Preparation of Recombinant TDP-43 Proteins
For preparation of TDP-43 proteins with an N-terminal His tag, cDNA of TDP-43 was cloned in a multiple cloning site (XhoI and BamHI) of a plasmid vector, pET15b (Novagen). After transfection of the plasmids in Escherichia coli (Rosetta), expression of His-tagged TDP-43 proteins was induced by the addition of 1 mm isopropyl β-d-thiogalactoside. Following incubation at 37 °C for 6 h, all TDP-43 proteins in this study were obtained as inclusion bodies, which were redissolved in PBS containing 6 m guanidine hydrochloride (GdnHCl) and then purified with Ni2+ affinity chromatography using the Proteus IMAC Midi kit (Pro-Chem Inc.). The bound TDP-43 proteins were washed with a wash buffer (pH 8.0) containing 50 mm Tris, 500 mm NaCl, 25 mm imidazole, and 6 m GdnHCl and then eluted from a spin column with an elution buffer (50 mm Tris, 200 mm NaCl, 125 mm imidazole, 6 m GdnHCl, pH 8.0).
For preparation of soluble and refolded TDP-43, TDP-43 proteins unfolded in the elution buffer (400 μm) were 40-fold diluted with a buffer (pH 8.0) containing 100 mm Na-Pi, 100 mm NaCl, 5 mm EDTA, 5 mm DTT, and 10% glycerol (buffer A), which, however, produced significant amounts of precipitates. These insoluble materials were removed by ultracentrifugation at 110,000 × g for 30 min, and we thus recovered the soluble TDP-43 proteins in a supernatant fraction. Protein concentration was spectroscopically determined from the absorbance at 280 nm by using the following extinction coefficients: 44,920 cm−1 m−1, TDP-43FL; 26,930 cm−1 m−1, TDP-43N; 17,990 cm−1 m−1, TDP-43C; 25,440 cm−1 m−1, TDP-43N-1; 19,480 cm−1 m−1, TDP-432-C.
Filter Binding Assay
TG- or AC-repeated DNA was modified with a biotin at the 5′-end (Operon Biotechnologies, Tokyo). To reduce retention of free single-stranded DNA, a nitrocellulose membrane (PROTRAN, 0.2 μm, Schleicher & Schuell) was presoaked for 10 min in 0.4 m
KOH, rinsed in H2
O, and then equilibrated in buffer A for an hour before use (
). The biotin-labeled TG- or AC-repeated DNA (biotin-(TG)12
, 0.1 μm
) was incubated with soluble TDP-43 proteins (0.1–1.0 μm
) in buffer A. After an hour at 37 °C, the mixture was filtered through a pretreated nitrocellulose membrane overlaid on a nylon membrane (Hybond-N+, 0.45 μm, Amersham Biosciences) in a 96-well slot-blot apparatus. After extensive washing of membranes with buffer A, the bound DNAs were cross-linked to the membranes by shortwave ultraviolet radiation (UV Stratalinker, Stratagene). Followed by blocking with 3% bovine serum albumin (BSA) in TBS with 0.1% Tween 20, the membranes were incubated with streptavidin-HRP (Promega), and the biotin-labeled DNAs on the membranes were developed with the SuperSignal West Dura chemiluminescent substrate (Pierce).
Biochemical and Morphological Analysis of in Vitro TDP-43 Aggregates
To examine aggregation of TDP-43 proteins, 1 μm soluble TDP-43 protein in buffer A was set in a 96-well plate and shaken at 37 °C at 1,200 rpm (BioShaker MBR-024, TAITEC). Turbidity was monitored at every 30 min in a plate reader (ARVO MX, PerkinElmer Life Sciences) by measuring absorbance around 405 nm. After agitation for 27 h, 25 μm thioflavin T was then added to the reaction mixture, and the fluorescence intensity was recorded by using a plate reader (ARVO MX) with a CW lamp filter (440 nm cutoff) and an emission filter (486 nm cutoff). Thioflavin T fluorescence before sample agitation was also examined. Final TDP-43 aggregates were obtained as a pellet fraction after ultracentrifugation of the reaction mixtures at 110,000 × g for 30 min.
For a seeding reaction, TDP-43 aggregates were resuspended in H2O, sheared with ultrasonication, and then added to a solution of 3 μm soluble TDP-43 proteins in buffer A containing 25 μm thioflavin T. The fluorescence was monitored by using SpectraMax M2 (Molecular Devices) at intervals of 2 min with 442 and 485 nm of excitation and emission wavelength, respectively. No plate shaking was performed during the reactions.
For morphological analysis, TDP-43 aggregates were first adsorbed on 400-mesh grids coated by a glow-charged supporting membrane. After washing with pure water, negative staining with 1% uranyl acetate was performed, and then images were obtained using an electron microscope (1200EX, JEOL).
Identification of Core Regions in TDP-43 Aggregates
22.5 μg of TDP-43 aggregates were first resuspended in 100 μl of a buffer (pH 8.0) containing 50 mm Tris, 100 mm NaCl, and 5 mm CaCl2 and mixed with 5 μg of Pronase (Calbiochem). After incubation at 37 °C for an hour, the mixture was ultracentrifuged at 110,000 × g for 30 min, and the supernatant fraction was discarded. The pellets were washed once with 150 μl of 50 mm Tris, 100 mm NaCl, pH 8.0 and redissolved in 20 μl of 50 mm Tris, 200 mm NaCl, 6 m GdnHCl, 5 mm EDTA, 5 mm DTT, pH 8.0. After desalting by using NuTip C-18 (Glygen Corp.), samples were mixed with a matrix, α-cyano-4-hydroxycinnamic acid, and MALDI-MS and MS/MS spectra were acquired using a 4800 Plus MALDI-TOF/TOF analyzer (Applied Biosystems). Identification of peptides based upon the observed m/z values was performed using MS-NonSpecific.
Plasmid Construction and Cell Culture
All plasmids for expression of TDP-43 proteins in cultured cells were constructed using pIRESneo3 (Clontech). Namely, cDNAs of full-length TDP-43 (TDP-43FL) and a C-terminal truncate of TDP-43 (TDP-432-C) that were fused with a C-terminal HA tag were cloned in a multiple cloning site using AgeI and BamHI sites.
Transduction of protein aggregates into cultured cells was performed by using the BioPORTER protein delivery reagent (Genlantis). On day 1, 1.2 × 105 cells of human embryonic kidney cells, HEK293T, in DMEM, 10% FBS were seeded on each well of a 12-well plate coated with polyethyleneimine and incubated at 37 °C. On the next day (day 2), 1.6 μg of plasmids was transfected per each well using Lipofectamine 2000 (Invitrogen). After 4 h of transfection, a culture medium was replaced with fresh DMEM, 10% FBS and incubated at 37 °C. On day 3, a culture medium was changed to Opti-MEM 1 h before aggregate transduction. A dried film of BioPORTER was first dissolved with 50 μl of PBS containing 10 μm protein aggregates and then mixed with 450 μl of Opti-MEM. By using this aggregate/BioPORTER-containing Opti-MEM, the transfected cells were further incubated at 37 °C for 4 h. The culture medium was then changed to DMEM, 0.5% FBS and again incubated at 37 °C overnight. On day 4, cells were harvested by PBS containing 1% sarkosyl with a protease inhibitor mixture, Complete, EDTA-free (Roche Diagnostics). Following lysis with ultrasonication, lysates were ultracentrifuged at 110,000 × g for 30 min to separate into supernatant (a soluble fraction) and pellets (an insoluble fraction). The pellets were redissolved in a 2% SDS-containing PBS with the same volume with that of the corresponding supernatant. The protein concentration in supernatant was determined by BCA assay using BSA as a standard.
Each of a soluble fraction containing 3 μg of total proteins and an insoluble fraction (10× volume of the corresponding soluble fraction) was mixed with an SDS-PAGE sample buffer containing β-mercaptoethanol and loaded on a 12.5% SDS-PAGE gel (PAGEL, ATTO) after boiling for 5 min. Following electrophoresis, proteins were electroblotted on a 0.2-μm PVDF membrane (Bio-Rad) and analyzed by Western blotting using rabbit polyclonal anti-HA (Y-11, Santa Cruz Biotechnology, 1:1,000), rabbit polyclonal anti-His probe (H-15, Santa Cruz Biotechnology, 1:1,000), rabbit polyclonal anti-ubiquitin (DAKO, 1:1,000), or mouse anti-TDP-43 phospho-Ser-409/410 antibody (Cosmo Bio, 1:1,000). Corresponding secondary antibodies that are conjugated with HRP were used as follows: anti-rabbit (1:5,000) and anti-mouse (1:1,000) antibodies. Blots were developed with the SuperSignal West Dura chemiluminescent substrate (Pierce), and images were obtained using LAS1000 (Fujifilm).
For immunostaining of HA-tagged TDP-43 proteins, cells were fixed with 4% paraformaldehyde containing 0.12 m sucrose in PBS for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 5 min, and blocked with 0.1% BSA in PBS for 30 min. Cells were then incubated with anti-HA-fluorescein, high affinity (3F10) (1:100 dilution, Roche Diagnostics) in PBS containing 0.1% BSA for an hour, washed once with 0.1% Triton X-100 in PBS, and washed twice with 0.1% BSA in PBS. For detection of ubiquitin, rabbit polyclonal anti-ubiquitin mouse polyclonal ubiquitin (DAKO, 1:300) and anti-rabbit IgG conjugated with Alexa Fluor 555 (Invitrogen, 1:1,000) were used. Cells were incubated with Hoechst 33342 (Invitrogen, 1:3,000 dilution) in PBS for 15 min and then washed twice with PBS and mounted onto glass slides.
For visualization of TDP-432-C seeds added to cells, TDP-432-C aggregates were incubated in the presence of 1 mm tris(2-carboxyethyl)phosphine with 1 mm Alexa Fluor 555 maleimide (Invitrogen) for an hour at room temperature. Modified aggregates were washed three times with PBS by ultracentrifugation at 110,000 × g for 30 min and used for transduction experiments. When modified aggregates were resolubilized in PBS containing 2% SDS and loaded on a SDS-PAGE gel, a red fluorescent band with a molecular weight corresponding to that of TDP-432-C was found (data not shown), which confirms the successful modification.
Confocal images with a slice thickness of ∼1 μm were obtained by a laser scanning microscope of LSM5 Exciter system (Carl Zeiss, Germany) using a 40× objective lens. Obtained images were processed and analyzed by LSM image examiner (Carl Zeiss).