Evaluation of microRNAs in the immunopathogenesis of microcephaly caused by Zika virus in experimental models

Author(s)

Carolina Manganeli Polonio

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2022-12-05

Examining board members

Jean Pierre Schatzmann Peron; Ana Campa; Marcelo Alves da Silva Mori; Maria Notomi Sato

• Advisor: Jean Pierre Schatzmann Peron

Abstract

The Zika Virus (ZIKV) is a flavivirus that leads to neurological impairment characterizing the Congenital ZIKV Syndrome (CZS). Although neuronal precursor cells and neurons are the most affected cells, astrocytes are more susceptible to ZIKV infection, once tolerate higher viral loads, suffer less apoptosis and are used for viral replication. Curiously, it is known that several regulators of biological processes are involved in the susceptibility to CZS, including microRNAs (miRNAs). MiRNAs are small non-coding RNAs, which work downstream of transcription factors by interacting with complementary mRNA target sequences for further post-transcriptional repression. In this context, the principal aim is investigated miRNAsmRNA interactions during vertical transmission of ZIKV, focusing on astrocytes. First, we demonstrated that ZIKV increases miRNAs expression in the brain of mice neonates from infected mothers, stimulating autophagy. Upregulation of miR-295 limits the expression of Bcl2l11, an autophagy regulator gene, in vivo and in astrocytes in vitro. Furthermore, comparing miRNAs profile in SJL astrocytes, we observed an increased expression of miR-295 and miR-302d. Using bioinformatics platforms, miRNAs mimics and inhibitors, and luciferase assay, we demonstrated that Ahrr, Neurod4 and Neurod6 genes are downregulated by miR- 295 and miR-302d. Interestingly, miR-295 and miR-302d are transcribed along with Nlrp12 and Larp7 genes, respectively. To better understand the mechanism used by ZIKV to induce miRNAs, we evaluated transcription factors for Nlrp12 and Larp7 transcription. Interestingly, ZIKV increased the expression of CEBP/b and NF-kB p65 subunit. Corroborating, ZIKV induced the recruitment of CEBP/b to Nlrp12 and Larp7 promote, p65 to Larp7 promoter. Moreover, Rela and Cebpb inhibition by siRNAs decreased Nlrp12, Larp7, premiR-295, premiR-302d, miR- 295 and miR-302d, while increased Ahrr, Neurod4 and Neurod6 gene expression. Following, we demonstrate that the activation of this described pathway is a susceptibility mechanism to CZS, since we did not observe increased expression of Cebpb, Rela, Nlrp12, premiR-295, Larp7, premiR-302d, miR-295 and miR-302d, and their target genes, Ahrr, Neurod4, Neurod6 in astrocytes from C57BL/6 WT resistant mice to microcephaly caused by ZIKV. Thus, we demonstrate that ZIKV activates transcription factors C/EBPb and NF-kB to increase transcription of Nlrp12 and Larp7, and consequently, miR-295 and miR-302d in SJL astrocytes. These miRNAs lead to decreased expression of Ahrr, Neurod4 and Neurod6 genes, contributing to CZS development in susceptible SJL mice, but not in resistant C57BL/6 WT mice. Together, we suggest a mechanism of susceptibility to CZS development. (AU)

• FAPESP's process: 17/11828-0 - Evaluation of the microRNAs role in the immunopathogenesis of microcephaly caused by Zika virus in experimental models
• Grantee: Carolina Manganeli Polonio
• Support Opportunities: Scholarships in Brazil - Doctorate