Unknown,Transcriptomics,Genomics,Proteomics

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Single-cell RNA sequencing on Drosophila melanogaster brain in an ALS/FTD model

ABSTRACT: The mutation in the C9orf72 gene with a hexanucleotide repeat has been reported multiple times to be the most common genetic cause of FTD and ALS (Frontotemporal Dementia and Amyotrophic Lateral Sclerosis), both of which are devastating neurodegenerative diseases having no cures currently. Our lab previously created fruit fly models expressing 36(C4G2) repeats, these are highly toxic to adult neurons of fruit flies. This is one of the most commonly used fly models of disease. Like many neurodegenerative diseases, FTD and ALS display selective neuronal vulnerability: only some neuronal populations succumb to disease, even though the toxic species are ubiquitously expressed. Our lab proposes to identify which neuronal populations are selectively depleted in response to the expression of the repeats and analyse which pathways are activated in vulnerable and resistant neuronal populations using our fly model of disease. This is done by scRNA sequencing across multiple time points, tracking disease development. The workflow was first having the flies ready and their brains being dissected. The brains were then dissociated by collagenase and dispase, and the cell suspensions were passed through a 10um cell strainer. The single-cell suspensions were checked for viability and the single-cell libraries were prepared with 10X Chromium 3' platform.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Dunxin Shen

PROVIDER: E-MTAB-12440 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:G4C2 hexanucleotide repeat expansions in a non-coding region of the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). G4C2 insertion length is variable, and patients can carry up to several thousand repeats. Dipeptide repeat proteins (DPR) translated from G4C2 transcripts are thought to be a main driver of toxicity. Experiments in model organisms with relatively short DPR have shown that arginine-rich DPR are most toxic, while polyGlycine-Alanine (GA) DPRs cause only mild toxicity. However, GA is the most abundant DPR in patient brains, and experimental work in animals has generally relied on the use of low numbers of repeats, with DPRs often tagged for in vivo tracking. Whether repeat length, or tagging, affect the toxicity of GA has not been systematically assessed. Therefore, we generated Drosophila fly lines expressing GA100, GA200 or GA400 specifically in adult neurons. Consistent with previous studies, expression of GA100 and GA200 caused only mild toxicity. In contrast, neuronal expression of GA400 drastically reduced climbing ability and survival of the flies, indicating that long GA DPRs can be highly toxic in vivo. This toxicity could be abolished by tagging GA400. Proteomics analysis of fly brains showed a repeat-length-dependent modulation of the brain proteome, with GA400 causing earlier and stronger changes than shorter GA proteins. PolyGA expression up-regulated proteins involved in ER to Golgi trafficking, and down-regulated proteins involved in insulin signalling. Experimental down-regulation of Tango1, a highly conserved regulator of ER-to Golgi transport, partially rescued GA400 toxicity, suggesting that misregulation of this process contributes to polyGA toxicity. Experimentally increasing insulin signaling also rescued GA toxicity. In summary, our data show that long polyGA proteins can be highly toxic in vivo, and that they may therefore contribute to ALS/FTD pathogenesis in humans patients.

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Single-cell RNA sequencing on Drosophila melanogaster brain in an ALS/FTD model

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