title: 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 Ca(2+) 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 Ca(2+)-binding and Ca(2+) 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 Ca(2+) channels in mock- and TMEV-infected cells, respectively, was demonstrated using voltage clamp experiments. TMEV infection in mouse astrocytes induced a Ca(2+) current with a density proportional to the amount of viral particles used for infection. The use of Ca(2+) channel blockers, nimodipine and ω-conotoxin-GVIA, showed that both functional L- and N-type Ca(2+) channels were upregulated in infected astrocytes. The upregulation of Ca(2+) channels in astrocytes after TMEV infection provides insight into the molecular processes and potential role of astrocyte Ca(2+) dysregulation in the pathophysiology of encephalomyelitis and is important for the development of novel therapeutic strategies leading to prevention of neurodegeneration.
authors.affiliation: Instituto Cajal, CSIC, Madrid, Spain.
authors.forename: N
authors.initials: N
authors.lastname: Rubio
authors.forename: A
authors.initials: A
authors.lastname: Almanza
authors.forename: F
authors.initials: F
authors.lastname: Mercado
authors.forename: M-Á
authors.initials: MÁ
authors.lastname: Arévalo
authors.forename: L M
authors.initials: LM
authors.lastname: Garcia-Segura
authors.forename: R
authors.initials: R
authors.lastname: Vega
authors.forename: E
authors.initials: E
authors.lastname: Soto
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism* / Q000821:virology*; D015220:Calcium Channels / Q000096:biosynthesis*; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D051379:Mice; D011051:Poliomyelitis / Q000378:metabolism* / Q000503:physiopathology / Q000662:veterinary; D017945:Theilovirus / Q000472:pathogenicity*; D015854:Up-Regulation / Q000502:physiology*
chemical_list: D015220:Calcium Channels
keywords: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; CNS; DMEM; Dulbecco’s modified Eagle medium; EAE; EDTA; EGTA; FCS; HEPES; HPLC; HVA; LVA; MS; SEM; TMEV; TMEV infection; Theiler virus; Theiler’s murine encephalomyelitis virus; central nervous system; encephalomyelitis; ethylene glycol tetraacetic acid; ethylenediaminetetraacetic acid; experimental autoimmune encephalomyelitis; fetal calf serum; high voltage-activated; high-pressure liquid chromatography; low voltage-activated; m.o.i.; multiple sclerosis; multiplicities of infection; neurodegeneration; qPCR; quantitative real time PCR; standard error of the mean