Plausible role of Nrf2- DJ-1 axis in Mouse Hepatitis Virus induced cellular stress in Microglia

Kundu, Soumya (2020) Plausible role of Nrf2- DJ-1 axis in Mouse Hepatitis Virus induced cellular stress in Microglia. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Soumya Kundu (14RS081)) 14RS081.pdf - Submitted Version Restricted to Repository staff only Download (14MB)

Abstract

Successful virus infection and completion of viral life cycle including replication of genome and assembly of functional virus particles is satisfied at the expense of host cell metabolism. A number of viruses have evolved the ability to alter host cellular environments by inducing cellular stress responses like the oxidative stress response, unfolded protein response (UPR) and heat shock response. Coronaviruses (CoVs) are a family of enveloped, positive-strand RNA viruses that have been associated with several animal and human diseases, including severe acquired respiratory syndrome (SARS). Mouse coronavirus (m-CoV) is an important member of the CoV family. The neurotropic strain of Mouse Hepatitis virus (a m-CoV), MHV-A59, is associated with the activation of cellular stress pathways in an experimental animal model, and this thesis aims to understand the interplay of such cellular stress pathways in this model. MHV-A59 infection results in glial cell activation and subsequent demyelination, although the underlying mechanism is not clear. To investigate the underlying molecular mechanisms, my work is focused on the regulation and interplay of stress response proteins related to oxidative stress, ER stress and heat shock response and their relation to an isogenic spike protein EGFP-tagged recombinant strain of MHV-A59, RSA59 induced glial infection. I focused our study on microglia and astrocyte and investigated the DJ-1-Nrf2 axis in the orchestration of the antioxidant pathway, BiP mediated UPR pathway, and HSF1 mediated heat shock response. The models used in my study are the C57BL/6 mouse model, microglia isolated from adult mice, primary cell culture model and established cell line studies. RSA59 infection in 4-week old C57BL/6 mice resulted in a loss in weight, and the development of disease severity increases through the acute stage of infection till day 9, followed by a recovery phase. Infectious viral load and expression of viral N gene were observed in the acute stage, which decreases with increasing day points p.i. Astrocyte and microglial activation were observed in different parts of the brain studied- cortex, caudate putamen, basal forebrain, hypothalamus, and midbrain. Upon RSA59 infection, Nrf2, HMOX-1, catalase, NQO1, txnrd1, and DJ-1 (oxidative stress response), XBP1, BiP, ERp29 and eIF2α (UPR), and HSF1 and Hsp70 (heat shock response) genes were upregulated at different time points p.i. As astrocytes and microglia, the CNS resident glial cells, gets activated upon RSA59 infection, it is important to understand how these cells are regulating cellular stress responses individually. To address this question, I followed a reductionist approach to investigate the changes in stress response pathway genes in microglia enriched from adult infected mouse brain, as well as primary glial cells enriched in primary astrocytes and primary microglia, and N9 microglia and DBT astrocytoma cell line studies. In cellular studies, DJ-1 was upregulated in microglia isolated from the adult infected mouse brain. Primary astrocytes and microglia revealed upregulation of different antioxidants (except HMOX-1 in primary microglia) and UPR genes upon RSA59 infection. RSA59 infection causes a decrease in cell viability, generation of oxidative stress, and differential regulation of different antioxidants, UPR and heat shock genes and proteins in cell culture models. Molecular insights into DJ-1 upregulation upon viral infection in N9 cells revealed the involvement of ER stress response protein XBP1 in DJ-1 transcription. Since previous literature had demonstrated the anti-oxidative and cellular protective role of these proteins, differential regulation of these genes could be considered as one of the mechanisms underlying the MHV-A59 infection in glial cells. In this work, in silico and in vitro analysis of the interaction between XBP1 protein and DJ-1 is performed. Molecular docking analysis revealed the binding between the DJ-1 promoter and a positively charged groove of XBP1, providing a possible implication of their role as interacting partners. Electrophoretic mobility shift assay and modified western blot analysis confirmed the interaction of DJ-1 promoter by XBP1 upon RSA59 infection. This study elucidates a novel communication between antioxidant mechanism and UPR by DJ-1 and XBP1 in the outcome of Coronavirus infection. These findings thus confirm the longstanding view that virus-induced cellular stress responses of the host are mutually dependent and open newer therapeutic possibilities targeted to the regulation of these cellular stress response pathways in the future.

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