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, 10 (1), 1218

Zika Virus Infection Leads to Mitochondrial Failure, Oxidative Stress and DNA Damage in Human iPSC-derived Astrocytes

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Zika Virus Infection Leads to Mitochondrial Failure, Oxidative Stress and DNA Damage in Human iPSC-derived Astrocytes

Pítia Flores Ledur et al. Sci Rep.

Abstract

Zika virus (ZIKV) has been extensively studied since it was linked to congenital malformations, and recent research has revealed that astrocytes are targets of ZIKV. However, the consequences of ZIKV infection, especially to this cell type, remain largely unknown, particularly considering integrative studies aiming to understand the crosstalk among key cellular mechanisms and fates involved in the neurotoxicity of the virus. Here, the consequences of ZIKV infection in iPSC-derived astrocytes are presented. Our results show ROS imbalance, mitochondrial defects and DNA breakage, which have been previously linked to neurological disorders. We have also detected glial reactivity, also present in mice and in post-mortem brains from infected neonates from the Northeast of Brazil. Given the role of glia in the developing brain, these findings may help to explain the observed effects in congenital Zika syndrome related to neuronal loss and motor deficit.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
ZIKV infection rate of human brain cells derived from iPSC. (AC). Cells were infected at a multiplicity of infection (MOI) = 1 and analyzed 72 hours post infection (hpi). (A). Immunofluorescence images of NSCs stained for ZIKV NS1 protein (green) and Nestin (red). (B) Astrocytes stained for ZIKV NS1 protein (green) and S100b (red). (C) Neurons stained for ZIKV NS1 protein (green) and beta tubulin III (red). Blue channel is DAPI staining for all cell types. Scale bar: 50 µm. (D). Quantification of the rate of infection according to cell type (Data is shown as average ± SEM. For NSCs, n = 3, derived from iPS lines GM23279A, CF1 and CF2, *p = 0.026; for astrocytes, n = 4, derived from iPS lines GM23279A, CF1, CF2 and C15, **p = 0.003; for neurons, n = 3, derived from iPS lines GM23279A, CF1 and CF2, non-significant). (E) Cell viability assay (MTT) for MOCK and ZIKV-infected NSCs, non-significant, n = 3. (F) MTT for MOCK and ZIKV-infected astrocytes, n = 3, ***p < 0.001. Data is shown as average ± SEM.
Figure 2
Figure 2
ZIKV-induced mitochondrial dysfunction. (A) Respirometry analysis of MOCK or ZIKV-infected astrocytes after 24 and 48 hpi. (B) Routine respiration, (C) ATP synthesis, (D) Reserve capacity of infected astrocytes at different time points post infection. N = 12 for MOCK and N = 3 for each time point of ZIKV infection. Astrocyte lines used were derived from iPSC lines GM23279A, C15, CF1 and CF2.
Figure 3
Figure 3
ROS production in ZIKV infection. (A) Staining of ROS superoxide indicator dye DHE in MOCK and ZIKV-infected astrocytes (untreated and treated with ascorbic acid 80 µM). Hoechst was used for nuclei staining. N = 3. Scale bar: 100 µm. (B) Quantification of DHE staining (shown as the percentage of DHE-positive cells) in MOCK and ZIKV-infected astrocytes treated or untreated with ascorbic acid. N = 3. (C) Quantification of mitoSOX ROS superoxide indicator dye staining (shown as the percentage of mitoSOX-positive cells) in MOCK and ZIKV-infected astrocytes untreated or treated with ascorbic acid. N = 3. Data is shown as average ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001. Astrocytes derived from iPSC lines GM23279A, CF1 and C15 were used.
Figure 4
Figure 4
ZIKV-induced DNA damage. (A) Alkaline comet assay shows an increase in DNA damage in ZIKV-infected astrocytes when compared to MOCK. N = 4. (B) γH2AX and 53BP1 immunostaining in MOCK- and ZIKV-infected astrocytes, 48 h.p.i.. DAPI for nuclei staining is shown in blue, γH2AX is shown in green, and 53BP1 is shown in red. Merged images are shown at the right. Scale bar: 50 µm. Quantification of images is shown on Fig. 5C. (C) Western blotting (WB) analysis of γH2AX and 53BP1 in MOCK and ZIKV-infected astrocytes at 24 and 48 hpi, MOI = 1. (D) Protein levels were quantified related to actin expression levels. N = 3. *p < 0.05, ***p < 0.001.
Figure 5
Figure 5
Ascorbic acid attenuates DNA damage. (A) Alkaline comet assay. N = 4. *p = 0.02. (B) Ascorbic acid treatment in ZIKV-infected astrocytes, stained for DDR proteins γH2AX and 53BP1. (C) Quantification of γH2AX and 53BP1 fluorescence levels in MOCK- and ZIKV-infected astrocytes treated and untreated with ascorbic acid. Over 200 nuclei were quantified per condition. Scale bar: 50 µM. (D) Number co-localized foci of γH2AX and 53BP1 were quantified in the same astrocyte nuclei analyzed in C. *p < 0.05, ***p < 0.001. Astrocytes derived from iPSC lines GM23279A, CF1 and C15 were used.
Figure 6
Figure 6
Astrogliosis induced by ZIKV infection. (A) Immunofluorescence images of MOCK-, ZIKV-infected iPSC-derived astrocytes and TNF-α condition as positive control. DAPI (blue) and GFAP (red). Scale bar: 50 μm. (B) Quantification of GFAP intensity in MOCK- and ZIKV-infected astrocytes at 4 dpi, MOI = 0.125. Quantification of TNF-α (10 ng/ml) condition as positive control of gliosis assay. N = 4. *p = 0.015, **p = 0.0085. (C) Immunofluorescence images of ZIKV-infected mice brains. DAPI (blue), ZIKV NS1 protein (green) and GFAP (red). Scale bar: 20 µm. (D) Quantification of GFAP intensity in MOCK- and ZIKV-infected 7 dpi mice cingulate cortex. N = 3. (E) Immunofluorescence images of control and infected post-mortem human brains. DAPI (blue), ZIKV NS1 protein (green) and GFAP (red). White arrows indicate GFAP positive infected cells. Scale bar: 50 µm. (F) Immunohistochemistry for GFAP in control and infected post-mortem human brains. (G,H) Quantification of GFAP intensity and average number of GFAP positive cells in post-mortem human tissue, respectively. Data is shown as average ± SEM; ***p < 0.0001.

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