Ethics statement

All iPSC lines were previously derived from skin biopsy fibroblasts, following signed informed consent, collected under ethical approval granted by the South Wales Research Ethics Committee (WA/12/0186) and the South Central Berkshire Research Ethics Committee (REC10/H0505/71) in the James Martin Stem Cell Facility, University of Oxford, under standardized protocols. The iPSC lines have all been published previously (see below and Supplementary Table 1).

iPSC culture

Three iPSC lines derived from three different ALS patients carrying a HRE in C9orf72 and as controls, an isogenic line, previously generated through CRISPR/Cas9 genome editing using homology directed repair¹⁸, and four sex- and age-matched healthy control iPSC lines, were used in this study. All iPSC lines have previously been published and extensively characterized (for details on demographics and characterization, see Supplementary Table 1). iPSC were cultured in mTeSR™1 (85850, StemCell Technologies) on Geltrex™ (A1413302, ThermoFisher) with daily medium changes. Cells were passaged using EDTA (0.5 mM), expanded to produce consistent, frozen cell stocks for the study (cryopreserved in 50% mTeSR™1, 30% embryonic stem cell fetal bovine serum (16141002, Merck), 10% KnockOut™ DMEM (10829018, ThermoFisher), 10% DMSO (D2650, Merck)) with a maximum of 3 subsequent passages during experimentation, and tested negative for mycoplasma using the MycoAlert^TM Mycoplasma Detection Kit (Lonza).

Differentiation of iPSC-derived motor neurons

iPSC were differentiated to MNs using a previously published protocol³⁹. Briefly, neural induction was performed using DMEM-F12/Neurobasal 50:50 supplemented with N2 (1X), B27 (1X), 2-Mercaptoethanol (1X), Antibiotic-Antimycotic (1X, all ThermoFisher), Ascorbic Acid (0.5 μM), Compound C (1 μM, both Merck), and Chir99021 (3 μM, R&D Systems). After two days in culture, Retinoic Acid (1 μM, Merck) and Smoothened Agonist (500 nM, R&D Systems) were added. After another 2 days, Compound C and Chir99021 were removed. On day in vitro (DIV)18, the cells were re-plated on polyethylenimine (PEI, 0.07%, Merck) and Geltrex™ (ThermoFisher) or PDL (Sigma-Aldrich)/ Laminin (R&D Systems)/ Fibronectin (Corning) in medium additionally supplemented with BDNF (10 ng/mL), GDNF (10 ng/mL), Laminin (0.5 μg/mL, all ThermoFisher), and DAPT (10 μM, R&D Systems). Three days later, the medium was changed to microglia medium as described below, and MNs were either co-cultured with microglia or maintained in monoculture. Half medium changes were performed every 2–4 days.

Differentiation of iPSC-derived microglia precursors

iPSC were differentiated to macrophage/microglia precursors as described previously^15,40. Briefly, embryoid body (EB) formation was induced by seeding iPSC into Aggrewell 800 wells (STEMCELL Technologies) in mTeSR™1 supplemented with BMP4 (50 ng/mL), VEGF (50 ng/mL, both Peprotech), and SCF (20 ng/mL, Miltenyi Biotec). After four to seven days with daily ¾ medium changes, EBs were transferred to T175 flasks and differentiated in X-VIVO15 (Lonza), supplemented with Interleukin-3 (25 ng/mL, R&D Systems), MCS-F (100 ng/mL), GlutaMAX (1X, both ThermoFisher), and 2-Mercaptoethanol (1X). Fresh medium was added weekly. After approximately two months, precursors were harvested by collecting the supernatant, passed through a cell strainer (40 μM, Falcon/Greiner), and either differentiated to microglia in monoculture or co-culture as described below.

Differentiation of iPSC-derived microglia

Microglia precursors were plated on PEI (0.07%) and Geltrex™ or PDL/Laminin/Fibronectin using our previously characterized microglia medium¹⁷ comprised of Advanced DMEM-F12 (ThermoFisher), GlutaMAX (1X), N2 (1X), Antibiotic-Antimycotic (1X), 2-Mercaptoethanol (1X), Interleukin-34 (100 ng/mL, Peprotech/BioLegend), BDNF (10 ng/mL), GDNF (10 ng/mL), and Laminin (0.5 μg/mL). Microglia were differentiated for approximately 14 days, with half medium changes every 2–4 days, and collected for RNA/protein extraction or used for assays.

Co-culture of iPSC-derived motor neurons and microglia

Co-cultures were generated as described before using the HC-2b line (Supplementary Table 1) to generate MNs¹⁷. Briefly, three days after the final re-plating of differentiating MNs (DIV21), microglia precursors were harvested. MNs were rinsed with PBS, and microglia precursors re-suspended in microglia medium were added to each well in a 1:1 ratio with the MNs. Co-cultures were maintained for at least 14 days, with half medium changes every 2–4 days. In select experiments, direct contact of the two cell types was prevented by plating microglia in tissue culture inserts (0.4 µm pore size, 83.3932.040, Sarstedt).

Priming of microglia with LPS and MMP9 inhibitor treatment

For microglial monocultures, all medium was aspirated, and LPS (100 ng/mL, L4391, Merck) in microglia medium was added for 48 h. Control wells received fresh microglia medium only. In select conditions, repetitive LPS treatments were performed on day 0, 2, and 4. For co-cultures, LPS treatments were started on DIV25 and performed for 10 days. Half of the medium was aspirated and replaced with LPS in microglia medium (final concentration: 100 ng/mL), with additional half medium changes with LPS-containing microglia medium on DIV29 and 33. In additional experiments, LPS treatments were started on DIV25 and performed for 20 days, with half media changes every other day. BDNF and GDNF were removed from the culture media for these experiments. To inhibit MMP9, cultures were concomitantly treated with LPS (100 ng/mL) and MMP9 inhibitor I (MMP9i, 3 μM, 444278, Merck) or DMSO in equal concentrations (0.3%) as vehicle control.

Priming of microglia with TNF/IL1B

All medium was aspirated from microglial monocultures, and TNF (50 ng/mL) and IL1B (20 ng/mL, both Peprotech) in microglia medium were added for 48 h. Control wells received fresh microglia medium only.

Treatment of MNs with rhMMP9 and MMP9 inhibitor

MNs were treated with recombinant active human MMP9 (rhMMP9, 30 ng/mL, PF024-5UG, Merck) and MMP9i (3 μM, 444278, Merck) or DMSO in equal concentrations (0.3%) as vehicle control. Treatments were performed on DIV21 and DIV25, and MN viability was determined by MTS assay on DIV29.

Live-imaging of phagocytosis and quantification

Unstimulated and LPS-primed (100 ng/mL for 48 h) microglial monocultures were stained with Hoechst in Live Cell Imaging Solution (1:10,000, ThermoFisher) for 10 min. pHrodo™ Red Zymosan Bioparticles™ (P35364, ThermoFisher) in Live Cell Imaging Solution (50 µg/mL) were added to each well and images were acquired after 150 min using an EVOS™ XL Core Cell Imaging System (ThermoFisher) equipped with a 10x objective. In selected conditions, cells were additionally treated with the phagocytosis/degradation inhibitors Cytochalasin D (10 μM, C2618, Merck), Bafilomycin A1 (1 μM, 1334, R&D Systems), or DMSO (vehicle), with a 1 h-pretreatment. Quantification was performed automatically using an analysis macro in Fiji and the area of phagocytosis particles per cell was obtained by dividing the area of the internalized phagocytosis particles by the number of Hoechst-positive microglia.

Patch-clamp electrophysiology

Whole-cell patch-clamp electrophysiology was performed as described previously¹⁷. Healthy control MNs on DIV 42–46 in co-culture with healthy control or C9orf72-ALS patient-derived microglia were maintained in an extracellular solution containing 167 mM NaCl, 2.4 mM KCl, 1 mM MgCl₂, 10 mM glucose, 10 mM HEPES, and 2 mM CaCl₂ adjusted to a pH of 7.4 and 300 mOsm. Electrodes with tip resistances between 7 and 12 MΩ were produced from borosilicate glass (0.86 mm inner diameter; 1.5 mm outer diameter). An intracellular solution was added to the electrode containing 140 mM K-Gluconate, 6 mM NaCl, 1 mM EGTA, 10 mM HEPES, 4 mM MgATP, 0.5 mM Na₃GTP, adjusted to pH 7.3 and 290 mOsm. Data acquisition was performed using a Multiclamp 700B amplifier, digidata 1550 A and clampEx 6 software. Series resistance (R_s) was maintained at <30 MΩ. Voltage gated channel currents were measured on voltage clamp where neurons were pre-pulsed for 250 ms with −140 mV pulse and a 10 mV-step voltage was applied from −70 mV to +70 mV. Induced action potentials (APs) were recorded on current clamp, neurons were held at −70 mV and 15 current steps of 10 pA were applied from −10 pA to 130 pA. Voltage gated channel currents and properties of induced action potentials (rheobase, max APs, APs over rheobase) were quantified using Clampfit 10.3 (pCLAMP Software suite, Molecular Devices). Max APs were quantified by determining the number of APs in sweep with the longest train. AP duration and amplitude were quantified in Matlab R2022a (MathWorks) using the first action potential recorded from each neuron.

MEA recordings

In all, 2–15 min recordings of neuronal activity were obtained for co-cultures in 48-well multi-electrode array (MEA) plates (M768-tMEA-48B, Axion BioSystems) using the Maestro system (Axion Biosystems). Analysis was performed using the AXIS software v2.5.2 (Axion BioSystems) with SD > 5.5 as the detection threshold. The mean firing rate per well was calculated by averaging all active (>1 spike/min) electrodes per well.

RNA isolation and RT-qPCR

RNA was extracted using an RNAeasy Mini Plus kit (Qiagen) and equal amounts of RNA (100 ng) were reverse-transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher). Quantitative real-time PCR was performed with Fast SYBR™ Green Master Mix (ThermoFisher) using a LightCycler® 480 PCR System (Roche) and the primers (ThermoFisher) listed in Supplementary Table 3. All kits were used according to the manufacturer’s instructions. Quantification of the relative fold gene expression was performed using the 2^–∆∆Ct method with normalization to the TBP reference gene (TBP Ct range 0.5) and the mean gene expression of the control microglia for each experiment.

RNA sequencing

Three healthy control and three C9orf72-ALS patient-derived iPSC lines were differentiated into microglia, with three independent differentiations per line. RNA was extracted as described above. All RNA samples displayed RIN values of at least 8.5. Preparation of samples and bioinformatical analysis was then performed as described before¹⁷. Briefly, samples were normalized to 100 ng prior to library preparation. Polyadenylated transcript enrichment and strand specific library preparation was completed using the NEBNext Ultra II mRNA kit (NEB) following the manufacturer’s instructions. Paired end sequencing (read length: 150 base pairs) was performed using a NovaSeq6000 platform (Illumina, NovaSeq 6000 S2/S4 reagent kit, v1.5, 300 cycles), generating a raw read count of a minimum of 30 M reads per sample. Paired-end reads were mapped to the human GRCh38.p13 reference genome (https://www.gencodegenes.org) using HISAT2 v2.2.1⁴¹. The counts table was obtained using FeatureCounts v2.0.1⁴². Normalization of counts and differential expression analysis was performed using DESeq2 v1.28.1⁴³ in RStudio 1.4.1103, including the biological sex in the model and with the Benjamini-Hochberg method for multiple testing correction. The three different differentiations per condition were collapsed into one datapoint unless indicated otherwise where the biological sex and differentiation replicate were included in the model. Exploratory data analysis was performed following variance-stabilizing transformation of the counts table, using principal component analysis. Log₂ fold change (log₂ fc) shrinkage was performed using the ‘normal’ approach. Genes with | log₂ fc| > 0.5 and adjusted p-value < 0.05 were defined as differentially expressed genes (DEG). Data was interpreted with annotations from the Gene Ontology database using clusterProfiler v3.16.1⁴⁴. We analyzed pathway enrichment by performing over-representation analysis (ORA) for all DEGs and gene set enrichment analyses (GSEA) on the whole transcriptome ranked by log₂fc, using the standard settings in clusterProfiler for ORA and GSEA (adjusted p-value < 0.05 using the Benjamini-Hochberg method).

Immunofluorescence

Cells cultured on coverslips were pre-fixed with 2% paraformaldehyde (PFA) in PBS for 2 min at room temperature (RT) and then fixed with 4% PFA in PBS for another 15 min at RT. After permeabilization and blocking with 5% donkey serum and 0.2% Triton X-100 in PBS for 1 h at RT, the coverslips were incubated with the primary antibodies listed in Supplementary Table 2, diluted in 1% donkey serum and 0.1% Triton X-100 in PBS, at 4 °C ON. After three washes with PBS-0.1% Triton X-100 for 5 min each, coverslips were incubated with corresponding fluorescent secondary antibodies Alexa Fluor® 488/568/647 donkey anti-mouse/rabbit/goat/guineapig (all 1:1000, ThermoFisher or Abcam). Coverslips were then washed twice with PBS-0.1% Triton X-100 for 5 min each and incubated with DAPI (1 µg/mL, Sigma-Aldrich) in PBS for 10 min. After an additional 5 min-washing step with PBS-0.1% Triton X-100, the coverslips were mounted onto microscopy slides using ProLong™ Diamond, Gold or Glass Antifade Mountant (ThermoFisher). Confocal microscopy was performed using an LSM 710, LSM 880 (both Zeiss) or Fluoview FV1000 (Olympus) microscope.

Analysis of microglial ramifications

Four to five z-stack images (z-interval: 1 μm) of randomly selected visual fields were taken at 60x magnification for each coverslip, and maximum intensity projections were generated in Fiji. To analyze the branching of IBA1-positive microglia in monoculture and co-culture, the cell volume, ramification index, average branch length, and number of branch points was automatically determined using 3DMorph as described elsewhere¹⁹.

Quantification of TDP-43 mislocalization in microglia

Four to five z-stack images of randomly selected visual fields were taken at 60x magnification for each coverslip, and maximum intensity projections were generated in Fiji. The mean intensity values for TDP-43 in the nucleus and cytoplasm were determined in a blinded fashion, with the DAPI and IBA1 signal used to identify the nuclear and cytoplasmic boundaries, respectively. The ratio of cytoplasmic to nuclear TDP-43 was then calculated.

Quantification of microglia marker expression in microglia monocultures

Three z-stack images of randomly selected visual fields were taken at 40x magnification for each coverslip, and maximum intensity projections were generated in Fiji. The number of DAPI-positive nuclei and IBA1⁺/DAPI⁺ and TMEM119⁺/DAPI⁺ cells was automatically quantified per view field for each line using a macro.

Quantification of microglia and motor neuron numbers and ratios in co-culture

Three to five z-stack images of randomly selected visual fields were taken at 10x or 40x magnification for each coverslip, and maximum intensity projections were generated in Fiji. For microglia, using a macro, an auto threshold was applied on the IBA1 channel to create a mask, and the number of microglial cells was determined for each view field by counting the number of DAPI-positive nuclei within the IBA1 mask using the ‘analyze particles’ function. For MNs, an auto threshold was applied on the TUJ1 channel to create a mask, and the number of neurons was determined for each view field by counting the number of DAPI-positive nuclei within the TUJ1 mask using the ‘analyze particles’ function. To calculate the microglia:MN ratio, the number of microglial cells was divided by the MN number. For the assessment of MN survival after long-term LPS exposure, the absolute MN number was normalized to the mean of the control lines to determine the relative MN survival by accounting for batch effects between differentiations after long-term culture.

Quantification of apoptotic marker expression in co-culture

Five z-stacks images of randomly selected visual fields were taken at 40x magnification for each coverslip. Maximum intensity projections were generated. Using a macro, an auto threshold was applied on the TUJ1 channel to create a mask, and the expression of the CC3 apoptotic marker within neurons was determined for each view field by quantifying the area of the CC3 signal within the TUJ1 mask using the ‘analyze particles’ function. To account for batch effects between differentiations, the fold change in expression was generated by normalizing the absolute measurements to the mean of the control lines.

Quantification of neurite outgrowth in co-culture

Four to five z-stacks of randomly selected view fields showing neurites but avoiding neuronal somas were obtained at 60x magnification for each coverslip. Maximum intensity projections were generated in Fiji. Using a macro, an auto threshold was applied to the TUJ1 staining, and the neurite outgrowth for each view field was determined by measuring the area of TUJ1-positive neurites per view field using the ‘analyze particles’ function in Fiji.

Analysis of neuronal synaptophysin expression

Four to five z-stacks images of randomly selected visual fields were taken at 60x magnification for each coverslip. Maximum intensity projections were generated in Fiji. Using a macro, an auto threshold was applied to the synaptophysin staining to analyze dot-like structures, and the number and area of synaptophysin particles was determined for each view field using the ‘analyze particles’ function in Fiji.

RNAscope

RNA foci were detected using the RNAscope Multiplex Fluorescent v2 Assay (323100, ACD) according to the manufacturer’s instructions. In brief, iPSC microglia fixed with 4% PFA were sequentially dehydrated by 50%, 70% and 100% ethanol for 1 min each, and stored at −20 °C. After rehydrating with 100%, 70% and 50% ethanol, the cells were incubated with 0.1% Tween-20 in PBS, hydrogen peroxide and Protease III (dilution ratio 1:15) for 10 min each, with PBS washes in between. Probes targeting the (G₄C₂)_n and (C₄G₂)_n expansions (ACD, 884351-C2 and 884361-C2) were added to separate coverslips and incubated for 2 h, followed by amplification and development steps according to the standard protocol. Sequential ICC was performed by incubation with primary antibody against IBA1 (1:200, 019-19741, Wako Fujifilm) for 2 h at RT and then according to the immunofluorescence protocol detailed above.

Quantification of RNA foci formation in microglia

Five z-stack images of randomly selected visual fields were taken at 40x or 63x magnification for each coverslip, and maximum intensity projections were generated in Fiji. The number of foci-positive cells and number of foci per foci-positive nucleus were determined by manual counting.

MTS viability assay

In select experiments, MN viability was determined using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS, G3580, Promega) according to the manufacturer’s instructions. Plate readings were performed after 90–120 min incubation with assay reagents.

Measurement of cytokine/chemokine and neurofilament release

Culture supernatants were collected and spun down at 1200 x g and 4 °C for 10 min. Pooled samples were analyzed using the Proteome Profiler Human XL Cytokine Array (ARY022B) or the Protease/Protease Inhibitor Array (ARY025, both R&D Systems) according to the manufacturer’s instructions. The signal was visualized on a ChemiDoc^TM MP imaging system (Bio-Rad) and analyzed using ImageStudioLite v5.2.5 (LI-COR). In addition, the Human DPP4 (DC260B), MMP9 (DMP900), CXCL10 Quantikine ELISAs (DIP100), and Active MMP9 Fluorokine E Kit (F9M00, all R&D Systems) were used according to the manufacturer’s instructions. Human neurofilament light chain (NfL) release was measured using a Meso Scale Discovery (MSD) assay (K1517XR-2) according to the manufacturer’s instructions.

Protein extraction

Cells were rinsed with PBS, and 100–200 μL of cold RIPA buffer (ThermoFisher) supplemented with complete^TM protease inhibitor cocktail and PhosSTOP^TM phosphatase inhibitor (both Merck) were added to each well. 10 1s-impulses with motor-driven pestles were applied per sample to homogenize the lysates. After incubation on ice for 30 min, samples were centrifuged at 12,000 rpm and 4^oC for 15 min, and the supernatants were stored at −80 °C. Quantification of the protein concentration was performed using the Pierce^TM Bicinchoninic Acid Protein Assay kit (ThermoFisher) according to the manufacturer’s instructions. Protein samples were then prepared at defined concentrations (0.3–1.0 μg/μL) by dilution in distilled water, NuPAGE^TM loading buffer and NuPAGE^TM sample reducing agent (both ThermoFisher). This was followed by incubation at 95 °C for 5 min to denature and reduce the samples.

Western blot

5–20 μg of each sample and Spectra Multicolour Broad Range Protein Ladder or PageRuler™ Prestained Protein Ladder (both ThermoFisher) were loaded onto pre-cast NuPAGE^TM Bis-Tris 4–12% gradient gels (ThermoFisher). The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using NuPAGE^TM MES running buffer (ThermoFisher) at 50–130 V. Proteins were then transferred to nitrocellulose membranes at 20–25 V for 7 min using an iBlot 2 Dry blotting system (both ThermoFisher). Following blocking with 5% milk or BSA in TBS/0.1% Tween-20 (TBS-T, ThermoFisher) at RT for one hour, membranes were incubated with the primary antibodies listed in Supplementary Table 4 diluted in 3% milk or BSA in TBS-T at 4 °C overnight. Membranes were washed three times with TBS-T for 7 min and then incubated with corresponding horseradish peroxidase (HRP)-coupled secondary antibodies at room temperature for 1 h. The following HRP-coupled secondary antibodies (all 1:5000) were used: sheep anti-mouse HRP (NA931V), donkey anti-rabbit HRP (NA934V, both GE Healthcare), donkey anti-goat HRP (PA1-28664, ThermoFisher). Membranes were again washed three times with TBS-T for 7 min and then incubated for one minute with enhanced chemiluminescence solution (Millipore). The ChemiDoc^TM MP imaging system was used to visualize the signal. Band intensities were quantified using Fiji v1.53q⁴⁵ and normalized to the housekeeping genes β-actin or GAPDH and the mean expression of the controls for each experiment. After development, membranes were washed with TBS-T two times for 5 min, followed by a 10 min-incubation with Restore^TM PLUS stripping buffer (ThermoFisher). After two additional 5 min-washes with TBS-T, membranes were blocked and incubated with new primary antibodies as described above.

Analysis of Poly(GA)/(GP) expression

Cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with 2× complete Mini, EDTA free protease inhibitor cocktail (Merck), and 2% SDS (ThermoFisher) and homogenized by sonication. After centrifugation at 17,000 × g at 16 °C for 20 min, the protein content of the supernatants was determined as described in the Supplementary Methods. The samples were adjusted to the same concentration (0.6–1.0 mg/mL) and MSD immunoassay was performed in singleplex using 96-well SECTOR plates (MSD) to quantify Poly(GA)/(GP) expression, as described previously²⁰. In brief, plates were coated with unlabeled anti-poly(GP) or anti-poly(GA) antibodies. After blocking, samples were loaded at 45 µg of protein per well for the poly(GP) immunoassay and at 27 µg of protein for poly(GA). Biotinylated anti-poly(GP) and anti-poly(GA) antibodies were used as detectors, followed by sulfo-tagged streptavidin (Meso Scale Discovery, R32AD). Plates were read with the MSD reading buffer (Meso Scale Discovery, R92TC) using the MSD Sector Imager 2400. Signals correspond to intensity of emitted light upon electrochemical stimulation of the assay plate. Prior to analysis, the average reading from a calibrator containing no peptide was subtracted from each reading. Negative signals were set to ‘0’.

Statistics and reproducibility

No statistical method was used to predetermine sample size. Investigators were blinded to allocation during analyses if permitted by the experimental design. Statistical analyses were conducted using GraphPad Prism 9. No data were excluded from the analyses. Data from multiple lines and differentiations were pooled to compare the overall effect of the C9orf72 HRE versus controls. Comparisons of two groups were performed by two-tailed unpaired t-tests and multiple group comparisons by one-way or two-way ANOVA with appropriate post-hoc tests as indicated in the figure legends. The number of independent experiments and lines are indicated in each figure legend. Data are presented as single data points and means ± SEM. Differences were considered significant when P < 0.05 (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant). Box plots show the median (center line), upper and lower quartiles (box limits), 1.5x interquartile range (whiskers), and outliers (points). GraphPad Prism 9 or RStudio 1.4.1103 were used to plot data. Final assembly of figures was done using Adobe Illustrator 25.4.1.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-023-41603-0