Search

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.

Background Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by either disruptions of the gene UBE3A or deletion of chromosome 15 at 15q11-q13, which encompasses UBE3A and several other genes, including GABRB3, GABRA5, GABRG3, encoding gamma-aminobutyric acid type A receptor subunits ( β 3, α 5, γ 3). Individuals with deletions are generally more impaired than those with other genotypes, but the underlying pathophysiology remains largely unknown. Here, we used electroencephalography (EEG) to test the hypothesis that genes other than UBE3A located on 15q11-q13 cause differences in pathophysiology between AS genotypes. Methods We compared spectral power of clinical EEG recordings from children (1–18 years of age) with a deletion genotype (n = 37) or a nondeletion genotype (n = 21) and typically developing children without Angelman syndrome (n = 48). Results We found elevated theta power (peak frequency: 5.3 Hz) and diminished beta power (peak frequency: 23 Hz) in the deletion genotype compared with the nondeletion genotype as well as excess broadband EEG power (1–32 Hz) peaking in the delta frequency range (peak frequency: 2.8 Hz), shared by both genotypes but stronger for the deletion genotype at younger ages. Conclusions Our results provide strong evidence for the contribution of non-UBE3A neuronal pathophysiology in deletion AS and suggest that hemizygosity of the GABRB3-GABRA5-GABRG3 gene cluster causes abnormal theta and beta EEG oscillations that may underlie the more severe clinical phenotype. Our work improves the understanding of AS pathophysiology and has direct implications for the development of AS treatments and biomarkers.

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.

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.

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 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 6,899 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, the extension of the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. There is preliminary but striking 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 main stock for a significant action of the genotyped inbred samples. We conclude that MiniMUGA is a valuable platform for genetic QC and important new tool to the increase rigor and reproducibility of mouse research.

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.

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 a ~300 F2 population of extreme seizure susceptibility and performed 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 in an F2 intercrosses of seizure susceptible and resistant strains 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 (e.g. 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 enables the identification of protective/risk genes and novel molecular mechanisms linked to complex seizure traits that are currently challenging to study and treat.

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.

Background Loss of UBE3A causes Angelman syndrome, whereas excess UBE3A activity appears to increase the risk for autism. Despite this powerful association with neurodevelopmental disorders, there is still much to be learned about UBE3A, including its cellular and subcellular organization in the human brain. The issue is important, since UBE3A’s localization is integral to its function. Methods We used light and electron microscopic immunohistochemistry to study the cellular and subcellular distribution of UBE3A in the adult human cerebral cortex. Experiments were performed on multiple tissue sources, but our results focused on optimally preserved material, using surgically resected human temporal cortex of high ultrastructural quality from nine individuals. Results We demonstrate that UBE3A is expressed in both glutamatergic and GABAergic neurons, and to a lesser extent in glial cells. We find that UBE3A in neurons has a non-uniform subcellular distribution. In somata, UBE3A preferentially concentrates in euchromatin-rich domains within the nucleus. Electron microscopy reveals that labeling concentrates in the head and neck of dendritic spines and is excluded from the PSD. Strongest labeling within the neuropil was found in axon terminals. Conclusions By highlighting the subcellular compartments in which UBE3A is likely to function in the human neocortex, our data provide insight into the diverse functional capacities of this E3 ligase. These anatomical data may help to elucidate the role of UBE3A in Angelman syndrome and autism spectrum disorder.

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.

Mutations or deletions of the transcription factorTCF4are linked to Pitt–Hopkins syndrome (PTHS) and schizophrenia, suggesting that the precise pathogenic mutations dictate cellular, synaptic, and behavioral consequences. Here, we generated two novel mouse models of PTHS, one that mimics the most common pathogenicTCF4point mutation (human R580W, mouse R579W) and one that deletes three pathogenic arginines, and explored phenotypes of these lines alongside models of pan-cellular or CNS-specific heterozygousTcf4disruption. We used mice of both sexes to show that impairedTcf4function results in consistent microcephaly, hyperactivity, reduced anxiety, and deficient spatial learning. All four PTHS mouse models demonstrated exaggerated hippocampal long-term potentiation (LTP), consistent with deficits in hippocampus-mediated behaviors. We further examined R579W mutant mice and mice with pan-cellularTcf4heterozygosity and found that they exhibited hippocampal NMDA receptor hyperfunction, which likely drives the enhanced LTP. Together, our data pinpoint convergent neurobiological features in PTHS mouse models and provide a foundation for preclinical studies and a rationale for testing whether NMDAR antagonists might be used to treat PTHS.SIGNIFICANCE STATEMENTPitt–Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder associated withTCF4mutations/deletions. Despite this genetic insight, there is a need to identify the function of TCF4 in the brain. Toward this goal, we developed two mouse lines, including one harboring the most prevalent pathogenic point mutation, and compared them with two existing models that conditionally deleteTcf4. Our data identify a set of overlapping phenotypes that may serve as outcome measures for preclinical studies of PTHS treatments. We also discovered penetrant enhanced synaptic plasticity across mouse models that may be linked to increased NMDA receptor function. These data reveal convergent neurobiological characteristics of PTHS mouse models and support the further investigation of NMDA receptor antagonists as a possible PTHS treatment.

Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of function of the maternally inherited UBE3A allele. It is currently unclear how the consequences of this genetic insult unfold to impair neurodevelopment. We reasoned that by elucidating the basis of microcephaly in AS, a highly penetrant syndromic feature with early postnatal onset, we would gain new insights into the mechanisms by which maternal UBE3A loss derails neurotypical brain growth and function. Detailed anatomical analysis of both male and female maternal Ube3a-null mice reveals that microcephaly in the AS mouse model is primarily driven by deficits in the growth of white matter tracts, which by adulthood are characterized by densely packed axons of disproportionately small caliber. Our results implicate impaired axon growth in the pathogenesis of AS and identify noninvasive structural neuroimaging as a potentially valuable tool for gauging therapeutic efficacy in the disorder.SIGNIFICANCE STATEMENT People who maternally inherit a deletion or nonfunctional copy of the UBE3A gene develop Angelman syndrome (AS), a severe neurodevelopmental disorder. To better understand how loss of maternal UBE3A function derails brain development, we analyzed brain structure in a maternal Ube3a knock-out mouse model of AS. We report that the volume of white matter (WM) is disproportionately reduced in AS mice, indicating that deficits in WM development are a major factor underlying impaired brain growth and microcephaly in the disorder. Notably, we find that axons within the WM pathways of AS model mice are abnormally small in caliber. This defect is associated with slowed nerve conduction, which could contribute to behavioral deficits in AS, including motor dysfunction.

Epilepsy is a neurological disorder with complex etiologies and genetic architecture. Animal models have a critical role in understanding the pathophysiology of epilepsy. Here we studied epilepsy utilizing a genetic reference population of Collaborative Cross (CC) mice with publicly available whole genome sequences. We measured multiple epilepsy traits in 35 CC strains, and we identified novel animal models that exhibit extreme outcomes in seizure susceptibility, seizure propagation, epileptogenesis, and sudden unexpected death in epilepsy. We performed QTL mapping in an F2 population and identified seven novel and one previously identified loci associated with seizure sensitivity. We combined whole genome sequence and hippocampal gene expression to pinpoint biologically plausible candidate genes and candidate variants associated with seizure sensitivity. These resources provide a powerful toolbox for studying complex features of seizures and for identifying genes associated with particular seizure outcomes, and hence will facilitate the development of new therapeutic targets for epilepsy.

Background Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by mutations affecting UBE3A gene expression. Previous studies in mice revealed distinct critical periods during neurodevelopment in which reactivation of Ube3a gene expression can prevent the onset of behavioral deficits. Whether UBE3A is required for brain function throughout life is unknown. Here, we address the importance of maintaining UBE3A expression after normal brain development. Findings Using a conditional mouse, we deleted the Ube3a gene at three ages spanning brain maturation. We assessed the consequences of Ube3a gene deletion by testing the mice in behavioral tasks previously shown to produce robust phenotypes in AS model mice. Early embryonic deletion of Ube3a recapitulated all behavioral deficits of AS mice. In contrast, Ube3a gene deletion at 3 or 12 weeks of age did not have a significant effect on most behavioral tasks and did not increase seizure sensitivity. Conclusions Taken together, these results emphasize that UBE3A critically impacts early brain development, but plays a more limited role in adulthood. Our findings provide important considerations for upcoming clinical trials in which UBE3A gene expression is reactivated and suggest that even transient UBE3A reinstatement during a critical window of early development is likely to prevent most adverse Angelman syndrome phenotypes. However, sustained UBE3A expression into adulthood is probably needed for optimal clinical benefit. Electronic supplementary material The online version of this article (10.1186/s13229-019-0277-1) contains supplementary material, which is available to authorized users.

Dendritic spines are a morphological feature of the majority of excitatory synapses in the mammalian neocortex and are motile structures with shapes and lifetimes that change throughout development. Proper cortical development and function, including cortical contributions to learning and memory formation, require appropriate experience-dependent dendritic spine remodeling. Dendritic spine abnormalities have been reported for many neurodevelopmental disorders, including Angelman syndrome (AS), which is caused by the loss of the maternally inheritedUBE3Aallele (encoding ubiquitin protein ligase E3A). Prior studies revealed that UBE3A protein loss leads to reductions in dendritic spine density and diminished excitatory synaptic transmission. However, the decrease in spine density could come from either a reduction in spine formation or an increase in spine elimination. Here, we used acute and longitudinalin vivotwo-photon microscopy to investigate developmental and experience-dependent changes in the numbers, dynamics, and morphology of layer 5 pyramidal neuron apical dendritic spines in the primary visual cortex of control and AS model mice (Ube3am−/p+mice). We found that neurons in AS model mice undergo a greater elimination of dendritic spines than wild-type mice during the end of the first postnatal month. However, when raised in darkness, spine density and dynamics were indistinguishable between control and AS model mice, which indicates that decreased spine density in AS model mice reflects impaired experience-driven spine maintenance. Our data thus demonstrate an experience-dependent anatomical substrate by which the loss of UBE3A reduces dendritic spine density and disrupts cortical circuitry.SIGNIFICANCE STATEMENTReduced dendritic spine densities are common in the neurodevelopmental disorder Angelman syndrome (AS). Because prior reports were based on postmortem tissue, it was unknown whether this anatomical deficit arises from decreased spine formation and/or increased spine elimination. Here, we usedin vivotwo-photon imaging to track spines over multiple days in a mouse model of AS. We found that spine formation is normal, but experience-dependent spine maintenance is reduced in the visual cortex of AS model mice. Our data pinpoint the anatomical process underlying the loss of dendritic spines, which can account for the decreased excitatory synaptic connectivity associated with AS. Therefore, normalizing spine maintenance is a potential therapeutic strategy.

Loss of maternal UBE3A causes Angelman syndrome (AS), a neurodevelopmental disorder associated with severe epilepsy. We previously implicated GABAergic deficits onto layer (L) 2/3 pyramidal neurons in the pathogenesis of neocortical hyperexcitability, and perhaps epilepsy, in AS model mice. Here we investigate consequences of selective Ube3a loss from either GABAergic or glutamatergic neurons, focusing on the development of hyperexcitability within L2/3 neocortex and in broader circuit and behavioral contexts. We find that GABAergic Ube3a loss causes AS-like increases in neocortical EEG delta power, enhances seizure susceptibility, and leads to presynaptic accumulation of clathrin-coated vesicles (CCVs) – all without decreasing GABAergic inhibition onto L2/3 pyramidal neurons. Conversely, glutamatergic Ube3a loss fails to yield EEG abnormalities, seizures, or associated CCV phenotypes, despite impairing tonic inhibition onto L2/3 pyramidal neurons. These results substantiate GABAergic Ube3a loss as the principal cause of circuit hyperexcitability in AS mice, lending insight into ictogenic mechanisms in AS.

SUMMARY 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., Graphical Abstract, In Brief Sidorov et al. demonstrate that patterned visual input can drive experience-dependent plasticity in the timing of neural responses in mouse anterior cingulate cortex.

Background Angelman syndrome (AS) is a severe neurodevelopmental disorder lacking effective therapies. AS is caused by mutations in ubiquitin protein ligase E3A (UBE3A), which is genomically imprinted such that only the maternally inherited copy is expressed in neurons. We previously demonstrated that topoisomerase I (Top1) inhibitors could successfully reactivate the dormant paternal allele of Ube3a in neurons of a mouse model of AS. We also previously showed that one such Top1 inhibitor, topotecan, could unsilence paternal UBE3A in induced pluripotent stem cell-derived neurons from individuals with AS. Although topotecan has been well-studied and is FDA-approved for cancer therapy, its limited CNS bioavailability will likely restrict the therapeutic use of topotecan in AS. The goal of this study was to identify additional Top1 inhibitors with similar efficacy as topotecan, with the expectation that these could be tested in the future for safety and CNS bioavailability to assess their potential as AS therapeutics. Methods We tested 13 indenoisoquinoline-derived Top1 inhibitors to identify compounds that unsilence the paternal allele of Ube3a in mouse neurons. Primary cortical neurons were isolated from embryonic day 14.5 (E14.5) mice with a Ube3a-YFP fluorescent tag on the paternal allele (Ube3a m+/pYFP mice) or mice that lack the maternal Ube3a allele and hence model AS (Ube3a m−/p+ mice). Neurons were cultured for 7 days, treated with drug for 72 h, and examined for paternal UBE3A protein expression by Western blot or fluorescence immunostaining. Dose responses of the compounds were determined across a log range of drug treatments, and cytotoxicity was tested using a luciferase-based assay. Results All 13 indenoisoquinoline-derived Top1 inhibitors unsilenced paternal Ube3a. Several compounds exhibited favorable paternal Ube3a unsilencing properties, similar to topotecan, and of these, indotecan (LMP400) was the most effective based on estimated Emax (maximum response of unsilencing paternal Ube3a) and EC50 (half maximal effective concentration). Conclusions We provide pharmacological profiles of indenoisoquinoline-derived Top1 inhibitors as paternal Ube3a unsilencers. All 13 tested compounds were effective at unsilencing paternal Ube3a, although with variable efficacy and potency. Indotecan (LMP400) demonstrated a better pharmacological profile of Ube3a unsilencing compared to our previous lead compound, topotecan. Taken together, indotecan and its structural analogues are potential AS therapeutics whose translational potential in AS treatment should be further assessed.

Individuals with Angelman syndrome (AS) suffer sleep disturbances that severely impair quality of life. Whether these disturbances arise from sleep or circadian clock dysfunction is currently unknown. Here, we explored the mechanistic basis for these sleep disorders in a mouse model of Angelman syndrome (Ube3am−/p+mice). Genetic deletion of the maternalUbe3aallele practically eliminates UBE3A protein from the brain ofUbe3am−/p+mice, because the paternal allele is epigenetically silenced in most neurons. However, we found that UBE3A protein was present in many neurons of the suprachiasmatic nucleus—the site of the mammalian circadian clock—indicating thatUbe3acan be expressed from both parental alleles in this brain region in adult mice. We found that whileUbe3am−/p+mice maintained relatively normal circadian rhythms of behavior and light-resetting, these mice exhibited consolidated locomotor activity and skipped the timed rest period (siesta) present in wild-type (Ube3am+/p+) mice. Electroencephalographic analysis revealed that alterations in sleep regulation were responsible for these overt changes in activity. Specifically,Ube3am−/p+mice have a markedly reduced capacity to accumulate sleep pressure, both during their active period and in response to forced sleep deprivation. Thus, our data indicate that the siesta is governed by sleep pressure, and thatUbe3ais an important regulator of sleep homeostasis. These preclinical findings suggest that therapeutic interventions that target mechanisms of sleep homeostasis may improve sleep quality in individuals with AS.SIGNIFICANCE STATEMENTAngelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of expression of the maternal copy of theUBE3Agene. Individuals with AS have severe sleep dysfunction that affects their cognition and presents challenges to their caregivers. Unfortunately, current treatment strategies have limited efficacy due to a poor understanding of the mechanisms underlying sleep disruptions in AS. Here we demonstrate that abnormal sleep patterns arise from a deficit in accumulation of sleep drive, uncovering theUbe3agene as a novel genetic regulator of sleep homeostasis. Our findings encourage a re-evaluation of current treatment strategies for sleep dysfunction in AS, and suggest that interventions that promote increased sleep drive may alleviate sleep disturbances in individuals with AS.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by mutation or deletion of the maternalUBE3Aallele. The maternalUBE3Aallele is expressed in nearly all neurons of the brain and spinal cord, whereas the paternalUBE3Aallele is repressed by an extremely long antisense transcript (UBE3A-ATS). Little is known about expression ofUBE3Ain the peripheral nervous system, where loss of maternalUBE3Amight contribute to AS phenotypes. Here we sought to examine maternal and paternalUbe3aexpression in DRGs neurons and to evaluate whether nociceptive responses were affected in AS model mice (global deletion of maternalUbe3aallele;Ube3am−/p+). We found that most large-diameter proprioceptive and mechanosensitive DRG neurons expressed maternalUbe3aand paternalUbe3a-ATS. In contrast, most small-diameter neurons expressedUbe3abiallelically and had low to undetectable levels ofUbe3a-ATS. Analysis of single-cell DRG transcriptomes further suggested thatUbe3ais expressed monoallelically in myelinated large-diameter neurons and biallelically in unmyelinated small-diameter neurons. Behavioral responses to some noxious thermal and mechanical stimuli were enhanced in male and female AS model mice; however, nociceptive responses were not altered by the conditional deletion of maternalUbe3ain the DRG. These data suggest that the enhanced nociceptive responses in AS model mice are due to loss of maternalUbe3ain the central, but not peripheral, nervous system. Our study provides new insights into sensory processing deficits associated with AS.SIGNIFICANCE STATEMENTAngelman syndrome (AS) is a neurodevelopmental disorder caused by loss or mutation of the maternalUBE3Aallele. While sensory processing deficits are frequently associated with AS, it is currently unknown whetherUbe3ais expressed in peripheral sensory neurons or whether maternal deletion ofUbe3aaffects somatosensory responses. Here, we found thatUbe3ais primarily expressed from the maternally inherited allele in myelinated large-diameter sensory neurons and biallelically expressed in unmyelinated small-diameter neurons. Nociceptive responses to select noxious thermal and mechanical stimuli were enhanced following global, but not sensory neuron-specific, deletion of maternalUbe3ain mice. These data suggest that maternal loss ofUbe3aaffects nociception via a central, but not peripheral mechanism, with implications for AS.

Injury of CNS nerve tracts remodels circuitry through dendritic spine loss and hyper-excitability, thus influencing recovery. Due to the complexity of the CNS, a mechanistic understanding of injury-induced synaptic remodeling remains unclear. Using microfluidic chambers to separate and injure distal axons, we show that axotomy causes retrograde dendritic spine loss at directly injured pyramidal neurons followed by retrograde presynaptic hyper-excitability. These remodeling events require activity at the site of injury, axon-to-soma signaling, and transcription. Similarly, directly injured corticospinal neurons in vivo also exhibit a specific increase in spiking following axon injury. Axotomy-induced hyper-excitability of cultured neurons coincides with elimination of inhibitory inputs onto injured neurons, including those formed onto dendritic spines. Netrin-1 downregulation occurs following axon injury and exogenous netrin-1 applied after injury normalizes spine density, presynaptic excitability, and inhibitory inputs at injured neurons. Our findings show that intrinsic signaling within damaged neurons regulates synaptic remodeling and involves netrin-1 signaling., Spinal cord injury can induce synaptic reorganization and remodeling in the brain. Here the authors study how severed distal axons signal back to the cell body to induce hyperexcitability, loss of inhibition and enhanced presynaptic release through netrin-1.

Autism spectrum disorder (ASD) is genetically heterogeneous with convergent symptomatology, suggesting common dysregulated pathways. We analyzed brain transcriptional changes in five mouse models of Pitt-Hopkins Syndrome (PTHS), a syndromic form of ASD caused by mutations in TCF4 (transcription factor 4, not TCF7L2 / T-Cell Factor 4). Analyses of differentially expressed genes (DEGs) highlighted oligodendrocyte (OL) dysregulation, which we confirmed in two additional mouse models of syndromic ASD (Ptenm3m4/m3m4 and Mecp2tm1.1Bird). The PTHS mouse models showed cell-autonomous reductions in OL numbers and myelination, functionally confirming OL transcriptional signatures. Next, we integrated PTHS mouse model DEGs with human idiopathic ASD postmortem brain RNA-seq data, and found significant enrichment of overlapping DEGs and common myelination-associated pathways. Importantly, DEGs from syndromic ASD mouse models, and reduced deconvoluted OL numbers, distinguished human idiopathic ASD cases from controls across three postmortem brain datasets. These results implicate disruptions in OL biology as a cellular mechanism in ASD pathology.

Autism spectrum disorder (ASD) affects approximately 1:68 individuals and has incalculable burdens on affected individuals, their families, and health care systems. While the genetic contributions to idiopathic ASD are heterogeneous and largely unknown, the causal mutations for syndromic forms of ASD - including truncations and copy number variants - provide a genetic toehold with which to gain mechanistic insights1-3. Models of these syndromic disorders have been used to better characterize the molecular and physiological processes disrupted by these mutations4. Two fundamental questions remain - how biologically similar are the mouse models of syndromic forms of ASD, and how relevant are these mouse models to their human analogs? To address these questions, we performed integrative transcriptomic analyses of seven independent mouse models of three syndromic forms of ASD generated across five laboratories, and assessed dysregulated genes and their pathways in human postmortem brain from patients with ASD and unaffected controls. These cross-species analyses converged on shared disruptions in myelination and axon development across both syndromic and idiopathic ASD, highlighting both the face validity of mouse models for these disorders and identifying novel convergent molecular phenotypes amendable to rescue with therapeutics.

Topotecan is a topoisomerase 1 (TOP1) inhibitor that is used to treat various forms of cancer. We recently found that topotecan reduces the expression of multiple long genes, including many neuronal genes linked to synapses and autism. However, whether topotecan alters synaptic protein levels and synapse function is currently unknown. Here we report that in primary cortical neurons, topotecan depleted synaptic proteins that are encoded by extremely long genes, including Neurexin-1, Neuroligin-1, Cntnap2, and GABA(A)β3. Topotecan also suppressed spontaneous network activity without affecting resting membrane potential, action potential threshold, or neuron health. Topotecan strongly suppressed inhibitory neurotransmission via pre- and postsynaptic mechanisms and reduced excitatory neurotransmission. The effects on synaptic protein levels and inhibitory neurotransmission were fully reversible upon drug washout. Collectively, our findings suggest that TOP1 controls the levels of multiple synaptic proteins and is required for normal excitatory and inhibitory synaptic transmission.

Genetic alterations of the maternal UBE3A allele result in Angelman syndrome (AS), a neurodevelopmental disorder characterized by severe developmental delay, lack of speech, and difficulty with movement and balance. The combined effects of maternal UBE3A mutation and cell type-specific epigenetic silencing of paternal UBE3A are hypothesized to result in a complete loss of functional UBE3A protein in neurons. However, the allelic specificity of UBE3A expression in neurons and other cell types in the brain has yet to be characterized throughout development, including the early postnatal period when AS phenotypes emerge. Here we define maternal and paternal allele-specific Ube3a protein expression throughout postnatal brain development in the mouse, a species which exhibits orthologous epigenetic silencing of paternal Ube3a in neurons and AS-like behavioral phenotypes subsequent to maternal Ube3a deletion. We find that neurons downregulate paternal Ube3a protein expression as they mature and, with the exception of neurons born from postnatal stem cell niches, do not express detectable paternal Ube3a beyond the first postnatal week. By contrast, neurons express maternal Ube3a throughout postnatal development, during which time localization of the protein becomes increasingly nuclear. Unlike neurons, astrocytes and oligodendrotyes biallelically express Ube3a. Notably, mature oligodendrocytes emerge as the predominant Ube3a-expressing glial cell type in the cortex and white matter tracts during postnatal development. These findings demonstrate the spatiotemporal characteristics of allele-specific Ube3a expression in key brain cell types, thereby improving our understanding of the developmental parameters of paternal Ube3a silencing and the cellular basis of AS.

Topoisomerases are expressed throughout the developing and adult brain and are mutated in some individuals with autism spectrum disorder (ASD). However, how topoisomerases are mechanistically connected to ASD is unknown. Here we find that topotecan, a topoisomerase 1 (TOP1) inhibitor, dose-dependently reduces the expression of extremely long genes in mouse and human neurons, including nearly all genes that are longer than 200 kilobases. Expression of long genes is also reduced after knockdown of Top1 or Top2b in neurons, highlighting that both enzymes are required for full expression of long genes. By mapping RNA polymerase II density genome-wide in neurons, we found that this length-dependent effect on gene expression was due to impaired transcription elongation. Interestingly, many high-confidence ASD candidate genes are exceptionally long and were reduced in expression after TOP1 inhibition. Our findings suggest that chemicals and genetic mutations that impair topoisomerases could commonly contribute to ASD and other neurodevelopmental disorders. Reducing topoisomerase activity in mouse and human neurons is found to reduce the expression of long genes by impairing transcription elongation: among genes affected are numerous high-confidence candidates for autism spectrum disorder. Topoisomerases, enzymes involved in DNA winding, are expressed throughout the brain, and mutations have been discovered in some individuals with autism spectrum disorders (ASD). Mark Zylka and colleagues show that reducing topoisomerase activity selectively reduces the expression of long genes in mouse and human neurons by impairing transcription elongation. The authors note that many ASD candidate genes, including Cntnap2, Nrxn1 and Cntn4, are exceptionally long and confirm that expression of several ASD candidate genes is reduced by topoisomerase inhibition. These findings suggest that chemicals and genetic mutations that impair topoisomerases — and possibly other components of the transcription machinery — could contribute to ASD and other neurodevelopmental disorders.

NMDA receptors (NMDARs) are required for experience-driven plasticity during formative periods of brain development and are critical for neurotransmission throughout postnatal life. Most NMDAR functions have been ascribed to postsynaptic sites of action, but there is now an appreciation that presynaptic NMDARs (preNMDARs) can modulate neurotransmitter release in many brain regions, including the neocortex. Despite these advances, the cellular mechanisms by which preNMDARs can affect neurotransmitter release are largely unknown. Here we interrogated preNMDAR functions pharmacologically to determine how these receptors promote spontaneous neurotransmitter release in mouse primary visual cortex. Our results provide three new insights into the mechanisms by which preNMDARs can function. First, preNMDARs can enhance spontaneous neurotransmitter release tonically with minimal extracellular Ca2+or with major sources of intracellular Ca2+blocked. Second, lowering extracellular Na+levels reduces the contribution of preNMDARs to spontaneous transmitter release significantly. Third, preNMDAR enhance transmitter release in part through protein kinase C signaling. These data demonstrate that preNMDARs can act through novel pathways to promote neurotransmitter release in the absence of action potentials.

Topoisomerase 1 (TOP1) inhibitors, including camptothecin and topotecan, covalently trap TOP1 on DNA, creating cleavage complexes (cc’s) that must be resolved before gene transcription and DNA replication can proceed. We previously found that topotecan reduces the expression of long (>100 kb) genes and unsilences the paternal allele of Ube3a in neurons. Here, we sought to evaluate overlap between TOP1cc-dependent and -independent gene regulation in neurons. To do this, we utilized Top1 conditional knockout mice, Top1 knockdown, the CRISPR-Cas9 system to delete Top1, TOP1 catalytic inhibitors that do not generate TOP1cc’s, and a TOP1 mutation (T718A) that stabilizes TOP1cc’s. We found that topotecan treatment significantly alters the expression of many more genes, including long neuronal genes, immediate early genes, and paternal Ube3a, when compared to Top1 deletion. Our data show that topotecan has a stronger effect on neuronal transcription than Top1 deletion, and identifies TOP1cc-dependent and -independent contributions to gene expression.

Mutations or deletions of the maternal allele of the UBE3A gene cause Angelman syndrome (AS), a severe neurodevelopmental disorder. The paternal UBE3A/Ube3a allele becomes epigenetically silenced in most neurons during postnatal development in humans and mice; hence, loss of the maternal allele largely eliminates neuronal expression of UBE3A protein. However, recent studies suggest that paternal Ube3a may escape silencing in certain neuron populations, allowing for persistent expression of paternal UBE3A protein. Here we extend evidence in AS model mice (Ube3am–/p+) of paternal UBE3A expression within the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Paternal UBE3A-positive cells in the SCN show partial colocalization with the neuropeptide arginine vasopressin (AVP) and clock proteins (PER2 and BMAL1), supporting that paternal UBE3A expression in the SCN is often of neuronal origin. Paternal UBE3A also partially colocalizes with a marker of neural progenitors, SOX2, implying that relaxed or incomplete imprinting of paternal Ube3a reflects an overall immature molecular phenotype. Our findings highlight the complexity of Ube3a imprinting in the brain and illuminate a subpopulation of SCN neurons as a focal point for future studies aimed at understanding the mechanisms of Ube3a imprinting.

Aberrant signaling through the Raf/MEK/ERK (ERK/MAPK) pathway causes pathology in a family of neurodevelopmental disorders known as 'RASopathies' and is implicated in autism pathogenesis. Here, we have determined the functions of ERK/MAPK signaling in developing neocortical excitatory neurons. Our data reveal a critical requirement for ERK/MAPK signaling in the morphological development and survival of large Ctip2+ neurons in layer 5. Loss of Map2k1/2 (Mek1/2) led to deficits in corticospinal tract formation and subsequent corticospinal neuron apoptosis. ERK/MAPK hyperactivation also led to reduced corticospinal axon elongation, but was associated with enhanced arborization. ERK/MAPK signaling was dispensable for axonal outgrowth of layer 2/3 callosal neurons. However, Map2k1/2 deletion led to reduced expression of Arc and enhanced intrinsic excitability in both layers 2/3 and 5, in addition to imbalanced synaptic excitation and inhibition. These data demonstrate selective requirements for ERK/MAPK signaling in layer 5 circuit development and general effects on cortical pyramidal neuron excitability. DOI: http://dx.doi.org/10.7554/eLife.11123.001, eLife digest In the nervous system, cells called neurons form networks that relay information in the form of electrical signals around the brain and the rest of the body. Typically, an electrical signal travels from branch-like structures at one end of the cell, through the cell body and then along a long fiber called an axon to reach junctions with another neurons. The connections between neurons start to form as the nervous system develops in the embryo, and any errors or delays in this process can cause severe neurological disorders and intellectual disabilities. For example, genetic mutations affecting a communication system within cells known as the ERK/MAPK pathway can lead to a family of syndromes called the “RASopathies”. Abnormalities in this pathway may also contribute to certain types of autism. However, it is not clear how alterations to the ERK/MAPK pathway cause these conditions. Xing et al. investigated whether ERK/MAPK signaling regulates the formation of connections between neurons and the activity of neurons in mouse brains. The experiments showed that the growth of axons that extend from an area of the brain called the cerebral cortex towards the spinal cord are particularly sensitive to changes in the level of signaling through the ERK/MAPK pathway. On the other hand, inhibiting the pathway has relatively little effect on the growth of axons within the cerebral cortex. Further experiments showed that many neurons in the cerebral cortex require the ERK/MAPK pathway to activate genes that alter neuronal activity and the strength of the connections between neurons. Xing et al.’s findings suggest that defects in the connections between the cerebral cortex and different regions of the nervous system may contribute to the symptoms observed in patients with conditions linked to alterations in ERK/MAPK activity. Future studies will focus on understanding the molecular mechanisms by which ERK/MAPK pathway influences the organization and activity of neuron circuits during the development of the nervous system. DOI: http://dx.doi.org/10.7554/eLife.11123.002

Motivated reward-seeking behaviours are governed by dopaminergic ventral tegmental area projections to the nucleus accumbens. In addition to dopamine, these mesoaccumbal terminals co-release other neurotransmitters including glutamate and GABA, whose roles in regulating motivated behaviours are currently being investigated. Here we demonstrate that loss of the E3-ubiquitin ligase, UBE3A, from tyrosine hydroxylase-expressing neurons impairs mesoaccumbal, non-canonical GABA co-release and enhances reward-seeking behaviour measured by optical self-stimulation., Mesoaccumbal terminals within the VTA are known to co-release both GABA and dopamine, although the functional role of the former has yet to be determined. Here, the authors find that non-canonical GABA release is regulated by the E3-ubiquitin ligase, UBE3A, and enhances optogenetic self-stimulation.

Key points • Cortical glycine receptors (GlyRs) flux chloride based on a local chloride gradient set by chloride transporters; this gradient changes with age. • In acute slices from the developing mouse visual cortex, tonic GlyR activity increases spontaneous excitatory neurotransmitter release in a calcium-dependent manner. • Glycine transporters, localized mainly to astrocytes, regulate this tonic activity. • After a critical period of early development, GlyRs are no longer tonically active and become hyperpolarizing, inhibiting spontaneous neurotransmitter release. • These results define mechanisms that contribute to baseline neurotransmission during critical periods of neuronal development, and help identify synaptic functions that could be impacted by GlyR dysfunction. Abstract Glycine receptors (GlyRs) are found in most areas of the brain, and their dysfunction can cause severe neurological disorders. While traditionally thought of as inhibitory receptors, presynaptic-acting GlyRs (preGlyRs) can also facilitate glutamate release under certain circumstances, although the underlying molecular mechanisms are unknown. In the current study, we sought to better understand the role of GlyRs in the facilitation of excitatory neurotransmitter release in mouse visual cortex. Using whole-cell recordings, we found that preGlyRs facilitate glutamate release in developing, but not adult, visual cortex. The glycinergic enhancement of neurotransmitter release in early development depends on the high intracellular to extracellular Cl− gradient maintained by the Na+–K+–2Cl− cotransporter and requires Ca2+ entry through voltage-gated Ca2+ channels. The glycine transporter 1, localized to glial cells, regulates extracellular glycine concentration and the activation of these preGlyRs. Our findings demonstrate a developmentally regulated mechanism for controlling excitatory neurotransmitter release in the neocortex.

Cancer drugs that can potentially treat Angelman syndrome are identified. Genomic imprinting disorders arise owing to a loss of function of non-imprinted alleles expressed from one parent. In Angelman syndrome, a neurodevelopmental disorder caused by dysfunction of the maternal allele of the Ube3a gene, the paternal allele remains intact but is epigenetically silenced. Benjamin Philpot and colleagues perform an unbiased drug screen on mouse cortical neurons expressing fluorescent Ube3a and identify topoisomerase inhibitors that are capable of activating paternal Ube3a, including topotecan, a cancer therapeutic approved by the US Food and Drug Administration. When the drug is delivered in vivo, paternal Ube3a is activated in multiple regions of the brain, and effects persist for several weeks after drug cessation. This demonstrates a potential method for reactivating dormant alleles of imprinted genes, which may be a therapeutic strategy in disorders such as Angelman syndrome. Angelman syndrome is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of the ubiquitin protein ligase E3A (UBE3A)1,2,3. In neurons, the paternal allele of UBE3A is intact but epigenetically silenced4,5,6, raising the possibility that Angelman syndrome could be treated by activating this silenced allele to restore functional UBE3A protein7,8. Using an unbiased, high-content screen in primary cortical neurons from mice, we identify twelve topoisomerase I inhibitors and four topoisomerase II inhibitors that unsilence the paternal Ube3a allele. These drugs included topotecan, irinotecan, etoposide and dexrazoxane (ICRF-187). At nanomolar concentrations, topotecan upregulated catalytically active UBE3A in neurons from maternal Ube3a-null mice. Topotecan concomitantly downregulated expression of the Ube3a antisense transcript that overlaps the paternal copy of Ube3a9,10,11. These results indicate that topotecan unsilences Ube3a in cis by reducing transcription of an imprinted antisense RNA. When administered in vivo, topotecan unsilenced the paternal Ube3a allele in several regions of the nervous system, including neurons in the hippocampus, neocortex, striatum, cerebellum and spinal cord. Paternal expression of Ube3a remained elevated in a subset of spinal cord neurons for at least 12 weeks after cessation of topotecan treatment, indicating that transient topoisomerase inhibition can have enduring effects on gene expression. Although potential off-target effects remain to be investigated, our findings suggest a therapeutic strategy for reactivating the functional but dormant allele of Ube3a in patients with Angelman syndrome.

Summary Neuronal activity regulates the transcription and translation of the immediate-early gene Arc/Arg3.1, a key mediator of synaptic plasticity. Proteasome-dependent degradation of Arc tightly limits its temporal expression, yet the significance of this regulation remains unknown. We disrupted the temporal control of Arc degradation by creating an Arc knockin mouse (ArcKR) where the predominant Arc ubiquitination sites were mutated. ArcKR mice had intact spatial learning but showed specific deficits in selecting an optimal strategy during reversal learning. This cognitive inflexibility was coupled to changes in Arc mRNA and protein expression resulting in a reduced threshold to induce mGluR-LTD and enhanced mGluR-LTD amplitude. These findings show that the abnormal persistence of Arc protein limits the dynamic range of Arc signaling pathways specifically during reversal learning. Our work illuminates how the precise temporal control of activity-dependent molecules, such as Arc, regulates synaptic plasticity and is crucial for cognition., Graphical Abstract, Highlights • Mutation of Arc ubiquitination sites slows Arc degradation • Arc knockin mice have enhanced mGluR-LTD • The persistence of Arc reduces cognitive flexibility, Ubiquitin-dependent proteasomal degradation of Arc tightly limits its temporal expression, yet the significance of this regulation remains unknown. We generated a mouse disrupting the temporal control of Arc removal (ArcKR). ArcKR mice exhibit enhanced mGluR-LTD and have impaired cognitive flexibility.

Bidirectional synaptic plasticity during development ensures that appropriate synapses in the brain are strengthened and maintained while inappropriate connections are weakened and eliminated. This plasticity is well illustrated in mouse visual cortex, where monocular deprivation during early postnatal development leads to a rapid depression of inputs from the deprived eye and a delayed strengthening of inputs from the non-deprived eye. The mechanisms that control these bidirectional synaptic modifications remain controversial. Here we demonstrate, both in vitro and in vivo, that genetic deletion or reduction of the NR2A NMDA receptor subunit impairs activity-dependent weakening of synapses and enhances the strengthening of synapses. Although brief monocular deprivation in juvenile WT mice normally causes a profound depression of the deprived-eye response without a change in the non-deprived eye response, NR2A-knockout mice fail to exhibit deprivation-induced depression and instead exhibit precocious potentiation of the non-deprived eye inputs. These data support the hypothesis that a reduction in the NR2A/B ratio during monocular deprivation is permissive for the compensatory potentiation of non-deprived inputs.

NMDA-type glutamate receptors (NMDARs) mediate many forms of synaptic plasticity. These tetrameric receptors consist of two obligatory NR1 subunits and two regulatory subunits, usually a combination of NR2A and NR2B. In the neonatal neocortex NR2B-containing NMDARs predominate, and sensory experience facilitates a developmental switch in which NR2A levels increase relative to NR2B. In this review, we clarify the roles of NR2 subunits in synaptic plasticity, and argue that a primary role of this shift is to control the threshold, rather than determining the direction, for modifying synaptic strength. We also discuss recent studies that illuminate the mechanisms regulating NR2 subunits, and suggest that the NR2A/NR2B ratio is regulated by multiple means, which may control the ratio both locally at individual synapses and globally in a cell-wide manner. Finally, we use the visual cortex as a model system to illustrate how activity-dependent modifications in the NR2A/NR2B ratio may contribute to the development of cortical functions.

The suggestion that NMDA receptor (NMDAR)-dependent plasticity is subunit specific, with NR2B-types required for long-term depression (LTD) and NR2A-types critical for the induction of long-term potentiation (LTP), has generated much attention and considerable debate. By investigating the suggested subunit-specific roles of NMDARs in the mouse primary visual cortex over development, we report several important findings that clarify the roles of NMDAR subtypes in synaptic plasticity. We observed that LTD was not attenuated by application of ifenprodil, an NR2B-type antagonist, or NVP-AAM007, a less selective NR2A-type antagonist. However, we were surprised that NVP-AAM007 completely blocked adult LTP (postnatal day (P) 45–90), while only modestly affecting juvenile LTP (P21-28). To assess whether this developmental transition reflected an increasing role for NR2A-type receptors with maturity, we characterized the specificity of NVP-AAM007. We found not only that NVP-AAM007 lacks discernable subunit specificity but also that the effects of NVP-AAM077 on LTP could be mimicked using subsaturating concentrations of APV, a global NMDAR antagonist. These results indicate that the effects of NVP-AAM077 on synaptic plasticity are largely explained by nonspecific blockade of NMDARs. Moreover our findings are the first to reveal a developmental increase in the sensitivity of LTP to NMDAR antagonism. We suggest that discrepant reports describing the effect of NVP-AAM077 on LTP may be partially explained by this developmental shift in the properties of LTP. These results indicate that the degree of NMDAR activation required for LTP increases with development, providing insight into a novel underlying mechanism governing the properties of synaptic plasticity.

During neuronal activity, extracellular potassium concentration ([K+]out) becomes elevated and, if uncorrected, causes neuronal depolarization, hyperexcitability, and seizures. Clearance of K+from the extracellular space, termed K+spatial buffering, is considered to be an important function of astrocytes. Results from a number of studies suggest that maintenance of [K+]outby astrocytes is mediated by K+uptake through the inward-rectifying Kir4.1 channels. To study the role of this channel in astrocyte physiology and neuronal excitability, we generated a conditional knock-out (cKO) of Kir4.1 directed to astrocytes via the human glial fibrillary acidic protein promotergfa2. Kir4.1 cKO mice die prematurely and display severe ataxia and stress-induced seizures. Electrophysiological recordings revealed severe depolarization of both passive astrocytes and complex glia in Kir4.1 cKO hippocampal slices. Complex cell depolarization appears to be a direct consequence of Kir4.1 removal, whereas passive astrocyte depolarization seems to arise from an indirect developmental process. Furthermore, we observed a significant loss of complex glia, suggestive of a role for Kir4.1 in astrocyte development. Kir4.1 cKO passive astrocytes displayed a marked impairment of both K+and glutamate uptake. Surprisingly, membrane and action potential properties of CA1 pyramidal neurons, as well as basal synaptic transmission in the CA1 stratum radiatum appeared unaffected, whereas spontaneous neuronal activity was reduced in the Kir4.1 cKO. However, high-frequency stimulation revealed greatly elevated posttetanic potentiation and short-term potentiation in Kir4.1 cKO hippocampus. Our findings implicate a role for glial Kir4.1 channel subunit in the modulation of synaptic strength.

Phosphatidylinositol transfer proteins (PITPs) bind phosphatidylinositol (PtdIns) and phosphatidylcholine and play diverse roles in coordinating lipid metabolism/signaling with intracellular functions. The underlying mechanisms remain unclear. Genetic ablation of PITPalpha in mice results in neonatal lethality characterized by intestinal and hepatic steatosis, spinocerebellar neurodegeneration, and glucose homeostatic defects. We report that mice expressing a PITPalpha selectively ablated for PtdIns binding activity (Pitpalpha(T59D)), as the sole source of PITPalpha, exhibit phenotypes that recapitulate those of authentic PITPalpha nullizygotes. Analyses of mice with graded reductions in PITPalpha activity reveal proportionately graded reductions in lifespan, demonstrate that intestinal steatosis and hypoglycemia are apparent only when PITPalpha protein levels are strongly reduced (>or=90%), and correlate steatotic and glucose homeostatic defects with cerebellar inflammatory disease. Finally, reconstitution of PITPalpha expression in the small intestine substantially corrects the chylomicron retention disease and cerebellar inflammation of Pitpalpha(0/0) neonates, but does not rescue neonatal lethality in these animals. These data demonstrate that PtdIns binding is an essential functional property of PITPalpha in vivo, and suggest a causal linkage between defects in lipid transport and glucose homeostasis and cerebellar inflammatory disease. Finally, the data also demonstrate intrinsic neuronal deficits in PITPalpha-deficient mice that are independent of intestinal lipid transport defects and hypoglycemia.

Presynaptic NMDA receptors (preNMDARs) play pivotal roles in excitatory neurotransmission and synaptic plasticity. They facilitate presynaptic neurotransmitter release and modulate mechanisms controlling synaptic maturation and plasticity during formative periods of brain development. There is an increasing understanding of the roles of preNMDARs in experience-dependent synaptic and circuit-specific computation. In this review, we summarize the latest understanding of compartment-specific expression and function of preNMDARs, and how they contribute to synapse-specific and circuit-level information processing.

Angelman syndrome (AS) is a severe neurological disorder that is associated with prominent movement and balance impairments that are widely considered to be due to defects of cerebellar origin. Here, using the cerebellar-specific vestibulo-ocular reflex (VOR) paradigm, we determined that cerebellar function is only mildly impaired in the Ube3a(m-/P+) mouse model of AS. VOR phase-reversal learning was singularly impaired in these animals and correlated with reduced tonic inhibition between Golgi cells and granule cells. Purkinje cell physiology, in contrast, was normal in AS mice as shown by synaptic plasticity and spontaneous firing properties that resembled those of controls. Accordingly, neither VOR phas-ereversal learning nor locomotion was impaired following selective deletion of Ube3a in Purkinje cells. However, genetic normalization of alpha CaMKII inhibitory phosphorylation fully rescued locomotor deficits despite failing to improve cerebellar learning in AS mice, suggesting extracerebellar circuit involvement in locomotor learning. We confirmed this hypothesis through cerebellum-specific reinstatement of Ube3a, which ameliorated cerebellar learning deficits but did not rescue locomotor deficits. This double dissociation of locomotion and cerebellar phenotypes strongly suggests that the locomotor deficits of AS mice do not arise from impaired cerebellar cortex function. Our results provide important insights into the etiology of the motor deficits associated with AS.

Increasing data suggest that glutamate might act as a cell-signaling molecule in non-neuronal tissues such as the skin. Here we demonstrate the presence of functional N-methyl-D-aspartate (NMDA)-type glutamate receptors in human keratinocytes. NMDA receptor expression strongly reflects the degree of cell-to-cell contact. Wounding polarizes the expression of NMDA receptors in keratinocytes involved in re-epithelialization, and the process of re-epithelialization is inhibited by NMDA receptor activation. We also demonstrate that squamous cell carcinomas lack NMDA receptors. Our data suggest that Ca2+ entry through NMDA receptors influences the cycle of keratinocyte proliferation, differentiation, and migration during epithelialization. Moreover, NMDA receptor activation might play a role in contact-mediated inhibition of growth, a process that is absent during neoplastic pathology. This receptor may serve as a pharmacological target for modulating keratinocyte behavior and treating cutaneous disorders.

Summary The quiescence of adult neural stem cells (NSCs) is regulated by local parvalbumin (PV) interneurons within the dentate gyrus (DG). Little is known about how local PV interneurons communicate with distal brain regions to regulate NSCs and hippocampal neurogenesis. Here, we identify GABAergic projection neurons from the medial septum (MS) as the major afferents to dentate PV interneurons. Surprisingly, dentate PV interneurons are depolarized by GABA signaling, which is in sharp contrast to most mature neurons hyperpolarized by GABA. Functionally, these long-range GABAergic inputs are necessary and sufficient to maintain adult NSC quiescence and ablating them leads to NSC activation and subsequent depletion of the NSC pool. Taken together, these findings delineate a GABAergic network involving long-range GABAergic projection neurons and local PV interneurons that couples dynamic brain activity to the neurogenic niche in controlling NSC quiescence and hippocampal neurogenesis.

Primary open-angle glaucoma (POAG) is characterized by progressive neurodegeneration of retinal ganglion cells (RGCs). Why RGCs degenerate in low-pressure POAG remains poorly understood. To gain mechanistic insights, we developed a novel mouse model based on a mutation in human optineurin associated with hereditary, low-pressure POAG. This mouse improves the design and phenotype of currently available optineurin mice, which showed high global overexpression. Although both 18-month-old optineurin and nontransgenic control mice showed an age-related decrease in healthy axons and RGCs, the expression of mutant optineurin enhanced axonal degeneration and decreased RGC survival. Mouse visual function was determined using visual evoked potentials, which revealed specific visual impairment in contrast sensitivity. The E50K optineurin transgenic mouse described here exhibited clinical features of POAG and may be useful for mechanistic dissection of POAG and therapeutic development.

The effort and cost of obtaining neurons for large-scale screens has limited drug discovery in neuroscience. To overcome these obstacles, we fabricated arrays of releasable polystyrene micro-rafts to generate thousands of uniform, mobile neuron mini-cultures. These mini-cultures sustain synaptically-active neurons which can be easily transferred, thus increasing screening throughput by >30-fold. Compared to conventional methods, micro-raft cultures exhibited significantly improved neuronal viability and sample-to-sample consistency. We validated the screening utility of these mini-cultures for both mouse neurons and human induced pluripotent stem cell-derived neurons by successfully detecting disease-related defects in synaptic transmission and identifying candidate small molecule therapeutics. This affordable high-throughput approach has the potential to transform drug discovery in neuroscience.

Genomic imprinting describes an epigenetic process through which genes can be expressed in a parent-of-origin-specific manner. The monoallelic expression of imprinted genes renders them particularly susceptible to disease causing mutations. A large proportion of imprinted genes are expressed in the brain, but little is known about their functions. Indeed, it has proven difficult to identify cell type-specific imprinted genes due to the heterogeneity of cell types within the brain. Here we used laser capture microdissection of visual cortical neurons and found evidence that sorting nexin 14 (Snx14) is a neuronally imprinted gene in mice. SNX14 protein levels are high in the brain and progressively increase during neuronal development and maturation. Snx14 knockdown reduces intrinsic excitability and severely impairs both excitatory and inhibitory synaptic transmission. These data reveal a role for monoallelic Snx14 expression in maintaining normal neuronal excitability and synaptic transmission.

Calcineurin is a calcium-dependent protein phosphatase that has been implicated in various aspects of synaptic plasticity. By using conditional gene-targeting techniques, we created mice in which calcineurin activity is disrupted specifically in the adult forebrain. At hippocampal Schaffer collateral-CA1 synapses, LTD was significantly diminished, and there was a significant shift in the LTD/LTP modification threshold in mutant mice. Strikingly, although performance was normal in hippocampus-dependent reference memory tasks, including contextual fear conditioning and the Morris water maze, the mutant mice were impaired in hippocampus-dependent working and episodic-like memory tasks, including the delayed matching-to-place task and the radial maze task. Our results define a critical role for calcineurin in bidirectional synaptic plasticity and suggest a novel mechanistic distinction between working/episodic-like memory and reference memory.

Optimal function of neuronal networks requires interplay between rapid forms of Hebbian plasticity and homeostatic mechanisms that adjust the threshold for plasticity, termed metaplasticity. Numerous forms of rapid synapse plasticity have been examined in detail. However, the rules that govern synaptic metaplasticity are much less clear. Here we demonstrate a local subunit-specific switch in NMDA receptors that alternately primes or prevents potentiation at single synapses. Prolonged suppression of neurotransmitter release enhances NMDA receptor currents, increases the number of functional NMDA receptors containing NR2B, and augments calcium transients at single dendritic spines. This local switch in NMDA receptors requires spontaneous glutamate release, but is independent of action potentials. Moreover, single inactivated synapses exhibit a lower induction threshold for both long-term synaptic potentiation and plasticity-induced spine growth. Thus, spontaneous glutamate release adjusts plasticity threshold at single synapses by local regulation of NMDA receptors, providing a novel spatially delimited form of synaptic metaplasticity.

Sensory experience is crucial in the refinement of synaptic connections in the brain during development. It has been suggested that some forms of experience-dependent synaptic plasticity in vivo are associated with changes in the complement of postsynaptic glutamate receptors, although direct evidence has been lacking. Here we show that visual experience triggers the rapid synaptic insertion of new NMDA receptors in visual cortex. The new receptors have a higher proportion of NR2A subunits and, as a consequence, different functional properties. This effect of experience requires NMDA receptor activation and protein synthesis. Thus, rapid regulation of postsynaptic glutamate receptors is one mechanism for developmental plasticity in the brain. Changes in NMDA receptor expression provide a mechanism by which brief sensory experience can regulate the properties of NMDA receptor-dependent plasticity in visual cortex.

Microtubule-associated protein 2 (MAP2) is a neuron-specific cytoskeletal protein, enriched in dendrites and cell bodies, that helps determine dendritic shape. MAP2 regulates microtubule stability in a phosphorylation-dependent manner. The present study used immunocytochemistry with phosphoepitope-specific and phosphorylation state-independent antibodies to examine experience-dependent changes in MAP2 expression during postnatal development of the olfactory bulb. Our results demonstrate that immunoreactivity reflecting total MAP2 expression reaches a maximal level by postnatal day 20 (P20). The degree of staining for phosphoindependent forms of MAP2 is relatively unaffected by blocking odorant passage to one half the nasal epithelium via unilateral naris closure, a manipulation that attenuates physiological activity in the bulb. However, olfactory restriction from P1 dramatically reduces immunoreactivity for antibody AP18, which recognizes MAP2 only when phosphorylated on Ser136. Quantification of staining in the granule cell layer indicates that the greatest difference (64%) between control and experimental bulbs occurs after occlusion from P1 to P30 compared with animals deprived from P1 to P10 or P1 to P20. The shift in MAP2 phosphorylation occurs even when deprivation is delayed until P30, after the sensitive period for experience-dependent changes in bulb volume. Thus, the degree of the phosphorylation shift depends on the duration but not the time of onset of naris closure.Because staining for phosphorylation-independent forms of MAP2 is unchanged by naris closure, the total amount of the protein per unit area is probably not significantly altered. However, the large reductions of AP18-immunoreactivity in the bulb after olfactory restriction suggest that there is an activity-dependent stimulation of MAP2 phosphorylation.