Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate Immune Receptor TLR3

Abstract

Emerging evidence from the current outbreak of Zika virus (ZIKV) indicates a strong causal link between Zika and microcephaly. To investigate how ZIKV infection leads to microcephaly, we used human embryonic stem cell-derived cerebral organoids to recapitulate early stage, first trimester fetal brain development. Here we show that a prototype strain of ZIKV, MR766, efficiently infects organoids and causes a decrease in overall organoid size that correlates with the kinetics of viral copy number. The innate immune receptor Toll-like-Receptor 3 (TLR3) was upregulated after ZIKV infection of human organoids and mouse neurospheres and TLR3 inhibition reduced the phenotypic effects of ZIKV infection. Pathway analysis of gene expression changes during TLR3 activation highlighted 41 genes also related to neuronal development, suggesting a mechanistic connection to disrupted neurogenesis. Together, therefore, our findings identify a link between ZIKV-mediated TLR3 activation, perturbed cell fate, and a reduction in organoid volume reminiscent of microcephaly.

Keywords: Toll-like receptors; Zika virus; microcephaly; organoids.

Figure 1

Characterization of cerebral organoids reveals recapitulation of fetal brain regions. (A) Bright-field image of representative organoids show development of neuroepithelial layer. Scale bar: 250µm. (B) DAPI stained organoid shows complex inner morphology including ventricle-like structures from 30-day-old organoids. Scale bar: 250µm. (C) Organoids immunostained for neuronal (TUJ1+) and neural progenitor cells (SOX1+) cells. TUJ1 shows generalized neuronal differentiation while neural progenitors are localized near inner ventricle-like structures in 30-day-old organoids. Immunostaining for forebrain (PAX6), dorsal cortex (EMX1), hippocampus (PROX1) and interneurons (CALB2) show differentiation of organoids into discrete brain regions 30-day-old organoids. Also see Figure S1. Scale bar: 100µm. (D) Calcium dye imaging of cerebral organoids using Fluo-4 shows functional neural activity.

Figure 2

RNA map of Cerebral Organoids development. (A) Heat map of transcriptome analysis from human embryonic stem cells (Group 1) and cerebral organoids after 1 month (Group 2) and 2 months (Group 3) in culture show 3226 and 3357 significantly differentially expressed genes with fold change >2, p-value <0.05. See also Figure S2A. See Table 1. (B) Gene ontology analysis show top 10 more enriched terms for upregulated (top) and downregulated (bottom) genes during cerebral organoid differentiation. (C) Grouped functional pathway analysis of differentially expressed genes during organoid formation. (D) Heat map of differentially expressed genes between organoids 1 month (Group 2) and 2 months (Group 3) in culture. Group 1 represents human embryonic stem cells. (E) Gene ontology analysis of differentially expressed genes in organoids 1 and 2 months old suggest formation of retinal tissue. (F–G) Spearman’s correlation heat map of cerebral organoid transcriptomes compared with regions of the fetal brain and age post conception weeks (pcw). See Table 2 for heat map brain region legend. Also see Figure S2B and S2C.

Figure 3

ZIKV results in attenuated organoid growth in cerebral organoids and neurospheres through activation of TLR3. (A) ZIKV infected Vero cells for virus expansion. Vero cells were seeded and infected at MOI of 1 and viral supernatant was collected 48 hours post inoculation. Scale bar 100µm. (B) Bright-field images of mouse neurospheres at Day 0 and Day 1 post-inoculation with ZIKV. Scale bar: 250µm. (C) Immunohistochemistry shows ZIKV can robustly infect mouse neurospheres. Scale bar: 50µm. (D) Representative bright-field images of individual human cerebral organoids treated with ZIKV over time. Scale bar: 250µm. (E) ZIKV viral copy count in organoid supernatant quantified by one-step RT-qPCR after ZIKV infection shows organoid susceptibility and viral permissiveness. *** p-value<0.001, Student’s t-test. (F) Quantification of organoid size over time with and without ZIKV infection. Bars represent the min, average and max relative organoid size. Individual organoids were measured over time relative to their respective Day 0 size from n=5 organoid samples * p-value<0.05, Student’s t-test. (G) Representative images of ZIKV treated organoids stained for ZIKV envelope protein and Nestin. Scale bar: 100µm.

Figure 4

ZIKV induces TLR3 and regulates pathways involved in apoptosis and neurogenesis. (A) Bright-field images of mouse neurospheres at Day 0 and Day 1 post-inoculation with ZIKV with or without TLR3 competitive inhibitor, or TLR3 agonist poly(I:C). Scale bar: 250µm. (B) Neurospheres show significant change in size 1 day post-inoculation with ZIKV with or without TLR3 competitive inhibitor, or TLR3 agonist poly(I:C) as quantified by ImageJ. Box and whiskers plot show 10–90 percentile. * p-value<0.05. *** p-value<0.001. n.s. = not significant, Student’s t-test. (C) Representative bright-field images of individual human cerebral organoids treated with ZIKV with or without TLR3 competitive inhibitor. Scale bar: 250µm. (D) Schematic of target selection for RT-qPCR analyses. Differentially genes involved in organoid formation (from Figure 2A) and TLR regulated genes (data not shown) were analyzed to identify common pathways activated upon ZIKV infection. The two significantly enriched pathways from this dataset were “positive regulation of nervous system development” and “regulation of synapse structure or activity.” (E) RT-qPCR analysis of TLR3 upregulation in organoids mock and ZIKV treated. Error bars represent SEM. (F) RT-qPCR analysis of differentially expressed genes, Ntn1 and EphB2, involved in TLR3 activation and neurogenesis in organoids treated with TLR3 agonist poly(I:C). Error bars represent SEM. * p-value<0.05. ** p-value<0.01, Student’s t-test. (G) RT-qPCR analysis of Ntn1 and EphB2 expression in human organoids upon ZIKV and ZIKV+ TLR3 competitive inhibitor. * p-value<0.05. *** p-value<0.001, Student’s t-test. (H) Model for ZIKV infection and TLR3 mediated downregulation of regulators of neurogenesis and upregulation of pro-apoptotic pathways in NPCs.