Peptidomimetic inhibitors targeting TrkB/PSD-95 signaling improves cognition and seizure outcomes in an Angelman Syndrome mouse model.

Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder with profoundly debilitating symptoms with no FDA-approved cure or therapeutic. Brain-derived neurotrophic factor (BDNF), and its receptor TrkB, have a well-established role as regulators of synaptic plasticity, dendritic outgrowth, dendritic spine formation and maintenance. Previously, we reported that the association of PSD-95 with TrkB is critical for intact BDNF signaling in the AS mouse model, as illustrated by attenuated PLCγ and PI3K signaling and intact MAPK pathway signaling. These data suggest that drugs tailored to enhance the TrkB-PSD-95 interaction may provide a novel approach for the treatment of AS and a variety of NDDs. To evaluate this critical interaction, we synthesized a class of high-affinity PSD-95 ligands that bind specifically to the PDZ3 domain of PSD-95, denoted as Syn3 peptidomimetic ligands. We evaluated Syn3 and its analog D-Syn3 (engineered using dextrorotary (D)-amino acids) in vivo using the Ube3a exon 2 deletion mouse model of AS. Following systemic administration of Syn3 and D-Syn3, we demonstrated improvement in the seizure domain of AS. Learning and memory using the novel object recognition assay also illustrated improved cognition following Syn3 and D-Syn3, along with restored long-term potentiation. Finally, D-Syn3 treated mice showed a partial rescue in motor learning. Neither Syn3 nor D-Syn3 improved gross exploratory locomotion deficits, nor gait impairments that have been well documented in the AS rodent models. These findings highlight the need for further investigation of this compound class as a potential therapeutic for AS and other genetic NDDs.

Surgical restabilization reduces the progression of post-traumatic osteoarthritis initiated by ACL rupture in mice.

OBJECTIVE: People who sustain joint injuries such as anterior cruciate ligament (ACL) rupture often develop post-traumatic osteoarthritis (PTOA). In human patients, ACL injuries are often treated with ACL reconstruction. However, it is still unclear how effective joint restabilization is for reducing the progression of PTOA. The goal of this study was to determine how surgical restabilization of a mouse knee joint following non-invasive ACL injury affects PTOA progression. DESIGN: In this study, 187 mice were subjected to non-invasive ACL injury or no injury. After injury, mice underwent restabilization surgery, sham surgery, or no surgery. Mice were then euthanized on day 14 or day 49 after injury/surgery. Functional analyses were performed at multiple time points to assess voluntary movement, gait, and pain. Knees were analyzed ex vivo with micro-computed tomography, RT-PCR, and whole-joint histology to assess articular cartilage degeneration, synovitis, and osteophyte formation. RESULTS: Both ACL injury and surgery resulted in loss of epiphyseal trabecular bone (-27-32%) and reduced voluntary movement at early time points. Joint restabilization successfully lowered OA score (-78% relative to injured at day 14, p < 0.0001), and synovitis scores (-37% relative to injured at day 14, p = 0.042), and diminished the formation of chondrophytes/osteophytes (-97% relative to injured at day 14, p < 0.001, -78% at day 49, p < 0.001). CONCLUSIONS: This study confirmed that surgical knee restabilization was effective at reducing articular cartilage degeneration and diminishing chondrophyte/osteophyte formation after ACL injury in mice, suggesting that these processes are largely driven by joint instability in this mouse model. However, restabilization was not able to mitigate the early inflammatory response and the loss of epiphyseal trabecular bone, indicating that these processes are independent of joint instability.

Hyperexcitability and translational phenotypes in a preclinical mouse model of SYNGAP1-Related Intellectual Disability.

Disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1-related intellectual disability (SRID). Without functional SynGAP1 protein, individuals are developmentally delayed and have prominent features of intellectual disability, motor impairments, and epilepsy. Over the past two decades, there have been numerous discoveries indicting the critical role of Syngap1. Several rodent models with a loss of Syngap1 have been engineered identifying precise roles in neuronal structure and function, as well as key biochemical pathways key for synapse integrity. Homozygous loss of SYNGAP1/Syngap1 is lethal. Heterozygous mutations of Syngap1 result in a broad range of behavioral phenotypes. Our in vivo functional data, using the original mouse model from the Huganir laboratory, corroborated behaviors including robust hyperactivity and deficits in learning and memory in young adults. Furthermore, we described impairments in the domain of sleep, characterized using neurophysiological data collected with wireless, telemetric electroencephalography (EEG). Syngap1+/- mice exhibited elevated spiking events and spike trains, in addition to elevated power, most notably in the delta power frequency. For the first time, we illustrated primary neurons from Syngap1+/- mice displayed increased network firing activity, greater bursts, and shorter inter-burst intervals between peaks by employing high density microelectrode arrays (HD-MEA). Our work bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate quantitative, translational biomarkers in vivo and in vitro that can be utilized for the development and efficacy assessment of targeted treatments for SRID.

Hyperexcitability and translational phenotypes in a preclinical model of SYNGAP1 mutations.

SYNGAP1 is a critical gene for neuronal development, synaptic structure, and function. Although rare, the disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1 -related intellectual disability. Without functional SynGAP1 protein, patients present with intellectual disability, motor impairments, and epilepsy. Previous work using mouse models with a variety of germline and conditional mutations has helped delineate SynGAP1's critical roles in neuronal structure and function, as well as key biochemical signaling pathways essential to synapse integrity. Homozygous loss of SYNGAP1 is embryonically lethal. Heterozygous mutations of SynGAP1 result in a broad range of phenotypes including increased locomotor activity, impaired working spatial memory, impaired cued fear memory, and increased stereotypic behavior. Our in vivo functional data, using the original germline mutation mouse line from the Huganir laboratory, corroborated robust hyperactivity and learning and memory deficits. Here, we describe impairments in the translational biomarker domain of sleep, characterized using neurophysiological data collected with wireless telemetric electroencephalography (EEG). We discovered Syngap1+/- mice exhibited elevated spike trains in both number and duration, in addition to elevated power, most notably in the delta power band. Primary neurons from Syngap1+/- mice displayed increased network firing activity, greater spikes per burst, and shorter inter-burst intervals between peaks using high density micro-electrode arrays (HD-MEA). This work is translational, innovative, and highly significant as it outlines functional impairments in Syngap1 mutant mice. Simultaneously, the work utilized untethered, wireless neurophysiology that can discover potential biomarkers of Syngap1 RI-D, for clinical trials, as it has done with other NDDs. Our work is substantial forward progress toward translational work for SynGAP1R-ID as it bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate multiple quantitative, translational biomarkers in vivo and in vitro for the development of treatments for SYNGAP1-related intellectual disability.

Hyperexcitability and translational phenotypes in a preclinical model of SYNGAP1 mutations.

SYNGAP1 is a critical gene for neuronal development, synaptic structure, and function. Although rare, the disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1-related intellectual disability. Without functional SynGAP1 protein, patients present with intellectual disability, motor impairments, and epilepsy. Previous work using mouse models with a variety of germline and conditional mutations has helped delineate SynGAP1's critical roles in neuronal structure and function, as well as key biochemical signaling pathways essential to synapse integrity. Homozygous loss of SYNGAP1 is embryonically lethal. Heterozygous mutations of SynGAP1 result in a broad range of phenotypes including increased locomotor activity, impaired working spatial memory, impaired cued fear memory, and increased stereotypic behavior. Our in vivo functional data, using the original germline mutation mouse line from the Huganir laboratory, corroborated robust hyperactivity and learning and memory deficits. Here, we describe impairments in the translational biomarker domain of sleep, characterized using neurophysiological data collected with wireless telemetric electroencephalography (EEG). We discovered Syngap1 +/- mice exhibited elevated spike trains in both number and duration, in addition to elevated power, most notably in the delta power band. Primary neurons from Syngap1 +/- mice displayed increased network firing activity, greater spikes per burst, and shorter inter-burst intervals between peaks using high density micro-electrode arrays (HD-MEA). This work is translational, innovative, and highly significant as it outlines functional impairments in Syngap1 mutant mice. Simultaneously, the work utilized untethered, wireless neurophysiology that can discover potential biomarkers of Syngap1R-ID, for clinical trials, as it has done with other NDDs. Our work is substantial forward progress toward translational work for SynGAP1R-ID as it bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate multiple quantitative, translational biomarkers in vivo and in vitro for the development of treatments for SYNGAP1-related intellectual disability.

Quantitative measures of motor development in Angelman syndrome.

Angelman Syndrome is a rare neurodevelopmental disorder characterized by developmental delay, lack of speech, seizures, intellectual disability, characteristic behavior, and movement disorders. Clinical gait analysis provides the opportunity for movement quantification to investigate an observed maladaptive change in gait pattern and offers an objective outcome of change. Pressure-sensor-based technology, inertial and activity monitoring, and instrumented gait analysis (IGA) were employed to define motor abnormalities in Angelman syndrome. Temporal-spatial gait parameters of persons with Angelman Syndrome (pwAS) show deficiencies in gait performance through walking speed, step length, step width, and walk ratio. pwAS walk with reduced step lengths, increased step width, and greater variability. Three-dimensional motion kinematics showed increased anterior pelvic tilt, hip flexion, and knee flexion. PwAS have a walk ratio more than two standard deviations below controls. Dynamic electromyography showed prolonged activation of knee extensors, which was associated with a decreased range of motion and the presence of hip flexion contractures. Use of multiple gait tracking modalities revealed that pwAS exhibit a change in gait pattern to a flexed knee gait pattern.  Cross-sectional studies of individuals with AS show a regression toward this maladaptive gait pattern over development in pwAS ages 4-11. PwAS unexpectedly did not have spasticity associated with change in gait pattern. Multiple quantitative measures of motor patterning may offer early biomarkers of gait decline consistent with critical periods of intervention, insight into appropriate management strategies, objective primary outcomes, and early indicators of adverse events.

Packed School Lunch Food Consumption: A Childhood Plate Waste Nutrient Analysis.

Packed school lunch consumption remains a sparsely studied aspect of childhood nutrition. Most American research focuses on in-school meals provided through the National School Lunch Program (NSLP). The wide variety of available in-home packed lunches are usually nutritionally inferior compared to the highly regulated in-school meals. The purpose of this study was to examine the consumption of home-packed lunches in a sample of elementary-grade children. Through weighing packed school lunches in a 3rd grade class, mean caloric intake was recorded at 67.3% (32.7% plate waste) of solid foods, while sugar-sweetened beverage intake reported a 94.6% intake. This study reported no significant consumption change in the macronutrient ratio. Intake showed significantly reduced levels of calories, sodium, cholesterol, and fiber from the home-packed lunches (p < 0.05). The packed school lunch consumption rates for this class were similar to those reported for the regulated in-school (hot) lunches. Calories, sodium, and cholesterol intake are within childhood meal recommendations. What is encouraging is that the children were not "filling up" on more processed foods at the expense of nutrient dense foods. Of concern is that these meals still fall short on several parameters, especially low fruit/vegetable intake and high simple sugar consumption. Overall, intake moved in a healthier direction compared to the meals packed from home.

Ictal vocalizations in the Scn1a+/- mouse model of Dravet syndrome.

OBJECTIVE: Ictal vocalizations have shown diagnostic utility in epilepsy patients. Audio recordings of seizures have also been used for seizure detection. The present study aimed to determine whether generalized tonic-clonic seizures in the Scn1a+/- mouse model of Dravet syndrome are associated with either audible mouse squeaks or ultrasonic vocalizations. METHODS: Acoustic recordings were captured from group-housed Scn1a+/- mice undergoing video-monitoring to quantify spontaneous seizure frequency. We generated audio clips (n = 129) during a generalized tonic-clonic seizure (GTCS) that included 30 seconds immediately prior to the GTCS (preictal) and 30 seconds following the conclusion of the seizure (postictal). Nonseizure clips (n = 129) were also exported from the acoustic recordings. A blinded reviewer manually reviewed the audio clips, and vocalizations were identified as either an audible (<20 kHz) mouse squeak or ultrasonic (>20 kHz). RESULTS: Spontaneous GTCS in Scn1a+/- mice were associated with a significantly higher number of total vocalizations. The number of audible mouse squeaks was significantly greater with GTCS activity. Nearly all (98%) the seizure clips contained ultrasonic vocalizations, whereas ultrasonic vocalizations were present in only 57% of nonseizure clips. The ultrasonic vocalizations emitted in the seizure clips were at a significantly higher frequency and were nearly twice as long in duration as those emitted in the nonseizure clips. Audible mouse squeaks were primarily emitted during the preictal phase. The greatest number of ultrasonic vocalizations was detected during the ictal phase. SIGNIFICANCE: Our study shows that ictal vocalizations are exhibited by Scn1a+/- mice. Quantitative audio analysis could be developed as a seizure detection tool for the Scn1a+/- mouse model of Dravet syndrome.

Transcriptional reprogramming restores UBE3A brain-wide and rescues behavioral phenotypes in an Angelman syndrome mouse model.

Angelman syndrome (AS) is a neurogenetic disorder caused by the loss of ubiquitin ligase E3A (UBE3A) gene expression in the brain. The UBE3A gene is paternally imprinted in brain neurons. Clinical features of AS are primarily due to the loss of maternally expressed UBE3A in the brain. A healthy copy of paternal UBE3A is present in the brain but is silenced by a long non-coding antisense transcript (UBE3A-ATS). Here, we demonstrate that an artificial transcription factor (ATF-S1K) can silence Ube3a-ATS in an adult mouse model of Angelman syndrome (AS) and restore endogenous physiological expression of paternal Ube3a. A single injection of adeno-associated virus (AAV) expressing ATF-S1K (AAV-S1K) into the tail vein enabled whole-brain transduction and restored UBE3A protein in neurons to ∼25% of wild-type protein. The ATF-S1K treatment was highly specific to the target site with no detectable inflammatory response 5 weeks after AAV-S1K administration. AAV-S1K treatment of AS mice showed behavioral rescue in exploratory locomotion, a task involving gross and fine motor abilities, similar to low ambulation and velocity in AS patients. The specificity and tolerability of a single injection of AAV-S1K therapy for AS demonstrate the use of ATFs as a promising translational approach for AS.

Unique Features of the Gut Microbiome Characterized in Animal Models of Angelman Syndrome.

A large subset of patients with Angelman syndrome (AS) suffer from concurrent gastrointestinal (GI) issues, including constipation, poor feeding, and reflux. AS is caused by the loss of ubiquitin ligase E3A (UBE3A) gene expression in the brain. Clinical features of AS, which include developmental delays, intellectual disability, microcephaly, and seizures, are primarily due to the deficient expression or function of the maternally inherited UBE3A allele. The association between neurodevelopmental delay and GI disorders is part of the increasing evidence suggesting a link between the brain and the gut microbiome via the microbiota-gut-brain axis. To investigate the associations between colonization of the gut microbiota in AS, we characterized the fecal microbiome in three animal models of AS involving maternal deletions of Ube3A, including mouse, rat, and pig, using 16S rRNA amplicon sequencing. Overall, we identified changes in bacterial abundance across all three animal models of AS. Specific bacterial groups were significantly increased across all animal models, including Lachnospiraceae Incertae sedis, Desulfovibrios sp., and Odoribacter, which have been correlated with neuropsychiatric disorders. Taken together, these findings suggest that specific changes to the local environment in the gut are driven by a Ube3a maternal deletion, unaffected by varying housing conditions, and are prominent and detectable across multiple small and large animal model species. These findings begin to uncover the underlying mechanistic causes of GI disorders in AS patients and provide future therapeutic options for AS patients. IMPORTANCE Angelman syndrome (AS)-associated gastrointestinal (GI) symptoms significantly impact quality of life in patients. In AS models in mouse, rat, and pig, AS animals showed impaired colonization of the gut microbiota compared to wild-type (healthy) control animals. Common changes in AS microbiomes across all three animal models may play a causal effect for GI symptoms and may help to identify ways to treat these comorbidities in patients in the future.

Touchscreen cognitive deficits, hyperexcitability and hyperactivity in males and females using two models of Cdkl5 deficiency.

Many neurodevelopmental disorders (NDDs) are the result of mutations on the X chromosome. One severe NDD resulting from mutations on the X chromosome is CDKL5 deficiency disorder (CDD). CDD is an epigenetic, X-linked NDD characterized by intellectual disability (ID), pervasive seizures and severe sleep disruption, including recurring hospitalizations. CDD occurs at a 4:1 ratio, with a female bias. CDD is driven by the loss of cyclin-dependent kinase-like 5 (CDKL5), a serine/threonine kinase that is essential for typical brain development, synapse formation and signal transmission. Previous studies focused on male subjects from animal models, likely to avoid the complexity of X mosaicism. For the first time, we report translationally relevant behavioral phenotypes in young adult (8-20 weeks) females and males with robust signal size, including impairments in learning and memory, substantial hyperactivity and increased susceptibility to seizures/reduced seizure thresholds, in both sexes, and in two models of CDD preclinical mice, one with a general loss-of-function mutation and one that is a patient-derived mutation.

Gait as a quantitative translational outcome measure in Angelman syndrome.

Angelman syndrome (AS) is a genetic neurodevelopmental disorder characterized by developmental delay, lack of speech, seizures, intellectual disability, hypotonia, and motor coordination deficits. Motor abilities are an important outcome measure in AS as they comprise a broad repertoire of metrics including ataxia, hypotonia, delayed ambulation, crouched gait, and poor posture, and motor dysfunction affects nearly every individual with AS. Guided by collaborative work with AS clinicians studying gait, the goal of this study was to perform an in-depth gait analysis using the automated treadmill assay, DigiGait. Our hypothesis is that gait presents a strong opportunity for a reliable, quantitative, and translational metric that can serve to evaluate novel pharmacological, dietary, and genetic therapies. In this study, we used an automated gait analysis system, in addition to standard motor behavioral assays, to evaluate components of motor, exploration, coordination, balance, and gait impairments across the lifespan in an AS mouse model. Our study demonstrated marked global motoric deficits in AS mice, corroborating previous reports. Uniquely, this is the first report of nuanced aberrations in quantitative spatial and temporal components of gait in AS mice compared to sex- and age-matched wildtype littermates followed longitudinally using metrics that are analogous in AS individuals. Our findings contribute evidence toward the use of nuanced motor outcomes (i.e., gait) as valuable and translationally powerful metrics for therapeutic development for AS, as well as other genetic neurodevelopmental syndromes. LAY SUMMARY: Movement disorders affect nearly every individual with Angelman Syndrome (AS). The most common motor problems include spasticity, ataxia of gait (observed in the majority of ambulatory individuals), tremor, and muscle weakness. This report focused on quantifying various spatial and temporal aspects of gait as a reliable, translatable outcome measure in a preclinical AS model longitudinally across development. By increasing the number of translational, reliable, functional outcome measures in our wheelhouse, we will create more opportunities for identifying and advancing successful medical interventions.

Emulating Near-Roadway Exposure to Traffic-Related Air Pollution via Real-Time Emissions from a Major Freeway Tunnel System.

Epidemiological and toxicological studies continue to demonstrate correlative and causal relationships between exposure to traffic-related air pollution and various metrics of adverse pulmonary, cardiovascular, and neurological health effects. The key challenge for in vivo studies is replicating real-world, near-roadway exposure dynamics in laboratory animal models that mimic true human exposures. The advantage of animal models is the accelerated time scales to show statistically significant physiological and/or behavioral response. This work describes a novel exposure facility adjacent to a major freeway tunnel system that provides a platform for real-time chronic exposure studies. The primary conclusion is that particulate matter (PM) concentrations at this facility are routinely well below the National Ambient Air Quality Standards (NAAQS), but studies completed to date still demonstrate significant neurological and cardiovascular effects. Internal combustion engines produce large numbers of ultrafine particles that contribute negligible mass to the atmosphere relative to NAAQS regulated PM2.5 but have high surface area and mobility in the body. It is posited here that current federal and state air quality standards are thus insufficient to fully protect human health, most notably the developing and aging brain, due to regulatory gaps for ultrafine particles.

Reconsidering animal models used to study autism spectrum disorder: Current state and optimizing future.

Neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD) and intellectual disability (ID), are pervasive, often lifelong disorders, lacking evidence-based interventions for core symptoms. With no established biological markers, diagnoses are defined by behavioral criteria. Thus, preclinical in vivo animal models of NDDs must be optimally utilized. For this reason, experts in the field of behavioral neuroscience convened a workshop with the goals of reviewing current behavioral studies, reports, and assessments in rodent models. Goals included: (a) identifying the maximal utility and limitations of behavior in animal models with construct validity; (b) providing recommendations for phenotyping animal models; and (c) guidelines on how in vivo models should be used and reported reliably and rigorously while acknowledging their limitations. We concluded by recommending minimal criteria for reporting in manuscripts going forward. The workshop elucidated a consensus of potential solutions to several problems, including revisiting claims made about animal model links to ASD (and related conditions). Specific conclusions included: mice (or other rodent or preclinical models) are models of the neurodevelopmental insult, not specifically any disorder (e.g., ASD); a model that perfectly recapitulates a disorder such as ASD is untenable; and greater attention needs be given to validation of behavioral testing methods, data analysis, and critical interpretation.

Early lysosome defects precede neurodegeneration with amyloid-ß and tau aggregation in NHE6-null rat brain.

Loss-of-function mutations in the X-linked endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome in males. Christianson syndrome involves endosome dysfunction leading to early cerebellar degeneration, as well as later-onset cortical and subcortical neurodegeneration, potentially including tau deposition as reported in post-mortem studies. In addition, there is reported evidence of modulation of amyloid-ß levels in experimental models wherein NHE6 expression was targeted. We have recently shown that loss of NHE6 causes defects in endosome maturation and trafficking underlying lysosome deficiency in primary mouse neurons in vitro. For in vivo studies, rat models may have an advantage over mouse models for the study of neurodegeneration, as rat brain can demonstrate robust deposition of endogenously-expressed amyloid-ß and tau in certain pathological states. Mouse models generally do not show the accumulation of insoluble, endogenously-expressed (non-transgenic) tau or amyloid-ß. Therefore, to study neurodegeneration in Christianson syndrome and the possibility of amyloid-ß and tau pathology, we generated an NHE6-null rat model of Christianson syndrome using CRISPR-Cas9 genome-editing. Here, we present the sequence of pathogenic events in neurodegenerating NHE6-null male rat brains across the lifespan. NHE6-null rats demonstrated an early and rapid loss of Purkinje cells in the cerebellum, as well as a more protracted neurodegenerative course in the cerebrum. In both the cerebellum and cerebrum, lysosome deficiency is an early pathogenic event, preceding autophagic dysfunction. Microglial and astrocyte activation also occur early. In the hippocampus and cortex, lysosome defects precede loss of pyramidal cells. Importantly, we subsequently observed biochemical and in situ evidence of both amyloid-ß and tau aggregation in the aged NHE6-null hippocampus and cortex (but not in the cerebellum). Tau deposition is widely distributed, including cortical and subcortical distributions. Interestingly, we observed tau deposition in both neurons and glia, as has been reported in Christianson syndrome post-mortem studies previously. In summary, this experimental model is among very few examples of a genetically modified animal that exhibits neurodegeneration with deposition of endogenously-expressed amyloid-ß and tau. This NHE6-null rat will serve as a new robust model for Christianson syndrome. Furthermore, these studies provide evidence for linkages between endolysosome dysfunction and neurodegeneration involving protein aggregations, including amyloid-ß and tau. Therefore these studies may provide insight into mechanisms of more common neurodegenerative disorders, including Alzheimer's disease and related dementias.

Developmental Exposure to a Human-Relevant Polychlorinated Biphenyl Mixture Causes Behavioral Phenotypes That Vary by Sex and Genotype in Juvenile Mice Expressing Human Mutations That Modulate Neuronal Calcium.

Polychlorinated biphenyls (PCBs) are putative environmental risks for neurodevelopmental disorders. Here, we tested two hypotheses: (1) developmental exposure to a human-relevant PCB mixture causes behavioral phenotypes relevant to neurodevelopmental disorders; and (2) expression of human mutations that dysregulate neuronal Ca2+ homeostasis influence sensitivity to behavioral effects of developmental PCB exposures. To test these hypotheses, we used mice that expressed a gain-of-function mutation (T4826I) in ryanodine receptor 1 (RYR1), the X-linked fragile X mental retardation 1 (FMR1) CGG repeat expansion or both mutations (double mutant; DM). Transgenic mice and wildtype (WT) mice were exposed to the MARBLES PCB mix at 0, 0.1, 1, and 6 mg/kg/day in the maternal diet throughout gestation and lactation. The MARBLES PCB mix simulates the relative proportions of the 12 most abundant PCB congeners found in the serum of pregnant women at increased risk for having a child with a neurodevelopmental disorder. We assessed ultrasonic vocalizations at postnatal day 7 (P7), spontaneous repetitive behaviors at P25-P30, and sociability at P27-P32. Developmental PCB exposure reduced ultrasonic vocalizations in WT litters in all dose groups, but had no effect on ultrasonic vocalizations in transgenic litters. Developmental PCB exposure significantly increased self-grooming and decreased sociability in WT males in the 0.1 mg/kg dose group, but had no effect on WT females in any dose group. Genotype alone influenced ultrasonic vocalizations, self-grooming and to a lesser extent sociability. Genotype alone also influenced effects of PCBs on sociability. PCB levels in the brain tissue of pups increased in a dose-dependent manner, but within any dose group did not differ between genotypes. In summary, developmental PCB exposure phenocopied social behavior phenotypes observed in mice expressing human mutations that modify intracellular Ca2+ dynamics, and expression of these mutations alleviated PCB effects on ultrasonic vocalizations and repetitive behavior, and modified the dose-response relationships and sex-dependent effects of PCB effects on social behavior. These findings suggest that: (1) developmental PCB exposure causes behavioral phenotypes that vary by sex and genotype; and (2) sex-specific responses to environmental factors may contribute to sex biases in the prevalence and/or severity of neurodevelopmental disorders.

Sex-specific acute and chronic neurotoxicity of acute diisopropylfluorophosphate (DFP)-intoxication in juvenile Sprague-Dawley rats.

Preclinical efforts to improve medical countermeasures against organophosphate (OP) chemical threat agents have largely focused on adult male models. However, age and sex have been shown to influence the neurotoxicity of repeated low-level OP exposure. Therefore, to determine the influence of sex and age on outcomes associated with acute OP intoxication, postnatal day 28 Sprague-Dawley male and female rats were exposed to the OP diisopropylfluorophosphate (DFP; 3.4 mg/kg, s.c.) or an equal volume of vehicle (∼80 µL saline, s.c.) followed by atropine sulfate (0.1 mg/kg, i.m.) and pralidoxime (2-PAM; 25 mg/kg, i.m.). Seizure activity was assessed during the first 4 h post-exposure using behavioral criteria and electroencephalographic (EEG) recordings. At 1 d post-exposure, acetylcholinesterase (AChE) activity was measured in cortical tissue, and at 1, 7, and 28 d post-exposure, brains were collected for neuropathologic analyses. At 1 month post-DFP, animals were analyzed for motor ability, learning and memory, and hippocampal neurogenesis. Acute DFP intoxication triggered more severe seizure behavior in males than females, which was supported by EEG recordings. DFP caused significant neurodegeneration and persistent microglial activation in numerous brain regions of both sexes, but astrogliosis occurred earlier and was more severe in males compared to females. DFP males and females exhibited pronounced memory deficits relative to sex-matched controls. In contrast, acute DFP intoxication altered hippocampal neurogenesis in males, but not females. These findings demonstrate that acute DFP intoxication triggers seizures in juvenile rats of both sexes, but the seizure severity varies by sex. Some, but not all, chronic neurotoxic outcomes also varied by sex. The spatiotemporal patterns of neurological damage suggest that microglial activation may be a more important factor than astrogliosis or altered neurogenesis in the pathogenesis of cognitive deficits in juvenile rats acutely intoxicated with OPs.

Excessive Laughter-like Vocalizations, Microcephaly, and Translational Outcomes in the Ube3a Deletion Rat Model of Angelman Syndrome.

Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder characterized by intellectual disabilities, motor and balance deficits, impaired communication, and a happy, excitable demeanor with frequent laughter. We sought to elucidate a preclinical outcome measure in male and female rats that addressed communication abnormalities of AS and other neurodevelopmental disorders in which communication is atypical and/or lack of speech is a core feature. We discovered, and herein report for the first time, excessive laughter-like 50 kHz ultrasonic emissions in the Ube3amat-/pat+ rat model of AS, which suggests an excitable, playful demeanor and elevated positive affect, similar to the demeanor of individuals with AS. Also in line with the AS phenotype, Ube3amat-/pat+ rats demonstrated aberrant social interactions with a novel partner, distinctive gait abnormalities, impaired cognition, an underlying LTP deficit, and profound reductions in brain volume. These unique, robust phenotypes provide advantages compared with currently available mouse models and will be highly valuable as outcome measures in the evaluation of therapies for AS.SIGNIFICANCE STATEMENT Angelman syndrome (AS) is a severe neurogenetic disorder for which there is no cure, despite decades of research using mouse models. This study used a recently developed rat model of AS to delineate disease-relevant outcome measures to facilitate therapeutic development. We found the rat to be a strong model of AS, offering several advantages over mouse models by exhibiting numerous AS-relevant phenotypes, including overabundant laughter-like vocalizations, reduced hippocampal LTP, and volumetric anomalies across the brain. These findings are unconfounded by detrimental motor abilities and background strain, issues plaguing mouse models. This rat model represents an important advancement in the field of AS, and the outcome metrics reported herein will be central to the therapeutic pipeline.

Insulin-like growth factor-2 does not improve behavioral deficits in mouse and rat models of Angelman Syndrome.

BACKGROUND: Angelman Syndrome (AS) is a rare neurodevelopmental disorder for which there is currently no cure or effective therapeutic. Since the genetic cause of AS is known to be dysfunctional expression of the maternal allele of ubiquitin protein ligase E3A (UBE3A), several genetic animal models of AS have been developed. Both the Ube3a maternal deletion mouse and rat models of AS reliably demonstrate behavioral phenotypes of relevance to AS and therefore offer suitable in vivo systems in which to test potential therapeutics. One promising candidate treatment is insulin-like growth factor-2 (IGF-2), which has recently been shown to ameliorate behavioral deficits in the mouse model of AS and improve cognitive abilities across model systems. METHODS: We used both the Ube3a maternal deletion mouse and rat models of AS to evaluate the ability of IGF-2 to improve electrophysiological and behavioral outcomes. RESULTS: Acute systemic administration of IGF-2 had an effect on electrophysiological activity in the brain and on a metric of motor ability; however the effects were not enduring or extensive. Additional metrics of motor behavior, learning, ambulation, and coordination were unaffected and IGF-2 did not improve social communication, seizure threshold, or cognition. LIMITATIONS: The generalizability of these results to humans is difficult to predict and it remains possible that dosing schemes (i.e., chronic or subchronic dosing), routes, and/or post-treatment intervals other than that used herein may show more efficacy. CONCLUSIONS: Despite a few observed effects of IGF-2, our results taken together indicate that IGF-2 treatment does not profoundly improve behavioral deficits in mouse or rat models of AS. These findings shed cautionary light on the potential utility of acute systemic IGF-2 administration in the treatment of AS.