Fig 2: DHCR7 inhibits TBK1 and IRF3 activation. A, B HEK293T cells were co-transfected with TBK1-coding plasmid together with DHCR7-expressing plasmid or empty vector (A). HEK293T cells were co-transfected with IRF3-ecoding plasmid, RIG-I encoding plasmid, together with DHCR7-expressing plasmid or empty vector (B). Cells were harvested at 24 ?h post-transfection and subjected to Western blot with indicated antibodies [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-P-IRF3(S396), anti-HA, and anti-GAPDH]. C U251 ?cells were transfected with the indicated dose of DHCR7 expressing plasmid or empty vector for 24 ?h. Cells were then infected with ZIKV (MOI ?= ?1) and harvested at 6 ?h post-infection and subjected to Western blot with indicated antibodies [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-P-IRF3(S396), anti-HA, and anti-GAPDH]. D U251 ?cells were transfected with HA-vector, HA-DHCR7 or HA-DHCR7 (G410S) for 24 ?h. Cells were then infected with ZIKV (MOI ?= ?1) and harvested at 6 ?h post-infection and subjected to Western blot with indicated antibodies [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-P-IRF3(S396), anti-HA, and anti-GAPDH]. E U251 ?cells were treated with the indicated dose of AY9944 for 2 ?h and infected with ZIKV (MOI ?= ?1) for 6 ?h, then subjected to Western blot with indicated antibodies [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-P-IRF3(S396), and anti-GAPDH]. Data are from at least three independent experiments and shown as representative data (A–E).

Fig 3: ALS SOD1-mutants damage mitochondria and release mtDNA to activate the IFN-I response(A and B) WT C20 and HASTR/ci35 cells were either treated with 1.0 µg/mL of tunicamycin for 8 h or transfected with indicated plasmids and 36 h after transfection, cells were subjected to fractionation and blotted with indicated antibodies (A). Total DNA extracted from cytosolic and nuclear fractions as treated in A. Cytosolic mtDNA genes was assessed via RT-qPCR (B).(C and D) Total DNA was harvested from the cytosolic and nuclear fractions as in A from whole brain and spinal cord lysates of WT or G85R mice at Week 60. Cytosolic mtDNA genes was assessed.(E) Primary microglia and astrocytes derived from WT or G85R mice at day-10 after birth were subjected to subcellular fractionation as in A. Cytosolic mtDNA genes were assessed.(F) Immunofluorescence staining of dsDNA in C20 cells transfected with plasmids encoding EGFP-SOD1 WT,-G85R, or-G93A.(G) Primary microglia and astrocytes derived from age-matched WT or G85R mice (day-10) were harvested and subjected to MMP assay.(H) C20 cells transfected either with HT-DNA (5.0 µg/mL), or Flag-MAVS/IRF3 5D plasmids (0.1 µg each) were either Sham, ddC, or EtBr treated. Ifnb1 mRNA was measured via RT-qPCR (left). Protein expression was analyzed through western blotting (right).[Note: MAVS in standard form (~52 kDa) are marked by asterisk (***); splicing variants, truncated forms, midi and mini-MAVS (<52 kDa) are marked by vertical blue line (Brubaker et al., 2014; Qi et al., 2017); MAVS in higher molecular weight (>52 kDa) are marked by vertical red line (Hou et al., 2011)].(I) C20 cells transfected with indicated plasmids were either sham or EtBr (150 ng/mL) treated for 4 days. Samples were subjected to fractionation and DNA extraction, followed by RT-qPCR analysis of mtDNA genes and the indicated mRNA. Statistical analysis corresponding to the same condition in the Sham treated group.(J) C20 cells transfected with the indicated plasmids were either sham or ddC (50 µg/mL) for 6-day. Samples were subjected to fractionation and DNA extraction, followed by RT-qPCR analysis of mtDNA gene and the indicated mRNA. Statistical analysis corresponds to the same conditions as the Sham treated group.(K) Knockdown efficiency of the Ppid gene in HASTR/ci35 cells was validated (left). Experimental design of HASTR/ci35 cells co-transfected with indicated siRNA (10 nM) and 0.01 µg plasmids (middle). 48 h later, cells were subjected to fractionation described in A and quantification of mtDNA genes.(L) Similar experimental design as in panel K using C20 cells. mtDNA genes and Ifnb1 mRNA was quantified.(M) C20 cells transfected with indicated plasmids were either vehicle (DMSO) or CsA (20 µM) treated. Samples were subjected to fractionation and DNA extraction, followed by measuring of Mt-co1. RT-qPCR of all mtDNA genes was normalized to genomic DNA, TERT. RT-qPCR of RNA samples was normalized to ß-actin. Data represent the mean of four independent experiments (mean ± SEM). Number of dots = number of mice. *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001; (B–E, G, and I–L: unpaired Student’s t test). (RFU = relative fluorescence unit; A.U. = arbitrary unit). See also Figure S5.

Fig 4: Activation of IFN-I response by ALS SOD1-mutants is dependent on the cGAS-STING signaling pathway(A) Knockout efficiency of Ddx58 (RIG-I), Ifih1 (MDA5), Mavs, or Ticam-1 (TRIF) gene in C20 cells examined via western blotting.(B) WT, DDX58, IFIH1, or MAVS-CRISPR/Cas9-deficient C20 cells either transfected with 0.01 µg Flag-SOD1 WT, G85R, G93A, or MAVS plasmids for 36 h, or 0.5 µg poly(I:C) for 8 h. Ifnb1 mRNA measured using RT-qPCR.(C) Similar conditions as in panel B performed using WT or MAVS-CRISPR/Cas9-deficient HASTR/ci35 cells, except human MAVS plasmid was replaced with human IRF3 5D plasmid. Ifnb1 mRNA assessed via RT-qPCR.(D–F) WT, TICAM, cGAS or STING-CRISPR/Cas9-deficient C20 cells either transfected with 0.01 µg Flag-SOD1 WT, G85R, G93A, or MAVS plasmids for 36 h, treated with LPS (100 ng/mL) for 4 h or transfected with HT-DNA (5.0 µg/mL) for 8 h. Ifnb1 mRNA measured via RT-qPCR. The knockout efficiency of cGAS, or STING assessed via western blot and functional assays.(G) WT C20 cells either treated with RU.521 (5.0 µg/mL) or H.151 (0.5 µg/mL) for 24 h. Cells were then transfected with 0.01 µg of Flag-SOD1 WT, G85R, G93A, or MAVS plasmids, or HT-DNA (1.0 µg/mL). Ifnb1 mRNA was measured via RT-qPCR.(H) Schematic diagram of Flag-tagged cGAS-WT and mutants (location indicated by asterisk), STING-WT and deletion mutants.(I) HEK293T cells stably expressing low amounts of Flag-STING-WT clones were first generated. The clones were further co-reconstituted with low expression of either Flag-cGAS-WT or point mutated plasmids. After selection, the double-positive clones cells were then transfected with 0.01 µg of Flag-SOD1 WT, G85R, or G93A plasmids. Ifnb1 mRNA was assessed via RT-qPCR. Protein expression for cGAS-WT and mutants from the 1.0 µg transfected samples were examined. Expression efficiency for each plasmid was evaluated via western blotting.(J) Similar conditions as in I were used but replaced with Flag-cGAS-WT plasmid co-transfected with either STING-WT or deletion mutant (?368). Ifnb1 mRNA was measured using RT-qPCR.(K) Protein lysates collected from C20 cells expressing the indicated plasmids were subjected to immunoblotting with the indicated antibodies.(L) Immunofluorescence staining of endogenous cGAS in primary astrocytes cells derived from day-10 WT, or G85R transgenic mice.(M) Both supernatant and lysates from C20 cells overexpressed with the indicated plasmids were collected and subjected to ELISA analysis of cGAMPs.(N) Sera was prepared from whole blood derived from SOD1 WT or G85R mice, followed by ELISA analysis of cGAMPs (day-10). All RT-qPCR of RNA samples were normalized to ß-actin. Data represent the mean of three independent experiments (mean ± SEM). *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001; (B–G, M, and N: unpaired Student’s t test). See also Figure S4.

Fig 5: Overexpression of ALS SOD1-mutants activate IFN-I response in vitro and confer resistance to viral infection(A and B) HeLa WT or murine RAW264.7 cells expressing indicated reporter were co-transfected with plasmids encoding EGFP-, human SOD1-WT,-G85R, or-G93A mutants. 36 h after transfection, cell lysates were collected and analyzed for luciferase activity.(C) THP-1-derived MF cells were transfected with 0.01 µg of indicated plasmids for 36 h, indicated mRNA levels were assessed via RT-qPCR.(D) Similar conditions in panel (C) were performed in an indicated neuronal cell lineage (H: Human; M: Murine).(E) C20 cells were transfected with indicated plasmids, followed by CVB3 (MOI = 1.0) infection at indicated time-points. CVB3 Vp1 mRNA was measured via RT-qPCR.(F) Supernatant from panel E were collected and subjected to the plaque assay to measure the release of viral particles.(G) Newly seeded C20 cells were pre-treated with supernatant collected from C20 cells expressing the indicated plasmids for 24 h. Cells were then infected with HCoV-OC43 (MOI = 1.0) for 72 h. In a parallel experiment, C20 cells were treated with 100 ng/mL of IFN-ß proteins for 24 h as positive control. HCoV-OC43 nucleocapsid RNA, Isg15, and Rsad2 mRNAs were measured using RT-qPCR.(H and I) C20 cells transfected with the indicated plasmid were infected with the indicated MOI of CVB3 and assessed 24 h later for cell viability with crystal violet staining and the LDH released assay.(J) NanoString analysis for gene expression of indicated family of transcription factors comparing whole brain of WT and SOD1-G85R mice (n = 3 for each genotype).(K) Immunoblot analysis of phosphorylated or total TBK1 and IRF3 or ß-actin in C20 cells after transfection with the indicated plasmids.(L) (1) TBK1, or IRF3-deficient C20 cells using CRISPR/Cas9 system expressing the indicated plasmids for 36 h. Ifnb1 and Rsad2 mRNA measured via RT-qPCR. Transfected poly(I:C) [p(I:C), 0.1 µg] served as positive control. (2) Knockout efficiency of the positive clones was confirmed by western blotting. Clone#1 of two lineage were selected for subsequent studies. Data represent the mean of three independent experiments with n = 3 biological replicates (mean ± SEM). *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001; (A–I, and L: two-tailed, unpaired Student’s t test). See also Figure S3.