Drosophila as a Model for MECP2 Gain of Function in Neurons

Methyl-CpG-binding protein 2 (MECP2) is a multi-functional regulator of gene expression. In humans loss of MECP2 function causes classic Rett syndrome, but gain of MECP2 function also causes mental retardation. Although mouse models provide valuable insight into Mecp2 gain and loss of function, the identification of MECP2 genetic targets and interactors remains time intensive and complicated. This study takes a step toward utilizing Drosophila as a model to identify genetic targets and cellular consequences of MECP2 gain-of function mutations in neurons, the principle cell type affected in patients with Rett-related mental retardation. We show that heterologous expression of human MECP2 in Drosophila motoneurons causes distinct defects in dendritic structure and motor behavior, as reported with MECP2 gain of function in humans and mice. Multiple lines of evidence suggest that these defects arise from specific MECP2 function. First, neurons with MECP2-induced dendrite loss show normal membrane currents. Second, dendritic phenotypes require an intact methyl-CpG-binding domain. Third, dendritic defects are amended by reducing the dose of the chromatin remodeling protein, osa, indicating that MECP2 may act via chromatin remodeling in Drosophila. MECP2-induced motoneuron dendritic defects cause specific motor behavior defects that are easy to score in genetic screening. In sum, our data show that some aspects of MECP2 function can be studied in the Drosophila model, thus expanding the repertoire of genetic reagents that can be used to unravel specific neural functions of MECP2. However, additional genes and signaling pathways identified through such approaches in Drosophila will require careful validation in the mouse model

High-Throughput Sequencing of mGluR Signaling Pathway Genes Reveals Enrichment of Rare Variants in Autism

Identification of common molecular pathways affected by genetic variation in autism is important for understanding disease pathogenesis and devising effective therapies. Here, we test the hypothesis that rare genetic variation in the metabotropic glutamate-receptor (mGluR) signaling pathway contributes to autism susceptibility. Single-nucleotide variants in genes encoding components of the mGluR signaling pathway were identified by high-throughput multiplex sequencing of pooled samples from 290 non-syndromic autism cases and 300 ethnically matched controls on two independent next-generation platforms. This analysis revealed significant enrichment of rare functional variants in the mGluR pathway in autism cases. Higher burdens of rare, potentially deleterious variants were identified in autism cases for three pathway genes previously implicated in syndromic autism spectrum disorder, TSC1, TSC2, and SHANK3, suggesting that genetic variation in these genes also contributes to risk for non-syndromic autism. In addition, our analysis identified HOMER1, which encodes a postsynaptic density-localized scaffolding protein that interacts with Shank3 to regulate mGluR activity, as a novel autism-risk gene. Rare, potentially deleterious HOMER1 variants identified uniquely in the autism population affected functionally important protein regions or regulatory sequences and co-segregated closely with autism among children of affected families. We also identified rare ASD-associated coding variants predicted to have damaging effects on components of the Ras/MAPK cascade. Collectively, these findings suggest that altered signaling downstream of mGluRs contributes to the pathogenesis of non-syndromic autism

The Insulin Receptor Substrate 1 (Irs1) in Intestinal Epithelial Differentiation and in Colorectal Cancer

Colorectal cancer (CRC) is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1) is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and in vitro polarizing Caco2 and HT29 cells. IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Analysis of IRS1 immunostaining in 24 cases of primary CRC with paired colonic epithelium and hepatic metastasis showed that staining intensity was significantly higher in metastases relative to both primary CRC (P<0.01) and colonic epithelium (P<0.01). Primary and metastatic CRCs, compared to colonic epithelium, contained significantly higher numbers of IRS1-positive cells (P = 0.013 and P = 0.014, respectively). Pathologic correlations in 163 primary CRCs revealed that diffuse IRS1 staining was associated with tumors combining differentiated phenotype and aggressive markers (high Ki67, p53, and ß-catenin). In Caco 2 IRS1 and InsR were maximally expressed after polarization, while IGF1R was highest in pre-polarized cells. No nuclear IRS1 was detected, while, with polarization, phosphorylated IRS1 (pIRS1) shifted from the lateral to the apical plasma membrane and was expressed in surface cells only. In HT29, that carry mutations constitutively activating survival signaling, IRS1 and IGF1R decreased with polarization, while pIRS1 localized in nuclear spots throughout the course. Overall, these data provide evidence that IRS1 is modulated according to CRC differentiation, and support a role of IRS1 in CRC progression and liver metastatization

Transcriptional Regulation of Rod Photoreceptor Homeostasis Revealed by In Vivo NRL Targetome Analysis

A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP–Seq) data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP–Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP–Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis

Whole-Exome Sequencing and Homozygosity Analysis Implicate Depolarization-Regulated Neuronal Genes in Autism

Although autism has a clear genetic component, the high genetic heterogeneity of the disorder has been a challenge for the identification of causative genes. We used homozygosity analysis to identify probands from nonconsanguineous families that showed evidence of distant shared ancestry, suggesting potentially recessive mutations. Whole-exome sequencing of 16 probands revealed validated homozygous, potentially pathogenic recessive mutations that segregated perfectly with disease in 4/16 families. The candidate genes (UBE3B, CLTCL1, NCKAP5L, ZNF18) encode proteins involved in proteolysis, GTPase-mediated signaling, cytoskeletal organization, and other pathways. Furthermore, neuronal depolarization regulated the transcription of these genes, suggesting potential activity-dependent roles in neurons. We present a multidimensional strategy for filtering whole-exome sequence data to find candidate recessive mutations in autism, which may have broader applicability to other complex, heterogeneous disorders

DIA1R Is an X-Linked Gene Related to Deleted In Autism-1

Background: Autism spectrum disorders (ASDs) are frequently occurring disorders diagnosed by deficits in three core functional areas: social skills, communication, and behaviours and/or interests. Mental retardation frequently accompanies the most severe forms of ASDs, while overall ASDs are more commonly diagnosed in males. Most ASDs have a genetic origin and one gene recently implicated in the etiology of autism is the Deleted-In-Autism-1 (DIA1) gene. Methodology/Principal Findings: Using a bioinformatics-based approach, we have identified a human gene closely related to DIA1, we term DIA1R (DIA1-Related). While DIA1 is autosomal (chromosome 3, position 3q24), DIA1R localizes to the X chromosome at position Xp11.3 and is known to escape X-inactivation. The gene products are of similar size, with DIA1 encoding 430, and DIA1R 433, residues. At the amino acid level, DIA1 and DIA1R are 62 % similar overall (28 % identical), and both encode signal peptides for targeting to the secretory pathway. Both genes are ubiquitously expressed, including in fetal and adult brain tissue. Conclusions/Significance: Examination of published literature revealed point mutations in DIA1R are associated with X-linked mental retardation (XLMR) and DIA1R deletion is associated with syndromes with ASD-like traits and/or XLMR. Together, these results support a model where the DIA1 and DIA1R gene products regulate molecular traffic through the cellular secretory pathway or affect the function of secreted factors, and functional deficits cause disorders with ASD-lik

Effects of eight neuropsychiatric copy number variants on human brain structure

peer reviewedMany copy number variants (CNVs) confer risk for the same range of neurodevelopmental symptoms and psychiatric conditions including autism and schizophrenia. Yet, to date neuroimaging studies have typically been carried out one mutation at a time, showing that CNVs have large effects on brain anatomy. Here, we aimed to characterize and quantify the distinct brain morphometry effects and latent dimensions across 8 neuropsychiatric CNVs. We analyzed T1-weighted MRI data from clinically and non-clinically ascertained CNV carriers (deletion/duplication) at the 1q21.1 (n = 39/28), 16p11.2 (n = 87/78), 22q11.2 (n = 75/30), and 15q11.2 (n = 72/76) loci as well as 1296 non-carriers (controls). Case-control contrasts of all examined genomic loci demonstrated effects on brain anatomy, with deletions and duplications showing mirror effects at the global and regional levels. Although CNVs mainly showed distinct brain patterns, principal component analysis (PCA) loaded subsets of CNVs on two latent brain dimensions, which explained 32 and 29% of the variance of the 8 Cohen’s d maps. The cingulate gyrus, insula, supplementary motor cortex, and cerebellum were identified by PCA and multi-view pattern learning as top regions contributing to latent dimension shared across subsets of CNVs. The large proportion of distinct CNV effects on brain morphology may explain the small neuroimaging effect sizes reported in polygenic psychiatric conditions. Nevertheless, latent gene brain morphology dimensions will help subgroup the rapidly expanding landscape of neuropsychiatric variants and dissect the heterogeneity of idiopathic conditions. © 2021, The Author(s)