Long 3'UTRs predispose neurons to inflammation by promoting immunostimulatory double-stranded RNA formation

Abstract

Loss of RNA homeostasis underlies numerous neurodegenerative and neuroinflammatory diseases. However, the molecular mechanisms that trigger neuroinflammation are poorly understood. Viral double-stranded RNA (dsRNA) triggers innate immune responses when sensed by host pattern recognition receptors (PRRs) present in all cell types. Here, we report that human neurons intrinsically carry exceptionally high levels of immunostimulatory dsRNAs and identify long 3'UTRs as giving rise to neuronal dsRNA structures. We found that the neuron-enriched ELAVL family of genes (ELAVL2, ELAVL3, and ELAVL4) can increase (i) 3'UTR length, (ii) dsRNA load, and (iii) activation of dsRNA-sensing PRRs such as MDA5, PKR, and TLR3. In wild-type neurons, neuronal dsRNAs signaled through PRRs to induce tonic production of the antiviral type I interferon. Depleting ELAVL2 in WT neurons led to global shortening of 3'UTR length, reduced immunostimulatory dsRNA levels, and rendered WT neurons susceptible to herpes simplex virus and Zika virus infection. Neurons deficient in ADAR1, a dsRNA-editing enzyme mutated in the neuroinflammatory disorder Aicardi-Goutières syndrome, exhibited intolerably high levels of dsRNA that triggered PRR-mediated toxic inflammation and neuronal death. Depleting ELAVL2 in ADAR1 knockout neurons led to prolonged neuron survival by reducing immunostimulatory dsRNA levels. In summary, neurons are specialized cells where PRRs constantly sense "self" dsRNAs to preemptively induce protective antiviral immunity, but maintaining RNA homeostasis is paramount to prevent pathological neuroinflammation.

Fig. 1.. Neurons have intrinsically high levels of dsRNA.

(A) Comparison of dsRNA levels across multiple human cell types. Cells were stained with DAPI (blue) and J2 immunostaining (green). (B) MFI quantification of (A). (C) Levels of total RNA and dsRNA compared between hESC and day 15 neurons. Total RNA (RNASelect, green) and dsRNA (J2, red). (D) Quantification of total RNA signal in (C). (E) Quantification of dsRNA signal normalized to total RNA signal in (C). (F) Immunofluorescent images of C57BL/6 mouse cortical brain, heart, and skin tissues. Tissue sections were stained with the 9D5 antibody (dsRNA, red) and neuron marker (NeuN, green). (G) Quantification of dsRNA signal in (F) colocalized with NeuN. (H) Mouse cerebral cortex stained with the 9D5 antibody (dsRNA, red) and NeuN (green). White dotted circle indicates neuron. Orange dotted circle indicates nonneuronal cells. All bar graphs are mean ± SD [n = 12 cells from biological triplicates in (B), (D), and (E); n = 4 images from biological triplicates in (G)]. All scale bars represent 10 μm, except for (F), which is 50 μm. (B) One-way ANOVA with Tukey corrected multiple comparisons. (D, E, and G) Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.. WT neurons constitutively produce tonic type I IFN through two dsRNA-sensing PRR pathways, MAVS and TRIF.

(A and B) Relative levels of type I IFN (IFN-β, pan–IFN-α, IFN-α21) and type III IFN (IFN-λ1) (A) and ISG (IFIT1, IFI44, ANGPTL1, STAT1) mRNA (B) in diverse human cell types. HLCs, cardio-myocytes, NPCs, neurons (day 15), and motor neurons (day 15) were all derived from WT hESCs (HUES8). (C) Type I IFN (IFN-β and IFN-α subtypes) expression in 54 nondiseased tissue sites analyzed by RNA-Seq. Data were derived from the GTEx database. TPM, transcript per million. (D) qPCR of IFN-β levels in WT neurons (day 20) after doxycycline (dox)–induced knockdown of the indicated immune genes using shRNA (sh). (E) qPCR of several ISGs in WT neurons (day 20) after doxycycline-induced knockdown of MAVS. For all qPCR, RPS11 was used as a housekeeping gene. All quantified data shown are mean ± SD (n = 3 biological replicates). (A and B) One-way ANOVA with Tukey corrected multiple comparisons. (D and E) Two-way ANOVA with Tukey corrected multiple comparisons, *P < 0.05, **P < 0.01, ***P < 0.001, and ****p < 0.0001.

Fig. 3.. ADAR1 regulates dsRNA burden in diverse cell types and tonic IFN production in neurons.

(A to C) WT and ADAR1 KO NPCs (derived from HUES8 cells) were differentiated to neurons using adherent monolayer culture methods. Neurons were stained for dsRNA (J2, green), a neuron marker (MAP2, red), and DAPI (blue) at different stages of differentiation (A). Bar graphs quantifying dsRNA signal (B) and IFN-β mRNA levels (C). (D to F) WT and ADAR1 KO hESCs (HUES8) were differentiated to motor neurons via embryoid bodies in cell suspension. Neurons were stained for dsRNA (J2, green), a neuron marker (TUJ1, red), and DAPI (blue) at different stages of differentiation (D). Bar graphs quantifying dsRNA signal (E) and IFN-β mRNA levels (F). (G) qPCR of IFN-β levels in ADAR1 KO neurons (day 20) after doxycycline-induced knockdown of the indicated immune genes using shRNA. (H) Immunoblot measuring PKR activation in WT and ADAR1 KO neurons over time during differentiation (ADAR1 KO neurons at day 20 were too unhealthy to obtain sufficient protein). p-PKR, phosphorylated PKR. (I) Relative levels of IFN-β mRNA in WT and ADAR1 KO HEK-293T, HeLa, hESCs, NPCs, and day 15 neurons. For all qPCR, RPS11 was used as a housekeeping gene. All quantified data shown are mean ± SD [n = 3 biological replicates in (C) and (F) to (H); n = 12 cells from biological triplicates in (B) and (E)]. Scale bars, 10 μm. Two-way ANOVAwith Tukey corrected multiple comparisons, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 4.. Neuron-enriched proteins HuB/C/D cooperate to induce 3′UTR elongation, dsRNA levels, and inflammation.

(A to C) WT HEK-293T cells were transfected with 100 ng of plasmid expressing FLAG-tagged HuB, HuC, or HuD or a combination of HuB (33 ng), HuC (33 ng), and HuD (33 ng). EV, empty vector. 3′UTR lengthening of select genes measured via qPCR (A). The long 3′UTR transcript isoform was normalized to total open reading frame (ORF)–containing transcripts. (B and C) Scatterplot of the PDUI value for genes in HEK-293T_HuB/C/D (n = 3 biological replicates) versus HEK-293T_EV (n = 3 biological replicates) (B) and HEK-293T_EV versus HEK-293T_Mock (n = 3 biological replicates) (C). PDUI values were derived via DaPars2 analysis. Significant change in 3′UTR length was defined as a change in PDUI greater than 1.5-fold. Genes with significantly lengthened (red, increase in PDUI) and shortened (blue, decrease in PDUI) 3′UTRs are colored (FDR ≤ 0.05). (D to G) WT, ADAR1 KO, or ADAR1/MDA5 double-knockout (DKO) HEK-293T cells were transfected as in (A). (D) Immunofluorescent images of dsRNA (J2, green) and DAPI (blue) in transfected cells. (E) Quantification of dsRNA in (D). (F) Relative IFN-β mRNA levels measured by qPCR. Housekeeping gene, RPS11. (G) Immunoblot measuring PKR activation. Scale bars, 10 μm. All quantified data shown are mean ± SD [n = 5 biological replicates in (A) and (F); n = 12 cells from biological triplicates in (E)]. (A) One-way ANOVA with Tukey corrected multiple comparisons. (E and F) Two-way ANOVA with Tukey corrected multiple comparisons, *P < 0.05, **P < 0.01, ***P < 0.001, and ****p < 0.0001.

Fig. 5.. Ectopic HuB/C/D expression in HEK-293T cells protects against SINV infection.

(A and B) Effects of HuB/C/D ectopic expression on the global transcriptome. (A) Volcano plot showing differential expression of genes between WT HEK-293T_HuB/C/D (n = 3 biological replicates) and WT HEK-293T_EV cells (n = 3 biological replicates). Up-regulated genes (red; FDR ≤ 0.001, CPM fold change ≥ 3) and down-regulated genes (blue; FDR ≤ 0.001, CPM fold change ≤ 0.333) are colored. Ectopically expressed ELAVL2, ELAVL3, and ELAVL4 (HuB, HuC, and HuD) and ISGs are marked with a black border. (B) Dot plot showing gene ontology (biological process) analysis of up-regulated genes in (A). The y axis represents different pathways, and the x axis represents the ratio of the DEGs. Darker red dots represent more significant enrichment. The circle size indicates the number of genes enriched in the pathway. (C to E) EV-, HuB-, or HuB/C/D-transfected WT HEK-293T cells were infected with a SINV dual reporter (MOI = 1); BFP reports for SINV genomic mRNA, and GFP reports for SINV subgenomic mRNA. For the pre–IFN-β set, cells were pretreated with 0.05 nM IFN-β for 24 hours before infection. For the post–IFN-β set, cells were treated with 0.05 nM IFN-β for 1 hour after infection until harvest. (C) Flow cytometry analysis of SINV-infected cells (GFP and BFP double-positive cells). Representative dot plots at 6 and 24 hours after infection. (D and E) Bar graph showing the frequency (%) of SINV-infected cells at 6 hours (D) and 24 hours (E) after infection. Data are shown as mean ± SD (n = 3 biological replicates). One-way ANOVA with Tukey corrected multiple comparisons, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 6.. HuB and HuC promote long 3′UTRs, dsRNA levels, and inflammation in neurons.

(A to G) Day 20 post-differentiation neurons derived from WT and ADAR1 KO hESCs were transduced with lentivirus containing the indicated doxycycline-inducible shRNA. (A) 3′UTR shortening of selected genes measured by qPCR. The long 3′UTR transcript isoform was normalized to total ORF-containing transcripts. (B) Immunofluorescent images of dsRNA (J2, green) and DAPI (blue) in transduced WT neurons. Expression of doxycycline-inducible shRNA is coupled with red fluorescent protein (RFP) expression (red). (C) Quantification of dsRNA in (B). (D) Relative IFN-β mRNA levels measured by qPCR. (E to G)Same as (B) to (D) above, except in ADAR1 KO neurons. For all qPCR, RPS11 was used as a housekeeping gene. All quantified data shown are mean ±SD [n = 3 biological replicates in (A), (D), and (G); n = 12 cells from biological triplicates in (C) and (F)]. Two-way ANOVA with Tukey corrected multiple comparisons, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 7.. HuB and HuC protect neurons from SINV, HSV-1, and ZIKV infection.

(A to E) Day 20 post-differentiation WT neurons were transduced with lentivirus containing doxycycline-inducible shRNA against HuB and HuC and then were infected with various viruses. (A and B) Neurons were infected with SINV (MOI = 0.1). (A) Fluorescent and brightfield images of neurons at 24 hours after infection. (B) Quantification of GFP-positive area in (A). (C and D) Neurons were infected with an HSV1 GFP reporter virus (MOI = 1). (C) Fluorescent and brightfield images of neurons at 24 hours after infection. (D) Quantification of GFP-positive area in (C). (E) Neurons were infected with ZIKV (MOI = 0.1), and infection was measured via qPCR of Zika RNA at both 24 (circle) and 48 (triangle) hours post-infection (hpi). (F) Summary graphic showing the main findings of this study. For Zika RNA qPCR, 18S RNA was used as a housekeeping gene. All quantified data shown are mean ± SD (n = 3 experimental replicates). Scale bars, 400 μm. (B and D) One-way ANOVA with Tukey corrected multiple comparisons. (E) Two-way ANOVA with Tukey corrected multiple comparisons, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.