Parechovirus-3 infection disrupts immunometabolism and leads to glutamate excitotoxicity in neural organoids

Figures

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  Fig. 1. Proteome analysis of neural organoids shows proteins, pathways, and metabolic dysregulation unique for HPeV infection. A, differences in clinical presentation of HPeV-1 and HPeV-3 infection B-D, Volcano plots showing protein abundance in neural organoids in B, HPeV-1 vs. mock C, HPeV-3 vs. mock, and D, HPeV-3 vs. HPeV-1 infection (adjusted P value < 0.1, Limma). The y-axis represents significance of protein abundance differences (negative log scale adjusted P-value) and the x-axis represents the fold change of the difference in abundance. E-F, Significantly regulated KEGG pathways linked to metabolism were analyzed where eight pathways are uniquely distinct for HPeV-3 infection in neural organoids. E, Pathway enrichment analysis results showing significantly regulated pathways (adjusted P value < 0.1, Limma) in the pair-wise proteomics analysis in neural organoids. This heatmap represents non, mixed, and distinct directionality (negative log scaled adjusted P value) of the pathways. The first column annotation denotes directionality of pathways and the second column denotes corresponding pair-wise comparison. F, Network visualization of within-pathway relationships between the…

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  Fig. 2. HPeV-3 infection results in significantly higher extracellular glutamate and cytotoxicity in neural organoids. A, Schematic representation of the proposed mechanisms of action of uniquely abundant proteins in HPeV-3 infection involving the glutamate cycle in a neuronal synapse. Glutamine is converted by glutaminase into glutamate, packed into vesicles, and released in a voltage dependent manner. After synaptic activation, the glutamate in the synaptic cleft is recycled predominantly by astrocytes and converted back to glutamine. Factors such as excess glutamate can trigger neurons to become reactive, which can induce loss of neuronal support and neurotoxicity. The proteins uniquely abundant in HPeV-3 infection are visualized in an orange box. B-C, Extracellular glutamate, D-E, extracellular LDH and F-G, extracellular NfL measured in the supernatant of HPeV-1, HPeV-3, or mock-infected neural organoids D,F,G, at 0, 5, and 10dpi, visualized as absolute data (RLU) or C, E,Gorcid.org/0000-0003-2986-4884, D,F) compared to mock using a one-way-ANOVA test with multiple comparisons or (, extracellular levels at 10dpi normalized to the amount of infectious viral particles (RLU/TCID50)…

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  Fig. 3. Astrocyte-neuronal co-cultures show significant increase in extracellular glutamate in the early phases of HPeV-3 infection and was followed by an increase in CC3 expression.A, Schematic representation of infection of the astrocyte-neuron co-culture with HPeV at a MOI of 0.5. B-C, Secretion levels at 0-, 2-, 4-, 6-, 24-, 48- and 72hpi measured in the supernatant of HPeV or mock-infected neural organoids as a percentage of maximum release control (Triton-X) of B, glutamate and C, LDH.Data corresponds to three technical replicates * represents significance of HPeV vs Mock, ◦ represents significance of HPeV-3 vs HPeV-1. D-F,Immunofluorescent images of HPeV-infected astrocyte-neuronal co-cultures stained for β-tubulin (magenta) and CC3 (green) at 72hpi. Scalebar represents 100µm.E-F, Quantification of mean fluorescent intensity of CC3 and β-tubulin relative to Hoechst as a fold change over MOCK. Data corresponds to the mean fluorescent intensity of representative images taken from multiple wells. B-C) Data are represented as the mean ± geometric standard deviation (SD) of three technical replicates. Statistical significance was analyzed compared to mock using a Kruskal–Wallis test…