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. 2018 Mar 7;26(3):730-743.
doi: 10.1016/j.ymthe.2018.01.008. Epub 2018 Jan 17.

microRNA-219 Reduces Viral Load and Pathologic Changes in Theiler's Virus-Induced Demyelinating Disease

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Free PMC article

microRNA-219 Reduces Viral Load and Pathologic Changes in Theiler's Virus-Induced Demyelinating Disease

Ana Lis Moyano et al. Mol Ther. .
Free PMC article

Abstract

Analysis of microRNA (miR) expression in the central nervous system white matter of SJL mice infected with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) revealed a significant reduction of miR-219, a critical regulator of myelin assembly and repair. Restoration of miR-219 expression by intranasal administration of a synthetic miR-219 mimic before disease onset ameliorates clinical disease, reduces neurogliosis, and partially recovers motor and sensorimotor function by negatively regulating proinflammatory cytokines and virus RNA replication. Moreover, RNA sequencing of host lesions showed that miR-219 significantly downregulated two genes essential for the biosynthetic cholesterol pathway, Cyp51 (lanosterol 14-α-demethylase) and Srebf1 (sterol regulatory element-binding protein-1), and reduced cholesterol biosynthesis in infected mice and rat CG-4 glial precursor cells in culture. The change in cholesterol biosynthesis had both anti-inflammatory and anti-viral effects. Because RNA viruses hijack endoplasmic reticulum double-layered membranes to provide a platform for RNA virus replication and are dependent on endogenous pools of cholesterol, miR-219 interference with cholesterol biosynthesis interfered virus RNA replication. These findings demonstrate that miR-219 inhibits TMEV-induced demyelinating disease through its anti-inflammatory and anti-viral properties.

Keywords: anti-viral; cholesterol; miRNA-219; microglia; myelin; oligodendrocytes; virus-induced demyelination.

Figures

Figure 1
Figure 1
Effects of BeAn Infection on OL-Related miRNAs (A and B) miRNA expression by qPCR of OL-related miRNAs, miR-17, miR-19b, miR-138, miR-219, miR-338, and astrocyte-related miR-146, in white matter of TMEV-infected mice at 30 days pi (A) and 90 days pi (B). One-way ANOVA followed by Dunnett’s test: (A) F (6, 30) = 5.472, *p < 0.05 and **p < 0.01 (n = 3 per group); and (B) F (6, 32) = 4.346, *p < 0.05, **p < 0.01, and ***p < 0.001 (n = 3 per group). (C) miRNA expression by qPCR of OL-related miRNAs: miR-17, miR-19b, miR-138, miR-219, miR-338, and astrocyte-related miR-146 in neural stem cells differentiated for 2 days (n = 3). (D) Cell viability (WST-1 assay) of CG-4 cells at 24 hr after TMEV infection (10 MOI) under proliferating (PDGFAA) or differentiating conditions (1 div or 3 div). MOI, multiplicity of infection; div, days in vitro. (E) Cell viability in differentiated (3 div) CG-4 cells treated with miR-219 or scrambled miR (50 nM) before or after TMEV infection (10 MOI); one-way ANOVA followed by Dunnett’s test, F (2, 6) = 10.65, *p < 0.05 (n = 3 per group). Data are expressed as mean ± SEM.
Figure 2
Figure 2
miR-219 Decreases Disease Severity, Improves Sensorimotor Functions and Peripheral Nerve Conduction, but Not Balance or Motor Coordination (A) Experimental design of weekly supplementation therapy by intranasal delivery of synthetic miR-219 mimics. (B) Temporal progression of cumulative clinical score over treatment period in healthy animals (control) and mice with chronic demyelination treated with vehicle (TMEV+veh), scrambled miR (TMEV+miR-scr), or miR-219 (TMEV+miR-219). Two-way ANOVA followed by Bonferroni’s test, F (3, 1,033) = 9,016, **p < 0.01 and ***p < 0.001 (n = 7 to 8 per group). (C) Cumulative clinical score over 22 weeks of treatment. One-way ANOVA followed by Tukey’s test, F (3, 1,078) = 281.3, ***p < 0.001 (n = 7 to 8 per group). Cumulative clinical score was calculated as the sum of the clinical score and body weight loss (see Materials and Methods). Data are expressed as mean ± SEM. (D) Clasping score: one-way ANOVA followed by Tukey’s test, F (3, 512) = 11.45, **p < 0.01 and ***p < 0.001. (E and F) Balance and motor coordination analyzed by rotarod test: latency to fall (sec) in the experimental groups treated for 6 weeks (E) or 22 weeks (F). One-way ANOVA followed by Tukey’s test in (F), F (3, 80) = 16.43, **p < 0.01. (G–I) Gait abnormalities assessed by footprint analysis: length (cm) of stride (G), sway (H), and stance (I) as an indication of motor coordination. (J) Motor nerve conduction velocity (MCV) (m/s) was determined on sciatic nerves. One-way ANOVA followed by Tukey’s test, F (3, 22) = 9.95, **p < 0.01 and ***p < 0.001. (K and L) Sensorimotor functions analyzed by hot plate (K) and tape test (L): latency (sec) to withdraw hindlimbs or forelimbs. One-way ANOVA followed by Tukey’s test: (I) F (3, 24) = 7.66 and (J) F (3, 20) = 15.19, *p < 0.05, **p < 0.01, and ***p < 0.001. All tests included controls (Ct) and mice with chronic demyelination treated with vehicle (TMEV+veh), scrambled miR (TMEV+miR-scr), or miR-219 (TMEV+miR-219). Data are expressed as the mean ± SEM, n = 7 to 8 mice per group.
Figure 3
Figure 3
Treatment with miR-219 Reduces TMEV-Mediated Demyelination (A and B) Representative micrographs of dorsal (A) and ventral (B) white matter in thoracic segments of the spinal cord; scale bar, 20 μm. (C–F) Demyelinated axons (% total axons) and myelin thickness (g ratio) were quantified in dorsal (C and E) and ventral (D and F) white matter from thoracic segments of the spinal cord; one-way ANOVA followed by Tukey’s test: (C) F (3, 27) = 157.1, (D) F (3, 29) = 44.5, (E) F (3, 850) = 81.4, and (F) F (3, 797) = 16.4, *p < 0.05, **p < 0.01, and ***p < 0.001. (C–F) n = 3 per group. (G) Representative micrographs of Luxol fast blue and cresyl violet staining of thoracic segments of the spinal cord show demyelinated areas (solid line) relative to total white matter (WM) (dashed line); scale bar, 100 μm. (H) Quantification of demyelinated areas by Luxol fast blue and cresyl violet staining; one-way ANOVA followed by Tukey’s test, F (3, 32) = 10.45, *p < 0.05 (n = 4 per group). Experimental groups: controls (Ct) and mice with chronic demyelination treated with vehicle (TMEV+veh), scrambled miR (TMEV+miR-scr), or miR-219 (TMEV+miR-219). Data are expressed as the mean ± SEM.
Figure 4
Figure 4
Treatment with miR-219 Improves Myelination in TMEV-Infected Mice (A–D) Confocal microscopy quantitative analysis of MBP (myelin, green) and NeuN (neurons, red) (A and B) and of CC1 (OLs, red, arrowheads) (C and D) in thoracic spinal cord sections. Background staining controls of anti-mouse Alexa 546/anti-rabbit Cy5 and control anti-mouse Alexa 546 are shown. Nuclei were stained with DAPI (blue); scale bar, 20 μm. One-way ANOVA followed by Tukey’s test, F (3, 151) = 11.11, *p < 0.05 and **p < 0.01 (n = 4 per group). (E and F) Western blot analysis of MBP levels in protein extracts from the thoracic spinal cord (E); one-way ANOVA followed by Tukey’s test (F), F (3, 23) = 11.01, *p < 0.05 and **p < 0.01 (n = 7 to 8 per group). (G and H) Pdgfrα+ cells were counted after RNA in situ hybridization (G); scale bar, 40 μm; one-way ANOVA followed by Tukey’s test (H), F (3, 97) = 9.97, *p < 0.05 (n = 4 per group). (I and J) Western blot analysis of PDGFRα levels in protein extracts from the thoracic spinal cord (I); one-way ANOVA followed by Tukey’s test (H), F (3, 28) = 16.25, *p < 0.05 and **p < 0.01 (n = 7 to 8 per group). (K and L) Olig2+ cells were counted after RNA in situ hybridization (n = 4 per group); scale bar, 40 μm. Experimental groups: controls (Ct) and mice with chronic demyelination treated with vehicle (TMEV+veh), scrambled miR (TMEV+miR-scr), or miR-219 (TMEV+miR-219). Data are expressed as the mean ± SEM.
Figure 5
Figure 5
miR-219 Reduces Astrogliosis, Microglia Activation, BeAn Copy Numbers, and Immunoreactivity (A) Astrogliosis was analyzed by western blotting of Serpina3n and GFAP in protein extracts from the thoracic spinal cord: (B) one-way ANOVA followed by Tukey’s test, F (3, 21) = 47.47, *p < 0.05 and ***p < 0.001 (n = 5–8 per group); and (C) one-way ANOVA followed by Tukey’s test, F (3, 21) = 17.03, ***p < 0.001 (n = 5–7 per group). (D) Microglia was quantified by confocal microscopy in thoracic segments of spinal cords; one-way ANOVA followed by Tukey’s test, F (3, 108) = 29.83, *p < 0.05 and ***p < 0.001 (n = 4 per group). (E) BeAn copy number by qPCR; one-way ANOVA followed by Tukey’s test, F (3, 26) = 7.62, *p < 0.05 (n = 7 to 8 per group). (F) BeAn capsid staining was quantified by confocal microscopy; one-way ANOVA followed by Tukey’s test, F (2, 72) = 19.15, **p < 0.01 and ***p < 0.001 (n = 4 per group). Experimental groups: control (Ct) and mice with chronic demyelination treated with vehicle (TMEV+veh), scrambled miR (TMEV+miR-scr), or miR-219 (TMEV+miR-219). Data are expressed as mean ± SEM.
Figure 6
Figure 6
miR-219 Reduces Proinflammatory Cytokines and Restores IL-1β Activation Quantitation of IL-10 (A), IL-4 (B), CXCL1 (C), IFN-γ (D), IL-2 (E), IL-6 (F), IL-1β (G), and TNF-α (J), respectively, in thoracic spinal cords analyzed by a multiplex ELISA: MSD proinflammatory panel; one-way ANOVA followed by Tukey’s test: IL-10 F (3, 17) = 32.62, IL-4 F (3, 16) = 14.95, CXCL1 F (3, 11) = 11.12, IFN-γ F (3, 19) = 18.43, IL-2 F (3, 22) = 8.16, IL-6 F (3, 19) = 26.23, IL-1β F (3, 18) = 59.66, and TNF-α F (3, 18) = 19.41, *p < 0.05, **p < 0.01 and ***p < 0.001 (n = 5–7 per group). (H and I) Representative Western blot of active (17 KDa) and total (31 KDa) IL-1β expression (H) and band analysis of activated IL-1β normalized to the total protein (I). One-way ANOVA followed by Tukey’s test F (3, 21) = 13.94, ***p < 0.001 (n = 6–7 per group). (K and L) Representative Western blot of active (17 KDa) and total (25 KDa) TNF-α expression (K) and band analysis of activated TNF-α normalized to total protein levels (L). Experimental groups: control (Ct) and mice with chronic demyelination treated with vehicle (TMEV+veh), scrambled miR (TMEV+miR-scr) or miR-219 (TMEV+miR-219). Data are expressed as the mean ± SEM.
Figure 7
Figure 7
miR-219 Potentiates Transcriptional Changes in Cholesterol-Related Genes (A–F) qPCR validation of genes from cluster 1 (see also Figures S3A and S3B) that are involved in cholesterol biosynthesis Hmgcs1: hydroxymethylglutaryl-CoA synthase (B), Srebf1: esterol regulatory element-binding protein 1 (C), Hmgcr: 3-hydroxy-3-methylglutaryl-coenzyme A reductase (E), and Srebf2: esterol regulatory element-binding protein 2 (F) and are also predicted targets of miR-219 (Cyp51: lanosterol 14-α demethylase, A, and Insig1: insulin-induced gene 1 protein, D); one-way ANOVA followed by Tukey’s test: Hmgcs1, F (3, 25) = 39.02; Srebf1, F (3, 26) = 6.14; Hmgcr, F (3, 27) = 24.91; Srebf2, F (3, 29) = 5.45; Cyp51, F (3, 23) = 77.44; and Insig1, F (3, 27) = 59.09; *p < 0.05, **p < 0.01, and ***p < 0.001 (n = 5–8 per group). Experimental groups: control (Ct) and mice with chronic demyelination treated with vehicle (TMEV+veh), scrambled miR (TMEV+miR-scr), or miR-219 (TMEV+miR-219). Data are expressed as mean ± SEM.
Figure 8
Figure 8
miR-219 Reduces Cholesterol Levels In Vivo and In Vitro (A) Cholesterol levels by the Amplex Red Cholesterol assay in thoracic spinal cords; one-way ANOVA followed by Tukey’s test, F (2, 21) = 10.44, *p < 0.05 and ***p < 0.001 (n = 6 to 7 per group). (B) Filipin (cholesterol) was measured after staining and analysis by confocal microscopy in thoracic spinal cord; one-way ANOVA followed by Tukey’s test, F (2, 66) = 4.05, *p < 0.05 (n = 4 per group). Experimental groups: control (Ct) and mice with chronic demyelination treated with vehicle (TMEV+veh), scrambled miR (TMEV+miR-scr), or miR-219 (TMEV+miR-219). (C and D) Micrographs of neuroglial CG-4 cells (C) and cell viability (WST-1 assay) (D), respectively, of CG-4 cells at 24 hr after TMEV infection (10 MOI) in differentiating conditions (3 div) treated with vehicle (TMEV+veh), scrambled miR (TMEV+miR-scr), miR-219 (TMEV+miR-219), or MBCD (1.5 mM) before infection; scale bar, 40 μm; one-way ANOVA followed by Tukey’s test, F (3, 13) = 9.08, **p < 0.01 (n = 3–6 per group). (E) Cholesterol levels measured by the Amplex Red Cholesterol assay in CG4 cells from (D). (F) Cholesterol levels by the Amplex Red Cholesterol assay of OL (n = 2) and astrocytes (n = 3) treated with miR-219 before TMEV infection. Student’s t test, t(4) = 5.55, **p = 0.005. Data are expressed as mean ± SEM.

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