Mouse model of Timothy syndrome recapitulates triad of autistic traits

Abstract

Autism and autism spectrum disorder (ASD) typically arise from a mixture of environmental influences and multiple genetic alterations. In some rare cases, such as Timothy syndrome (TS), a specific mutation in a single gene can be sufficient to generate autism or ASD in most patients, potentially offering insights into the etiology of autism in general. Both variants of TS (the milder TS1 and the more severe TS2) arise from missense mutations in alternatively spliced exons that cause the same G406R replacement in the Ca(V)1.2 L-type calcium channel. We generated a TS2-like mouse but found that heterozygous (and homozygous) animals were not viable. However, heterozygous TS2 mice that were allowed to keep an inverted neomycin cassette (TS2-neo) survived through adulthood. We attribute the survival to lowering of expression of the G406R L-type channel via transcriptional interference, blunting deleterious effects of mutant L-type channel overactivity, and addressed potential effects of altered gene dosage by studying Ca(V)1.2 knockout heterozygotes. Here we present a thorough behavioral phenotyping of the TS2-neo mouse, capitalizing on this unique opportunity to use the TS mutation to model ASD in mice. Along with normal general health, activity, and anxiety level, TS2-neo mice showed markedly restricted, repetitive, and perseverative behavior, altered social behavior, altered ultrasonic vocalization, and enhanced tone-cued and contextual memory following fear conditioning. Our results suggest that when TS mutant channels are expressed at levels low enough to avoid fatality, they are sufficient to cause multiple, distinct behavioral abnormalities, in line with the core aspects of ASD.

Fig. 1.

TS2-neo mice showed normal diurnal rhythm and locomotor activity but decreased locomotion in a novel environment. (A) Basic activity monitored over 5 d in a home-cage with shelter and water/food. n (each genotype) = 12. (B) Time spent moving: average of five 24-h dark/light cycles (gray/white background, respectively) (Left) and pooled data for five dark periods (Right) reveal no differences between genotypes. (C) Distance moved: TS2-neo mice traveled the same average distance as WT littermates during five dark cycles. (D) Monitoring of ambulatory activity over 5 min in activity chamber. n (each genotype) = 31. (E) Time spent moving was slightly but significantly smaller in TS2-neo mice (P = 0.03 for genotype effect, ANOVA) (Left); likewise for cumulative time spent moving (P = 0.03, Student's t test) (Right). (F) Distance moved: TS2-neo mice traveled a slightly but significantly smaller distance (P = 0.04, Student's t test).

Fig. 2.

TS2-neo mice exhibited no sign of increased anxiety in light/dark box and elevated zero maze. (A) Light/dark box: mice were exposed to an activity chamber with dark and bright sides for 10 min. (B) Percentage of time spent in dark side. No significant difference between TS2-neo and WT mice for time course (P > 0.8 for genotype effect, ANOVA) (Left) or cumulative time (P > 0.8, Student's t test) (Right). n (WT) = 13, n (TS2-neo) = 12. (C) Elevated zero maze: mice were exposed to elevated, annular platform with two closed and two open quadrants for 8 min. n (WT) = 9, n (TS2-neo) = 13. (D) No significant difference between TS2-neo and WT littermates for time course (P > 0.6 for genotype effect, ANOVA) (Left) and cumulative time spent in closed quadrants (P > 0.2, Mann–Whitney u test) (Right).

Fig. 3.

TS2-neo mice showed decreased approach behavior to a novel environment. (A) After a 5-d exposure to home-cage environment with a shelter and water/food, NE (additional tube and chamber) was attached for 15 min. (B) TS2-neo mice showed twofold increases in latency to initiate contact with NE (P = 0.02, Mann–Whitney u test) (Left) and to enter it (P = 0.03, Mann–Whitney u test) (Right) and entered it significantly fewer times (C, P = 0.01, Student's t test). (D) A minute-to-minute comparison of time spent in NE showed significant genotype effect (P = 0.02, ANOVA) (Left). TS2-neo mice spent less than half as much time in NE than WT (P = 0.02, Student's t test) (Right) but a correspondingly longer time in the shelter (E, Left, P = 0.01, Student's t test). (E, Right) No genotype difference for time spent in the open area of the home-cage. n (each genotype) = 12.

Fig. 4.

Repetitive marble burying and perseveration in searching for previous escape platform location in MWM and water Y-maze. (A) Marble burying: TS2-neo mice buried twice as many marbles as WT littermates during a 30-min exposure to 20 marbles (P = 0.03, Student's t test). n (WT) = 23, n (TS2-neo) = 23. (B) MWM: during the first four trials of reversal learning, TS-neo mice spent significantly more time in previous target quadrant than WT (P < 0.03, Student's t test). n (WT) = 27, n (TS2-neo) = 29. (C) Water Y-maze: percentage of correct arm choices per trial block during acquisition (day 1 of experiment), test (day 2), reversal (day 3), and forced training (day 3). During reversal training, TS2-neo mice made significantly fewer correct choices than control mice (P = 0.02 for genotype effect, ANOVA). n (WT) = 11, n (TS2-neo) = 11. (D) Color-coded graph depicting individual trial outcomes in water Y-maze (white/green for wrong/correct arm choices, respectively). Forced training (incorrect arm blocked): three TS2-neo mice never made correct choice, whereas the WT mouse entered correct arm from first trial onward.

Fig. 5.

TS2-neo mice displayed decreased preference for a social object in an automated social home-cage assay. (A) After a 4-d exposure to a home-cage with shelter and water/food, empty and occupied corrals were presented in opposite corners. (B) During initial 10 min, both genotypes preferred to stay close to occupied corral than near empty corral (P < 0.0001 for corral effect, ANOVA). (C) Whereas WT mice displayed significant preference for the occupied vs. the empty corral during the first full hour (P < 0.01, Bonferroni post hoc test), and a trend for similar preference over the last 3 h, TS2-neo mice showed a trend for preference for the occupied corral during the first full hour and opposite preference for the last 3 h. (D) Intensity map of last 2 h, depicting spatial distribution of TS2-neo:WT time ratio (red/blue for increased/decreased TS2-neo dwell time, respectively). (E) Cumulative distribution of preference index for last 2 h. TS2-neo mice showed significantly less preference for the occupied corral than WT mice (P = 0.04, Student's t test). n (each genotype) = 12. (Inset) Ca_V1.2^+/− mice show the same preference for the occupied corral as WT. n (WT) = 9, n (Ca_V1.2^+/−) = 10.

Fig. 6.

TS2-neo pups emit shorter ultrasonic vocalization calls. (A) Starting at PND 2, pups were separated from their dam and litter and their USVs were recorded for 5 min. (B) The duration of calls was significantly decreased in TS2-neo mice compared with WT littermates (P = 0.007 for genotype effect, ANOVA). n (WT) = 6, n (TS2-neo) = 4.

Fig. 7.

TS2-neo mice exhibit increased persistence of tone-cued and contextual fear memory. (A) Protocol: on day 1 (acquisition), mice were exposed to five tone-shock pairings in context A. On day 2 (tone memory), mice were exposed to the tone in a different context (context B), and on day 3 (context memory) to the same context (context A) without tone. Long-term tone memory and context memory were retested on days 8 and 15 or 9 and 16, respectively. (B) Acquisition: TS2-neo mice froze at the same level as WT littermates (P = 0.2, Student's t test). (C) Tone memory: TS2-neo froze significantly more on day 15 (P < 0.02 for genotype effect, ANOVA; day 15: P < 0.05, Bonferroni post hoc test). (D) Context memory: TS2-neo froze significantly more on day 16 (P < 0.02 for genotype effect, ANOVA; day 16: P < 0.05, Bonferroni post hoc test). The decrease in freezing was significant in WT (P < 0.0001 for day effect, ANOVA; day 3 vs. day 9: P < 0.05; day 3 vs. day 16: P < 0.001, Bonferroni post hoc test) but not TS2-neo mice. n (WT) = 10, n (TS2-neo) = 11.