C9orf72repeat expansion-carrying iPSC-microglia from FTD patients show increased phagocytic activity concomitantly with decreased number of autophagosomal-lysosomal vesicles

Abstract C9orf72 hexanucleotide repeat expansion (HRE) is a major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The role of microglia in these C9orf72 HRE-associated diseases is understudied. To elucidate effects of C9orf72 HRE on microglia, we have characterized human induced pluripotent stem cell-derived microglia (iMG) from behavioral variant frontotemporal dementia (bvFTD) patients carrying the C9orf72 HRE. C9orf72 HRE iMG were compared to iMG from healthy controls and sporadic bvFTD patients. The phenotypes of iMG were analyzed using bulk RNA sequencing, biochemical and immunofluorescence analyses, and live cell imaging. C9orf72 HRE-carrying iMG showed nuclear RNA foci and poly-GP dipeptide repeat proteins but no decreased C9orf72 mRNA or protein expression. TDP-43 pathology was absent from all bvFTD iMG. As compared to healthy control iMG, quantitative immunofluorescence analyses indicated that all bvFTD iMG had reduced number, size, and intensity of LAMP2-A-positive vesicles. C9orf72 HRE-carrying iMG additionally showed decreased number, size, and intensity of p62/SQSTM1-positive vesicles. These changes were accompanied by increased phagocytic activity of the C9orf72 HRE-carrying iMG. Serum starvation increased phagocytic activity also in the iMG of sporadic bvFTD patients. RNA sequencing revealed that iMG of C9orf72 HRE-carrying bvFTD patients as compared to the iMG of sporadic bvFTD patients showed differential gene expression in pathways related to RNA and protein regulation and mitochondrial metabolism. Our data suggest potential alterations in the autophagosomal/lysosomal pathways in bvFTD patient iMG, which are further reinforced by the C9orf72 HRE and functionally manifest as increased phagocytic activity. Competing Interest Statement The authors have declared no competing interest. Footnotes hannah.rostalski{at}uef.fi (H.R.); tomi.hietanen{at}uef.fi (T.H.); dorit.hoffmann{at}uef.fi (D.H.); nadine.huber{at}uef.fi (N.H.); sohvi.ohtonen{at}uef.fi (S.O.); henna.jantti{at}uef.fi (H.J.); viivi.pekkala{at}uef.fi (V.P.); stina.leskela{at}uef.fi (S.L.); sarka.lehtonen{at}uef.fi (Š.L.); tarja.malm{at}uef.fi (T.M.); annakaisa.haapasalo{at}uef.fi (A.H.); hannah.rostalski{at}bric.ku.dk (H.R.) ; sami.heikkinen{at}uef.fi (S.H.); teemu.kuulasmaa{at}uef.fi (T.K.); petra.makinen{at}uef.fi (P.M.); teemu.natunen{at}uef.fi (T.N.); henna.martiskainen{at}uef.fi (H.M.); mari.takalo{at}uef.fi (M.T.); mikko.hiltunen{at}uef.fi (M.H.); ashutosh.dhingra{at}dzne.de (A.D.); salvador.rodriguez-nieto{at}dzne.de (S.R.); kasper.katisko{at}uef.fi (K.K.); eino.solje{at}uef.fi (E.S.); paivi.hartikainen{at}kuh.fi (P.H.); jari.koistinaho{at}helsinki.fi (J.K.); anne.portaankorva{at}helsinki.fi (A.M.P.) Abbreviations - ab - Antibody - AIF1 - Allograft inflammatory factor 1 - ALS - Amyotrophic lateral sclerosis - ANOVA - Analysis of variance - ARM - Active response microglia - AUC - Area under the curve - BSA - Bovine serum albumin - bvFTD - Behavioral variant frontotemporal dementia - C1QA - Complement C1q subcomponent subunit A - C9orf72 - Chromosome 9 open reading frame 72 - CCL2 - C-C motif chemokine ligand 2 - CD - Cluster of differentiation - CHIT1 - Chitinase 1 - CNS - Central nervous system - CORO1C - Coronin 1C - COX-2 - Cyclooxygenase-2 - CSF - Cerebrospinal fluid - CX3CR1 - CX3C chemokine receptor 1 - CXCL10 - C-X-C motif chemokine ligand 10 - DAPI - 4′,6-diamidino-2-phenylindole - DMSO - Dimethyl sulfoxide - DNA - Deoxyribonucleic acid - DPBS - Dulbecco’s phosphate-buffered saline - DPR - Dipeptide repeat - EDTA - Ethylenediaminetetraacetic acid - FBS - Fetal bovine serum - FISH - Fluorescence in situ hybridization - FTD - Frontotemporal dementia - GAS6 - Growth arrest-specific 6 - GPR34 - G-protein coupled receptor 34 - HC - Healthy control - HEXB - Beta-hexosaminidase subunit beta - HRE - Hexanucleotide repeat expansion - HSP90AA1 - Heat shock protein 90 alpha family class A member 1 - IBA1 - Ionized calcium binding adapter molecule 1 - IL - Interleukin - iMG - iPSC-derived microglia - iPSC - induced pluripotent stem cell - IRM - Interferon response microglia - kDa - kilo Dalton - LAMP - Lysosome-associated membrane glycoprotein - MAP1LC3B/LC3B - Microtubule-associated proteins 1A/1B light chain 3B - MDMi - monocyte derived microglia - MERTK - MER Proto-Oncogene, Tyrosine Kinase - MSD - Meso Scale Discovery - NANOG - Nanog homeobox - ORM1 - Orosomucoid 1 - P2RY12 - P2Y purinoceptor 12 - PCR - Polymerase chain reaction - PDL - Poly-D-lysine - POU5F1 - POU domain, class 5, transcription factor 1 - PROS1 - Protein S - PSEN2 - Presenilin 2 - PTGS2 - Prostaglandin-endoperoxide synthase 2 - RAC2 - Ras-related C3 botulinum toxin substrate 2 - RCU - Red calibrated unit - RNA - Ribonucleic acid - RT - Room temperature - S100A8 - S100 calcium-binding protein A8 - SDS - Sodium dodecyl sulphate - SOD1 - Superoxide dismutase 1 - SOX2 - SRY (sex determining region Y)-box 2 - SQSTM1 - Sequestosome 1 - SSEA4 - Stage-specific embryonic antigen 4 - TARDBP - TAR DNA Binding Protein - TDP-43 - TAR DNA-binding protein 43 - TMEM119 - Transmembrane protein 119 - TNF - Tumor necrosis factor - TRA181 - Tumor-related Antigen-1-81 - TREM2 - Triggering receptor expressed on myeloid cells 2 - TUBA1 - Tubulin Alpha 1b - TYROBP - TYRO protein tyrosine kinase-binding protein - UMAP - Uniform Manifold Approximation and Projection - WB - Western blotting