Dissecting the autism-associated 16p11.2 locus identifies multiple drivers in neuroanatomical phenotypes and unveils a male-specific role for the major vault protein.

BACKGROUND: Using mouse genetic studies and systematic assessments of brain neuroanatomical phenotypes, we set out to identify which of the 30 genes causes brain defects at the autism-associated 16p11.2 locus. RESULTS: We show that multiple genes mapping to this region interact to regulate brain anatomy, with female mice exhibiting far fewer brain neuroanatomical phenotypes. In male mice, among the 13 genes associated with neuroanatomical defects (Mvp, Ppp4c, Zg16, Taok2, Slx1b, Maz, Fam57b, Bola2, Tbx6, Qprt, Spn, Hirip3, and Doc2a), Mvp is the top driver implicated in phenotypes pertaining to brain, cortex, hippocampus, ventricles, and corpus callosum sizes. The major vault protein (MVP), the main component of the vault organelle, is a conserved protein found in eukaryotic cells, yet its function is not understood. Here, we find MVP expression highly specific to the limbic system and show that Mvp regulates neuronal morphology, postnatally and specifically in males. We also recapitulate a previously reported genetic interaction and show that Mvp+/-;Mapk3+/- mice exhibit behavioral deficits, notably decreased anxiety-like traits detected in the elevated plus maze and open field paradigms. CONCLUSIONS: Our study highlights multiple gene drivers in neuroanatomical phenotypes, interacting with each other through complex relationships. It also provides the first evidence for the involvement of the major vault protein in the regulation of brain size and neuroanatomy, specifically in male mice.

YWHAE loss of function causes a rare neurodevelopmental disease with brain abnormalities in human and mouse.

PURPOSE: Miller-Dieker syndrome is caused by a multiple gene deletion, including PAFAH1B1 and YWHAE. Although deletion of PAFAH1B1 causes lissencephaly unambiguously, deletion of YWHAE alone has not clearly been linked to a human disorder. METHODS: Cases with YWHAE variants were collected through international data sharing networks. To address the specific impact of YWHAE loss of function, we phenotyped a mouse knockout of Ywhae. RESULTS: We report a series of 10 individuals with heterozygous loss-of-function YWHAE variants (3 single-nucleotide variants and 7 deletions <1 Mb encompassing YWHAE but not PAFAH1B1), including 8 new cases and 2 follow-ups, added with 5 cases (copy number variants) from literature review. Although, until now, only 1 intragenic deletion has been described in YWHAE, we report 4 new variants specifically in YWHAE (3 splice variants and 1 intragenic deletion). The most frequent manifestations are developmental delay, delayed speech, seizures, and brain malformations, including corpus callosum hypoplasia, delayed myelination, and ventricular dilatation. Individuals with variants affecting YWHAE alone have milder features than those with larger deletions. Neuroanatomical studies in Ywhae-/- mice revealed brain structural defects, including thin cerebral cortex, corpus callosum dysgenesis, and hydrocephalus paralleling those seen in humans. CONCLUSION: This study further demonstrates that YWHAE loss-of-function variants cause a neurodevelopmental disease with brain abnormalities.

Characterization of Two Mouse Chd7 Heterozygous Loss-of-Function Models Shows Dysgenesis of the Corpus Callosum and Previously Unreported Features of CHARGE Syndrome.

CHARGE syndrome is a rare congenital disorder frequently caused by mutations in the chromodomain helicase DNA-binding protein-7 CHD7. Here, we developed and systematically characterized two genetic mouse models with identical, heterozygous loss-of-function mutation of the Chd7 gene engineered on inbred and outbred genetic backgrounds. We found that both models showed consistent phenotypes with the core clinical manifestations seen in CHARGE syndrome, but the phenotypes in the inbred Chd7 model were more severe, sometimes having reduced penetrance and included dysgenesis of the corpus callosum, hypoplasia of the hippocampus, abnormal retrosplenial granular cortex, ventriculomegaly, hyperactivity, growth delays, impaired grip strength and repetitive behaviors. Interestingly, we also identified previously unreported features including reduced levels of basal insulin and reduced blood lipids. We suggest that the phenotypic variation reported in individuals diagnosed with CHARGE syndrome is likely due to the genetic background and modifiers. Finally, our study provides a valuable resource, making it possible for mouse biologists interested in Chd7 to make informed choices on which mouse model they should use to study phenotypes of interest and investigate in more depth the underlying cellular and molecular mechanisms.

Haploinsufficiency of the HIRA gene located in the 22q11 deletion syndrome region is associated with abnormal neurodevelopment and impaired dendritic outgrowth.

The 22q11.2 deletion syndrome (22q11DS) is associated with a wide spectrum of cognitive and psychiatric symptoms. Despite the considerable work performed over the past 20 years, the genetic etiology of the neurodevelopmental phenotype remains speculative. Here, we report de novo heterozygous truncating variants in the HIRA (Histone cell cycle regulation defective, S. Cerevisiae, homolog of, A) gene associated with a neurodevelopmental disorder in two unrelated patients. HIRA is located within the commonly deleted region of the 22q11DS and encodes a histone chaperone that regulates neural progenitor proliferation and neurogenesis, and that belongs to the WD40 Repeat (WDR) protein family involved in brain development and neuronal connectivity. To address the specific impact of HIRA haploinsufficiency in the neurodevelopmental phenotype of 22q11DS, we combined Hira knock-down strategies in developing mouse primary hippocampal neurons, and the direct study of brains from heterozygous Hira+/- mice. Our in vitro analyses revealed that Hira gene is mostly expressed during neuritogenesis and early dendritogenesis stages in mouse total brain and in developing primary hippocampal neurons. Moreover, shRNA knock-down experiments showed that a twofold decrease of endogenous Hira expression level resulted in an impaired dendritic growth and branching in primary developing hippocampal neuronal cultures. In parallel, in vivo analyses demonstrated that Hira+/- mice displayed subtle neuroanatomical defects including a reduced size of the hippocampus, the fornix and the corpus callosum. Our results suggest that HIRA haploinsufficiency would likely contribute to the complex pathophysiology of the neurodevelopmental phenotype of 22q11DS by impairing key processes in neurogenesis and by causing neuroanatomical defects during cerebral development.

A knock-in mouse model for KCNQ2-related epileptic encephalopathy displays spontaneous generalized seizures and cognitive impairment.

OBJECTIVE: Early onset epileptic encephalopathy with suppression-burst is one of the most severe epilepsy phenotypes in human patients. A significant proportion of cases have a genetic origin, and the most frequently mutated gene is KCNQ2, encoding Kv7.2, a voltage-dependent potassium channel subunit, leading to so-called KCNQ2-related epileptic encephalopathy (KCNQ2-REE). To study the pathophysiology of KCNQ2-REE in detail and to provide a relevant preclinical model, we generated and described a knock-in mouse model carrying the recurrent p.(Thr274Met) variant. METHODS: We introduced the p.(Thr274Met) variant by homologous recombination in embryonic stem cells, injected into C57Bl/6N blastocysts and implanted in pseudopregnant mice. Mice were then bred with 129Sv Cre-deleter to generate heterozygous mice carrying the p.(Thr274Met), and animals were maintained on the 129Sv genetic background. We studied the development of this new model and performed in vivo electroencephalographic (EEG) recordings, neuroanatomical studies at different time points, and multiple behavioral tests. RESULTS: The Kcnq2Thr274Met/+ mice are viable and display generalized spontaneous seizures first observed between postnatal day 20 (P20) and P30. In vivo EEG recordings show that the paroxysmal events observed macroscopically are epileptic seizures. The brain of the Kcnq2Thr274Met/+ animals does not display major structural defects, similar to humans, and their body weight is normal. Kcnq2Thr274Met/+ mice have a reduced life span, with a peak of unexpected death occurring for 25% of the animals by 3 months of age. Epileptic seizures were generally not observed when animals grew older. Behavioral characterization reveals important deficits in spatial learning and memory in adults but no gross abnormality during early neurosensory development. SIGNIFICANCE: Taken together, our results indicate that we have generated a relevant model to study the pathophysiology of KCNQ2-related epileptic encephalopathy and perform preclinical research for that devastating and currently intractable disease.

Large-scale neuroanatomical study uncovers 198 gene associations in mouse brain morphogenesis.

Brain morphogenesis is an important process contributing to higher-order cognition, however our knowledge about its biological basis is largely incomplete. Here we analyze 118 neuroanatomical parameters in 1,566 mutant mouse lines and identify 198 genes whose disruptions yield NeuroAnatomical Phenotypes (NAPs), mostly affecting structures implicated in brain connectivity. Groups of functionally similar NAP genes participate in pathways involving the cytoskeleton, the cell cycle and the synapse, display distinct fetal and postnatal brain expression dynamics and importantly, their disruption can yield convergent phenotypic patterns. 17% of human unique orthologues of mouse NAP genes are known loci for cognitive dysfunction. The remaining 83% constitute a vast pool of genes newly implicated in brain architecture, providing the largest study of mouse NAP genes and pathways. This offers a complementary resource to human genetic studies and predict that many more genes could be involved in mammalian brain morphogenesis.

A Method for Parasagittal Sectioning for Neuroanatomical Quantification of Brain Structures in the Adult Mouse.

In this article, we present a standardized protocol for fast and robust neuroanatomical phenotyping of the adult mouse brain, which complements a previously published article (doi: 10.1002/cpmo.12) in Current Protocols in Mouse Biology. It is aimed at providing an experimental pipeline within an academic research setting from experimental work to data analysis. Our analysis focuses on one single parasagittal plane, covering the majority of brain regions involved in higher order cognitions such as the cortex, hippocampus, and cerebellum, for a total of 166 parameters of area, length, and cell-level measurements in contrast to 78 parameters in our previously published coronal screen. Benefits of using parasagittal analysis for large-scale neuroanatomic screens are discussed. © 2018 by John Wiley & Sons, Inc.

WD40-repeat 47, a microtubule-associated protein, is essential for brain development and autophagy.

The family of WD40-repeat (WDR) proteins is one of the largest in eukaryotes, but little is known about their function in brain development. Among 26 WDR genes assessed, we found 7 displaying a major impact in neuronal morphology when inactivated in mice. Remarkably, all seven genes showed corpus callosum defects, including thicker (Atg16l1, Coro1c, Dmxl2, and Herc1), thinner (Kif21b and Wdr89), or absent corpus callosum (Wdr47), revealing a common role for WDR genes in brain connectivity. We focused on the poorly studied WDR47 protein sharing structural homology with LIS1, which causes lissencephaly. In a dosage-dependent manner, mice lacking Wdr47 showed lethality, extensive fiber defects, microcephaly, thinner cortices, and sensory motor gating abnormalities. We showed that WDR47 shares functional characteristics with LIS1 and participates in key microtubule-mediated processes, including neural stem cell proliferation, radial migration, and growth cone dynamics. In absence of WDR47, the exhaustion of late cortical progenitors and the consequent decrease of neurogenesis together with the impaired survival of late-born neurons are likely yielding to the worsening of the microcephaly phenotype postnatally. Interestingly, the WDR47-specific C-terminal to LisH (CTLH) domain was associated with functions in autophagy described in mammals. Silencing WDR47 in hypothalamic GT1-7 neuronal cells and yeast models independently recapitulated these findings, showing conserved mechanisms. Finally, our data identified superior cervical ganglion-10 (SCG10) as an interacting partner of WDR47. Taken together, these results provide a starting point for studying the implications of WDR proteins in neuronal regulation of microtubules and autophagy.

Histomorphological Phenotyping of the Adult Mouse Brain.

This article describes a series of standard operating procedures for morphological phenotyping of the mouse brain using basic histology. Many histological studies of the mouse brain use qualitative approaches based on what the human eye can detect. Consequently, some phenotypic information may be missed. Here we describe a quantitative approach for the assessment of brain morphology that is simple and robust. A total of 78 measurements are made throughout the brain at specific and well-defined regions, including the cortex, the hippocampus, and the cerebellum. Experimental design and timeline considerations, including strain background effects, the importance of sectioning quality, measurement variability, and efforts to correct human errors are discussed. © 2016 by John Wiley & Sons, Inc.