Tissue- and cell-type-specific molecular and functional signatures of 16p11.2 reciprocal genomic disorder across mouse brain and human neuronal models.

Chromosome 16p11.2 reciprocal genomic disorder, resulting from recurrent copy-number variants (CNVs), involves intellectual disability, autism spectrum disorder (ASD), and schizophrenia, but the responsible mechanisms are not known. To systemically dissect molecular effects, we performed transcriptome profiling of 350 libraries from six tissues (cortex, cerebellum, striatum, liver, brown fat, and white fat) in mouse models harboring CNVs of the syntenic 7qF3 region, as well as cellular, transcriptional, and single-cell analyses in 54 isogenic neural stem cell, induced neuron, and cerebral organoid models of CRISPR-engineered 16p11.2 CNVs. Transcriptome-wide differentially expressed genes were largely tissue-, cell-type-, and dosage-specific, although more effects were shared between deletion and duplication and across tissue than expected by chance. The broadest effects were observed in the cerebellum (2,163 differentially expressed genes), and the greatest enrichments were associated with synaptic pathways in mouse cerebellum and human induced neurons. Pathway and co-expression analyses identified energy and RNA metabolism as shared processes and enrichment for ASD-associated, loss-of-function constraint, and fragile X messenger ribonucleoprotein target gene sets. Intriguingly, reciprocal 16p11.2 dosage changes resulted in consistent decrements in neurite and electrophysiological features, and single-cell profiling of organoids showed reciprocal alterations to the proportions of excitatory and inhibitory GABAergic neurons. Changes both in neuronal ratios and in gene expression in our organoid analyses point most directly to calretinin GABAergic inhibitory neurons and the excitatory/inhibitory balance as targets of disruption that might contribute to changes in neurodevelopmental and cognitive function in 16p11.2 carriers. Collectively, our data indicate the genomic disorder involves disruption of multiple contributing biological processes and that this disruption has relative impacts that are context specific.

Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing.

Epigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi Syndrome (PWS) is caused by loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.

Convergent coexpression of autism-associated genes suggests some novel risk genes may not be detectable in large-scale genetic studies.

Autism spectrum disorder (ASD) is a heritable neurodevelopmental disorder characterized by deficits in social interactions and communication. Protein-altering variants in many genes have been shown to contribute to ASD; however, understanding the convergence across many genes remains a challenge. We demonstrate that coexpression patterns from 993 human postmortem brains are significantly correlated with the transcriptional consequences of CRISPR perturbations in human neurons. Across 71 ASD risk genes, there was significant tissue-specific convergence implicating synaptic pathways. Tissue-specific convergence was further demonstrated across schizophrenia and atrial fibrillation risk genes. The degree of ASD convergence was significantly correlated with ASD association from rare variation and differential expression in ASD brains. Positively convergent genes showed intolerance to functional mutations and had shorter coding lengths than known risk genes even after removing association with ASD. These results indicate that convergent coexpression can identify potentially novel genes that are unlikely to be discovered by sequencing studies.

Transcriptional consequences of MBD5 disruption in mouse brain and CRISPR-derived neurons.

BACKGROUND: MBD5, encoding the methyl-CpG-binding domain 5 protein, has been proposed as a necessary and sufficient driver of the 2q23.1 microdeletion syndrome. De novo missense and protein-truncating variants from exome sequencing studies have directly implicated MBD5 in the etiology of autism spectrum disorder (ASD) and related neurodevelopmental disorders (NDDs). However, little is known concerning the specific function(s) of MBD5. METHODS: To gain insight into the complex interactions associated with alteration of MBD5 in individuals with ASD and related NDDs, we explored the transcriptional landscape of MBD5 haploinsufficiency across multiple mouse brain regions of a heterozygous hypomorphic Mbd5+/GT mouse model, and compared these results to CRISPR-mediated mutations of MBD5 in human iPSC-derived neuronal models. RESULTS: Gene expression analyses across three brain regions from Mbd5+/GT mice showed subtle transcriptional changes, with cortex displaying the most widespread changes following Mbd5 reduction, indicating context-dependent effects. Comparison with MBD5 reduction in human neuronal cells reinforced the context-dependence of gene expression changes due to MBD5 deficiency. Gene co-expression network analyses revealed gene clusters that were associated with reduced MBD5 expression and enriched for terms related to ciliary function. LIMITATIONS: These analyses included a limited number of mouse brain regions and neuronal models, and the effects of the gene knockdown are subtle. As such, these results will not reflect the full extent of MBD5 disruption across human brain regions during early neurodevelopment in ASD, or capture the diverse spectrum of cell-type-specific changes associated with MBD5 alterations. CONCLUSIONS: Our study points to modest and context-dependent transcriptional consequences of Mbd5 disruption in the brain. It also suggests a possible link between MBD5 and perturbations in ciliary function, which is an established pathogenic mechanism in developmental disorders and syndromes.

Paradoxical effect of baclofen on social behavior in the fragile X syndrome mouse model.

INTRODUCTION: Fragile X syndrome (FXS) is a common monogenetic cause of intellectual disability, autism spectrum features, and a broad range of other psychiatric and medical problems. FXS is caused by the lack of the fragile X mental retardation protein (FMRP), a translational regulator of specific mRNAs at the postsynaptic compartment. The absence of FMRP leads to aberrant synaptic plasticity, which is believed to be caused by an imbalance in excitatory and inhibitory network functioning of the synapse. Evidence from studies in mice demonstrates that GABA, the major inhibitory neurotransmitter in the brain, and its receptors, is involved in the pathogenesis of FXS. Moreover, several FXS phenotypes, including social behavior deficits, could be corrected in Fmr1 KO mice after acute treatment with GABAB agonists. METHODS: As FXS would probably require a lifelong treatment, we investigated the effect of chronic treatment with the GABAB agonist baclofen on social behavior in Fmr1 KO mice on two behavioral paradigms for social behavior: the automated tube test and the three-chamber sociability test. RESULTS: Unexpectedly, chronic baclofen treatment resulted in worsening of the FXS phenotypes in these behavior tests. Strikingly, baclofen treatment also affected wild-type animals in both behavioral tests, inducing a phenotype similar to that of untreated Fmr1 KO mice. CONCLUSION: Altogether, the disappointing results of recent clinical trials with the R-baclofen enantiomer arbaclofen and our current results indicate that baclofen should be reconsidered and further evaluated before its application in targeted treatment for FXS.

A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function.

Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5' untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.

Translational endpoints in fragile X syndrome.

Fragile X syndrome (FXS) occurs in less than 10% of the intellectually disabled (ID) population. The cause of FXS is a CGG trinucleotide repeat longer than 200 CGG units within the first exon of the FMR1 gene, which leads to hypermethylation and consequently silencing of the FMR1 gene. The lack of FMR1's gene product, the fragile X mental retardation protein (FMRP) in neurons is the cause of the ID in patients with FXS. FMRP plays an important role in local protein synthesis at the synapse including modulation of synaptic plasticity. The advancing knowledge about the cellular function of FMRP has led to the identification of translational endpoints for future therapeutic intervention strategies. This review highlights the challenging routes to the identification of reliable outcome measures in preclinical studies using both cellular models and Fmr1 knockout mice. Finally, clinical studies carried out to correct intellectual and behavioral deficits in patients with FXS, using a variety of existing and new drugs, are discussed.

Rescue of dendritic spine phenotype in Fmr1 KO mice with the mGluR5 antagonist AFQ056/Mavoglurant.

Fragile X syndrome (FXS) is the leading monogenic cause of intellectual disability and autism. The disease is a result of lack of expression of the fragile X mental retardation protein. Brain tissues of patients with FXS and mice with FMRP deficiency have shown an abnormal dendritic spine phenotype. We investigated the dendritic spine length and density of hippocampal CA1 pyramidal neurons in 2-, 10-, and 25-week-old Fmr1 knockout (KO). Next, we studied the effects of long-term treatment with an mGluR5 antagonist, AFQ056/Mavoglurant, on the spine phenotype in adult Fmr1 KO mice. We observed alterations in the spine phenotype during development, with a decreased spine length in 2-week-old Fmr1 KO mice compared with age-match wild-type littermates, but with increased spine length in Fmr1 KO mice compared with 10- and 25-week-old wild-type controls. No difference was found in spine density at any age. We report a rescue of the abnormal spine length in adult Fmr1 KO mice after a long-term treatment with AFQ056/Mavoglurant. This finding suggests that long-term treatment at later stage is sufficient to reverse the structural spine abnormalities and represents a starting point for future studies aimed at improving treatments for FXS.

Epigenetic characterization of the FMR1 promoter in induced pluripotent stem cells from human fibroblasts carrying an unmethylated full mutation.

Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an unmethylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 promoter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene.

Chronic administration of AFQ056/Mavoglurant restores social behaviour in Fmr1 knockout mice.

Fragile X syndrome is caused by lack of FMR1 protein (FMRP) leading to severe symptoms, including intellectual disability, hyperactivity and autistic-like behaviour. FMRP is an RNA binding protein involved in the regulation of translation of specific target mRNAs upon stimulation of metabotropic glutamate receptor 5 (mGluR5) at the synapse. The absence of FMRP leads to enhanced activity of mGluR5 signal transduction pathways. Many conflicting results have been reported regarding social behaviour deficits in Fmr1 knockout mice, and little is known about the involvement of mGluR5 pathways on social behaviour. In this study, a three-chambered task was used to determine sociability and preference for social novelty in Fmr1 knockout mice. Disruption of Fmr1 functioning resulted in enhanced interaction with stranger mouse during sociability while no significant changes were observed during preference for social novelty assay. Chronic administration of a specific mGluR5 antagonist, AFQ056/Mavoglurant, was able to restore sociability behaviour of Fmr1 knockout mice to levels of wild type littermates. These results support the importance of mGluR5 signalling pathways on social interaction behaviour and that AFQ056/Mavoglurant might be useful as potential therapeutic intervention to rescue various behavioural aspects of the fragile X phenotype.

Delay and impairment in brain development and function in rat offspring after maternal exposure to methylmercury.

Maternal exposure to the neurotoxin methylmercury (MeHg) has been shown to have adverse effects on neural development of the offspring in man. Little is known about the underlying mechanisms by which MeHg affects the developing brain. To explore the neurodevelopmental defects and the underlying mechanism associated with MeHg exposure, the cerebellum and cerebrum of Wistar rat pups were analyzed by [(18)F]FDG PET functional imaging, field potential analysis, and microarray gene expression profiling. Female rat pups were exposed to MeHg via maternal diet during intrauterinal and lactational period (from gestational day 6 to postnatal day (PND)10), and their brain tissues were sampled for the analysis at weaning (PND18-21) and adulthood (PND61-70). The [(18)F]FDG PET imaging and field potential analysis suggested a delay in brain activity and impaired neural function by MeHg. Genome-wide transcriptome analysis substantiated these findings by showing (1) a delay in the onset of gene expression related to neural development, and (2) alterations in pathways related to both structural and functional aspects of nervous system development. The latter included changes in gene expression of developmental regulators, developmental phase-associated genes, small GTPase signaling molecules, and representatives of all processes required for synaptic transmission. These findings were observed at dose levels at which only marginal changes in conventional developmental toxicity endpoints were detected. Therefore, the approaches applied in this study are promising in terms of yielding increased sensitivity compared with classical developmental toxicity tests.