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.

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.

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.

Functional rescue in an Angelman syndrome model following treatment with lentivector transduced hematopoietic stem cells.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by impaired communication skills, ataxia, motor and balance deficits, intellectual disabilities, and seizures. The genetic cause of AS is the neuronal loss of UBE3A expression in the brain. A novel approach, described here, is a stem cell gene therapy which uses lentivector-transduced hematopoietic stem and progenitor cells to deliver functional UBE3A to affected cells. We have demonstrated both the prevention and reversal of AS phenotypes upon transplantation and engraftment of human CD34+ cells transduced with a Ube3a lentivector in a novel immunodeficient Ube3amat-/pat+ IL2rg-/y mouse model of AS. A significant improvement in motor and cognitive behavioral assays as well as normalized delta power measured by electroencephalogram was observed in neonates and adults transplanted with the gene modified cells. Human hematopoietic profiles observed in the lymphoid organs by detection of human immune cells were normal. Expression of UBE3A was detected in the brains of the adult treatment group following immunohistochemical staining illustrating engraftment of the gene-modified cells expressing UBE3A in the brain. As demonstrated with our data, this stem cell gene therapy approach offers a promising treatment strategy for AS, not requiring a critical treatment window.

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.

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.

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.

Sexually dimorphic neuroanatomical differences relate to ASD-relevant behavioral outcomes in a maternal autoantibody mouse model.

Immunoglobulin G (IgG) autoantibodies reactive to fetal brain proteins in mothers of children with ASD have been described by several groups. To understand their pathologic significance, we developed a mouse model of maternal autoantibody related ASD (MAR-ASD) utilizing the peptide epitopes from human autoantibody reactivity patterns. Male and female offspring prenatally exposed to the salient maternal autoantibodies displayed robust deficits in social interactions and increased repetitive self-grooming behaviors as juveniles and adults. In the present study, neuroanatomical differences in adult MAR-ASD and control offspring were assessed via high-resolution ex vivo magnetic resonance imaging (MRI) at 6 months of age. Of interest, MAR-ASD mice displayed significantly larger total brain volume and of the 159 regions examined, 31 were found to differ significantly in absolute volume (mm3) at an FDR of <5%. Specifically, the absolute volumes of several white matter tracts, cortical regions, and basal nuclei structures were significantly increased in MAR-ASD animals. These phenomena were largely driven by female MAR-ASD offspring, as no significant differences were seen with either absolute or relative regional volume in male MAR-ASD mice. However, structural covariance analysis suggests network-level desynchronization in brain volume in both male and female MAR-ASD mice. Additionally, preliminary correlational analysis with behavioral data relates that volumetric increases in numerous brain regions of MAR-ASD mice were correlated with social interaction and repetitive self-grooming behaviors in a sex-specific manner. These results demonstrate significant sex-specific effects in brain size, regional relationships, and behavior for offspring prenatally exposed to MAR-ASD autoantibodies relative to controls.

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.

Imprinting effects of UBE3A loss on synaptic gene networks and Wnt signaling pathways.

Ubiquitin E3 ligase 3A (UBE3A) encodes an E3 ubiquitin ligase whose loss from the maternal allele causes the neurodevelopmental disorder Angelman syndrome (AS). Previous studies of UBE3A function have not examined full Ube3a deletion in mouse, the complexity of imprinted gene networks in brain nor the molecular basis of systems-level cognitive dysfunctions in AS. We therefore utilized a systems biology approach to elucidate how UBE3A loss impacts the early postnatal brain in a novel CRISPR/Cas9-engineered rat Angelman model of a complete Ube3a deletion. Strand-specific transcriptome analysis of offspring from maternally or paternally inherited Ube3a deletions revealed the expected parental expression patterns of Ube3a sense and antisense transcripts by postnatal day 2 (P2) in hypothalamus and day 9 (P9) in cortex, compared to wild-type littermates. The dependency of genome-wide effects on parent-of-origin, Ube3a genotype and time (P2 and P9) was investigated through transcriptome (RNA sequencing of cortex and hypothalamus) and methylome (whole-genome bisulfite sequencing of hypothalamus). Weighted gene co-expression and co-methylation network analyses identified co-regulated networks in maternally inherited Ube3a deletion offspring enriched in postnatal developmental processes including Wnt signaling, synaptic regulation, neuronal and glial functions, epigenetic regulation, ubiquitin, circadian entrainment and splicing. Furthermore, we showed that loss of the paternal Ube3a antisense transcript resulted in both unique and overlapping dysregulated gene pathways with maternal loss, predominantly at the level of differential methylation. Together, these results provide a holistic examination of the molecular impacts of UBE3A loss in brain, supporting the existence of interactive epigenetic networks between maternal and paternal transcripts at the Ube3a locus.

Autism-specific maternal autoantibodies produce behavioral abnormalities in an endogenous antigen-driven mouse model of autism.

Immune dysregulation has been noted consistently in individuals with autism spectrum disorder (ASD) and their families, including the presence of autoantibodies reactive to fetal brain proteins in nearly a quarter of mothers of children with ASD versus <1% in mothers of typically developing children. Our lab recently identified the peptide epitope sequences on seven antigenic proteins targeted by these maternal autoantibodies. Through immunization with these peptide epitopes, we have successfully created an endogenous, antigen-driven mouse model that ensures a constant exposure to the salient autoantibodies throughout gestation in C57BL/6J mice. This exposure more naturally mimics what is observed in mothers of children with ASD. Male and female offspring were tested using a comprehensive sequence of behavioral assays, as well as measures of health and development highly relevant to ASD. We found that MAR-ASD male and female offspring had significant alterations in development and social interactions during dyadic play. Although 3-chambered social approach was not significantly different, fewer social interactions with an estrous female were noted in the adult male MAR-ASD animals, as well as reduced vocalizations emitted in response to social cues with robust repetitive self-grooming behaviors relative to saline treated controls. The generation of MAR-ASD-specific epitope autoantibodies in female mice prior to breeding created a model that demonstrates for the first time that ASD-specific antigen-induced maternal autoantibodies produced alterations in a constellation of ASD-relevant behaviors.

Persistent behavior deficits, neuroinflammation, and oxidative stress in a rat model of acute organophosphate intoxication.

Current medical countermeasures for organophosphate (OP)-induced status epilepticus (SE) are not effective in preventing long-term morbidity and there is an urgent need for improved therapies. Rat models of acute intoxication with the OP, diisopropylfluorophosphate (DFP), are increasingly being used to evaluate therapeutic candidates for efficacy in mitigating the long-term neurologic effects associated with OP-induced SE. Many of these therapeutic candidates target neuroinflammation and oxidative stress because of their implication in the pathogenesis of persistent neurologic deficits associated with OP-induced SE. Critical to these efforts is the rigorous characterization of the rat DFP model with respect to outcomes associated with acute OP intoxication in humans, which include long-term electroencephalographic, neurobehavioral, and neuropathologic effects, and their temporal relationship to neuroinflammation and oxidative stress. To address these needs, we examined a range of outcomes at later times post-exposure than have previously been reported for this model. Adult male Sprague-Dawley rats were given pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to administration of DFP (4 mg/kg, sc), which was immediately followed by atropine sulfate (2 mg/kg, im) and pralidoxime (25 mg/kg, im). This exposure paradigm triggered robust electroencephalographic and behavioral seizures that rapidly progressed to SE lasting several hours in 90% of exposed animals. Animals that survived DFP-induced SE (~70%) exhibited spontaneous recurrent seizures and hyperreactive responses to tactile stimuli over the first 2 months post-exposure. Performance in the elevated plus maze, open field, and Pavlovian fear conditioning tests indicated that acute DFP intoxication reduced anxiety-like behavior and impaired learning and memory at 1 and 2 months post-exposure in the absence of effects on general locomotor behavior. Immunohistochemical analyses revealed significantly increased expression of biomarkers of reactive astrogliosis, microglial activation and oxidative stress in multiple brain regions at 1 and 2 months post-DFP, although there was significant spatiotemporal heterogeneity across these endpoints. Collectively, these data largely support the relevance of the rat model of acute DFP intoxication as a model for acute OP intoxication in the human, and support the hypothesis that neuroinflammation and/or oxidative stress represent potential therapeutic targets for mitigating the long-term neurologic sequelae of acute OP intoxication.

Pathogenic WDFY3 variants cause neurodevelopmental disorders and opposing effects on brain size.

The underpinnings of mild to moderate neurodevelopmental delay remain elusive, often leading to late diagnosis and interventions. Here, we present data on exome and genome sequencing as well as array analysis of 13 individuals that point to pathogenic, heterozygous, mostly de novo variants in WDFY3 (significant de novo enrichment P = 0.003) as a monogenic cause of mild and non-specific neurodevelopmental delay. Nine variants were protein-truncating and four missense. Overlapping symptoms included neurodevelopmental delay, intellectual disability, macrocephaly, and psychiatric disorders (autism spectrum disorders/attention deficit hyperactivity disorder). One proband presented with an opposing phenotype of microcephaly and the only missense-variant located in the PH-domain of WDFY3. Findings of this case are supported by previously published data, demonstrating that pathogenic PH-domain variants can lead to microcephaly via canonical Wnt-pathway upregulation. In a separate study, we reported that the autophagy scaffolding protein WDFY3 is required for cerebral cortical size regulation in mice, by controlling proper division of neural progenitors. Here, we show that proliferating cortical neural progenitors of human embryonic brains highly express WDFY3, further supporting a role for this molecule in the regulation of prenatal neurogenesis. We present data on Wnt-pathway dysregulation in Wdfy3-haploinsufficient mice, which display macrocephaly and deficits in motor coordination and associative learning, recapitulating the human phenotype. Consequently, we propose that in humans WDFY3 loss-of-function variants lead to macrocephaly via downregulation of the Wnt pathway. In summary, we present WDFY3 as a novel gene linked to mild to moderate neurodevelopmental delay and intellectual disability and conclude that variants putatively causing haploinsufficiency lead to macrocephaly, while an opposing pathomechanism due to variants in the PH-domain of WDFY3 leads to microcephaly.

Neuronal overexpression of Ube3a isoform 2 causes behavioral impairments and neuroanatomical pathology relevant to 15q11.2-q13.3 duplication syndrome.

Maternally derived copy number gains of human chromosome 15q11.2-q13.3 (Dup15q syndrome or Dup15q) cause intellectual disability, epilepsy, developmental delay, hypotonia, speech impairments, and minor dysmorphic features. Dup15q syndrome is one of the most common and penetrant chromosomal abnormalities observed in individuals with autism spectrum disorder (ASD). Although ∼40 genes are located in the 15q11.2-q13.3 region, overexpression of the ubiquitin-protein E3A ligase (UBE3A) gene is thought to be the predominant molecular cause of the phenotypes observed in Dup15q syndrome. The UBE3A gene demonstrates maternal-specific expression in neurons and loss of maternal UBE3A causes Angelman syndrome, a neurodevelopmental disorder with some overlapping neurological features to Dup15q. To directly test the hypothesis that overexpression of UBE3A is an important underlying molecular cause of neurodevelopmental dysfunction, we developed and characterized a mouse overexpressing Ube3a isoform 2 in excitatory neurons. Ube3a isoform 2 is conserved between mouse and human and known to play key roles in neuronal function. Transgenic mice overexpressing Ube3a isoform 2 in excitatory forebrain neurons exhibited increased anxiety-like behaviors, learning impairments, and reduced seizure thresholds. However, these transgenic mice displayed normal social approach, social interactions, and repetitive motor stereotypies that are relevant to ASD. Reduced forebrain, hippocampus, striatum, amygdala, and cortical volume were also observed. Altogether, these findings show neuronal overexpression of Ube3a isoform 2 causes phenotypes translatable to neurodevelopmental disorders.

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

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.

Behavioral and neuroanatomical approaches in models of neurodevelopmental disorders: opportunities for translation.

PURPOSE OF REVIEW: This review highlights the invaluable contribution of in-vivo rodent models in dissecting the underlying neurobiology for numerous neurodevelopmental disorders. Currently, models are routinely generated with precision genomics and characterized for research on neurodevelopmental disorders. In order to impact translation, outcome measures that are translationally relevant are essential. This review emphasizes the importance of accurate neurobehavioral and anatomical analyses. RECENT FINDINGS: Numerous well validated assays for testing alterations across behavioral domains with sensitivity and throughput have become important tools for studying the effects of genetic mutations on neurodevelopment. Recent work has highlighted relationships and links between behavioral outcomes and various anatomical metrics from neuroimaging via magnetic resonance. These readouts are biological markers and outcome measures for translational research and will be have important roles for genetic or pharmacologic intervention strategies. SUMMARY: Combinatorial approaches that leverage translationally relevant behavior and neuroanatomy can be used to develop a platform for assessment of cutting edge preclinical models. Reliable, robust behavioral phenotypes in preclinical model systems, with clustering of brain disease will lead to well informed, precise biochemical mechanistic hypotheses. Ultimately, these steadfast workhorse techniques will accelerate the progress of developing and testing targeted treatments for multiple neurodevelopmental disorders.

Persistent neuroinflammation and cognitive impairment in a rat model of acute diisopropylfluorophosphate intoxication.

BACKGROUND: Acute intoxication with organophosphorus (OP) cholinesterase inhibitors can trigger convulsions that progress to life-threatening status epilepticus. Survivors face long-term morbidity including mild-to-severe decline in memory. It is posited that neuroinflammation plays a key role in the pathogenesis of OP-induced neuropsychiatric deficits. Rigorous testing of this hypothesis requires preclinical models that recapitulate relevant phenotypic outcomes. Here, we describe a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP) that exhibits persistent neuroinflammation and cognitive impairment. METHODS: Neuroinflammation, neurodegeneration, and cognitive function were compared in adult male Sprague Dawley rats injected with an acutely toxic dose of DFP vs. vehicle controls at multiple time points up to 36 days post-exposure. Neuroinflammation was quantified using immunohistochemical biomarkers of microglia (ionized calcium-binding adapter molecule 1, IBA1) and activated astrocytes (glial fibrillary acidic protein, GFAP) and positron emission tomography (PET) imaging of [11C]-(R)-PK11195, a ligand for the 18-kDa mitochondrial membrane translocator protein (TSPO). FluoroJade-B staining was used to assess neurodegeneration; Pavlovian conditioning, to assess cognitive function. RESULTS: Animals exhibited moderate-to-severe seizures within minutes of DFP injection that continued for up to 6 h post-injection. As indicated by IBA1 and GFAP immunoreactivity and by PET imaging of TSPO, acute DFP intoxication triggered neuroinflammation in the hippocampus and cortex during the first 3 days that peaked at 7 days and persisted to 21 days post-exposure in most animals. Neurodegeneration was detected in multiple brain regions from 1 to 14 days post-exposure. All DFP-intoxicated animals exhibited significant deficits in contextual fear conditioning at 9 and 20 days post-exposure compared to vehicle controls. Whole-brain TSPO labeling positively correlated with seizure severity score, but did not correlate with performance in the contextual fear-conditioning task. CONCLUSIONS: We describe a preclinical model in which acute DFP intoxication causes seizures, persistent neuroinflammation, neurodegeneration, and memory impairment. The extent of the neuroinflammatory response is influenced by seizure severity. However, the observation that a subset of animals with moderate seizures and minimal TSPO labeling exhibited cognitive deficits comparable to those of animals with severe seizures and significant TSPO labeling suggests that DFP may impair learning and memory circuitry via mechanisms independent of seizures or neuroinflammation.

Behavioural phenotyping assays for mouse models of autism.

Autism is a heterogeneous neurodevelopmental disorder of unknown aetiology that affects 1 in 100-150 individuals. Diagnosis is based on three categories of behavioural criteria: abnormal social interactions, communication deficits and repetitive behaviours. Strong evidence for a genetic basis has prompted the development of mouse models with targeted mutations in candidate genes for autism. As the diagnostic criteria for autism are behavioural, phenotyping these mouse models requires behavioural assays with high relevance to each category of the diagnostic symptoms. Behavioural neuroscientists are generating a comprehensive set of assays for social interaction, communication and repetitive behaviours to test hypotheses about the causes of autism. Robust phenotypes in mouse models hold great promise as translational tools for discovering effective treatments for components of autism spectrum disorders.

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.

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.