Mouse Hepatitis Virus Induced Remodeling of Gap Junction Intercellular Communication Plays a Major Role in Demyelination

Basu, Rahul (2017) Mouse Hepatitis Virus Induced Remodeling of Gap Junction Intercellular Communication Plays a Major Role in Demyelination. PhD thesis, Indian Institute of Science Education and Research Kolkata.

PDF (PhD thesis of Rahull Basu (11RS010)) Final_Thesis_Rahul_Basu.pdf - Submitted Version Restricted to Repository staff only Download (5MB)

Abstract

Gap junctions (GJs) are membrane channels that form intercellular communication or unopposed hemichannels to facilitate the exchange of small molecules (< 1 kDa in size) between neighboring cells and extracellular environment. GJs are comprised of tetraspan membrane proteins, namely connexins (Cxs). Hexamers of Cx proteins form hemichannels on the cell surface and a pair of apposing hemichannels from two neighboring cells form a GJ channel (GJC), which further aggregate into higher order arrays named GJ plaques (GJPs). Astrocytes, the most abundant glial cells, communicate to other astrocytes and glial cells to form a functional “panglial syncytium”. Astrocytes mainly express GJ protein Cx43 and Cx30, of which Cx43 is most abundantly expressed. Cx43, being the most abundant Cx in CNS, forms homotypic Cx43/Cx43 mediated GJIC between two astrocytes and heterotypic Cx43/Cx47 mediated GJICs between astrocytes and oligodendrocytes. These GJCs perform homeostatic function like maintaining K+ buffering and nutrient homeostasis in CNS, and have crucial impact on maintenance of CNS myelin. In human CNS demyelinating disease multiple sclerosis (MS) and other dysmyelinating genetic diseases, the alteration of GJCs are proposed to have important role in disease pathogenesis. In MS, the way in which this alteration of GJ is initiated and how it is associated with progression of the disease is not well understood. In a viral model of MS, demyelinating strain of mouse hepatitis virus (MHV), MHV-A59, causes hepatitis and meningoencephalitis in the acute phase and results in demyelination and axonal loss in the chronic phase of infection. Astrocytes, the most abundant glial cells in CNS, are infected by MHV-A59 but the role of astrocytic MHV-A59 infection induced neuroinflammation remains to be explored. Thus, it was evaluated whether MHV-A59 infection in astrocytes could alter the expression, localization and function of Cx43, in vitro, in primary astrocyte culture. Uninfected astrocytes showed that profuse amount of Cx43 was localized at the cell surface. In contrast, upon infection with MHV-A59, most of the Cx43 was retained in the intercellular compartment like the endoplasmic reticulum (ER)/endoplasmic reticulum Golgi intermediate complex (ERGIC). Upon MHV-A59 infection Cx43 was also reduced in total protein and RNA level, which further inhibited a significant amount of Cx43 mediated detergent insoluble GJP formation on the cell surface. The small molecule permeable, functional channel formation between primary astrocytes was also reduced significantly. The mechanistic aspects of MHVA59 induced Cx43 retention in the ER/ERGIC of primary astrocytes were further investigated. Cx43 is delivered to the cell surface with the help of the microtubule (MT) network. Demyelinating strains of MHV also specifically use the MT-network as a conduit to reach the cell surface. Hence, it is hypothesized that MHV-A59 viral particle trafficking in primary astrocytes directly restricted Cx43 delivery to the cell surface. Colchicine induced destabilization of MT-network affected Cx43 delivery to the cell surface. The double-label immunofluorescence studies showed that the colocalization between Cx43 and MT-threads (immunostained for β- tubulin) was also reduced. In consistence, the colocalization between viral particle and MTthreads was observed, specifically at the cell surface. The direct protein-protein interaction between Cx43/β-tubulin was significantly depleted upon MHV-A59 infection and viralnucleocapsid (N) protein was coimmunoprecipitated with β-tubulin in primary astrocytes. Finally, whether the alteration of Cx43 in primary astrocytes also affected GJ expression in vivo was assessed. C57Bl/6 mice were infected with MHV-A59 and sacrificed at the peak of inflammation (acute phase, day 5 p.i.) and at the peak of demyelination (chronic phase, day 30 p.i.), and mock infected mice were generated in parallel. Upon successful viral infection, Cx43 protein expression was depleted at day 5 p.i., specifically in virus-infected brains. The downregulation of Cx43 protein was also associated with reduction in Cx43 mRNA expression. Cx43 expression was similar in mock and MHV-A59 infected brains at the chronic phase at day 30 p.i. In contrast, oligodendrocytic coupling partner of Cx43, Cx47, was persistently downregulated both at acute and chronic phase. This sustained loss of Cx47 was associated with loss of myelin marker proteolipid protein (PLP) in the white matter areas in chronic infected CNS. This study elucidated the mechanism of initial GJ alteration induced by MHV-A59 infection and provided a link to loss of myelin protein observed due to sustained loss of GJ proteins.

Actions (login required)

View Item