Characterization of early markers of disease in the mouse model of mucopolysaccharidosis IIIB.

BACKGROUND: Mucopolysaccharidosis (MPS) IIIB, also known as Sanfilippo Syndrome B, is a devastating childhood disease. Unfortunately, there are currently no available treatments for MPS IIIB patients. Yet, animal models of lysosomal storage diseases have been valuable tools in identifying promising avenues of treatment. Enzyme replacement therapy, gene therapy, and bone marrow transplant have all shown efficacy in the MPS IIIB model systems. A ubiquitous finding across rodent models of lysosomal storage diseases is that the best treatment outcomes resulted from intervention prior to symptom onset. Therefore, the aim of the current study was to identify early markers of disease in the MPS IIIB mouse model as well as examine clinically-relevant behavioral domains not yet explored in this model. METHODS: Using the MPS IIIB mouse model, we explored early developmental trajectories of communication and gait, and later social behavior, fear-related startle and conditioning, and visual capabilities. In addition, we examined brain structure and function via magnetic resonance imaging and diffusion tensor imaging. RESULTS: We observed reduced maternal isolation-induced ultrasonic vocalizations in MPS IIIB mice relative to controls, as well as disruption in a number of the spectrotemporal features. MPS IIIB also exhibited disrupted thermoregulation during the first two postnatal weeks without any differences in body weight. The developmental trajectories of gait were largely normal. In early adulthood, we observed intact visual acuity and sociability yet a more submissive phenotype, increased aggressive behavior, and decreased social sniffing relative to controls. MPS IIIB mice showed greater inhibition of startle in response to a pretone with a decrease in overall startle response and reduced cued fear memory. MPS IIIB also weighed significantly more than controls throughout adulthood and showed larger whole brain volumes and normalized regional volumes with intact tissue integrity as measured with magnetic resonance and diffusion tensor imaging, respectively. CONCLUSIONS: Together, these results indicate disease markers are present as early as the first two weeks postnatal in this model. Further, this model recapitulates social, sensory and fear-related clinical features. Our study using a mouse model of MPS IIIB provides essential baseline information that will be useful in future evaluations of potential treatments.

Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits.

Phenotypic heterogeneity in monogenic neurodevelopmental disorders can arise from differential severity of variants underlying disease, but how distinct alleles drive variable disease presentation is not well understood. Here, we investigate missense mutations in DNA methyltransferase 3A (DNMT3A), a DNA methyltransferase associated with overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity. We generate a Dnmt3aP900L/+ mouse mimicking a mutation with mild to moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. P900L mutants exhibit core growth and behavioral phenotypes shared across models but show subtle epigenomic changes, while R878H mutants display extensive disruptions. We identify mutation-specific dysregulated genes that may contribute to variable disease severity. Shared transcriptomic disruption identified across mutations overlaps dysregulation observed in other developmental disorder models and likely drives common phenotypes. Together, our findings define central drivers of DNMT3A disorders and illustrate how variable epigenomic disruption contributes to phenotypic heterogeneity in neurodevelopmental disease.

A comprehensive assay of social motivation reveals sex-specific roles of autism-associated genes and oxytocin.

Social motivation is critical to the development of typical social functioning. Social motivation, specifically one or more of its components (e.g., social reward seeking or social orienting), could be relevant for understanding phenotypes related to autism. We developed a social operant conditioning task to quantify effort to access a social partner and concurrent social orienting in mice. We established that mice will work for access to a social partner, identified sex differences, and observed high test-retest reliability. We then benchmarked the method with two test-case manipulations. Shank3B mutants exhibited reduced social orienting and failed to show social reward seeking. Oxytocin receptor antagonism decreased social motivation, consistent with its role in social reward circuitry. Overall, we believe that this method provides a valuable addition to the assessment of social phenotypes in rodent models of autism and the mapping of potentially sex-specific social motivation neural circuits.

Extensive characterization of a Williams syndrome murine model shows Gtf2ird1-mediated rescue of select sensorimotor tasks, but no effect on enhanced social behavior.

Williams syndrome is a rare neurodevelopmental disorder exhibiting cognitive and behavioral abnormalities, including increased social motivation, risk of anxiety and specific phobias along with perturbed motor function. Williams syndrome is caused by a microdeletion of 26-28 genes on chromosome 7, including GTF2IRD1, which encodes a transcription factor suggested to play a role in the behavioral profile of Williams syndrome. Duplications of the full region also lead to frequent autism diagnosis, social phobias and language delay. Thus, genes in the region appear to regulate social motivation in a dose-sensitive manner. A "complete deletion" mouse, heterozygously eliminating the syntenic Williams syndrome region, has been deeply characterized for cardiac phenotypes, but direct measures of social motivation have not been assessed. Furthermore, the role of Gtf2ird1 in these behaviors has not been addressed in a relevant genetic context. Here, we have generated a mouse overexpressing Gtf2ird1, which can be used both to model duplication of this gene alone and to rescue Gtf2ird1 expression in the complete deletion mice. Using a comprehensive behavioral pipeline and direct measures of social motivation, we provide evidence that the Williams syndrome critical region regulates social motivation along with motor and anxiety phenotypes, but that Gtf2ird1 complementation is not sufficient to rescue most of these traits, and duplication does not decrease social motivation. However, Gtf2ird1 complementation does rescue light-aversive behavior and performance on select sensorimotor tasks, perhaps indicating a role for this gene in sensory processing or integration.

Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits.

Phenotypic heterogeneity is a common feature of monogenic neurodevelopmental disorders that can arise from differential severity of missense variants underlying disease, but how distinct alleles impact molecular mechanisms to drive variable disease presentation is not well understood. Here, we investigate missense mutations in the DNA methyltransferase DNMT3A associated with variable overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity in neurodevelopmental disease. We generate a DNMT3A P900L/+ mouse model mimicking a disease mutation with mild-to-moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. We show that the P900L mutation leads to disease-relevant overgrowth, obesity, and social deficits shared across DNMT3A disorder models, while the R878H mutation causes more extensive epigenomic disruption leading to differential dysregulation of enhancers elements. We identify distinct gene sets disrupted in each mutant which may contribute to mild or severe disease, and detect shared transcriptomic disruption that likely drives common phenotypes across affected individuals. Finally, we demonstrate that core gene dysregulation detected in DNMT3A mutant mice overlaps effects in other developmental disorder models, highlighting the importance of DNMT3A-deposited methylation in neurodevelopment. Together, these findings define central drivers of DNMT3A disorders and illustrate how variable disruption of transcriptional mechanisms can drive the spectrum of phenotypes in neurodevelopmental disease.

Extensive characterization of a Williams Syndrome murine model shows Gtf2ird1 -mediated rescue of select sensorimotor tasks, but no effect on enhanced social behavior.

Williams Syndrome is a rare neurodevelopmental disorder exhibiting cognitive and behavioral abnormalities, including increased social motivation, risk of anxiety and specific phobias along with perturbed motor function. Williams Syndrome is caused by a microdeletion of 26-28 genes on chromosome 7, including GTF2IRD1 , which encodes a transcription factor suggested to play a role in the behavioral profile of Williams Syndrome. Duplications of the full region also lead to frequent autism diagnosis, social phobias, and language delay. Thus, genes in the region appear to regulate social motivation in a dose-sensitive manner. A 'Complete Deletion' mouse, heterozygously eliminating the syntenic Williams Syndrome region, has been deeply characterized for cardiac phenotypes, but direct measures of social motivation have not been assessed. Furthermore, the role of Gtf2ird1 in these behaviors has not been addressed in a relevant genetic context. Here, we have generated a mouse overexpressing Gtf2ird1 , which can be used both to model duplication of this gene alone and to rescue Gtf2ird1 expression in the Complete Deletion mice. Using a comprehensive behavioral pipeline and direct measures of social motivation, we provide evidence that the Williams Syndrome Critical Region regulates social motivation along with motor and anxiety phenotypes, but that Gtf2ird1 complementation is not sufficient to rescue most of these traits, and duplication does not decrease social motivation. However, Gtf2ird1 complementation does rescue light-aversive behavior and performance on select sensorimotor tasks, perhaps indicating a role for this gene in sensory processing or integration.

Perinatal oxycodone exposure causes long-term sex-dependent changes in weight trajectory and sensory processing in adult mice.

RATIONALE: In utero opioid exposure is associated with lower weight and a neonatal opioid withdrawal syndrome (NOWS) at birth, along with longer-term adverse neurodevelopmental outcomes and mood disorders. While NOWS is sometimes treated with continued opioids, clinical studies have not addressed if long-term neurobehavioral outcomes are worsened with continued postnatal exposure to opioids. In addition, pre-clinical studies comparing in utero only opioid exposure to continued post-natal opioid administration for withdrawal mitigation are lacking. OBJECTIVES: Here, we sought to understand the impact of continued postnatal opioid exposure on long term behavioral consequences. METHODS: We implemented a rodent perinatal opioid exposure model of oxycodone (Oxy) exposure that included Oxy exposure until birth (short Oxy) and continued postnatal opioid exposure (long Oxy) spanning gestation through birth and lactation. RESULTS: Short Oxy exposure was associated with a sex-specific increase in weight gain trajectory in adult male mice. Long Oxy exposure caused an increased weight gain trajectory in adult males and alterations in nociceptive processing in females. Importantly, there was no evidence of long-term social behavioral deficits, anxiety, hyperactivity, or memory deficits following short or long Oxy exposure. CONCLUSIONS: Our findings suggest that offspring with prolonged opioid exposure experienced some long-term sequelae compared to pups with opioid cessation at birth. These results highlight the potential long-term consequences of opioid administration as a mitigation strategy for clinical NOWS symptomology and suggest alternatives should be explored.

A MYT1L syndrome mouse model recapitulates patient phenotypes and reveals altered brain development due to disrupted neuronal maturation.

Human genetics have defined a new neurodevelopmental syndrome caused by loss-of-function mutations in MYT1L, a transcription factor known for enabling fibroblast-to-neuron conversions. However, how MYT1L mutation causes intellectual disability, autism, ADHD, obesity, and brain anomalies is unknown. Here, we developed a Myt1l haploinsufficient mouse model that develops obesity, white-matter thinning, and microcephaly, mimicking common clinical phenotypes. During brain development we discovered disrupted gene expression, mediated in part by loss of Myt1l gene-target activation, and identified precocious neuronal differentiation as the mechanism for microcephaly. In contrast, in adults we discovered that mutation results in failure of transcriptional and chromatin maturation, echoed in disruptions in baseline physiological properties of neurons. Myt1l haploinsufficiency also results in behavioral anomalies, including hyperactivity, muscle weakness, and social alterations, with more severe phenotypes in males. Overall, our findings provide insight into the mechanistic underpinnings of this disorder and enable future preclinical studies.

Ontogenetic Oxycodone Exposure Affects Early Life Communicative Behaviors, Sensorimotor Reflexes, and Weight Trajectory in Mice.

Nationwide, opioid misuse among pregnant women has risen four-fold from 1999 to 2014, with commensurate increase in neonates hospitalized for neonatal abstinence syndrome (NAS). NAS occurs when a fetus exposed to opioids in utero goes into rapid withdrawal after birth. NAS treatment via continued post-natal opioid exposure has been suggested to worsen neurodevelopmental outcomes. We developed a novel model to characterize the impact of in utero and prolonged post-natal oxycodone (Oxy) exposure on early behavior and development. Via subcutaneous pump implanted before breeding, C57BL/6J dams were infused with Oxy at 10 mg/kg/day from conception through pup-weaning. At birth, in utero oxy-exposed pups were either cross-fostered (paired with non-Oxy exposed dams) to model opioid abstinence (in utero Oxy) or reared by their biological dams still receiving Oxy to model continued post-natal opioid exposure (prolonged Oxy). Offspring from vehicle-exposed dams served as cross-fostered (in utero Veh) or biologically reared (prolonged Veh) controls. In utero Oxy exposure resulted in sex-dependent weight reductions and altered spectrotemporal features of isolation-induced ultrasonic vocalization (USV). Meanwhile, prolonged Oxy pups exhibited reduced weight and sex-differential delays in righting reflex. Specifically, prolonged Oxy female offspring exhibited increased latency to righting. Prolonged Oxy pups also showed decreases in number of USV calls and changes to spectrotemporal USV features. Overall, ontogenetic Oxy exposure was associated with impaired attainment of gross and sensorimotor milestones, as well as alterations in communication and affective behaviors, indicating a need for therapeutic interventions. The model developed here will enable studies of withdrawal physiology and opioid-mediated mechanisms underlying these neurodevelopmental deficits.

Functions of Gtf2i and Gtf2ird1 in the developing brain: transcription, DNA binding and long-term behavioral consequences.

Gtf2ird1 and Gtf2i are two transcription factors (TFs) among the 28 genes deleted in Williams syndrome, and prior mouse models of each TF show behavioral phenotypes. Here we identify their genomic binding sites in the developing brain and test for additive effects of their mutation on transcription and behavior. GTF2IRD1 binding targets were enriched for transcriptional and chromatin regulators and mediators of ubiquitination. GTF2I targets were enriched for signal transduction proteins, including regulators of phosphorylation and WNT. Both TFs are highly enriched at promoters, strongly overlap CTCF binding and topological associating domain boundaries and moderately overlap each other, suggesting epistatic effects. Shared TF targets are enriched for reactive oxygen species-responsive genes, synaptic proteins and transcription regulators such as chromatin modifiers, including a significant number of highly constrained genes and known ASD genes. We next used single and double mutants to test whether mutating both TFs will modify transcriptional and behavioral phenotypes of single Gtf2ird1 mutants, though with the caveat that our Gtf2ird1 mutants, like others previously reported, do produce low levels of a truncated protein product. Despite little difference in DNA binding and transcriptome-wide expression, homozygous Gtf2ird1 mutation caused balance, marble burying and conditioned fear phenotypes. However, mutating Gtf2i in addition to Gtf2ird1 did not further modify transcriptomic or most behavioral phenotypes, suggesting Gtf2ird1 mutation alone was sufficient for the observed phenotypes.

The trajectory of gait development in mice.

OBJECTIVE: Gait irregularities are prevalent in neurodevelopmental disorders (NDDs). However, there is a paucity of information on gait phenotypes in NDD experimental models. This is in part due to the lack of understanding of the normal developmental trajectory of gait maturation in the mouse. MATERIALS AND METHODS: Using the DigiGait system, we have developed a quantitative, standardized, and reproducible assay of developmental gait metrics in commonly used mouse strains that can be added to the battery of mouse model phenotyping. With this assay, we characterized the trajectory of gait in the developing C57BL/6J and FVB/AntJ mouse lines. RESULTS: In both lines, a mature stride consisted of 40% swing and 60% stance in the forelimbs, which mirrors the mature human stride. In C57BL/6J mice, developmental trajectories were observed for stance width, paw overlap distance, braking and propulsion time, rate of stance loading, peak paw area, and metrics of intraindividual variability. In FVB/AntJ mice, developmental trajectories were observed for percent shared stance, paw overlap distance, rate of stance loading, and peak paw area, although in different directions than C57 mice. By accounting for the impact of body length on stride measurements, we demonstrate the importance of considering body length when interpreting gait metrics. CONCLUSION: Overall, our results show that aspects of mouse gait development parallel a timeline of normal human gait development, such as the percent of stride that is stance phase and swing phase. This study may be used as a standard reference for developmental gait phenotyping of murine models, such as models of neurodevelopmental disease.

Examining the Reversibility of Long-Term Behavioral Disruptions in Progeny of Maternal SSRI Exposure.

Serotonergic dysregulation is implicated in numerous psychiatric disorders. Serotonin plays widespread trophic roles during neurodevelopment; thus perturbations to this system during development may increase risk for neurodevelopmental disorders. Epidemiological studies have examined association between selective serotonin reuptake inhibitor (SSRI) treatment during pregnancy and increased autism spectrum disorder (ASD) risk in offspring. It is unclear from these studies whether ASD susceptibility is purely related to maternal psychiatric diagnosis, or if treatment poses additional risk. We sought to determine whether maternal SSRI treatment alone or in combination with genetically vulnerable background was sufficient to induce offspring behavior disruptions relevant to ASD. We exposed C57BL/6J or Celf6+/- mouse dams to fluoxetine (FLX) during different periods of gestation and lactation and characterized offspring on tasks assessing social communicative interaction and repetitive behavior patterns including sensory sensitivities. We demonstrate robust reductions in pup ultrasonic vocalizations (USVs) and alterations in social hierarchy behaviors, as well as perseverative behaviors and tactile hypersensitivity. Celf6 mutant mice demonstrate social communicative deficits and perseverative behaviors, without further interaction with FLX. FLX re-exposure in adulthood ameliorates the tactile hypersensitivity yet exacerbates the dominance phenotype. This suggests acute deficiencies in serotonin levels likely underlie the abnormal responses to sensory stimuli, while the social alterations are instead due to altered development of social circuits. These findings indicate maternal FLX treatment, independent of maternal stress, can induce behavioral disruptions in mammalian offspring, thus contributing to our understanding of the developmental role of the serotonin system and the possible risks to offspring of SSRI treatment during pregnancy.