Protein expression pattern in cerebellum of Cav2.1 mutant, tottering-6j mice

Abstract

Neuronal voltage-gated Cav2.1 channel controls a broad array of functions, including neurotransmitter release, neuronal excitability, activity-dependent gene expression, and neuronal survival. The Cav2.1 channel is molecular complexes consisting of several subunits: α1, α2/δ, β, and γ. The pore-forming subunit, α1, is encoded by the Cacna1a gene. Tottering-6j mice, generated by the Neuroscience Mutagenesis Facility at The Jackson Laboratory, are a recessive mutant strain in which the mutation has been chemically induced by ethylnitrosourea. In tottering-6j mice, mutation in the Cacna1a gene results in a base substitution (C-to-A) in the consensus splice acceptor sequence, which results in deletion of a part of the S4-S5 linker, S5, and a part of S5-S6 linker domain I in the α1 subunit of Cav2.1 channel. The mice display motor dysfunctions and absence-like seizures. However, protein expression in the cerebellum of tottering-6j mice has not been investigated. Real-time quantitative reverse transcription polymerase chain reaction and histological analyses of the cerebellum of tottering-6j mice revealed high expression levels of tyrosine hydroxylase, zebrin II, and ryanodine receptor 3 compared with those of wild-type mice. Conversely, a low level of calretinin expression was found compared with wild-type mice. These results indicate that Cacna1a mutation plays a significant role in protein expression patterns and that the tottering-6j mouse is a useful model for understanding protein expression mechanisms.

Fig. 1.

mRNA expression of calbindin D-28K (Calb1), calretinin (Calb2), tyrosine hydroxylase (TH), Zebrin II (ZebrinII), ryanodine receptor 1 (Ryr1), ryanodine receptor 2 (Ryr2), and ryanodine receptor 3 (Ryr3) genes in the cerebellum of wild-type and tottering-6j mice. The data are presented as means ± SEM. * P<0.05, ** P<0.01, compared with the appropriate control (Dunnett’s test).

Fig. 2.

Cytoarchitecture of the cerebellum. Tottering-6j mice had normal cytoarchitecture compared with +/+ mice. There was no Purkinje cell degeneration, and the length of the molecular layer was normal. The scale bar represents 200 µm.

Fig. 3.

Expression of TH in the cerebellum. TH expression was increased in the cerebellum of tottering-6j mice compared with +/+ mice. The scale bar represents 50 µm. M: molecular layer, P: Purkinje cell layer, Gr: granular layer.

Fig. 4.

Expression of Ryr3 in the cerebellum. Ryr3 expression was increased in tottering-6j mice compared with +/+ mice. The scale bar represents 50 µm. M: molecular layer, P: Purkinje cell layer, Gr: granular layer.

Fig. 5.

Expression of Calb2 in the cerebellum. Calb2 expression was decreased in tottering-6j mice compared with +/+ mice. The scale bar represents 50 µm. M: molecular layer, P: Purkinje cell layer, Gr: granular layer.

Fig. 6.

Apoptotic cell death in the cerebellum. The number of apoptotic cells in tottering-6j mice was greater than that of +/+ mice. Apoptotic cells were found in the granular layer. Apoptotic cells are indicated with arrowheads. The scale bar represents 200 µm.