Interaction of RNA with a C-terminal fragment of the amyotrophic lateral sclerosis-associated TDP43 reduces cytotoxicity

A hallmark of amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease, is formation of inclusion bodies (IBs) from misfolded proteins in neuronal cells. TAR RNA/DNA-binding protein 43 kDa (TDP43) is an ALS-causative protein forming IBs in ALS patients. The relation between localization of the IBs and neurotoxicity remains largely unknown. We characterized aggregation of fluorescently tagged TDP43 and its carboxyl-terminal fragments (CTFs) by analytical fluorescence imaging techniques. Quantitative time-lapse analysis in individual live cells showed that fluorescent-protein-tagged TDP43 was cleaved and a 35 kDa TDP43 CTF (TDP35) formed ubiquitin (Ub)-negative cytoplasmic IBs. Although TDP35 formed mildly toxic Ub-negative IBs in the cytoplasm, TDP25, another type of a TDP43 CTF, efficiently formed sufficiently toxic Ub-positive IBs. One- or two-color fluorescence correlation spectroscopy (FCS/FCCS) revealed that coaggregation of TDP25 with TDP43 was initiated by depletion of the RNA that binds to TDP25. Moreover, nuclear localization tagging TDP25 reduced the rate of neuronal cell death. These observations point to the need to elucidate the novel sequestration mechanism and details of the toxicity of the misfolded and aggregation-prone TDP43 CTFs (as well as the RNA binding and nuclear retention) in order to identify possible preventive interventions against ALS.


Time-lapse analysis during apoptosis
Neuro2A cells were grown in a glass-based dish (#3910-035, AGC Techno glass, Shizuoka, Japan) for 16 h before the transfection. A plasmid mixture consisting of 300 ng of the R-TDP43-G-coding plasmid, 400 ng of H2B-CFP, and 300 ng of pCAGGS was transfected into the Neuro2A cells with 2.5 L of Lipofectamine 2000 (Thermo Fisher Scientific). For detection of caspase 3 activation, the plasmid mixture for transfection was changed to the one consisting of 300 ng T-TDP43-Y, 500 ng LSSmOrange-DEVD-mKate2, and 200 ng H2B-iRFP. At 24 h after the transfection, the medium was replaced with the maintaining medium containing 0.5 M STS. Immediately after the addition of STS, time-lapse analysis was started on an LSM510 META confocal microscope (Carl Zeiss) through a Plan-Neofluar 20×/0.5NA objective for R-TDP43-G, or a C-Apochromat 40×/1.2NA W Korr. UV-VIS-IR water immersion objective for T-TDP43-Y at 37°C and 5% CO2. The microscope was operated on the AIM 3.2 software platform (Carl Zeiss). For examination of R-TDP43-G and H2B-CFP, we excited CFP, GFP, or RFP at 458, 488, or 543 nm, respectively. The excitation beams were split by an HFT488/543 filter for GFP and RFP, or an HFT458 filter for CFP. GFP and RFP fluorescent signals were separated by a dichroic mirror (NFT545) and collected through a BP505-530 band pass filter and an LP585 long pass filter, respectively. Fluorescence from CFP was collected through a BP474-525 band pass filter. The pinhole size for CFP, GFP, and RFP was set to 81, 84, and 98 m, respectively.
The zoom factor was set to 1×. X-and Y-scanning sizes were each 512 pixels. For analysis of T-TDP43-Y, LSSmOrange-DEVD-mKate2, and H2B-iRFP, we excited mTFP1, LSSmOrange, and mKate2 at 458 nm, and YFP and iRFP at 514 and 633 nm, respectively. The excitation beams were split by an HFT458 filter for mTFP1, LSSmOrange, and mKate2, by an HFT458/514 filter for YFP, or HFT514/633 filter for iRFP. mTFP1, LSSmOrange, and mKate2 fluorescent signals were simultaneously collected through a spectrophotodetector (META) at 477-499, 584-595, and 659-702 nm, respectively. YFP and iRFP fluorescence was collected through a META at 531-584 and 659-798 nm, respectively. The pinhole size for all conditions was set to 200 m. The zoom factor was set to 2×. X-and Y-scanning sizes were each 1024 pixels. The acquired images were processed in the ImageJ 1.47v software (National Institutes of Health, Bethesda, MD) and Photoshop CS4 (Adobe Systems, San Jose, CA).

Nuclear-cytoplasmic fractionation and western blotting
Neuro2A cells transiently expressing R-TDP43-G and these cells in the time-lapse condition were grown on a 3.5-cm NUNC plastic tissue culture dishes (#150318; Thermo Fisher Scientific). The cells were lysed at 1 h intervals up to 7 h after the addition of STS. After the cells were washed in 2 mL of PBS, 200 L of hypo-osmotic lysis buffer consisting of 10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 0.1% NP-40, 1 mM DTT, and 1% protease inhibitor cocktail (Sigma-Aldrich) was added, and the mixture was incubated for 5 min at 4°C. After the lysates were centrifuged at 800 × g for 5 min at 4°C, the supernatants were recovered as a cytoplasmic fraction. Hyperosmotic lysis buffer consisting of 20 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 400 mM NaCl, 0.1 mM EDTA, 0.1% NP-40, 10% glycerol, 0.01 U/L benzonase (Sigma-Aldrich), 1 mM DTT, and 1% protease inhibitor cocktail (Sigma-Aldrich) was added, and the pellets were subjected to shaker conditions at 1,800 rpm for 60 min at 4°C (#CM-1000; Tokyo Rikakikai Co, Ltd., Tokyo, Japan), followed by washing in 200 L of hypo-osmotic buffer once. After centrifugation at 20,400 × g for 5 min at 4°C, the supernatants were recovered as a nuclear fraction. After 1/3 volume of 4× Laemmli sample buffer was added to each sample, the samples were incubated at 98C for 2 min. The samples were applied to a 10-20% e-PAGEL (#2331740; ATTO, Tokyo, Japan) and subjected to electrophoresis in SDS-containing buffer. The proteins were blotted on a polyvinylidene difluoride (PVDF) membrane (For GFP, RFP, and tubulin detection, Hybond-P was used instead [purchased from GE Healthcare, Logan, UT]; and for caspase 3 detection, Immobilon-P SQ was used instead; purchased from Merck Millipore, Darmstadt, Germany) using a mini-trans blot cell (Bio-Rad, Hercules, CA).
The membranes were blocked in 5% skim milk in PBS-T (PBS containing 0.05% Tween 20). After the membranes were washed in PBS-T 3 times, a primary anti-GFP antibody (GF200; Nacalai Tesque, Kyoto, Japan), anti-RFP (#R10367; Life Technologies), anti-TDP43 antibody (#3448; Cell Signaling Technology, Danvers, MA), anti-caspase 3 (#9662; Cell Signaling Technology, Danvers, MA), or an -tubulin antibody (DM1A; Merck Millipore) was allowed to react with the membrane in the blocking buffer or Can Get Signal Solution 1 (TOYOBO, Osaka, Japan). As secondary antibodies, anti-mouse or rabbit IgG antibody conjugated with horse radish peroxidase (The Jackson Laboratory, Bar Harbor, ME) was incubated with the membranes in the blocking solution. Images of the luminescent signals were acquired on a LAS 4000 mini (Fujifilm, Tokyo, Japan) with an ECL reagent (GE Healthcare).
The images were processed in the ImageJ 1.47v software (National Institutes of Health) and Photoshop CS4 software (Adobe Systems).

The assay of solubility of TDP43 and the CTFs
Neuro2A cells transiently expressing GFP, TDP43-GFP, GFP-TDP35, GFP-TDP25, or GFP-NLS-TDP25 and these cells under the conditions of the cell viability assay were grown in 3.5-cm NUNC plastic tissue-culture dishes (#150318; Thermo Fisher Scientific). After the cells were washed in 2 mL of PBS, 200 L of lysis buffer consisting of 25 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 0.01 U/L benzonase, and 1% protease inhibitor cocktail (Sigma-Aldrich) was added, and the mixture was incubated for 5 min at 4°C. After the lysates were centrifuged at 20,400 × g for 10 min at 4°C, the supernatants were recovered as a SDS-soluble fraction.
The pellets was solubilized in 20 L of 1 M urea buffered with PBS for 30 min at room temperature followed by washing in 200 L of PBS. For dilution of urea, 180 L of PBS was added to the ureasolubilized samples. After 1/3 volume of 4× Laemmli sample buffer was added to each lysate, the samples were incubated at 98°C for 5 min. The samples were applied to a 10-20% e-PAGEL (#2331740; ATTO) and subjected to electrophoresis in SDS-containing buffer. The proteins tagged with GFP were detected with an anti-GFP antibody (GF200; Nacalai Tesque), similarly to detection in the nuclear-cytoplasmic fractionation assay.

The assay of cleavage efficiency of TDP43 during apoptosis
Neuro2A cells expressing no transfected plasmids, R-TDP43-G, or T-TDP43-Y were prepared. For STS-untreated cells were trypsinized and suspended in the culturing medium. For STS-treated cells for 24 h, cells were suspended using pipetting. After the centrifugation of the cells, cell pellets were washed in PBS once followed by the pellets were freezed at -80°C. After the pellets were thawed, cells were suspended in PBS containing 1% SDS, 0.01 U/L benzonase, and 1% protease inhibitor cocktail (Sigma-Aldrich) and then incubated for 10 min. at 25°C. After each soluble fractions were recovered after centrifugation at 20,400 × g for 10 min at 25°C, 1/3 volume of 4× Laemmli sample buffer was added to each lysate and then incubated at 98°C for 5 min. The samples were applied to a 10-20% e-PAGEL (#2331740; ATTO) and subjected to electrophoresis in SDS-containing buffer. The proteins were detected with an anti-TDP43 antibody (#3448; Cell Signaling Technology), similarly to detection in the nuclear-cytoplasmic fractionation assay.

Immunofluorescence staining
Neuro2A cells expressing GFP-or FLAG-tagged proteins were fixed in 4% paraformaldehyde buffered with 100 mM Hepes-KOH (pH7.5) at 37°C. After cells were washed in Tris-buffer saline (TBS) three times, PBS containing 0.5% Triton X-100 and 0.5% Saponin was treated for membrane permeabilization. Non-specific binding of antibodies were blocked in PBS containing 5% normal goat serum (DAKO, Glostrup, Denmark) and 10% glycerol (Blocking buffer). Primary and secondary antibodies were reacted in the blocking buffer. Cells were mounted in ProLong Gold (Thermo Fisher Scientific).

Confocal fluorescence microscopy
Neuro2A cells were transfected with GFP-tagged TDP43 or the CTFs as described for other experiments above, except for addition of a plasmid coding 100 ng RFP-Ub or 500 ng TDP43-RFP by means of Lipofectamine 2000. By the addition of a plasmid encoding RFP-Ub or TDP43-RFP, the amount of the plasmid encoding GFP-TDP25 was reduced to normalize the total amount of transfected DNA to 1.0 g. After incubation for 1 day, the media were replaced with the one containing 2 M MG-132 (Peptide Institute, Osaka, Japan) or 0.4 L DMSO (Nacalai Tesque). After incubation for 16 h, confocal fluorescence microscopy was conducted on an LSM 510 META through a C-Apochromat 40×/1.2NA W Korr. UV-VIS-IR (Carl Zeiss) at 37°C and 5% CO2. The microscope was operated on the AIM 3.2 software platform (Carl Zeiss). GFP and RFP were excited at 488 or 543 nm, respectively.
The excitation beams were split by an HFT488/543 filter. GFP and RFP fluorescent signals were separated by a dichroic mirror (NFT545) and collected through a BP505-530 band pass filter and an LP585 long pass filter, respectively. Pinhole sizes for GFP and RFP were set to 70 and 82 m, respectively. The zoom factor was set to 4×. X-and Y-scanning sizes were each 1024 pixels. The images were reconstructed using an average value of 4-line scanning. The images were processed in the ImageJ 1.47v software (National Institutes of Health) and Photoshop CS4 software (Adobe Systems).

RNA staining in live cells
Neuro2A cells expressing RFP-TDP25 were stained with medium containing 5 M SYTO RNA Select  The zoom factor was set to 5×. X-and Y-scanning sizes were each 512 pixels. Interval time for image acquisition was set to 15 or 10 s (for cytoplasmic IB or nucleoli, respectively). The photobleaching period was 3.2 or 1.3 s (for cytoplasmic IB or nucleoli, respectively). Relative fluorescence intensity was measured in the AIM3.2 software (Carl Zeiss) and calculated according to Eq. 1.
where IBL(t) and where I(t) is the intensity at time point t, I0 is the base line intensity, A is the maximum recovery rate, and k is recovery constant.

Airyscan (confocal super-resolution) fluorescence microscopy
where I(t+) is the fluorescence intensity obtained by the single-photon counting method in a detection volume at delay time  (angular brackets denote ensemble averages). A multicomponent diffusion model with a triplet state for curve-fitting is given by Eq. 5: where  RCA values for estimation of interaction strength were obtained using Eq. 9 9 .
where GC(0) and GR(0) are the cross-correlation function and autocorrelation function of DNA at delay time zero, respectively.

Gel-Shift assay
Flp-In TReX Neuro2A expressing TDP43-GFP, GFP-TDP25, or GFP by addition of doxycycline in the culturing medium for 48 h was lysed according to the experiment of RNase treatment followed by FCS measurement. Concentration of each proteins tagged with GFP was measured using a ConfoCor2 (Carl Zeiss) 10 , according to the Eq. 10.
where, C is a concentration of the GFP-tagged protein, N is the number of fluorescence molecule from FCS measurement (N = [G(0) -1] -1 ), w is radius of the confocal volume, z is half height of the confocal volume. Here,   w 2 z is an effective confocal volume. NA is the Avogadro number. The radius (w) is obtained using the Eq. 11. The z was calculated using structure parameter (s = z/w).
where, Rh6G is the diffusion time of Rhodamine 6G from FCS measurement, DRh6G is the diffusion coefficient of Rhodamine 6G (414 m 2 /s). Each lysates were diluted as 2 nM GFP-tagged proteins was included using lysis buffer. Total RNA was extracted from Neuro2A cells using TRIzol Plus RNA Purification kit (Thermo Fisher Scientific) and the concentration was quantified using a spectrophotometer (Shimadzu, Kyoto, Japan). Total RNA (150 ng) as the same volume to the diluted lysates was added to the lysates, and 4× sample buffer containing 200 mM Hepes-KOH (pH7.9) and 40% Glycerol was subsequently added. Samples were separated using a native-gel comprising 4.5% polyacrylamide and 375 mM Hepes-KOH (pH7.5) in an electrophoresis running buffer comprising 35 mM Hepes and 43 mM imidazole for approximately 3 h (120 V) at 4°C. GFP-tagged proteins were exited at 488 nm and visualized using a Typhoon (GE Healthcare). For confirmation of RNA existence in the gel, RNA was stained using SYBR Gold (Thermo Fisher Scientific) in the electrophoresis running buffer followed by visualization using a UV transilluminator (ATTO).
After centrifugation at 20,400 × g for 15 min, supernatants were collected as a soluble fraction (s).
Neuro2A cells expressing FLAG-tagged proteins were lysed in a lysis buffer containing 0.1% SDS.
After centrifugation at 20,400 × g for 15 min, supernatants were collected as a soluble fraction (S). Normalized G(τ)