Introduction
Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders. Despite the fact that FTD presents with changes in behavior, personality and language and ALS is a motor neuron disease which leads to progressive paralysis, there is genetic, neuropathological and clinical overlap between them. Not only do FTD and ALS frequently occur in the same family, many ALS patients develop FTD-like cognitive and behavioral impairments [
22,
39,
54], and as many as half of FTD patients develop motor neuron dysfunction [
39]. Neuropathologically, neuronal and glial inclusions of TDP-43 are found in most ALS cases, as well as in the most common pathological subtype of FTD [frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP)]. Because of this overlap, ALS and FTLD-TDP are considered part of a disease spectrum. This concept was recently reinforced with the discovery that a G
4C
2·G
2C
4 repeat expansion in a non-coding region of the
C9ORF72 gene is the most common genetic cause of ALS and FTLD-TDP [
14,
41,
58].
How the repeat expansion in
C9ORF72 causes “c9FTD/ALS” is not yet definitively known, but many advances have been made since the discovery of this mutation in 2011 (see [
21] for review). Potential mechanisms include loss of C9ORF72 function due to epigenetic changes resulting in decreased
C9ORF72 mRNA expression [
5,
73]. In addition, repeat-containing RNAs bidirectionally transcribed from the expanded repeat are thought to contribute to disease pathogenesis. The binding of these transcripts by various RNA-binding proteins (RBPs) may impair the ability of these RBPs to interact with their respective RNA targets. Because the repeat-containing transcripts form nuclear RNA foci, RBPs that bind these transcripts may be sequestered therein, also resulting in their loss of function. Furthermore, we and others have shown that transcripts of expanded G
4C
2 and G
2C
4 repeats undergo repeat-associated non-ATG (RAN) translation [
3,
20,
43,
44,
78], an unconventional mode of translation that occurs in the absence of an initiating ATG and in all possible reading frames, first described by Ranum and colleagues [
77]. RAN translation of expanded G
4C
2 and G
2C
4 repeats leads to the synthesis of 6 “c9RAN proteins” of repeating dipeptides: poly(GA) and poly(GR) from sense G
4C
2 repeats, poly(PR) and poly(PA) from antisense G
2C
4 repeats, and poly(GP) proteins from both sense and antisense transcripts.
Neuronal inclusions of c9RAN proteins are now considered a hallmark of c9FTD/ALS. While this implicates RAN translation as a mechanism of disease, confirmatory data are lacking. The Ranum group has shown that poly(PR) and poly(GP) proteins induce cellular toxicity in cultured cells independently of the accumulation of RNA foci [
78], suggesting that c9RAN protein expression may indeed be detrimental. However, given that inclusions of poly(GP) proteins are present in some, but not all, affected regions of the central nervous system (CNS) in c9FTD/ALS [
3,
20], and a recent study showing that poly(GA) pathology, unlike TDP-43 pathology, does not correlate with the degree of neurodegeneration in c9FTD/ALS [
40], put into question the contribution of c9RAN proteins to disease pathogenesis. Conversely, the discovery of a c9FTD kindred with early intellectual disability and extensive poly(GA) inclusions but little, if any, TDP-43 pathology [
56], provides compelling evidence that c9RAN proteins, or at least poly(GA) proteins, are harmful. Like poly(GP) inclusions, inclusions of poly(GA) appear to be abundant in c9FTD/ALS [
37,
40,
43,
44,
56], perhaps because of the hydrophobic nature of the protein. Using various models, the present study thus sought to evaluate the neurotoxic potential of poly(GA) protein expression and aggregation, as well as the mechanism(s) driving this toxicity.
Materials and methods
Generation of plasmids
To generate expression vectors for GFP-(GA)
50, GFP-(GP)
47, GFP-(GR)
50, GFP-(PR)
50 or GFP-(PA)
50, gene fragments containing individual dipeptide repeats (Table
1) were synthesized by GeneArt and ligated to the HindIII and BamHI restriction sites of a pEGFP-C1 vector (Clontech Laboratories) in frame with the EGFP coding sequence. To generate the AAV1-GFP-(GA)
50 expression vector, the EGFP coding sequence with restriction sites identical to those in pEGFP-C1 and containing a stop codon in each frame downstream of the multiple cloning site was cloned into the AAV expression vector pAM/CBA-pl-WPRE-BGH (“pAAV”). Gene fragments from the gene synthesis were cloned into the HindIII and BamHI restriction sites of the pAAV EGFP fusion vector in frame with the EGFP coding sequence. To generate the GST-(GA)
50 vector, PCR was performed to synthesize cDNA of a (GA)
50 fragment, which was then inserted into a pGEX-6P-1 vector (GE Healthcare) using BamHI and XhoI cloning sites. To generate the ATG-(GA)
50-V5 vector, cDNA of the (GA)
50 fragment with an ATG start codon was inserted into a pcDNA6-V5-His vector (Life Technologies) using HindIII and XhoI cloning sites. To generate the GFP-c9(GA)
50 expression vector, a gene fragment containing 50 pathological G
4C
2 repeats was produced as previously described [
33], and then ligated to EcoRV restriction sites of a pcDNA6-V5-His vector (Life Technologies). The GFP sequence was inserted upstream of (G
4C
2)
50 using HindIII and EcoRI cloning sites to drive protein expression in the GA reading frame. To generate pAG3-6 × stops-(±)ATG-(GA)
50-3T vectors, cDNA of the (GA)
50 fragment with or without an ATG start codon was inserted in a pAG3-6 × stops-3T vector between the 6 stop codons and the 3 tags (Online Resource 4a). The sequence of all plasmids was verified by sequence analysis.
Table 1
Synthetic cDNA sequences used for expression vectors
Purification of recombinant proteins and generation of anti-poly(GA) antibody
GST or GST-(GA)50 plasmids were used for transformation in Rosetta™(DE3)pLysS competent cells (EMD4Biosciences). To induce expression of recombinant proteins, bacteria were cultured overnight at 16 °C in the presence of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). After centrifugation, the bacteria pellet was washed with phosphate-buffered saline (PBS), and then lysed on ice for 30 min with PBS containing 1 % Triton X-100 (PBST). After sonication, the lysates were centrifuged at 18,000g for 30 min. The resulting supernatant was applied to a Glutathione Sepharose 4B column (GE Healthcare). After washing the column with PBST, the recombinant proteins were eluted from the column using Tris–HCl (50 mM, pH 8.0) containing 20 mM reduced glutathione. Afterwards, the solution was dialyzed with 50 mM Tris–HCl (50 mM, pH 8.0) to remove glutathione, and then concentrated. Recombinant GST-(GA)50 protein was used as the antigen to produce rabbit polyclonal antibodies. Pre-immune serum from rabbits was tested against brain tissue from c9FTD/ALS cases by immunohistochemistry and confirmed negative. Antiserum was used directly for Western blot and immunohistochemistry studies.
Immunohistochemistry and semi-quantitative analysis of poly(GA) inclusions in c9FTD/ALS
Neuropathological assessment was performed on 10 cases with the expanded
C9ORF72 hexanucleotide repeat. These cases were neuropathologically diagnosed as frontotemporal lobar degeneration (FTLD;
N = 4), amyotrophic lateral sclerosis (ALS;
N = 4), or both (FTLD–MND;
N = 2). Four regions of formalin-fixed paraffin-embedded tissue from the left hemi-brain were selected: the midfrontal gyrus of the frontal lobe, the posterior hippocampus at the level of the lateral geniculate nucleus (including occipitotemporal gyrus), the thalamus at the level of the subthalamic nucleus, and the cerebellum with the dentate nucleus. Tissue was cut into 6-µm-thick sections, mounted on glass slides, and dried overnight. Slides were subsequently deparaffinized and immunohistochemistry was performed using 30 min of antigen retrieval (steam), Dako EnVision+ reagents and Autostainer (DAKO), and the anti-poly(GA) rabbit polyclonal antibody (1:50,000). Following immunohistochemistry, all slides were counterstained with Lerner’s hematoxylin, dehydrated, and coverslipped. Slides were analyzed semi-quantitatively (Table
2) on an Olympus BX40 microscope (Olympus America). Neuropathologic burden was graded on a 4-point scale: sparse (±), mild (+), moderate (++), and severe (+++). In densely neuronal-populated regions, such as the internal granule cell layer of the cerebellum and the dentate fascia of the hippocampus, the pathologic grade was validated using averaged counts in 40× and 20× magnification microscopic fields, respectively. Slides were imaged using a Zeiss AxioImager Z1 microscope (Carl Zeiss Microscopy).
Table 2
Poly(GA) neuropathology in c9FTD/ALS cases
FTLD | 1 | ++ | ++ | ++ | ++ | ++ | ++ | +++ |
FTLD | 2 | + | + | ++ | ++ | ± | + | ++ |
FTLD | 3 | + | + | + | + | ++ | ++ | + |
FTLD | 4 | ++ | ++ | ++ | + | ++ | + | +++ |
FTLD–MND | 5 | ++ | ++ | +++ | +++ | + | ++ | +++ |
FTLD–MND | 6 | + | + | + | ++ | ++ | ± | ++ |
ALS | 7 | ++ | + | ++ | ++ | ++ | + | ** |
ALS | 8 | +++ | + | ++ | ++ | + | ++ | +++ |
ALS | 9 | ++ | ++ | ++ | ++ | ± | ++ | +++ |
ALS | 10 | ++ | ++ | ++ | ++ | + | ++ | ++ |
Electron microscopy (EM) and immuno-electron microscopy (immuno-EM)
To examine the filamentous structure of recombinant GST-(GA)
50 proteins, recombinant GST or GST-(GA)
50 proteins were diluted to 1 µg/µl in 20 mM Tris–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl
2 in a final volume of 30 µl. The samples were incubated at 30 °C for 6 h, and then diluted to 0.1 µg/µl by reaction buffer and loaded onto grids for regular EM analysis. For immuno-EM analysis, mouse monoclonal anti-GST antibody (1:20, Thermo Scientific) was used as primary antibody, and goat anti-mouse IgG conjugated with 6 nm colloidal gold particles (1:20, Jackson ImmunoResearch Laboratories) was used as the secondary antibody. To examine the filamentous structure of poly(GA) proteins in c9FTD/ALS patients, small pieces (1.5 × 1.5 × 1 mm) of cerebellar folia or hippocampus from formalin-fixed brains were dissected and processed for routine electron microscopy (EM) or post-embedding immunogold EM as previously described [
34]. Rabbit polyclonal anti-poly(GA) antibody (1:50) was used as a primary antibody and goat anti-rabbit IgG conjugated with 18 nm colloidal gold particles (1:20, Jackson ImmunoResearch Laboratories) was used as the secondary antibody. Thin sections stained with uranyl acetate and lead citrate were examined with a Philips 208S electron microscope (FEI) fitted with a Gatan 831 Orius CCD camera (Gatan). Digital images were processed with Adobe Photoshop CS5 software.
Preparation of urea fractions and dot blot analysis of frozen cerebellar tissue
The urea fraction of human tissues was prepared as previously described [
46,
75]. In brief, ~100 mg frozen postmortem cerebellar tissue from carriers and non-carriers of the
C9ORF72 repeat expansion was subjected to a sequential extraction protocol using low-salt buffer, high salt–Triton X-100 buffer, myelin floatation buffer, and sarkosyl buffer. Sarkosyl-insoluble material was finally extracted in urea buffer and saved as the urea fraction. For dot blots, urea fractions (2 µl per sample) were dotted directly onto nitrocellulose membrane. After incubation at 37 °C for 30 min, the membrane was blocked with 5 % nonfat dry milk in Tris-buffered saline containing 0.1 % Triton X-100 (TBST) for 1 h, then incubated with rabbit polyclonal anti-poly(GA) antibody (1:1000) overnight at 4 °C. The membrane was washed in TBST, and then incubated with donkey anti-rabbit IgG conjugated to horseradish peroxidase (1:5000, Jackson ImmunoResearch Laboratories) for 1 h. Protein expression was visualized by enhanced chemiluminescence treatment and exposure to a film.
Preparation of cell lysates
HEK293T cells grown in 6-well plates were transfected for 48 h with 1 µg of an expression vector [GFP or GFP-(GA)50] or 5 µg of an expression vector [ATG-(GA)50-V5, ATG-(GA)100-V5, ATG-(GA)50-3T or (GA)50-3T] using Lipofectamine 2000 (Life Technologies). To prepare Triton X-100 soluble and insoluble fractions, cell pellets expressing GFP or GFP-(GA)50 were lysed in Co-IP buffer (50 mM Tris–HCl, pH 7.4, 300 mM NaCl, 1 % Triton X-100, 5 mM EDTA) containing PMSF as well as protease and phosphatase inhibitors. Lysates were sonicated on ice, and then centrifuged at 16,000g for 20 min. Supernatants were saved as Triton X-100-soluble fractions. The insoluble pellets were dissolved in Co-IP buffer plus 2 % SDS, PMSF, and both a protease and phosphatase inhibitor mixture. After sonication, lysates were centrifuged at 16,000g for 20 min, and these supernatants were saved as Triton X-100-insoluble fractions. To prepare total cell lysates, pellets from cells transfected with ATG-(GA)50-V5, ATG-(GA)100-V5, ATG-(GA)50-3T or (GA)50-3T constructs were lysed in Co-IP buffer plus 2 % SDS, PMSF, and both protease and phosphatase inhibitor mixture. After sonication, lysates were centrifuged at 16,000g for 20 min. The protein concentration of supernatants was determined by BCA assay (Thermo Scientific) prior to Western blot analysis.
Fluorescence in situ hybridization (FISH)
To examine RNA foci, cells were subjected to FISH. In brief, HEK293T cells grown on glass coverslips in 24-well plates were transfected with 0.5 µg GFP-(GA)50 or GFP-c9(GA)50 expression vectors made, respectively, with a synthetic sequence [(GGXGCX)50; GFP-(GA)50] or a pathological sequence [(GGGGCC)50; GFP-c9(GA)50]. After 24 h, cells were fixed in 4 % paraformaldehyde for 20 min, permeabilized in DEPC-treated PBS-0.2 % Triton X-100 for 10 min, and washed 3 times with DEPC-treated PBS. Cells were hybridized with denatured Cy3-conjugated probe (5′-Cy3/GGCUCCAGCACCAGCACCUGCGCC-3′, 2 ng/µl) for the synthetic sequence, or (5′-Cy3/GGCCCCGGCCCCGGCCCCGGCCCC-3′, 2 ng/µl) for the pathological sequence, overnight at 37 °C. The composition of the hybridization buffer was 50 % formamide, 10 % dextran sulfate, 0.1 mg/ml yeast tRNA, 2 × SSC, 50 mM sodium phosphate. Cells were then washed once with 50 % formamide/1 × SSC for 30 min at 37°C, and twice with DEPC-PBS at room temperature for 5 min. Then, the cells were counterstained with Hoechst 33258 (1 µg/ml, Life Technologies). Immunostained cells were visualized using a Zeiss LSM 510 META confocal microscope.
Cell culture, immunofluorescence staining and quantification of activated caspase-3-positive cells
HEK293T cells grown on glass coverslips in 24-well plates were transfected with 0.5 µg of an expression vector [GFP, GFP-(GA)5, GFP-(GA)50, ATG-(GA)50-V5, ATG-(GA)100-V5, ATG-(GA)50-3T or (GA)50-3T] using Lipofectamine 2000 (Life Technologies). After 48 h, media was collected for analysis using an LDH assay (Promega) to assess cell toxicity. In addition, cells were fixed with 4 % paraformaldehyde in PBS for 15 min, and then permeabilized with PBS–0.5 % Triton X-100 for 10 min. To examine caspase-3 activation, cells were blocked with 5 % nonfat milk for 1 h at room temperature, then incubated overnight at 4 °C with rabbit polyclonal anti-activated caspase-3 antibody (9661, 1:250, Cell signaling) and mouse monoclonal anti-HA antibody (clone 3F10, 1:1000, Roche; used for cells expressing HA-tagged proteins) or mouse monoclonal anti-V5 antibody (R960-25, 1:2000, Life Technologies; used for cells expressing V5-tagged proteins). To determine whether poly(GA) inclusions are ubiquitin- and p62-positive, cells expressing ATG-(GA)100-V5 were incubated with rabbit polyclonal anti-V5 antibody (V8137, 1:2000, Sigma) and mouse monoclonal anti-ubiquitin (clone Ubi-1, 1:100, EMD Millipore) or mouse monoclonal anti-p62 (610833, 1:100, BD Biosciences) antibodies. To examine the ER-Golgi distribution of GFP-(GA)50 inclusions, cells were subjected to immunofluorescence staining using rabbit polyclonal anti-giantin antibody (ab24586, 1:250, Abcam) or mouse monoclonal anti-KDEL antibody (10C3, 1:500, StressGen). After washing, cells were incubated with the corresponding Alexa Fluor 488 donkey anti-mouse and Alexa 568-conjugated donkey anti-rabbit secondary antibody (1:500, Molecular Probes) at room temperature for 2 h. Hoechst 33258 (1 µg/ml, Life Technologies) was used to stain cellular nuclei. Images were obtained on a Zeiss LSM 510 META confocal microscope. To quantify the number of cells positive for activated caspase-3 staining, coverslips mounted on slides were scanned by Aperio ScanScope. Sixteen fields were randomly selected under 20× magnification. For each field, the number of activated caspase-3-positive cells and the number of GFP-positive cells were counted in a blinded fashion using MetaMorph software. These counts were used to calculate the average percentage of activated caspase-3-positive cells in cells expressing GFP, GFP-(GA)5 or GFP-(GA)50.
Primary neuronal culture preparation, quantification of neurite outgrowth and immunofluorescence staining
Primary neuronal cultures were prepared as previously described [
76]. In brief, the cortex from embryonic day 18 (E18) mice was dissected and digested. Following centrifugation to collect the cell pellet, cells were resuspended in Neurobasal A (Life Technologies) supplemented with B27, GMAX, gentamicin and bFGF (Life Technologies). Neurons were seeded at a density of 9 × 10
3 cells/well in 96-well plates, 2 × 10
4 cells/coverslip in 24-well plates, or 7 × 10
5 cells/well in 6-well plates. At day in vitro 4, neurons were transduced to express GFP, GFP-(GA)
50, or ATG-(GA)
100-V5. The following numbers of rAAV1 genome particles were used for 96-, 24- and 6-well plates, respectively: 1 × 10
9, 1.2 × 10
9, and 1 × 10
10. Quantification of neurite outgrowth was performed as previously described [
19,
76], using neurons in 96-well plates immunostained 5 days post-transduction with mouse monoclonal anti-MAP2 antibody (M9942, 1:1000, Sigma) and rabbit polyclonal anti-V5 antibody (V8137, 1:2000, Sigma) (used for cells expressing V5-tagged proteins). The average total neurite length per neuron was calculated for all groups using counts obtained in a blinded fashion. Caspase-3 activation was evaluated using neurons on glass coverslips in 24-well plates. Seven days post-transduction, neurons were fixed and immunostained using a rabbit polyclonal anti-activated caspase-3 antibody (9661, 1:250, Cell signaling) and/or mouse monoclonal anti-V5 antibody (R960-25, 1:2000, Life Technologies). Western blot analysis and LDH studies were conducted using neurons grown in 6-well plates. Seven days post-transduction, media was collected for the LDH assay, and cells were lysed in Co-IP buffer with 2 % SDS, PMSF, as well as protease and phosphatase inhibitors. Lysates were sonicated and centrifuged at 16,000
g for 20 min. Supernatants were saved and the protein concentration was determined by BCA assay. Samples were analyzed by Western blot. Some cultures were exposed to different treatments. Non-transduced neurons were treated with tunicamycin (10 µg/µl, Sigma) or MG-132 (10 µM, EMD4Biosciences) at day in vitro 6, then harvested the next day. Neurons expressing GFP-(GA)
50 were treated with a fresh preparation of TUDCA (0.25 or 0.5 mM, EMD4Biosciences) 1 day after transduction or salubrinal (2.5 or 5.0 µM, Sigma) 3 days after transduction. Seven days after transduction, TUDCA- or salubrinal-treated neurons were harvested for analysis.
Proteasome activity assay
Proteasome activity assays were performed as previously described [
61]. In brief, pellets of neurons subjected to different treatments were lysed in 350 μl of assay buffer (10 mM Tris–HCl, 0.5 mM DDT, 5 mM ATP, 0.035 % SDS, 5 mM MgCl
2, pH 7.8). After homogenization, cell lysates were centrifuged at 1,000
g for 10 min. The supernatants were collected and the protein concentration was determined by Bradford assay (Thermo Scientific). Then, the proteasome substrate Suc-Leu-Leu-Val-Tyr-AMC was added to 300 μl of lysate at a final concentration of 40 μM. The reaction was started by incubating samples at 37°C for 30 min, and then terminated by adding 75 μl H
2O and 11.25 μl ethanol to quench the substrate. After centrifugation at 1,000
g for 3 min, 100 μl of each sample was added into a black 96-well plate in triplicate. Fluorescence was measured at 360 nm excitation and 465 nm emission. Data was normalized to the GFP control group.
Live cell imaging
HEK293T cells grown in 96-well plates were transfected with 0.1 µg of GFP-(GA)50 vector using Lipofectamine 2000 (Life Technologies). To monitor inclusion formation 12 h post-transduction, images of live cells were obtained every 5 min for 5 h using the BD pathway 855. The resulting images were combined to create the video shown in the Online Resources section.
Western blot analysis
Western blot analysis was performed as previously described [
75,
76]. In brief, samples were heated in Laemmli’s buffer, and equal amounts of protein were loaded into 10-well 10 % or 4–20 % Tris–glycine gels (Novex). After transfer, blots were blocked with 5 % nonfat dry milk in TBST for 1 h, then incubated with a rabbit polyclonal anti-GFP antibody (A-6455, 1:2000, Life Technologies), rabbit polyclonal anti-activated caspase-3 antibody (9661, 1:1000, Cell signaling), mouse monoclonal anti-V5 antibody (R960-25, 1:2000, Life Technologies), rabbit polyclonal anti-poly(GA) serum (1:4000), mouse monoclonal anti-GST antibody (MA4-004, 1:2000, Thermo Scientific), mouse monoclonal anti-HA antibody (clone 3F10, 1:1000, Roche), mouse monoclonal anti-Myc antibody (clone 9E10, 1:1000, Roche), mouse monoclonal anti-Flag antibody (F3165, 1:1000, Sigma), rabbit polyclonal anti-ubiquitin antibody (Z0458, 1:1000, Dako), mouse monoclonal anti-BIP antibody (610978, 1:1000, BD Biosciences), rabbit polyclonal anti-CHOP antibody (sc-575, 1:200, Santa Cruz Biotechnology), rabbit polyclonal anti-phospho-PERK (sc-32577, 1:500, Santa Cruz Biotechnology), rabbit polyclonal anti-PERK (sc-13073, 1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti-ATF6 (sc-22799, 1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti-phospho-eIF2α (9721, 1:500, Cell signaling) or mouse monoclonal GAPDH antibody (H86504 M, 1:10000, Meridian Life Science) overnight at 4°C. Membranes were washed in TBST and incubated with donkey anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (1:5000; Jackson ImmunoResearch) for 1 h. Protein expression was visualized by enhanced chemiluminescence treatment and exposure to film.
Total RNA was extracted from HEK293T cells, neurons or frozen postmortem frontal cortex tissues using the RNAeasy Plus Mini Kit (QIAGEN) as per the manufacturer’s instructions, combined with an in-column DNase I digestion step. RNA integrity was obtained using the Agilent 2100 Bioanalyzer (Agilent Technologies). cDNA was obtained after reverse-transcription polymerase chain reactions using 500–2000 ng of RNA with random primers and the High Capacity cDNA Transcription Kit (Applied Biosystems) as per the manufacturer’s instructions. To examine XBP1 splicing in neurons, 2 μl of cDNA was used in a 20 μl reaction according to the manufacturer’s protocol for a Taq PCR Core kit (Qiagen). The PCR primers for XBP1 and GAPDH were used as in previously described [
28]. The amplification conditions consisted of an initial denaturation step at 95 °C for 5 min; 10 cycles of 94 °C for 20 s, 65–55 °C touchdown for 20 s, and 72 °C for 30 s; 35 cycles of 94 °C for 20 s, 58 °C for 20 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min. The PCR products were run on 2 % agarose gels for 35 min at 135 V. The intensity of unspliced and spliced XBP1 bands was quantified by densitometric analysis using Fujifilm MultiGauge software, and then the percentage of unspliced or spliced XBP1 was calculated.
To quantify mRNA levels of GFP, GFP-(GA)
5 and GFP-(GA)
50 in HEK293T cells or neurons, quantitative PCR (qRT-PCR) was conducted in triplicate for all samples using SYBR green assay (Life Technologies) on an ABI Prism 7900HT Fast Real-Time PCR System (Applied Biosystems). The primers used were: GFP: 5′-GAAGCGCGATCACATGGT-3′ and 5′-CCATGCCGAGAGTGATCC-3′; GAPDH: 5′-CATGGCCTTCCGTGTTCCTA-3′ and 5′-CCTGCTTCACCACCTTCTTGAT-3′. The mRNA values of GFP, GFP-(GA)
5 and GFP-(GA)
50 were normalized to GAPDH values, an endogenous transcript control. To quantify ER stress markers in frontal cortex tissues from ALS patients with or without the
C9ORF72 repeat expansion (Table
3), qRT-PCR was performed using TaqMan assays (Life Technologies) for ATF4 (Hs00909569_g1), CHOP (Hs00358796_g1), or BIP (Hs00946350_g1), or a SYBR green assay for RPLP0 (primers: 5′-TCTACAACCCTGAAGTGCTTGAT-3′ and 5′-CAATCTGCAGACAGACACTGG-3′). The mRNA values of ATF4, CHOP, and BIP were normalized to RPLP0 values, an endogenous transcript control. Since it has been reported that GAPDH expression is altered in neurodegenerative diseases [
11], we evaluated several endogenous controls by qRT-PCR in the frontal cortex regions and RPLP0 was chosen because this transcript presented overall low C
t values and lowest C
t variation (unpublished data). In addition, RPLP0 has been used as a stable reference gene in previous studies [
1,
16,
31].
Table 3
ALS cases with or without the C9ORF72 repeat expansion used for qPCR analysis of ER stress markers
c9ALS | 7 | Y | M | 52 | 58 |
c9ALS | 9 | Y | F | n/a | 49 |
c9ALS | 10 | Y | F | n/a | 41 |
c9ALS | 11 | Y | F | 61 | 64 |
c9ALS | 12 | Y | M | 56 | 58 |
c9ALS | 13 | Y | M | 49 | 53 |
c9ALS | 14 | Y | F | n/a | 50 |
c9ALS | 15 | Y | F | n/a | 50 |
ALS | 16 | N | M | 45 | 50 |
ALS | 17 | N | M | 53 | 58 |
ALS | 18 | N | F | n/a | 53 |
ALS | 19 | N | M | n/a | 47 |
ALS | 20 | N | F | n/a | 60 |
ALS | 21 | N | F | n/a | 65 |
ALS | 22 | N | F | n/a | 69 |
ALS | 23 | N | F | n/a | 70 |
ALS | 24 | N | F | n/a | 72 |
ALS | 25 | N | F | n/a | 79 |
ALS | 26 | N | F | n/a | 80 |
Discussion
RAN translation is becoming a well-established phenomenon in many repeat expansion disorders [
3,
44,
65,
77]. Inclusions composed of RAN-translated proteins are now considered a neuropathological hallmark of c9FTD/ALS [
3,
20,
43,
44,
78], but the contribution of this unconventional form of translation to disease pathogenesis has so far been enigmatic. While each c9RAN protein may influence neuronal health differently, herein we provide evidence that poly(GA) proteins are neurotoxic and could thus be implicated in the neurodegenerative processes of c9FTD/ALS.
Using cultured cells as a model to express c9RAN proteins of 50 repeating dipeptides, we observed that poly(GA) proteins formed ubiquitin- and p62-positive cytoplasmic inclusions, recapitulating the poly(GA) pathology of c9FTD/ALS [
2,
44]. In contrast, GFP-(PA)
50 and GFP-(GP)
47 remained diffusely distributed, whereas GFP-(GR)
50 and GFP-(PR)
50 aggregated in the nucleus, perhaps as a result of repeat length and the fact that arginine (R)-rich regions can function as a nuclear localization signal. Because most poly(GR) and poly(PR) inclusions in c9FTD/ALS patients are cytoplasmic [
20,
43,
44,
78], the exclusively nuclear localization of GFP-(GR)
50 and GFP-(PR)
50 inclusions in cultured cells does not accurately reflect c9FTD/ALS neuropathology. These studies indicate that poly(GA) proteins are highly aggregation-prone compared to the other c9RAN proteins, and that, depending on the model, longer repeat lengths should be used to evaluate poly(GP), poly(GR), poly(PA), and poly(PR) aggregation and toxicity to better mimic c9FTD/ALS.
To study the neuropathologic profile of poly(GA) inclusions in c9FTD/ALS, we generated a novel anti-poly(GA) antibody using as the antigen-purified recombinant GST-(GA)
50, which formed high molecular weight material and filaments in vitro. Consistent with previous reports [
40,
44,
56], we found that poly(GA) proteins were highly insoluble in c9FTD/ALS brain tissue, and that anti-poly(GA) immunoreactive inclusions were abundant throughout the CNS. To our knowledge, we are the first to report that poly(GA) proteins form filamentous structures in c9FTD/ALS brain tissue. Similar filamentous structures were produced in experimental models; we show that poly(GA) proteins with as little as 50 dipeptide-repeats self-assembled and formed filaments in vitro, as well as in cultured cells and primary neurons. It was with some surprise that we found, using fluorescent microscopy of live cells, that poly(GA) proteins quickly (within mere minutes) converted from a diffuse distribution to compact, small aggregates. Perhaps this rapid transformation occurs when the concentration of poly(GA) proteins within a localized area meets a critical threshold. These small aggregates may then serve to seed the aggregation of soluble poly(GA) proteins, thus causing larger inclusions to form. Poly(GA) may similarly seed the aggregation of other proteins, including c9RAN proteins, given that poly(GA) proteins have been shown to co-localize with various c9RAN proteins in neuronal inclusions in c9FTD/ALS [
43,
44]. Like poly(GA) proteins, polyA proteins form high molecular weight species and inclusions in spinocerebellar ataxia type 8 (SCA8) [
77], further indicating that proteins with long stretches of hydrophobic residues are especially aggregation-prone.
Cultured cell and primary neuron models provide evidence that expression and aggregation of poly(GA) proteins cause cellular toxicity. Compared to cells expressing GFP-(GA)
5, which remained diffusely localized, there was enhanced cytotoxicity in cells expressing GFP-(GA)
50, which formed cytoplasmic inclusions. The majority of GFP-(GA)
50-expressing cells immunopositive for active caspase-3 contained such inclusions, suggesting that poly(GA) aggregation drives cell death. Nonetheless, it remains possible that soluble and insoluble poly(GA) oligomers also play a toxic role, as is speculated to be the case of inclusion-forming proteins, such as tau and TDP-43, in other neurodegenerative diseases. Whatever the neurotoxic poly(GA) species may be, our studies demonstrate that this c9RAN protein impairs function of the UPS in primary neurons. Compared to neurons expressing GFP alone, proteasome activity was significantly decreased in neurons expressing GFP-(GA)
50, and this was accompanied with a dramatic accumulation of ubiquitinated proteins. These findings are consistent with reports demonstrating that protein aggregates and/or oligomers can impair the UPS [
6,
7,
30,
35,
71].
ER-associated protein degradation (ERAD), which controls protein homeostasis in the ER, is dependent on proper UPS activity [
10,
12]; consequently, inhibition of the UPS impairs ERAD, leading to the accumulation of misfolded proteins in the ER and ER stress [
17,
32,
47,
50,
53,
74]. Under conditions of ER stress, BIP, a master regulator of the unfolded protein response (UPR) that normally binds to and inhibits activity of ER stress sensors, PERK, ATF6 and IRE1, dissociates from these proteins. This disassociation leads to the activation of different downstream signaling pathways such as PERK auto-phosphorylation, ATF6 cleavage and XBP-1 splicing [
25,
67]. Of note, the PERK–CHOP pathway was selectively activated in primary neurons expressing GFP-(GA)
50, as evidenced by increased levels of the ER sensor phospho-PERK, and its downstream target CHOP. Selective activation of the PERK–CHOP pathway was similarly observed in non-transduced neurons treated with the proteasome inhibitor, MG-132. Other similarities between GFP-(GA)
50 expression and MG-132-induced proteasome inhibition include the accumulation of ubiquitinated proteins, decreased proteasome activity, and caspase-3 activation. In contrast, treatment of neurons with tunicamycin, an ER stress inducer, activated both PERK–CHOP and IRE–XBP1 pathways without impairing proteasome activity. Together, our findings suggest that poly(GA) proteins indirectly induce ER stress through proteasome inhibition. Of importance, we show that mRNA levels of ER stress markers of the PERK–CHOP pathway, ATF4 and CHOP, are significantly increased in c9ALS cases compared to sporadic ALS cases without the
C9ORF72 repeat expansion. While no difference in BIP mRNA levels were observed, this may be due to the shared TDP-43 pathology between
C9ORF72-positive and -negative ALS, which has been associated with ER stress [
68,
70]. In addition to our findings using human postmortem samples, it is also worth noting that a recent study reported that iPSC-derived motor neurons from c9ALS patient, which contain RNA foci and c9RAN proteins but no TDP-43 pathology [
15], exhibit an increased vulnerability to tunicamycin [
23], supporting the association between the
C9ORF72 repeat expansion and ER stress. Future studies investigating whether, like poly(GA) proteins, RNA foci and other c9RAN proteins inhibit proteasome activity and cause ER stress are warranted.
ER stress activation of the PERK–CHOP pathway is well documented (see [
25,
67] for review). In brief, PERK is activated through auto-phosphorylation upon release from BIP. Upon activation, PERK phosphorylates eIF2α to attenuate global protein synthesis as a pro-survival mechanism. However, phosphorylation of eIF2α selectively allows the translation of ATF4 and CHOP, which induce cell death under prolonged ER stress. This can occur through multiple mechanisms that include expression of pro-apoptotic genes, promotion of protein synthesis, production of reactive oxygen species (ROS) and ATP depletion [
18,
24,
25,
42,
51,
63]. For example, ATF4 and CHOP up-regulate levels of GADD34, which forms a complex with PP1C to dephosphorylate eIF2α; the dephosphorylation of eIF2α in turn enhances protein synthesis, ATP depletion and ROS production, which consequently induce cell death [
24,
42]. The importance of this pathway is further validated by studies showing selective inhibitors of the GADD34–PP1C complex protect cells from ER stress-induced cell death [
9,
66]. Consistent with these findings, we show that salubrinal, a selective inhibitor of the GADD34–PP1C complex [
9,
36,
60], restored eIF2α phosphorylation, reduced ER stress and increased cell survival in neurons expressing poly(GA) proteins. In addition, TUDCA, an inhibitor of ER stress [
13,
38,
52,
72], blocked the increase in phospho-PERK and CHOP normally observed in poly(GA)-expressing neurons, and provided neuroprotection against poly(GA)-induced toxicity. Of interest, the levels of poly(GA) and ubiquitinated proteins were not themselves decreased after salubrinal and TUDCA treatment, suggesting that proteasome inhibition, the culprit responsible for inducing ER stress, remained present in these cells. This may explain why levels of BIP, which correlate with the amount of misfolded proteins in the ER, were not altered following treatment of GFP-(GA)
50-expressing cells with TUDCA. As a chemical chaperone, TUDCA may have bound to these misfolded proteins and caused the release of BIP, which then became free to interact with PERK and inhibit PERK auto-phosphorylation and activation. As a result, CHOP levels decreased as did caspase-3 activation. However, BIP levels remain unchanged because TUDCA did not eliminate the accumulation of misfolded proteins, but rather targeted downstream events only. Conversely, salubrinal treatment would have decreased protein translation by enhancing eIF2α phosphorylation, thus reducing the misfolded protein load in the ER. Consequently, this would cause a decrease in levels of BIP and phospho-PERK, and increase cell survival. That CHOP levels were not also decreased by salubrinal treatment may result from the increase in eIF2α phosphorylation, which regulates CHOP expression.
In summary, we have generated a novel poly(GA) antibody and confirmed that abundant poly(GA) neuronal inclusions are detected throughout the CNS of c9FTD/ALS cases. We show that poly(GA) proteins are highly aggregation-prone and form filamentous structures in experimental models and c9FTD/ALS brain tissue. Of particular importance, our data provide compelling evidence that poly(GA) proteins contribute to the neurodegeneration in c9FTD/ALS. The expression of poly(GA) proteins causes neurotoxicity; this toxicity occurs in the absence of RNA foci, and is associated with impairment of the UPS and induction of ER stress. ER stress is believed to play an important role in several neurodegenerative diseases, including sporadic ALS [
4,
26,
59,
69], and familiar ALS caused by mutations in Cu/Zn superoxide dismutase [
8,
29,
48,
55] or vesicle-associated membrane protein-associated protein B [
27,
45,
62]. Our data extend the list of diseases involving ER stress, and suggest that targeting the ER, using small molecules such as salubrinal and TUDCA, may be a promising therapeutic approach for c9FTD/ALS.