The first knockin mouse model of episodic ataxia type 2

Abstract

Episodic ataxia type 2 (EA2) is an autosomal dominant disorder associated with attacks of ataxia that are typically precipitated by stress, ethanol, caffeine or exercise. EA2 is caused by loss-of-function mutations in the CACNA1A gene, which encodes the α1A subunit of the CaV2.1 voltage-gated Ca(2+) channel. To better understand the pathomechanisms of this disorder in vivo, we created the first genetic animal model of EA2 by engineering a mouse line carrying the EA2-causing c.4486T>G (p.F1406C) missense mutation in the orthologous mouse Cacna1a gene. Mice homozygous for the mutated allele exhibit a ~70% reduction in CaV2.1 current density in Purkinje cells, though surprisingly do not exhibit an overt motor phenotype. Mice hemizygous for the knockin allele (EA2/- mice) did exhibit motor dysfunction measurable by rotarod and pole test. Studies using Cre-flox conditional genetics explored the role of cerebellar Purkinje cells or cerebellar granule cells in the poor motor performance of EA2/- mice and demonstrate that manipulation of either cell type alone did not cause poor motor performance. Thus, it is possible that subtle dysfunction arising from multiple cell types is necessary for the expression of certain ataxia syndromes.

Keywords: Ataxia; CACNA1A; Cerebellar granule cell; Channelopathy; Cre; EA2; Episodic ataxia type 2; Knockin; Purkinje cell; Voltage-gated calcium channel.

Fig. 1

Generation and molecular characterization of EA2 knockin mice. (A) The EA2 knockin construct contained a c.4486T>G substitution, coding a p.F1406C missense in exon 26 of Cacna1a and a neomycin resistance cassette (neo^r), flanked by two loxP sites, upstream of exon 27. The targeting construct was introduced into C57BL/6J–derived ES cells via homologous recombination and cells carrying the construct were injected into blastocysts. (B) A series of PCRs from the DNA of F1 mice were used to verify correct homologous recombination. Reactions from primers P1 and P3 illustrate proper insertion of the 5’ end of the construct and reactions from primers P4 and P6 verify 3’ recombination. (C) DNA and mRNA sequences illustrate the presence of the c.4486T>G substitution in the PCR product of genomic DNA and RT-PCR product of brain mRNA from +/+ and EA2/EA2 mice. (D) Western blot shows the normal expression of Ca_V2.1 in frontal cortex and cerebellum.

Fig. 2

Physiological properties of EA2 knockin (p.F1406C) Ca_V2.1 (A) Depolarizing voltage pulses (100 ms) ranging from −60 to + 40 mV (top) were applied to acutely dissociated PCs in the presence of Ca_V1.2 and Ca_V2.2 antagonists, and Ba²⁺ currents (I_Ba) were measured. Representative traces from +/+ and EA2/EA2 mice are shown (bottom). (B) Current-voltage (I-V) relationships were plotted for +/+ (n=7), EA2/+ (n=3), and EA2/EA2 (n=4) PCs using the voltage protocol shown in (A). Symbols represent mean + SEM. (C) Peak I_Ba vs. time was recorded for +/+ and (D) EA2/EA2 PCs during 100 ms depolarizations from −60 to −20 mV. Arrows indicate the sequential addition of Ca²⁺ channel blockers (5 µM nimodipine (Ca_V1.2), 3 µM ω-conotoxin MVIIC (Ca_V2.1), 1 µM ω-conotoxin GVIA (Ca_V2.2)).

Fig. 3

Normal cytoarchitecture in the EA2 mutant cerebellum. Examination of cerebella from EA2 mutants stained for Nissl substance (A-J) and calbindin (K-O) revealed no evidence of degeneration (Scale bar A-E=500 µm, F-O=25 µm).

Fig. 4

Motor phenotype of EA2 mutant mice. Mice carrying various combinations of wild-type (+), knockin (EA2), and knockout (-) Cacna1a were tested for their acquisition of motor skills with the accelerating rotarod (A) and pole test (B) as well as grip strength as assessed by the cling test (C) and baseline locomotor activity (D). EA2/- mice (n=17), but not EA2/+ (n=11), +/−(n=10), or EA2/EA2 (n=11) mice showed reduced performance compared to +/+ (n=16) on the rotarod (***p<0.001, Tukey’s test) and pole test (*p<0.05, Tukey’s test). No significant differences between genotypes were found for cling test (F_4,44=0.97, P>0.4) or locomotor activity (F_4,27=0.27, P>0.5).

Fig. 5

Response of EA2 mutant mice to caffeine and EtOH. (A) Rotarod performance was assessed following 15 mg/kg caffeine or saline in +/+ (n=14), EA2/+ (n=11), EA2/EA2 (n=11), and EA2/− (n=6) mice previously trained on rotarod. No main effect of caffeine (F_1,38=0.24, P>0.5) or interaction between genotype and caffeine (F_3,38=0.82, P>0.4) were found. (B) Rotarod performance was assessed 20 minutes following EtOH (1,1.5, and 2g/kg) in the same cohort of mice. A significant effect of genotype (F_3,38=4.73, P<0.01, *p<0.05 for EA2/-) and EtOH dose was found (F_3,114=167, P<0.001, *p<0.05 for 1g/kg, ***p<0.001 for 1.5g/kg and 2g/kg), but no interaction was found between EtOH and genotype (F_9,114=0.66, P>0.5).

Fig. 6

X-Gal staining as a reporter for Cre recombinase activity. The Math1-Cre and L7-Cre transgenes were bred onto the Cre reporter line, Rosa26, which expresses β-galactosidase in the presence of Cre recombinase. X-Gal staining, in blue, was located in PCs in L7-Cre expressing mice (A), and in GCs in Math1-Cre expressing mice exposed to tamoxifen at E16.5 (B), but not control mice that did not carry a Cre transgene (C). (Scale bar = 200 µm)

Fig. 7

Rotarod performance of cell-type specific EA2/- mice. (A) PC-specific EA2/- mice (EA2/flox; L7-Cre/-, n=9) did not exhibit motor dysfunction detectable by rotarod (F_2,23=0.17, P>0.5) compared to control mice (EA2/+; L7-Cre/-, n=10, EA2/flox, n=7). Similarly, (B) GC-specific EA2/- mice (EA2/flox; Math1-Cre/-, n=10) did not exhibit a motor dysfunction detectable by rotarod (F_2,36=0.84, P>0.25) compared to control mice (EA2/+; Math1-Cre/-, n=11, EA2/flox, n=14).