The Perineuronal Net Protein Brevican Acts in Nucleus Accumbens Parvalbumin-Expressing Interneurons of Adult Mice to Regulate Excitatory Synaptic Inputs and Motivated Behaviors.

BACKGROUND: Experience-dependent functional adaptation of nucleus accumbens (NAc) circuitry underlies the development and expression of reward-motivated behaviors. Parvalbumin-expressing GABAergic (gamma-aminobutyric acidergic) interneurons (PVINs) within the NAc are required for this process. Perineuronal nets (PNNs) are extracellular matrix structures enriched around PVINs that arise during development and have been proposed to mediate brain circuit stability. However, their function in the adult NAc is largely unknown. Here, we studied the developmental emergence and adult regulation of PNNs in the NAc of male and female mice and examined the cellular and behavioral consequences of reducing the PNN component brevican in NAc PVINs. METHODS: We characterized the expression of PNN components in mouse NAc using immunofluorescence and RNA in situ hybridization. We lowered brevican in NAc PVINs of adult mice using an intersectional viral and genetic method and quantified the effects on synaptic inputs to NAc PVINs and reward-motivated learning. RESULTS: PNNs around NAc PVINs were developmentally regulated and appeared during adolescence. In the adult NAc, PVIN PNNs were also dynamically regulated by cocaine. Transcription of the gene that encodes brevican was regulated in a cell type- and isoform-specific manner in the NAc, with the membrane-tethered form of brevican being highly enriched in PVINs. Lowering brevican in NAc PVINs of adult mice decreased their excitatory inputs and enhanced both short-term novel object recognition and cocaine-induced conditioned place preference. CONCLUSIONS: Regulation of brevican in NAc PVINs of adult mice modulates their excitatory synaptic drive and sets experience thresholds for the development of motivated behaviors driven by rewarding stimuli.

Genome binding properties of Zic transcription factors underlie their changing functions during neuronal maturation.

Background: The Zic family of transcription factors (TFs) promote both proliferation and maturation of cerebellar granule neurons (CGNs), raising the question of how a single, constitutively expressed TF family can support distinct developmental processes. Here we use an integrative experimental and bioinformatic approach to discover the regulatory relationship between Zic TF binding and changing programs of gene transcription during CGN differentiation. Results: We first established a bioinformatic pipeline to integrate Zic ChIP-seq data from the developing mouse cerebellum with other genomic datasets from the same tissue. In newborn CGNs, Zic TF binding predominates at active enhancers that are co-bound by developmentally-regulated TFs including Atoh1, whereas in mature CGNs, Zic TF binding consolidates toward promoters where it co-localizes with activity-regulated TFs. We then performed CUT&RUN-seq in differentiating CGNs to define both the time course of developmental shifts in Zic TF binding and their relationship to gene expression. Mapping Zic TF binding sites to genes using chromatin looping, we identified the set of Zic target genes that have altered expression in RNA-seq from Zic1 or Zic2 knockdown CGNs. Conclusion: Our data show that Zic TFs are required for both induction and repression of distinct, developmentally regulated target genes through a mechanism that is largely independent of changes in Zic TF binding. We suggest that the differential collaboration of Zic TFs with other TF families underlies the shift in their biological functions across CGN development.

Imaging the binding of MECP2 to DNA.

Mutations in the methyl-DNA binding domain of MECP2 cause Rett syndrome; however, distinct mutations are associated with different severity of the disease. Live-cell imaging and single-molecule tracking are sensitive methods to quantify the DNA binding affinity and diffusion dynamics of nuclear proteins. In this issue of Genes & Development, Zhou and colleagues (pp. 883-900) used these imaging methods to quantitatively describe the partial loss of DNA binding resulting from a novel pathological MECP2 mutation with intermediate disease severity. These data demonstrate how single-molecule tracking can advance understanding of the molecular mechanisms connecting MECP2 mutations with Rett syndrome pathophysiology.

Single-Nucleus Transcriptional Profiling of GAD2-Positive Neurons From Mouse Lateral Habenula Reveals Distinct Expression of Neurotransmission- and Depression-Related Genes.

Background: Glutamatergic projection neurons of the lateral habenula (LHb) drive behavioral state modulation by regulating the activity of midbrain monoaminergic neurons. Identifying circuit mechanisms that modulate LHb output is of interest for understanding control of motivated behaviors. Methods: A small population of neurons within the medial subnucleus of the mouse LHb express the GABAergic (gamma-aminobutyric acidergic)-synthesizing enzyme GAD2, and they can inhibit nearby LHb projection neurons; however, these neurons lack markers of classic inhibitory interneurons, and they coexpress the vesicular glutamate transporter VGLUT2. To determine the molecular phenotype of these neurons, we genetically tagged the nuclei of GAD2-positive cells and used fluorescence-activated nuclear sorting to isolate and enrich these nuclei for single-nucleus RNA sequencing. Results: Our data confirm that GAD2+/VGLUT2+ neurons intrinsic to the LHb coexpress markers of both glutamatergic and GABAergic transmission and that they are transcriptionally distinct from either GABAergic interneurons or habenular glutamatergic neurons. We identify gene expression programs within these cells that show sex-specific differences in expression and that are implicated in major depressive disorder, which has been linked to LHb hyperactivity. Finally, we identify the Ntng2 gene encoding the cell adhesion protein netrin-G2 as a marker of LHb GAD2+/VGLUT2+ neurons and a gene product that may contribute to their target projections. Conclusions: These data show the value of using genetic enrichment of rare cell types for transcriptome studies, and they advance understanding of the molecular composition of a functionally important class of GAD2+ neurons in the LHb.

Bidirectional regulation of postmitotic H3K27me3 distributions underlie cerebellar granule neuron maturation dynamics.

The functional maturation of neurons is a prolonged process that extends past the mitotic exit and is mediated by the chromatin-dependent orchestration of gene transcription programs. We find that expression of this maturation gene program in mouse cerebellar granule neurons (CGNs) requires dynamic changes in the genomic distribution of histone H3 lysine 27 trimethylation (H3K27me3), demonstrating a function for this chromatin modification beyond its role in cell fate specification. The developmental loss of H3K27me3 at promoters of genes activated as CGNs mature is facilitated by the lysine demethylase and ASD-risk gene, Kdm6b. Interestingly, inhibition of the H3K27 methyltransferase EZH2 in newborn CGNs not only blocks the repression of progenitor genes but also impairs the induction of mature CGN genes, showing the importance of bidirectional H3K27me3 regulation across the genome. These data demonstrate that H3K27me3 turnover in developing postmitotic neurons regulates the temporal coordination of gene expression programs that underlie functional neuronal maturation.

Single-nucleus transcriptional profiling of GAD2-positive neurons from mouse lateral habenula reveals distinct expression of neurotransmission- and depression-related genes.

Glutamatergic projection neurons of the lateral habenula (LHb) drive behavioral state modulation by regulating the activity of midbrain monoaminergic neurons. Identifying circuit mechanisms that modulate LHb output is of interest for understanding control of motivated behaviors. A small population of neurons within the medial subnucleus of the mouse LHb express the GABAergic synthesizing enzyme GAD2, and they can inhibit nearby LHb projection neurons; however, these neurons lack markers of classic inhibitory interneurons and they co-express the vesicular glutamate transporter VGLUT2. To determine the molecular phenotype of these neurons, we genetically tagged the nuclei of GAD2-positive cells and used fluorescence-activated nuclear sorting to isolate and enrich these nuclei for single nuclear RNA sequencing (FANS-snRNAseq). Our data confirm that GAD2+/VGLUT2+ neurons intrinsic to the LHb co-express markers of both glutamatergic and GABAergic transmission and that they are transcriptionally distinct from either GABAergic interneurons or habenular glutamatergic neurons. We identify gene expression programs within these cells that show sex-specific differences in expression and that are implicated in major depressive disorder (MDD), which has been linked to LHb hyperactivity. Finally, we identify the Ntng2 gene encoding the cell adhesion protein Netrin-G2 as a marker of LHb GAD2+/VGLUT+ neurons and a gene product that may contribute to their target projections. These data show the value of using genetic enrichment of rare cell types for transcriptome studies, and they advance understanding of the molecular composition of a functionally important class of GAD2+ neurons in the LHb.

Cell-type specific transcriptional adaptations of nucleus accumbens interneurons to amphetamine.

Parvalbumin-expressing (PV+) interneurons of the nucleus accumbens (NAc) play an essential role in the addictive-like behaviors induced by psychostimulant exposure. To identify molecular mechanisms of PV+ neuron plasticity, we isolated interneuron nuclei from the NAc of male and female mice following acute or repeated exposure to amphetamine (AMPH) and sequenced for cell type-specific RNA expression and chromatin accessibility. AMPH regulated the transcription of hundreds of genes in PV+ interneurons, and this program was largely distinct from that regulated in other NAc GABAergic neurons. Chromatin accessibility at enhancers predicted cell-type specific gene regulation, identifying transcriptional mechanisms of differential AMPH responses. Finally, we assessed expression of PV-enriched, AMPH-regulated genes in an Mecp2 mutant mouse strain that shows heightened behavioral sensitivity to psychostimulants to explore the functional importance of this transcriptional program. Together these data provide novel insight into the cell-type specific programs of transcriptional plasticity in NAc neurons that underlie addictive-like behaviors.

Mutations of the histone linker H1-4 in neurodevelopmental disorders and functional characterization of neurons expressing C-terminus frameshift mutant H1.4.

Rahman syndrome (RMNS) is a rare genetic disorder characterized by mild to severe intellectual disability, hypotonia, anxiety, autism spectrum disorder, vision problems, bone abnormalities and dysmorphic facies. RMNS is caused by de novo heterozygous mutations in the histone linker gene H1-4; however, mechanisms underlying impaired neurodevelopment in RMNS are not understood. All reported mutations associated with RMNS in H1-4 are small insertions or deletions that create a shared frameshift, resulting in a H1.4 protein that is both truncated and possessing an abnormal C-terminus frameshifted tail (H1.4 CFT). To expand understanding of mutations and phenotypes associated with mutant H1-4, we identified new variants at both the C- and N-terminus of H1.4. The clinical features of mutations identified at the C-terminus are consistent with other reports and strengthen the support of pathogenicity of H1.4 CFT. To understand how H1.4 CFT may disrupt brain function, we exogenously expressed wild-type or H1.4 CFT protein in rat hippocampal neurons and assessed neuronal structure and function. Genome-wide transcriptome analysis revealed ~ 400 genes altered in the presence of H1.4 CFT. Neuronal genes downregulated by H1.4 CFT were enriched for functional categories involved in synaptic communication and neuropeptide signaling. Neurons expressing H1.4 CFT also showed reduced neuronal activity on multielectrode arrays. These data are the first to characterize the transcriptional and functional consequence of H1.4 CFT in neurons. Our data provide insight into causes of neurodevelopmental impairments associated with frameshift mutations in the C-terminus of H1.4 and highlight the need for future studies on the function of histone H1.4 in neurons.

Transgenic mice for in vivo epigenome editing with CRISPR-based systems.

CRISPR-Cas9 technologies have dramatically increased the ease of targeting DNA sequences in the genomes of living systems. The fusion of chromatin-modifying domains to nuclease-deactivated Cas9 (dCas9) has enabled targeted epigenome editing in both cultured cells and animal models. However, delivering large dCas9 fusion proteins to target cells and tissues is an obstacle to the widespread adoption of these tools for in vivo studies. Here, we describe the generation and characterization of two conditional transgenic mouse lines for epigenome editing, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. By targeting the guide RNAs to transcriptional start sites or distal enhancer elements, we demonstrate regulation of target genes and corresponding changes to epigenetic states and downstream phenotypes in the brain and liver in vivo, and in T cells and fibroblasts ex vivo. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development.

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Demonstrating trustworthiness when collecting and sharing genomic data: public views across 22 countries.

BACKGROUND: Public trust is central to the collection of genomic and health data and the sustainability of genomic research. To merit trust, those involved in collecting and sharing data need to demonstrate they are trustworthy. However, it is unclear what measures are most likely to demonstrate this. METHODS: We analyse the 'Your DNA, Your Say' online survey of public perspectives on genomic data sharing including responses from 36,268 individuals across 22 low-, middle- and high-income countries, gathered in 15 languages. We examine how participants perceived the relative value of measures to demonstrate the trustworthiness of those using donated DNA and/or medical information. We examine between-country variation and present a consolidated ranking of measures. RESULTS: Providing transparent information about who will benefit from data access was the most important measure to increase trust, endorsed by more than 50% of participants across 20 of 22 countries. It was followed by the option to withdraw data and transparency about who is using data and why. Variation was found for the importance of measures, notably information about sanctions for misuse of data-endorsed by 5% in India but almost 60% in Japan. A clustering analysis suggests alignment between some countries in the assessment of specific measures, such as the UK and Canada, Spain and Mexico and Portugal and Brazil. China and Russia are less closely aligned with other countries in terms of the value of the measures presented. CONCLUSIONS: Our findings highlight the importance of transparency about data use and about the goals and potential benefits associated with data sharing, including to whom such benefits accrue. They show that members of the public value knowing what benefits accrue from the use of data. The study highlights the importance of locally sensitive measures to increase trust as genomic data sharing continues globally.

TRPing into excitotoxic neuronal death.

It is a striking paradox that the activation of NMDA-type glutamate receptors (NMDARs) can both promote neuronal survival and induce excitotoxic cell death. Yet the molecular mechanisms that distinguish these cellular consequences have remained obscure. A recent study by Yan et al. (2020) reveals a novel interaction between NMDARs and TRPM4 that is required for NMDAR-induced neuronal death. Small molecule disruption of this interaction reduces excitotoxicity in stroke without blocking physiological NMDAR signaling.

Global Public Perceptions of Genomic Data Sharing: What Shapes the Willingness to Donate DNA and Health Data?

Analyzing genomic data across populations is central to understanding the role of genetic factors in health and disease. Successful data sharing relies on public support, which requires attention to whether people around the world are willing to donate their data that are then subsequently shared with others for research. However, studies of such public perceptions are geographically limited and do not enable comparison. This paper presents results from a very large public survey on attitudes toward genomic data sharing. Data from 36,268 individuals across 22 countries (gathered in 15 languages) are presented. In general, publics across the world do not appear to be aware of, nor familiar with, the concepts of DNA, genetics, and genomics. Willingness to donate one's DNA and health data for research is relatively low, and trust in the process of data's being shared with multiple users (e.g., doctors, researchers, governments) is also low. Participants were most willing to donate DNA or health information for research when the recipient was specified as a medical doctor and least willing to donate when the recipient was a for-profit researcher. Those who were familiar with genetics and who were trusting of the users asking for data were more likely to be willing to donate. However, less than half of participants trusted more than one potential user of data, although this varied across countries. Genetic information was not uniformly seen as different from other forms of health information, but there was an association between seeing genetic information as special in some way compared to other health data and increased willingness to donate. The global perspective provided by our "Your DNA, Your Say" study is valuable for informing the development of international policy and practice for sharing genomic data. It highlights that the research community not only needs to be worthy of trust by the public, but also urgent steps need to be taken to authentically communicate why genomic research is necessary and how data donation, and subsequent sharing, is integral to this.

The NMDA receptor subunit GluN3A regulates synaptic activity-induced and myocyte enhancer factor 2C (MEF2C)-dependent transcription.

N-Methyl-d-aspartate type glutamate receptors (NMDARs) are key mediators of synaptic activity-regulated gene transcription in neurons, both during development and in the adult brain. Developmental differences in the glutamate receptor ionotropic NMDA 2 (GluN2) subunit composition of NMDARs determines whether they activate the transcription factor cAMP-responsive element-binding protein 1 (CREB). However, whether the developmentally regulated GluN3A subunit also modulates NMDAR-induced transcription is unknown. Here, using an array of techniques, including quantitative real-time PCR, immunostaining, reporter gene assays, RNA-Seq, and two-photon glutamate uncaging with calcium imaging, we show that knocking down GluN3A in rat hippocampal neurons promotes the inducible transcription of a subset of NMDAR-sensitive genes. We found that this enhancement is mediated by the accumulation of phosphorylated p38 mitogen-activated protein kinase in the nucleus, which drives the activation of the transcription factor myocyte enhancer factor 2C (MEF2C) and promotes the transcription of a subset of synaptic activity-induced genes, including brain-derived neurotrophic factor (Bdnf) and activity-regulated cytoskeleton-associated protein (Arc). Our evidence that GluN3A regulates MEF2C-dependent transcription reveals a novel mechanism by which NMDAR subunit composition confers specificity to the program of synaptic activity-regulated gene transcription in developing neurons.

Neurobiological functions of transcriptional enhancers.

Transcriptional enhancers are regulatory DNA elements that underlie the specificity and dynamic patterns of gene expression. Over the past decade, large-scale functional genomics projects have driven transformative progress in our understanding of enhancers. These data have relevance for identifying mechanisms of gene regulation in the CNS, elucidating the function of non-coding regulatory sequences in neurobiology and linking sequence variation within enhancers to genetic risk for neurological and psychiatric disorders. However, the sheer volume and complexity of genomic data presents a challenge to interpreting enhancer function in normal and pathogenic neurobiological processes. Here, to advance the application of genome-scale enhancer data, we offer a primer on current models of enhancer function in the CNS, we review how enhancers regulate gene expression across the neuronal lifespan, and we suggest how emerging findings regarding the role of non-coding sequence variation offer opportunities for understanding brain disorders and developing new technologies for neuroscience.

Activity-Dependent Transcription Collaborates with Local Dendritic Translation to Encode Stimulus-Specificity in the Genome Binding of NPAS4.

A new study in Cell (Brigidi et al., 2019) shows that local dendritic versus somatic translation of the neuronal activity-inducible transcription factor NPAS4 drives the formation of distinct heterodimers that enable stimulus-specificity to be encoded into the pattern of NPAS4 binding across the genome.

Transcribing Memories in Genome Architecture.

Whether dynamic changes in genome architecture underlie transcriptional and functional plasticity in mature neurons has been technically challenging to address. A recent study (Yamada et al., Nature, 2019) exploited experimental advantages of the cerebellum to reveal cell type-specific changes in chromatin architecture that coordinate neural activity-induced changes in gene transcription and contribute to sensorimotor learning.

A striatal interneuron circuit for continuous target pursuit.

Most adaptive behaviors require precise tracking of targets in space. In pursuit behavior with a moving target, mice use distance to target to guide their own movement continuously. Here, we show that in the sensorimotor striatum, parvalbumin-positive fast-spiking interneurons (FSIs) can represent the distance between self and target during pursuit behavior, while striatal projection neurons (SPNs), which receive FSI projections, can represent self-velocity. FSIs are shown to regulate velocity-related SPN activity during pursuit, so that movement velocity is continuously modulated by distance to target. Moreover, bidirectional manipulation of FSI activity can selectively disrupt performance by increasing or decreasing the self-target distance. Our results reveal a key role of the FSI-SPN interneuron circuit in pursuit behavior and elucidate how this circuit implements distance to velocity transformation required for the critical underlying computation.