Figure 1.

Basal activation of pro-inflammatory cytokines, chemokines, and IFN-I response in the SOD1-G85R in vivo model

(A) Whole brain tissues were isolated from WT and G85R mice at day-10 after birth. Purified whole RNA was subjected to NanoString multiplex gene analysis using the murine neuroinflammation panel that consists of 770 genes. Heat-map representation of differentially expressed genes with upregulated related genes (fold-change >2.0 as the cut-off threshold) by G85R expressed as fold changes relative to WT controls.

(B) The upregulated genes from (A) were subjected to unbiased protein network and enrichment analysis via the STRING-CytoScape.

(C) Representative heatmap of upregulated immune mediator profile (fold-change ≥2.0) in G85R mice after clustering into their respective families.

(D) Spinal cord from WT or G85R mouse models were isolated and dissected into three sections as depicted (left). RNA from each section were harvested and subjected to analysis of the indicated mRNA (right).

(E) Von Kossa and immunohistochemistry staining of brain tissue from WT or G85R mice with the indicated antibodies. Quantification of staining was performed via optical density determination.

(F) Kaplan-Meier survival plots of age-matched G93A mice challenged either with vehicle or MCP-1 neutralizing antibody (500 μg/week). Number of mice that survived at the endpoint are indicated.

(G) Primary cells were isolated from the brain of WT or G85R mice at day-10 after birth through immunopanning procedures. RNA was purified and subjected to RT-qPCR analysis.

(H) Newborn WT or G85R mice were intracranially injected with vehicle (DMEM), 500, 10,000, or 50,000 plaque-forming units (pfus) of CVB3 at day-8. The brain and the entire blood was harvested at two time-points (Day 10, and Week 60), respectively. Vp1 and 2A mRNA were quantified via RT-qPCR.

(I) C20 cells were first pre-treated with serum harvested from WT or G85R mice, followed by infection with CVB3 (MOI = 1.0). Vp1, Rsad2, and Isg15 mRNA were measured via RT-qPCR.

(J) Serum collected from endpoint WT or G85R mice (week-60) were subjected to ELISA analysis for IFN-β protein. All RT-qPCR assays are normalized to β-actin. Data represent the mean of at least three independent experiments with n = 3 biological replicates (mean ± SEM). Number of dots = number of mice/biological replicates. ∗p< 0.05, ∗∗p< 0.01, ∗∗∗p< 0.001, ∗∗∗∗p< 0.0001; (D, E, and G–J: unpaired Student’s t test; F: Log rank Mantel-Cox test). A.U. = arbitrary units. See also Figure S1.