c9orf72 Disease-Related Foci are Each Composed of One Mutant Expanded Repeat RNA
SUMMARY The chromosome 9 open reading frame 72 (c9orf72) gene contains a hexanucleotide (GGGGCC) repeat expansion responsible for many cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The mutant intronic RNA forms \xe2\x80\x9cfoci\xe2\x80\x9d within nuclei, but the connection between transcript expression, foci, and biochemical disease mechanisms is unclear. Knowing the absolute numbers of cellular RNAs, in any system, is important for understanding the molecular mechanisms of natural physiology, disease, and drug action. Absolute numbers, however, are rarely determined and this absence is a major impediment to understanding complex systems. Using quantitative methods, we demonstrate foci are single RNA molecules. Most cells have no foci while 1 \xe2\x80\x93 2 % have greater than ten. Knowing the number of disease causing molecules may contribute to understanding ALS and FTD pathology and successful drug discovery. More broadly, our data suggest that small numbers of RNA molecules may have a big impact on disease.
INTRODUCTION<!>Cell Culture<!>Primers, Probes, and in vitro transcription<!>qPCR and ddPCR<!>Statistical analysis<!>Quantifying RNA<!>Copy number of c9orf72 in fibroblast cells<!>Copy number of c9orf72 in astrocytes and motor neurons<!>Distribution of foci in patient-derived fibroblast cells<!>Implications of c9orf72 RNA foci and copy number
<p>An expanded hexanucleotide repeat has been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (Renton et al., 2011; DeJesus-Hernandez et al., 2011; Haeusler et al., 2016). This repeat expansion occurs in the first intron of the chromosome 9 open reading frame 72 (c9orf72) gene. It accounts for one-third of familial ALS and a quarter of familial FTD. The sequence of the repeat within c9orf72 pre-mRNA is GGGGCC. Patients with ALS or FTD typically have one mutant c9orf72 allele that contains 700-1600 repeats, while unaffected individuals have fewer than 24 repeats in both alleles (DeJesus-Hernandez et al., 2011). There is also an antisense transcript at the c9orf72 loci that overlaps the repeat expansion (producing a CCCCGG repeat) and this antisense transcript has also been implicated in disease (Gendron et al., 2013; Zu et al., 2013).</p><p>The identification of mutant RNA as a contributor to ALS and FTD was important because mutant c9orf72 provides a starting point for developing agents to treat both diseases and for understanding the molecular mechanism of pathogenesis (Haessler et al., 2014). For c9orf72 FTD/ALS, one potential mechanism involves non-AUG dependent translation of repetitive RNA (RAN translation) generating dipeptides that can interact with low complexity domains within cellular proteins and induce toxicity (Ash et al. 2013; Mori et al., 2013; Wen et al., 2014; Lin et al., 2016; Lee et al., 2016). A second mechanism involves the sequestration of proteins through binding the expanded repeat (Lee et al., 2013) although the identity of major interacting proteins and the extent of their contribution remains in dispute (Gitler and Tsuji, 2016). Another possible contribution to mechanism is the potential for the expanded repeats with mutant intron 1 (GGGGCC) or that overlapping antisense transcript (CCCCGG) to form stable secondary structures (Haeusler et al., 2014; Dodd et al., 2016).</p><p>Mutant intronic repeat RNAs form foci that are detectable by fluorescence in situ hybridization (FISH) (Taneja et al., 1995) and these foci are a hallmark molecular pathology of c9orf72 ALS/FTD (DeJesus-Hernandez et al., 2011; Zu et al. 2013). Foci provide a visual starting point for understanding where mutant intronic RNA is localized inside cells and how its quantity varies between cells. It is unknown, however, whether foci are composed of multiple aggregating mutant intronic RNA molecules or whether each focus is composed of a single mutant intronic RNA.</p><p>One key characterization is missing from almost all studies investigating novel mechanisms of action for c9orf72 mutant transcripts and other noncoding RNAs – the number of RNA molecules per cell. This feature is important because the number of RNA molecules forms the foundation for understanding mechanism (Schwanhausser et al., 2011; Bosson et al. 2014; Denzler et al., 2014). For c9orf72 ALS/FTD, the number of sense intronic RNA or antisense RNA transcripts will help define the limits on: 1) the number of proteins that can be sequestered by the mutant expanded repeats; and 2) the production of toxic peptides.</p><p>Here we measure the number of transcripts associated with mutant c9orf72 transcription per cell. Mutant intronic RNA or antisense RNA containing the GGGGCC or CCCCGG expanded repeats is primarily localized in the nucleus. There is an approximately a one to one correspondence between mutant intronic RNA or antisense RNA per cell and foci per cell, suggesting that each focus is a single RNA molecule. Because of the equivalence between RNA copy number and foci, fluorescent in situ hybridization is a single-molecule detection strategy for visualizing the cellular localization and distribution of cellular mutant c9orf72 RNA. Mutant c9orf72 transcripts are not evenly distributed among cells, supporting the hypothesis that disease pathogenesis involves non-cell autonomous mechanisms (Ilieva et al., 2009) in which a minority of cells are responsible for observed phenotypes.</p><!><p>c9orf72 ALS patient-derived fibroblast cell lines (C9-4, C9-5, C9-18) and healthy control fibroblast cell lines (Con-1, Con-3, Con-4) are gifts from Dr. John Ravits (Univ. of California, San Diego). Healthy control fibroblast cell line GM02153 was obtained from Coriell institute (Camden, NJ, USA). Cells were cultured as described (Hu et al., 2015). Astrocyte and motor neuron differentiated from ALS patient-derived ips cell lines were prepared as described (Zhang et al., 2015; Li et al., 2015). Nuclear and cytoplasmic fractions were prepared as described (Gagnon et al., 2014). In situ hybridization was performed as described (Hu et al., 2015).</p><!><p>Primer sequences, probe sequences, and protocols for in vitro transcription are provided as Supplementary Information.</p><!><p>After reverse transcription, PCR reaction solution was reconstituted to a final volume of 20 μL containing one twentieth of cDNA, 10 ul of 2X PCR mixture, 1 ul of 20X probe and 1ul of gene specific primers. Unless otherwise noted final primer and probe concentrations were 0.5 μM and 0.25 μM respectively. PCR of antisense transcript of c9orf72 used strand gene specific reverse primer (ASRP) and LK FP.</p><p>qPCR was performed on a CFX96 Touch™ Real-Time PCR Detection System (Bio-rad) using iTaq Supermix with Rox (Bio-Rad). PCR reactions were done in triplicate at 55 °C 2 min, 95 °C 3 min and 95 °C 30 s, 60 °C 30 s for 4 0 cycles in an optical 96-well plate.</p><p>ddPCR reaction was assembled using same batch of cDNA for qPCR. 70 μL of droplet generation oil and 20 μL of ddPCR reaction (containing 1× ddPCR supermix for probes (no dUTP) (Bio-Rad), 0.5 uM primer, 0.25 uM probe, one twentieth of cDNA to final volume of 20 μl) were used to form droplet in a QX100 droplet generator. The partitioned emulsion containing at least 14,000 droplets was then slowly transferred to a twin.tec 96-well semi-skirted PCR plate (Eppendorf). After heat-sealed with foil, the plates containing the droplets was PCR cycled to final point under conditions at 95°C , 5 min, 95°C 30 s and 60°C 30 s for 40 cycles, 72°C for 30 s, then held at 12°C. Following PCR, samples were read on a BioRad QX100 reader and data were analyzed using Quantasoft software (Bio-Rad, USA). Data obtained were converted to copies per cell based on RT efficiency obtained from standard curve and the known cell numbers corresponding to the input cDNA.</p><!><p>Statistical significance was analysed using a two-tailed unpaired t-test. Data are presented as means ± s.d. of three or more independent experimental results.</p><!><p>We used two strategies to evaluate the absolute numbers of transcripts associated with expression of the c9orf72 gene per cell, including mRNA, mutant expanded GGGGCC repeat intronic RNA, and mutant expanded CCCCGG repeat antisense transcript. One strategy was quantitative PCR (qPCR). qPCR relies on an increase in fluorescent signal that is proportional to the amplified product. Cycle threshold (Ct) values provide a relative numerical value for abundance. The second strategy was droplet digital PCR (ddPCR) (Dodd et al., 2012), a highly quantitative and precise method that directly measures copy numbers per cell. In this strategy RNA molecules are converted to cDNA and each cDNA is enclosed individually within droplets and amplified. If a DNA is present within a sample, that presence will be detected and counted. Primers were designed to amplify either the c9orf72 intronic (intron 1) transcript, the overlapping antisense transcript, or c9orf72 mRNA (Sup. Figure S1) (Figure 1A). These primers were used in similar workflows regardless of whether ddPCR or qPCR was used (Figure 1B).</p><p>Neither qPCR nor ddPCR directly provides insights into the number of RNA molecules per cell. To obtain absolute numbers it is necessary to count the number of input cells prior to harvesting RNA. It is also necessary to use an RNA template of equivalent sequence and known concentration as a standard to account for varying efficiencies during preparation of cDNA or during amplification. We prepared the standards by in vitro reverse transcription of a synthetic DNA containing a short sequence sequence from c9orf72 intron 1 or from the mature mRNA and measured their concentrations (Sup. Figure S2). A similar standard was prepared containing a sequence from GAPDH to allow GAPDH mRNA to be used for comparison. We then obtained standard curves relating the number of RNA molecules to measured qPCR Ct value or to the number of molecules detected by ddPCR (Supplemental Figure S3) (Boeuf et al., 2005; Larionov et al., 2005; Svec et al., 2015). These standard curves were used to estimate the number of intronic sense, antisense, or mRNA molecules in samples obtained from cells.</p><!><p>We used our independent ddPCR and qPCR strategies to determine the copy number of c9orf72 mRNA, intronic transcript, and antisense transcript in patient derived fibroblast cell line C9-4. GAPDH was used as a benchmark control gene for comparison. ddPCR and qPCR reported the presence 4276 and 4528 molecules of GAPDH mRNA per cell (Figure 2A), suggesting our two approaches were yielding similar and consistent data. These measurements for GAPDH mRNA per cell are consistent with those determined by other methods (Marinov et al., 2014; White et al., 2011; Marcus et al., 2006).</p><p>Both qPCR and ddPCR revealed that c9orf72 mRNA was present at 15-16 copies per cell, a much lower copy number compared to GAPDH mRNA (Figure 2B). The intronic sense transcript was present at an even lower number, measured at approximately three copies per cell by either approach. The antisense transcript was also present at a lower number, measured at 2.5 or 6 copies by qPCR or ddPCR, respectively.</p><p>We fractionated cells and examined the subcellular distribution of transcripts. Both the c9orf72 intronic sense and antisense hexanucleotide repeat transcripts could be detected in cytoplasm, but the majority of transcripts containing the hexanucleotide repeats were present in the nucleus (Figure 2C). Relative to the intronic sense and antisense transcripts, c9orf72 mRNA was more highly expressed in both nuclei and cytoplasm but expression was more balanced between the two compartments.</p><p>To generalize our findings, we compared transcript copy numbers between additional disease-free and patient-derived cell lines (Figure 2D-G). Because our initial experiments had indicated that qPCR and ddPCR revealed similar numbers for RNA molecules per cell (Fig. 2AB), we focused on qPCR to provide quantitation for the remainder of this study. For the intronic c9orf72 transcript, the mutant cell lines contained about four copies of the transcript per cell and the wild-type cell lines contained less than one copy (Figure 2DG). For the antisense c9orf72 transcript we observed approximately three copies per cell and approximately one copy per wild-type cell (Figure 2EG). Both the intronic and antisense transcripts were significantly more highly expressed in patient-derived cells than they were in non-disease cells (Figure 2G). For comparison, in mutant cell lines c9orf72 mRNA was present at fifteen to twenty copies per cell and 5-10 copies per cell in non-disease cell lines (Figure 2FG).</p><p>When evaluating our data, it is important to realize that the patient-derived cells used in these experiments were heterozygous for mutant c9orf72. Our qPCR and ddPCR detection methods will detect both mutant and wild-type c9orf72 intron 1 (primers cannot detect the GGGGCC or CCCCGG repeats directly because of the highly structured nature of both sequences). As a result, experiments that detect 3 – 4 copies of c9orf72 intronic RNA are probably detecting less than 3-4 copies of mutant intronic RNA encoding the expanded repeat.</p><!><p>To move towards a more relevant biological context, we examined the copy number of transcripts associated with c9orf72 gene expression (mRNA, intronic/intron 1, antisense) in human motor neurons and astrocytes (Figure 3). To obtain these cells we differentiated induced pluripotent stem (iPS) cells (Donnelly et al., 2012). Astrocytes are glial cells that provide a variety of support functions. We used qPCR to measure RNA molecules per cell and found that the mRNA copy number was approximately twelve (Figure 3A). As observed for fibroblasts, in astrocytes we observed lower copy numbers of intronic and antisense transcripts than mRNA transcripts. Both intronic and antisense transcript were present at approximately one copy per cell.</p><p>For motor neurons derived from ALS patient iPS cells, we observed approximately one half to one copy of the intronic transcript per cell regardless of whether cells were mutant or derived from healthy individuals (Figure 3B). More antisense transcript was detected in mutant cells with over nine copies measured. As observed for fibroblasts and astrocytes, more copies of mRNA were detected than antisense or intronic transcripts. In all cases, expression of the transcripts was higher in patient-derived cells than non-disease control cells. With the exception of higher detection of the antisense transcription in neuronal cells, these results confirm that cultured neural cells show the same trends in mutant intronic c9orf72 expression as fibroblast cells.</p><!><p>qPCR and ddPCR allow quantitation of RNA in terms of RNA molecules per cell within a population of cells. These methods do not provide insights into whether the distribution among individual cells is homogeneous or heterogeneous. In our previous studies we had visualized foci and noted that there were only a few foci per cell (Hu et al., 2015). This low number of foci, together with the low RNA copy number observed by ddPCR and qPCR, suggested that each focus may represent a single RNA molecule. To investigate that hypothesis more quantitatively we calculated the percentage of cells that contained foci, the average number of foci per cell, and the distribution of different numbers of foci among cell.</p><p>c9orf72 mutant intronic or antisense RNA foci inside patient derived cells can be detected by Fluorescence In Situ
Hybridization (FISH) using probes that are complementary to either the GGGGCC expanded repeat within intron1 or the CCCCGG expanded repeat within the antisense transcript (Figure 4A). Approximately 40% of cells had observable foci in either the sense or antisense direction with the remainder having no detectable foci (Figure 4B). The average number of foci for the entire cell population was approximately 0.7 per cell for mutant intronic transcript and 1.4 per cell for the antisense transcript (Figure 4C). This low number masked a broad divergence among cells. For cells possessing mutant intronic or mutant antisense foci, the majority had one or two but a small percentage of cells (1-2 %) had more than ten foci (Figure 4DE) and visually stood out from the mass of other cells examined by FISH (Figure 4A). The results for foci formed by mutant intronic transcripts are similar to those observed previously by Baloh and coworkers (Sareen et al., 2013).</p><!><p>RNA is a versatile macromolecule with diverse functions inside cells (Mattick and Morris, 2014). For some classes of RNA, such as mRNA, our understanding of how sequence translates into function is relatively mature. For other classes, such as micro RNAs (miRNA) or long noncoding RNAs (lncRNA), our understanding of the mechanism that connects RNA sequence to function is incomplete (Matsui and Corey, 2016).</p><p>In this study we examine the mutant intronic and mutant antisense transcripts encoding expanded GGGGCC or CCGGGG repeats respectively at the c9orf72 locus. For mutant intronic transcripts, regardless of method or cell line, we estimate that one to four copies are present per cell. For mutant antisense transcripts, our estimate is one to six copies per cell. We conclude that c9orf72 is not a highly expressed gene and that its effects on ALS/FTD pathology must take its low expression level into account.</p><p>To put the copy number of the c9orf72 transcripts into context, we can compare it to the previously determined transcript number for the well-studied noncoding RNA MALAT1 (Dodd et a., 2013). We had observed ~3000 copies per cell, with approximately 500 in the cytoplasm and 2500 in the nucleus. MALAT1 also forms foci and dozens were present in each cell nuclei (Gagnon et al., 2014). The comparison of c9orf72 and MALAT1 demonstrates that foci-forming noncoding RNAs can vary dramatically in their levels of expression from low (i.e. c9orf72) to high (i.e. MALAT1).</p><p>Foci are a visual manifestation for expression of sense or antisense c9orf72 transcripts. For both mutant intronic and mutant antisense transcripts, we detected approximately one focus per fibroblast cell. This number is similar to the ddPCR or qPCR estimate of one to six copies per cell detected in various cell lines - an estimate that includes expression of the non-mutant allele. We conclude, therefore, that each foci represents a single molecule of mutant c9orf72 RNA regardless of whether FISH is designed to detect the expanded repeats within mutant intronic (GGGGCC) or antisense transcripts (CCCCGG).</p><p>FISH permits single-molecule imaging of expanded mutant repeats and reveals both transcript location and number within individual cells. Approximately half of the c9orf72 ALS/FTD cells had no detectable foci, while the remainder had one or more. 1-2 % of cells had > 10 sense or antisense foci (Figure 4DE). The small fraction of cells that were well-endowed with mutant foci may play a role in disease that is disproportionate to their number, consistent with non-cell autonomous mechanisms similar to those described for other neurodegenerative diseases (Ilieva et al., 2009). It is also possible that these few cells are represent those in an advanced diseased state. As more cells reach this state, the disease might progress even though only a few cells have an large number of foci at any one time.</p><p>One mechanism to explain the role of mutant RNA in the pathogenesis of c9orf72 ALS/FTD involves the sequestration of functionally significant proteins (Gitler and Tsujii, 2016). This mechanism has been well characterized for myotonic dystrophy, a trinucleotide repeat disease (Wheeler et al., 2009; Konieczny et al. 2014). The number of repeats within mutant c9orf72 has been estimated to be between 700 and 1600 (DeJesus-Hernandez et al., 2011) which would lead to an expanded repeat region between 4200 and 9600 nucleotides. While the exact number proteins that can bind to this region is unknown, it is likely to range from hundreds per transcript to more than a thousand in those cells that possess >10 foci. Given the wide variety of proteins implicated as binding partners for the expanded c9orf72 repeats (Gitler and Tsujii, 2016) it is likely that the binding sites are divided between a heterogeneous assortment of proteins. Binding of proteins to a relative handful of transcripts would need to be sufficient to produce a physiologic effect.</p><p>A second mechanism involves toxicity due to AUG-independent translation of dipeptide proteins from either the sense or antisense transcripts (Green et al., 2016). The amount of Repeat Associated Non-standard translation (RAN) products is limited by the amount of mutant RNA that reaches the cytoplasm for translation. Our data reveal that a small fraction of c9orf72 mutant intronic or antisense transcripts can be detected in the cytoplasm (Figure 2C). In iPSC-derived neuronal cells, just 0.5 mutant intronic transcripts were detected per cell so the average number in the cytoplasm will be less (Figure 3C). Over one hundred protein products, however, can be produced per mRNA per hour (Schwanhausser et al., 2011). RAN protein might accumulate over time and eventually provoke deleterious effects. Accumulation of RAN products may be encouraged by the stability of the products and their presence in protease-resistant cytoplasmic aggregates. Visual inspection of published microscopy detailing RAN protein accumulation reveals a minority of cells with visible cytoplasmic inclusions (Zu et al., 2013; Ash et al., 2013; Gendron et al., 2013). These data are consistent with a heterogeneous cell population with differing c9orf72 expression (Figure 4DE).</p><p>Because there appear to be so few RNA molecules, it may be valuable to consider whether RNA or dipeptide expression are involved in disease and to search for alternate causes. One alternative hypothesis is that the expanded repeat affects the structure of the chromosome in the vicinity of the c9orf72 locus. This might cause subtle changes in noncoding RNA expression, the expression of nearby genes, or the three dimensional architecture of the genome. R-loops formation, in which the mutant transcript hybridizes directly to chromosomal DNA, may also affect gene expression (Haeusler et al., 2014) and would require just one RNA transcript.</p><p>All RNA species are continually being transcribed and degraded. Ultimately, the biochemical equilibrium defined by these processes will govern the half-lives of mutant c9orf72 mutant intronic and mutant antisense transcripts. Our data suggest that each foci is a single mutant RNA molecule. It is possible that the half-life of these RNA species is extended by binding of proteins to the expanded repeats or the ability of either the GGGGCC or CCCCGG repeats to from stable intramolecular structures (Haeusler et al., 2014; Dodd et al., 2016).</p><p>The linkage between disease and the GGGGCC repeat expansion within intronic c9orf72 provides a starting point for drug discovery for ALS/FTD and knowledge of absolute transcript number will affect the development of treatment options. Approaches to treatment include the use of antisense oligonucleotides (Donnelly et al., 2012; Sareen et al., 2012; Jiang et al., 2016; Lagier-Tourenne et al., 2013), duplex RNAs (Hu et al., 2015), or small molecules (Su et al., 2014) that either block the expanded repeat within intronic c9or72 RNA or that target the intron outside the expansion. The small number of target transcripts suggests that relatively low numbers of inhibitors are needed to achieve the excess compound needed for potent inhibition of the delirious effects of mutant c9orf72. Many other diseases are caused by expansions of repetitive regions and the analysis presented here may broadly useful.</p><p>Our results lead to several testable hypotheses. The most fundamental hypothesis, one that applies broadly to RNA biology, is that measuring the number of RNA molecules will provides insights into mechanism. The number of RNA molecules will be especially important when those RNA molecules have been implicated in disease causation. For c9orf72, we hypothesize that small numbers of RNA molecules are responsible for disease. Increasing expression of mutant intronic c9orf72 should increase foci number and speed onset of disease. If this outcome is not observed, further consideration should be given to mutant genomic DNA as a direct cause independent of protein or RNA expression.</p>