Introduction
An expanded GGGGCC hexanucleotide repeat within a non-coding region of the
C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two devastating multisystem neurodegenerative disorders with significant genetic, neuropathological, and clinical overlap [
11,
30]. FTD, a common form of early-onset dementia, is characterized clinically by abnormalities in behavior and language, whereas ALS is characterized by upper and lower motor neuron signs, which include weakness and muscle atrophy. FTD-like cognitive and behavioral impairments are also present in up to 50 % of ALS patients [
16,
19,
27], and as many as half of FTD patients develop motor neuron dysfunction (MND) reminiscent of ALS [
19].
The mechanisms by which the
C9ORF72 hexanucleotide repeat expansion causes “c9FTD/ALS” are not definitively known, but at least three pathogenic pathways may be at play. Several groups have shown that mRNA levels of certain
C9ORF72 variants are decreased in c9FTD/ALS [
11,
15,
25,
30], suggesting loss of C9ORF72 function as a potential neurotoxic mechanism. In addition, RNA transcripts containing the expanded repeat may cause neurodegeneration by two means: through their accumulation into discrete structures in the nucleus, termed RNA foci, and by serving as a template for the synthesis of aberrantly expressed, aggregation-prone proteins by repeat-associated non-ATG (RAN) translation. Indeed, (GGGGCC)
exp RNA foci are observed in c9FTD/ALS [
11], and the accumulation of neuronal inclusions composed of “c9RAN proteins” is a pathological hallmark of c9FTD/ALS [
3,
25].
RNA foci are thought to cause cellular toxicity by sequestering specific RNA-binding proteins in a sequence-dependent manner, consequently disrupting their function [
4]. In myotonic dystrophy type 1 (DM1), for instance, RNA foci formed of CAG·CTG repeat transcripts bind and inactivate the splicing factor muscleblind-like 1 protein (MBNL1) [
23,
32,
33]. This sequestration of MBNL1 results in the mis-splicing of a subset of pre-mRNA targets that account for some of the characteristic features of disease [
10,
20]. Transcripts of (GGGGCC)
exp that accumulate as nuclear foci in c9FTD/ALS may similarly sequester RNA-binding proteins and cause the misregulation of crucial downstream RNA targets leading to cellular dysfunction [
24,
29,
38].
In addition to foci formation, transcripts of expanded repeats may be susceptible to RAN translation, an unconventional mode of translation that occurs across expanded repeat tracts despite the absence of an initiating codon. First described by Ranum and colleagues for expanded trinucleotide CAG·CTG repeats [
39], transcripts of expanded CGG repeats [
34], and the aforementioned GGGGCC repeats [
3,
25] are now known to be RAN translated. Because RAN translation can occur in all possible reading frames, various products can be synthesized from a given transcript. Recently, we, as well as the Edbauer group and colleagues, independently reported that RAN translation of (GGGGCC)
exp RNA in c9FTD/ALS results in the production of poly(GP), poly(GA), and poly(GR) proteins [
3,
25]. The presence of neuronal inclusions composed of these c9RAN proteins throughout the central nervous system is now considered pathognomonic of c9FTD/ALS [
3,
25].
For many microsatellite expansion disorders, the expanded repeat is bidirectionally transcribed [
4]. Detectable expression of both sense and antisense transcripts containing the hexanucleotide repeat in
C9ORF72 patients indicates that bidirectional transcription also occurs in c9FTD/ALS [
25]. Consequently, not only do (GGGGCC)
exp transcripts form foci and undergo RAN translation, so too may (CCCCGG)
exp transcripts resulting from bidirectional transcription of the
C9ORF72 expanded repeat. The goal of this study was thus to examine whether (CCCCGG)
exp-derived RNA foci and c9RAN translation proteins are present in c9FTD/ALS.
Materials and methods
Secondary structure prediction and model building of CCCCGG repeats
Folding of the RNA sequence comprising CCCCGG repeats of 10 (60 bases), 50 (300 bases), and 200 (1,200 bases) was carried out as previously described to determine secondary structure motifs [
3]. The secondary structure prediction and modeling was built by examining the output from several RNA prediction packages: MFOLD, Sfold, Vienna RNA Package (RNAfold) [
12,
17,
18,
21,
22,
31,
40,
41]. MFOLD utilizes a minimum free energy RNA structure prediction algorithm, Sfold utilizes statistical sampling of all possible structures, while Vienna Package has several options, including an minimum free energy calculation. Equivalent structures were given from each package for the ten repeat cases. MFOLD was used as the primary secondary structure prediction package for consistency across all models. Free energies were calculated for each secondary structure prediction at the level of decomposition (base pairing) and global energy [
36]. MFOLD verifies each secondary structure prediction generated for valid structure [
36]. MFOLD parameters include the following: (1) linear RNA sequence; (2) zero constraints, forces, or prohibitions on all bases allowing maximal sampling; (3) folding temperature of 37 °C; (4) physiological ionic conditions; (5) structure draw mode: untangle with loop fix; and (6) the remainder of MFOLD settings were set to default.
Generation of antibodies
For each of the peptide antigens (C-Ahx-(PR)8-amide, C-Ahx-(GP)8-amide and C-Ahx-(PA)8-amide), two rabbits were immunized. Pre-immune serum from each rabbit was tested against peptide antigens and tissue from c9FTD/ALS cases by Western blot and immunohistochemistry, respectively, and confirmed negative. Antiserum was used directly or affinity purified before use.
Meso Scale Discovery immunoassays
Peptides diluted in Tris-buffered saline (TBS) were added to duplicate wells (35 μl/well) of a 96-well MSD assay plate at final concentrations 0.1 μg/well. Following overnight incubation at 4 °C, wells were washed with TBS containing 0.2 % Tween 20 (TBSTw), and blocked with TBSTw+3 % non-fat milk. Antibody solution (25 μl/well) containing the indicated anti-PR, anti-GP, anti-PA, anti-GA or anti-GR antibodies (1:1,000) and SULFO-TAG™-rabbit secondary antibody (0.5 μg/ml, in blocking buffer) was added. Following a 2-h incubation and final washes, antibody binding to immobilized peptides was evaluated by adding MSD Read Buffer and measuring light emission at 620 nm upon electrochemical stimulation using the MSD Sector Imager 2400.
Western blot analysis and immunofluorescence staining for antibody characterization
HEK293T cells were transfected with Lipofectamine™ 2000 with pEGFP-C1 vector only, or pEGFP-C1 (Clontech) plasmids into which oligonucleotides of five repeats of PR, GP, PA, GA or GR were inserted. For Western blotting, cell lysates collected 2 days post-transfection were resolved by 10 % Tris–Glycine SDS-PAGE (Invitrogen) and transferred to nitrocellulose membranes for probing with anti-PR, anti-GP, anti-PA, anti-GA or anti-GR, or with anti-GFP (Abcam, 1:2,000). For immunostaining, coverslips were fixed, permeabilized and blocked, then probed with the indicated antibodies followed by anti-rabbit-AF594 (Alexa Fluor) and Hoechst [
3].
Cloning of CCCCGG expression vectors
To generate the antisense (CCCCGG)2 and (CCCCGG)66 expression vectors, we first generated sense (GGGGCC)2 and (GGGGCC)66 expression vectors. Toward this end, genomic DNA from muscle or spleen from a C9ORF72 expanded repeat carrier was used as a template in a nested PCR strategy using ThermalAce DNA Polymerase (Invitrogen) to amplify the (GGGGCC)n repeat region, including 113 bp of 5′ and 99 bp of 3′ flanking sequence. The upstream primer used was 5′-AAGGAAGCTTAGTACTCGCTGAGGGTGAAC-3′; downstream primers used were 5′-GCTTGGATCCCCCACTCGCCACCGCCTG-3′ and 5′-GTCAGAGAAATGAGAGGGAAAG-3′. The PCR products were cloned into the pAG3 expression vector (kindly provided by Dr. T. Golde, University of Florida) using restriction sites HindIII and BamHI. The pAG3 expression vector has a pcDNA3.0 backbone and a CMV-enhanced chicken B-actin promoter. The clones containing (GGGGCC)2 or (GGGGCC)66 were screened by colony PCR, and further verified by hairpin sequence analysis. The plasmids of (GGGGCC)2 or (GGGGCC)66 were digested using HindIII, and then the overhangs were filled-in using DNA Polymerase I (Klenow) Fragment. After purification, the fragments were digested with BamHI. The inserts with (GGGGCC)2 or (GGGGCC)66, including the 5′ and 3′ flanking sequences, were re-cloned in the antisense orientation into a pAG3 expression vector using BamHI and EcoRV to generate the (CCCCGG)2 and (CCCCGG)66 expression constructs. The sequence of antisense vectors was verified by hairpin sequence analysis. Note that the DNA sequence of the CCCCGG repeat is provided in Online Resource 1, which also highlights the regions included in the generation of pAG3-(CCCCGG)2 and pAG3-(CCCCGG)66 expression vectors.
RNA fluorescence in situ hybridization of cultured cells expressing (CCCCGG)n expression vectors
Evaluation of foci formation in HeLa cells transfected with (CCCCGG)2 or (CCCCGG)66 expression vectors was carried out by RNA fluorescence in situ hybridization (FISH). In brief, cells grown on glass coverslips were transfected with 0.5 μg of the (CCCCGG)2 or (CCCCGG)66 constructs. After 36 h, cells were fixed and permeabilized in 4 % paraformaldehyde + 20 % acetic acid + 2 mM ribonucleoside vanadyl complex (Sigma) for 10 min at room temperature. Cells were then washed with phosphate buffered saline treated with diethylpyrocarbonate (DEPC–PBS), and hybridized with denatured Cy3-conjugated (GGGGCC)4 probe (2 ng/μl) in hybridization buffer (50 % formamide, 10 % dextran sulfate, 0.1 mg/ml yeast tRNA, 2X saline–sodium citrate buffer (SSC), 50 mM sodium phosphate buffer) overnight at 37 °C. After washing once with 40 % formamide/1XSSC for 30 min at 37 °C, and twice with DEPC–PBS for 5 min at room temperature, nuclei were counterstained with Hoechst 33258 (1 μg/ml, Invitrogen) prior to mounting coverslips. Images were obtained on a Zeiss LSM 510 META confocal microscope.
Western blot analysis of cultured cells expressing (CCCCGG)n expression vectors
To determine whether ectopic expression of expanded CCCCGG transcripts leads to RAN translation, HEK293T cells were transfected with 5 μg of the (CCCCGG)2 or (CCCCGG)66 constructs. After 36 h, cells were harvested and washed with ice-cold PBS (pH 7.4), then cell pellets were lysed in buffer (50 mM Tris–HCl, pH 7.4, 300 mM NaCl, 1 % Triton-X-100, 5 mM EDTA, 2 % sodium dodecyl sulfate (SDS), plus phenylmethylsulfonyl fluoride (PMSF) and both a protease and phosphatase inhibitor mixture). After centrifugation at 16,000g for 20 min at 4 °C, the supernatant was collected and protein concentration determined by BCA assay. For Western blot analysis, samples were prepared in Laemmli’s buffer, heated for 5 min at 95 °C, and equal amounts of protein (30 μg) were loaded into Novex® 10–20 % Tricine gels. After transfer, blots were blocked with 5 % non-fat dry milk in TBST for 1 h, and then incubated with the purified anti-PR, anti-GP or anti-PA (1:1,000), or mouse monoclonal GAPDH antibody (1:10,000, Biodesign) overnight at 4 °C. Membranes were washed then incubated with anti-species horseradish peroxidase-linked secondary antibodies (1:5,000; Jackson ImmunoResearch) for 1 h. Protein expression was visualized by enhanced chemiluminescence treatment and exposure to film.
Human case material
All cases examined in this study were selected from a series of autopsied brains submitted to the neuropathology laboratory at Mayo Clinic in Jacksonville. The sources of this case material include the Mayo Clinic Florida ALS Center (
n = 11), referral to the Parkinson disease brain bank (
n = 7), the State of Florida Alzheimer’s Disease Initiative (
n = 2), Florida Alzheimer’s Disease Research Center (
n = 1) and CurePSP/Society of Progressive Supranuclear Palsy brain bank (
n = 1). The presence or absence of
C9ORF72 repeat expansion was determined using frozen cerebellar tissue from the right hemibrain and a previously described repeat-primed polymerase chain reaction (PCR) method [
11]. In addition, repeat length was estimated using Southern blotting techniques as previously described [
35] (Table
1). In brief, 7–10 μg of high-quality genomic DNA extracted from frozen frontal cortex and cerebellum was digested with
XbaI, and electrophoresed in a 0.8 % agarose gel. DNA was then transferred to a positively charged nylon membrane (Roche), cross-linked, and subsequently hybridized with a DIG-labeled probe. Expansions were visualized with anti-DIG antibody (Roche) and CDP-star substrate (Roche) on X-ray film after multiple exposures. The most abundant expansion sizes were estimated using AlphaEaseFC (Alpha Innotech) based on their position relative to DNA molecular weight markers.
Table 1
C9ORF72 antisense transcript RAN translation cohort
1 | Y | FTLD-TDP | 66 | M | 16.0 | 12.5 | ± | + | +++ | ± | + | +++ |
2 | Y | FTLD-TDP | 71 | M | 25.2 | 11.4 | ± | ± | ++ | ± | ± | ++ |
3 | Y | FTLD-TDP | 86 | M | 50.0 | 17.5 | + | ± | +++ | ± | ± | +++ |
4 | Y | FTLD-MND | 68 | M | 25.6 | 12.8 | ± | ± | ++ | ± | ± | +++ |
5 | Y | FTLD-MND | 70 | M | 38.4 | 10.1 | + | + | +++ | + | + | +++ |
6 | Y | FTLD-MND | 61 | F | 35.6 | 13.7 | + | + | +++ | ± | ± | +++ |
7 | Y | ALS | 53 | M | 46.8 | 14.9 | + | + | +++ | + | + | +++ |
8 | Y | ALS | 49 | F | 23.8 | 10.0 | + | + | +++ | ± | ± | +++ |
9 | Y | ALS | 41 | F | 22.3 | 10.9 | + | + | +++ | + | + | +++ |
10 | N | FTLD-TDP | 88 | F | − | − | − | − | − | − | − | − |
11 | N | FTLD-TDP | 65 | M | − | − | − | − | − | − | − | − |
12 | N | FTLD-TDP | 83 | F | − | − | − | − | − | − | − | − |
13 | N | FTLD-MND | 64 | M | − | − | − | − | − | − | − | − |
14 | N | FTLD-MND | 72 | F | − | − | − | − | − | − | − | − |
15 | N | FTLD-MND | 66 | F | − | − | − | − | − | − | − | − |
16 | N | ALS | 60 | M | − | − | − | − | − | − | − | − |
17 | N | ALS | 61 | F | − | − | − | − | − | − | − | − |
18 | N | ALS | 53 | F | − | − | − | − | − | − | − | − |
19 | N | HD | 80 | F | − | − | − | − | − | − | − | − |
20 | N | HD | 70 | F | − | − | − | − | − | − | − | − |
21 | N | Kennedy’s | 80 | M | − | − | − | − | − | − | − | − |
22 | N | SCA3 | 53 | M | − | − | − | − | − | − | − | − |
Fluorescence in situ hybridization and immunofluorescence staining of human tissue
Formalin-fixed, paraffin-embedded frontal cortex, spinal cord, and cerebellum sections were cut at a 5-μm thickness and mounted on glass slides, then subjected to RNA FISH followed by immunofluorescence staining. For FISH, slides were deparaffinized and rehydrated, incubated with pepsin (4 mg/ml in 0.9 % NaCl, pH 1.5) for 20 min at 37 °C, rinsed in water, then immersed in ice-cold 20 % acetic acid for 90 s, prior to dehydration. A Cy3-tagged (GGGGCC)4 probe (IDT) which hybridizes to the expanded CCCCGG repeat was applied to the tissue, which was then sealed under a coverslip. Prior to use, the probe was diluted to 5 ng/μl in hybridization buffer (10 % dextran sulfate, 50 % formamide, 2XSSC, 50 mM sodium phosphate buffer, 10 ng/ml tRNA pH 7.0), then heated at 80 °C for 10 min, placed on ice for 5 min, then heated at 37 °C for 10 min. Alternatively, to detect foci formed of (GGGGCC)exp RNA, a TYE563-labeled LNA probe (5′TYE563-CCCGGCCCCGGCCCC-3′TYE563; Exiqon, Inc) was applied to the tissue. This probe was diluted to 0.4 ng/ml in hybridization buffer (10 % dextran sulfate, 50 % formamide, 2XSSC, 50 mM sodium phosphate buffer, 10 ng/ml tRNA, pH 7.0), then heated at 80 °C for 75 s, according the manufacturer’s instructions. Following a 2-day hybridization at 37 or 55 °C for the antisense and sense probe, respectively, coverslips were removed and slides were washed: once in 2X SSC, three times in 50 % formamide/2X SCC at 37 °C, and three times in 1X SSC at 37 °C. Slides were subsequently subjected to immunofluorescence staining: slides were blocked with DAKO Serum-Free Protein Block and then incubated with anti-GP (1:3,000), choline acetyltransferase antibody (ChAT; 1:200; Chemicon AB144P), microtubule-associated protein 2 antibody (MAP2; 1:750, clone AP-20) or glial fibrillary acidic protein antibody (GFAP; 1:500, clone GA-5) overnight at 4 °C. The following day, slides were incubated with an Alexa Fluor 488-conjugated secondary antibody (1:500, Molecular Probes) for 1.5 h at room temperature. Slides were then treated with a solution of Sudan Black for 2 min to block auto-fluorescence, and coverslipped using Vectashield-DAPI mounting medium (Vector Laboratories).
Imaging was performed using a Zeiss Axio Imager Z1 microscope to visualize foci and poly(GP) inclusions. Although it is difficult to distinguish binding of the probe to an individual RNA transcript given that the fluorescence resulting thereof is not sufficiently strong to yield a signal above background, the FISH method employed is well suited to examine nuclear foci. The presence of multiple labeled transcripts in a compact location, as is the case for foci, results in a highly distinct, punctate fluorescent signal much brighter than background. Note that, to validate specificity of the probes targeting GGGGCC and CCCCGG RNA, FISH was carried out on frontal cortex and cerebellar tissue from ALS, frontotemporal lobar degeneration (FTLD), and FTLD-MND patients with normal C9ORF72 repeat length. In addition, a (CAGG)6 probe targeting the CCTG repeat was tested and shown also to be negative.
To determine what percentage of affected cells (i.e., cells having either foci or a poly(GP) inclusion) have both nuclear foci and poly(GP) pathology, we analyzed frontal cortex and cerebellar sections of four c9FTD/ALS cases co-stained for either sense or antisense foci and poly(GP) inclusions. For each section, we examined 25 cells with inclusions by fluorescence microscopy, and then changed the excitation filter to determine whether they also had foci; we then examined 25 cells with foci and determined whether they had inclusions. In this manner, a total of 50 cells were examined per section. For each group (Group 1—frontal cortex probed for antisense foci and poly(GP) inclusions; Group 2—cerebellum probed for antisense foci and poly(GP) inclusions; Group 3—frontal cortex probed for sense foci and poly(GP) inclusions; Group 4—cerebellum probed for sense foci and poly(GP) inclusions), the percentage of cells having both foci and inclusions was calculated and compared by two-way ANOVA. In addition, we compared the frequency of sense and antisense foci in the frontal cortex of the four examined cases. For each section, the number of cells with foci and the number of total cells were counted in 12 randomly selected, non-overlapping fields from layers I–III. The average percentage of cells with antisense foci among the four cases was compared to the average percentage of cells with sense foci by paired, two-tailed t test.
Immunohistochemistry
For immunohistochemical analysis, 9 cases exhibiting an expanded
C9ORF72 repeat (3 FTLD-TDP, 3 FTLD-MND, 3 ALS) were matched with 13 negative control cases that lacked the expanded repeat, including 3 FTLD-TDP, 3 FTLD-MND, 3 ALS, and 4 cases with CAG repeat disorders, including 2 cases with Huntington’s disease, 1 case with Kennedy’s disease, and 1 case with spinocerebellar ataxia type 3 (Table
1). For the 9 c9FTD/ALS cases and the 9 FTD/ALS matched controls, immunohistochemistry was performed on formalin-fixed paraffin-embedded tissue from the motor cortex, hippocampus (including temporal cortex), basal forebrain (including amygdala), thalamus (at the level of the subthalamic nucleus), medulla, and cerebellum. For the four trinucleotide repeat disorders, sections from the basal forebrain (2 Huntington’s disease cases), medulla (Kennedy’s disease case) or pons (spinocerebellar ataxia type 3 case) were used. Five-micron-thick tissue sections were cut from paraffin blocks, mounted on charged glass slides, and allowed to dry overnight at 65 °C. The following day, slides were deparaffinized and rehydrated in serial washes in xylene and alcohol before steaming the slides for 30 min in 1X Tris–EDTA (pH 9) buffer solution. Immunohistochemistry was performed using the Dako Autostainer and the Dako EnVision™ + Rabbit (DAB) kits. Immunostaining was performed with the following antibodies: anti-PA (Rb8604, 1:2,500), anti-GP (Rb7379, 1:10,000), and anti-PR (Rb8736, affinity purified, 1:100). Following immunohistochemistry, slides were counterstained with Lerner’s hematoxylin, dehydrated, and coverslipped. All imaging was conducted using the Zeiss Axio Imager Z1 microscope.
Discussion
The discovery of new neuropathologic features specific to c9FTD/ALS, namely the formation of RNA foci and the production of c9RAN proteins resulting from the synthesis of antisense transcripts of the expanded C9ORF72 repeat, provides additional insight into the pathobiology of c9FTD/ALS. Through the production of both sense and antisense expanded repeat RNA and five distinct c9RAN proteins, the repeat expansion leads to the production of seven potentially toxic biomolecules.
To determine whether antisense transcripts of the expanded
C9ORF72 repeat are RAN translated, we generated novel rabbit polyclonal antibodies for the detection of poly(GP), poly(PR) and poly(PA) proteins. Examination of RAN translation in cultured cell models showed that poly(GP) and poly(PR) proteins were synthesized in (CCCCGG)
66-expressing cells, but not in cells expressing only two CCCCGG repeats. That poly(PA) proteins were not detected in (CCCCGG)
66-expressing cells may be because a crucial upstream sequence necessary for translation of poly(PA) peptides is missing from the (CCCCGG)
66 expression vector. Alternatively, it may be due to the fact that RAN translation is repeat length-dependent, with different reading frames having different length thresholds [
9,
39]. Nonetheless, our immunohistochemical analysis of c9RAN proteins in human tissue indicates either this sequence is present, or the repeat length threshold is met, in c9FTD/ALS patients.
Because poly(GP) proteins can be synthesized from sense and antisense transcripts of the expanded C9ORF72 repeat, their exact origin is not definitely known. Yet, the presence of poly(PA) and poly(PR) neuronal inclusions in post-mortem c9FTD/ALS brain tissue is indicative of RAN translation of the antisense transcript. These inclusions are specific to c9FTD/ALS cases, not being found in matched FTD/ALS controls lacking the C9ORF72 expanded repeat, or in other repeat disorders. We did note a difference between poly(PA) and poly(PR) pathology in comparison to poly(GP) pathology, the latter being markedly more frequent. For example, poly(GP) pathology is extensive in granule cells and primary neurons of the cerebellum, but cerebellar poly(PA) and poly(PR) inclusions are sparse in these same populations.
While we cannot rule-out the possibility that poly(GP) inclusions appear more abundant because of differences in antibody affinities, their increased frequency may be due to the fact that poly(GP) proteins are synthesized from both sense and antisense transcripts. In addition, the antisense transcript may be less efficiently translated than the sense transcript. CCCCGG repeats are predicted to fold into an imperfect hairpin akin to what we have previously shown for GGGGCC repeats [
3]. However, compared to the GGGGCC repeat, which has an optimal organization to maximize base pairing, the CCCCGG repeat has fewer base pairs and more frequent sets of four unpaired C nucleotides within the stem, and thus relies more on base stacking effects. While the stability of both CCCCGG and GGGGCC repeat transcripts increases with increasing repeat length, the CCCCGG repeat is predicted to be less stable than its GGGGCC counterpart, which may decrease its susceptibility to RAN translation. It should be mentioned that two previous studies report that r(GGGGCC) repeats form an intramolecular G-quadraplex structure [
13,
29]. As has been suggested for other quadruplexes [
8] and r(CGG) repeats, the r(GGGGCC) repeat, and perhaps the r(CCCCGG) repeat, may adopt two conformations that are in equilibrium: an extended hairpin structure and a quadruplex.
Another possible explanation for the higher frequency of poly(GP) inclusions in c9FTD/ALS may be due to ATG-initiated translation. While no sequence has been reported for the antisense transcript, resorting to analysis of the genomic DNA consensus sequence for C9ORF72 has revealed no ATG codons for sense transcript-derived c9RAN proteins, or for the antisense transcript-derived poly(PA) protein. However, one and three potential ATG start codons were found in the antisense poly(PR) and poly(GP) frames, respectively (Online Resource 1). Nonetheless, whether ATG codons are in fact present in RNA transcripts is not yet known, and our cell culture data provide evidence that poly(PR) and poly(GP) proteins can be translated from (CCCCGG)exp in the absence of an ATG initiation site.
Whether aberrant expression of c9RAN proteins, and the inclusions formed thereof, influence disease pathogenesis remains to be elucidated. Nevertheless, the formation of abnormal proteinaceous inclusions is associated with neurotoxicity in various neurodegenerative diseases. The inclusions may sequester other proteins causing loss of function, impair/overwhelm protein degradation systems, displace cytoplasmic organelles, and may themselves have neurotoxic properties. It is noteworthy that polyA and polyG proteins synthesized by RAN translation of expanded CAG·CTG repeats accumulate in disease-relevant tissues of patients with spinocerebellar ataxia type 8 and DM1, and that their expression in cultured cells is sufficient to cause apoptotic cell death [
26]. In addition, in both DM1 patients and in mice expressing (CUG)
exp, polyG aggregates co-localize with caspase-8, an early indicator of polyG-induced apoptosis [
26]. In c9FTD/ALS, neuronal inclusions of c9RAN proteins are similarly present in vulnerable areas (e.g., neurons of neocortex and hippocampus), but there is a paucity of such inclusions in certain affected areas, such as the spinal cord [
3,
25]. As with other aggregation-prone proteins involved in neurodegeneration (e.g., tau [
14]), it remains unclear whether c9RAN inclusions per se are toxic, or whether RAN translation of transcripts from the
C9ORF72 repeat contributes to neurodegeneration through the formation of toxic, soluble oligomers.
As with RAN translation, the consequence of foci formation in c9FTD/ALS is currently under investigation. Taking cues from other repeat expansion disorders, it is anticipated that foci will sequester select RNA-binding proteins, and cause the misregulation of crucial downstream RNA targets. To date, studies have identified several proteins that bind GGGGCC transcripts [
24,
29,
38]. The findings herein highlight the necessity to similarly identify proteins bound and sequestered by CCCCGG foci.
Examination of foci formed of sense or antisense transcripts of the expanded
C9ORF72 repeat revealed that, in addition to the frontal cortex and spinal cord, RNA foci accumulate in the cerebellum. Together with other studies, it is now evident that numerous pathological features are present in the cerebellum of c9FTD/ALS patients, including nuclear RNA foci, c9RAN protein inclusions, as well as p62-positive inclusions [
1,
3,
6,
25,
26]. Furthermore, FTD cases caused by the
C9ORF72 repeat expansion show atrophy of the parietal lobe and cerebellum, in addition to frontal and temporal lobe atrophy [
37]. In fact, c9FTD is characterized by greater cerebellar atrophy than sporadic FTD, as well as FTD caused by mutations in
MAPT [
37]. Consequently, cerebellar atrophy, nuclear RNA foci, and proteinaceous inclusions may be considered characteristic features of c9FTD/ALS. Future studies of c9FTD/ALS should therefore encompass evaluations of the cerebellum which, to date, has been largely neglected.
Of interest, foci and poly(GP) inclusions were seldom observed in the same cell in the frontal cortex and cerebellum of c9FTD/ALS patients. Although it is possible that foci too small to be easily detected are present in cells with poly(GP) inclusions, our findings suggest that only those (GGGGCC)
exp or (CCCCGG)
exp transcripts that escape being sequestered as foci, and are instead exported to the cytoplasm, become available for RAN translation. That RNA foci are found in both neuronal and glial cells (Figs.
4,
5), while poly(GP) inclusions are neuronal [
3], coupled with the fact that RNA foci in the cerebellum are found in greatest abundance in cells in proximity to the Purkinje cell layer, whereas poly(GP) inclusions are widely expressed throughout the molecular and granule layers, does provide a basis for the lack of co-occurrence of these two features in a given cell. While it remains to be determined whether other c9RAN proteins are more frequently found in the same cells as foci, and whether both sense and antisense foci are present within the same cell, these findings support the notion that foci and inclusions represent two distinct pathogenic mechanisms for
C9ORF72 repeat expansions.
Since the 2011 discovery that the expanded hexanucleotide repeat in
C9ORF72 causes chromosome 9p-linked FTD and ALS [
11,
30], several neuropathological features unique to c9FTD/ALS have been identified [
1,
3,
5,
7,
11,
24,
25,
28]. The findings from the present study expand this list and highlight the need to broaden our view of potential disease mechanisms to include toxicity potential stemming from the antisense transcript. Going forward, it will be critical to distinguish if and how RNA transcripts and c9RAN proteins contribute to the pathogenesis of disease, and whether the frequency or regional localization of RNA foci and c9RAN inclusions correlate with distinct clinical features. While these questions are being investigated, c9RAN proteins should be explored as a biomarker for c9FTD/ALS, as should treatment strategies aimed at eradicating the putatively toxic sense and antisense transcripts responsible for both foci formation and RAN translation.
Acknowledgments
We are grateful to all patients, family members, and caregivers who agreed to brain donation, without which these studies would have been impossible. We also acknowledge expert technical assistance of Linda Rousseau and Virginia Phillips for histology, and Beth Marten, Pamela Desaro, Amelia Johnston and Kristin Staggs-Douberly for brain banking. This work was supported by Mayo Clinic Foundation; National Institutes of Health/National Institute on Aging [R01 AG026251 (LP, RR), P01 AG003949 (DWD), P50 AG016574 (DWD, RR)]; National Institutes of Health/National Institute of Neurological Disorders and Stroke [R21 NS074121 (TFG, KB), R21 NS079807 (YZ), R01 NS080882 (RR), R01 NS063964 (LP); R01 NS077402 (LP), P50 NS072187 (DWD, RR); R21 NS084528 (LP)]; National Institute of Environmental Health Services [R01 ES20395 (LP)]; Amyotrophic Lateral Sclerosis Association (KB, LP); ALS Therapy Alliance (RR).