Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS

Abstract

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders with clinical, genetic, and neuropathological overlap. A hexanucleotide (GGGGCC) repeat expansion in a non-coding region of C9ORF72 is the major genetic cause of both diseases. The mechanisms by which this repeat expansion causes "c9FTD/ALS" are not definitively known, but RNA-mediated toxicity is a likely culprit. RNA transcripts of the expanded GGGGCC repeat form nuclear foci in c9FTD/ALS, and also undergo repeat-associated non-ATG (RAN) translation resulting in the production of three aggregation-prone proteins. The goal of this study was to examine whether antisense transcripts resulting from bidirectional transcription of the expanded repeat behave in a similar manner. We show that ectopic expression of (CCCCGG)66 in cultured cells results in foci formation. Using novel polyclonal antibodies for the detection of possible (CCCCGG)exp RAN proteins [poly(PR), poly(GP) and poly(PA)], we validated that (CCCCGG)66 is also subject to RAN translation in transfected cells. Of importance, foci composed of antisense transcripts are observed in the frontal cortex, spinal cord and cerebellum of c9FTD/ALS cases, and neuronal inclusions of poly(PR), poly(GP) and poly(PA) are present in various brain tissues in c9FTD/ALS, but not in other neurodegenerative diseases, including CAG repeat disorders. Of note, RNA foci and poly(GP) inclusions infrequently co-occur in the same cell, suggesting these events represent two distinct ways in which the C9ORF72 repeat expansion may evoke neurotoxic effects. These findings provide mechanistic insight into the pathogenesis of c9FTD/ALS, and have significant implications for therapeutic strategies.

Fig. 1

Schematic representation of the possible proteins generated by RAN translation of expanded GGGGCC and CCCCGG repeats in all possible reading frames

Fig. 2

Antibody characterization for c9RAN proteins. a The immunoreactivity of antibodies to c9RAN proteins towards (PA)₈, (PR)₈ and (GP)₈ peptides was measured by adsorbing peptides onto carbon electrodes in 96-well MSD plates, and co-incubating wells with anti-PA, anti-PR or anti-GP antibodies, and a SULFO-tagged anti-rabbit secondary antibody. Antibody binding to respective peptides was quantified by measuring the intensity of emitted light upon electrochemical stimulation of the plate using the MSD Sector Imager 2400. For each pair of antibodies, binding responses were normalized to the signal of the antibody showing the highest binding to its respective antigen. Error bars indicate standard deviations from duplicate wells. b Western blot analysis of lysates from HEK293T cells transfected to express enhanced GFP-tagged (PA)₅, (PR)₅ or (GP)₅. Blots were probed with the indicated antibodies. c Immunofluorescence staining of HEK293T cells transfected to express the indicated enhanced GFP (green)-tagged peptides using anti-PA, anti-PR or anti-GP (red) antibodies. Nuclei are stained with Hoechst (blue). Scale bar 10 μm. Note that similar studies were conducted to test potential cross-reactivity of antibodies to poly(GA) and poly(GR), as shown in Online Resource 3

Fig. 3

Expression of expanded CCCCGG repeats in cultured cells leads to foci formation and expression of c9RAN proteins. a HEK293T cells transfected to express (CCCCGG)₂ or (CCCCGG)₆₆ were subjected to RNA fluorescence in situ hybridization using a probe against CCCCGG repeat transcripts. Note the foci (red) in Hoechst-stained nuclei (blue) of (CCCCGG)₆₆-expressing cells, but not (CCCCGG)₂-expressing cells. Scale bar 5 μm. b Western blot analysis of lysates from (CCCCGG)_n-expressing cells shows that poly(PR) and poly(GP) proteins, but not poly(PA) proteins, are expressed in cells transfected with (CCCCGG)₆₆. No c9RAN protein was detected in control cells expressing non-expanded (CCCCGG)₂

Fig. 4

C9ORF72 hexanucleotide repeat transcripts form nuclear RNA foci in frontal cortex, spinal cord and cerebellum in c9FTD/ALS. Fluorescence in situ hybridization (FISH) of c9FTD/ALS frontal cortex (a) and c9ALS spinal cord (b) tissue using a probe against the CCCCGG repeat transcripts shows RNA foci (red) in the nucleus (stained with DAPI, blue) of cells. In (b), note that foci are observed in motor neurons that stain positively for ChAT. c Cerebellar sections of c9FTD/ALS cases were subjected to FISH using a probe against CCCCGG repeat transcripts or GGGGCC repeat transcripts. In most instances, foci-bearing cells within the cerebellum were found in proximity to the Purkinje cell layer separating the molecular and granular layers. However, RNA foci were also observed in cells of the molecular layer, deep within the granular layer, in Purkinje cells, and in cells within the white matter. Scale bars 10 μm

Fig. 5

Nuclear RNA foci are present in both neurons and glia. Fluorescence in situ hybridization of c9FTD/ALS cerebellar tissue using a probe against the GGGGCC repeat was followed by immunofluorescence staining with the neuronal marker, MAP2, or the astrocytic marker, GFAP. Note that nuclear RNA foci are present in MAP2-positive and MAP2-negative cells, as well as GFAP-positive and GFAP-negative cells. Scale bar 10 μm

Fig. 6

Antisense c9RAN proteins in human post-mortem tissue. Immunohistochemistry reveals poly(PA)- [left column], poly(PR)- [middle column], and poly(GP)- [right column] reactive lesions throughout the central nervous system, including the hippocampus (endplate-CA3 on the top left, dentate fascia on the bottom right), cerebellum, amygdala, thalamus, motor cortex (layers 2–3), and medulla (inferior olivary nucleus). Lesions are often neuronal cytoplasmic inclusions (NCI) with a star-shaped morphology, but can also appear as dense NCI, small neuronal intranuclear inclusions, or diffuse neuronal “pre-inclusions”. Anti-GP, which detects poly(GP) proteins that can be made from both sense and antisense transcripts of the C9ORF72 expanded repeat, reveal greater pathologic burden compared to the anti-PA and PR antibodies. Case numbers correspond to c9FTD/ALS cases in Table 1. Scale bar 10 μm

Fig. 7

Nuclear RNA foci and poly(GP) inclusions are seldom observed in the same cell. Fluorescence in situ hybridization of frontal cortex and cerebellar tissue of c9FTD/ALS cases using a probe against sense and antisense C9ORF72 hexanucleotide repeat transcripts was followed by immunofluorescence staining to detect poly(GP) inclusions, which may result from RAN translation of both sense and antisense transcripts. Though infrequent, both foci and poly(GP) inclusions can co-occur in the same cell (cells indicated by an asterisk in a and b). Scale bars 10 μm. AS antisense foci, S sense foci. c To determine the percentage of cells having both foci and poly(GP) inclusions in the frontal cortex and cerebellum, quantitative analysis was undertaken on four cases co-stained for poly(GP) inclusions and either sense or antisense foci. For each section, we examined 25 cells with inclusions and determined whether they had foci, and examined 25 cells with foci and determined whether they had inclusions, for a total of 50 cells per section. Data are presented as mean ± SEM, n = 4. The brain region sampled (frontal cortex vs. cerebellum, P = 0.0292) and foci type (antisense vs. sense, P = 0.0011) both significantly affect the percentage of cells having foci and inclusions, as assessed by two-way ANOVA. To determine the percentage of cells with antisense and sense foci in the frontal cortex, the number of cells with foci and the total number of cells were counted in 12 randomly selected, non-overlapping fields for each case. Data are presented as mean ± SEM, n = 4. No significant difference between the percentage of cells with sense or antisense foci was detected, as assessed by paired two-tailed t test (P = 0.1916) (d). n.s. not significant