Mutations of the transcriptional regulator PHF6 cause the X-linked intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS), but the pathogenesis of BFLS remains poorly understood. Here, we report a mouse model of BFLS, generated using a CRISPR-Cas9 approach, in which cysteine 99 within the PHD domain of PHF6 is replaced with phenylalanine (C99F). Mice harboring the patient-specific C99F mutation display deficits in cognitive functions, emotionality, and social behavior, as well as reduced threshold to seizures. Electrophysiological studies reveal that the intrinsic excitability of entorhinal cortical stellate neurons is increased in PHF6 C99F mice. Transcriptomic analysis of the cerebral cortex in C99F knockin mice and PHF6 knockout mice show that PHF6 promotes the expression of neurogenic genes and represses synaptic genes. PHF6-regulated genes are also overrepresented in gene signatures and modules that are deregulated in neurodevelopmental disorders of cognition. Our findings advance our understanding of the mechanisms underlying BFLS pathogenesis.

Local translation in neurites is a phenomenon that enhances the spatial segregation of proteins and their functions away from the cell body, yet it is unclear how local translation varies across neuronal cell types. Further, it is unclear whether differences in local translation across cell types simply reflect differences in transcription or whether there is also a cell type-specific post-transcriptional regulation of the location and translation of specific mRNAs. Most of the mRNAs discovered as being locally translated have been identified from hippocampal neurons because their laminar organization facilitates neurite-specific dissection and microscopy methods. Given the diversity of neurons across the brain, studies have not yet analyzed how locally translated mRNAs differ across cell types. Here, we used the SynapTRAP method to harvest two broad cell types in the mouse forebrain: GABAergic neurons and layer 5 projection neurons. While some transcripts overlap, the majority of the local translatome is not shared across these cell types. In addition to differences driven by baseline expression levels, some transcripts also exhibit cell type-specific post-transcriptional regulation. Finally, we provide evidence that GABAergic neurons specifically localize mRNAs for peptide neurotransmitters, including somatostatin and cortistatin, suggesting localized production of these key signaling molecules in the neurites of GABAergic neurons. Overall, this work suggests that differences in local translation in neurites across neuronal cell types are poised to contribute substantially to the heterogeneity in neuronal phenotypes.

Preclinical work has long focused on male animals, though biological sex clearly influences risk for certain diseases, including many psychiatric disorders. Such disorders are often treated by drugs targeting the CNS norepinephrine system. Despite roles for noradrenergic neurons in behavior and neuropsychiatric disease models, their molecular characterization has lagged. We profiled mouse noradrenergic neurons in vivo, defining over 3,000 high-confidence transcripts expressed therein, including druggable receptors. We uncovered remarkable sex differences in gene expression, including elevated expression of the EP3 receptor in females-which we leverage to illustrate the behavioral and pharmacologic relevance of these findings-and of Slc6a15 and Lin28b, both major depressive disorder (MDD)-associated genes. Broadly, we present a means of transcriptionally profiling locus coeruleus under baseline and experimental conditions. Our findings underscore the need for preclinical work to include both sexes and suggest that sex differences in noradrenergic neurons may underlie behavioral differences relevant to disease.

Mice produce ultrasonic vocalizations (USV) in multiple communicative contexts, including adult social interaction (e.g., male to female courtship), as well as pup calls when separated from the dam. Assessment of pup USV has been widely applied in models of social and communicative disorders, dozens of which have shown alterations to this conserved behavior. However, features such as call production rate can vary substantially even within experimental groups and it is unclear to what extent aspects of USV represent stable trait-like influences or are vulnerable to an animal's state. To address this question, we have employed a mixed modeling approach to describe consistency in USV features across time, leveraging multiple large cohorts recorded from two strains, and across ages/times. We find that most features of pup USV show consistent patterns within a recording session, but inconsistent patterns across postnatal development. This supports the conclusion that pup USV is most strongly influenced by "state"-like variables. In contrast, adult USV call rate and call duration show higher consistency across sessions and may reflect a stable "trait." However, spectral features of adult song such as the presence of pitch jumps do not show this level of consistency, suggesting that pitch modulation is more susceptible to factors affecting the animal's state at the time of recording. Overall, the utility of this work is three-fold. First, as variability necessarily affects the sensitivity of the assay to detect experimental perturbation, we hope the information provided here will be used to help researchers plan sufficiently powered experiments, as well as prioritize specific ages to study USV behavior and to decide which features to consider most strongly in analysis. Second, via the mouseTube platform, we have provided these hundreds of recordings and associated data to serve as a shared resource for other researchers interested in either benchmark data for these strains or in developing algorithms for studying features of mouse song. Finally, we hope that this work informs both interpretation of USV studies in models of developmental disorder, and helps to further research into understanding the neural processes that contribute to the production and predictability of USV behavior.

Comparative analysis can provide important insights into complex biological systems. As demonstrated in the accompanying paper, translating ribosome affinity purification (TRAP) permits comprehensive studies of translated mRNAs in genetically defined cell populations after physiological perturbations. To establish the generality of this approach, we present translational profiles for 24 CNS cell populations and identify known cell-specific and enriched transcripts for each population. We report thousands of cell-specific mRNAs that were not detected in whole-tissue microarray studies and provide examples that demonstrate the benefits deriving from comparative analysis. To provide a foundation for further biological and in silico studies, we provide a resource of 16 transgenic mouse lines, their corresponding anatomic characterization, and translational profiles for cell types from a variety of central nervous system structures. This resource will enable a wide spectrum of molecular and mechanistic studies of both well-known and previously uncharacterized neural cell populations.

We have recently developed a novel method for the affinity purification of the complete suite of translating mRNA from genetically labeled cell populations. This method permits comprehensive quantitative comparisons of the genes employed by each specific cell type. We provide a detailed description of tools for analysis of data generated with this and related methodologies. An essential question that arises from these data is how to identify those genes that are enriched in each cell type relative to all others. Genes relatively specifically employed by a cell type may contribute to the unique functions of that cell, and thus may become useful targets for development of pharmacological tools for cell-specific manipulations. We describe here a novel statistic, the specificity index, which can be used for comparative quantitative analysis to identify genes enriched in specific cell populations across a large number of profiles. This measure correctly predicts in situ hybridization patterns for many cell types. We apply this measure to a large survey of CNS cell-specific microarray data to identify those genes that are significantly enriched in each population Data and algorithms are available online (www.bactrap.org).

Mice produce ultrasonic vocalizations (USVs) in a variety of social situations, and USVs have been leveraged to study many neurological diseases including verbal dyspraxia, depression, autism and stuttering. Pups produce isolation calls, a common USV, spontaneously when they are isolated from their mother during the first 2 weeks of life. Several genetic manipulations affect (and often reduce) pup isolation calls in mice. To facilitate the use of this assay as a means of testing whether significant functional differences in genotypes exist instead of contextual differences, we test the variability inherent in many commons measures of mouse vocalizations. Here we use biological consistency as a way of determining which are reproducible in mouse pup vocalizations. We present a comprehensive analysis of the normal variability of these vocalizations in groups of mice, individual mice and different strains of mice. To control for maturation effects, we recorded pup isolation calls in the same group of C57BL/6J 5 days old mice twice, with 1 h of rest in between recordings. In almost all cases, the group averages between the first and second recordings were the same. We also found that there were high correlations in some parameters in individual mice across recording while others were not well correlated. These findings could be replicated for the majority of features in a separate group of C57BL/6J mice and a group of 129/SvEvBrd-C57BL/6J mice. The averages of these mouse USV features are highly consistent and represent a robust assay to test the effects of genetic and other interventions in the experimental setting.

Nociceptin and its receptor are widely distributed throughout the brain in regions associated with reward behavior, yet how and when they act is unknown. Here, we dissected the role of a nociceptin peptide circuit in reward seeking. We generated a prepronociceptin (Pnoc)-Cre mouse line that revealed a unique subpopulation of paranigral ventral tegmental area (pnVTA) neurons enriched in prepronociceptin. Fiber photometry recordings during progressive ratio operant behavior revealed pnVTAPnoc neurons become most active when mice stop seeking natural rewards. Selective pnVTAPnoc neuron ablation, inhibition, and conditional VTA nociceptin receptor (NOPR) deletion increased operant responding, revealing that the pnVTAPnoc nucleus and VTA NOPR signaling are necessary for regulating reward motivation. Additionally, optogenetic and chemogenetic activation of this pnVTAPnoc nucleus caused avoidance and decreased motivation for rewards. These findings provide insight into neuromodulatory circuits that regulate motivated behaviors through identification of a previously unknown neuropeptide-containing pnVTA nucleus that limits motivation for rewards.

Norepinephrine (NE), and specific adrenoceptors, have been reported to influence distinct aspects of adult hippocampal neurogenesis, including latent stem cell activation, progenitor proliferation, and differentiation. These findings are predominantly based on the use of pharmacological approaches in both in vitro and in vivo systems. Here, we sought to assess the consequences of genetic ablation of NE on adult hippocampal neurogenesis, by examining dopamine β hydroxylase knockout (Dbh -/-) mice, which lack NE from birth. We find that Dbh -/- mice exhibit no difference in adult hippocampal progenitor proliferation and survival. Further, the number of immature newborn neurons, labeled using stage-specific developmental markers within the hippocampal neurogenic niche, was also unaltered in Dbh -/- mice. In contrast, the noradrenergic neurotoxin DSP-4, which had previously been shown to reduce adult hippocampal neurogenesis in rats, also resulted in a decline in hippocampal progenitor proliferation in C57/Bl6N mice. These findings indicate that pharmacological lesioning of noradrenergic afferents in adulthood, but not the complete genetic loss of NE from birth, impairs adult hippocampal neurogenesis in mice.

Within eukaryotic cells, translation is regulated independent of transcription, enabling nuanced, localized, and rapid responses to stimuli. Neurons respond transcriptionally and translationally to synaptic activity. Although transcriptional responses are documented in astrocytes, here we test whether astrocytes have programmed translational responses. We show that seizure activity rapidly changes the transcripts on astrocyte ribosomes, some predicted to be downstream of BDNF signaling. In acute slices, we quantify the extent to which cues of neuronal activity activate translation in astrocytes and show that this translational response requires the presence of neurons, indicating that the response is non-cell autonomous. We also show that this induction of new translation extends into the periphery of astrocytes. Finally, synaptic proteomics show that new translation is required for changes that occur in perisynaptic astrocyte protein composition after fear conditioning. Regulation of translation in astrocytes by neuronal activity suggests an additional mechanism by which astrocytes may dynamically modulate nervous system functioning.

Williams Syndrome is a rare neurodevelopmental disorder exhibiting cognitive and behavioral abnormalities, including increased social motivation, risk of anxiety and specific phobias along with perturbed motor function. Williams Syndrome is caused by a microdeletion of 26-28 genes on chromosome 7, including GTF2IRD1 , which encodes a transcription factor suggested to play a role in the behavioral profile of Williams Syndrome. Duplications of the full region also lead to frequent autism diagnosis, social phobias, and language delay. Thus, genes in the region appear to regulate social motivation in a dose-sensitive manner. A 'Complete Deletion' mouse, heterozygously eliminating the syntenic Williams Syndrome region, has been deeply characterized for cardiac phenotypes, but direct measures of social motivation have not been assessed. Furthermore, the role of Gtf2ird1 in these behaviors has not been addressed in a relevant genetic context. Here, we have generated a mouse overexpressing Gtf2ird1 , which can be used both to model duplication of this gene alone and to rescue Gtf2ird1 expression in the Complete Deletion mice. Using a comprehensive behavioral pipeline and direct measures of social motivation, we provide evidence that the Williams Syndrome Critical Region regulates social motivation along with motor and anxiety phenotypes, but that Gtf2ird1 complementation is not sufficient to rescue most of these traits, and duplication does not decrease social motivation. However, Gtf2ird1 complementation does rescue light-aversive behavior and performance on select sensorimotor tasks, perhaps indicating a role for this gene in sensory processing or integration.

Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.

Aquaporin-4 (AQP4) is a water channel protein that links astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, that preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel mouse AQP4 line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wildtype (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to wildtype, and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. Increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two were unchanged. However, at 100% readthrough, AQP4x localization and formation of higher-order complexes was disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for increased endothelial cell vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels.

Motor symptoms in Parkinson's disease (PD) are caused by degeneration of dopamine (DA) neurons of the substantia nigra (SN), while early non-motor symptoms such as anxiety and sleep disturbances are likely mediated by dysfunction of locus coeruleus (LC) norepinephrine (NE) neurons. The LC develops α-synuclein pathology prior to SN DA neurons in PD, and later undergoes degeneration, but the mechanisms responsible for its vulnerability are unknown. The SN and LC are the only structures in the brain that produces appreciable amounts of neuromelanin (NM), a dark cytoplasmic pigment. It has been proposed that NM initially plays a protective role by sequestering toxic catecholamine metabolites and heavy metals, but may become harmful during aging and PD as they overwhelm cellular machinery and are released during neurodegeneration. Rodents do not naturally produce NM, limiting the study of causal relationships between NM and PD-associated LC pathology. Adapting a viral-mediated approach for expression of human tyrosinase, the enzyme responsible for peripheral melanin production, we successfully promoted pigmentation in mouse LC neurons that recapitulates key features of endogenous NM found in primates, including eumelanin and pheomelanin, lipid droplets, and a double-membrane encasement. Pigment expression results in mild neurodegeneration, reduced NE levels, transcriptional changes, and novelty-induced anxiety phenotypes as early as 1-week post-injection. By 6-weeks, NM accumulation is associated with severe LC neurodegeneration and a robust neuroinflammatory response. These phenotypes are reminiscent of LC dysfunction in PD, validating this model for studying the consequences of pigment accumulation in the LC as it relates to neurodegenerative disease.

In vitro studies indicate the neurodevelopmental disorder gene myelin transcription factor 1-like (MYT1L) suppresses non-neuronal lineage genes during fibroblast-to-neuron direct differentiation. However, MYT1L's molecular and cellular functions in the adult mammalian brain have not been fully characterized. Here, we found that MYT1L loss leads to up-regulated deep layer (DL) gene expression, corresponding to an increased ratio of DL/UL neurons in the adult mouse cortex. To define potential mechanisms, we conducted Cleavage Under Targets & Release Using Nuclease (CUT&RUN) to map MYT1L binding targets and epigenetic changes following MYT1L loss in mouse developing cortex and adult prefrontal cortex (PFC). We found MYT1L mainly binds to open chromatin, but with different transcription factor co-occupancies between promoters and enhancers. Likewise, multiomic data set integration revealed that, at promoters, MYT1L loss does not change chromatin accessibility but increases H3K4me3 and H3K27ac, activating both a subset of earlier neuronal development genes as well as Bcl11b, a key regulator for DL neuron development. Meanwhile, we discovered that MYT1L normally represses the activity of neurogenic enhancers associated with neuronal migration and neuronal projection development by closing chromatin structures and promoting removal of active histone marks. Further, we showed that MYT1L interacts with HDAC2 and transcriptional repressor SIN3B in vivo, providing potential mechanisms underlying repressive effects on histone acetylation and gene expression. Overall, our findings provide a comprehensive map of MYT1L binding in vivo and mechanistic insights into how MYT1L loss leads to aberrant activation of earlier neuronal development programs in the adult mouse brain.

Sex differences in the brain may play an important role in sex-differential prevalence of neuropsychiatric conditions. In order to understand the transcriptional basis of sex differences, we analyzed multiple, large-scale, human postmortem brain RNA-seq datasets using both within-region and pan-regional frameworks. We find evidence of sex-biased transcription in many autosomal genes, some of which provide evidence for pathways and cell population differences between chromosomally male and female individuals. These analyses also highlight regional differences in the extent of sex-differential gene expression. We observe an increase in specific neuronal transcripts in male brains and an increase in immune and glial function-related transcripts in female brains. Integration with single-cell data suggests this corresponds to sex differences in cellular states rather than cell abundance. Integration with case-control gene expression studies suggests a female molecular predisposition towards Alzheimer's disease, a female-biased disease. Autism, a male-biased diagnosis, does not exhibit a male predisposition pattern in our analysis. Finally, we provide region specific analyses of sex differences in brain gene expression to enable additional studies at the interface of gene expression and diagnostic differences.

Williams syndrome (WS) is a neurodevelopmental disorder caused by a 1.5-1.8 Mbp deletion on chromosome 7q11.23, affecting the copy number of 26-28 genes. Phenotypes of WS include cardiovascular problems, craniofacial dysmorphology, deficits in visual-spatial cognition and a characteristic hypersocial personality. There are still no genes in the region that have been consistently linked to the cognitive and behavioral phenotypes, although human studies and mouse models have led to the current hypothesis that the general transcription factor 2 I family of genes, GTF2I and GTF2IRD1, are responsible. Here we test the hypothesis that these two transcription factors are sufficient to reproduce the phenotypes that are caused by deletion of the WS critical region (WSCR). We compare a new mouse model with loss of function mutations in both Gtf2i and Gtf2ird1 to an established mouse model lacking the complete WSCR. We show that the complete deletion (CD) model has deficits across several behavioral domains including social communication, motor functioning and conditioned fear that are not explained by loss of function mutations in Gtf2i and Gtf2ird1. Furthermore, transcriptome profiling of the hippocampus shows changes in synaptic genes in the CD model that are not seen in the double mutants. Thus, we have thoroughly defined a set of molecular and behavioral consequences of complete WSCR deletion and shown that genes or combinations of genes beyond Gtf2i and Gtf2ird1 are necessary to produce these phenotypic effects.

Alternative translation initiation and stop codon readthrough in a few well-studied cases have been shown to allow the same transcript to generate multiple protein variants. Because the brain shows a particularly abundant use of alternative splicing, we sought to study alternative translation in CNS cells. We show that alternative translation is widespread and regulated across brain transcripts. In neural cultures, we identify alternative initiation on hundreds of transcripts, confirm several N-terminal protein variants, and show the modulation of the phenomenon by KCl stimulation. We also detect readthrough in cultures and show differential levels of normal and readthrough versions of AQP4 in gliotic diseases. Finally, we couple translating ribosome affinity purification to ribosome footprinting (TRAP-RF) for cell-type-specific analysis of neuronal and astrocytic translational readthrough in the mouse brain. We demonstrate that this unappreciated mechanism generates numerous and diverse protein isoforms in a cell-type-specific manner in the brain.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder marked by the loss of motor neurons (MNs) in the brain and spinal cord, leading to fatally debilitating weakness. Because this disease predominantly affects MNs, we aimed to characterize the distinct expression profile of that cell type to elucidate underlying disease mechanisms and to identify novel targets that inform on MN health during ALS disease time course. microRNAs (miRNAs) are short, noncoding RNAs that can shape the expression profile of a cell and thus often exhibit cell-type-enriched expression. To determine MN-enriched miRNA expression, we used Cre recombinase-dependent miRNA tagging and affinity purification in mice. By defining the in vivo miRNA expression of MNs, all neurons, astrocytes, and microglia, we then focused on MN-enriched miRNAs via a comparative analysis and found that they may functionally distinguish MNs postnatally from other spinal neurons. Characterizing the levels of the MN-enriched miRNAs in CSF harvested from ALS models of MN disease demonstrated that one miRNA (miR-218) tracked with MN loss and was responsive to an ALS therapy in rodent models. Therefore, we have used cellular expression profiling tools to define the distinct miRNA expression of MNs, which is likely to enrich future studies of MN disease. This approach enabled the development of a novel, drug-responsive marker of MN disease in ALS rodents.SIGNIFICANCE STATEMENT Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons (MNs) in the brain and spinal cord are selectively lost. To develop tools to aid in our understanding of the distinct expression profiles of MNs and, ultimately, to monitor MN disease progression, we identified small regulatory microRNAs (miRNAs) that were highly enriched or exclusive in MNs. The signal for one of these MN-enriched miRNAs is detectable in spinal tap biofluid from an ALS rat model, where its levels change as disease progresses, suggesting that it may be a clinically useful marker of disease status. Furthermore, rats treated with ALS therapy have restored expression of this MN RNA marker, making it an MN-specific and drug-responsive marker for ALS rodents.

The overwhelming success of exome- and genome-wide association studies in discovering thousands of disease-associated genes necessitates developing novel high-throughput functional genomics approaches to elucidate the molecular mechanisms of these genes. Here, we have coupled multiplexed repression of neurodevelopmental disease-associated genes to single-cell transcriptional profiling in differentiating human neurons to rapidly assay the functions of multiple genes in a disease-relevant context, assess potentially convergent mechanisms, and prioritize genes for specific functional assays. For a set of 13 autism spectrum disorder (ASD)-associated genes, we show that this approach generated important mechanistic insights, revealing two functionally convergent modules of ASD genes: one that delays neuron differentiation and one that accelerates it. Five genes that delay neuron differentiation (ADNP, ARID1B, ASH1L, CHD2, and DYRK1A) mechanistically converge, as they all dysregulate genes involved in cell-cycle control and progenitor cell proliferation. Live-cell imaging after individual ASD-gene repression validated this functional module, confirming that these genes reduce neural progenitor cell proliferation and neurite growth. Finally, these functionally convergent ASD gene modules predicted shared clinical phenotypes among individuals with mutations in these genes. Altogether, these results show the utility of a novel and simple approach for the rapid functional elucidation of neurodevelopmental disease-associated genes.