Cognitive deficits in the Snord116 deletion mouse model for Prader-Willi syndrome

Abstract

Prader-Willi syndrome (PWS) is an imprinted neurodevelopmental disease caused by a loss of paternal genes on chromosome 15q11-q13. It is characterized by cognitive impairments, developmental delay, sleep abnormalities, and hyperphagia often leading to obesity. Clinical research has shown that a lack of expression of SNORD116, a paternally expressed imprinted gene cluster that encodes multiple copies of a small nucleolar RNA (snoRNA) in both humans and mice, is most likely responsible for many PWS symptoms seen in humans. The majority of previous research using PWS preclinical models focused on characterization of the hyperphagic and metabolic phenotypes. However, a crucial understudied clinical phenotype is cognitive impairments and thus we investigated the learning and memory abilities using a model of PWS, with a heterozygous deletion in Snord116. We utilized the novel object recognition task, which doesn't require external motivation, or exhaustive swim training. Automated findings were further confirmed with manual scoring by a highly trained blinded investigator. We discovered deficits in Snord116+/- mutant mice in the novel object recognition, location memory and tone cue fear conditioning assays when compared to age-, sex- matched, littermate control Snord116+/+ mice. Further, we confirmed that despite physical neo-natal developmental delays, Snord116+/- mice had normal exploratory and motor abilities. These results show that the Snord116+/- deletion murine model is a valuable preclinical model for investigating learning and memory impairments in individuals with PWS without common confounding phenotypes.

Keywords: Animal model; Behavior; Cognitive; Genetics; Learning and memory; Neurodevelopment; Prader-Willi; Snord116.

Fig. 1.

Snord116 +/– displayed robust impairments on the novel object recognition (NOR) assay in two independent cohorts by manual and automated quantification measures. This version of NOR started with animal habituation to the open field testing arena for 30-min. After 24-hr, the mouse received another 10-min habituation session. Next, the subjects were given a 10-min familiarization session during which time spent sniffing each object was recorded. The objects were then cleaned and after a 1-hr inter-trial interval, the mouse was placed back into the arena with one familiar object and one novel object. (A) Snord116 +/– mice do not spend more time sniffing the novel object as compared to the familiar object. Snord116 +/+ litter- mates spend more time sniffing the novel object as compared to familiar object. (B) During the familiarization phase, manual scoring of objects revealed no left-right side bias in either Snord116 + /+ or Snord116 +/–. (C) Automated scoring via Noldus Ethovision 9.0XT illustrated Snord116 +/– mice do not spend more time in the zone defined around the novel object versus the zone defined around the familiar object, while Snord116 +/+ exhibit this typical object preference (p = 0.07) during the 5-min testing phase. (D) Similar to manual data, in the familiarization phase, automated tracings confirmed the absence of a left-right side bias in Snord116 +/+ and Snord116 +/– mice. (E) Manual scoring of the 5-min testing phase in Cohort 2 confirmed that Snord116 +/– mice do not spend more time sniffing the novel object as compared to the familiar object. Once again, Snord116 +/+ littermates spend more time sniffing the novel object when compared to familiar object. (F) Reproducing Cohort 1, during the familiarization phase, manual scoring of objects revealed no left-right side bias in Snord116+/+ and Snord116 +/–. (G) Similar to manual data and Cohort 1, automated scoring via Noldus Ethovision 9.0XT illustrated Snord116 + /– mice do not spend more time in the zone defined around the novel object versus the zone defined around the familiar object, while Snord116 +/+ exhibited a trend to toward novel object preference (p = 0.13) during the 5-min testing phase. (H) In the familiarization phase, automated tracings repeatedly confirmed the absence of a left-right side bias in either Snord116 +/+ or Snord116 +/– mice. *p < 0.05, novel object versus familiar object, paired t-test within genotype.

Fig. 2.

Snord116 +/− displayed impairments on the object location memory (OLM) task by manual and automated quantification measures. This version of the OLM used the same apparatus that was used for testing NOR and used the same habituation and familiarization procedures. However, the subject was exposed to different objects. Two identical objects were placed 12-cm away from the wall and 18-cm from each other) for a 10-min familiarization session. (A) Snord116 +/− mice do not spend more time sniffing the displaced object in the new location as compared to the object in the original location. Snord116 +/+ littermates spend more time sniffing the displaced object in the new location when compared to familiar location. (B) Automated scoring via Noldus Ethovision 9.0XT illustrated Snord116 +/− mice do not spend more time in the zone defined around the displaced object versus the zone defined around the familiar location, while Snord116 +/+ exhibit this object location memory (p = 0.09) during the 5-min testing phase.

Fig. 3.

Snord116 +/− displayed impairments in learning and memory in cued but not contextual fear conditioning. Learning and memory was evaluated by Pavlovian fear conditioning using two components, contextual cues and an auditory tone, with assessments of freezing time before and after the presentation of three tone-shock pairings. (A) Normal levels of freezing post-training indicate associations between tone and shock were made in all genotypes. In addition, both genotypes showed typical basal levels of freezing suggesting no confounds of deficits in sensory reactivity or pain threshold. (B) The 24-hr contextual component illustrated typical levels freezing, indicating no abnormal contextual learning and memory in either Snord116 + /+ or Snord116 +/− mice. (C) Cued conditioning freeze time 48-hr after initial training, before and after, the presentation of three tone-cues is illustrated. Snord116 +/− mice had lower freezing scores compared to Snord116 +/+ following the auditory reminder tone cue. *p < 0.05 versus Snord116 +/+ by student’s unpaired t-tests.

Fig. 4.

General motor ability and coordination in a novel open field and a balance beam assay were normal in Snord116 +/− mice. Activity and motor abilities are essential controls because hypolocomotion could confound the interpretation of result of interaction with objects, arena and object exploration, and sniffing and investigation times. Motor abilities were measured by (A) total distance traversed, (B) horizontal activity, (C) vertical activity and (D) time spent in the center of an arena over the course of a 30-min trial. Data are shown in 5-min time bins. (E) Summed activity over the course of the 30-min session highlights similar exploratory levels in Snord116 +/+ and Snord116 +/− mice. (F) Both groups showed longer latencies to cross the rod-shaped beams as they became thinner and more difficult to traverse. However, no genotype effects were observed in latency to traverse any of the various widths of beams, regardless of difficulty.

Fig. 5.

Anxiety-like behavioral testing illustrated inconsistent and confounded behavioral phenotypes in Snord116+/− mice. Anxiety-like behaviors were assessed in two gold standard assays: elevated plus-maze and light↔dark conflict. (A) During the elevated plus-maze, percent time spent on the open arm and (B) number of total arm entries (open+closed) were fewer in Snord116+/− mice as compared to Snord116+/ +. (C) In the light↔dark conflict assay, time spent in the dark chamber and (D) transitions between chambers did not differ between Snord116+/+ and Snord116+/− mice. *p < 0.05 versus Snord116+/+ by student’s unpaired t-test.