Abstract
One of the family of voltage-gated calcium channels (VGCC), the N-type Ca(2+) channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca(2+) channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca(2+) channel α(1B)-deficient (α(1B)(-/-)) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35-55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α(1B)(-/-) mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α(1B)(-/-) mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α(1B)(-/-) mice and was significantly inhibited by a selective N-type Ca(2+) channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca(2+). These results suggest that the N-type Ca(2+) channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.
Highlights
Multiple sclerosis (MS) is an immune-mediated disorder of the CNS leading to a progressive decline of motor and sensory functions and permanent disability
The body weight loss in wild type (WT) mice was significantly more severe than in ␣1BϪ/Ϫ mice (Fig. 1B). Clinical parameters of this above acute phase experiment, we evaluated the delayed type hypersensitivity (DTH) response before sacrifice
Numerous studies have demonstrated that the calcium ion is critical in the neuronal damage seen in neurodegenerative disease models such as trauma [26], ischemia [27], and EAE [28]
Summary
Experimental Animals—All animal procedures conformed to the Japanese regulations for animal care and use, following the Guidelines for Animal Experimentation of the Japanese Association for Laboratory of Animal Science, and were approved by the Animal Care and Use Committee of Eisai Co., Ltd. and Chiba University. For the present experiments, ␣1BϪ/Ϫ female mice (backcrossed generation into C57BL/6NCrlCrlj) were kept in specific pathogen-free facilities and maintained at constant temperature (22 °C Ϯ 2 °C) and humidity (55 Ϯ 5%) with a 12:12 h light:dark cycle. WT (filled circle) and ␣1BϪ/Ϫ (KO: open circle) mice were immunized for EAE with MOG35–55 emulsified in complete Freund’s adjuvant and intravenously injected with pertussis toxin on days 0 and 2. They were monitored daily for clinical score (A) and body weight (B) until day 58. The difference between the two groups was statistically significant (*, p Ͻ 0.05) as determined by repeated measures of analysis of variance
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