The carboxyl termini of RAN translated GGGGCC nucleotide repeat expansions modulate toxicity in models of ALS/FTD

Abstract

An intronic hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This repeat is thought to elicit toxicity through RNA mediated protein sequestration and repeat-associated non-AUG (RAN) translation of dipeptide repeat proteins (DPRs). We generated a series of transgenic Drosophila models expressing GGGGCC (G₄C₂) repeats either inside of an artificial intron within a GFP reporter or within the 5' untranslated region (UTR) of GFP placed in different downstream reading frames. Expression of 484 intronic repeats elicited minimal alterations in eye morphology, viability, longevity, or larval crawling but did trigger RNA foci formation, consistent with prior reports. In contrast, insertion of repeats into the 5' UTR elicited differential toxicity that was dependent on the reading frame of GFP relative to the repeat. Greater toxicity correlated with a short and unstructured carboxyl terminus (C-terminus) in the glycine-arginine (GR) RAN protein reading frame. This change in C-terminal sequence triggered nuclear accumulation of all three RAN DPRs. A similar differential toxicity and dependence on the GR C-terminus was observed when repeats were expressed in rodent neurons. The presence of the native C-termini across all three reading frames was partly protective. Taken together, these findings suggest that C-terminal sequences outside of the repeat region may alter the behavior and toxicity of dipeptide repeat proteins derived from GGGGCC repeats.

Keywords: Amyotrophic lateral sclerosis; C9ORF72; Carboxyl terminus; Dipeptide repeat proteins (DPRs); Drosophila; Repeat-associated non-AUG translation (RAN).

Fig. 1

Intronic G4C2 repeats influence splicing and elicit RNA foci in Drosophila.a Schematic of constructs used to generate transgenic fly lines. The Prospero fly intron 1 (gray lines) was introduced into UAS-GFP (blue lines). Either three G4C2 repeats or serial (G4C2)21–28 repeat units separated by 14 nt interruptions were serially inserted into the intron. b Locations of the primer pairs used for measuring unspliced, spliced, and total mRNA are show in the schematic. Quantification of the expression of the mature (left) and unspliced (right) GFP product in the indicated fly lines (right), n = 9. c Western blot of lysates from heads of G4C2 intronic repeat flies (left) with quantification of GFP normalized to beta-tubulin (right), n = 5. d Quantification of RNA foci in the retina of the indicated intronic fly, n = 10. e Representative eye phenotypes from flies of the indicated genotypes crossed to GMR-GAL4 to drive expression in developing ommatidia (left) and quantification of eye phenotype scores (right). f Quantification of the number of progeny carrying the transgene after flies of the indicated genotypes were crossed to a ubiquitous driver (act5c-GAL4). g Quantification of the distance crawled by 3rd instar larvae from the crosses of the indicated fly genotypes to a larval motor neuron specific driver (OK6-GAL4). h Flies carrying the indicated transgenes were crossed to a Tubulin Geneswitch driver (Tub5) to allow ubiquitous expression post eclosion. Adult male flies were placed on food containing RU-486 to activate gene expression and their viability was tracked over 50 days. Graphs represent means ± SEM. * p < 0.05; ** p < 0.01 by Kruskal-Wallis after Dunn’s correction for multiple comparisons. The number of flies for each genotype for E, F, G and H was > 100

Fig. 2

Drosophila 5’UTR G4C2 repeat models. a Schematic of constructs used to generate transgenic fly lines. (G4C2)28 repeats were inserted into the 5’UTR of GFP without an upstream start codon in all three reading frames relative to the GFP reading frame. b Western blot (left) of lysates from heads of G4C2 exonic repeat flies normalized to β-tubulin (right), n = 6. c Quantification of RNA foci in the retina of the indicated fly lines, n = 10. Graphs represent means ± SEM. * p < 0.05; ** p < 0.01 by Kruskal-Wallis after Dunn’s correction for multiple comparisons

Fig. 3

5’UTR G4C2 repeats with different carboxyl termini elicit differential toxicity in Drosophilaa Representative eye phenotypes from flies of the indicated genotypes crossed to GMR-GAL4 to drive expression in developing ommatidia (right) and quantification of eye phenotype scores (right). b The number of progeny carrying the transgene was determined after flies of the indicated genotypes were crossed to a ubiquitous driver (act5c-GAL4). c Quantification of the distance crawled by 3rd instar larvae from the crosses of the indicated fly genotypes to a larval motor neuron specific driver (OK6-GAL4). d Flies carrying the indicated transgenes were crossed to a Tubulin Geneswitch driver (Tub5) to allow ubiquitous expression post eclosion. Adult male flies were placed on food containing RU-486 to activate gene expression and their viability was tracked over 50 days. Graphs represent means ± SEM. ** p < 0.01 by Kruskal-Wallis after Dunn’s correction for multiple comparisons. Log-rank (Mantel-Cox) test for survival, * p < 0.05, ** p < 0.01. The number of flies for each genotype was > 100

Fig. 4

G4C2 repeats toxicity is associated with nuclear accumulation of RAN translation peptides. a Representative images of transverse retinal sections from Drosophila of the indicated genotypes probed with antibody against GA dipeptide repeats. b Quantification of total GA repeat staining by immunofluorescence (left) and the percentile of cells with significant nuclear accumulation of GA DPR staining for the indicated genotypes (right). c Representative Immunofluorescence images of Drosophila retinal sections probed with antibodies against the GP dipeptide repeats. d Quantification of total GP repeat staining by immunofluorescence (left) and the percentile of cells with significant nuclear accumulation of GP DPR staining for the indicated genotypes (right). E) Representative Immunofluorescence image of transverse retinal sections from Drosophila of the indicated genotypes probed with antibody against GR dipeptide repeats. F) Quantification of total GR repeat staining by immunofluorescence (left) and the percentile of cells with significant nuclear accumulation of GR DPR staining for the indicated genotypes. Graphs represent means ± SEM. * p < 0.05; ** p < 0.01 by Kruskal-Wallis after Dunn’s correction for multiple comparisons. The number of flies analyzed for each respective genotype in B, D and F was 10

Fig. 5

Native carboxyl terminal sequences reduce GGGGCC repeat toxicity in rodent neurons and in flies. a Top: Schematic of the constructs transfected into neurons, with GFP placed in different reading frames relative to the GGGGCC repeat. Bottom: Neuronal survival of neurons expressing these constructs is graphed as the cumulative risk of death (Y axis, with higher values reflecting increased death) over time (X-axis, measured in hours). b Top: Schematic of the constructs transfected into neurons with shorter or expanded G4C2 repeats and with or without the native Cterminus. Bottom: Cumulative risk of death of neurons. c Top: Schematic of the constructs used to generate transgenic fly lines. Bottom: Representative eye phenotypes from flies of the indicated genotypes crossed to GMR-GAL4 to drive expression in developing ommatidia (left) and quantification of eye phenotype scores (right). Graphs represent means ± SEM. For Panels A and B, n = number of neurons. **p < 0.01 difference in survival measured by Cox proportional hazard analysis. Each graph represents at least 3 independent experiments. For panel C, ** p < 0.01 by Kruskal-Wallis after Dunn’s correction for multiple comparisons compared to control group. † p < 0.01 by Kruskal-Wallis after Dunn’s correction for multiple comparisons compared to (G4C2)69 CT group. The number of flies analyzed in panel C was > 100

Fig. 6

Native carboxyl terminal sequences decrease GR dipeptide repeat licited toxicity in rodent neurons. a Top: Schematic of constructs generated to evaluate impact of including an AUG codon in each individual reading frame of the repeat while maintaining the native C-terminal sequence across all 3 reading frames. Bottom: Survival of neurons expressing the indicated constructs as measured by cumulative risk of death. b Schematic of constructs used in panels c-e. c Cumulative risk of death of neurons transfected with constructs containing an AUG codon in the GA reading frame with or without the native C-terminus. d) Cumulative risk of death of neurons transfected with constructs containing an AUG codon in the GP reading frame with or without the native C-terminus. e Cumulative risk of death of neurons transfected with constructs containing an AUG codon in the GR reading frame with or without the native C-terminus. *p < 0.05 and ** p < 0.01 indicate a difference in survival as determined by Cox proportional hazard analysis. The number of neurons for each analysis were as indicated in the respective lines. Each graph represents at least 3 independent experiments