The therapeutic potential of Connexin 43 and its chaperone in restricting murine-β-coronavirus-induced neuroinflammatory demyelination

Kasle, Grishma Deepak (2025) The therapeutic potential of Connexin 43 and its chaperone in restricting murine-β-coronavirus-induced neuroinflammatory demyelination. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Grishma Deepak Kasle (20RS083)) 20RS083.pdf - Submitted Version Restricted to Repository staff only Download (12MB)

Abstract

Mouse hepatitis virus MHV-A59, a neurotropic murine β-coronavirus, serves as a valuable experimental model for understanding the mechanisms underlying virus-induced neuroinflammation and demyelination. Inoculation of MHV-A59 in the mouse central nervous system (CNS) causes infection of neurons, astrocytes, and oligodendrocytes, with cell-to-cell fusion playing a critical role in the induction of infection. These neuroglial cells are interconnected through gap junction intercellular communication (GJIC), forming a pan-glial syncytium that facilitates metabolic coupling, which is essential in maintaining CNS homeostasis. These Gap Junctions (GJs) are composed of connexin proteins, with different glial cells expressing different connexins. Connexin 43 (Cx43) is one of the most predominant connexins in the CNS, mainly expressed by astrocytes, and its coupling partner, Cx47, is expressed on oligodendrocytes. The Cx43-Cx47 coupling between the astrocytes and oligodendrocytes is of utmost importance since it has been implicated in demyelination. Previous studies from the lab have demonstrated that MHV-A59 infection leads to altered expression of Cx43 in the CNS, which also destabilises and downregulates the expression of its coupling partner Cx47. A similar downregulation of Cx43 is also observed in MHV-A59-infected murine primary astrocytes, accompanied by altered trafficking of Cx43 to the cell surface in infected cells. From a mechanistic standpoint, it was shown that MHV-A59 infection induces endoplasmic reticulum (ER) stress in infected cells, thereby reducing the expression of ER-resident chaperone ERp29, known to be important for Cx43 trafficking. Over-expression of ERp29 in DBT cells showed improved Cx43 trafficking and restriction of MHV-A59 infectivity and viral spread. With a vision to test the therapeutic potential of improved Cx43 expression in restricting MHV-A59 infectivity and spread in vivo, this study tested a pharmacological modulator of Cx43, 4-phenylbutyrate (4-PBA). In the current study, a reductionist approach was used to demonstrate that treatment of MHV-infected primary astrocytes with 4-PBA restored Cx43 transport to the cell surface and restricted MHV-A59 infectivity and spread. Using the Cx43 mimetic peptide, Gap 27, to block Cx43, the current study demonstrated that the antiviral potential of 4-PBA was mitigated upon blocking of Cx43 channels. Further, in vivo, the treatment of MHV-A59-infected mice with 4-PBA reduced acute MHV-A59 infectivity and viral spread in the brain. 4-PBA treatment in mice also modulated the glial cell response to infection, mounting a robust inflammatory response. Further demonstrating that the reduced viral spread is attributed to the 4-PBA-mediated improved expression of Cx43 in the CNS.4-PBA treatment also protected mice against MHV-A59-mediated chronic progressive demyelination. Furthermore, previous studies have demonstrated that inflammatory cytokines can modulate the expression of Cx43.To understand the effect of MHV-infection-induced neuroinflammation on Cx43 expression, the mRNA expression of a battery of cytokines in vivo in the mouse brain, as well as in vitro in the murine primary astrocytes upon infection, was studied, which revealed that TNF-α is an important candidate cytokine that may affect Cx43 expression. Further, treatment of primary astrocytes with exogenous TNF-α downregulated the expression of Cx43. Finally, the study demonstrates that sequestering the TNF-α released by primary astrocytes upon MHV-A59 infection using TNF-α neutralising antibodies mitigated the downregulation of Cx43. Overall, these findings highlight the importance of GJIC in exerting an antiviral effect in the CNS of mice and support a Cx43-dependent mechanism that can be therapeutically targeted to protect against β-coronavirus infection and chronic demyelination.

Actions (login required)

View Item