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

## TL;DR

The study shows that microglia in C9ORF72-related ALS/FTD have reduced function, and restoring PU.1 can help recover their normal activity.

## Contribution

The paper identifies PU.1 as a key factor in microglial dysfunction in C9-ALS/FTD and demonstrates its functional restoration in a 3D organoid model.

## Key findings

- C9-oMGs show reduced phagocytic and immune response activity in neural organoids.
- PU.1 regulon is the most strongly downregulated transcription factor network in C9-oMGs.
- Viral overexpression of PU.1 rescues phagocytosis and gene expression defects in C9-microglia.

## 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, 3D 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.

Accumulating evidence implicates non-neuronal cells in ALS pathogenesis. Using patient-derived cerebral organoids, Ljubikj et al. reveal reduced microglial activity in C9ORF72-ALS/FTD and identify PU.1 as a key deregulated factor, restoration of which helps to rescue microglial function.