Differential Expression of DJ-1 in Demyelinating Strain Versus Non-demyelinating Strain of Mouse Hepatitis Virus (MHV) Infection

Kumar, Rahul (2017) Differential Expression of DJ-1 in Demyelinating Strain Versus Non-demyelinating Strain of Mouse Hepatitis Virus (MHV) Infection. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Rahul Kumar (12MS104)) Rahul_Kumar_Master's_Thesis_DBS.pdf - Submitted Version Restricted to Repository staff only Download (3MB)

Abstract

One of the experimental animal models to study the neurological disease, Multiple Sclerosis (MS) is Mouse Hepatitis Virus (MHV) infection in mice. Microglial infection with the neurotropic strain of MHV (MHV-A59) results in activated microglia/macrophage-mediated myelin stripping from the axon. One of the possible mechanisms of microglial activation is the induction of oxidative stress. DJ-1 has been one of the key players involved in the cellular defense mechanism against oxidative stress. However, much light has not been shed upon the role of DJ-1 in viral induced neural damage, making it important to assess its regulation in neural cells upon viral infection. In our experiments, we have used three cell lines- N9 microglial cell line, DBT astrocytoma cell line and Neuro2a neuroblastoma cell line. Previous experiments in our lab showed an association of oxidative stress with MHV infection particularly in N9 cell line. Also, a marked upregulation of DJ-1 mRNA and protein level has been observed in N9 cell line upon viral infection as compared to the DBT and Neuro2a cell lines. Thus, there is a cell-specific regulation of DJ-1 protein underlying MHV infection. In order to perform successive experiments, optimal viral infection time point was determined using MTT assay. Also, DCFDA Assay was used to detect time dependent ROS generation upon viral infection. To determine the relationship between ROS induction and DJ-1 upregulation, N9 cells were treated with tert-Butyl hydroperoxide (TBHP)- ROS. Increase in ROS concentration results in DJ-1 mRNA upregulation, indicating a direct correlation between them in N9 cell line. DJ-1 being a ROS-quencher protein, its upregulation may help cells to combat oxidative stress. We have also found an increase in DJ-1 (mRNA level) in N9 cells treated with either TBHP or RSA59 whereas a significant decrease has been found in combined treatment (both TBHP and RSA59). In addition, downregulation of DJ-1 mRNA and protein level has been associated with late viral infection (24 hours) as opposed to early viral infection (12 and 20 hours). The increase in DJ-1 (mRNA level) has also been validated in microglia-enriched cell population isolated from mice brain and primary microglia isolated from neonatal mice. In contrary to this, no such change, with respect to DJ-1 protein level with time in N9 cells, have been observed upon infection with an MHV2 strain of virus (a non-demyelinating strain of MHV). Further validation of the relationship between oxidative stress and DJ-1 and regulation of possible signaling players might provide an indication of the signaling process underlying DJ-1 regulation as a response to viral induced oxidative stress.

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