The identification of novel genes underlying complex mouse behavioral traits remains an important step in understanding normal brain function and its dysfunction in mental health disorders. To identify dominant mutations that influence locomotor activity, we performed a mouse N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen and mapped several loci as quantitative traits. Here we describe the fine-mapping and positional cloning of a hyperactivity locus mapped to the medial portion of mouse chromosome four. We employed a modified recombinant progeny testing approach to fine-map the confidence interval from ≈20 Mb down to ≈5 Mb. Whole exome resequencing of all exons in this region revealed a single missense mutation in the adhesion G protein-coupled receptor brain-specific angiogenesis inhibitor 2 (Bai2). This mutation, R619W, is located in a critical extracellular domain that is a hotspot for mutations in this receptor class. We find that in two different mammalian cell lines, surface expression of Bai2 R619W is markedly reduced relative to wild-type Bai2, suggesting that R619W is a loss-of-function mutation. Our results highlight the powerful combination of ENU mutagenesis and next-generation sequencing to identify specific mutations that manifest as subtle behavioral phenotypes.

During development of the central nervous system, precise synaptic connections between presynaptic and postsynaptic neurons are formed. While significant progress has been made in our understanding of AMPA receptor trafficking during synaptic plasticity, less is known about the molecules that recruit AMPA receptors to nascent synapses during synaptogenesis. Here we identify a type II transmembrane protein (SynDIG1) that regulates AMPA receptor content at developing synapses in dissociated rat hippocampal neurons. SynDIG1 colocalizes with AMPA receptors at synapses and at extrasynaptic sites and associates with AMPA receptors in heterologous cells and brain. Altered levels of SynDIG1 in cultured neurons result in striking changes in excitatory synapse number and function. SynDIG1-mediated synapse development is dependent on association with AMPA receptors via its extracellular C terminus. Intriguingly, SynDIG1 content in dendritic spines is regulated by neuronal activity. Altogether, we define SynDIG1 as an activity-regulated transmembrane protein that regulates excitatory synapse development.

Altering AMPA receptor (AMPAR) content at synapses is a key mechanism underlying the regulation of synaptic strength during learning and memory. Previous work demonstrated that SynDIG1 (synapse differentiation-induced gene 1) encodes a transmembrane AMPAR-associated protein that regulates excitatory synapse strength and number. Here we show that the related protein SynDIG4 (also known as Prrt1) modifies AMPAR gating properties in a subunit-dependent manner. Young SynDIG4 knockout (KO) mice have weaker excitatory synapses, as evaluated by immunocytochemistry and electrophysiology. Adult SynDIG4 KO mice show complete loss of tetanus-induced long-term potentiation (LTP), while mEPSC amplitude is reduced by only 25%. Furthermore, SynDIG4 KO mice exhibit deficits in two independent cognitive assays. Given that SynDIG4 colocalizes with the AMPAR subunit GluA1 at non-synaptic sites, we propose that SynDIG4 maintains a pool of extrasynaptic AMPARs necessary for synapse development and function underlying higher-order cognitive plasticity.

Modification of the strength of excitatory synaptic connections is a fundamental mechanism by which neural circuits are refined during development and learning. Synapse Differentiation Induced Gene 1 (SynDIG1) has been shown to play a key role in regulating synaptic strength in vitro. Here, we investigated the role of SynDIG1 in vivo in mice with a disruption of the SynDIG1 gene rather than use an alternate loxP-flanked conditional mutant that we find retains a partial protein product. The gene-trap insertion with a reporter cassette mutant mice shows that the SynDIG1 promoter is active during embryogenesis in the retina with some activity in the brain, and postnatally in the mouse hippocampus, cortex, hindbrain, and spinal cord. Ultrastructural analysis of the hippocampal CA1 region shows a decrease in the average PSD length of synapses and a decrease in the number of synapses with a mature phenotype. Intriguingly, the total synapse number appears to be increased in SynDIG1 mutant mice. Electrophysiological analyses show a decrease in AMPA and NMDA receptor function in SynDIG1-deficient hippocampal neurons. Glutamate stimulation of individual dendritic spines in hippocampal slices from SynDIG1-deficient mice reveals increased short-term structural plasticity. Notably, the overall levels of PSD-95 or glutamate receptors enriched in postsynaptic biochemical fractions remain unaltered; however, activity-dependent synapse development is strongly compromised upon the loss of SynDIG1, supporting its importance for excitatory synapse maturation. Together, these data are consistent with a model in which SynDIG1 regulates the maturation of excitatory synapse structure and function in the mouse hippocampus in vivo.

A subset of medulloblastomas, the most common brain tumor in children, is hypothesized to originate from granule neuron precursors (GNPs) in which the sonic hedgehog (SHH) pathway is over-activated. MXD3, a basic helix-look-helix zipper transcription factor of the MAD family, has been reported to be upregulated during postnatal cerebellar development and to promote GNP proliferation and MYCN expression. Mxd3 is upregulated in mouse models of medulloblastoma as well as in human medulloblastomas. Therefore, we hypothesize that MXD3 plays a role in the cellular events that lead to medulloblastoma biogenesis. In agreement with its proliferative role in GNPs, MXD3 knock-down in DAOY cells resulted in decreased proliferation. Sustained overexpression of MXD3 resulted in decreased cell numbers due to increased apoptosis and cell cycle arrest. Structure-function analysis revealed that the Sin3 interacting domain, the basic domain, and binding to E-boxes are essential for this activity. Microarray-based expression analysis indicated up-regulation of 84 genes and down-regulation of 47 genes. Potential direct MXD3 target genes were identified by ChIP-chip. Our results suggest that MXD3 is necessary for DAOY medulloblastoma cell proliferation. However, increased level and/or duration of MXD3 expression ultimately reduces cell numbers via increased cell death and cell cycle arrest.

The transcription factor MXD3 is an atypical member of the MYC/MAX/MXD transcriptional network and has been previously shown to be an important regulator of cell proliferation. MXD3 has been shown to be overexpressed and to be required for medulloblastoma and acute lymphoblastic leukemia cell proliferation. In this study we leveraged datasets from The Cancer Genome Atlas to examine MXD3 across several cancers. We find that MXD3 transcripts are significantly overexpressed in ~72% of the available datasets. The gene itself is not frequently altered, while the promoter appears to be hypomethylated. We examine the possibility that aberrant regulation of the MXD3 message is the cause of abnormal MXD3 expression across cancers. Specifically, we looked at MXD3 alternative splicing in glioblastoma multiforme (GBM) and find notable functional differences between the splice variants. The 3'UTR confers differential message stability. Furthermore, the different coding sequences lead to different protein stabilities and localizations. Altogether, these data extend our knowledge of MXD3 in the context of human cancers while characterizing a previously unstudied splice variant of MXD3.

Consistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis. Asymmetric gene expression of the TGFβ superfamily member Nodal related 1 (Nr1) in the left lateral mesoderm plate is a highly conserved step regulating the situs of the heart and viscera. In Xenopus, movement of maternal serotonin (5HT) through gap-junctional paths at cleavage stages dictates asymmetry upstream of Nr1. However, the mechanisms linking earlier biophysical asymmetries with this transcriptional control point are not known.

Regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) at synapses is a predominant mechanism for regulating synaptic strength. We identified the transmembrane protein synapse differentiation-induced gene 1 (SynDIG1; SD1) as an AMPAR interacting protein that regulates excitatory synaptic strength and AMPAR number both in vitro and in vivo. The related protein SynDIG4 (SD4; also known as PRRT1) was identified in several independent proteomic screens in complex with AMPARs, suggesting that it may function as an AMPAR auxiliary factor. Here, we show that the co-expression of SD4 with GluA1 or GluA2 homomeric AMPARs in COS cells leads to a 50 or 33% increase in the mean area of AMPAR puncta, respectively. This effect is accentuated when AMPAR puncta are stratified for co-localization with SD4, resulting in a 100 and 65% increase in GluA1 and GluA2 puncta, respectively. Chimeric proteins expressing only the membrane bound domain of SD4 co-expressed with full-length GluA1 or GluA2 recapitulated the effects of wild-type (WT) SD4. Additionally, the mean puncta area of GluA1 or GluA2 chimeras expressing the membrane and C-terminal domains increased significantly when co-localized with WT SD4. Similarly, the co-expression of GluA1 or GluA2 with SD4 results in a significant increase in the mean area of SD4 puncta co-localized with GluA1 or GluA2, respectively. Last, we observed a significant increase in the co-localization of SD4 with GluA1 after glycine induced long-term potentiation (LTP). The mean size of GluA1 puncta was significantly increased when stratified, indicating that co-localization with SD4 increases synaptic GluA1 cluster size during LTP. These data indicate mutually dependent clustering of SD4 and AMPAR subunits both in COS cells and primary hippocampal neurons, suggesting a mechanism for increased synaptic strength during chemical LTP.

The modulation of AMPA receptor (AMPAR) content at synapses is thought to be an underlying molecular mechanism of memory and learning. AMPAR content at synapses is highly plastic and is regulated by numerous AMPAR accessory transmembrane proteins such as TARPs, cornichons, and CKAMPs. SynDIG (synapse differentiation-induced gene) defines a family of four genes (SynDIG1-4) expressed in distinct and overlapping patterns in the brain. SynDIG1 was previously identified as a novel transmembrane AMPAR-associated protein that regulates synaptic strength. The related protein SynDIG4 [also known as Prrt1 (proline-rich transmembrane protein 1)] has recently been identified as a component of AMPAR complexes. In this study, we show that SynDIG1 and SynDIG4 have distinct yet overlapping patterns of expression in the central nervous system, with SynDIG4 having especially prominent expression in the hippocampus and particularly within CA1. In contrast to SynDIG1 and other traditional AMPAR auxiliary subunits, SynDIG4 is de-enriched at the postsynaptic density and colocalizes with extrasynaptic GluA1 puncta in primary dissociated neuron culture. These results indicate that, although SynDIG4 shares sequence similarity with SynDIG1, it might act through a unique mechanism as an auxiliary factor for extrasynaptic GluA1-containing AMPARs. J. Comp. Neurol. 524:2266-2280, 2016. © 2015 Wiley Periodicals, Inc.

The many diverse functions executed by the central nervous system (CNS) are mirrored in the diverse shapes, connections, and firing patterns of its individual neuronal cell types. Furthermore, distinct neurological diseases are the result of defects in specific neuronal cell types. However, despite the significance of this cellular diversity underlying brain function and disease, we know relatively little about the genes that contribute to purposeful differences among regions and cell types within the brain. A major challenge in this endeavor is the paucity of markers that define the many regions and cell types thought to exist. Cataloging the neuronal cell types and cell- and region-specific marker genes requires novel avenues that enable researchers to define gene expression profiles of brain regions and individual neurons and to apply this information to understand functional and structural properties in the normal and diseased brain. Functional genomic approaches such as gene expression profiling offers the exclusive opportunity to glimpse the detailed inner workings of distinct neuronal cell types. Recent studies have applied microarray technology in unique and novel ways to understand the molecular mechanisms that underlie such neuronal diversity and their potential role in brain diseases.