Comparative profiling of white matter development in the human and mouse brain reveals volumetric deficits and delayed myelination in Angelman syndrome.

Background: Angelman syndrome (AS), a severe neurodevelopmental disorder resulting from the loss of the maternal UBE3A gene, is marked by changes in the brain's white matter (WM). The extent of WM abnormalities seems to correlate with the severity of clinical symptoms, but these deficits are still not well characterized or understood. This study provides the first large-scale measurement of WM volume reduction in children with AS. Furthermore, we probed the underlying neuropathology by examining the progression of myelination in an AS mouse model. Methods: We conducted magnetic resonance imaging (MRI) on children with AS (n=32) and neurotypical controls (n=99) aged 0.5-12 years. In parallel, we examined myelination in postnatal Ube3a maternal-null mice (Ube3a m-/p+; AS model), Ube3a paternal-null mice (Ube3a m+/p-), and wildtype controls (Ube3a m+/p+) using immunohistochemistry, Western blotting, and electron microscopy. Results: Our data revealed that AS individuals exhibit significant reductions in brain volume by ~1 year of age, with WM reduced by 26% and gray matter by 21% by 6-12 years of age-approximately twice the reductions observed in the adult AS mouse model. In our AS mouse model, we saw a global delay in the onset of myelination, which normalized within days (likely corresponding to months or years in human development). This myelination delay is caused by the loss of UBE3A in neurons rather than UBE3A haploinsufficiency in oligodendrocytes. Interestingly, ultrastructural analyses did not reveal any abnormalities in myelinated or unmyelinated axons. Limitations: It is difficult to extrapolate the timing and duration of the myelination delay observed in AS model mice to individuals with AS. Conclusions: This study reveals WM deficits as a hallmark in children with AS, demonstrating for the first time that these deficits are already apparent at 1 year of age. Parallel studies in a mouse model of AS show that these deficits may be associated with delayed onset of myelination due to the loss of neuronal (but not glial) UBE3A. These findings emphasize the potential of WM as both a therapeutic target for interventions and a valuable biomarker for tracking the progression of AS and the effectiveness of potential treatments.

Neuronal UBE3A substrates hold therapeutic potential for Angelman syndrome.

Emerging therapies for Angelman syndrome, a severe neurodevelopmental disorder, are focused on restoring UBE3A gene expression in the brain. Further therapeutic opportunities may arise from a better understanding of how UBE3A gene products-both long and short isoforms of the ubiquitin ligase E3A (UBE3A)-function in neurons. Great strides have been made recently toward identifying ubiquitin substrates of UBE3A in vitro and in heterologous expression systems. From this work, a particularly close relationship between UBE3A and subunits of the 19S regulatory particle of the proteasome has become evident. We propose that further research cognizant of isoform-specific UBE3A functional roles will be instrumental in elucidating key UBE3A/substrate relationships within distinct neuronal compartments, lending to the discovery of novel therapeutic targets and valuable clinical biomarkers for the treatment of Angelman syndrome.

Ube3a unsilencer for the potential treatment of Angelman syndrome.

Deletion of the maternal UBE3A allele causes Angelman syndrome (AS); because paternal UBE3A is epigenetically silenced by a long non-coding antisense (UBE3A-ATS) in neurons, this nearly eliminates UBE3A protein in the brain. Reactivating paternal UBE3A holds promise for treating AS. We previously showed topoisomerase inhibitors can reactivate paternal UBE3A, but their therapeutic challenges prompted our search for small molecule unsilencers with a different mechanism of action. Here, we found that (S)-PHA533533 acts through a novel mechanism to significantly increase paternal Ube3a mRNA and UBE3A protein levels while downregulating Ube3a-ATS in primary neurons derived from AS model mice. Furthermore, peripheral delivery of (S)-PHA533533 in AS model mice induces widespread neuronal UBE3A expression. Finally, we show that (S)-PHA533533 unsilences paternal UBE3A in AS patient-derived neurons, highlighting its translational potential. Our findings provide a lead for developing a small molecule treatment for AS that could be safe, non-invasively delivered, and capable of brain-wide unsilencing of paternal UBE3A.

Sleep EEG signatures in mouse models of 15q11.2-13.1 duplication (Dup15q) syndrome.

BACKGROUND: Sleep disturbances are a prevalent and complex comorbidity in neurodevelopmental disorders (NDDs). Dup15q syndrome (duplications of 15q11.2-13.1) is a genetic disorder highly penetrant for NDDs such as autism and intellectual disability and it is frequently accompanied by significant disruptions in sleep patterns. The 15q critical region harbors genes crucial for brain development, notably UBE3A and a cluster of gamma-aminobutyric acid type A receptor (GABAAR) genes. We previously described an electrophysiological biomarker of the syndrome, marked by heightened beta oscillations (12-30 Hz) in individuals with Dup15q syndrome, akin to electroencephalogram (EEG) alterations induced by allosteric modulation of GABAARs. Those with Dup15q syndrome exhibited increased beta oscillations during the awake resting state and during sleep, and they showed profoundly abnormal NREM sleep. This study aims to assess the translational validity of these EEG signatures and to delve into their neurobiological underpinnings by quantifying sleep physiology in chromosome-engineered mice with maternal (matDp/ + mice) or paternal (patDp/ + mice) inheritance of the full 15q11.2-13.1-equivalent duplication, and mice with duplication of just the UBE3A gene (Ube3a overexpression mice; Ube3a OE mice) and comparing the sleep metrics with their respective wildtype (WT) littermate controls. METHODS: We collected 48-h EEG/EMG recordings from 35 (23 male, 12 female) 12-24-week-old matDp/ + , patDp/ + , Ube3a OE mice, and their WT littermate controls. We quantified baseline sleep, sleep fragmentation, spectral power dynamics during sleep states, and recovery following sleep deprivation. Within each group, distinctions between Dup15q mutant mice and WT littermate controls were evaluated using analysis of variance (ANOVA) and student's t-test. The impact of genotype and time was discerned through repeated measures ANOVA, and significance was established at p < 0.05. RESULTS: Our study revealed that across brain states, matDp/ + mice mirrored the elevated beta oscillation phenotype observed in clinical EEGs from individuals with Dup15q syndrome. Time to sleep onset after light onset was significantly reduced in matDp/ + and Ube3a OE mice. However, NREM sleep between Dup15q mutant and WT littermate mice remained unaltered, suggesting a divergence from the clinical presentation in humans. Additionally, while increased beta oscillations persisted in matDp/ + mice after 6-h of sleep deprivation, recovery NREM sleep remained unaltered in all groups, thus suggesting that these mice exhibit resilience in the fundamental processes governing sleep-wake regulation. CONCLUSIONS: Quantification of mechanistic and translatable EEG biomarkers is essential for advancing our understanding of NDDs and their underlying pathophysiology. Our study of sleep physiology in the Dup15q mice underscores that the beta EEG biomarker has strong translational validity, thus opening the door for pre-clinical studies of putative drug targets, using the biomarker as a translational measure of drug-target engagement. The unaltered NREM sleep may be due to inherent differences in neurobiology between mice and humans. These nuanced distinctions highlight the complexity of sleep disruptions in Dup15q syndrome and emphasize the need for a comprehensive understanding that encompasses both shared and distinct features between murine models and clinical populations.

Regional and cellular organization of the autism-associated protein UBE3A/E6AP and its antisense transcript in the brain of the developing rhesus monkey.

Angelman syndrome (AS) is a neurogenetic disorder caused by mutations or deletions in the maternally-inherited UBE3A allele, leading to a loss of UBE3A protein expression in neurons. The paternally-inherited UBE3A allele is epigenetically silenced in neurons during development by a noncoding transcript (UBE3A-ATS). The absence of neuronal UBE3A results in severe neurological symptoms, including speech and language impairments, intellectual disability, and seizures. While no cure exists, therapies aiming to restore UBE3A function-either by gene addition or by targeting UBE3A-ATS-are under development. Progress in developing these treatments relies heavily on inferences drawn from mouse studies about the function of UBE3A in the human brain. To aid translational efforts and to gain an understanding of UBE3A and UBE3A-ATS biology with greater relevance to human neurodevelopmental contexts, we investigated UBE3A and UBE3A-ATS expression in the developing brain of the rhesus macaque, a species that exhibits complex social behaviors, resembling aspects of human behavior to a greater degree than mice. Combining immunohistochemistry and in situ hybridization, we mapped UBE3A and UBE3A-ATS regional and cellular expression in normal prenatal, neonatal, and adolescent rhesus macaque brains. We show that key hallmarks of UBE3A biology, well-known in rodents, are also present in macaques, and suggest paternal UBE3A silencing in neurons-but not glial cells-in the macaque brain, with onset between gestational day 48 and 100. These findings support proposals that early-life, perhaps even prenatal, intervention is optimal for overcoming the maternal allele loss of UBE3A linked to AS.

Heterogeneity in the progression of retinal pathologies in mice harboring patient mimicking Impg2 mutations.

Biallelic mutations in interphotoreceptor matrix proteoglycan 2 (IMPG2) in humans cause retinitis pigmentosa (RP) with early macular involvement, albeit the disease progression varies widely due to genetic heterogeneity and IMPG2 mutation type. There are currently no treatments for IMPG2-RP. To aid preclinical studies toward eventual treatments, there is a need to better understand the progression of disease pathology in appropriate animal models. Toward this goal, we developed mouse models with patient mimicking homozygous frameshift (T807Ter) or missense (Y250C) Impg2 mutations, as well as mice with a homozygous frameshift mutation (Q244Ter) designed to completely prevent IMPG2 protein expression, and characterized the trajectory of their retinal pathologies across postnatal development until late adulthood. We found that the Impg2T807Ter/T807Ter and Impg2Q244Ter/Q244Ter mice exhibited early onset gliosis, impaired photoreceptor outer segment maintenance, appearance of subretinal deposits near the optic disc, disruption of the outer retina, and neurosensorial detachment, whereas the Impg2Y250C/Y250C mice exhibited minimal retinal pathology. These results demonstrate the importance of mutation type in disease progression in IMPG2-RP and provide a toolkit and preclinical data for advancing therapeutic approaches.

Human retinal organoids harboring IMPG2 mutations exhibit a photoreceptor outer segment phenotype that models advanced retinitis pigmentosa.

Interphotoreceptor matrix proteoglycan 2 (IMPG2) mutations cause a severe form of early-onset retinitis pigmentosa (RP) with macular involvement. IMPG2 is expressed by photoreceptors and incorporated into the matrix that surrounds the inner and outer segments (OS) of rods and cones, but the mechanism of IMPG2-RP remains unclear. Loss of Impg2 function in mice produces a mild, late-onset photoreceptor phenotype without the characteristic OS loss that occurs in human patients. We generated retinal organoids (ROs) from patient-derived induced pluripotent stem (iPS) cells and gene-edited embryonic stem cells to model human IMPG2-RP in vitro. All ROs harboring IMPG2 mutations lacked an OS layer, in contrast to isogenic controls. Subsequent protein analyses revealed that this phenotype arises due to a loss of IMPG2 expression or its inability to undergo normal post-translational modifications. We hypothesized that loss of IMPG2 function destabilizes the interphotoreceptor matrix and renders the OS vulnerable to physical stressors, which is accentuated in the tissue culture environment. In support of this mechanism, transplantation of IMPG2 mutant ROs into the protected subretinal space of immunocompromised rodents restored OS production. Beyond providing a robust platform to study IMPG2-RP, this human RO model system may serve a broader role in honing strategies to treat advanced photoreceptor-based diseases.

Molecular and behavioral consequences of Ube3a gene overdosage in mice.

Chromosome 15q11.2-q13.1 duplication syndrome (Dup15q syndrome) is a severe neurodevelopmental disorder characterized by intellectual disability, impaired motor coordination, and autism spectrum disorder. Chromosomal multiplication of the UBE3A gene is presumed to be the primary driver of Dup15q pathophysiology, given that UBE3A exhibits maternal monoallelic expression in neurons and that maternal duplications typically yield far more severe neurodevelopmental outcomes than paternal duplications. However, studies into the pathogenic effects of UBE3A overexpression in mice have yielded conflicting results. Here, we investigated the neurodevelopmental impact of Ube3a gene overdosage using bacterial artificial chromosome-based transgenic mouse models (Ube3aOE) that recapitulate the increases in Ube3a copy number most often observed in Dup15q. In contrast to previously published Ube3a overexpression models, Ube3aOE mice were indistinguishable from wild-type controls on a number of molecular and behavioral measures, despite suffering increased mortality when challenged with seizures, a phenotype reminiscent of sudden unexpected death in epilepsy. Collectively, our data support a model wherein pathogenic synergy between UBE3A and other overexpressed 15q11.2-q13.1 genes is required for full penetrance of Dup15q syndrome phenotypes.

Rescue of behavioral and electrophysiological phenotypes in a Pitt-Hopkins syndrome mouse model by genetic restoration of Tcf4 expression.

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by monoallelic mutation or deletion in the transcription factor 4 (TCF4) gene. Individuals with PTHS typically present in the first year of life with developmental delay and exhibit intellectual disability, lack of speech, and motor incoordination. There are no effective treatments available for PTHS, but the root cause of the disorder, TCF4 haploinsufficiency, suggests that it could be treated by normalizing TCF4 gene expression. Here, we performed proof-of-concept viral gene therapy experiments using a conditional Tcf4 mouse model of PTHS and found that postnatally reinstating Tcf4 expression in neurons improved anxiety-like behavior, activity levels, innate behaviors, and memory. Postnatal reinstatement also partially corrected EEG abnormalities, which we characterized here for the first time, and the expression of key TCF4-regulated genes. Our results support a genetic normalization approach as a treatment strategy for PTHS, and possibly other TCF4-linked disorders.

Ictal neural oscillatory alterations precede sudden unexpected death in epilepsy.

Sudden unexpected death in epilepsy is the most catastrophic outcome of epilepsy. Each year there are as many as 1.65 cases of such death for every 1000 individuals with epilepsy. Currently, there are no methods to predict or prevent this tragic event, due in part to a poor understanding of the pathologic cascade that leads to death following seizures. We recently identified enhanced seizure-induced mortality in four inbred strains from the genetically diverse Collaborative Cross mouse population. These mouse models of sudden unexpected death in epilepsy provide a unique tool to systematically examine the physiological alterations during fatal seizures, which can be studied in a controlled environment and with consideration of genetic complexity. Here, we monitored the brain oscillations and heart functions before, during, and after non-fatal and fatal seizures using a flurothyl-induced seizure model in freely moving mice. Compared with mice that survived seizures, non-survivors exhibited significant suppression of brainstem neural oscillations that coincided with cortical epileptic activities and tachycardia during the ictal phase of a fatal seizure. Non-survivors also exhibited suppressed delta (0.5-4 Hz)/gamma (30-200 Hz) phase-amplitude coupling in cortex but not in brainstem. A connectivity analysis revealed elevated synchronization of cortex and brainstem oscillations in the delta band during fatal seizures compared with non-fatal seizures. The dynamic ictal oscillatory and connectivity features of fatal seizures provide insights into sudden unexpected death in epilepsy and may suggest biomarkers and eventual therapeutic targets.

Evaluation of electroencephalography biomarkers for Angelman syndrome during overnight sleep.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss-of-function mutations in the maternal copy of the UBE3A gene. AS is characterized by intellectual disability, impaired speech and motor skills, epilepsy, and sleep disruptions. Multiple treatment strategies to re-express functional neuronal UBE3A from the dormant paternal allele were successful in rodent models of AS and have now moved to early phase clinical trials in children. Developing reliable and objective AS biomarkers is essential to guide the design and execution of current and future clinical trials. Our prior work quantified short daytime electroencephalograms (EEGs) to define promising biomarkers for AS. Here, we asked whether overnight sleep is better suited to detect AS EEG biomarkers. We retrospectively analyzed EEGs from 12 overnight sleep studies from individuals with AS with age and sex-matched Down syndrome and neurotypical controls, focusing on low frequency (2-4 Hz) delta rhythms and sleep spindles. Delta EEG rhythms were increased in individuals with AS during all stages of overnight sleep, but overnight sleep did not provide additional benefit over wake in the ability to detect increased delta. Abnormal sleep spindles were not reliably detected in EEGs from individuals with AS during overnight sleep, suggesting that delta rhythms represent a more reliable biomarker. Overall, we conclude that periods of wakefulness are sufficient, and perhaps ideal, to quantify delta EEG rhythms for use as AS biomarkers. LAY SUMMARY: Electroencephalography (EEG) is a safe and reliable way of measuring abnormal brain activity in Angelman syndrome. We found that low-frequency "delta" EEG rhythms are increased in individuals with Angelman syndrome during all stages of overnight sleep. Delta rhythms can be used as a tool to measure improvement in future clinical trials.

Dual-isoform hUBE3A gene transfer improves behavioral and seizure outcomes in Angelman syndrome model mice.

Loss of the maternal UBE3A allele causes Angelman syndrome (AS), a debilitating neurodevelopmental disorder. Here, we devised an AS treatment strategy based on reinstating dual-isoform expression of human UBE3A (hUBE3A) in the developing brain. Kozak sequence engineering of our codon-optimized vector (hUBE3Aopt) enabled translation of both short and long hUBE3A protein isoforms at a near-endogenous 3:1 (short/long) ratio, a feature that could help to support optimal therapeutic outcomes. To model widespread brain delivery and early postnatal onset of hUBE3A expression, we packaged the hUBE3Aopt vector into PHP.B capsids and performed intracerebroventricular injections in neonates. This treatment significantly improved motor learning and innate behaviors in AS mice, and it rendered them resilient to epileptogenesis and associated hippocampal neuropathologies induced by seizure kindling. hUBE3A overexpression occurred frequently in the hippocampus but was uncommon in the neocortex and other major brain structures; furthermore, it did not correlate with behavioral performance. Our results demonstrate the feasibility, tolerability, and therapeutic potential for dual-isoform hUBE3A gene transfer in the treatment of AS.

The Impact of SST and PV Interneurons on Nonlinear Synaptic Integration in the Neocortex.

Excitatory synaptic inputs arriving at the dendrites of a neuron can engage active mechanisms that nonlinearly amplify the depolarizing currents. This supralinear synaptic integration is subject to modulation by inhibition. However, the specific rules by which different subtypes of interneurons affect the modulation have remained largely elusive. To examine how inhibition influences active synaptic integration, we optogenetically manipulated the activity of the following two subtypes of interneurons: dendrite-targeting somatostatin-expressing (SST) interneurons; and perisomatic-targeting parvalbumin-expressing (PV) interneurons. In acute slices of mouse primary visual cortex, electrical stimulation evoked nonlinear synaptic integration that depended on NMDA receptors. Optogenetic activation of SST interneurons in conjunction with electrical stimulation resulted in predominantly divisive inhibitory gain control, reducing the magnitude of the supralinear response without affecting its threshold. PV interneuron activation, on the other hand, had a minimal effect on the supralinear response. Together, these results delineate the roles for SST and PV neurons in active synaptic integration. Differential effects of inhibition by SST and PV interneurons likely increase the computational capacity of the pyramidal neurons in modulating the nonlinear integration of synaptic output.

A small-molecule screen reveals novel modulators of MeCP2 and X-chromosome inactivation maintenance.

BACKGROUND: Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MeCP2) gene. While MeCP2 mutations are lethal in most males, females survive birth but show severe neurological defects. Because X-chromosome inactivation (XCI) is a random process, approximately 50% of the cells silence the wild-type (WT) copy of the MeCP2 gene. Thus, reactivating the silent WT copy of MeCP2 could provide therapeutic intervention for RTT. METHODS: Toward this goal, we screened ~ 28,000 small-molecule compounds from several libraries using a MeCP2-luciferase reporter cell line and cortical neurons from a MeCP2-EGFP mouse model. We used gain/increase of luminescence or fluorescence as a readout of MeCP2 reactivation and tested the efficacy of these drugs under different drug regimens, conditions, and cellular contexts. RESULTS: We identified inhibitors of the JAK/STAT pathway as XCI-reactivating agents, both by in vitro and ex vivo assays. In particular, we show that AG-490, a Janus Kinase 2 (JAK2) kinase inhibitor, and Jaki, a pan JAK/STAT inhibitor, are capable of reactivating MeCP2 from the inactive X chromosome, in different cellular contexts. CONCLUSIONS: Our results suggest that inhibition of the JAK/STAT pathway is a new potential pathway to reinstate MeCP2 gene expression as an efficient RTT treatment.

Deficits in higher visual area representations in a mouse model of Angelman syndrome.

BACKGROUND: Sensory processing deficits are common in individuals with neurodevelopmental disorders. One hypothesis is that deficits may be more detectable in downstream, "higher" sensory areas. A mouse model of Angelman syndrome (AS), which lacks expression of the maternally inherited Ube3a allele, has deficits in synaptic function and experience-dependent plasticity in the primary visual cortex. Thus, we hypothesized that AS model mice have deficits in visually driven neuronal responsiveness in downstream higher visual areas (HVAs). METHODS: Here, we used intrinsic signal optical imaging and two-photon calcium imaging to map visually evoked neuronal activity in the primary visual cortex and HVAs in response to an array of stimuli. RESULTS: We found a highly specific deficit in HVAs. Drifting gratings that changed speed caused a strong response in HVAs in wildtype mice, but this was not observed in littermate AS model mice. Further investigation with two-photon calcium imaging revealed the effect to be largely driven by aberrant responses of inhibitory interneurons, suggesting a cellular basis for higher level, stimulus-selective cortical dysfunction in AS. CONCLUSION: Assaying downstream, or "higher" circuitry may provide a more sensitive measure for circuit dysfunction in mouse models of neurodevelopmental disorders. TRIAL REGISTRATION: Not applicable.

Content and Performance of the MiniMUGA Genotyping Array: A New Tool To Improve Rigor and Reproducibility in Mouse Research.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.

Visual Sequences Drive Experience-Dependent Plasticity in Mouse Anterior Cingulate Cortex.

Mechanisms of experience-dependent plasticity have been well characterized in mouse primary visual cortex (V1), including a form of potentiation driven by repeated presentations of a familiar visual sequence ("sequence plasticity"). The prefrontal anterior cingulate cortex (ACC) responds to visual stimuli, yet little is known about if and how visual experience modifies ACC circuits. We find that mouse ACC exhibits sequence plasticity, but in contrast to V1, the plasticity expresses as a change in response timing, rather than a change in response magnitude. Sequence plasticity is absent in ACC, but not V1, in a mouse model of a neurodevelopmental disorder associated with intellectual disability and autism-like features. Our results demonstrate that simple sensory stimuli can be used to reveal how experience functionally (or dysfunctionally) modifies higher-order prefrontal circuits and suggest a divergence in how ACC and V1 encode familiarity.

Region and Cell Type Distribution of TCF4 in the Postnatal Mouse Brain.

Transcription factor 4 is a class I basic helix-loop-helix transcription factor regulating gene expression. Altered TCF4 gene expression has been linked to non-syndromic intellectual disability, schizophrenia, and a severe neurodevelopmental disorder known as Pitt-Hopkins syndrome. An understanding of the cell types expressing TCF4 protein in the mouse brain is needed to help identify potential pathophysiological mechanisms and targets for therapeutic delivery in TCF4-linked disorders. Here we developed a novel green fluorescent protein reporter mouse to visualize TCF4-expressing cells throughout the brain. Using this TCF4 reporter mouse, we observed prominent expression of TCF4 in the pallial region and cerebellum of the postnatal brain. At the cellular level, both glutamatergic and GABAergic neurons express TCF4 in the cortex and hippocampus, while only a subset of GABAergic interneurons express TCF4 in the striatum. Among glial cell groups, TCF4 is present in astrocytes and immature and mature oligodendrocytes. In the cerebellum, cells in the granule and molecular layer express TCF4. Our findings greatly extend our knowledge of the spatiotemporal and cell type-specific expression patterns of TCF4 in the brain, and hence, lay the groundwork to better understand TCF4-linked neurological disorders. Any effort to restore TCF4 functions through small molecule or genetic therapies should target these brain regions and cell groups to best recapitulate TCF4 expression patterns.

Collaborative Cross mice reveal extreme epilepsy phenotypes and genetic loci for seizure susceptibility.

OBJECTIVE: Animal studies remain essential for understanding mechanisms of epilepsy and identifying new therapeutic targets. However, existing animal models of epilepsy do not reflect the high level of genetic diversity found in the human population. The Collaborative Cross (CC) population is a genetically diverse recombinant inbred panel of mice. The CC offers large genotypic and phenotypic diversity, inbred strains with stable genomes that allow for repeated phenotypic measurements, and genomic tools including whole genome sequence to identify candidate genes and candidate variants. METHODS: We evaluated multiple complex epileptic traits in a sampling of 35 CC inbred strains using the flurothyl-induced seizure and kindling paradigm. We created an F2 population of 297 mice with extreme seizure susceptibility and performed quantitative trait loci (QTL) mapping to identify genomic regions associated with seizure sensitivity. We used quantitative RNA sequencing from CC hippocampal tissue to identify candidate genes and whole genome sequence to identify genetic variants likely affecting gene expression. RESULTS: We identified new mouse models with extreme seizure susceptibility, seizure propagation, epileptogenesis, and SUDEP (sudden unexpected death in epilepsy). We performed QTL mapping and identified one known and seven novel loci associated with seizure sensitivity. We combined whole genome sequencing and hippocampal gene expression to pinpoint biologically plausible candidate genes (eg, Gabra2) and variants associated with seizure sensitivity. SIGNIFICANCE: New mouse models of epilepsy are needed to better understand the complex genetic architecture of seizures and to identify therapeutics. We performed a phenotypic screen utilizing a novel genetic reference population of CC mice. The data we provide enable the identification of protective/risk genes and novel molecular mechanisms linked to complex seizure traits that are currently challenging to study and treat.