All experiments were conducted in accordance with relevant guidelines and followed protocols approved by the Massachusetts General Hospital Institutional Review Board, Institutional Animal Care and Use Committee, and Partners Institutional Biosafety Committee. Studies performed in postmortem human tissue were approved by the Massachusetts Alzheimer’s Disease Research Center (ADRC) and the Veterans Affairs Biorepository Brain Bank (VABBB).

Analysis of publicly available ALS datasets

FASTQ files (GSE143743 [1] and GSE76220 [49]) were obtained from the NCBI gene expression omnibus (GEO) database or were previously generated by our lab (dbGAP: phs002440.v2.p1 [35]). FASTQ files were aligned to GRCh38 using STAR (2.7.3a) [19], and read counts were generated using –quantMode GeneCounts in STAR. Differentially expressed genes were determined using DESeq2 (1.36.0) [57] in R (4.0.3). For the reanalysis of postmortem sALS SMNs [49], a model of design =  ~ Patient Sex + Disease was used to normalize for effects by patient sex as in [35]. Genes with adjusted p-value < 0.05 met statistical significance for differential expression. Gene set enrichment analysis (GSEA) (fgsea 1.22.0) [92] was performed in R (4.2.1) using log2(fold change) values.

Postmortem human and mouse tissue immunohistochemistry and immunostaining

Postmortem motor cortex tissue included sections from C9orf72 repeat expansion with ALS (five ALS alone and three ALS/FTD), p-TDP-43-negative AD (8), and non-neurological disease controls (6) provided by the ADRC, as well as TDP-43, FUS, PFN1, KIF5A, NEK1 fALS, and four non-neurological controls provided by the VABBB. Information on human samples is provided in Supplementary Table 1. Briefly, formalin-fixed, paraffin-embedded (FFPE) slides from postmortem human primary motor cortex and occipital cortex were de-paraffinized with xylene followed by a descending ethanol series. Peroxidase quenching was performed by incubating slides in 3% hydrogen peroxide solution for 30 min. Sections were rinsed with PBS, and antigen retrieval was then performed by microwaving sections in 10 mM citrate buffer, pH 6.0, and allowing them to cool on ice for 30 min. The sections were permeabilized using 0.4% Triton X-100 (Millipore Sigma, 9400) for 8 min at room temperature (RT) and incubated with a blocking solution containing 2.5% normal horse serum (Vectastain ABC kit) for 90 min at RT. The sections were then incubated with primary antibody anti-STING (1/50, R&D systems, AF6516) overnight at 4 °C. Immunolabeling was then visualized using the biotinylated secondary antibody sheep IgG horseradish peroxidase (HRP) for 90 min at RT and incubated with the avidin–biotin enzyme complex for 30 min. Stains were visualized by incubation with the Vector peroxidase substrate kit DAB (VectorLabs). Slides were then dehydrated in ascending ethanol/xylene series and coverslipped with Cytoseal X. Immunohistochemical negative controls included incubation of sections with no primary or secondary antibody but with all other immunoperoxidase-DAB steps unchanged. Investigators were blinded throughout the processing and analysis of IHC staining. Imaging of immunoperoxidase sections was done using a 40 × objective on a Nanozoomer.

For C9orf72 mouse tissue, FFPE sagittal brains from four AAV (G4C2)2 (two males and two females) and five AAV (G4C2)149 (three males and two females) 1-year-old mice were sliced into 6 μm sections using a microtome. For STING antibody validation, 1-year-old wild-type (WT) and goldenticket (gt) STINGgt/gt mice were transcardially perfused with cold 0.01 M PBS, followed by cold 4% PFA in 0.01 M PBS. Their colon and spleen were collected and cut on a vibratome into coronal sections of 40 μm.

For tissue immunofluorescence staining, FFPE human and mouse sections were de-paraffinized with xylene and re-hydrated in descending ethanol series. Antigen retrieval was then performed by microwaving sections in 10 mM citrate buffer, pH 6.0, and allowing them to cool on ice for 30 min. Sections were permeabilized using 0.4% Triton X-100 (Millipore Sigma, 9400) for 8 min at RT and incubated with a blocking solution containing 10% Normal Donkey Serum and 0.1% Triton X-100 for 90 min at RT. Human sections were then incubated with primary antibodies including anti-STING (1/50, R&D systems, AF6516), anti-CRYM (1/250, Abcam, ab220085), anti-p-IRF3 (1/50, Cell Signaling Technology, 29047S), and anti-HuC/HuD (1/2000, Sigma-Aldrich, MABN153) overnight at 4 °C. Mouse sections were incubated with primary antibodies including anti-STING (1/100, Proteintech, 19,851–1-AP), anti-p-IRF3 (1/50, Cell Signaling Technology, 29047S), anti-CTIP2 (1/100, Abcam, ab18465), and anti-p-NF-κB (1/50, Santa Cruz Biotechnology, sc-136548) overnight at 4 °C (Supplementary Table 2). After three washes with PBS, the slides were incubated with species-specific Alexa Fluor (− 488, − 647, − 568; Thermo Fisher Scientific) secondary antibodies at a 1/500 dilution for 90 h at RT. Finally, the slides were incubated for 5 min with DAPI solution (Thermo Fisher Scientific, D1306) for nuclear staining and washed three times in PBS. After 5 min incubation in 70% ethanol, the sections were immersed in Autofluorescence Eliminator Reagent (Millipore, 2160) for 5 min. The sections were coverslipped with ProLong Diamond Antifade solution (Life Technologies, P36962). Confocal images were taken using a 20 × objective on a Zeiss LSM 900 Confocal microscope. Investigators were blinded throughout the processing and quantitative analysis of immunofluorescence staining of CTIP2-positive and STING-positive neurons in mouse sections.

Human iPSCs

Human iPSC lines were obtained from the Target ALS Repository, Harvard University, Jackson Laboratory, and the NINDS Human Cell and Data Repository (NHCDR). Human iPSC lines included non-isogenic control lines 11a [9], ND50003 (FA0000010) and KOLF2.1 J [75] and five fALS lines harboring mutations in C9orf72: 19f [45], NDS00268 (ND50074), NDS00269 (ND50075), NDS00270 (ND50076), and NDS00273 (ND50080). An isogenic pair of lines consisting of an unedited control TDP-43+/+ line and an edited TDP-43+/G298S iPSC line (harboring a single fALS mutation) was previously generated [77]. iPSCs were cultured as described previously [65]. Briefly, iPSCs were maintained on Matrigel-coated 6 well-plates (Corning, 354,277, and Falcon, 353,046) in mTeSR media (Stemcell Technologies, 85,850) for SMN differentiation or mTeSR Plus media (Stemcell Technologies, 100–0274) for NGN2 induction at 37 °C with 5% CO2.

Cell culturePrimary mouse cortical neurons

Primary cortical neurons were isolated from cerebral cortices of embryonic day 13–14 mouse embryos (Charles River Laboratories) as previously described [93] with modifications. Briefly, pregnant female mice were euthanized between embryonic days 13 and 14, and embryonic cortices were dissected and enzymatically dissociated using a papain dissociation kit (Worthington Biochemical, LK003178). Neurons were plated on poly-D-lysine-coated 384- (Corning Life Sciences, 354,663) and 24-well plates (Falcon, 08-772-1) at densities of 1 × 104 and 2.5 × 105 viable neurons, respectively. The neurons were maintained at 37 °C with 5% CO2 in Neurobasal medium (Life Technologies, 21,103–049) supplemented with 1% GlutaMAX (Thermo Fisher Scientific, 35,050–061), 2% B-27 supplement (Life Technologies, 17,504–044), and 1% penicillin/streptomycin (Life Technologies, 15,070–063). Primary cortical neurons were grown for 5 days before receiving described drug treatments and analysis.

SMN differentiation from iPSCs

SMN differentiation from iPSCs followed a previously described protocol [87] with modification [65]. Briefly, iPSCs were enzymatically dissociated into single cells for 10 min at 37 °C with StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific, A1110501) and counted using a Countess II FL Automated Cell Counter (Thermo Fisher Scientific, AMQAF1000). Cells were neuralized as a suspension culture at a density of 2 × 106 cells/ml using SB431542 (20 µM, Sigma-Aldrich, S4317), LDN-193189 (0.1 µM, Stemgent 040019), CHIR-99021 (3 µM, Tocris, 99,021), FGF2 (10 ng/mL, Thermo Fisher Scientific, 13,256,029), and ascorbic acid (10 µM) (Sigma, A4403) in N2/B27 media (1:1 mixture of Neurobasal and Advanced DMEM/F12 (Life Technologies, 12,634–028) supplemented with 1% GlutaMAX, 100 µM β-mercaptoethanol (Life Technologies, 31,340–010), 2% B27-supplement, 1% N2-supplement (Gibco, 17,502–048), and 1% penicillin/streptomycin. On day 2, neural spheres were patterned to spinal cord identity by treating with retinoic acid (0.1 µM, Sigma, R2625), smoothened agonist (SAG, 500 nM, EMD Calbiochem, 566,660), along with SB-431542, LDN-193189, and CHIR-99021 in N2/B27 media for an additional 5 days. On day 7, brain-derived neurotrophic factor (BDNF, 10 ng/ml, Life Technologies, PHC7074) was added to the N2/B27 media to generate SMN progenitors. On day 9, SMN progenitors were cultured in the previous media and additionally supplemented with DAPT (10 µM, Tocris Bioscience, 2634) for 5–7 days. After 16 total days in culture, embryoid bodies were dissociated using 0.05% Trypsin–EDTA, pre-mixed with Matrigel, and seeded on poly-D-lysine-coated plates at either 1 × 104 cells per well (384-well plates, immunostaining) or 2.5 × 105 cells per well (24-well plates, RNA). The following day, the neurons were treated with uridine/5-fluoro-deoxyuridine (U/FDU, 1 µM) to remove residual proliferating cells and cultured in Neurobasal media supplemented with 1% GlutaMAX, 1% non-essential amino acids (Corning 25–025-CI), ß-mercaptoethanol (100 µM), B27-supplement (1%), N2-supplement (1%), retinoic acid (1 µM), ascorbic acid (2.5 µM), BDNF (10 ng/ml), glial-derived neurotrophic factor (GDNF, 10 ng/ml, Life Technologies PHC7044), ciliary neurotrophic factor (CNTF, 10 ng/ml, Life Technologies PHC7015), insulin-like growth factor (IGF-I, 10 ng/ml, R&D systems 291-G1-200), and penicillin/streptomycin (1%). Media was changed every 2–3 days.

NGN2 induction from iPSCs

NGN2 neurons were generated using a PiggyBac strategy [35, 75, 103]. PiggyBac plasmids were generated with tet-inducible transcription factors NGN2 [104] and nucleofected into iPSC lines together with a PiggyBac transposase plasmid using the Nucleofector I (Lonza, A-23) and the Human Stem Cell Nucleofector Kit 1 (Lonza, VPH-5012). Cells were plated on Vitronectin XF (Stemcell Technologies, 07180) and maintained in mTeSR Plus media supplemented with CET (Chroman1 (50 µM, MedChem Express, HY-15392), Emricasan (5 mM, Selleckchem, S7775), and Trans-ISRIB (0.7 mM, Tocris, 5284)) [12] for 24 h and selected using puromycin (10ug/mL, InvivoGen, ant-pr-1) for 48 h based on the presence of nuclear BFP2 signal, indicative of donor plasmid integration.

Selected iPSCs were then differentiated into post-mitotic neurons. In brief, 12 million iPSCs were plated in a T175 flask coated with Matrigel and cultured using induction media (DMEM/F12 medium, Life Technologies, 11,320–082; 1% N2-supplement; 1% non-essential amino acids; 1% GlutaMAX; and 1% penicillin/streptomycin) supplemented with CET and doxycycline (2 μg/mL, Sigma Aldrich, D9891-1G). A full media change was performed on day 2. On day 3, differentiated NGN2 neurons were dissociated using Accutase and frozen (1 million cells/vial) in induction media, supplemented with 40% FBS [Hyclone, SH30910.03HI) and 10% DMSO (Sigma, D2650)].

For individual experiments, NGN2 neurons were pre-mixed with Matrigel and plated in poly-D-lysine-coated plates, at either 10,000 cells/well (384-well plate, immunostaining) or 250,000 cells per well (24-well plate, RNA) in induction media supplemented with CET. After 24 h, the neurons were maintained at 37 °C with 5% CO2 in Neurobasal supplemented with 2% B27-supplement, NaCl (50 mM), 1% GlutaMAX, NT-3 (10 μg/mL, PeproTech, 450–03), BDNF (10 μg/mL), and 1% penicillin/streptomycin. The following day, the neurons were cultured in long-term media supplemented with aphidicolin (5uM, Cell Signaling Technology, 32,774). After 2 days, media was exchanged with fresh long-term media without aphidicolin. Every 3 days, 50% of the media was replaced with fresh media and maintained up to day 30 in culture. The neurons were grown for 10 to 30 days for time course analyses and for 20 days before receiving drug treatments.

Lentiviral shRNA neuronal transduction

Two separate lentiviral shRNAs directed against TDP-43 and a scrambled shRNA control were obtained from Origene (TL308946V). For shRNA lentiviral particle transduction, isoTDP-43+/+ NGN2 neurons were incubated for 4 h with media containing lentivirus particles for GFP-shScramble or GFP-shTDP-43 and cultured for 48 h. Transduction efficiency was assessed by GFP expression. Neuronal cultures were analyzed for STING and γH2AX levels 48 h post-transduction.

DNA-damaging agents and STING modulators

For induction of DNA damage, cells were incubated with etoposide (Sigma, E1383) and glutamate (Sigma, G1251) at 5 µM and 10 µM, respectively. For STING-activation experiments, 2′3′-cGAMP (Sigma, 5,318,890,001) was used at 20 μM in human iPSC-derived neurons, and DMXAA (Sigma, D5817) was used at 20 μg/ml in primary mouse cortical neurons. For STING-inhibition experiments, 1 μM H151 (Invitrogen, inh-h151) or 10 μM RU.521 (Sigma, SML2347) was used.

RT-qPCR

RNA was extracted from cells using Trizol reagent (Life Technologies, 15,596–018) and purified according to the manufacturer’s instructions. Purified RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4,368,814) according to the manufacturer’s instructions. RT-qPCR was carried out using iQ™ SYBR Green Supermix (Biorad, 1,708,882) on a Biorad CFX96 instrument with the following program: initial denaturation at 95 °C for 360 s; 40 cycles of 95 °C for 30 s, 55 °C for 60 s, and a melt curve step. The quantification cycle for the mRNAs of interest was normalized to TATA-box binding protein (TBP), hypoxanthine phosphoribosyltransferase (HPRT), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference mRNA, and data were expressed as fold change or log2 fold change over vehicle treatment. Gene-specific primer sequences are listed in Supplementary Table 3.

Immunocytochemistry, image acquisition, and analysis

iPSC-derived neurons and primary cortical neuron cultures were fixed for 20 min with 4% paraformaldehyde (Thermo Fisher Scientific, 28,908) at RT. Three gentle washes with PBS (Thermo Fisher Scientific, 10,010,049) were followed by cell membrane permeabilization using 0.25% Triton X-100 (Millipore Sigma, 9400) for 8 min at RT. Cells were incubated with a blocking solution containing 5% bovine serum albumin (BSA) and 10% Normal Donkey Serum for 45 min at RT. The blocking solution was removed, and cells were incubated with primary antibodies diluted in fresh blocking solution overnight at 4 °C. Primary antibodies included anti-TUJ1 (1/250, Aves Lab, TUJ), anti-CTIP2 (1/100, abcam, ab18465), anti-p-IRF3 (1/50, Cell Signaling Technology, 29047S), anti-p-NF-κB (1/50, Santa Cruz Biotechnology, sc-136548), anti-phospho-histone H2A.X (γH2AX, 1/50, Cell Signaling Technology, 9718 T), anti-calnexin (1/50, Novus Biological, NB300-518), anti-STING (1/100, Proteintech, 19,851–1-AP; mouse cells), and anti-STING (1/50, R&D systems, AF6516; human cells). Antibodies are listed in Supplementary Table 2. After three washes with PBS, cells were incubated with species-specific Alexa Fluor (− 488, − 647, − 568; Thermo Fisher Scientific) secondary antibodies at a 1/500 dilution for 1 h at RT. Finally, the cells were incubated for 5 min with DAPI solution for nuclear staining and washed three times in PBS. Confocal images were acquired with an Image X-Press Micro Confocal (Molecular Devices) or LSM900 confocal microscope (Zeiss). Four z-stacks were acquired per field (1.6 μm step size), 15 to 25 fields per well using a 40 × objective for iPSC-derived neurons and a 20 × objective for primary mouse cortical neurons. Within each experiment, all groups were imaged with the same acquisition settings. Automatic quantifications were performed using a custom Fiji/ImageJ-based plugin (National Institutes of Health, version 1.53c) [86] for all cellular analyses except TDP-43 depletion, which was performed by blinded manual quantifications of γH2AX and STING-positive neurons performed on 80 to 100 cells per well. For area and integrated intensity measurements of stained cytoplasmic STING and nuclear p-IRF3 and p-NF-kB, and γH2AX, human iPSC-neurons and primary mouse cortical neurons were analyzed using a custom Fiji/ImageJ-based plugin (National Institutes of Health). To investigate ER-activated STING, the nuclear mask was dilated (10 iterations), and the perinuclear stained STING area and integrated intensity were analyzed within the dilated nuclear mask.

Quantification and statistical analysis

All data were plotted and statistically analyzed on R-studio software (version 4.2.1). Boxplots and bars represented the mean and standard error of the mean. Unpaired two-tailed Student’s t tests (R-studio) were used to compare between groups. Differences were considered statistically significant for p values < 0.05. p values were depicted in graphs as *, **, ***, **** representing p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.

Data and code availability

Custom scripts used to analyze immunofluorescence and RNA-seq of published datasets are available at https://github.com/waingerlab/STING. Any additional information reported in this paper will be shared by the lead contact upon request.