Multiple autism-like behaviors in a novel transgenic mouse model

Abstract

Autism spectrum disorder (ASD) diagnoses are behaviorally based with no defined universal biomarkers, occur at a 1:110 ratio in the population, and predominantly affect males compared to females at approximately a 4:1 ratio. One approach to investigate and identify causes of ASD is to use organisms that display abnormal behavioral responses that model ASD-related impairments. This study describes a novel transgenic mouse, MALTT, which was generated using a forward genetics approach. It was determined that the transgene integrated within a non-coding region on the X chromosome. The MALTT line exhibited a complete repertoire of ASD-like behavioral deficits in all three domains required for an ASD diagnosis: reciprocal social interaction, communication, and repetitive or inflexible behaviors. Specifically, MALTT male mice showed deficits in social interaction and interest, abnormalities in pup and juvenile ultrasonic vocalization communications, and exhibited a repetitive stereotypy. Abnormalities were also observed in the domain of sensory function, a secondary phenotype prevalently associated with ASD. Mapping and expression studies suggested that the Fam46 gene family may be linked to the observed ASD-related behaviors. The MALTT line provides a unique genetic model for examining the underlying biological mechanisms involved in ASD-related behaviors.

Figure 1. Initial characterization of the MALTT transgenic line

(a) A fluorescently labeled (FITC) anti-digoxigenin antibody indicated the location of the TyBS minigene insertion (left) and Geimsa staining indicated (black arrow) the labeled chromosome was X (right). (b) A schematic diagram of the mouse X chromosome indicates at minimum two copies of 4.4kb minigene construct (0.4kb MuLV sequence+1.8kb promoter region+2.2kb tyrosinase (tyr) cDNA) inserted in the non-coding region between genes Gm732 and Brwd3, as well as the 5’ integration sequence (black lettering: X chromosome, gray lettering:minigene). A Southern blot probe against the minigene is indicated in red. (c) A Southern hybridization image indicates 2 minigene positive bands (0.8kb, ~2.9kb) apparent in MALTT (lane 1) but not WT (lane 2) BanI-digested gDNA (left). (d) A single band (minimum 20kb) was apparent in MALTT (lane 1) but not WT (lane 2) BbsI-digested gDNA (right). (e) Exemplary sagittal and coronal Nissl-stained images of WT and MALTT brain sections are shown.

Figure 2. MALTT mice are hyperactive and exhibit a rotational stereotypy

Total distance was measured during 10 minutes of open field exploration for mice aged 17, 22, 28, and 45 days. (a) Male total distance is shown. (b) Stereotypies in males were measured as total revolutions/total distance traveled for male mice. (c) Average absolute numbers of revolutions for males on PND45 are shown. (d) Female total distance is shown. All data are presented ± SEM. Males: WT n = 9–17, TG n = 9–13; females: WT n = 12–17, TG n = 11–17; * p ≤ 0.05, ** p ≤ 0.01

Figure 3. MALTT mice show altered pup ultrasonic vocalization patterns

Pup ultrasonic vocalizations (USVs), when separated from mother and littermates, were assessed on PND 3–14. (a) Male pup USV development curve is shown, WT n = 21; TG n = 24. (b) Female pup USV development curve is shown, WT n = 19; TG n = 22. All data are presented ± SEM.

Figure 4. Juvenile MALTT mice show decreased social interest

Three-chamber partition task: (a) Diagram of the testing apparatus with two outer side chambers, each housing a novel object or stranger mouse behind perforated Plexiglas, and the center chamber where the subject is started. Two doorways allowed the subject to move freely between all chambers. (b) A MALTT or WT subject was allowed to explore the apparatus. Mean total duration each subject spent per chamber (including time at partitions within side chambers) is shown. choose to spend time investigating a stranger mouse or novel object through separate partitions, WT n = 8; TG n = 9. (c) Mean total duration at stranger or object partition is indicated. (d) Frequency of subject entry into each side chamber from the center chamber is shown. All data are presented ± SEM. * p ≤ 0.05

Figure 5. Juvenile MALTT mice show social and social communication-related deficits

Direct social interaction task one: Two stranger mice were allowed to directly interact for 10 minutes while USVs were concomitantly recorded. (a) Mean duration of active, passive, and nonsocial behaviors for the subject mouse within a pair is shown. The subject of the pair was a WT in a WT + FVB/NJ stranger dyad, n = 8 pairs, or a TG in a TG + FVB/NJ stranger dyad, n = 9 pairs; (a–d). (b) Mean total USVs emitted by pairs is shown. (c) Representative event plot indicating (foreground) a pair’s USV events as they overlapped the subject mouse’s behavioral state (background). (d) Shown is the mean number of USVs emitted from the pair during the WT or TG subjects’ respective behavioral states. All data are presented ± SEM. * p ≤ 0.05, ** p ≤ 0.01

Figure 6. Juvenile MALTT mice show minimal vocalization in a social pair

Direct social interaction task two: MALTT + MALTT partner pairs or WT + WT partner pairs were analyzed for USVs during direct social interaction; MALTT n = 9 pairs; WT n = 8 pairs. (a) Mean total USVs emitted from same genotype pairs is shown. Individual pair data is indicated (black circles), with the exception of TG:TG data point “0” which includes 7 pairs’ values. (b) Mean duration of social and nonsocial behaviors for same genotype pairs is shown. All data are presented ± SEM. ** p ≤ 0.01

Figure 7. Juvenile male MALTT mice can detect and differentiate nonsocial and social odors

A habituation/ dishabituation olfactory test was used to assess sense of smell. WT n = 13; TG n = 11. (a) Nonsocial odor 1 (banana or almond) was presented repeatedly followed by a second nonsocial odor (banana or almond) and mice were assessed for dishabituation on first presentation of novel odor and habituation by the third presentation of the odor. (b) Same task as (a) but nonsocial odors were replaced with social odors. For both tasks odor 1 and odor 2 were alternated pseudorandomly between subjects. All data are presented ± SEM. + denotes p≤0.05 for habituation to odor for both genotypes, # denotes p≤0.05 for detection of novel odor for both genotypes, open circle denotes Tg, closed circle denotes WT, ** p ≤ 0.01 for a between-subjects effect

Figure 8. MALLT mice show aberrant secondary ASD-associated behaviors

(a) A tactile sensitivity assay indicates minimum threshold for hind paw plantar surface detection of force for male mice, WT n = 8; TG n = 7. (b) Mean percent prepulse inhibition (PPI) response for male mice is shown. Data is collapsed over ages 17, 22, 28, 45 days. The same subjects tested for OFA were tested second on PPI. (c) Mean percent PPI response for female mice is shown collapsed over ages 17, 22, 28, 45 days. (d) Percent of mice exhibiting seizure activity in response to an audiogenic stimulus is shown for males WT n = 11, TG n = 19; females WT n = 9, TG n = 13. All data are presented ± SEM. * p ≤ 0.05, ** p ≤ 0.01

Figure 9. Genes analyzed in the vicinity of the TyBS minigene integration site