Zika virus infection leads to mitochondrial failure, oxidative stress and DNA damage in human iPSC-derived astrocytes

www.nature.com/scientificreports OPEN Zika virus infection leads to mitochondrial failure, oxidative stress and DNA damage in human iPSC-derived astrocytes Pítia Flores Ledur1,9, Karina Karmirian1,2,9, Carolina da Silva Gouveia Pedrosa1, Leticia Rocha Quintino Souza1, Gabriela Assis-de-Lemos3, Thiago Martino Martins2, Jéssica de Cassia Cavalheiro Gomes Ferreira2, Gabriel Ferreira de Azevedo Reis4, Eduardo Santos Silva4, Débora Silva5, José Alexandre Salerno1,2, Isis Moraes Ornelas1, Sylvie Devalle1, Rodrigo Furtado Madeiro da Costa 1, Livia Goto-Silva1, Luiza Mendonça Higa6, Adriana Melo8, Amilcar Tanuri6, Leila Chimelli5, Marcos Massao Murata4, Patrícia Pestana Garcez 2, Eduardo Cremonese Filippi-Chiela7, Antonio Galina3, Helena Lobo Borges 2 & Stevens Kastrup Rehen 1,2* 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. Zika virus (ZIKV) was discovered in 1947 in Africa and remained quite neglected until occurrences of Zika infection were reported in Micronesia (2007), French Polynesia (2013) and South America (2013)1–5. ZIKV had not been tied to developmental disorders back then. In 2015–16, however, the virus was linked to an outbreak of congenital malformations in Brazil and declared a Public Health Emergency of International Concern1. Sequencing analysis defined two main strains, the African and the Asian, the latter which thrived in the Americas and was reported to be linked to congenital malformations and Guillain-Barré Syndrome6–9. ZIKV neurotropism is known since its discovery and first description in Uganda2. The virus deadly effects have been shown in neural stem cells, in addition to growth impairment of human neurospheres and brain organoids10–12. Almost all studies have described ZIKV infection and consequences in neural progenitor cells (NPCs), the first cell target extensively investigated. ZIKV infection disrupts NPC proliferation and differentiation, crucial processes correlated to congenital malformations12,13. On the other hand, astrocytes play important roles during neural development and multiple pathological findings in ZIKV-infected fetuses raised questions of possible glial disturbances14–16. Recently, studies have focused on the role of astrocytes during ZIKV infection: Retallack 1 D’Or Institute for Research and Education, Rio de Janeiro, Brazil. 2Institute of Biomedical Sciences, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil. 3Institute of Medical Biochemistry Leopoldo De Meis, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 4Insitute of Biology, Department of Biophysics and Biometrics, State University of Rio de Janeiro (UERJ), Rio de Janeiro, RJ, Brazil. 5Laboratory of Neuropathology, State Institute of Brain Paulo Niemeyer, Rio de Janeiro, RJ, Brazil. 6Institute of Biology, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil. 7Institute of Health Sciences, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil. 8Research Institute Prof. Joaquim Amorim Neto (IPESQ), Campina Grande, PB, Brazil. 9These authors contributed equally: Pítia Flores Ledur and Karina Karmirian. *email: Scientific Reports | (2020) 10:1218 | https://doi.org/10.1038/s41598-020-57914-x 1 www.nature.com/scientificreports/ www.nature.com/scientificreports and collaborators observed that ZIKV preferentially infected cells with glial and astrocytic markers in human organotypic cultures17. Simonin and colleagues described an increased ZIKV infection rate in a human astrocyte cell line when compared to human iPSC-derived NSCs18. More recently, primary human astrocytes were shown to be susceptible to ZIKV, with an infection rate of ~ 60% (MOI = 1)19. Human fetal astrocytes were also found to be more infected than human fetal neurons, with a higher viral production showing that they probably work as a viral reservoir20. Still, little is known about the specific cellular mechanisms disrupted by ZIKV19–22. The study of ZIKV biology can be greatly benefitted from the use of human induced pluripotent stem cells (iPSC), which allows the modelling of human development through differentiation into multiple cell types. ZIKV belongs to the Flaviviridae family. Members of this family replicate in the endoplasmic reticulum (ER), with evidences of ZIKV interaction with this organelle23–25. The replication of hepatitis C virus (HCV), also a member of Flaviviridae family, leads to Ca+2 release from the ER lumen to the cytoplasm, increasing the overall production of reactive oxygen species (ROS). Mitochondria might uptake this Ca+2, leading to more ROS production26. ROS is another common consequence of flavivirus infections27, and its increase has been described as a result of ZIKV infection in yeast28 and in brains of infected mice29. Other cellular processes, such as proliferation and mitosis, have been described as affected by ZIKV11,30,31. Mitosis defects happen due to deficient karyokinesis or chromosomal breakage, often leading to mitotic catastrophe or apoptosis to avoid chromosomal instability32. DNA breakage, in turn, can be caused by ROS generated during the viral replication process and can affect one strand of DNA (single-strand break, SSB) or both (double-strand break, DSB)33. The DNA damage response (DDR) activates repair pathways leading to the expression of genes used to monitor DNA damage. Viral structures can also trigger DDR by interacting with host DNA33. ZIKV has been shown to trigger DDR in human neural stem cells (NSCs)34, and proteomics and RNA-seq analysis demonstrated that ZIKV-infected neurospheres upregulate BRCA1 and MRE11A35. BRCA1 has been linked to DSBs36, and it interacts with phosphorylated histone H2AX (γH2AX), a classical DSB marker37. Here, cellular consequences of ZIKV infection in iPSC-derived astrocytes are investigated. Astrocytes are one of the cell types with the highest infection rate in the brain. ZIKV triggers mitochondrial damage and increased ROS levels, which culminate in DNA breaks, probably the virus final denouement leading to cell death. Moreover, ROS sc (...truncated)