Environmental Control of Astrocyte Pathogenic Activities in CNS Inflammation

Abstract

Genome-wide studies have identified genetic variants linked to neurologic diseases. Environmental factors also play important roles, but no methods are available for their comprehensive investigation. We developed an approach that combines genomic data, screens in a novel zebrafish model, computational modeling, perturbation studies, and multiple sclerosis (MS) patient samples to evaluate the effects of environmental exposure on CNS inflammation. We found that the herbicide linuron amplifies astrocyte pro-inflammatory activities by activating signaling via sigma receptor 1, inositol-requiring enzyme-1α (IRE1α), and X-box binding protein 1 (XBP1). Indeed, astrocyte-specific shRNA- and CRISPR/Cas9-driven gene inactivation combined with RNA-seq, ATAC-seq, ChIP-seq, and study of patient samples suggest that IRE1α-XBP1 signaling promotes CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, MS. In summary, these studies define environmental mechanisms that control astrocyte pathogenic activities and establish a multidisciplinary approach for the systematic investigation of the effects of environmental exposure in neurologic disorders.

Keywords: IRE1α; Sigmar1; XBP1; astrocyte; glia; inflammation; multiple sclerosis; neurodegeneration; zebrafish.

Figure 1.. Induction of glial nos2a expression in a zebrafish model of CNS inflammation.

A) Zebrafish neuroinflammation model. B) qPCR of mpz expression in zebrafish. n=2 per condition per timepoint. Two-way ANOVA, Bonferroni post-test. C) qPCR analysis 48h after treatment. n=4 per condition, n=3 for il17a/f1 in LPS/cuprizone. Two-way ANOVA, Bonferroni post-test. D) qPCR in EGFP⁺ cells from gfap::egfp fish. n=4 per condition. Two-way ANOVA, Bonferroni post-test. E) Environmental chemical screen flowchart. F) qPCR of nos2a expression in response to environmental chemicals (see Table S1). Red bars indicate increase over baseline (dashed line). n=2 per condition. G) qPCR of nos2a expression from F. n=3 per condition. One-way ANOVA, Holm-Sidak post-test relative to vehicle. H) qPCR of Nos2 expression in neonatal primary mouse astrocytes treated for 24h. Control, n=8; Vehicle, n=8; Linuron, n=6; PFNA, n=6; Vinclozolin, n=9; Methyl carbamate, n=6; Naphthalene, n=4. One-way ANOVA, Bonferroni post-test relative to vehicle on ln-normalized data. See also Table S1.

Figure 2.. Environmental chemicals boost astrocyte pathogenicity.

A) Pathway analysis of ToxCast-identified genes for Linuron and the unfolded protein response. B) Western blot and quantification for treated neonatal astrocytes. n=3 P-p65, n=3 P-IRE1α, n=4 XBP1. Kruskal-Wallis non-parametric ANOVA, uncorrected Dunn’s post-test. C) Western blot. n=4 per condition. Unpaired two-tailed t-test. D) qPCR. n=6 per condition. Unpaired two-tailed t-test. E) Thioflavin T (ThT) fluorescence in neonatal astrocytes treated for 24 hours. n=5–6 images per condition from N=3 cultures. One-way ANOVA, Dunnett post-test. F) (Left) Quantification of Xbp1 splicing. (Right-top) Schematic of Xbp1 splicing assay. (Right-bottom) Splicing assay gel. n=5–6 per condition at 4 hours; n=9 per condition at 24 hours except Linuron (n=3). One-way ANOVA, Bonferroni post-test. G) qPCR in neonatal astrocytes after 24h treatment. n=6–9 per condition, n=4 for Csf2 Linuron condition. One-way ANOVA, Bonferroni post-test, performed on ln-normalized data for Nos2, Il6, and Csf2. H) qPCR of Nos2 expression in neonatal primary mouse astrocytes. n=6 per condition, n=3 for GSK2656157 condition. One-way ANOVA, Holm-Sidak post-test. I) (Left) Predicted XBP1 and p65 binding sites in the murine Nos2 and Csf2 promoters. (Right) ChIP-qPCR of XBP1 and p65 recruitment to Nos2 and Csf2 promoters in neonatal primary mouse astrocytes. n=5 for p65 vehicle, n=2–3 for Linuron, n=6 otherwise. Kruskal-Wallis non-parametric ANOVA, uncorrected Dunn’s test (Csf2, XBP1 site 2) or one-sample t-test (otherwise). J) HEK293 luciferase reporter assay at the indicated hours post transfection (hpt). n=5–6 per group. One-way ANOVA, Dunnett post-test. See also Figure S1 and Table S2.

Figure 3.. Linuron boosts IRE1α/XBP1 signaling via Sigmar1.

A) Schematic of Sigmar1 protein domains. B) Co-immunoprecipitation of Sigmar1 and IRE1α in HEK293 cells. C) Western blot for NF-κB, P-IRE1α and XBP1 activation in neonatal murine astrocytes treated for 1 hour. n=3–7 per condition. One-way ANOVA, Sidak post-test. D) Xbp1 splicing in murine astrocytes activated for 24 hours (top). Representative images of agarose Xbp1 splicing gels (bottom). n=4–9 per condition. One-way ANOVA, Dunnett post-test. E) qPCR in neonatal murine astrocytes activated for 24 hours. n=7–16 per condition; n=4 for BD-1063 alone. One-way ANOVA, Tukey post-test. F) HEK293 luciferase reporter assay. n=4 per condition. One-way ANOVA, Tukey post-test on ln-normalized data.

Figure 4.. Genomic control of disease-promoting astrocyte activities by IRE1α-XBP1 signaling.

A) qPCR in sorted adult astrocytes from naïve and EAE mice. n=3 per group. Unpaired two-tailed t-test per gene. B) qPCR of Nos2 expression (left) or XBP1 binding to the Nos2 promoter by ChIP (right) in sorted adult astrocytes from naïve and EAE mice. n=3 naïve, n=8 EAE (qPCR) or n=2 (ChIP) per group. Unpaired two-tailed t-test on data ln-normalized (qPCR) or not (ChIP). C) Schematic of shRNA lentiviral vector and EAE development in transduced mice. n=13 shScrmbl (left), n=13 shXbp1, n=10 shScrmbl (right), n=9 shErn1. Two-way repeated measures ANOVA, Holm-Sidak post-test. LTR=long terminal repeats, GfaABC₁D=Gfap promoter, EGFP=enhanced green fluorescent protein, pA=polyadenylation signal, WPRE=woodchuck hepatitis virus post-transcriptional regulatory element. D) qPCR of Nos2 expression in EAE astrocytes. n=9 per condition. Unpaired non-parametric Kolmogorov-Smirnov t-test. E) RNA-seq analysis of astrocytes from EAE mice. Columns are n=4 biological replicates. Genes with p<0.05 are shown. F) Pathway analysis of astrocyte signaling in Xbp1 knockdown. G) Gene set enrichment analysis for astrocyte RNA-seq. H) ATAC-seq peaks in astrocytes. n=4 shScrmbl, n=3 shXbp1. Scale bars show read density. Transcriptional schematics shown above plots. I) EAE scores. n=7 Vehicle, n=16 sgScrmbl, n=9 sgXbp1, n=10 sgSigmar1. Two-way repeated measures ANOVA, Holm-Sidak post-test. U6=human U6 promoter, sgRNA=single guide RNA, 2A=self-cleaving peptide. J-K) Astrocyte Nanostring analyses from I. RNA pooled from n=4 mice (sgScrmbl) or n=7 mice (sgXbp1) in J, n=5 mice per group in K. See also Figures S2–S5 and Tables S3–S6.

Figure 5.. XBP1 in astrocytes controls monocytes and microglia during EAE.

A) Model of XBP1+ astrocyte pro-inflammatory signaling in the CNS. B) Flow cytometry plots (left) and quantification (right) of pro-inflammatory monocytes in CNS during EAE. n=10 per group. Unpaired two-tailed t-test. C) Pathway analysis of monocyte RNA-seq data. Columns are n=2 biological replicates. D) Heatmap of monocyte RNA-seq data (p<0.05). E) Gene set enrichment analysis of monocyte pathways. F) Heatmap of microglia RNA-seq data (p<0.05). n=4 shScrmbl, n=3 shXbp1. (G and H) Gene set enrichment analysis and pathway analysis of microglia RNA-seq. Ct=Gfap::shScrmbl, KD=Gfap::shXbp1. I) Nanostring analysis of EAE microglia from 4I. RNA pooled from n=5 mice, n=7 for sgXbp1. See also Tables S4–S5.

Figure 6.. IRE1α-XBP1 signaling in astrocytes is upregulated in MS.

A) qPCR of NOS2 in human fetal astrocytes treated for 48 hours. n=3 per condition. One-way ANOVA, Fisher’s LSD test. B) (Top) Quantification of XBP1 splicing. (Bottom) Gel of XBP1 splicing assay. n=3 per condition. One-way ANOVA, Bonferroni post-test on ln-transformed data. C) XBP1 splicing of MS patient tissue as quantification (top) and gel (bottom). n=3 healthy controls (HCs), n=4 otherwise. One-way ANOVA, Bonferroni post-test relative to control. pre-active=pre-active lesion, active=active lesion. White bars indicated spliced lanes. (D-F), Immunostaining of astrocyte colocalization with P-IRE1α (D-E) and XBP1s (F-G) in MS patients and HCs. NAGM=normally appearing gray matter. In (E) n=7 HC WM, n=6 NAWM, n=3 lesion, n=8 HC GM, n=8 NAGM images from N=3 MS patients and N=4 healthy controls. One-way ANOVA, Dunnett post-test relative to HC (left). Unpaired two-tailed t-test (right). In (G) n=11 HC WM, n=15 NAWM, n=10 lesion, n=6 HC GM, n=10 GM images from N=4 MS patients (NAWM, NAGM), N=3 MS patients (lesion), and N=2 HCs. One-way ANOVA, Dunnett post-test relative to HCs (left). Unpaired two-tailed t-test with Welch’s correction (right). (H-I) Quantification of P-IRE1α+ and XBP1s+ cells by cell type and MS pathology. n=30–54 cells per condition. See also Figure S6.