Expanded GGGGCC (G₄C₂) repeats in C9ORF72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How RNAs containing expanded G₄C₂ repeats are transcribed in human neurons is largely unknown. Here we describe a Drosophila model in which poly(GR) expression in adult neurons causes axonal and locomotor defects and premature death without apparent TDP-43 pathology. In an unbiased genetic screen, partial loss of Lilliputian (Lilli) activity strongly suppresses poly(GR) toxicity by specifically downregulating the transcription of GC-rich sequences in Drosophila. Knockout of AFF2/FMR2 (one of four mammalian homologues of Lilli) with CRISPR-Cas9 decreases the expression of the mutant C9ORF72 allele containing expanded G₄C₂ repeats and the levels of repeat RNA foci and dipeptide repeat proteins in cortical neurons derived from induced pluripotent stem cells of C9ORF72 patients, resulting in rescue of axonal degeneration and TDP-43 pathology. Thus, AFF2/FMR2 regulates the transcription and toxicity of expanded G₄C₂ repeats in human C9ORF72-ALS/FTD neurons.

Summary: While motor and cortical neurons are affected in C9orf72 ALS/FTD, it remains still largely unknown if and how non-neuronal cells induce or exacerbate neuronal damage. We generated C9orf72 ALS/FTD patient-derived induced pluripotent stem cells differentiated into microglia (iPSC-MG) and examined their intrinsic phenotypes. Similar to iPSC motor neurons, C9orf72 ALS/FTD iPSC-MG mono-cultures form G ₄ C ₂ repeat RNA foci, exhibit reduced C9orf72 protein levels and generate dipeptide repeat proteins. Healthy control and C9orf72 iPSC-MG equivalently express microglial specific genes and display microglial functions including inflammatory cytokine release and phagocytosis of extracellular toxic cargos such as synthetic amyloid beta peptides and healthy human brain synaptoneurosomes. Select C9orf72 iPSC-MG patient lines show inability to efficiently remove phagocytosed contents, suggesting dysfunction of the endosomal-lysosomal pathways. Finally, RNA sequencing revealed overall transcriptional changes in diseased microglia yet no significant differentially expressed microglial-enriched genes. These minimal differences in cellular, molecular and functional characteristics of microglial mono-cultures suggest that a diseased microenvironment is associated with microglial activation and subsequent regulation of neuronal dysfunction.