as described previously (18Cooper A.A. Gitler A.D. Cashikar A. Haynes C.M. Hill K.J. Bhullar B. Liu K. Xu K. Strathearn K.E. Liu F. Cao S. Caldwell K.A. Caldwell G.A. Marsischky G. Kolodner R.D. Labaer J. Rochet J.C. Bonini N.M. Lindquist S. Science. 2006; 313: 324-328Crossref PubMed Scopus (1090) Google Scholar). Briefly, after induction of empty vector, wild-type (WT) or mutant TDP-43 in 2% galactose, survivorship was determined at the indicated time points by harvesting cells at an A600 nm of 1, diluting them 1:1000, and plating 300 μl of these cells on synthetic media containing 2% glucose (represses TDP-43 expression). Plates were incubated at 30 °C for 2 days. Colony forming units were then determined. Yeast lysates were subjected to SDS-PAGE (4–12% gradient, Invitrogen) and transferred to a polyvinylidene difluoride membrane (Invitrogen). Membranes were blocked with 5% nonfat dry milk in PBS for 1 h at room temperature or overnight at 4 °C. Primary antibody incubations were performed at room temperature for 1 h. After washing with PBS, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, followed by washing in PBS plus 0.1% Tween 20 (PBST). Proteins were detected with Immobilon Western HRP Chemiluminescent Substrate (Millipore). The anti-GFP monoclonal antibody (Roche Applied Science) was used at 1:10,000, and phosphoglycerate kinase 1 antibody (Invitrogen) at 1:500. The HRP-conjugated anti-mouse secondary antibody was used at 1:5000. For immunocytochemistry experiments, yeast cells expressing untagged TDP-43 constructs were grown to a final A600 of 0.2–0.8 and then fixed with 3.7% formaldehyde for 1 h. Cells were collected by centrifugation at 2000 rpm for 5 min and washed in PBS. The cells were diluted and washed once before resuspending in 1 ml of Solution A (0.5 mm MgCl2, 1.2 m Sorbitol, 40 mm K3PO4, pH 6.5). Cells were incubated with 10 μl of 2-mercaptoethanol and 25 μl of 10 mg/ml lyticase for 15 min at 37 °C. Spheroplasted cells were collected at 4000 rpm for 5 min, washed twice with Solution A and once with PBS, and resuspended in PBS+BSA (1× PBS, 1 mg/ml bovine serum albumin). Spheroplasts were diluted 1:5 and incubated on Teflon-covered slides treated with 1 mg/ml polylysine. Wells were blocked with PBS+BSA for 30 min at room temperature. Primary antibody incubations using 1:200 anti-TDP-43 mouse polyclonal antibody (Novus) were performed for 1.5 h at room temperature. After washing with PBS+BSA, wells were incubated with 1:2000 Alexa 488 nm donkey anti-mouse polyclonal antibody (Invitrogen) for 1.5 h at room temperature. After washing with PBS+BSA, wells were incubated with Vectashield mounting medium containing 1.5 μg/ml 4′,6-diamidino-2-phenylindole (Vector Labs) for 5 min before visualization using fluorescence microscopy. We performed sedimentation analysis as described in a previous study (19Kaganovich D. Kopito R. Frydman J. Nature. 2008; 454: 1088-1095Crossref PubMed Scopus (698) Google Scholar). Cells expressing either YFP-tagged TDP-43 constructs or CFP-tagged polyglutamine-expanded huntingtin constructs for 4 or 6 h were lysed in 1× native yeast lysis buffer (30 mm HEPES, pH 8.0, 150 mm NaCl, 1% glycerol, 1 mm dithiothreitol, 0.5% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 50 mm N-ethylmaleimide, 1× protease inhibitor mixture (Roche Applied Science)). Cells were disrupted in a bead beater for 3 min at 4 °C. Cellular debris was removed by centrifugation at 6,000 × g for 5 min at 4 °C. The yeast lysate was separated into a total fraction and a pellet fraction. After the pellet fraction was spun at either 16,000 × g or 85,000 rpm in a TLA 100.1 rotor for 30 min at 4 °C the supernatant was recovered and designated the soluble fraction. The pellet fraction was resolubilized by boiling in 50 μl of 1× SDS sample buffer. The total and soluble fractions were boiled in equal volumes of 4× SDS sample buffer. 20% of the pellet fraction and 10% of soluble and total fractions were resolved by SDS-PAGE followed by immunoblotting with anti-GFP antibody. For fluorescence microscopy experiments, single colony isolates of the yeast strains were grown to mid-log phase in SRaf/-Ura media at 30 °C. Cultures were spun down and resuspended in the same volume of SGal/-Ura to induce expression of the TDP-43-YFP constructs. Cultures were induced with galactose for 6 h before being fixed with 70% ethanol and stained with 4′,6-diamidino-2-phenylindole in Vectashield mounting medium (Vector Laboratories) to visualize nuclei. Images were obtained using a Zeiss Axioplan Upright Microscope and a Zeiss AxioCam HRm high resolution monochrome charge-coupled device camera. The images were deblurred using a nearest neighbor algorithm in the AxioVision 4.5 software, and representative cells were chosen for figures. To assess differences in aggregation between wild-type and mutant TDP-43, yeast cultures were grown, induced, and processed as described above after having normalized all yeast cultures to A600 nm = 0.2 prior to galactose induction. After 6 h of induction, the identities of the samples were blinded to the observer before being examined. Several fields of cells were randomly chosen using the 4′,6-diamidino-2-phenylindole filter to prevent any bias toward populations of cells with increased amounts of aggregation in addition to obtaining the total number of cells in any given field. At least 200 cells per sample were counted for each replicate. Only cells with >3 foci under the YFP channel were considered as cells with aggregating TDP-43. TDP-43 and various missense (G294A, M337V, or Q331K) or deletion mutants (1–275 or 188–414) were expressed and purified from E. coli as either His-tagged or GST-tagged proteins. For His-tagged preparations, TDP-43 was cloned into pCOLD I (Takara) and overexpressed in E. coli BL21(RIL). Cells were lysed by sonication on ice in 40 mm Hepes-KOH, pH 7.4, 500 mm KCl, 20 mm MgCl2, 10% glycerol, 20 mm imidazole, 2 mm β-mercaptoethanol, and protease inhibitors (Complete, EDTA-free, Roche Applied Science). The proteins were purified over a nickel-nitrilotriacetic acid column (Qiagen). For GST-tagged preparations, TDP-43 was cloned into GV13 to yield a tobacco etch virus (TEV) cleavable GST-TDP-43 fusion protein, GST-TEV-TDP-43, and overexpressed in E. coli BL21(DE3)RIL or Rosetta2 (Novagen). Protein was purified over a glutathione-Sepharose column (Amersham Biosciences) according to the manufacturer's instructions. GST was then removed by cleavage with TEV protease (Invitrogen), and protease and GST were removed using nickel-nitrilotriacetic acid and glutathione-Sepharose. His-tagged and untagged TDP-43 proteins were ∼95% pure as assessed by SDS-PAGE. His-tagged and untagged proteins aggregated with identical kinetics and formed aggregates with very similar morphologies. After purification, proteins were buffer exchanged into assembly buffer (AB): 40 mm HEPES-KOH pH 7.4, 150 mm KCl, 20 mm MgCl2, 1 mm dithiothreitol. Proteins were filtered through a 0.22-μm filter. After filtration, the protein concentration was determined by Bradford assay (Bio-Rad), and the proteins were used immediately for aggregation reactions. For size-exclusion chromatography, a Superdex-200 10/300 GL analytical gel-filtration column (Amersham Biosciences) was calibrated with thyroglobulin (669 kDa), ferritin (440 kDa), bovine serum albumin (67 kDa), β-lactoglobulin (35 kDa), ribonuclease A (13.7 kDa), and aprotinin (6.5 kDa). TDP-43 was incubated in AB at 25 °C with agitation for 5 min, and any insoluble material was removed by centrifugation at 16,100 × g for 5 min. Protein was loaded onto the calibrated Superdex-200 10/300 GL column equilibrated in AB and eluted at 0.4 ml/min. Oligomeric fractions were pooled and processed for electron microscopy (see below). Filtered, purified TDP-43 was used immediately for aggregation assays. TDP-43 or missense mutant TDP-43 or deletion mutants (3 μm) were incubated at 25 °C in AB for 0–120 min with agitation at 1400 rpm in an Eppendorf Thermomixer. Turbidity was used to assess aggregation by measuring absorbance at 395 nm. For sedimentation analysis, reactions were centrifuged at 16,100 × g for 30 min at 25 °C. Supernatant and pellet fractions were then resolved by SDS-PAGE and stained with Coomassie Brilliant Blue, and the amount in either fraction was determined by densitometry in comparison to known quantities of TDP-43. Alternatively, reactions were processed for Congo Red binding or Thioflavin-T fluorescence as described before (20Chernoff Y.O. Uptain S.M. Lindquist S.L. Methods Enzymol. 2002; 351: 499-538Crossref PubMed Scopus (96) Google Scholar). For electron microscopy (EM) of in vitro aggregation reactions, TDP-43 protein samples (10 μl of a 3 μm solution) were adsorbed onto glow-discharged 300-mesh Formvar/carbon-coated copper grid (Electron Microscopy Sciences) and stained with 2% (w/v) aqueous uranyl acetate. Excess liquid was removed, and grids were allowed to air dry. Samples were viewed using a JEOL 1010 transmission electron microscope. Images were captured with a Hamamatsu digital camera using AMT acquisition software. Conventional EM was performed as previously described (21Rieder S.E. Banta L.M. Köhrer K. McCaffery J.M. Emr S.D. Mol. Biol. Cell. 1996; 7: 985-999Crossref PubMed Scopus (241) Google Scholar). Briefly, the cells were fixed in 3% glutaraldehyde contained in 0.1 m sodium cacodylate, pH 7.4, 5 mm CaCl2, 5 mm MgCl2, and 2.5% sucrose for 1 h at 25 °C with gentle agitation; spheroplasted; embedded in 2% ultra low temperature agarose (prepared in water); cooled; and subsequently cut into small pieces (∼1 mm3). The cells are then post-fixed in 1% OsO4/1% potassium ferrocyanide contained in 0.1 m cacodylate/5 mm CaCl2, pH 7.4, for 30 min at room temperature. The blocks are washed thoroughly 4× with ddH2O, 10 min total; transferred to 1% thiocarbohydrazide at room temperature for 3 min; washed in ddH2O (4×, 1 min each); and transferred to 1% OsO4/1% potassium ferrocyanide in cacodylate buffer, pH 7.4, for an additional 3 min at room temperature. The cells are then washed 4× with ddH2O (15 min total); en bloc stained in Kellenberger's uranyl acetate for 2 h to overnight; dehydrated through a graded series of ethanol; and subsequently embedded in Spurr resin. Sections were cut on a Reichert Ultracut T ultramicrotome; post stained with Kellenberger's uranyl acetate and lead citrate; and observed on a Philips TEM 420 at 80 kV. Images were recorded with a Soft Imaging System Megaview III digital camera, and figures were assembled in Adobe Photoshop 10.0. To test whether TDP-43 is inherently aggregation-prone, bacterially expressed recombinant TDP-43 was purified as a soluble protein under native conditions. Upon incubation at 25 °C with agitation, TDP-43 rapidly aggregated after a lag phase of ∼5–10 min, as determined by an increase in turbidity (Fig. 1B) and by the amount that entered the pellet fraction after centrifugation (Fig. 1C). Several control proteins, including BSA, soybean trypsin inhibitor, creatine kinase, and GFP, did not aggregate under identical conditions. After 30 min, no further TDP-43 aggregation occurred (Fig. 1, B and C). This timeframe for TDP-43 aggregation is extended to several hours if we omit agitation during incubation (data not shown). Thus, TDP-43 is an inherently aggregation-prone protein. It is likely that sophisticated cellular proteostasis mechanisms (17Balch W.E. Morimoto R.I. Dillin A. Kelly J.W. Science. 2008; 319: 916-919Crossref PubMed Scopus (1787) Google Scholar, 22Morimoto R.I. Genes Dev. 2008; 22: 1427-1438Crossref PubMed Scopus (705) Google Scholar), not reconstituted here, prevent such rapid TDP-43 aggregation in vivo. However, age-associated decline in proteostatic control in concert with environmental factors might enable TDP-43 to aggregate in disease. Regardless of the triggers of TDP-43 aggregation in disease, in vitro assays similar to the one we report here have been tremendously powerful tools in exploring basic mechanisms underpinning the aggregation events in Parkinson disease (PD) and Alzheimer disease (23Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1271) Google Scholar, 24Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 571-576Crossref PubMed Scopus (1347) Google Scholar, 25Lashuel H.A. Hartley D.M. Petre B.M. Wall J.S. Simon M.N. Walz T. Lansbury Jr., P.T. J. Mol. Biol. 2003; 332: 795-808Crossref PubMed Scopus (207) Google Scholar). Aggregates formed by pure TDP-43 did not react with the amyloid-diagnostic dyes Congo Red and Thioflavin-T, in contrast to those formed by Sup35-NM, the prion domain of the yeast prion protein Sup35 (26Shorter J. Lindquist S. Nat. Rev. Genet. 2005; 6: 435-450Crossref PubMed Scopus (423) Google Scholar) (Fig. 1, D and E). Thus, pure TDP-43 aggregates are likely to be non-amyloid just like aggregated species of TDP-43 in ALS and FTLD-U patients (27Kwong L.K. Uryu K. Trojanowski J.Q. Lee V.M. Neurosignals. 2008; 16: 41-51Crossref PubMed Scopus (100) Google Scholar). In ALS and FTLD-U, TDP-43 is ubiquitinated, phosphorylated, and proteolytically cleaved (4Neumann 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. Science. 2006; 314: 130-133Crossref PubMed Scopus (4595) Google Scholar). The relative extent and contribution of these modifications to the pathogenicity of TDP-43 remain to be defined. Our in vitro aggregation assays will provide the foundation for future studies aimed at determining the effects of TDP-43 post-translational modification and processing on aggregation. Next, we determined which regions of TDP-43 are critical for aggregation in vitro. We purified TDP-43 fragments: 1–275, which comprises the N-terminal domain, RRM1 and RRM2, and 188–414, which comprises RRM2 and the C-terminal domain (Fig. 1A). 1–275 is soluble, whereas 188–414 is the minimal fragment able to confer toxicity and aggregation in a yeast model of TDP-43 proteinopathies (28Johnson B.S. McCaffery J.M. Lindquist S. Gitler A.D. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6439-6444Crossref PubMed Scopus (327) Google Scholar). Importantly, pure 1–275 did not aggregate, whereas 188–414 aggregated with similar kinetics to full-length TDP-43 (Fig. 1B). Thus, the C-terminal domain plays an important role in TDP-43 aggregation, which is striking because of the >25 recently reported ALS-linked TDP-43 mutations, all but one are within this domain (Fig. 2A) (12Banks G.T. Kuta A. Isaacs A.M. Fisher E.M. Mamm. Genome. 2008; 19: 299-305Crossref PubMed Scopus (62) Google Scholar). Furthermore, similar aggregated C-terminal fragments accumulate in ALS and FTLD-U (4Neumann 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. Science. 2006; 314: 130-133Crossref PubMed Scopus (4595) Google Scholar). Therapeutic strategies aimed at targeting this region, which we have defined as responsible for driving aggregation, may be efficacious. Having established that TDP-43 is inherently aggregation-prone, we next asked if ALS-linked TDP-43 mutations affect aggregation in vivo. We have developed a yeast TDP-43 proteinopathy model to investigate mechanisms of TDP-43 aggregation and toxicity (28Johnson B.S. McCaffery J.M. Lindquist S. Gitler A.D. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6439-6444Crossref PubMed Scopus (327) Google Scholar). This model recapitulates several important features seen in human disease. In yeast, TDP-43 is initially localized to the nucleus but eventually forms cytoplasmic inclusions (28Johnson B.S. McCaffery J.M. Lindquist S. Gitler A.D. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6439-6444Crossref PubMed Scopus (327) Google Scholar), mimicking the pathobiology of TDP-43 in human neurons (4Neumann 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. Science. 2006; 314: 130-133Crossref PubMed Scopus (4595) Google Scholar). Importantly, expressing high levels of TDP-43 is toxic to yeast (28Johnson B.S. McCaffery J.M. Lindquist S. Gitler A.D. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6439-6444Crossref PubMed Scopus (327) Google Scholar), thus possibly modeling, in a simple cell, features of neurodegeneration. We tested the effects of seven recently reported ALS-linked mutations (6Kabashi E. Valdmanis P.N. Dion P. Spiegelman D. McConkey B.J. Vande Velde C. Bouchard J.P. Lacomblez L. Pochigaeva K. Salachas F. Pradat P.F. Camu W. Meininger V. Dupre N. Rouleau G.A. Nat. Genet. 2008; 40: 572-574Crossref PubMed Scopus (1242) Google Scholar, 7Sreedharan J. Blair I.P. Tripathi V.B. Hu X. Vance C. Rogelj B. Ackerley S. Durnall J.C. Williams K.L. Buratti E. Baralle F. de Belleroche J. Mitchell J.D. Leigh P.N. Al-Chalabi A. Miller C.C. Nicholson G. Shaw C.E. Science. 2008; 319: 1668-1672Crossref PubMed Scopus (1992) Google Scholar, 8Van Deerlin V.M. Leverenz J.B. Bekris L.M. Bird T.D. Yuan W. Elman L.B. Clay D. Wood E.M. Chen-Plotkin A.S. Martinez-Lage M. Steinbart E. McCluskey L. Grossman M. Neumann M. Wu I.L. Yang W.S. Kalb R. Galasko D.R. Montine T.J. Trojanowski J.Q. Lee V.M. Schellenberg G.D. Yu C.E. Lancet Neurol. 2008; 7: 409-416Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 9Rutherford N.J. Zhang Y.J. Baker M. Gass J.M. Finch N.A. Xu Y.F. Stewart H. Kelley B.J. Kuntz K. Crook R.J. Sreedharan J. Vance C. Sorenson E. Lippa C. Bigio E.H. Geschwind D.H. Knopman D.S. Mitsumoto H. Petersen R.C. Cashman N.R. Hutton M. Shaw C.E. Boylan K.B. Boeve B. Graff-Radford N.R. Wszolek Z.K. Caselli R.J. Dickson D.W. Mackenzie I.R. Petrucelli L. Rademakers R. PLoS Genet. 2008; 4: e1000193Crossref PubMed Scopus (371) Google Scholar, 11Yokoseki A. Shiga A. Tan C.F. Tagawa A. Kaneko H. Koyama A. Eguchi H. Tsujino A. Ikeuchi T. Kakita A. Okamoto K. Nishizawa M. Takahashi H. Onodera O. Ann. Neurol. 2008; 63: 538-542Crossref PubMed Scopus (329) Google Scholar) (Fig. 2A) on TDP-43 aggregation in this system. Wild-type (WT) and mutant TDP-43-YFP were expressed from a low copy (CEN) plasmid, under control of a galactose-inducible promoter, and cells were visualized by fluorescence microscopy. We confirmed that the TDP-43 proteins were expressed at comparable levels (Fig. 2B). We compared aggregation in cells expressing WT TDP-43 to those expressing each of the seven mutants. YFP alone was diffusely distributed between the c" @default.
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• W2020159880 title "TDP-43 Is Intrinsically Aggregation-prone, and Amyotrophic Lateral Sclerosis-linked Mutations Accelerate Aggregation and Increase Toxicity" @default.
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