key: cord-0983360-94j92mta
authors: Kundu, Soumya; Saadi, Fareeha; Sengupta, Sourodip; Antony, Gisha Rose; Raveendran, Vineeth A.; Kumar, Rahul; Kamble, Mithila Ashok; Sarkar, Lucky; Burrows, Amy; Pal, Debnath; Sen, Ganes C.; Sarma, Jayasri Das
title: DJ-1-Nrf2 axis is activated upon murine β-coronavirus infection in the CNS
date: 2021-09-05
journal: Brain Disord
DOI: 10.1016/j.dscb.2021.100021
sha: 6bebfa463d9ac6defb54a5336b7411ab21e41882
doc_id: 983360
cord_uid: 94j92mta

Coronaviruses have emerged as alarming pathogens owing to their inherent ability of genetic variation and cross-species transmission. Coronavirus infection burdens the endoplasmic reticulum (ER.), causes reactive oxygen species production and induces host stress responses, including unfolded protein response (UPR) and antioxidant system. In this study, we have employed a neurotropic murine β-coronavirus (M-CoV) infection in the Central Nervous System (CNS) of experimental mice model to study the role of host stress responses mediated by an interplay of DJ-1 and XBP1. DJ-1 is an antioxidant molecule with established functions in neurodegeneration. However, its regulation in virus-induced cellular stress response is less explored. Our study showed that M-CoV infection activated the glial cells and induced antioxidant and UPR genes during the acute stage when the viral titer peaks. As the virus particles decreased and acute neuroinflammation diminished at day ten p.i., a significant upregulation in UPR responsive XBP1, antioxidant DJ-1, and downstream signaling molecules, including Nrf2, was recorded in the brain tissues. Additionally, preliminary in silico analysis of the binding between the DJ-1 promoter and a positively charged groove of XBP1 is also investigated, thus hinting at a mechanism behind the upregulation of DJ-1 during MHV-infection. The current study thus attempts to elucidate a novel interplay between the antioxidant system and UPR in the outcome of coronavirus infection.

Successful virus infection and completion of the viral life cycle, including replicating genome and assembly of functional virus particles, are satisfied at the expense of host cell metabolism [1] . A number of viruses have evolved the ability to alter host cellular environments by inducing cellular stress responses like endoplasmic reticulum (E.R.) stress and oxidative stress responses, and unfolded protein response (UPR) [2] . Coronaviruses (CoVs) are a family of enveloped, positive-strand RNA viruses that have been associated with several animal and human diseases [3, 4] . The Coronavirus Disease 2019 (COVID19), originated in Wuhan, China, is an ongoing pandemic that has aggressively spread worldwide [5, 6] . Interestingly, this is the third example of zoonotic transmission and the coronavirus outbreak after SARS-CoV in 2003 and MERS-CoV in 2012 [7] . Thus, the highly contagious nature of CoVs and their ability to spill over different hosts makes them an important subject of investigation even in terms of host stress responses [8] . Coronavirus infection is known to upregulate the expression of E.R. stress markers like GRP78, GRP94 in the host cells [9] [10] [11] .

It is hypothesized that CoVs might cause E.R. stress in several ways [12] [13] [14] [15] [16] and to reinstate E.R. homeostasis, and the host cells have evolved UPR.

Another host stress response is the generation of reactive oxygen species (ROS). ROS are produced at all times, even during homeostatic conditions, as oxygen serves as the final electron acceptor in the mitochondrial energy metabolism. In fact, a substantial amount of ROS is generated as a by-product of protein synthesis and folding in the E.R., which can have detrimental effects on the host cell [17] . Therefore, the body has developed several defence mechanisms to keep the ROS in check. However, increased viral load and cellular stress can shift the redox balance between the prooxidants and antioxidants towards oxidative stress. On the one hand, this oxidative stress induces UPR to promote cell survival and preserve host cell functions [18] . On the other hand, an imbalance in the redox potential can directly or indirectly affect the E.R. protein folding process. Studies have shown that overexpression of SARS-CoV 3CL-Proprotein induces ROS and NFκB expression [19] . SARS-CoV accumulation in the E.R. can trigger all three major UPR signalling pathways, promoting an inflammatory response [20] . Recent findings on SARS-CoV-2 also report the induction of an inflammatory cytokine storm in response to rapid viral replication [21] . Nuclear factor erythroid 2-related factor 2 (Nrf2), which is also called the master regulator of cellular redox homeostasis, has shown implications as a therapeutic target in SARS-CoV-2 infection owing to its reported antioxidant, anti-inflammatory effects, and transcriptional repressor activity [22, 23] .

Our study is focused on identifying the putative role of DJ-1, a ROS sensing molecule, upon M-CoV (murine-CoV) infection of the glial cells in the outcome of E.R. and oxidative stress responses. Neurotropic M-CoV MHV-infection in mice is widely used as an experimental animal model to study viral-induced direct neuroglial dystrophy, innate neuroinflammation and chronic progressive CNS myelin pathology and axonal loss. DJ-1 is a 189 amino acid long dimeric protein ubiquitously expressed in all cells [24] . It is encoded by the Park7 gene and is involved in a variety of signalling cascades [25] . DJ-1 has been found to play essential roles in neuroprotection under several disease conditions like Parkinson's disease, Alzheimer's disease, and Multiple Sclerosis [26] [27] [28] . Upon activation, DJ-1 stimulates Nrf2 [29] , which regulates the expression of several antioxidant genes like Heme oxygenase 1 (HMOX-1), Catalase, Thioredoxin reductase 1 (txnrd1), NAD(P)H dehydrogenase (quinone) 1 (Nqo1) [30] , as well as heat shock response related heat shock factor 1 (HSF1). [31] HSF1 serves as a major transcription factor for several heat shock proteins. DJ-1 activation is also known to play essential roles in E.R. stress and UPR by modulating activating transcription factor 4 (ATF4) and decreasing the expression of its downstream signalling molecules CHOP and BiP [32] . While both DJ-1 and Nrf2 are relatively unexplored in CoV infections, X-box protein 1 (XBP1), a UPR transcription factor, has profound and established functions in modulating high E.R. stress upon CoV infection both in vitro and in vivo [16] . XBP1 works in the IRE1 branch of the UPR, the most conserved UPR signalling pathway that is also known to regulate oxidative stress [33] . Upon activation, XBP1 translocates to the nucleus, where it enhances the expression of various UPR genes, including molecular chaperons like HSF1 and Hsp70, that help to restore E.R. stress [33] .

In the current study, we have investigated the Nrf2-DJ-1-XBP1 axis during Mouse Hepatitis Virus (MHV-M-CoV) infection. RSA59, an isogenic spike gene recombinant strain of MHV-A59, is a well-established prototypic group 2-murine β coronavirus (M-CoV). It infects the liver and CNS and is a CoV model used to investigate the mechanisms of neuroinflammation, viral pathogenesis, host stress responses, as well as anti-viral immune responses [34, 35] . Intracranial infection of M-CoV causes a biphasic disease in C57Bl/6 mice, acute stage characterized by meningoencephalomyelitis (days 5/6 post-infection) and chronic stage demyelination and axonal loss [34] . Our previous studies have also shown that M-CoV infection induces optic neuritis by promoting infiltration of peripheral inflammatory cells of mixed populations during the acute stage [36] . Optic nerves from M-CoV infected mice develop demyelination and axonal loss during the chronic stage, accompanied by a significant loss of retinal ganglionic cells in the optic nerve [37] . Further, M-CoV leads to mitochondrial ROS accumulation in the infected optic nerves during peak inflammation and 30 days post-infection. ROS accumulation in infected mice correlates with decreased levels of proteins associated with mitochondrial function and biogenesis [37] .

The results show that M-CoV infection in the mouse brain results in profuse viral replication and increased viral transcript denoted by nucleocapsid gene, activation of astrocytes and microglia during acute infection, upregulation of inflammatory cytokines, and chemokines, and the upregulation of antioxidant and UPR genes. As the virus titer starts to decline and inflammation is resolved at day 10 p.i., a significant progressive increase was observed in the ROS sensing molecule DJ-1 along with the upregulation of its downstream effectors, selected heat shock responsive genes, and UPR marker XBP1. We have also attempted an in silico approach to show the interaction of DJ-1 promoter with XBP1. The result of such analysis will throw some light on the mechanism of DJ-1 upregulation and the dynamic communication between ROS, cellular stress, and UPR during M-CoV infection.

RSA59 viruses were used in the study. MHV-A59 is a dual tropic murine β Coronavirus that infects the liver and the CNS. Intracranial inoculation of MHV-A59 in C57BL/6 mice is used as an experimental animal model to understand the mechanisms of neurodegeneration and demyelination as observed in the human neurological disorder Multiple Sclerosis (M.S.).

RSA59 is an isogenic recombinant, demyelinating strain of MHV-A59 from our previous studies where the spike gene is introduced by targeted RNA recombination. Spike gene encodes an envelope glycoprotein that mediates virus-host attachment and various biological properties of MHV, including virus-cell and cell-cell fusion, viral spread, anti-viral host responses, and pathogenicity. Additionally, in RSA59, gene 4 is replaced by enhanced green fluorescence protein (EGFP) described elsewhere [38] [39] [40] . Throughout the paper, RSA59 is referred as M-CoV.

The animals and all experimental procedures were reviewed and approved by the Indian Institute of Science Education and Research-Kolkata (IISERK). Animal protocols were followed according to the Committee's guidelines for the purpose of control and supervision of experiments on animals (CPCSEA), India.

Four-week-old, M-CoV-seronegative C57BL/6 male mice were intracranially inoculated with 50% of the LD50 dose of M-CoV (20000 PFU) [38, 39] . Mice were monitored daily for clinical disease symptoms. Parallel controls were inoculated with PBS-BSA (Mock).

Mice were sacrificed on day 5 post-infection (peak of inflammation) for histopathological analyses. For RNA and protein studies, mice were sacrificed on days 3, 5 10 post-infection.

Mice were transcardially perfused with DEPC (diethyl polycarbonate) treated PBS, and liver, brain, and spinal cord tissues were harvested for experimentation. For immunofluorescence analysis, tissues were postfixed in 4% PFA for 48-72 hours, processed, and embedded in paraffin [38, 39] .

5µm thick serial sections from mock and RSA59 infected brains were subjected to immunofluorescence analysis. Briefly, tissue sections were deparaffinized and dehydrated and subjected to antigen unmasking (Vector Laboratories, , Burlingame, California, USA).

After blocking in blocking solution (1XPBS+ 2.5% Goat Serum + 0.5% TritonX-100) for 1 hour at 37ᴼC, the sections were incubated overnight at 4ᴼC with primary antibody. The primary antibody was washed in 1X PBS, and subsequently, the sections were labeled with a secondary antibody for 90 minutes at 37ᴼC. All incubations were carried out in a humidified chamber. After PBS washing, the sections were mounted with mounting medium containing Reactions were performed in quadruplets. The sequence of primers used is given in Table 1 .

Del Ct values were used to analyze changes in the expression pattern of respective genes. 

To look into the role of XBP1 in the regulation of DJ-1 expression, in silico analysis of interaction between XBP1 protein and the DJ-1 promoter is attempted (supplementary section). XBP1 protein structure was generated by homology modeling using I-TASSER software [41] . XBP1 sequence with sequence accession number 035426 (protein database from NCBI website (https://www.ncbi.nlm.nih.gov/protein/O35426.2)) was used for the modeling with no additional restraints or assigned templates. Along with that, no particular template was excluded from the I-TASSER template library, and no secondary structure was assigned for specific residues. Following this, molecular dynamics simulation was performed using GROMACS 5.1 software [42] . In summary, the protein model was solvated in the SPC water model inside a cube with a 1 nm distance from the edges. Charge neutralization was performed using eight Chloride ions and energy minimization was performed using the Steepest Descent Method. The obtained structure was subjected to 2 ns of NVT (number, volume, temperature) equilibration using leapfrog integrator and modified Berendsen thermostat, followed by 2 ns of NPT (number, pressure, temperature) equilibration with the addition of Parrinello-Rahman barostat for pressure coupling. After this, Molecular dynamics simulation was performed using the OPLS-AA force field under periodic boundary conditions for 50 ns extracting frames every 2.5 ns, and the surface charge distribution of XBP1 protein was calculated using APBS plugin for PyMOL [43] .

The DJ-1 promoter region was chosen from -456 to -427 bp (DJ-1 promoter sequence was taken from gene id: 57320 -nucleotide database from NCBI website (https://www.ncbi.nlm.nih.gov/gene/?term=57320)), and the B-DNA structure was generated by using 3D DART software [44] , with no specific nucleic acid modeling parameters. Initial docking of the two obtained structures was done using NP Dock Server according to previously published methodology [45] , was saved. The best-refined structure was selected, and the interacting structures were analyzed, and images were obtained using PyMOL (http://www.pymol.org). 

Upon M-CoV infection, 4-week-old C57BL/6 mice developed acute stage hepatitis illustrated by necrotic and non-necrotic lesions, inflammation in the meninges (meningitis) and encephalitis characterized by perivascular cuffing and microglial nodule as described previously [38, 39] . Neuroinflammation induced by M-CoV, RSA59, provides a stage for studying the nexus between oxidative stress and the UPR pathway at different days postinfection.

On day 5 p.i., M-CoV infected brain tissue sections were subjected to immunolabelling by 

Viral N gene expression is observed on days 3, 5, and 10 p.i. ( Figure 1A ). Viral titer, as assessed by routine plaque assay, increased at the onset of inflammation (day 3 p.i) and

reached its peak at day 5 p.i. The virus titre starts to decline by day 7 p.i. as reported earlier ( Figure 1B ) [38, 47] . During the acute phase, i.e., days 3, 5, and 10 p.i. until the viral titer drops down below detection limit, RT-qPCR analysis was performed for Nrf2, HMOX-1, Catalase, Nqo1, txnrd1, HSF1, Hsp70. Genes showed differential regulation at different times post-infection. Results revealed that at early time points during the peak of inflammation (days 3 and 5 p.i.), antioxidant genes Nrf2, HMOX-1, Nqo1, and catalase showed most significant upregulation when infectious viral particles were below the detection limit and acute neuroinflammation is resolved( Figure 1C , D, E, G). However, antioxidant gene txnrd1 did not show any change upon M-CoV infection ( Figure 1F ). The heat shock responsive genes (HSF1 and Hsp70) showed significant upregulation, ( Figure 1H , I) [1] . Thus, the most striking increase in the cellular stress pathway genes is detected when infection and acute inflammation are resolved. This also marks the bridging between innate and adaptive immunity. The fold change in mRNA expressions is shown in Figure 1J .

To understand the glial cell-specific cellular response in a reductionist approach, we investigated antioxidant response genes' expression in mixed glial cultures enriched in microglia or astrocytes. Both primary microglia and astrocytes were characterized by the expression of CD11b or GFAP respectively (Figure 2A , E) and infected with M-CoV at 2 MOI ( Figure 2B , F) as discussed in the materials and methods section. At 24 hours p.i., RT-qPCR analysis was performed for antioxidant response pathway markers Nrf2 and HMOX-1, which showed significant upregulation in infected mouse brain. Results revealed a significant upregulation of Nrf2 in both primary microglia and astrocytes ( Figure 2C, G) . However, HMOX-1 was upregulated in primary astrocytes but down-regulated in microglia ( Figure 2D , H). The fold change in mRNA expression is shown in Figure 2I .

The regulatory role of ROS sensing molecule DJ-1 and UPR pathway marker, XBP1 was assessed both in brain and individually in microglia and astrocytes by examining the mRNA expression. Such roles will be important in understanding the possible crosstalk between the oxidative stress and UPR pathways in the process of microglial activation. Results showed significant upregulation of DJ-1 and XBP1 mRNA with days of post infection until day 10 as observed in mice brains ( Figure 3A , B) and fold change in mRNA expression is shown in Figure 3C . Likewise, both primary microglia ( Figure 3D , E) and primary astrocytes ( Figure   3G , 

Our results have shown that M-CoV infection causes an upregulation of the UPR pathway associated XBP1 and antioxidant pathway DJ-1. A previous report [48] showed that XBP1 activation was involved in the upregulation of DJ-1 mRNA by direct interaction with the DJ-1 promoter to prevent cell death. Therefore, we used the in silico approach to identify DJ-1 and XBP1 as interaction partners in modulating host stress response to M-CoV.

For in silico analysis, we performed molecular dynamics simulation on the structure of XBP1 protein generated by iterative threading-based modeling using I-TASSER software.

Molecular dynamics simulation was performed using GROMACS 5.1 for 50 ns, and frames were extracted every 2.5 ns. The protein structure was stable, with an average backbone rootmean-square-deviation (RMSD) value of 0.4097±0.02982 Å, and showed a highly positively charged groove (denoted by blue region). Encouraged by the highly positive charged site in the XBP1 protein capable of DNA binding, we generated a B-DNA model of the DJ-1 promoter region. Following this, docking was performed using NP Dock Server (http://genesilico.pl/NPDock). Since NP Dock performs rigid docking, we needed to consider the DNA binding grove's intrinsic flexibility. This was achieved by using XBP1 protein structures extracted every 2.5 ns during the 50 ns M.D. simulation. Docking using the NP Dock server gave several docked models. Still, the best-refined structure, as provided by the software, showed stable binding between the DJ-1 promoter and the positively charged groove of XBP1 protein at the same site. The fit of the structure of the DJ-1 promoter in the XBP1 protein groove was visually analyzed using PyMOL (Supplementary Figure 2) .

Our recent findings on the upregulation of DJ-1 and XBP1 both in vivo and in vitro in combination with in silico study predicts that XBP1 may promote DJ-1 expression and provide cytoprotective function.

M-CoV induced glial cell activation heightens the secretion of a complex array of chemokines, cytokines, and production of oxygen radicals directing the host anti-viral response [49] [50] [51] [52] [53] . In turn, activated glial cells fine-tune E.R. stress and oxidative stress responses to preserve the host tissues' integrity [18, 54, 55] . [27, 29, 58] , where DJ-1 is shown to play a prominent protective role by balancing the cellular redox potential [59] . Likewise, as expected, DJ-1 upregulation is associated with the increase in its downstream signalling molecule Nrf2, a master regulator of oxidative stress and detoxification processes [29] .

Moreover, our findings reveal the increased expression of cytoprotective and detoxifying genes, including Nqo1, Catalase, and HMOX-1, upon M-CoV infection, which is induced by Nrf2 and is in line with previous findings [30] .

Many studies in M-CoV infection have acknowledged the functional importance of XBP1, which is a transcription regulator of UPR [60] known to be induced in response to the accumulation of unfolded proteins and other stress factors by IRE1 mediated splicing [33] .

IRE1-XBP1 is the most conserved UPR signalling pathway known to regulate oxidative stress [33] . In our studies, we observed an upregulation of XBP1 along with DJ-1 on day 10 p.i. However, the demonstration of possible crosstalk between the oxidative stress response and UPR in M-CoV infection in mounting host protection is the highlight of our study.

Previous research had identified consensus sites on XBP1 for DJ-1 binding in humans and mice [48] . However, we have taken an in silico approach to gain insight into the charge 

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Origin and evolution of pathogenic coronaviruses

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Coronavirus-induced ER stress response and its involvement in regulation of coronavirus-host interactions

Coronavirus infection, ER stress, apoptosis and innate immunity. Front Microbiol

Ultrastructural characterization of SARS coronavirus. Emerging infectious diseases

Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex

Regulation of Stress Responses and Translational Control by Coronavirus. Viruses

Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease

Interplay between the unfolded protein response and reactive oxygen species: a dynamic duo

Severe acute respiratory syndrome coronavirus 3C-like protease-induced apoptosis. FEMS immunology and medical microbiology

Modulation of the unfolded protein response by the severe acute respiratory syndrome coronavirus spike protein

The trinity of COVID-19: immunity, inflammation and intervention

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DJ-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras

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Increased DJ-1 expression under oxidative stress and in Alzheimer's disease brains

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Gene expression profiling of NRF2-mediated protection against oxidative injury. Free radical biology & medicine

NRF2 transcriptionally activates the heat shock factor 1 promoter under oxidative stress and affects survival and migration potential of MCF7 cells

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Divergence and Conservation of the Major UPR Branch IRE1-bZIP Signaling Pathway across

A mechanism of virus-induced demyelination

Microglia-mediated neuroinflammation is an amplifier of virus-induced neuropathology

Experimental Optic Neuritis Induced by a Demyelinating Strain of Mouse Hepatitis Virus

SIRT1 Activating compounds reduce oxidative stress mediated neuronal loss in viral induced CNS demyelinating disease

CD4 deficiency causes poliomyelitis and axonal blebbing in murine coronavirus induced neuroinflammation

Mechanisms of Primary Axonal Damage in a Viral Model of Multiple Sclerosis

Enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system

I-TASSER server for protein 3D structure prediction

GROMACS: High performance molecular stimulations through multilevel parallelism from laptops to supercomputers. SoftwareX

PyMOL: An Open-Source Molecular Graphics Tool

3D-DART: a DNA structure modelling server

NPDock: a web server for protein-nucleic acid docking

Interplay Between the Unfolded Protein Response and Immune Function in the Development of Neurodegenerative Diseases

Enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system

ER-stress-associated functional link between Parkin and DJ-1 via a transcriptional cascade involving the tumor suppressor p53 and the spliced X-box binding protein XBP-1

Keeping it in check: chronic viral infection and antiviral immunity in the brain

Microglia and neuroinflammation: a pathological perspective

Differential regulation of innate and adaptive immune responses in viral encephalitis

Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia

Coronavirus neurovirulence correlates with the ability of the virus to induce proinflammatory cytokine signals from astrocytes and microglia

Microglia Polarization and Endoplasmic Reticulum Stress in Chronic Social Defeat Stress Induced Depression Mouse

Endoplasmic reticulum stress differentially modulates the IL-6 family of cytokines in murine astrocytes and macrophages

Redox control of microglial function: molecular mechanisms and functional significance

Astrocytes Do Not Forfeit Their Neuroprotective Roles After Surviving Intense Oxidative Stress. Frontiers in Molecular Neuroscience

DJ-1 and prevention of oxidative stress in Parkinson's disease and other age-related disorders

The role of cysteine oxidation in DJ-1 function and dysfunction

XBP1 mRNA Is Induced by ATF6 and Spliced by IRE1 in Response to ER Stress to Produce a Highly Active Transcription Factor

Coronavirus infection, ER stress, apoptosis and innate immunity

The authors declare that they have no competing interests. Quantitative real-time PCR analysis of the brain tissues of M-CoV infected mice at day 5 p.i. was performed for cytokines IL-6, IL-1β, IFNα, IFNβ, IFNγ, and CSF2 (I) and chemokines CCl2, CCl5, CXCL9, and CXCL10 (J). Solid lines represented the differences in mean values. Asterix (*) indicated values that were statistically significant by One-Way ANOVA analysis and uncorrected Fisher's LSD Multiple Comparison Test (*P<0.05, **P<0.01).

promoter. XBP1 protein structure was generated using I-TASSER homology modeling, and the DJ-1 promoter was generated using 3D DART software. The surface charge distribution of XBP1 protein was analyzed using the APBS plugin (PyMOL). The observed structure revealed a highly positively charged groove (demarcated by blue region) in the XBP1 protein, where the DJ-1 promoter can bind. The stable XBP1 protein structure was confirmed by molecular dynamics simulation using GROMACS software, and its interaction with the DJ-1 promoter was confirmed using the NP Dock server. The fit of the structure of the DJ-1 promoter in the XBP1 protein groove was visually analyzed using PyMOL (A).

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.