MEK signaling represents a viable therapeutic vulnerability of KRAS-driven somatic brain arteriovenous malformations.

Brain arteriovenous malformations (bAVMs) are direct connections between arteries and veins that remodel into a complex nidus susceptible to rupture and hemorrhage. Most sporadic bAVMs feature somatic activating mutations within KRAS, and endothelial-specific expression of the constitutively active variant KRASG12D models sporadic bAVM in mice. By leveraging 3D-based micro-CT imaging, we demonstrate that KRASG12D-driven bAVMs arise in stereotypical anatomical locations within the murine brain, which coincide with high endogenous Kras expression. We extend these analyses to show that a distinct variant, KRASG12C, also generates bAVMs in predictable locations. Analysis of 15,000 human patients revealed that, similar to murine models, bAVMs preferentially occur in distinct regions of the adult brain. Furthermore, bAVM location correlates with hemorrhagic frequency. Quantification of 3D imaging revealed that G12D and G12C alter vessel density, tortuosity, and diameter within the mouse brain. Notably, aged G12D mice feature increased lethality, as well as impaired cognition and motor function. Critically, we show that pharmacological blockade of the downstream kinase, MEK, after lesion formation ameliorates KRASG12D-driven changes in the murine cerebrovasculature and may also impede bAVM progression in human pediatric patients. Collectively, these data show that distinct KRAS variants drive bAVMs in similar patterns and suggest MEK inhibition represents a non-surgical alternative therapy for sporadic bAVM.

Magel2 truncation alters select behavioral and physiological outcomes in a rat model of Schaaf-Yang syndrome.

Previous studies in mice have utilized Magel2 gene deletion models to examine the consequences of its absence. We report the generation, molecular validation and phenotypic characterization of a novel rat model with a truncating Magel2 mutation modeling variants associated with Schaaf-Yang syndrome-causing mutations. Within the hypothalamus, a brain region in which human MAGEL2 is paternally expressed, we demonstrated, at the level of transcript and peptide detection, that rat Magel2 exhibits a paternal, parent-of-origin effect. In evaluations of behavioral features across several domains, juvenile Magel2 mutant rats displayed alterations in anxiety-like behavior and sociability measures. Moreover, the analysis of peripheral organ systems detected alterations in body composition, cardiac structure and function, and breathing irregularities in Magel2 mutant rats. Several of these findings are concordant with reported mouse phenotypes, indicating the conservation of MAGEL2 function across rodent species. Our comprehensive analysis revealing impairments across multiple domains demonstrates the tractability of this model system for the study of truncating MAGEL2 mutations.

Identifying genetic determinants of inflammatory pain in mice using a large-scale gene-targeted screen.

ABSTRACT: Identifying the genetic determinants of pain is a scientific imperative given the magnitude of the global health burden that pain causes. Here, we report a genetic screen for nociception, performed under the auspices of the International Mouse Phenotyping Consortium. A biased set of 110 single-gene knockout mouse strains was screened for 1 or more nociception and hypersensitivity assays, including chemical nociception (formalin) and mechanical and thermal nociception (von Frey filaments and Hargreaves tests, respectively), with or without an inflammatory agent (complete Freund's adjuvant). We identified 13 single-gene knockout strains with altered nocifensive behavior in 1 or more assays. All these novel mouse models are openly available to the scientific community to study gene function. Two of the 13 genes (Gria1 and Htr3a) have been previously reported with nociception-related phenotypes in genetically engineered mouse strains and represent useful benchmarking standards. One of the 13 genes (Cnrip1) is known from human studies to play a role in pain modulation and the knockout mouse reported herein can be used to explore this function further. The remaining 10 genes (Abhd13, Alg6, BC048562, Cgnl1, Cp, Mmp16, Oxa1l, Tecpr2, Trim14, and Trim2) reveal novel pathways involved in nociception and may provide new knowledge to better understand genetic mechanisms of inflammatory pain and to serve as models for therapeutic target validation and drug development.

Soft windowing application to improve analysis of high-throughput phenotyping data.

MOTIVATION: High-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors. RESULTS: Here we introduce 'soft windowing', a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources. AVAILABILITY AND IMPLEMENTATION: The method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Rigor and reproducibility in rodent behavioral research.

Behavioral neuroscience research incorporates the identical high level of meticulous methodologies and exacting attention to detail as all other scientific disciplines. To achieve maximal rigor and reproducibility of findings, well-trained investigators employ a variety of established best practices. Here we explicate some of the requirements for rigorous experimental design and accurate data analysis in conducting mouse and rat behavioral tests. Novel object recognition is used as an example of a cognitive assay which has been conducted successfully with a range of methods, all based on common principles of appropriate procedures, controls, and statistics. Directors of Rodent Core facilities within Intellectual and Developmental Disabilities Research Centers contribute key aspects of their own novel object recognition protocols, offering insights into essential similarities and less-critical differences. Literature cited in this review article will lead the interested reader to source papers that provide step-by-step protocols which illustrate optimized methods for many standard rodent behavioral assays. Adhering to best practices in behavioral neuroscience will enhance the value of animal models for the multiple goals of understanding biological mechanisms, evaluating consequences of genetic mutations, and discovering efficacious therapeutics.

Postnatal colonization with human "infant-type" Bifidobacterium species alters behavior of adult gnotobiotic mice.

Accumulating studies have defined a role for the intestinal microbiota in modulation of host behavior. Research using gnotobiotic mice emphasizes that early microbial colonization with a complex microbiota (conventionalization) can rescue some of the behavioral abnormalities observed in mice that grow to adulthood completely devoid of bacteria (germ-free mice). However, the human infant and adult microbiomes vary greatly, and effects of the neonatal microbiome on neurodevelopment are currently not well understood. Microbe-mediated modulation of neural circuit patterning in the brain during neurodevelopment may have significant long-term implications that we are only beginning to appreciate. Modulation of the host central nervous system by the early-life microbiota is predicted to have pervasive and lasting effects on brain function and behavior. We sought to replicate this early microbe-host interaction by colonizing gnotobiotic mice at the neonatal stage with a simplified model of the human infant gut microbiota. This model consortium consisted of four "infant-type" Bifidobacterium species known to be commensal members of the human infant microbiota present in high abundance during postnatal development. Germ-free mice and mice neonatally-colonized with a complex, conventional murine microbiota were used for comparison. Motor and non-motor behaviors of the mice were tested at 6-7 weeks of age, and colonization patterns were characterized by 16S ribosomal RNA gene sequencing. Adult germ-free mice were observed to have abnormal memory, sociability, anxiety-like behaviors, and motor performance. Conventionalization at the neonatal stage rescued these behavioral abnormalities, and mice colonized with Bifidobacterium spp. also exhibited important behavioral differences relative to the germ-free controls. The ability of Bifidobacterium spp. to improve the recognition memory of both male and female germ-free mice was a prominent finding. Together, these data demonstrate that the early-life gut microbiome, and human "infant-type" Bifidobacterium species, affect adult behavior in a strongly sex-dependent manner, and can selectively recapitulate the results observed when mice are colonized with a complex microbiota.

Loss of MeCP2 in the rat models regression, impaired sociability and transcriptional deficits of Rett syndrome.

Mouse models of the transcriptional modulator Methyl-CpG-Binding Protein 2 (MeCP2) have advanced our understanding of Rett syndrome (RTT). RTT is a 'prototypical' neurodevelopmental disorder with many clinical features overlapping with other intellectual and developmental disabilities (IDD). Therapeutic interventions for RTT may therefore have broader applications. However, the reliance on the laboratory mouse to identify viable therapies for the human condition may present challenges in translating findings from the bench to the clinic. In addition, the need to identify outcome measures in well-chosen animal models is critical for preclinical trials. Here, we report that a novel Mecp2 rat model displays high face validity for modelling psychomotor regression of a learned skill, a deficit that has not been shown in Mecp2 mice. Juvenile play, a behavioural feature that is uniquely present in rats and not mice, is also impaired in female Mecp2 rats. Finally, we demonstrate that evaluating the molecular consequences of the loss of MeCP2 in both mouse and rat may result in higher predictive validity with respect to transcriptional changes in the human RTT brain. These data underscore the similarities and differences caused by the loss of MeCP2 among divergent rodent species which may have important implications for the treatment of individuals with disease-causing MECP2 mutations. Taken together, these findings demonstrate that the Mecp2 rat model is a complementary tool with unique features for the study of RTT and highlight the potential benefit of cross-species analyses in identifying potential disease-relevant preclinical outcome measures.

Fmr1 and Nlgn3 knockout rats: novel tools for investigating autism spectrum disorders.

Animal models are critical for gaining insights into autism spectrum disorder (ASD). Despite their apparent advantages to mice for neural studies, rats have not been widely used for disorders of the human CNS, such as ASD, for the lack of convenient genome manipulation tools. Here we describe two of the first transgenic rat models for ASD, developed using zinc-finger nuclease (ZFN) methodologies, and their initial behavioral assessment using a rapid juvenile test battery. A syndromic and nonsyndromic rat model for ASD were created as two separate knockout rat lines with heritable disruptions in the genes encoding Fragile X mental retardation protein (FMRP) and Neuroligin3 (NLGN3). FMRP, a protein with numerous proposed functions including regulation of mRNA and synaptic protein synthesis, and NLGN3, a member of the neuroligin synaptic cell-adhesion protein family, have been implicated in human ASD. Juvenile subjects from both knockout rat lines exhibited abnormalities in ASD-relevant phenotypes including juvenile play, perseverative behaviors, and sensorimotor gating. These data provide important first evidence regarding the utility of rats as genetic models for investigating ASD-relevant genes.

Genetic reduction of muscarinic M4 receptor modulates analgesic response and acoustic startle response in a mouse model of fragile X syndrome (FXS).

INTRODUCTION: The G-protein coupled muscarinic acetylcholine receptors, widely expressed in the CNS, have been implicated in fragile X syndrome (FXS). Recent studies have reported an overactive signaling through the muscarinic receptors in the Fmr1KO mouse model. Hence, it was hypothesized that reducing muscarinic signaling might modulate behavioral phenotypes in the Fmr1KO mice. Pharmacological studies from our lab have provided evidence for this hypothesis, with subtype-preferring muscarinic M1 and M4 receptor antagonists modulating select behaviors in the Fmr1KO mice. Since the pharmacological antagonists were not highly specific, we investigated the specific role of M4 receptors in the Fmr1KO mouse model, using a genetic approach. METHODS: We created a double mutant heterozygous for the M4 receptor gene and hemizygous for the Fmr1 gene and examined the mutants on various behaviors. Each animal was tested on a behavior battery comprising of open-field activity (activity), light-dark (anxiety), marble burying (perseverative behavior), prepulse inhibition (sensorimotor gating), rotarod (motor coordination), passive avoidance (learning and memory) and hotplate (analgesia). Animals were also tested on the audiogenic seizure protocol and testis weights were measured. RESULTS: Reduction of M4 receptor expression in the heterozygotes completely rescued the analgesic response and partly rescued the acoustic startle response phenotype in the Fmr1KO mice. However, no modulation was observed in a number of behaviors including learning and memory, activity, perseverative behavior and audiogenic seizures. CONCLUSION: Reducing M4 receptor signaling altered only select behavioral phenotypes in the Fmr1KO mouse model, suggesting that other targets are involved in the modulation of fragile X behaviors.

The modulation of fragile X behaviors by the muscarinic M4 antagonist, tropicamide.

Muscarinic acetylcholine receptors (mAChR) are G protein-coupled receptors (M1-M5), grouped together into two functional classes, based on their G protein interaction. Although ubiquitously expressed in the CNS, the M4 protein shows highest expression in the neostriatum, cortex, and hippocampus. Electrophysiological and biochemical studies have provided evidence for overactive mAChR signaling in the fragile X knock-out (Fmr1KO) mouse model, and this has been hypothesized to contribute to the phenotypes seen in Fmr1KO mice. To address this hypothesis we used an M4 antagonist, tropicamide, to reduce the activity through the M4 mAChR and investigated the behavioral response in the Fmr1KO animals. Data from the marble-burying assay have shown that tropicamide treatment resulted in a decreased number of marbles buried in the wild-type (WT) and in the knockout (KO) animals. Results from the open field assay indicated that tropicamide increases activity in both the WT and KO mice. In the passive avoidance assay, tropicamide treatment resulted in the improvement of performance in both the WT and the KO animals at the lower doses (2 and 5 mg/kg), and the drug was shown to be important for the acquisition and not the consolidation process. Lastly, we observed that tropicamide causes a significant decrease in the percentage of audiogenic seizures in the Fmr1KO animals. These results suggest that pharmacological antagonism of the M4 receptor modulates select behavioral responses in the Fmr1KO mice.

Modulation of behavioral phenotypes by a muscarinic M1 antagonist in a mouse model of fragile X syndrome.

RATIONALE: Muscarinic acetylcholine receptors (mAChR) are G protein-coupled receptors, widely expressed in the CNS. Electrophysiological and molecular studies have provided evidence for overactive M1 receptor signaling in the fragile X knockout (Fmr1 KO) mouse model, suggesting the involvement of the M1 receptors in fragile X syndrome. Overactive signaling through the M1 receptor has been hypothesized to contribute to the phenotypes seen in fragile X mice. OBJECTIVE: We investigated the modulation of behavioral responses in the Fmr1 KO animals by reducing the activity through the muscarinic M1 receptor using the pharmacological agent dicyclomine, an M1 antagonist. METHODS: The behavioral assays used to investigate the pharmacological effects include marble burying (perseverative behavior), open-field exploration (activity), passive avoidance (learning and memory), prepulse inhibition (sensorimotor gating), and audiogenic seizures. RESULTS: Data from the marble-burying assay suggests that treatment with dicyclomine results in a decrease in the number of marbles buried in the wild-type and in the KO animals. To examine the possibility of drug-induced sedation, overall activity was measured in an open-field chamber. Dicyclomine only increases activity at a dose of 20 mg/kg in the wild-type mice but did not affect exploration in the KO animals. Lastly, we observed that dicyclomine causes a significant decrease in the percentage of audiogenic seizures in the Fmr1 KO animals. CONCLUSION: Our findings suggest that pharmacologically reducing the activity through the mAChR M1 alters select behavioral responses in the Fmr1 KO mice.