C9orf72-Associated Arginine-Rich Dipeptide Repeat Proteins Reduce the Number of Golgi Outposts and Dendritic Branches in Drosophila Neurons

Abstract

Altered dendritic morphology is frequently observed in various neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the cellular and molecular basis underlying these pathogenic dendritic abnormalities remains largely unclear. In this study, we investigated dendritic morphological defects caused by dipeptide repeat protein (DPR) toxicity associated with G4C2 expansion mutation of C9orf72 (the leading genetic cause of ALS and FTD) in Drosophila neurons and characterized the underlying pathogenic mechanisms. Among the five DPRs produced by repeat-associated non-ATG translation of G4C2 repeats, we found that arginine-rich DPRs (PR and GR) led to the most significant reduction in dendritic branches and plasma membrane (PM) supply in Class IV dendritic arborization (C4 da) neurons. Furthermore, expression of PR and GR reduced the number of Golgi outposts (GOPs) in dendrites. In Drosophila brains, expression of PR, but not GR, led to a significant reduction in the mRNA level of CrebA, a transcription factor regulating the formation of GOPs. Overexpressing CrebA in PR-expressing C4 da neurons mitigated PM supply defects and restored the number of GOPs, but the number of dendritic branches remained unchanged, suggesting that other molecules besides CrebA may be involved in dendritic branching. Taken together, our results provide valuable insight into the understanding of dendritic pathology associated with C9-ALS/FTD.

Keywords: C9orf72; CrebA; Golgi outposts; amyotrophic lateral sclerosis; dendrites.

Fig. 1. Arginine-rich DPRs induce dendritic defects in Drosophila C4 da neurons.

(A) Representative images of dendrites of C4 da neurons of control (Ctrl) or expressing the denoted transgenes (Genotype: Ctrl, +/+;ppk^1a> UAS-CD4-tdGFP/+; RO288, UAS-RO288/+;ppk^1a> UAS-CD4-tdGFP/+; PA36, UAS-PA36/+;ppk^1a> UAS-CD4-tdGFP/+; GA36, UAS-GA36/+;ppk^1a> UAS-CD4-tdGFP/+; PR36, UAS-PR36/+;ppk^1a> UAS-CD4-tdGFP/+; GR36, UAS-GR36/+;ppk^1a> UAS-CD4-tdGFP/+). Scale bar = 20 µm. (B) Quantification of the dendritic branch points of neurons expressing the denoted transgenes described in Fig. 1A. *P < 0.05, **P < 0.01, ****P < 0.0001 by two-tailed t-test; error bars ± SEM; n ≥ 6 neurons. (C) Quantification of the dendritic length of neurons expressing the denoted transgenes described in Fig. 1A. ***P < 0.001, ****P < 0.0001 by two-tailed t-test; error bars ± SEM; n ≥ 6 neurons. (D) Quantification of the number of dendritic branch points in C4 da neurons of Ctrl or expressing PR36 at 1 day after eclosion (Genotype: Ctrl, +/+;GS-ppk^1a,ppk-UAS-CD4-tdTOM/+; PR36, UAS-PR36/+;GS-ppk^1a,ppk-UAS-CD4-tdTOM/+). N.S., not significant, P = 0.0918 by two-tailed t-test; error bars ± SEM; n ≥ 3 neurons. (E) Quantification of the number of dendritic branch points in C4 da neurons of Ctrl or expressing PR36 at 15 day after RU486-mediated transcriptional activation of gal4. ****P < 0.0001 by two-tailed t-test; error bars ± SEM; n = 6 neurons.

Fig. 2. Arginine-rich DPRs impair PM supply and reduce the number of GOPs in C4 da neurons.

(A) Representative CD4-tdGFP-labeled dendrite images (pseudo-colored) in C4 da neurons expressing denoted DPR transgenes (Genotype: Ctrl, +/+;ppk^1a> UAS-CD4-tdGFP/+; PR36, UAS-PR36/+;ppk^1a> UAS-CD4-tdGFP/+; GR36, UAS-GR36/+;ppk^1a> UAS-CD4-tdGFP/+). White arrowheads indicate soma of neurons. Scale bar = 20 µm. (B) Quantification of the intensity value of membrane-targeted fluorescence proteins (CD4-tdGFP) in C4 da neurons expressing denoted DPR transgenes described in Fig. 2A. ***P = 0.0001, ****P < 0.0001 by two-tailed t-test; error bars ± SEM; n = 6 neurons. (C) Representative images of GOPs (marked by medial-Golgi marker ManII-eGFP) in C4 da neurons of Ctrl or expressing PR36 (Genotype: Ctrl, UAS-ManII-eGFP/+;ppk^1a> UAS-CD4-tdTOM/+; PR36, UAS-PR36/UAS-ManII-eGFP;ppk^1a>UAS-CD4-tdTOM/+). White arrowheads indicate GOPs. Scale bar = 10 µm. (D) Quantification of the number of GOPs in C4 da neurons of Ctrl or expressing PR36 described in Fig. 2C. ***P = 0.0006 by two-tailed t-test; error bars ± SEM; n ≥ 3 neurons.

Fig. 3. Nuclear PR36 toxicity down-regulates CrebA mRNA expression.

(A) Subcellular localization of PR36 (magenta) in C4 da neurons expressing PR36 (Genotype: UAS-PR36/+;ppk^1a-gal4/+). HRP (green) indicate membrane marker. Scale bar = 5 µm. (B) The Intensitiy profiles of fluorescent signals representing PR36 (magenta) and HRP (green) across cell bodies along yellow arrow presented at Fig. 3A. The gray dashed lines indicate border of nuclei. (C) The amount of mRNA level of CrebA and 18S rRNA in the brain of Ctrl or expressing PR36 (Genotype: Ctrl, +/+;elav-gal4/+; PR36, UAS-PR36/+;elav-gal4/+). (D) Quantification of the mRNA level of CrebA. The values were normalized by those of 18S rRNA. **P = 0.0036 by two-tailed t-test; error bars ± SEM; n = 4 replicates.

Fig. 4. Up-regulation of CrebA restores the number of GOPs and PM supply in C4 da neurons expressing PR36.

(A) Representative images of GOPs (marked by medial-Golgi marker ManII-eGFP) in C4 da neurons of Ctrl or expressing PR36, CrebA, or PR36 + CrebA (Genotype: Ctrl, UAS-ManII-eGFP/+;ppk^1a> UAS-CD4-tdTOM/+; PR36, UAS-PR36/UAS-ManII-eGFP;ppk^1a> UAS-CD4-tdTOM/+; CrebA, UAS-CrebA/UAS-ManII-eGFP;ppk^1a> UAS-CD4-tdTOM/+; PR36 + CrebA, UAS-PR36/UAS-CrebA,UAS-ManII-eGFP;ppk^1a> UAS-CD4-tdTOM/+). White arrowheads indicate GOPs. Scale bar = 10 µm. (B) Quantification of the number of GOPs in C4 da neurons of Ctrl or expressing PR36, CrebA, or PR36 + CrebA as described in Fig. 4A. ****P < 0.0001 by two-tailed t-test; error bars ± SEM; n = 13 neurons. (C) Representative CD4-tdGFP-labeled dendrite images (pseudo-colored) in C4 da neurons expressing the denoted transgenes (Genotype: Ctrl, +/+;ppk^1a> UAS-CD4-tdGFP/+; PR36, UAS-PR36/+;ppk^1a> UAS-CD4-tdGFP/+; CrebA, UAS-CrebA/+;ppk^1a> UAS-CD4-tdGFP/+; PR36 + CrebA, UAS-PR36/UAS-CrebA;ppk^1a> UAS-CD4-tdGFP/+). White arrowheads indicate soma of neurons. Scale bar = 20 µm. (D) Quantification of the intensity value of CD4-tdGFP in C4 da neurons expressing the denoted transgenes described in Fig. 4C. ****P < 0.0001 by two-tailed t-test; error bars ± SEM; n ≥ 3 neurons.