Activated iPSC-microglia from C9orf72 ALS/FTD patients exhibit endosomal-lysosomal dysfunction

Background A mutation in the C9orf72 gene is the most common genetic mutation of familial and sporadic ALS, as well as familial FTD. While prior studies have focused on elucidating the mechanisms of neuronal dysfunction and neurodegeneration associated with this genetic mutation, the contribution of microglia to disease pathogenesis in the ALS/FTD disease spectrum remains poorly understood. Methods Here, we generated a new disease model consisting of cultured C9orf72 ALS/FTD patient-derived induced pluripotent stem cells differentiated into microglia (iPSC-MG). We used this model to study the intrinsic cellular and molecular phenotypes of microglia triggered by the C9orf72 gene mutation. Results We show that C9orf72 ALS/FTD iPSC-MG have a similar transcriptional profile compared to control iPSC-MG, despite the presence of C9orf72 -associated phenotypes including reduced C9orf72 protein levels and dipeptide-repeat protein translation. Interestingly, C9orf72 ALS/FTD iPSC-MG exhibit intrinsic dysfunction of phagocytic activity upon exposure to Aβ or brain synaptoneurosomes and display a heightened inflammatory response. Detailed analysis of the endosomal and lysosomal pathways revealed altered expression of endosomal marker early endosome antigen 1 and lysosomal associated membrane protein 1 in C9orf72 ALS/FTD iPSC-MG, which was confirmed in patient postmortem tissues. Conclusions These findings demonstrate that unstimulated C9orf72 iPSC-MG mono-cultures share a largely similar transcriptome profile with control microglia, despite the presence of C9orf72 disease phenotypes. The dysfunction of the endosomal-lysosomal pathway as demonstrated by aberrant microglia phagocytosis and engulfment of cellular debris and brain pathogens suggests that disease-related microglia phenotypes are not intrinsic but instead require microglia to be activated. In summary, the C9orf72 iPSC-MG culture system provides a novel human disease model to study the role of microglia in C9orf72 ALS/FTD disease pathogenesis. ### Competing Interest Statement The authors have declared no competing interest. * iPSCs : induced pluripotent stem cell iPSC-MG : induced pluripotent stem cells derived microglia ALS/FTD : amyotrophic lateral sclerosis/frontotemporal dementia C9orf72 : chromosome 9 open reading frame 72 HRE : hexanucleotide repeat expansion G4C2 : GGGGCC DPR : dipeptide repeat TDP-43 : TAR-DNA binding protein 43 AD : Alzheimer’s disease CNS : central nervous system sALS : sporadic ALS PC : principal component LPS : lipopolysaccharide EEA1 : early endosome antigen 1 Lamp1 : lysosomal associated membrane protein 1 CD43+ : cluster of differentiating 43 positive HPCs : hematopoietic progenitor cells GFs : growth factors MBM : microglia basal media IL-34 : interleukin-34 M-CSF : macrophage colony-stimulating factor TGFβ1 : transforming growth factor β1 CD200 : cluster of differentiation 200 CX3CL1 : fracktaline chemokine C-X3-C motif ligand 1 EBs : embryoid bodies FB-NIM : Forebrain Neural Induction Media FB-DM : forebrain neuronal differentiation media hSN-rodo : human brain synaptoneurosomes fluorescently tagged with pHrodo succinimidyl ester P2ry12 : purinergic surface receptor P2Y12 CX3CR1 : C-X3-C Motif Chemokine Receptor 1 TREM2 : triggering receptor expressed on myeloid cells 2 TMEM119 : transmembrane protein 119 OPC : oligodendrocyte precursor cells RAN : repeat associated non-AUG N/C : nucleocytoplasmic ADAR2 : adenosine deaminase acting on double stranded RNA 2 IL-1 : interleukin-1 IL-6 : interleukin-6 TNF-α : tumor necrosis factor-α IL-4 : interleukin-4 IL-10 : interleukin-10 RabGEF : Rab guanine nucleotide exchange factor Iba-1 : ionized calcium binding adaptor molecule 1 DAM : disease-associated microglia iMNs : induced motor neurons C1q : complement component 1q