Introduction
Frontotemporal lobar degeneration (FTLD) is a common cause of young-onset dementia [
15,
24], characterised by atrophy of the frontal and temporal lobes. The three major genetic causes of FTLD are mutations in
MAPT [
16,
23,
27],
GRN [
4,
9], and
C9orf72 [
10,
13,
25]. FTLD shares clinical, genetic and pathological features with amyotrophic lateral sclerosis (ALS), and
C9orf72 mutation is the most common known cause of ALS [
21]. FTLD has several neuropathological subtypes, the most common of which are cases with tau inclusions (FTLD-tau) and those with TDP-43-positive inclusions (FTLD-TDP) [
7,
19]. FTLD-TDP can be further subdivided based on the anatomical and subcellular localisation of the inclusions [
18]. Pathologically,
C9orf72 cases fall into two of the four FTLD-TDP subtypes: FTLD-TDP type A, being characterised by many TDP-43-positive neuronal cytoplasmic inclusions and short dystrophic neurites, the majority of which are found in layer 2 of the cortex; or FLTD-TDP type B, characterised by moderate numbers of neuronal cytoplasmic inclusions and dystrophic neurites in all cortical layers [
18,
20]. In addition to TDP-43 pathology small neuronal p62 positive ‘star-like inclusions’ are observed predominantly in the hippocampus and cerebellum [
2].
The FTD- and ALS-causing mutation in
C9orf72 is an expanded GGGGCC repeat, located upstream of the translation start codon [
10,
13,
25]. Expanded repeats in non-coding regions of other genes also cause other diseases with neurodegenerative features, including myotonic dystrophy and certain spinocerebellar ataxias [
8]. These diseases are characterised by the aggregation of the expanded repeat RNA into nuclear RNA foci [
29]. Intriguingly, it has also been shown that despite being in non-coding regions of the gene, expanded repeats are able to initiate their own translation, a phenomenon termed repeat-associated non-ATG (RAN) translation [
30]. The
C9orf72 GGGGCC repeat expansion causes RAN translation, leading to the formation of neuronal inclusions containing RAN protein products generated from sense transcripts [
3,
22]. Nuclear RNA foci containing repeats transcribed in the sense direction were reported in post-mortem tissue of four
C9orf72 cases in one study [
9], but this was not replicated in another study [
26].
C9orf72 antisense repeat transcripts have been reported [
22], but whether antisense transcripts contribute to pathology by generating RNA foci and RAN products, and whether sense RNA foci are a characteristic feature of
C9orf72 FTLD (C9FTLD) are unknown. We report here, in a series of C9FTLD cases and controls, that sense and antisense RNA foci are a characteristic and specific feature of C9FTLD.
Materials and methods
Cases
Brain specimens (described in Table
1) were obtained from the Queen Square Brain Bank for Neurological Disorders, UCL Institute of Neurology, London and the MRC London Neurodegenerative Diseases Brain Bank, Institute of Psychiatry, King’s College London. The samples were fixed in 10 % buffered formalin for histopathology and immunohistochemistry. Histological sections from the frontal cortex, hippocampus and cerebellum were analysed. Seven cases had
C9orf72 expansion repeat confirmed by repeat primed PCR (rp-PCR) analysis and Southern blot as previously described [
5,
12]. Three cases have been reported previously (cases 11, 12 [
20], and case 13, which has a homozygous repeat expansion [
12]). Four cases were rp-PCR analysed for this study (cases 14–17). One case (case 18) was determined pathologically using the characteristic p62 pathology found only in
C9orf72 expansion carriers. Additional cases used as controls were three FTLD-TDP type B cases and four FTLD-TDP type A cases without a known genetic mutation (5 of these 7 cases had DNA available and were confirmed by rp-PCR to have normal
C9orf72 repeat length); three Alzheimer’s disease cases all pathologically diagnosed with Braak and Braak stage VI pathology and Thal phase 5 [
6,
28]; ten neurologically normal controls. The neuropathological diagnosis was determined using established diagnostic criteria, in line with consensus recommendations for the FTLD spectrum [
18]. Immunohistochemistry was performed as previously described [
17]. This study was approved by the UCL Institute of Neurology and National Hospital for Neurology and Neurosurgery Local Research Ethics Committee.
Table 1
Details of cases used in this study
1 | Normal control | M | NA | 85 | NA |
2 | Normal control | F | NA | 82 | NA |
3 | Normal control | M | NA | 57 | NA |
4 | Normal control | F | NA | 79 | NA |
5 | Normal control | F | NA | 94 | NA |
6 | Normal control | M | NA | 71 | NA |
7 | Normal control | F | NA | 68 | NA |
8 | Normal control | F | NA | 80 | NA |
9 | Normal control | M | NA | 69 | NA |
10 | Normal control | F | NA | 93 | NA |
11 | C9FTLD-TDP type A | M | 62 | 72 | 10 |
12 | C9FTLD-TDP type A | F | 66 | 74 | 8 |
13 | C9FTLD-TDP type A | M | 43 | 45 | 2 |
14 | C9FTLD-TDP type B | F | 64 | 66 | 2 |
15 | C9FTLD-TDP type A | M | 53 | 63 | 10 |
16 | C9FTLD-TDP type A | M | 62 | 68 | 6 |
17 | C9FTLD-TDP type A | F | 56 | 67 | 11 |
18 | C9FTLD-TDP type A | M | 54 | 60 | 6 |
19 | FTLD-TDP type A | M | 57 | 62 | 5 |
20 | FTLD-TDP type A | M | 75 | 79 | 4 |
21 | FTLD-TDP type A | F | 75 | 78 | 3 |
22 | FTLD-TDP type A | F | 83 | 87 | 4 |
23 | FTLD-TDP type B | F | 75 | 77 | 2 |
24 | FTLD-TDP type B | F | 63 | 67 | 4 |
25 | FTLD-TDP type B | M | 67 | 69 | 2 |
26 | AD | F | 67 | 76 | 9 |
27 | AD | M | 60 | 66 | 6 |
28 | AD | F | 51 | 62 | 11 |
RNA fluorescence in situ hybridisation (FISH) with protein immunostaining
Supplier and catalogue numbers for FISH reagents are detailed in Supplementary Table 1. 7 μm-thick paraffin sections were dewaxed, followed by antigen retrieval in citrate buffer (0.1 M, pH 6) for 20 min in a microwave. Sections were then dehydrated in a graded series of alcohols, air dried and rehydrated in phosphate-buffered saline (PBS), briefly washed in 2×SSC and incubated for 30 min in pre-hybridisation solution (50 % formamide/2×SSC) at 80 °C. Hybridisation solution (50 % formamide, 2×SSC, 0.8 mg/ml tRNA, 0.8 mg/ml salmon sperm DNA, 0.16 % BSA, 8 % dextran sulphate, 1.6 mM ribonucleoside vanadyl complex, 5 mM EDTA, 0.2 ng/μl probe) was heated at 80 °C for 5 min prior to incubation with sections for 2 h at 80 °C. Sections were firstly washed three times for 30 min each with a high-stringency wash solution (50 % formamide/0.5×SSC) at 80 °C, and then three times for 10 min at room temperature in 0.5×SSC. After a brief wash in PBS sections were blocked in 10 % foetal bovine serum/PBS for 30 min, and then incubated with primary antibody (diluted in PBS) overnight at 4 °C, washed with PBS, and then incubated with appropriate secondary antibodies (all Alexa Fluor, Life Technologies at 1:500 in PBS) for 1 h at room temperature. Autofluorescent background was reduced using Sudan black B (0.2 % in 70 % ethanol/PBS, 10 min). After further washes in PBS, sections were mounted with ProlongGold containing DAPI (Life Technologies). Images were obtained using a LSM710 META confocal microscope (Zeiss). For FISH with both probes, the sense probe was applied first as above, and after the three 30 min 80 °C washes in 50 % formamide/0.5×SSC, the antisense probe was added for 2 h, followed by the protocol above. For DNase and RNase treatments the standard protocol above was followed with additional steps: after rehydration in PBS, sections were incubated in 0.5 % Triton/PBS for 10 min, washed once in PBS, and treated with either RNase A (1 mg/ml in PBS/0.05 % Tween-20) or Turbo DNase (Ambion, 200 U/ml in supplied buffer) for 2 h at 37 °C. After the treatment sections were washed three times in PBS, once in 2×SSC and then placed in pre-hybridisation solution and the standard FISH protocol resumed. 2′-O-methyl RNA probe (Integrated DNA Technologies) sequences were (GGCCCC)4, (GGGGCC)4 or (CAG)7, 5′ labelled with either Cy3 or Alexa488. Primary antibodies were for NeuN (1:250, Millipore ABN78), GFAP (1:250, Abcam ab4674), Iba1 (1:500, Wako 019-19741), CAII (1:2000, Abcam ab6621), p62 (1:200, Abnova H00008878-M01) and phospho TDP-43 (1:500, Cosmo Bio, CAC-TIP-PTD-P01).
Quantification of RNA Foci, TDP-43 and p62
Three to six 40× z-stack images (45,176 μm2 per image) were taken at high resolution (2048 × 2048 pixels) using a 1.4 NA objective, in the anterior frontal cortex, two in the dentate fascia of the hippocampus and two in the granule cell layer of the cerebellum, to ensure a minimum of 100 neurons were quantified in each brain region (approximately 120 neurons in frontal cortex, 350 in hippocampus and 1,200 in cerebellum). Quantification was performed blinded. RNA foci were counted in maximum intensity projections of z-stack images using the touch count tool in Volocity image analysis software (Perkin Elmer), and then scored for co-occurrence with staining of the nucleus and cell type markers. RNA foci were determined to be on the edge of the nucleus if they were touching the edge of DAPI staining, and in the cytoplasm if they were outside of the nucleus but within the staining of the cell type markers. Cytoplasmic localisation was not determined for astrocytes or microglia, or neurons of the cerebellum as cytoplasm could not be clearly identified. Cytoplamsic p62 and phospho TDP-43 inclusions were identified by Volocity image analysis software using the same threshold for all images. Inclusions positive for both p62 and phospho TDP-43 were only included in the TDP-43 group, to enable specific quantification of p62-positive, TDP-43-negative inclusions. All data is presented as the mean ± standard error of the mean. The homozygous C9FTLD case is shown on all graphs, but is excluded from means and statistical analysis. Statistical testing was performed with GraphPad Prism software using either a paired t test or one-way ANOVA and post hoc Bonferroni test.
Discussion
We provide the first definitive evidence for the presence of both sense and antisense RNA foci in C9FTLD. This has important implications for disease mechanism. Firstly, it suggests that either or both sense and antisense RNA foci could play a pathogenic role in C9FTLD; presumably through sequestration of RNA-binding proteins, as has been observed in other non-coding repeat expansion diseases [
8]. The identities of the sequestered proteins in patient RNA foci are currently unknown. Our detection of antisense as well as sense RNA foci means identification of proteins sequestered by antisense transcripts will be of great interest. We observed that fewer neurons contained antisense than sense foci, but that there were a greater number of antisense RNA foci per neuron. However, it appeared that the antisense foci were generally smaller than the sense foci, so it is unclear whether one type of foci has the potential to sequester more protein than the other. We have previously shown that the sense (GGGGCC)n RNA forms a G-quadruplex structure [
11], which could mediate sequestration of proteins. Cytosine-rich sequences can form a distinct four-stranded structure termed an i-motif [
14]. It will be important to determine whether the (GGCCCC)
n sequence forms an i-motif and whether this has biological significance. The identification of cytoplasmic antisense RNA foci raises the possibility that antisense RAN translation products will be produced, as well as the sense RAN translation products already reported [
3,
22], further increasing the potential pathogenic species in C9FTLD.
Sense RNA foci were initially reported in 25 % of cells in the frontal cortex of 2 C9FTLD cases and the spinal cord of 2 C9ALS cases [
10], but a second study was unable to replicate these findings and observed RNA foci in controls [
26]. Sense RNA foci have also been detected in C9FTLD-derived induced pluripotent stem cells [
1]. We report the first RNA FISH study on a series of C9FTLD cases, and both normal and neurodegenerative disease controls, and show that using our optimised FISH protocol, both GGGGCC sense and GGCCCC antisense RNA foci are specific to C9FTLD. We observed sense and antisense foci in 37 and 26 % of frontal cortical neurons, respectively, and a total frontal cortical neuronal foci burden (sense and antisense) of 51 %, which shows that foci are a major pathology in C9FTLD. We used a much higher hybridisation temperature than the previous studies (possible due to the extremely high melting temperature of our 2′-
O-methyl RNA probes), and stringent washes, which might explain the greater specificity. We also note that the RNA foci were not visible using a standard fluorescence microscope, but were readily visible with a confocal microscope, which may also explain differences with previous studies. It is likely that the enhanced signal to noise provided by high-resolution microscopy enabled sensitive detection of the RNA foci, which are, therefore, likely to be small structures. Our use of
z-stacks to image the entire volume of the cell allowed a more accurate estimation of RNA foci per cell and the percentage of neurons containing foci than using confocal images from a single plane, which, given the small size of foci, would lead to an underestimation of foci numbers.
We identified sense and antisense RNA foci in astrocytes, microglia and oligodendrocytes, but at lower frequency than in neurons. This suggests neurons are the primary sites of foci pathology but also raises the possibility of impaired glial function due to RNA foci formation. It will be interesting to determine whether the same repertoires of RNA-binding proteins are sequestered in these different cell types, and to understand why neurons have a greater RNA foci burden. Although foci were present in all three brain regions, the highest burden of foci was consistently found in the frontal cortex, which is the region that suffers the greatest neuronal loss in FTLD. A previous study on the homozygous case described the clinical and pathological features as severe, but not completely outside of the usual disease spectrum [
12]. Our data show that the homozygous case displays severe sense and antisense RNA foci pathology when compared to the heterozygous cases, with a higher proportion of neurons containing foci and also more foci per cell. This difference was most striking in the frontal cortex and hippocampus, with no apparent difference in the cerebellum. The severe foci burden and early age at onset of the homozygous case are consistent with our observation that foci burden inversely correlates with age at onset. However, it will be important to replicate this finding in a larger series.
The majority of RNA foci in all cell types were located in the nucleus, consistent with a role in nuclear RNA-binding protein sequestration. In the different brain regions between 10 and 22 % of neuronal foci were located at the very edge of the nucleus, with the cerebellum having a significantly higher proportion than the frontal cortex. The significance of foci on the edge of the nucleus is unclear but it would be interesting to determine whether they are in proximity to nuclear pores and whether they have the potential to block nuclear RNA import or export. 9 % of sense RNA foci in the frontal cortex were cytoplasmic, consistent with RAN translation of the repeat transcripts, which would require the cytoplasmic translation machinery. Significantly fewer cytoplasmic antisense foci were found in the frontal cortex, which may reflect differential nuclear export or turnover. Neither sense nor antisense foci showed an obvious association with p62- or TDP-43-positive inclusions, although cytoplasmic foci were occasionally found within both types of inclusion in all brain regions where they are found. This suggests that the presence of RNA foci does not predict the presence of TDP-43- or p62-positive inclusions. Co-localisation analyses with antibodies specific for each RAN product will be required to determine whether individual sense or antisense RAN products are associated specifically with sense or antisense RNA foci.
Our identification of sense and antisense RNA foci, and the significant correlation between sense foci burden and age at onset, add weight to the possibility that toxic RNA gain of function is involved in C9FTLD pathogenesis, but does not rule out a role for loss of C9orf72 function or RAN translation products. The identification of proteins sequestered by RNA foci will be a key step in confirming the validity of the toxic RNA hypothesis. In conclusion, we have identified both GGGGCC sense and GGCCCC antisense RNA foci as a consistent and specific feature of C9FTLD in a series of cases and controls. These findings have important implications for understanding disease mechanism and may help inform the drug discovery process.
Acknowledgments
We would like to thank Priya Gami for her technical assistance. We thank the MRC London Neurodegenerative Diseases Brain Bank, Institute of Psychiatry, King’s College London and the Queen Square Brain Bank for Neurological Disorders, UCL Institute of Neurology, London for providing tissue. AMI was funded by Alzheimer’s Research UK (ARUK), the MHMS General Charitable Trust and the UK Medical Research Council, TL is supported by an ARUK fellowship and PF by a Medical Research Council/Motor Neurone Disease Association Lady Edith Wolfson Fellowship.