Sublethal enteroviral infection exacerbates disease progression in an ALS mouse model

Figures

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  Fig. 1. Sublethal CVB3 infection exacerbates ALS-related phenotypes and decreases the lifespan of SOD1G85R mice. A Schematic diagram illustrating intracerebroventricular injections of neonatal SOD1G85R and C57BL/6J mice with either 500 pfu of CVB3 or an equal volume of DMEM (mock infection). Mouse motor functions were monitored weekly starting at week 20 post-infection (PI) until the mice were approaching the humane endpoint or at the experimental endpoint of week 60 PI. Mouse tissues were collected at day 10, week 20 and week 60 PI or at the humane endpoint. B Kaplan–Meier plots comparing survival rates among C57BL/6J Mock (n = 12; male = 4 and female = 8), SOD1G85R Mock (n = 10; male = 3 and female = 7); C57BL/6J CVB3 (n = 9; male = 4 and female = 5), and SOD1G85R CVB3 (n = 8; male = 2 and female = 6) mice. P = 0.002 between SOD1G85R mice inoculated with CVB3 and mock-infected. C Relative body weight measured weekly after infection. Body weight at day 0 was arbitrarily set to 1. D Mouse motor function measurement, including hindlimb reflex score, inverted grid time, footprint time, and stride distance, assessed weekly after infection. Quantifications are presented as mean ± standard…

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  Fig. 2. Sublethal CVB3 infection leads to ALS-related pathologies in vivo. A Representative images of viral protein VP1 immunohistochemical and H&E histological staining in the hippocampus regions of the brain from mock- and CVB3-infected C57BL/6J or SOD1G85R mice at day 10 PI. The red boxes indicate the region of the magnification. Black boxes on the top right illustrate the enlarged images. B Representative images of TDP-43 immunohistochemical staining in the hippocampus regions of the brain from mock- and CVB3-injected C57BL/6J or SOD1G85R mice at day 10 PI or week 60 PI (or humane endpoint) as indicated. The red arrows indicate TDP-43 mislocalization. Black boxes on the top right illustrate the enlarged images. Scale bar of magnified images = 100 μm. Scale bar of whole brain images = 1000 μm

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  Fig. 3. Sublethal CVB3 infection triggers microglia/astrocyte activation and immune infiltration in mice. Representative images of GFAP, Iba1, CD19 and NK1.1 immunohistochemical staining in the hippocampus regions of the brain from mock- or CVB3-infected C57BL/6J or SOD1G85R mice at day 10 PI (A) or week 60 PI (or humane endpoint) (B). Similar observation was made in the regions of infected cerebral cortex (data not shown). Images for other targets (CD68, CD79A, CD4, can CD8) can be found in the Additional file 2: Fig S2. Immune infiltrations were quantified by optical density based on the IHC staining in the hippocampus and cerebral cortex regions. Black boxes on the top right illustrate the enlarged images. Quantifications are presented as mean ± SEM (n = 3–4 individual mouse brain samples for each target, timepoint and experimental condition). Scale bar = 100 μm. Statistical analysis was carried out using two-way ANOVA, followed by Bonferroni’s multiple comparison test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

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  Fig. 4. CVB3 infection promotes proinflammatory responses in the CNS of SOD1G85R mice. A–B Gene expression comparison of different proinflammatory cytokines/chemokines conducted using NanoString mouse immunology panel on the brain and spinal cord samples harvested from mock- (n = 3) and CVB3-infected SOD1G85R mice (n = 3) at week 20 PI. Panel (A) shows the Gene Ontology analysis of significantly expressed genes (p < 0.05, via student’s t test) in the brain (top) and spinal cord (bottom) of CVB3-infected SOD1G85R mice normalized to mock-infected counterparts. Panel (B) shows differentially expressed cytokine/chemokine genes (in relative Log2 fold changes) in the brain (top) and spinal cord (bottom) of CVB3-infected SOD1G85R mice normalized to mock-infected counterparts. C RT-qPCR validation of four significantly increased gene expression of TNFA, NFKB2, CXCL10 and CCL2 in the same brain samples (week 20 PI) as above. Quantifications are presented as mean ± SEM (n = 3–4 for each group). Statistical analysis was carried out using student’s t test. *, p < 0.05; **, p < 0.01

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  Fig. 5. CVB3-accelerated ALS progression is mitigated by early antiviral treatment. A Schematic diagram illustrating the experimental plan for CVB3 injection, ribavirin administration, and the endpoint. B Kaplan–Meier plots comparing mouse survivals among SOD1G85R CVB3 (n = 8; male = 2 and female = 6), SOD1G85R CVB3 day 1 ribavirin (n = 5; male = 2 and female = 3), and SOD1G85R CVB3 day 15 ribavirin (n = 5; male = 2 and female = 3) mice. P = 0.0037 between SOD1G85R CVB3 and SOD1G85R CVB3 day 1 ribavirin groups. C Mouse motor function measured weekly (i.e., hindlimb reflex score, inverted grid time, footprint time, and stride distance) starting at week 20 PI until humane or experimental endpoint. The vertical black line indicates the point of deviation between the two groups. D–E RT-qPCR evaluation of proinflammatory genes (D) and viral RNA (E) in the brain tissues collected at week 60 PI or the humane endpoints. F Representative TDP-43 IHC staining images in the hippocampus regions of the brain collected at week 60 PI or the humane endpoint. The red arrows indicate TDP-43 mislocalization. Scale bar = 100 μm. Quantifications are presented as mean ± SEM (n = 3–4). Statistical analysis was…

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  Fig. 6. Schematic summary of mechanisms that enteroviral infection exacerbates disease phenotype in an ALS mouse model. Sublethal infection of enteroviruses, such as CVB3 modelled in this study, promotes early onset and progression of ALS-like phenotype, and decreases the lifespan of mice in SOD1G85R ALS mouse model. This exacerbation is associated with at least three molecular and pathological phenotypes induced by CVB3 infection: (1) direct tissue damages or neuronal cell death, (2) dysregulated immune responses, and (3) TDP-43 pathologies. It appears that sublethal viral infection alone is not sufficient to provoke ALS-like phenotypes in C57BL/6J mice. However, together with SOD1G85R-mediated toxicities (e.g., increase proinflammatory response, oxidative stress, protein aggregates and neuronal cell death), viral infection accelerates disease progression and reduces mouse lifespan in SOD1G85R ALS mice