Upregulation of voltage-gated Ca2+ channels in mouse astrocytes infected with Theiler’s murine encephalomyelitis virus (TMEV)

Abstract

Theiler’s murine encephalomyelitis virus (TMEV) induces demyelination in susceptible strains of mice through a CD4+ Th1 T cell-mediated immunopathological process. TMEV infection produces a syndrome in mice that resembles multiple sclerosis. In this work, we focused on the increased expression of the genes encoding voltage-gated Ca2+ channel subunits in SJL/J mouse astrocytes infected in culture with a BeAn strain of TMEV. Affymetrix DNA murine genome U74v2 DNA microarray hybridized with cRNA from mock- and TMEV-infected astrocytes revealed the upregulation of four sequences encoding Ca2+-binding and Ca2+ channel subunit proteins. The DNA hybridization results were further validated using conventional RT-PCR and quantitative RT-PCR, demonstrating the increased expression of mRNA encoding channel subunit proteins. Western blotting also showed the increased synthesis of L- and N-type channel subunit specific proteins after infection. The reduced expression and the functional upregulation of functional voltage-gated Ca2+ channels in mock- and TMEV-infected cells, respectively, was demonstrated using voltage clamp experiments. TMEV infection in mouse astrocytes induced a Ca2+ current with a density proportional to the amount of viral particles used for infection. The use of Ca2+ channel blockers, nimodipine and ω-conotoxin-GVIA, showed that both functional L- and N-type Ca2+ channels were upregulated in infected astrocytes. The upregulation of Ca2+ channels in astrocytes after TMEV infection provides insight into the molecular processes and potential role of astrocyte Ca2+ dysregulation in the pathophysiology of encephalomyelitis and is important for the development of novel therapeutic strategies leading to prevention of neurodegeneration.