FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions

Abstract

C9ORF72 repeat expansion is the most common genetic mutation in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Abnormal dipeptide repeat proteins (DPRs) generated from repeat-associated non-AUG (RAN) translation of repeat-containing RNAs are thought to be pathogenic; however, the mechanisms are unknown. Here we report that (GR)80 and (PR)80 are toxic in neuronal and non-neuronal cells in Drosophila. In contrast to reported shorter poly(GR) forms, (GR)80 is mostly localized throughout the cytosol without detectable accumulation in the nucleolus, accompanied by suppression of Notch signaling and cell loss in the wing. Some Notch target genes are also downregulated in brains and iPSC-derived cortical neurons of C9ORF72 patients. Increased Notch expression largely suppressed (GR)80-induced cell loss in the wing. When co-expressed in Drosophila, HeLa cells, or human neurons, (GA)80 recruited (GR)80 into cytoplasmic inclusions, partially decreasing the toxicity of (GR)80 and restoring Notch signaling in Drosophila. Thus, different DPRs have opposing roles in cell loss and we identify the Notch pathway as one of the receptor signaling pathways that might be compromised in C9ORF72 FTD/ALS.

Keywords: ALS; DPR; Drosophila; FTD; Inclusion; Motor neuron; Notch; Poly(GA); Poly(GR); Poly(PR); RAN translation.

Fig. 1

Toxicity of different FTD/ALS-associated dipeptides in the Drosophila eye. a Schematic representation of DNA constructs that express Flag-tagged (GA)₈₀, (PR)₈₀, and (GR)₈₀ under the control of the UAS elements. In control lines, the AUG start codon was replaced by a UAG or UAA stop codon to block translation. X at the third codon can be randomly any one of the four nucleotides. b Representative images of Drosophila eyes with different genotypes. Neither GMR-Gal4 nor any of the UAS transgenic lines showed an eye phenotype. Expression of (PR)₈₀ or (GR)₈₀ in the eye by the GMR-Gal4 resulted in grossly deformed eyes

Fig. 2

(GR)₈₀ is toxic in neuronal and non-neuronal cells in Drosophila. a Effects of (GA)₈₀, (GR)₈₀, and (PR)₈₀ on locomotor activity of adult flies. (GA)₈₀, (GR)₈₀, and (PR)₈₀ were expressed in motor neurons by OK371-Gal4 at 18 °C, and surviving 3-day-old adult flies (10 flies of each genotype) were tested for climbing activity. Forty OK371-Gal4 flies and 10 flies expressing (GA) ₈₀ mRNA but with a stop codon instead of the ATG start codon (see Table S1) were also examined. Values are mean ± SEM. **p value <0.01, ***p value <0.001, by Student’s t test. b Effects of (GA)₈₀, (GR)₈₀, and (PR)₈₀ expression on dendritic branching of ddaE sensory neurons. ddaE neurons were labeled with mCD8-GFP driven by 221-Gal4. The number of neuron analyzed for each genotype is listed on each column. Values are mean ± SD. **p value <0.01, by single-factor ANOVA. c Effects of (GA)₈₀, (GR)₈₀, and (PR)₈₀ on the survival of wing margin cells. The expression of (GR)₈₀, and (PR)₈₀ resulted in the wing notching phenotype. d The percentage of flies with or without wing notching phenotype is shown for each genotype. More than 300 flies of both sexes were scored for each genotype

Fig. 3

Notch expression suppresses (GR)₈₀ toxicity. a Representative images of Drosophila wings with margin defects of different severities. The image of a normal wing was from a UAS-(GR) ₈₀/+ fly; other wing images were from N ⁵⁴¹⁹/+; Vg-Gal4, UAS-(GR) ₈₀/+ flies. b Quantification of wing margin defects in female flies heterozygous for the N ⁵⁴¹⁹ allele, expressing (GR) ₈₀ driven by Vg-Gal4 recombined to the same second chromosome and expressing (GR) ₈₀ on the N ⁵⁴¹⁹/+ background by Vg-Gal4. Only female flies were examined, and the total number of flies for each genotype from three experiments is listed above each column. c Representative images of wing margin defects in Vg-Gal4, UAS-(GR) ₈₀ /+ flies. d Representative wing images of Vg-Gal4, UAS-(GR) ₈₀ /+; UAS-N ^FL7 flies showing suppression of the wing margin defects by ectopic expression of full-length Notch. UAS-GFP was used as the control for UAS-N ^FL7. e Quantification of Notch suppression of wing margin defects caused by (GR)₈₀. The total number of flies of both sexes examined for each genotype from 3 experiments is listed above each column

Fig. 4

(GR)₈₀ downregulates notch signaling in Drosophila. GFP expression controlled by the Notch-responsive element (NRE) at the dorsoventral boundary (green arrowheads) of a control wing disc (a) and a wing disc expressing (GR)₈₀ driven by Vg-Gal4 (b). The areas indicated by red rectangles are enlarged and presented as the two right panels. Wg immunostaining at the dorsoventral boundary (red arrowheads) of a control wing disc (c) and a wing disc expressing (GR)₈₀ (d). Expression levels of the Notch targets HES1 (e) and HEY1 (f) in iPSC-derived neurons and brain tissues of subjects with C9ORF72 repeat expansion. The number of iPSC lines or brain samples analyzed is indicated in each column. Values are mean ± SEM. *p value <0.05, **p value <0.01 by Student’s t test

Fig. 5

Subcellular localization of (GR)₈₀ and (GA)₈₀. a Flag-tagged (GA)₈₀ forms inclusions mostly in the cytosol of Drosophila salivary gland cells. Some inclusions are highlighted by red arrowheads. b (GR)₈₀ is largely present throughout the cytoplasm of salivary gland cells, and their nucleoli are larger than those of cells expressing the (GR)₈₀-control construct. These are confocal images, and one or two (GR)₈₀-positive dots (yellow arrowhead) were observed on chromatin in each cell at different confocal planes. Scale bar 20 μm. c HA-tagged (GA)₈₀ recruits Flag-tagged (GR)₈₀ into cytoplasmic inclusions when the two are co-expressed. All inclusions contain both (GA)₈₀ and (GR)₈₀ (some are indicated by yellow arrowheads)

Fig. 6

(GA)₈₀ suppresses (GR)₈₀ toxicity through inclusion formation. a HA-tagged (GA)₈₀ forms inclusion in HeLa cells. b (GR)₈₀ expression alone in HeLa cells shows diffuse cytoplasmic localization. c When (GA)₈₀ and (GR)₈₀ are co-expressed in HeLa cells, (GR)₈₀ is recruited into (GA)₈₀ inclusions (yellow arrowhead). d HA-tagged (GA)₈₀ forms inclusion in iPSC-derived human neurons. e (GR)₈₀ expression alone in iPSC-derived human neurons shows cytoplasmic localization. f When (GA)₈₀ and (GR)₈₀ are co-expressed in iPSC-derived human neurons, (GR)₈₀ is recruited into (GA)₈₀ inclusions (yellow arrowhead). Scale bar 10 μm. g (GA)₈₀ partially suppresses (GR)₈₀ toxicity in wing disc cells, resulting in a less severe wing notching phenotype. In this experiment, Vg-Gal4 and UAS-(GR) ₈₀ were recombined onto the same chromosome. Wg expression at the dorsoventral boundary of wing discs expressing UAS-(GA) ₈₀ control (Table S1) and UAS-(GR) ₈₀ (h) or both UAS-(GA) ₈₀ and UAS-(GR) ₈₀ (i). UAS-(GR) ₈₀ on the third chromosome. The brackets indicate the dorsoventral boundary. j Wg expression levels in wing discs of flies with genotypes described in panels h and i. Scale bar in panels a–f, h, i: 20 μm. Values are mean ± SEM. *p value <0.05 by single-factor ANOVA