Western Blots

To extract proteins, cells were centrifuged at 3000 × g for 4 min and washed twice with PBS. Cells were subsequently incubated in ice for 20 min in M-PER (Pierce) with protease and phosphatase inhibitors. After incubation, the lysate was centrifuged at 4 °C for 30 min at maximum speed, and the supernatant was removed and stored for subsequent analysis. Proteins were resolved by SDS-PAGE (10% BisTris from Invitrogen) under reducing conditions and transferred to nitrocellulose membranes, which were then incubated in a 5% solution of not-fat milk for 1 h at 20 °C. After overnight incubation at 4 °C with the appropriate primary antibody, the blots were washed with Tween/Tris-buffered saline for 20 min and incubated at 20 °C with secondary antibody. Blots were subsequently washed with Tween/Tris-buffered saline for 20 min and incubated for 5 min with SuperSignal (Pierce).

Immunofluorescence

For fluorescent labeling, cells were first washed with PBS and then fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were washed three times with PBS and treated with antigen retrieval buffer (100 mm Tris and 5% urea, pH 9.5) for 10 min at 95 °C. After being washed three times with PBS, cells were incubated for 15 min at room temperature in 0.1% Triton X-100, followed by a 1-h incubation at room temperature in 10% goat serum. Cells were then placed in the appropriate primary antibody diluted in blocking solution overnight at 4 °C. Following three rinses with PBS, cells were placed in the appropriate secondary antibodies conjugated to Alexa 488, 555, or 619 fluorophores (Molecular Probes). Following a 1-h incubation, cells were rinsed three times and coverslipped using Fluoromount-G (SouthernBiotech).

Cloning

Full-length TDP-43 (FL-TDP) in pcDNA3.1 plasmid was a gift from Dr. Michael J. Strong (University of Western Ontario, London, Ontario, Canada). To clone the sequence encoding the last 199 amino acids of TDP-43 (C199-TDP) under the control of the cytomegalovirus promoter, we used a homologous recombination-based approach (BD Biosciences InFusion system). C199-TDP was amplified by PCR using a proof-checking DNA polymerase with the following primers: forward, 5′-GTTTAAACTTAAGCTTCCACCATGGATGTGATGGATGTCTTC-3′; and reverse, 5′-GCCCTCTAGACTCGAGCTAGTGATTCATTCCCCAGCCAG-3′. Both primers included 16 bp of homology to the site of insertion within the pcDNA3.1 multiple cloning site. In addition, the forward primers included a Kozak consensus site (CCACC) and the ATG start codon. To elicit recombination between the linearized pcDNA3.1 plasmid and the C199-TDP fragment, we combined 100 ng of the linearized pcDNA3.1 plasmid and 50 ng of the C199-TDP fragment with 10 μl of water; the mixture was added to the InFusion Dry-Down mixture (BD Biosciences) and incubated at room temperature for 30 min. The InFusion mixture contains a proprietary recombinase enzyme that catalyzes the specific recombination of two double-stranded homologous regions of DNA.

Metabolic Labeling and Immunoprecipitation

Pulse-chase labeling experiments with [³⁵S]methionine were performed as described (37). Briefly, N2A cells stably transfected with FL-TDP or C199-TDP were plated on 6-well plates (100,000 cells/well), incubated for 1–2 days, and washed with methionine-free Dulbecco's modified Eagle's medium. Cells were subsequently pulse-labeled with [³⁵S]methionine (0.1 mCi/well) at 37 °C for 2 h. Thereafter, cells were washed and subjected to 3-, 6-, and 24-h chases in complete medium with or without 3-methyladenine (3-MA) or rapamycin. TDP-43 was immunoprecipitated from cell lysates using an anti-TDP-43 polyclonal antibody (Proteintech Group).

Real-time PCR

Total RNA was extracted using an RT² qPCR-grade RNA isolation kit (SABiosciences) according to the manufacturer's protocol and quantified using a BioSpec-1601 spectrophotometer (Shimadzu). 1 μg of total RNA was used to make the cDNA by using an RT² First Strand kit (SABiosciences) in a total volume of 20 μl according to the manufacturer's protocol. After reverse transcription-PCR, 1 μl of cDNA was amplified and quantified in 96-well plates using an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). For PCR, the amplification conditions consisted of an initial activation step at 95 °C for 10 min and 40 cycles at 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s. The fluorescence of the double-stranded products accumulated was monitored in real time. The relative mRNA levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase reference housekeeping gene. For the low molecular mass neurofilament (NFL) gene, the forward primer was 5′-GAGTGGCTTTCGGCTTGCT-3′, and the reverse primer was 5′-CACATTGCCGTAGATCCTGAACT-3′. Glyceraldehyde-3-phosphate dehydrogenase was amplified using primers from SABiosciences.

Overexpression of C199-TDP Leads to Cytoplasmic TDP-43 Accumulation

A recent work by Petrucelli and co-workers (24) showed that progranulin mediates the proteolytic cleavage of TDP-43 to generate ~35- and ~25-kDa species, a process mediated by caspase activation. Notably, the ~25-kDa fragment has been consistently isolated from affected brain regions of FTLD-U patients, indicating that it may be involved in the disease pathogenesis.

We therefore sought to determine the role of the ~25-kDa C-terminal fragment (encoded by the last 199 amino acids of TDP-43, referred to as C199-TDP) (Fig. 3A) in TDP-43 aggregation. Using a homologous recombination-based approach, we cloned the sequence encoding the last 199 amino acids of TDP-43 under the control of the cytomegalovirus promoter. An ATG start codon and a Kozak sequence were also added at the 5′-end of the sequence (data not shown). We used this construct to transfect N2A and SH-SY5Y cells. Twenty-four hours after transfection, cells were stained with anti-TDP-43 monoclonal antibody 2E2-D3. We found that overexpression of C199-TDP led to an increase in cytoplasmic TDP-43 immunoreactivity, which was associated with a reduction in nuclear TDP-43 staining (Fig. 3, B–D). These data are consistent with the results showing that accumulation of cytoplasmic TDP-43, after deletion of its nuclear localization signals, leads to a redistribution of endogenous TDP-43 from the nucleus to the cytoplasm (40).

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FIGURE 3.

C199-TDP is sufficient to induce nuclear TDP-43 depletion and cytoplasmic TDP-43 accumulation. A, amino acid sequence of TDP-43 (NCBI accession number NP_031401). The putative 199-amino acid sequence of the ~25-kDa fragment is underlined and in boldface. Notably, this fragment has been isolated from FTLD-U and ALS brains. B, representative microphotographs of cells transfected with C199-TDP or vector alone. The arrows point to cytoplasmic TDP-43-positive accumulation. Note the reduction in nuclear staining (arrowhead), which is also evident by reduced TDP-43 localization with 4′,6-diamidino-2-phenylindole. C, three-dimensional reconstruction of a z-stack showing that the TDP-43 immunoreactivity (green) is predominately outside the nucleus (blue). D, quantitative assessment of the number of cells with cytosolic TDP-43 staining after C199-TDP expression. *, p < 0.01 as determined by t test analysis. E, representative Western blot of the nuclear fraction of cells transfected with C199-TDP, further confirming the decrease in endogenous TDP-43 levels in the nucleus. F, quantitative assessment of the Western blots shows an ~60% decrease in nuclear steady-state levels of TDP-43 after C199-TDP transfection. *, p < 0.01 as determined by t test analysis.

Notably, we found that the accumulation of cytoplasmic TDP-43 was associated with a decrease in endogenous nuclear TDP-43 immunoreactivity (Fig. 3B, arrowhead). To better quantify these changes, we directly measured TDP-43 levels in nuclear fractions of C199-TDP-transfected and control cells. Although the cytoplasmic levels of TDP-43 did not change after expression of C199-TDP (data not shown), we found an ~60% decrease in nuclear TDP-43 steady-state levels (Fig. 3, E and F). These data suggest that the overexpression of C199-TDP is sufficient to cause cytoplasmic TDP-43 accumulation, which leads to a redistribution of endogenous TDP-43 from the nucleus to the cytoplasm. This is directly relevant to FTLD-U and ALS, as it has been shown that, in these diseases, there is a significant reduction in the levels of nuclear TDP-43 (8).

Rapamycin Reduces TDP-43 Mislocalization

Taken together, the data presented here provide compelling evidence for a role of autophagy in the turnover of C-terminal fragments of TDP-43, which are sufficient to initiate TDP-43 aggregation. We next sought to determine the effect of increasing autophagy on TDP-43 aggregation.

Twenty-four hours after being transfected with C199-TDP, cells were treated for 24 h with rapamycin (0.5 μg/ml). Rapamycin is a selective inhibitor of mTOR, a negative regulator of autophagy. We found that, after 24 h of rapamycin treatment, the LC3-II/LC3-I ratio was significantly increased in the rapamycin-treated cells compared with control cells, indicating an increase in autophagosomal formation (Fig. 4, A and B). Phospho-p70^S6K is a mitogen-activated Ser/Thr protein kinase whose activity is directly controlled by mTOR (41). Indeed, mTOR-mediated phosphorylation of p70^S6K at Thr³⁸⁹ closely correlates with p70^S6K activity in vivo (42), and it is normally used to infer mTOR activity (43). To determine whether the increase in autophagy after rapamycin treatment was mediated by mTOR, we measured the levels of phospho-p70^S6K and total p70^S6K and found that the ratio of phospho-p70^S6K to total p70^S6K was significantly reduced in cells treated with rapamycin, indicating a decrease in mTOR activity (Fig. 4, A and C). Taken together, these data show that rapamycin administration increases autophagy via an mTOR-mediated mechanism.

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FIGURE 4.

Rapamycin administration increases autophagy by decreasing mTOR activity. After transfection with C199-TDP, cells were treated with rapamycin or vehicle as described under “Experimental Procedures.” A, representative Western blots probed with LC3 (to measure autophagy induction) and phospho-p70^S6K and total p70^S6K (to measure mTOR activity). β-Actin was used as a loading control. B, quantitative assessment of the Western blots shows that, after rapamycin administration, there was a significant increase in the LC3-II/LC3-I ratio, which indicates an increase in autophagy induction. p = 0.0250 as determined by t test analysis. C, quantitative assessment of the Western blots shows that, after rapamycin administration, there was a significant decrease in p70^S6K, which indicates a decrease in mTOR activity. p = 0.0343 as determined by t test analysis.

We next determined the effect of the rapamycin-mediated increase in autophagy on TDP-43 mislocalization. Cells transfected with C199-TDP and treated with rapamycin or vehicle were stained with a TDP-43-specific antibody. We found a significant decrease in the number of cells with prominent cytoplasmic TDP immunoreactivity (Fig. 5, A and B). The reduction in cytosolic TDP-43 immunoreactivity was associated with a reduction in C199-TDP steady-state levels as measured by Western blotting (Fig. 5C). Notably, after rapamycin administration, we could not detect TDP-43 low molecular mass species (Fig. 5C).

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FIGURE 5.

Rapamycin reduces cytosolic TDP-43 immunoreactivity. Cells transfected with C199-TDP were treated with rapamycin or vehicle as described under “Experimental Procedures.” A, representative microphotographs of cells stained with 2E2-D3. Remarkably, rapamycin administration significantly reduced TDP-43 mislocalization as denoted by the increased TDP-43 immunoreactivity (green) in the nucleus (blue). B, quantitative assessment of the number of cells showing cytosolic TDP-43 immunoreactivity after C199-TDP expression. Rapamycin reduced the number of cells showing cytosolic TDP-43 immunoreactivity by ~60%. p = 0.0016 as determined by t test analysis. C, representative Western blot probed with anti-TDP-43 monoclonal antibody 2E2-D3 showing a significant decrease in the steady-state levels of the C-terminal fragments of TDP-43. D, representative Western blot after pulse-chase experiments. After being pulse-labeled for 2 h, cells were chased for the indicated time points. E and F, quantitative assessment of the FL-TDP and C199-TDP levels after the pulse-chase experiments indicates that the FL-TDP levels decreased similarly in the control (CTL) and rapamycin-treated cells (p > 0.05). In contrast, C199-TDP levels were significantly lower in rapamycin-treated cells compared with control cells at 6 and 24 h, indicating that rapamycin facilitates the turnover of C199-TDP. *, p < 0.01; **, p < 0.001 as determined using one-way analysis of variance, followed by the post hoc Bonferroni test to determine individual differences in groups. DAPI, 4′,6-diamidino-2-phenylindole; AU, arbitrary units.

To better understand the effects of rapamycin on TDP-43 metabolism, N2A cells stably transfected with C199-TDP were pulse-labeled with [³⁵S]methionine for 2 h and chased in excess amounts of unlabeled methionine with or without rapamycin. Subsequently, the cell lysate was immunoprecipitated with a polyclonal antibody raised against TDP-43. Although rapamycin had no effects on FL-TDP, these experiments showed that rapamycin significantly facilitated the turnover of the ~25-kDa C-terminal fragment of TDP-43 (Fig. 5, D and F). Taken together, these results show the involvement of the autophagic system in TDP-43 metabolism and suggest a potential beneficial effect for rapamycin.