C9orf72-ALS human iPSC microglia are pro-inflammatory and toxic to co-cultured motor neurons via MMP9

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss, with additional pathophysiological involvement of non-neuronal cells such as microglia. The commonest ALS-associated genetic variant is a hexanucleotide repeat expansion (HRE) mutation in *C9orf72*. Here, we study its consequences for microglial function using human iPSC-derived microglia. By RNA-sequencing, we identify enrichment of pathways associated with immune cell activation and cyto-/chemokines in *C9orf72* HRE mutant microglia versus healthy controls, most prominently after LPS priming. Specifically, LPS-primed *C9orf72* HRE mutant microglia show consistently increased expression and release of matrix metalloproteinase-9 (MMP9). LPS-primed *C9orf72* HRE mutant microglia are toxic to co-cultured healthy motor neurons, which is ameliorated by concomitant application of an MMP9 inhibitor. Finally, we identify release of dipeptidyl peptidase-4 (DPP4) as a marker for MMP9-dependent microglial dysregulation in co-culture. These results demonstrate cellular dysfunction of *C9orf72* HRE mutant microglia, and a non-cell-autonomous role in driving *C9orf72*-ALS pathophysiology in motor neurons through MMP9 signaling.

Björn F. Vahsen, Sumedha Nalluru, Georgia R. Morgan, Lucy Farrimond, Emily Carroll, Yinyan Xu, Kaitlyn M. L. Cramb, Benazir Amein, Jakub Scaber, Antigoni Katsikoudi, Ana Candalija, Mireia Carcolé, Ruxandra Dafinca, Adrian M. Isaacs, Richard Wade-Martins, Elizabeth Gray, Martin R. Turner, Sally A. Cowley, Kevin Talbot