- Open Access
Bidirectional nucleolar dysfunction in C9orf72 frontotemporal lobar degeneration
- Sarah Mizielinska†1, 2,
- Charlotte E. Ridler†1,
- Rubika Balendra†1, 3,
- Annora Thoeng1,
- Nathan S. Woodling3,
- Friedrich A. Grässer4,
- Vincent Plagnol5,
- Tammaryn Lashley6,
- Linda Partridge3, 7 and
- Adrian M. Isaacs1Email author
© The Author(s). 2017
Received: 5 April 2017
Accepted: 6 April 2017
Published: 18 April 2017
An intronic GGGGCC expansion in C9orf72 is the most common known cause of both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat expansion leads to the generation of sense and antisense repeat RNA aggregates and dipeptide repeat (DPR) proteins, generated by repeat-associated non-ATG translation. The arginine-rich DPR proteins poly(glycine-arginine or GR) and poly(proline-arginine or PR) are potently neurotoxic and can localise to the nucleolus when expressed in cells, resulting in enlarged nucleoli with disrupted functionality. Furthermore, GGGGCC repeat RNA can bind nucleolar proteins in vitro. However, the relevance of nucleolar stress is unclear, as the arginine-rich DPR proteins do not localise to the nucleolus in C9orf72-associated FTLD/ALS (C9FTLD/ALS) patient brain. We measured nucleolar size in C9FTLD frontal cortex neurons using a three-dimensional, volumetric approach. Intriguingly, we found that C9FTLD brain exhibited bidirectional nucleolar stress. C9FTLD neuronal nucleoli were significantly smaller than control neuronal nucleoli. However, within C9FTLD brains, neurons containing poly(GR) inclusions had significantly larger nucleolar volumes than neurons without poly(GR) inclusions. In addition, expression of poly(GR) in adult Drosophila neurons led to significantly enlarged nucleoli. A small but significant increase in nucleolar volume was also observed in C9FTLD frontal cortex neurons containing GGGGCC repeat-containing RNA foci. These data show that nucleolar abnormalities are a consistent feature of C9FTLD brain, but that diverse pathomechanisms are at play, involving both DPR protein and repeat RNA toxicity.
An intronic GGGGCC expansion in C9orf72 is the most common known cause of both frontotemporal lobar dementia (FTLD) and amyotrophic lateral sclerosis (ALS) [7, 27]. Healthy individuals have fewer than 30 repeats, whereas patients have several hundred to several thousand repeats [2, 7, 33]. The repeat expansion mutation might cause pathogenesis by loss of function of the C9orf72 protein, or gain-of-function mechanisms from i) sense and antisense repeat RNA and/or ii) the dipeptide repeat proteins poly(GA), poly(GP), poly(GR), poly(PR) and poly(AP), which are generated by repeat-associated non-ATG translation .
Previously, over-expression of poly(GR) and poly(PR) were reported to be extremely toxic to adult Drosophila neurons and primary rat neurons [19, 34]. Over-expression of poly(GR) or poly(PR) repeats in cell models leads to their localisation in the nucleolus, and results in enlarged nucleoli and altered ribosomal RNA processing [13, 32, 34]. Additionally, nucleolar proteins modify poly(PR) toxicity in yeast . C9orf72 repeat RNA has been shown to bind nucleolar proteins in vitro, suggesting that RNA toxicity may also contribute to nucleolar dysfunction [5, 10]. Dispersal of the nucleolar protein nucleolin was observed within neurons of adult C9orf72 BAC transgenic mice, but no consequent change in ribosomal RNA biogenesis was detected . However, enlarged nucleoli and altered ribosomal RNA processing have been reported in cells derived from patients with a C9orf72 repeat expansion, including lymphocytes, fibroblasts and induced pluripotent stem cells differentiated into neurons . Increases in nucleolar size and number are generally considered to be a consequence of cell demand for ribosome biogenesis, and are a hallmark of tumour cells in cancer . However, disruption of nucleolar structure and ribosomal RNA transcription have also been reported in several neurodegenerative diseases, both in post-mortem patient tissue and animal models .
Recent proteomic studies have found that the binding partners of the arginine-rich DPR proteins are enriched in proteins containing low-complexity domains (LCDs), which are often found in membraneless organelles such as the nucleolus [14, 15]. The LCDs facilitate liquid-liquid phase separation, enabling cellular partitioning of membraneless organelles. The nucleolar protein nucleophosmin has an LCD that is bound by poly(GR) and poly(PR), altering its phase-separation properties and leading to altered nucleolar dynamics in cell culture assays . These data suggest that disruption of the function of membraneless organelles is an important pathway in C9FTLD/ALS pathogenesis, and consequently confirmation of these findings in patient tissue is a key next step.
The relevance of nucleolar stress to disease pathogenesis has been questioned, as poly(GR) and poly(PR) inclusions do not localise to the nucleolus in C9FTLD/ALS patient brain and nucleolar size was reported to be unaffected in a small sample of C9FTLD/ALS brains . To provide clarity to this important issue, we measured nucleolar size in C9FTLD brains using a three-dimensional, volumetric approach, rather than single-plane area measurements. We show here, for the first time, that nucleolar stress does occur in C9FTLD patient brain in a bidirectional manner and is associated with both repeat RNA and poly(GR) pathology.
Materials and methods
Brain specimens (described in Additional file 1: Table S1) were obtained from Queen Square Brain Bank for Neurological Disorders, UCL Institute of Neurology, London. Samples were fixed in 10% buffered formalin for histopathology and immunohistochemistry. Histological sections from the anterior frontal F1-F2 region were analysed. We analysed eight controls with no known neurodegenerative disease, eight cases with heterozygous C9orf72 repeat expansions, and one homozygous repeat expansion case. Seven expansion cases (cases 9, 12, 14–18) were previously described , including the homozygous repeat expansion case (case 17) . The neuropathological diagnosis was determined using established diagnostic criteria, in line with consensus recommendations for the FTLD spectrum . This study was approved by the UCL Institute of Neurology and National Hospital for Neurology and Neurosurgery Local Research Ethics Committee.
Fly stocks and husbandry
All fly stocks were maintained and experiments conducted at 25 °C on a 12 h:12 h light:dark cycle at 60% constant humidity, on standard sugar-yeast food containing 15 g/L agar, 50 g/L sugar, 100 g/L brewer’s yeast, 30 ml/L nipagin (10% in ethanol) and 3 ml/L propionic acid). RU486 (Sigma) dissolved in ethanol was added to a final concentration of 200 μM. Published transgenic fly lines expressing 100 repeats of the DPR proteins GR and glycine-alanine (GA), under the upstream activating sequence (UAS) promoter, were used . Expression of these DPR proteins was restricted to adult neurons using the inducible elav-GeneSwitch driver . Two days after eclosion adult elav-GeneSwitch > (GR)100 and elav-GeneSwitch > (GA)100 flies were fed with food containing 200 μM RU486 to induce expression of the DPR proteins, or with no RU486 as a control, for 7 days.
Human post-mortem brain
Twenty micrometer-thick paraffin-embedded sections of human post-mortem frontal cortex tissue were dewaxed in xylene and rehydrated in graded alcohols, followed by antigen-retrieval in proteinase K (S3020, Dako) for 2 mins, and pressure cooking in citrate buffer (0.1 M, pH6) for 10 mins. Following a PBS wash, sections were blocked in 10% foetal bovine serum in PBS for 30 mins, and then incubated with primary antibody in PBS overnight at 4 °C. The following primary antibodies were used: nucleophosmin (1:1000, Abcam, ab10530), nucleolin (1:50, Santa Cruz, sc-8031), NeuN (1:1000, Millipore, ABN78), and 5H9 antibody against poly(GR) (1:25, ). Samples were then washed in PBS and incubated with the appropriate secondary antibodies (Alexa Fluor, Life Technologies, 1:500 in PBS) for 1 h at room temperature. To reduce autofluorescent background, a 10 min incubation was carried out in Sudan black B (Sigma, 0.2% in 70% ethanol/30% PBS) followed by additional PBS washes. Samples were mounted using ProLong® Gold Antifade Mountant with DAPI (Life Technologies).
Brains were dissected in PBS containing 0.3% Triton-X (PBST) and then fixed in 4% PFA in PBST at room temperature for 15 min. Brains were washed three times in PBST, blocked in 10% bovine serum albumin (BSA) in PBST for 1 h at room temperature, and then incubated in primary antibody in 10% BSA in PBST at 4 °C for 48 h. The following primary antibodies were used: poly(GR) (1:25, 5H9 - as above), poly(GA) (1:300, Cosmobio, CAC-TIP-C9-P01), fibrillarin (1:400, Abcam, ab4566). Brains were washed in PBST and incubated with appropriate secondary antibodies (Alexa Fluor, Life Technologies, 1:250 in 10% BSA in PBST) at 4 °C overnight. Brains were then washed three times in PBST and whole mounted onto glass slides in VECTASHIELD® mounting medium with DAPI (Vector Laboratories, H-1200).
RNA fluorescent in situ hybridisation with immunofluorescence
Twenty micrometer-thick paraffin-embedded sections of human post-mortem frontal cortex tissue were dewaxed followed by antigen-retrieval as above. The protocol was then continued as per Mizielinska et al., 2013 . Briefly, sections were washed in 2 × SSC and incubated for 30 min in pre-hybridisation solution (50% formamide/2 × SSC) at 80 °C, and hybridised with a (GGCCCC)4 2′-O-methyl RNA probe labelled with Cy3 (Integrated DNA Technologies) for 2 h at 80 °C in 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). Sections were washed three times for 30 min each in 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, immunostaining was continued from blocking as above.
Image acquisition and quantification of nucleolar volume
Images of human post-mortem brain were acquired using an LSM710 confocal microscope (Zeiss) using a plan-apochromat 40×/1.4 NA oil immersion objective. Fluorescence intensity was set to peak for each patient to account for case-to-case variability. Images of Drosophila brains were acquired with an LSM700 confocal microscope (Zeiss) using a plan-apochromat 63× oil/1.4 NA immersion objective and the same settings for all images.
Immunofluorescence of human post-mortem brain
Twenty z-stack images were acquired per sample, consisting of twelve 2 μm z-planes of 2084 × 2084 pixels, over a 20 μm depth (of which approximately 14 μm contained tissue after processing). A minimum of 200 neurons were analysed (range 217–973), including a minimum of 16 poly(GR) aggregate-bearing neurons (range 16–139) per C9FTLD frontal cortex. For detection of nuclei, nucleoli and NeuN, gain was adjusted to peak intensity for each patient. Three-dimensional volumetric analysis of confocal images was performed using Volocity image analysis software (Perkin Elmer). Nucleoli (identified by either nucleophosmin or nucleolin), DAPI-stained nuclei and NeuN-positive neurons were identified by fluorescence-intensity threshold a set number of standard deviations above mean voxel intensity for each image. As poly(GR) aggregates are present in C9FTLD patient sections but not control sections, an absolute intensity threshold was used for detection. Low-level poly(GR) signal was observed inside the nucleolus in both cases and controls and was consequently excluded as background. No intra-nucleolar poly(GR) aggregates were observed by eye. Objects defined as nucleoli, poly(GR) aggregates and nuclei were compartmentalised into NeuN-positive neurons to give the volume of each stain present in each neuron. Occasionally neurons contained more than one nucleoli; the volumes of these structures were combined for analysis. Neurons in which no nucleolar stain was detected were excluded from analysis.
Immunofluorescence of Drosophila
Four z-stack images covering a 15 μm depth were taken from each Drosophila brain, achieving a minimum of 174 nucleoli per brain (range 174–920), including a minimum of five poly(GR) (range 5–63) or eight poly(GA) aggregates (range 8–53). Image acquisition and volumetric analysis of DAPI-stained nuclei, nucleoli and poly(GR) or poly(GA) aggregates was performed similarly to human post-mortem brain, except that no exclusion of background nucleolar staining of aggregates was required and aggregates were assigned to nucleoli by proximity of the centroids. Occasional low-level staining of poly(GR) or poly(GA) was observed in uninduced flies owing to the known leaky expression of the elav-GeneSwitch driver ; nucleoli associated with these aggregates were excluded from the subsequent analysis.
RNA fluorescent in situ hybridisation with immunofluorescence in human post-mortem brain
Ten to seventeen z-stack images at 2084 × 2084 pixels covering a 20 μm depth were acquired to ensure a minimum of 100 neurons were analysed (range 134–232), including a minimum of 38 RNA foci-bearing neurons (range 38–88) per C9FTLD frontal cortex. Image acquisition and volumetric analysis of nucleoli and poly(GR) aggregates was performed similarly to human post-mortem brain immunofluorescence analysis described above; RNA foci were additionally counted using the touch count tool; neurons were determined either by NeuN-positive staining or by morphology (size and DAPI staining).
Data are presented as median nucleolar or nuclear volume per individual patient or fly owing to the non-normal distribution of volumes in all data sets. Unpaired t tests were carried out to compare median nucleolar and nuclear volumes between control and C9FTLD patient brain, and between uninduced and induced (GR)100 or (GA)100 transgenic Drosophila. Paired regression analysis was carried out between neurons with and without pathology (DPR protein inclusions or RNA foci) in C9FTLD patient brain or Drosophila.
Nucleolar volume is reduced in C9FTLD patient brain
Nucleolar volume is increased in poly(GR) inclusion-bearing neurons in C9FTLD patient brain
Nucleolar enlargement can be evoked by expression of poly(GR) protein in vivo
Nucleolar volume is increased in RNA foci-bearing neurons in C9FTLD patient brain
Here, we present evidence for C9orf72 mutation-associated nucleolar alterations in patient brain. Using three-dimensional volumetric imaging, we have unveiled bidirectional changes in nucleolar size in C9FTLD patient brain. When comparing all frontal cortical neurons irrespective of the presence of RNA or protein pathology, C9FTLD cells had a smaller nucleophosmin-positive nucleolar volume than those in non-neurodegenerative control brains. However, within C9FTLD neurons, the presence of poly(GR) inclusions or RNA foci resulted in significant enlargement of nucleoli.
Thus far striking in vitro evidence exists for increased nucleolar size in both cells overexpressing the arginine-rich DPR proteins [13, 32, 34], and cells derived from C9FTLD/ALS patients . However, it is perhaps unsurprising that neurons in C9FTLD frontal cortex displayed an overall reduction in nucleolar size, as this type of nucleolar stress has been observed in several other neurodegenerative diseases . Nucleolar shrinkage would be expected to reduce ribosome biogenesis and generally decrease cell metabolism. Indeed, reduced ribosomal RNA maturation has been reported in C9ALS patient lymphocytes and motor cortex . Acutely, this change might be protective under conditions of cell stress by limiting energy expenditure, but chronically would result in cell damage and ultimately cell death . The nucleolar protein nucleophosmin is known to be downregulated upon excitotoxic stimuli in neurons and can increase levels of the classic nucleolar stress marker p53; however, nucleophosmin-induced cell death appears to be p53-independent .
In agreement with previous in vitro evidence (detailed above), we also unveiled a second type of nucleolar stress in C9FTLD neurons containing inclusions of the arginine-rich DPR protein poly(GR), in which nucleolar volume was almost double that of neurons without inclusions. This increased volume could be expected to increase ribosomal biogenesis; however, initial studies in vitro show reduced ribosomal RNA production when the arginine-rich DPR proteins are overexpressed in cells [13, 32]. These findings could suggest that the enlarged nucleoli are dysfunctional rather than implying an increased metabolism. Increases in nucleophosmin-positive nucleolar size owing to the presence of poly(GR) inclusions were replicated with another nucleolar protein nucleolin, although no difference was detected between control and C9FTLD cases. Both nucleophosmin and nucleolin localise to the nucleolus, but nucleophosmin is predominantly found in the granular centre of nucleoli, and nucleolin in both the dense fibrillary centre and the granular centre of nucleoli. Volume measurements for nucleolin were lower than those for nucleophosmin, which might reflect differences in detection sensitivity. Further work using super-resolution microscopy would be required to investigate these sub-nucleolar structures in more detail.
One discrepancy between cell models of C9FTLD/ALS and patient tissue is the localisation of the poly(GR) protein inclusions. In cells, short poly(GR) peptides readily localise to the nucleolus where they exert the aforementioned nucleolar enlargement and impaired ribosome biogenesis . By contrast, in patient brain, poly(GR) protein is detected primarily in large cytoplasmic inclusions and occasionally in small dot-like perinucleolar inclusions [1, 22]. Here, we have provided evidence linking cytoplasmic poly(GR) inclusions in patient tissue to changes in nucleolar volume, which has previously only been detected in cell models. Although poly(GR) protein is not detected in the nucleolus in C9FTLD patient brain, cytoplasmic inclusions might be present primarily in neurons that have a high poly(GR) protein load, including soluble protein, undetectable by the immunofluorescence methods used in this study.
It is likely that a proportion of the cytoplasmic inclusions that are detected with poly(GR) immunostaining also contain some of the other dipeptide repeat proteins, as the DPR proteins have previously been shown to co-occur within individual inclusions . The most commonly occurring DPR protein pathology is inclusions containing poly(GA) protein, which also evoke neurotoxicity [18, 19, 35, 36]. However, we specifically investigated the effect of poly(GR) and poly(GA) proteins on neuronal nucleoli in vivo, and observed a much higher capability for poly(GR) to evoke these changes than poly(GA) protein. Poly(GR) protein is thus highly likely to be responsible for the nucleolar enlargement associated with the poly(GR) inclusion pathology that we have detected in C9FTLD patient tissue.
As functional consequences of pathologies are difficult to ascertain in post-mortem patient brain, whether proteinaceous inclusions are toxic or whether they represent a protective mechanism by sequestering more toxic soluble protein species is often debated. Indeed, one study found that although nucleolar size is decreased in the brains of patients with Alzheimer’s disease compared with controls, it is increased in patients with asymptomatic Alzheimer’s disease, who exhibit Aβ plaques and neurofibrillary tangles but normal cognitive function , suggesting that increased nucleolar volume could also represent an early neuroprotective mechanism.
Surprisingly, we also uncovered a third association, between C9orf72 repeat RNA pathology and nucleolar volume, albeit an association with a smaller effect size (1.2-fold increase) than the change from the presence of poly(GR) inclusions (1.8-fold increase). This nucleolar enlargement was predominantly mediated by direct association of RNA foci with the nucleolar structure, evidenced by a larger increase in volume in nucleoli that colocalised with foci. C9orf72 sense repeat RNA has previously been shown to be able to interact with the nucleolar proteins in vitro [5, 10]. Depletion of RNA-binding proteins by RNA foci is hypothesised to be a key pathomechanism in other non-coding repeat expansion disorders, which are characterised by certain RNA-binding proteins being found within RNA foci. However, the localisation pattern of RNA foci and nucleophosmin that we detected did not recapitulate these findings, as RNA foci were generally detected on the edge of nucleolar structures.
Rather than nucleolar enlargement representing an increase in cell metabolism, it might infer a dispersal of the normal physiological structure of the organelle. Membraneless structures such as nucleoli are dynamic and respond to environmental conditions and cell stress, consequently C9orf72 arginine-rich DPR proteins or repeat RNA could either disrupt or prevent reformation of these structures. Indeed, recent studies that examined the arginine-rich DPR proteins interacting with LCD-containing proteins showed perturbation of phase separation, a process thought to recapitulate the formation of membraneless organelles [14, 15]. In addition, the arginine-rich DPRs themlseves can phase separate and alter the phase separation dynamics of other proteins . Alterations in membraneless organelles other than the nucleolus have also been observed in studies of C9FTD/ALS. Overexpression of C9orf72 repeats in cells causes an increase in the percentage of cells containing stress granules . In addition overexpressed poly(GR) or poly(PR) in cells can localise to [3, 35] and prevent the formation of stress granules subjected to a stressor . In C9FTD/ALS patient cells repeat RNA is localised to FMRP-positive transport granules , and both the number of stress granules  and P bodies  are increased. In the case of repeat RNA, nucleolar disruption might occur by sequestration of nucleolar proteins by soluble RNA species which would not be detected using the methodology in this study. The presence of RNA foci could reflect cell nuclei in which the concentration of retained repeat RNA is the highest, and thus a nuclear environment in which this scenario is more likely to occur.
In addition to effect size, the proportion of the C9FTLD neuronal populations affected by the bidirectional nucleolar changes that we have observed are also important to consider. Overall, in C9FTLD frontal cortical neurons a 25% reduction in nucleolar size exists compared with controls. However, nucleolar size within these neurons is clearly heterogeneous. The third of neurons that contain RNA foci show a 1.2-fold nucleolar enlargement, and the 6% of neurons that contain poly(GR) inclusions show a 1.8-fold enlargement, compared with neurons without these pathologies. While we can speculate on how this change affects neuronal function within these subsets of neurons, how functionality of the frontal cortex is affected at a network level is unknown.
In conclusion, we have provided the first evidence for nucleolar stress in C9FTLD patient brain, by using three-dimensional volumetric imaging. Bidirectional changes in nucleolar volume dependent on the presence or absence of C9orf72 repeat RNA or protein pathologies show the heterogeneity of pathomechanisms in patient neurons, but support findings in current experimental models and have important implications for understanding the complex disease processes involved in C9FTLD/ALS.
We thank 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 Motor Neurone Disease Association and the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (648716 - C9ND), LP was funded by the Wellcome Trust (098565/Z/12/Z) and the Max Plank Society, TL is supported by an ARUK fellowship, and RB is a Leonard Wolfson Clinical Research Training Fellow and funded by a Wellcome Trust Research Training Fellowship (107196/Z/14/Z).
SM, CER, RB and AMI wrote the manuscript; SM, AMI and TL designed and interpreted the human studies; RB, NSW, AMI and LP designed and interpreted the Drosophila studies; SM, CER, RB and AT performed immunostaining, imaging and analysis; FAG generated the 5H9 antibody; VP performed the statistical analyses. All authors read and approved the final manuscript.
The authors declare no competing interests.
Ethics approval and consent to participate
This study was approved by the UCL Institute of Neurology and National Hospital for Neurology and Neurosurgery Local Research Ethics Committee, reference LREC 03/N154.
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