Supplementary Figure 5: ZIKV triggers apoptosis via induction of terminal UPR and expression of Chop.
From: Stress-induced unfolded protein response contributes to Zika virus–associated microcephaly
a-b, Data showing number of Ctip2+ (F2,19=2.750, P=0.2614) a and Cux1+ (F2,19=7.665, P=0.0140) b cortical neurons in E18.5 brains after treatment with inhibitors as indicated, and presented as boxplots of median ± first to third quartiles while whiskers extend to maxima and minima with each symbol representing one independent sample (n = 4 to 8) as indicated on boxplots, and analyzed by Kruskal-Wallis followed by Dunn post-hoc tests. c, Representative images of immunolabeled coronal sections from brains of E18.5 embryos infected by ZIKV at E12.5 treated by co-injection of DMSO or PERKi with detection of Dapi (blue), ZIKV (red), PDI (green) (n = 5 independent embryonic brains per condition). d-e, Suppression of Chop, a terminal downstream target of PERK pathway, improves survival of ZIKV-infected cortical cells. Boxplot demonstrating Chop siRNA suppresses Chop expression in transiently transfected Neuro2A cells followed by qRT-PCR (n = 4 independent experiment per condition, F4,20=16.53, P=0.0024) d. Boxplot demonstrating the comparison of the percentages of ac-caspase 3+ cells in E16.5 mouse brains, after ICV injection of either ZIKV or ZIKV+PERKi at E12.5 followed by in utero co-electroporation of either Chop or control siRNA with pCAGGS-GFP (n = 5 embryonic brains per condition, F5,34 = 24.69, P = 0.0002) e. Data presented as boxplots of median ± first to third quartiles while whiskers extend to maxima and minima with each symbol representing one biologically independent sample as indicated on boxplots, and analyzed by Kruskal-Wallis followed by Dunn post hoc comparison test. f, Representative images of immunolabeled coronal sections from brains of E18.5 embryos infected by ZIKV at E12.5. ZIKV+control siRNA or ZIKV+Chop siRNA E18.5 brains with detection of Dapi (blue), ZIKV (red), GFP (green) and ac-caspase 3. g, Schematic representation of the mechanisms triggered by ZIKV in the developing cortex. By targeting the APs, ZIKV generates ER stress that raises UPR, leading to an overall reduction of the neuronal output as a result of increased direct neurogenesis (red Arrow). Infected projection neurons suffer from chronic ER stress that activates the UPR target Chop to promote apoptosis (target). Single in vivo injection of PERKi at the time of infection partially releases both pathophysiological events and prevents microcephlay in mouse embryos. Scale bar represent 100 µm c, or 50 µm f. ⋆⋆ P < 0.01, and ⋆ P < 0.05; ns means not significant. ROI = region of interest.
