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
]. 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
]. 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
]. 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
]. 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
]. 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
]. 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
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. , ), 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
Since the 2011 discovery that the expanded hexanucleotide repeat in C9ORF72
causes chromosome 9p-linked FTD and ALS [11
], several neuropathological features unique to c9FTD/ALS have been identified [1
]. 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.