PU.1 restores microglial dysfunction caused by C9ORF72 repeat expansions in neural organoids.

Abstract

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by loss of upper and lower motor neurons and progressive muscle wasting. Accumulating evidence indicates a role for non-neuronal cells in ALS pathogenesis, but their exact role and mechanism-of-action remain incompletely understood. A hexanucleotide (GGGGCC) repeat expansion (HRE) in C9ORF72 is the most common genetic cause of ALS (C9-ALS) and a frequent cause of frontotemporal dementia (FTD). Several lines of experimental evidence support a role for the immune system and microglia in C9-ALS/FTD, and, depending on experimental settings and species used, both reduced and increased microglial activity have been reported. To further study microglia in C9-ALS/FTD in the context of a complex, three-dimensional disease environment, we developed cerebral organoids that innately develop microglia derived from induced pluripotent stem cells (iPSCs) of C9-ALS/FTD patients and controls. Here we show reduced cellular complexity and transcriptional changes in C9 neural organoid-derived microglia (C9-oMGs), involving phagocytic, lysosomal and immune response pathways. The release of inflammatory cues from C9-ALS/FTD organoids is decreased and LAMP1 expression in C9-oMGs is reduced. Functional analysis using live imaging reveals impaired phagocytosis by C9-oMGs and reduced engulfment of the post-synaptic protein PSD-95 by C9-oMGs in organoids. Finally, our transcriptomics analysis identifies a PU.1 (encoded by SPI1) regulon as the most strongly downregulated transcription factor network in C9-oMGs. Viral overexpression of PU.1 rescues phagocytosis and gene expression defects in C9-microglia. Overall, our data demonstrate reduced microglial functions in a complex cellular disease environment and identify PU.1 as a potential target for restoring microglia changes in C9-ALS/FTD.