Non-Polio Enterovirus A71 and D68 Infection of Human Neuromuscular Organoids Reveals Distinct Mechanisms of Neuromuscular Impairment

Figures

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  Figure 1. NMOs recapitulate neuromuscular architecture and support EV infection. (A) Representative immunofluorescence image of an IMR90-4-derived NMO stained for Hoechst, β-III-tubulin (TUJ1, neurons) and fast myosin heavy chain (Fast MyHC; muscle). Scale bars: 300 µm. (B) Confocal z-stack of a NMJ in an IMR90-4-derived NMO stained with Hoechst, α-bungarotoxin (α-BTX; nAChRs), β-III-tubulin (TUJ1), and Fast MyHC. The main panel shows the XY projection, with the orthogonal YZ view displayed on the bottom edge. Scale bars: 50 µm and 20 µm. (C) Relative increase in viral RNA copy numbers in the supernatant at indicated time points following infection with EV-A71 (0 vs. 24 hpi: p = 0.21; 0 vs. 48 hpi: *** p = 0.0003; 0 vs. 72 hpi: **** p < 0.0001) or EV-D68 (0 vs. 24 hpi: p = 0.16; 0 vs. 48 hpi: *** p = 0.0001; 0 vs. 72 hpi: **** p < 0.0001). Data represent the geometric mean of three technical replicates per condition, calculated separately for each hiPSC line: IMR90-4 (circles), Gibco® A18945 (squares), and M001 (triangles). Statistical significance was assessed per virus using a Kruskal–Wallis test with Dunn’s multiple comparisons test. (D) Viral titres of EV-A71 (* p < 0.05) and EV-D68 (p…

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  Figure 2. EV-A71 and EV-D68 infection downregulate neuronal and muscular pathways in NMOs. (A) PCA of transcriptomic profiles from mock, EV-A71-, and EV-D68-infected NMOs. (B) Heatmap of scaled gene expression profiles from IMR90-4- and M001-derived NMOs under mock and infected conditions. (C) Volcano plots showing log2 fold change versus –log10 adjusted p values for EV-A71- and EV-D68-infected NMOs compared to mock controls. Significantly upregulated and downregulated genes (adjusted p < 0.05, Benjamini–Hochberg correction) are shown in blue and pink, respectively. Each dot represents an individual gene. (D) Distribution of significantly suppressed neuronal and muscular Gene Ontology (GO) biological processes identified by gene set enrichment analysis (GSEA). Pie charts indicate the relative proportion of downregulated pathways in infected NMOs compared to mock controls. (E) GSEA dot plots showing the main altered neuronal and muscular pathways following EV-A71 or EV-D68 infection.

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  Figure 3. SNAP25 expression and cleavage in NMOs upon EV infection. (A) Representative immunofluorescence image of an IMR90-4-derived NMO stained for Hoechst, SNAP25, Fast MyHC, and β-III-tubulin (TUJ1). Scale bars: 300 µm. (B) Densitometric analysis of SNAP25 protein abundance in NMOs derived from two hiPSC lines (IMR90-4, circles; M001, triangles) following EV-A71 (p = 0.47) or EV-D68 (p = 0.99) infection. SNAP25 band intensities were quantified from Western blots and normalised to the loading control and mock condition (set to 1). Data represent the geometric mean of three technical replicates from two independent experiments. Statistical significance was assessed per virus using a Kruskal–Wallis test with Dunn’s multiple comparisons test. (C) Representative Western blot showing SNAP25 cleavage in M001-derived NMOs at 96 hpi following infection with EV-A71 or EV-D68.

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  Figure 4. Caspase-3 activation in NMOs following staurosporine treatment or EV infection. (A) Representative immunofluorescence image of an IMR90-4-derived NMO stained for Hoechst, cleaved CC3, green, Fast MyHC, and β-III-tubulin (TUJ1); (B–D) NMOs treated with staurosporine (B) or infected with EV-A71 (C) or EV-D68 (D) for 72 h and stained for Hoechst, cleaved CC3, green, Fast MyHC, and β-III-tubulin (TUJ1). Scale bars: 200 µm.