Publications by authors named "Joseph D Dougherty"

79 Publications

Loss of Quaking RNA binding protein disrupts the expression of genes associated with astrocyte maturation in mouse brain.

Nat Commun 2021 03 9;12(1):1537. Epub 2021 Mar 9.

Department of Genetics, Washington University School of Medicine, Saint Louis, MO, 63110, USA.

Quaking RNA binding protein (QKI) is essential for oligodendrocyte development as myelination requires myelin basic protein mRNA regulation and localization by the cytoplasmic isoforms (e.g., QKI-6). QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we define the targets of QKI in the mouse brain via CLIPseq and we show that QKI-6 binds 3'UTRs of a subset of astrocytic mRNAs. Binding is also enriched near stop codons, mediated partially by QKI-binding motifs (QBMs), yet spreads to adjacent sequences. Using a viral approach for mosaic, astrocyte-specific gene mutation with simultaneous translating RNA sequencing (CRISPR-TRAPseq), we profile ribosome associated mRNA from QKI-null astrocytes in the mouse brain. This demonstrates a role for QKI in stabilizing CLIP-defined direct targets in astrocytes in vivo and further shows that QKI mutation disrupts the transcriptional changes for a discrete subset of genes associated with astrocyte maturation.
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http://dx.doi.org/10.1038/s41467-021-21703-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7943582PMC
March 2021

Shared developmental gait disruptions across two mouse models of neurodevelopmental disorders.

J Neurodev Disord 2021 Mar 20;13(1):10. Epub 2021 Mar 20.

Department of Psychiatry, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO, 63110-1093, USA.

Background: Motor deficits such as abnormal gait are an underappreciated yet characteristic phenotype of many neurodevelopmental disorders (NDDs), including Williams Syndrome (WS) and Neurofibromatosis Type 1 (NF1). Compared to cognitive phenotypes, gait phenotypes are readily and comparably assessed in both humans and model organisms and are controlled by well-defined CNS circuits. Discovery of a common gait phenotype between NDDs might suggest shared cellular and molecular deficits and highlight simple outcome variables to potentially quantify longitudinal treatment efficacy in NDDs.

Methods: We characterized gait using the DigiGait assay in two different murine NDD models: the complete deletion (CD) mouse, which models hemizygous loss of the complete WS locus, and the Nf1 mouse, which models a NF1 patient-derived heterozygous germline NF1 mutation. Longitudinal data were collected across four developmental time points (postnatal days 21-30) and one early adulthood time point.

Results: Compared to wildtype littermate controls, both models displayed markedly similar spatial, temporal, and postural gait abnormalities during development. Developing CD mice also displayed significant decreases in variability metrics. Multiple gait abnormalities observed across development in the Nf1 mice persisted into early adulthood, including increased stride length and decreased stride frequency, while developmental abnormalities in the CD model largely resolved by adulthood.

Conclusions: These findings suggest that the subcomponents of gait affected in NDDs show overlap between disorders as well as some disorder-specific features, which may change over the course of development. Our incorporation of spatial, temporal, and postural gait measures also provides a template for gait characterization in other NDD models and a platform to examining circuits or longitudinal therapeutics.
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http://dx.doi.org/10.1186/s11689-021-09359-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980331PMC
March 2021

Ontogenetic Oxycodone Exposure Affects Early Life Communicative Behaviors, Sensorimotor Reflexes, and Weight Trajectory in Mice.

Front Behav Neurosci 2021 22;15:615798. Epub 2021 Feb 22.

Department of Psychiatry, Washington University in St. Louis, St. Louis, MO, United States.

Nationwide, opioid misuse among pregnant women has risen four-fold from 1999 to 2014, with commensurate increase in neonates hospitalized for neonatal abstinence syndrome (NAS). NAS occurs when a fetus exposed to opioids goes into rapid withdrawal after birth. NAS treatment via continued post-natal opioid exposure has been suggested to worsen neurodevelopmental outcomes. We developed a novel model to characterize the impact of and prolonged post-natal oxycodone (Oxy) exposure on early behavior and development. Via subcutaneous pump implanted before breeding, C57BL/6J dams were infused with Oxy at 10 mg/kg/day from conception through pup-weaning. At birth, oxy-exposed pups were either cross-fostered (paired with non-Oxy exposed dams) to model opioid abstinence ( Oxy) or reared by their biological dams still receiving Oxy to model continued post-natal opioid exposure (prolonged Oxy). Offspring from vehicle-exposed dams served as cross-fostered ( Veh) or biologically reared (prolonged Veh) controls. Oxy exposure resulted in sex-dependent weight reductions and altered spectrotemporal features of isolation-induced ultrasonic vocalization (USV). Meanwhile, prolonged Oxy pups exhibited reduced weight and sex-differential delays in righting reflex. Specifically, prolonged Oxy female offspring exhibited increased latency to righting. Prolonged Oxy pups also showed decreases in number of USV calls and changes to spectrotemporal USV features. Overall, ontogenetic Oxy exposure was associated with impaired attainment of gross and sensorimotor milestones, as well as alterations in communication and affective behaviors, indicating a need for therapeutic interventions. The model developed here will enable studies of withdrawal physiology and opioid-mediated mechanisms underlying these neurodevelopmental deficits.
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http://dx.doi.org/10.3389/fnbeh.2021.615798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937712PMC
February 2021

Extended amygdala-parabrachial circuits alter threat assessment and regulate feeding.

Sci Adv 2021 Feb 26;7(9). Epub 2021 Feb 26.

Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

An animal's evolutionary success depends on the ability to seek and consume foods while avoiding environmental threats. However, how evolutionarily conserved threat detection circuits modulate feeding is unknown. In mammals, feeding and threat assessment are strongly influenced by the parabrachial nucleus (PBN), a structure that responds to threats and inhibits feeding. Here, we report that the PBN receives dense inputs from two discrete neuronal populations in the bed nucleus of the stria terminalis (BNST), an extended amygdala structure that encodes affective information. Using a series of complementary approaches, we identify opposing BNST-PBN circuits that modulate neuropeptide-expressing PBN neurons to control feeding and affective states. These previously unrecognized neural circuits thus serve as potential nodes of neural circuitry critical for the integration of threat information with the intrinsic drive to feed.
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http://dx.doi.org/10.1126/sciadv.abd3666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909877PMC
February 2021

CLIP and Massively Parallel Functional Analysis of CELF6 Reveal a Role in Destabilizing Synaptic Gene mRNAs through Interaction with 3' UTR Elements.

Cell Rep 2020 Dec;33(12):108531

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Psychiatry, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

CELF6 is a CELF-RNA-binding protein, and thus part of a protein family with roles in human disease; however, its mRNA targets in the brain are largely unknown. Using cross-linking immunoprecipitation and sequencing (CLIP-seq), we define its CNS targets, which are enriched for 3' UTRs in synaptic protein-coding genes. Using a massively parallel reporter assay framework, we test the consequence of CELF6 expression on target sequences, with and without mutating putative binding motifs. Where CELF6 exerts an effect on sequences, it is largely to decrease RNA abundance, which is reversed by mutating UGU-rich motifs. This is also the case for CELF3-5, with a protein-dependent effect on magnitude. Finally, we demonstrate that targets are derepressed in CELF6-mutant mice, and at least two key CNS proteins, FOS and FGF13, show altered protein expression levels and localization. Our works find, in addition to previously identified roles in splicing, that CELF6 is associated with repression of its CNS targets via the 3' UTR in vivo.
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http://dx.doi.org/10.1016/j.celrep.2020.108531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780154PMC
December 2020

DNMT3A Haploinsufficiency Results in Behavioral Deficits and Global Epigenomic Dysregulation Shared across Neurodevelopmental Disorders.

Cell Rep 2020 Nov;33(8):108416

Department of Neuroscience, Washington University School of Medicine, St. Louis, MO 63110-1093, USA. Electronic address:

Mutations in DNA methyltransferase 3A (DNMT3A) have been detected in autism and related disorders, but how these mutations disrupt nervous system function is unknown. Here, we define the effects of DNMT3A mutations associated with neurodevelopmental disease. We show that diverse mutations affect different aspects of protein activity but lead to shared deficiencies in neuronal DNA methylation. Heterozygous DNMT3A knockout mice mimicking DNMT3A disruption in disease display growth and behavioral alterations consistent with human phenotypes. Strikingly, in these mice, we detect global disruption of neuron-enriched non-CG DNA methylation, a binding site for the Rett syndrome protein MeCP2. Loss of this methylation leads to enhancer and gene dysregulation that overlaps with models of Rett syndrome and autism. These findings define the effects of DNMT3A haploinsufficiency in the brain and uncover disruption of the non-CG methylation pathway as a convergence point across neurodevelopmental disorders.
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http://dx.doi.org/10.1016/j.celrep.2020.108416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716597PMC
November 2020

DeepH&M: Estimating single-CpG hydroxymethylation and methylation levels from enrichment and restriction enzyme sequencing methods.

Sci Adv 2020 Jul 1;6(27). Epub 2020 Jul 1.

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA.

Increased appreciation of 5-hydroxymethylcytosine (5hmC) as a stable epigenetic mark, which defines cell identity and disease progress, has engendered a need for cost-effective, but high-resolution, 5hmC mapping technology. Current enrichment-based technologies provide cheap but low-resolution and relative enrichment of 5hmC levels, while single-base resolution methods can be prohibitively expensive to scale up to large experiments. To address this problem, we developed a deep learning-based method, "DeepH&M," which integrates enrichment and restriction enzyme sequencing methods to simultaneously estimate absolute hydroxymethylation and methylation levels at single-CpG resolution. Using 7-week-old mouse cerebellum data for training the DeepH&M model, we demonstrated that the 5hmC and 5mC levels predicted by DeepH&M were in high concordance with whole-genome bisulfite-based approaches. The DeepH&M model can be applied to 7-week-old frontal cortex and 79-week-old cerebellum, revealing the robust generalizability of this method to other tissues from various biological time points.
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http://dx.doi.org/10.1126/sciadv.aba0521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458459PMC
July 2020

High-throughput single-cell functional elucidation of neurodevelopmental disease-associated genes reveals convergent mechanisms altering neuronal differentiation.

Genome Res 2020 09 4;30(9):1317-1331. Epub 2020 Sep 4.

Department of Genetics, Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110, USA.

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 (, , , , and ) 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.
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http://dx.doi.org/10.1101/gr.262295.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545139PMC
September 2020

Massively Parallel Reporter Assays: Defining Functional Psychiatric Genetic Variants Across Biological Contexts.

Biol Psychiatry 2021 Jan 18;89(1):76-89. Epub 2020 Jun 18.

Department of Genetics, Washington University School of Medicine in St. Louis, St. Louis, Missouri; Department of Psychiatry, Washington University School of Medicine in St. Louis, St. Louis, Missouri. Electronic address:

Neuropsychiatric phenotypes have long been known to be influenced by heritable risk factors, directly confirmed by the past decade of genetic studies that have revealed specific genetic variants enriched in disease cohorts. However, the initial hope that a small set of genes would be responsible for a given disorder proved false. The more complex reality is that a given disorder may be influenced by myriad small-effect noncoding variants and/or by rare but severe coding variants, many de novo. Noncoding genomic sequences-for which molecular functions cannot usually be inferred-harbor a large portion of these variants, creating a substantial barrier to understanding higher-order molecular and biological systems of disease. Fortunately, novel genetic technologies-scalable oligonucleotide synthesis, RNA sequencing, and CRISPR (clustered regularly interspaced short palindromic repeats)-have opened novel avenues to experimentally identify biologically significant variants en masse. Massively parallel reporter assays (MPRAs) are an especially versatile technique resulting from such innovations. MPRAs are powerful molecular genetics tools that can be used to screen thousands of untranscribed or untranslated sequences and their variants for functional effects in a single experiment. This approach, though underutilized in psychiatric genetics, has several useful features for the field. We review methods for assaying putatively functional genetic variants and regions, emphasizing MPRAs and the opportunities they hold for dissection of psychiatric polygenicity. We discuss literature applying functional assays in neurogenetics, highlighting strengths, caveats, and design considerations-especially regarding disease-relevant variables (cell type, neurodevelopment, and sex), and we ultimately propose applications of MPRA to both computational and experimental neurogenetics of polygenic disease risk.
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http://dx.doi.org/10.1016/j.biopsych.2020.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938388PMC
January 2021

Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells.

Cell 2020 08 24;182(4):992-1008.e21. Epub 2020 Jul 24.

Department of Genetics, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA; Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2020.06.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510185PMC
August 2020

The trajectory of gait development in mice.

Brain Behav 2020 06 24;10(6):e01636. Epub 2020 Apr 24.

Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA.

Objective: Gait irregularities are prevalent in neurodevelopmental disorders (NDDs). However, there is a paucity of information on gait phenotypes in NDD experimental models. This is in part due to the lack of understanding of the normal developmental trajectory of gait maturation in the mouse.

Materials And Methods: Using the DigiGait system, we have developed a quantitative, standardized, and reproducible assay of developmental gait metrics in commonly used mouse strains that can be added to the battery of mouse model phenotyping. With this assay, we characterized the trajectory of gait in the developing C57BL/6J and FVB/AntJ mouse lines.

Results: In both lines, a mature stride consisted of 40% swing and 60% stance in the forelimbs, which mirrors the mature human stride. In C57BL/6J mice, developmental trajectories were observed for stance width, paw overlap distance, braking and propulsion time, rate of stance loading, peak paw area, and metrics of intraindividual variability. In FVB/AntJ mice, developmental trajectories were observed for percent shared stance, paw overlap distance, rate of stance loading, and peak paw area, although in different directions than C57 mice. By accounting for the impact of body length on stride measurements, we demonstrate the importance of considering body length when interpreting gait metrics.

Conclusion: Overall, our results show that aspects of mouse gait development parallel a timeline of normal human gait development, such as the percent of stride that is stance phase and swing phase. This study may be used as a standard reference for developmental gait phenotyping of murine models, such as models of neurodevelopmental disease.
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http://dx.doi.org/10.1002/brb3.1636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303394PMC
June 2020

Functions of Gtf2i and Gtf2ird1 in the developing brain: transcription, DNA binding and long-term behavioral consequences.

Hum Mol Genet 2020 06;29(9):1498-1519

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA.

Gtf2ird1 and Gtf2i are two transcription factors (TFs) among the 28 genes deleted in Williams syndrome, and prior mouse models of each TF show behavioral phenotypes. Here we identify their genomic binding sites in the developing brain and test for additive effects of their mutation on transcription and behavior. GTF2IRD1 binding targets were enriched for transcriptional and chromatin regulators and mediators of ubiquitination. GTF2I targets were enriched for signal transduction proteins, including regulators of phosphorylation and WNT. Both TFs are highly enriched at promoters, strongly overlap CTCF binding and topological associating domain boundaries and moderately overlap each other, suggesting epistatic effects. Shared TF targets are enriched for reactive oxygen species-responsive genes, synaptic proteins and transcription regulators such as chromatin modifiers, including a significant number of highly constrained genes and known ASD genes. We next used single and double mutants to test whether mutating both TFs will modify transcriptional and behavioral phenotypes of single Gtf2ird1 mutants, though with the caveat that our Gtf2ird1 mutants, like others previously reported, do produce low levels of a truncated protein product. Despite little difference in DNA binding and transcriptome-wide expression, homozygous Gtf2ird1 mutation caused balance, marble burying and conditioned fear phenotypes. However, mutating Gtf2i in addition to Gtf2ird1 did not further modify transcriptomic or most behavioral phenotypes, suggesting Gtf2ird1 mutation alone was sufficient for the observed phenotypes.
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http://dx.doi.org/10.1093/hmg/ddaa070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526791PMC
June 2020

A viral toolkit for recording transcription factor-DNA interactions in live mouse tissues.

Proc Natl Acad Sci U S A 2020 05 16;117(18):10003-10014. Epub 2020 Apr 16.

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110;

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF-binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type specificity through conditional expression of the TF-enzyme fusion. Here, we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF-binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.
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http://dx.doi.org/10.1073/pnas.1918241117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7211997PMC
May 2020

Human iPSC-Derived Neurons and Cerebral Organoids Establish Differential Effects of Germline NF1 Gene Mutations.

Stem Cell Reports 2020 04 2;14(4):541-550. Epub 2020 Apr 2.

Department of Neurology, Washington University School of Medicine, Box 8111, 660 S. Euclid Avenue, St. Louis, MO 63110, USA. Electronic address:

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by a spectrum of distinct germline NF1 gene mutations, traditionally viewed as equivalent loss-of-function alleles. To specifically address the issue of mutational equivalency in a disease with considerable clinical heterogeneity, we engineered seven isogenic human induced pluripotent stem cell lines, each with a different NF1 patient NF1 mutation, to identify potential differential effects of NF1 mutations on human central nervous system cells and tissues. Although all mutations increased proliferation and RAS activity in 2D neural progenitor cells (NPCs) and astrocytes, we observed striking differences between NF1 mutations on 2D NPC dopamine levels, and 3D NPC proliferation, apoptosis, and neuronal differentiation in developing cerebral organoids. Together, these findings demonstrate differential effects of NF1 gene mutations at the cellular and tissue levels, suggesting that the germline NF1 gene mutation is one factor that underlies clinical variability.
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http://dx.doi.org/10.1016/j.stemcr.2020.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160375PMC
April 2020

CNS microRNA profiles: a database for cell type enriched microRNA expression across the mouse central nervous system.

Sci Rep 2020 03 18;10(1):4921. Epub 2020 Mar 18.

Department of Neurology, Washington University School of Medicine in St. Louis, St. Louis, MO, 63110, USA.

microRNAs are short, noncoding RNAs that can regulate hundreds of targets and thus shape the expression landscape of a cell. Similar to mRNA, they often exhibit cell type enriched expression and serve to reinforce cellular identity. In tissue with high cellular complexity, such as the central nervous system (CNS), it is difficult to attribute microRNA changes to a particular cell type. To facilitate interpretation of microRNA studies in these tissues, we used previously generated data to develop a publicly accessible and user-friendly database to enable exploration of cell type enriched microRNA expression. We provide illustrations of how this database can be utilized as a reference as well as for hypothesis generation. First, we suggest a putative role for miR-21 in the microglial spinal injury response. Second, we highlight data indicating that differential microRNA expression, specifically miR-326, may in part explain regional differences in inflammatory cells. Finally, we show that miR-383 expression is enriched in cortical glutamatergic neurons, suggesting a unique role in these cells. These examples illustrate the database's utility in guiding research towards unstudied regulators in the CNS. This novel resource will aid future research into microRNA-based regulatory mechanisms responsible for cellular phenotypes within the CNS.
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http://dx.doi.org/10.1038/s41598-020-61307-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080788PMC
March 2020

An inducible Cre mouse line to sparsely target nervous system cells, including Remak Schwann cells.

Neural Dev 2020 02 20;15(1). Epub 2020 Feb 20.

Department of Genetics, Washington University School of Medicine, Campus Box 8232, 4566 Scott Ave, St. Louis, MO, 63110-1093, USA.

Nerves of the peripheral nervous system contain two classes of Schwann cells: myelinating Schwann cells that ensheath large caliber axons and generate the myelin sheath, and Remak Schwann cells that surround smaller axons and do not myelinate. While tools exist for genetic targeting of Schwann cell precursors and myelinating Schwann cells, such reagents have been challenging to generate specifically for the Remak population, in part because many of the genes that mark this population in maturity are also robustly expressed in Schwann cell precursors. To circumvent this challenge, we utilized BAC transgenesis to generate a mouse line expressing a tamoxifen-inducible Cre under the control of a Remak-expressed gene promoter (Egr1). However, as Egr1 is also an activity dependent gene expressed by some neurons, we flanked this Cre by flippase (Flpe) recognition sites, and coinjected a BAC expressing Flpe under control of a pan-neuronal Snap25 promoter to excise the Cre transgene from these neuronal cells. Genotyping and inheritance demonstrate that the two BACs co-integrated into a single locus, facilitating maintenance of the line. Anatomical studies following a cross to a reporter line show sparse tamoxifen-dependent recombination in Remak Schwann cells within the mature sciatic nerve. However, depletion of neuronal Cre activity by Flpe is partial, with some neurons and astrocytes also showing evidence of Cre reporter activity in the central nervous system. Thus, this mouse line will be useful in mosaic loss-of-function studies, lineage tracing studies following injury, live cell imaging studies, or other experiments benefiting from sparse labeling.
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http://dx.doi.org/10.1186/s13064-020-00140-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031956PMC
February 2020

Maternal Fluoxetine Exposure Alters Cortical Hemodynamic and Calcium Response of Offspring to Somatosensory Stimuli.

eNeuro 2019 Nov/Dec;6(6). Epub 2019 Dec 27.

Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110.

Epidemiological studies have found an increased incidence of neurodevelopmental disorders in populations prenatally exposed to selective serotonin reuptake inhibitors (SSRIs). Optical imaging provides a minimally invasive way to determine if perinatal SSRI exposure has long-term effects on cortical function. Herein we probed the functional neuroimaging effects of perinatal SSRI exposure in a fluoxetine (FLX)-exposed mouse model. While resting-state homotopic contralateral functional connectivity was unperturbed, the evoked cortical response to forepaw stimulation was altered in FLX mice. The stimulated cortex showed decreased activity for FLX versus controls, by both hemodynamic responses [oxyhemoglobin (HbO)] and neuronal calcium responses (-GCaMP6f fluorescence). Significant alterations in both cortical HbO and calcium response amplitude were seen in the cortex ipsilateral to the stimulated paw in FLX as compared to controls. The cortical regions of largest difference in activation between FLX and controls also were consistent between HbO and calcium contrasts at the end of stimulation. Taken together, these results suggest a global loss of response signal amplitude in FLX versus controls. These findings indicate that perinatal SSRI exposure has long-term consequences on somatosensory cortical responses.
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http://dx.doi.org/10.1523/ENEURO.0238-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978917PMC
September 2020

The TMEM106B FTLD-protective variant, rs1990621, is also associated with increased neuronal proportion.

Acta Neuropathol 2020 01 27;139(1):45-61. Epub 2019 Aug 27.

Department of Psychiatry, BJC Institute of Heath, Washington University School of Medicine, 425 S. Euclid Ave., Box 8134, St. Louis, MO, 63110, USA.

Apart from amyloid β deposition and tau neurofibrillary tangles, Alzheimer's disease (AD) is a neurodegenerative disorder characterized by neuronal loss and astrocytosis in the cerebral cortex. The goal of this study is to investigate genetic factors associated with the neuronal proportion in health and disease. To identify cell-autonomous genetic variants associated with neuronal proportion in cortical tissues, we inferred cellular population structure from bulk RNA-Seq derived from 1536 individuals. We identified the variant rs1990621 located in the TMEM106B gene region as significantly associated with neuronal proportion (p value = 6.40 × 10) and replicated this finding in an independent dataset (p value = 7.41 × 10) surpassing the genome-wide threshold in the meta-analysis (p value = 9.42 × 10). This variant is in high LD with the TMEM106B non-synonymous variant p.T185S (rs3173615; r = 0.98) which was previously identified as a protective variant for frontotemporal lobar degeneration (FTLD). We stratified the samples by disease status, and discovered that this variant modulates neuronal proportion not only in AD cases, but also several neurodegenerative diseases and in elderly cognitively healthy controls. Furthermore, we did not find a significant association in younger controls or schizophrenia patients, suggesting that this variant might increase neuronal survival or confer resilience to the neurodegenerative process. The single variant and gene-based analyses also identified an overall genetic association between neuronal proportion, AD and FTLD risk. These results suggest that common pathways are implicated in these neurodegenerative diseases, that implicate neuronal survival. In summary, we identified a protective variant in the TMEM106B gene that may have a neuronal protection effect against general aging, independent of disease status, which could help elucidate the relationship between aging and neuronal survival in the presence or absence of neurodegenerative disorders. Our findings suggest that TMEM106B could be a potential target for neuronal protection therapies to ameliorate cognitive and functional deficits.
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http://dx.doi.org/10.1007/s00401-019-02066-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942643PMC
January 2020

Gtf2i and Gtf2ird1 mutation do not account for the full phenotypic effect of the Williams syndrome critical region in mouse models.

Hum Mol Genet 2019 10;28(20):3443-3465

Department of Genetics.

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.
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http://dx.doi.org/10.1093/hmg/ddz176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343053PMC
October 2019

A Paranigral VTA Nociceptin Circuit that Constrains Motivation for Reward.

Cell 2019 07;178(3):653-671.e19

Departments of Anesthesiology, Division of Basic Research, Anatomy and Neurobiology, and Washington University Pain Center, Washington University School of Medicine, St. Louis, MO, USA; Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA; Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, MO, USA; Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA. Electronic address:

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 pnVTA neurons become most active when mice stop seeking natural rewards. Selective pnVTA neuron ablation, inhibition, and conditional VTA nociceptin receptor (NOPR) deletion increased operant responding, revealing that the pnVTA nucleus and VTA NOPR signaling are necessary for regulating reward motivation. Additionally, optogenetic and chemogenetic activation of this pnVTA 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.
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http://dx.doi.org/10.1016/j.cell.2019.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001890PMC
July 2019

Loss of CELF6 RNA binding protein impairs cocaine conditioned place preference and contextual fear conditioning.

Genes Brain Behav 2019 09 19;18(7):e12593. Epub 2019 Jun 19.

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri.

In addition to gene expression differences in distinct cell types, there is substantial post-transcriptional regulation driven in part by RNA binding proteins (RBPs). Loss-of-function RBP mutations have been associated with neurodevelopmental disorders, such as Fragile-X syndrome and syndromic autism. Work performed in animal models to elucidate the influence of neurodevelopmental disorder-associated RBPs on distinct behaviors has showed a connection between normal post-transcriptional regulation and conditioned learning. We previously reported cognitive inflexibility in a mouse model null for the RBP CUG-BP, Elav-like factor 6 (CELF6), which we also found to be associated with human autism. Specifically, these mice failed to potentiate exploratory hole-poking behavior in response to familiarization to a rewarding stimuli. Characterization of Celf6 gene expression showed high levels in monoaminergic populations such as the dopaminergic midbrain populations. To better understand the underlying behavioral disruption mediating the resistance to change exploratory behavior in the holeboard task, we tested three hypotheses: Does Celf6 loss lead to global restricted patterns of behavior, failure of immediate response to reward or failure to alter behavior in response to reward (conditioning). We found the acute response to reward was intact, yet Celf6 mutant mice exhibited impaired conditioned learning to both reward and aversive stimuli. Thus, we found that the resistance to change by the Celf6 mutant in the holeboard was most parsimoniously explained as a failure of conditioning, as the mice had blunted responses even to potent rewarding stimuli such as cocaine. These findings further support the role of RBPs in conditioned learning.
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http://dx.doi.org/10.1111/gbb.12593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059558PMC
September 2019

Erroneous inference based on a lack of preference within one group: Autism, mice, and the social approach task.

Autism Res 2019 08 11;12(8):1171-1183. Epub 2019 Jun 11.

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri.

The Social Approach Task is commonly used to identify sociability deficits when modeling liability factors for autism spectrum disorder (ASD) in mice. It was developed to expand upon existing assays to examine distinct aspects of social behavior in rodents and has become a standard component of mouse ASD-relevant phenotyping pipelines. However, there is variability in the statistical analysis and interpretation of results from this task. A common analytical approach is to conduct within-group comparisons only, and then interpret a difference in significance levels as if it were a group difference, without any direct comparison. As an efficient shorthand, we named this approach EWOCs: Erroneous Within-group Only Comparisons. Here, we examined the prevalence of EWOCs and used simulations to test whether this approach could produce misleading inferences. Our review of Social Approach studies of high-confidence ASD genes revealed 45% of papers sampled used only this analytical approach. Through simulations, we then demonstrate how a lack of significant difference within one group often does not correspond to a significant difference between groups, and show this erroneous interpretation increases the rate of false positives up to 25%. Finally, we define a simple solution: use an index, like a social preference score, with direct statistical comparisons between groups to identify significant differences. We also provide power calculations to guide sample size in future studies. Overall, elimination of EWOCs and adoption of direct comparisons should result in more accurate, reliable, and reproducible data interpretations from the Social Approach Task across ASD liability models. Autism Res 2019, 12: 1171-1183. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: The Social Approach Task is widely used to assess social behavior in mice and is frequently used in studies modeling autism. However, reviewing published studies showed nearly half do not use correct comparisons to interpret these data. Using simulated and original data, we argue the correct statistical approach is a direct comparison of scores between groups. This simple solution should reduce false positives and improve consistency of results across studies.
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http://dx.doi.org/10.1002/aur.2154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688965PMC
August 2019

The Differences in Local Translatome across Distinct Neuron Types Is Mediated by Both Baseline Cellular Differences and Post-transcriptional Mechanisms.

eNeuro 2018 Nov-Dec;5(6). Epub 2019 Feb 4.

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110.

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.
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http://dx.doi.org/10.1523/ENEURO.0320-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361723PMC
March 2019

Cell-Type-Specific Profiling of Alternative Translation Identifies Regulated Protein Isoform Variation in the Mouse Brain.

Cell Rep 2019 01;26(3):594-607.e7

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Psychiatry, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2018.12.077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392083PMC
January 2019

Neurodevelopmental disease genes implicated by de novo mutation and copy number variation morbidity.

Nat Genet 2019 01 17;51(1):106-116. Epub 2018 Dec 17.

Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA.

We combined de novo mutation (DNM) data from 10,927 individuals with developmental delay and autism to identify 253 candidate neurodevelopmental disease genes with an excess of missense and/or likely gene-disruptive (LGD) mutations. Of these genes, 124 reach exome-wide significance (P < 5 × 10) for DNM. Intersecting these results with copy number variation (CNV) morbidity data shows an enrichment for genomic disorder regions (30/253, likelihood ratio (LR) +1.85, P = 0.0017). We identify genes with an excess of missense DNMs overlapping deletion syndromes (for example, KIF1A and the 2q37 deletion) as well as duplication syndromes, such as recurrent MAPK3 missense mutations within the chromosome 16p11.2 duplication, recurrent CHD4 missense DNMs in the 12p13 duplication region, and recurrent WDFY4 missense DNMs in the 10q11.23 duplication region. Network analyses of genes showing an excess of DNMs highlights functional networks, including cell-specific enrichments in the D1 and D2 spiny neurons of the striatum.
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http://dx.doi.org/10.1038/s41588-018-0288-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309590PMC
January 2019

Characterization of a Mouse Model of Börjeson-Forssman-Lehmann Syndrome.

Cell Rep 2018 11;25(6):1404-1414.e6

Department of Neuroscience, Washington University School of Medicine, St. Louis, MO 63110, USA; Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2018.10.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261530PMC
November 2018

Altered social behavior in mice carrying a cortical Foxp2 deletion.

Hum Mol Genet 2019 03;28(5):701-717

Inserm, Institut du Fer à Moulin, Sorbonne Université, Paris, France.

Genetic disruptions of the forkhead box transcription factor FOXP2 in humans cause an autosomal-dominant speech and language disorder. While FOXP2 expression pattern are highly conserved, its role in specific brain areas for mammalian social behaviors remains largely unknown. Here we studied mice carrying a homozygous cortical Foxp2 deletion. The postnatal development and gross morphological architecture of mutant mice was indistinguishable from wildtype (WT) littermates. Unbiased behavioral profiling of adult mice revealed abnormalities in approach behavior towards conspecifics as well as in the reciprocal responses of WT interaction partners. Furthermore mutant mice showed alterations in acoustical parameters of ultrasonic vocalizations, which also differed in function of the social context. Cell type-specific gene expression profiling of cortical pyramidal neurons revealed aberrant regulation of genes involved in social behavior. In particular Foxp2 mutants showed the downregulation of Mint2 (Apba2), a gene involved in approach behavior in mice and autism spectrum disorder in humans. Taken together these data demonstrate that cortical Foxp2 is required for normal social behaviors in mice.
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http://dx.doi.org/10.1093/hmg/ddy372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381386PMC
March 2019

Examining the Reversibility of Long-Term Behavioral Disruptions in Progeny of Maternal SSRI Exposure.

eNeuro 2018 Jul-Aug;5(4). Epub 2018 Jul 9.

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110.

Serotonergic dysregulation is implicated in numerous psychiatric disorders. Serotonin plays widespread trophic roles during neurodevelopment; thus perturbations to this system during development may increase risk for neurodevelopmental disorders. Epidemiological studies have examined association between selective serotonin reuptake inhibitor (SSRI) treatment during pregnancy and increased autism spectrum disorder (ASD) risk in offspring. It is unclear from these studies whether ASD susceptibility is purely related to maternal psychiatric diagnosis, or if treatment poses additional risk. We sought to determine whether maternal SSRI treatment alone or in combination with genetically vulnerable background was sufficient to induce offspring behavior disruptions relevant to ASD. We exposed C57BL/6J or mouse dams to fluoxetine (FLX) during different periods of gestation and lactation and characterized offspring on tasks assessing social communicative interaction and repetitive behavior patterns including sensory sensitivities. We demonstrate robust reductions in pup ultrasonic vocalizations (USVs) and alterations in social hierarchy behaviors, as well as perseverative behaviors and tactile hypersensitivity. mutant mice demonstrate social communicative deficits and perseverative behaviors, without further interaction with FLX. FLX re-exposure in adulthood ameliorates the tactile hypersensitivity yet exacerbates the dominance phenotype. This suggests acute deficiencies in serotonin levels likely underlie the abnormal responses to sensory stimuli, while the social alterations are instead due to altered development of social circuits. These findings indicate maternal FLX treatment, independent of maternal stress, can induce behavioral disruptions in mammalian offspring, thus contributing to our understanding of the developmental role of the serotonin system and the possible risks to offspring of SSRI treatment during pregnancy.
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http://dx.doi.org/10.1523/ENEURO.0120-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071194PMC
February 2019

Exome sequencing of 85 Williams-Beuren syndrome cases rules out coding variation as a major contributor to remaining variance in social behavior.

Mol Genet Genomic Med 2018 09 15;6(5):749-765. Epub 2018 Jul 15.

National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.

Background: Large, multigenic deletions at chromosome 7q11.23 result in a highly penetrant constellation of physical and behavioral symptoms known as Williams-Beuren syndrome (WS). Of particular interest is the unusual social-cognitive profile evidenced by deficits in social cognition and communication reminiscent of autism spectrum disorders (ASD) that are juxtaposed with normal or even relatively enhanced social motivation. Interestingly, duplications in the same region also result in ASD-like phenotypes as well as social phobias. Thus, the region clearly regulates human social motivation and behavior, yet the relevant gene(s) have not been definitively identified.

Method: Here, we deeply phenotyped 85 individuals with WS and used exome sequencing to analyze common and rare variation for association with the remaining variance in social behavior as assessed by the Social Responsiveness Scale.

Results: We replicated the previously reported unusual juxtaposition of behavioral symptoms in this new patient collection, but we did not find any new alleles of large effect in the targeted analysis of the remaining copy of genes in the Williams syndrome critical region. However, we report on two nominally significant SNPs in two genes that have been implicated in the cognitive and social phenotypes of Williams syndrome, BAZ1B and GTF2IRD1. Secondary discovery driven explorations focusing on known ASD genes and an exome wide scan do not highlight any variants of a large effect.

Conclusions: Whole exome sequencing of 85 individuals with WS did not support the hypothesis that there are variants of large effect within the remaining Williams syndrome critical region that contribute to the social phenotype. This deeply phenotyped and genotyped patient cohort with a defined mutation provides the opportunity for similar analyses focusing on noncoding variation and/or other phenotypic domains.
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http://dx.doi.org/10.1002/mgg3.429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160704PMC
September 2018

Motor neuron-derived microRNAs cause astrocyte dysfunction in amyotrophic lateral sclerosis.

Brain 2018 09;141(9):2561-2575

Department of Neurology, Washington University School of Medicine; St. Louis, MO, USA.

We recently demonstrated that microRNA-218 (miR-218) is greatly enriched in motor neurons and is released extracellularly in amyotrophic lateral sclerosis model rats. To determine if the released, motor neuron-derived miR-218 may have a functional role in amyotrophic lateral sclerosis, we examined the effect of miR-218 on neighbouring astrocytes. Surprisingly, we found that extracellular, motor neuron-derived miR-218 can be taken up by astrocytes and is sufficient to downregulate an important glutamate transporter in astrocytes [excitatory amino acid transporter 2 (EAAT2)]. The effect of miR-218 on astrocytes extends beyond EAAT2 since miR-218 binding sites are enriched in mRNAs translationally downregulated in amyotrophic lateral sclerosis astrocytes. Inhibiting miR-218 with antisense oligonucleotides in amyotrophic lateral sclerosis model mice mitigates the loss of EAAT2 and other miR-218-mediated changes, providing an important in vivo demonstration of the relevance of microRNA-mediated communication between neurons and astrocytes. These data define a novel mechanism in neurodegeneration whereby microRNAs derived from dying neurons can directly modify the glial phenotype and cause astrocyte dysfunction.
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http://dx.doi.org/10.1093/brain/awy182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113638PMC
September 2018