Antisense RNA foci in the motor neurons of C9ORF72-ALS patients are associated with TDP-43 proteinopathy

Abstract

GGGGCC repeat expansions of C9ORF72 represent the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We and others have proposed that RNA transcribed from the repeat sequence is toxic via sequestration of RNA-binding factors. Both GGGGCC-repeat (sense) and CCCCGG-repeat (antisense) molecules are detectable by fluorescence in situ hybridisation as RNA foci, but their relative expression pattern within the CNS and contribution to disease has not been determined. Blinded examination of CNS biosamples from ALS patients with a repeat expansion of C9ORF72 showed that antisense foci are present at a significantly higher frequency in cerebellar Purkinje neurons and motor neurons, whereas sense foci are present at a significantly higher frequency in cerebellar granule neurons. Consistent with this, inclusions containing sense or antisense derived dipeptide repeat proteins were present at significantly higher frequency in cerebellar granule neurons or motor neurons, respectively. Immunohistochemistry and UV-crosslinking studies showed that sense and antisense RNA molecules share similar interactions with SRSF2, hnRNP K, hnRNP A1, ALYREF, and hnRNP H/F. Together these data suggest that, although sense and antisense RNA molecules might be expected to be equally toxic via their shared protein binding partners, distinct patterns of expression in various CNS neuronal populations could lead to relative differences in their contribution to the pathogenesis of neuronal injury. Moreover in motor neurons, which are the primary target of pathology in ALS, the presence of antisense foci (χ (2), p < 0.00001) but not sense foci (χ (2), p = 0.75) correlated with mislocalisation of TDP-43, which is the hallmark of ALS neurodegeneration. This has implications for translational approaches to C9ORF72 disease, and furthermore interacting RNA-processing factors and transcriptional activators responsible for antisense versus sense transcription might represent novel therapeutic targets.

Fig. 1

RNA FISH reveals the distribution of sense and antisense RNA foci in five neuronal populations. Representative images show that antisense RNA foci are more numerous in cerebellar Purkinje neurons and motor neurons; in contrast sense, RNA foci are more numerous in cerebellar granule neurons; neither population is more abundant in dentate gyrus neurons and CA4 subfield neurons of the hippocampus (a). Smaller foci are highlighted by arrowheads. As has been previously demonstrated for sense foci, antisense foci are occasionally present in the cytoplasm of mature motor neurons (b, arrowhead, the nuclear border is indicated by a dotted line). Scale bar 3 µm

Fig. 2

Immunohistochemistry reveals the distribution of dipeptide repeat protein containing inclusions consisting of species derived from sense and antisense repeat RNAs in two neuronal populations. Representative images showing that poly-GA and poly-GR containing inclusions are more numerous in cerebellar granule neurons, whereas poly-PA and poly-PR containing inclusions are more numerous in motor neurons. Staining was carried out individually for each protein (a) and then poly-GA and poly-PA were examined by dual staining (b). Inclusions are highlighted by arrowheads. Scale bar 3 µm

Fig. 3

Combined RNA FISH and IHC demonstrate colocalisation of nucleolin and nuclear speckle components with antisense RNA foci in Purkinje neurons from C9ORF72-ALS patients and the distribution of these proteins in Purkinje neurons from control individuals. SRSF2 (a), hnRNP A1 (b), hnRNP H/F (c), ALYREF (d), and hnRNP K (e) are observed to colocalise with antisense RNA foci (arrows) in Purkinje neurons from C9ORF72-ALS patients. A large scale view is shown to the left of a zoomed-in image. Colocalisation events are enlarged including orthogonal views, and unmerged protein and RNA foci are shown for comparison. There was not a significant difference between the staining of these proteins in controls and C9ORF72+ individuals, but no antisense RNA foci are observed in controls. Nucleolin was not observed to colocalise with antisense RNA foci (f); moreover, the distribution of nucleolin was variable in C9ORF72+ Purkinje neurons. In some cells, nucleolin was prominently nucleolar (f, left panel) and in other cells it was dispersed throughout the nucleus (f, right panel, RNA focus is indicated by an arrowhead). The dotted line illustrates the nuclear border in images a–e and the nucleolar border in image f. Scale bar 3 µm

Fig. 3

Combined RNA FISH and IHC demonstrate colocalisation of nucleolin and nuclear speckle components with antisense RNA foci in Purkinje neurons from C9ORF72-ALS patients and the distribution of these proteins in Purkinje neurons from control individuals. SRSF2 (a), hnRNP A1 (b), hnRNP H/F (c), ALYREF (d), and hnRNP K (e) are observed to colocalise with antisense RNA foci (arrows) in Purkinje neurons from C9ORF72-ALS patients. A large scale view is shown to the left of a zoomed-in image. Colocalisation events are enlarged including orthogonal views, and unmerged protein and RNA foci are shown for comparison. There was not a significant difference between the staining of these proteins in controls and C9ORF72+ individuals, but no antisense RNA foci are observed in controls. Nucleolin was not observed to colocalise with antisense RNA foci (f); moreover, the distribution of nucleolin was variable in C9ORF72+ Purkinje neurons. In some cells, nucleolin was prominently nucleolar (f, left panel) and in other cells it was dispersed throughout the nucleus (f, right panel, RNA focus is indicated by an arrowhead). The dotted line illustrates the nuclear border in images a–e and the nucleolar border in image f. Scale bar 3 µm

Fig. 3

Combined RNA FISH and IHC demonstrate colocalisation of nucleolin and nuclear speckle components with antisense RNA foci in Purkinje neurons from C9ORF72-ALS patients and the distribution of these proteins in Purkinje neurons from control individuals. SRSF2 (a), hnRNP A1 (b), hnRNP H/F (c), ALYREF (d), and hnRNP K (e) are observed to colocalise with antisense RNA foci (arrows) in Purkinje neurons from C9ORF72-ALS patients. A large scale view is shown to the left of a zoomed-in image. Colocalisation events are enlarged including orthogonal views, and unmerged protein and RNA foci are shown for comparison. There was not a significant difference between the staining of these proteins in controls and C9ORF72+ individuals, but no antisense RNA foci are observed in controls. Nucleolin was not observed to colocalise with antisense RNA foci (f); moreover, the distribution of nucleolin was variable in C9ORF72+ Purkinje neurons. In some cells, nucleolin was prominently nucleolar (f, left panel) and in other cells it was dispersed throughout the nucleus (f, right panel, RNA focus is indicated by an arrowhead). The dotted line illustrates the nuclear border in images a–e and the nucleolar border in image f. Scale bar 3 µm

Fig. 4

Specific and direct interactions between (GGGGCC)₅ and/or (CCCCGG)₅ and hnRNP A1, hnRNP F, SFRS2, and ALYREF but not hnRNP K or Magoh (negative control). Magoh, SRSF2 9-101, ALYREF, hnRNP A1-like2, hnRNP K, and hnRNP F were expressed in E. coli and purified (see Supplementary Table 4). (GGGGCC)₅ (sense) and (CCCCGG)₅ (antisense) RNA oligonucleotides were end labelled with polynucleotide kinase using [ɣ-32P]-ATP, prior to incubation with purified proteins. RNA was covalently bound (+) or not (−) following UV irradiation. The absence of radioactive signal (right panel, PhosphoImage) in the absence of UV irradiation demonstrates specificity of direct binding observed after UV treatment. All gels shown in the different panels were exposed simultaneously for the same amount of time (4 h). Note that a high molecular weight band is also observed for ALYREF due to oligomerisation properties [10]

Fig. 5

TDP-43 IHC and RNA FISH demonstrate that antisense RNA foci are significantly associated with nuclear clearance of TDP-43 in motor neurons. Representative images showing that antisense RNA foci (arrowheads) are significantly associated with nuclear clearance of TDP-43 in motor neurons of C9ORF72-ALS patients; split channel images are provided for comparison. Cleared TDP-43 may be present within a cytoplasmic inclusion (upper panels; RNA focus is indicated by the arrowhead, a compact inclusion is arrowed) or simply present in the cytoplasm (middle panels; RNA foci are indicated by arrowheads). In contrast, the absence of antisense RNA foci is significantly associated with the presence of nuclear TDP-43 (lower panels). Scale bar 3 µm