Viktoriya D. Nikolova

Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65

At least 120 non-olfactory G-protein-coupled receptors in the human genome are ‘orphans’ for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.

Behavioral deficits in an Angelman syndrome model: Effects of genetic background and age

Angelman syndrome (AS) is a severe neurodevelopmental disorder associated with disruption of maternally inherited UBE3A (ubiquitin protein ligase E3A) expression. At the present time, there is no effective treatment for AS. Mouse lines with loss of maternal Ube3a (Ube3a(m-/p+)) recapitulate multiple aspects of the clinical AS profile, including impaired motor coordination, learning deficits, and seizures. Thus, these genetic mouse models could serve as behavioral screens for preclinical efficacy testing, a critical component of drug discovery for AS intervention. However, the severity and consistency of abnormal phenotypes reported in Ube3a(m-/p+) mice can vary, dependent upon age and background strain, which is problematic for the detection of beneficial drug effects. As part of an ongoing AS drug discovery initiative, we characterized Ube3a(m-/p+) mice on either a 129S7/SvEvBrd-Hprt(b-m2) (129) or C57BL/6J (B6) background across a range of functional domains and ages to identify reproducible and sufficiently large phenotypes suitable for screening therapeutic compounds. The results from the study showed that Ube3a(m-/p+) mice have significant deficits in acquisition and reversal learning in the Morris water maze. The findings also demonstrated that Ube3a(m-/p+) mice exhibit motor impairment in a rotarod task, hypoactivity, reduced rearing and marble-burying, and deficient fear conditioning. Overall, these profiles of abnormal phenotypes can provide behavioral targets for evaluating effects of novel therapeutic strategies relevant to AS.

Prosocial effects of oxytocin in two mouse models of autism spectrum disorders

Clinical evidence suggests that oxytocin treatment improves social deficits and repetitive behavior in autism spectrum disorders (ASDs). However, the neuropeptide has a short plasma half-life and poor ability to penetrate the blood-brain barrier. In order to facilitate the development of more bioavailable oxytocinergic compounds as therapeutics to treat core ASD symptoms, small animal models must be validated for preclinical screens. This study examined the preclinical utility of two inbred mouse strains, BALB/cByJ and C58/J, that exhibit phenotypes relevant to core ASD symptoms. Mice from both strains were intraperitoneally administered oxytocin, using either acute or sub-chronic regimens. Acute oxytocin did not increase sociability in BALB/cByJ; however, sub-chronic oxytocin had significant prosocial effects in both BALB/cByJ and C58/J. Increased sociability was observed 24 h following the final oxytocin dose in BALB/cByJ, while prosocial effects of oxytocin emerged 1-2 weeks post-treatment in C58/J. Furthermore, acute oxytocin decreased motor stereotypy in C58/J and did not induce hypoactivity or anxiolytic-like effects in an open field test. This study demonstrates that oxytocin administration can attenuate social deficits and repetitive behavior in mouse models of ASD, dependent on dose regimen and genotype. These findings provide validation of the BALB/cByJ and C58/J models as useful platforms for screening novel drugs for intervention in ASDs and for elucidating the mechanisms contributing to the prosocial effects of oxytocin.

Repetitive behavior profile and supersensitivity to amphetamine in the C58/J mouse model of autism

Restricted repetitive behaviors are core symptoms of autism spectrum disorders (ASDs). The range of symptoms encompassed by the repetitive behavior domain includes lower-order stereotypy and self-injury, and higher-order indices of circumscribed interests and cognitive rigidity. Heterogeneity in clinical ASD profiles suggests that specific manifestations of repetitive behavior reflect differential neuropathology. The present studies utilized a set of phenotyping tasks to determine a repetitive behavior profile for the C58/J mouse strain, a model of ASD core symptoms. In an observational screen, C58/J demonstrated overt motor stereotypy, but not over-grooming, a commonly-used measure for mouse repetitive behavior. Amphetamine did not exacerbate motor stereotypy, but had enhanced stimulant effects on locomotion and rearing in C58/J, compared to C57BL/6J. Both C58/J and Grin1 knockdown mice, another model of ASD-like behavior, had marked deficits in marble-burying. In a nose poke task for higher-order repetitive behavior, C58/J had reduced holeboard exploration and preference for non-social, versus social, olfactory stimuli, but did not demonstrate cognitive rigidity following familiarization to an appetitive stimulus. Analysis of available high-density genotype data indicated specific regions of divergence between C58/J and two highly-sociable strains with common genetic lineage. Strain genome comparisons identified autism candidate genes, including Cntnap2 and Slc6a4, located within regions divergent in C58/J. However, Grin1, Nlgn1, Sapap3, and Slitrk5, genes linked to repetitive over-grooming, were not in regions of divergence. These studies suggest that specific repetitive phenotypes can be used to distinguish ASD mouse models, with implications for divergent underlying mechanisms for different repetitive behavior profiles.

Disruption of social approach by MK-801, amphetamine, and fluoxetine in adolescent C57BL/6J mice

Autism is a severe neurodevelopmental disorder, diagnosed on the basis of core behavioral symptoms. Although the mechanistic basis for the disorder is not yet known, genetic analyses have suggested a role for abnormal excitatory/inhibitory signaling systems in brain, including dysregulation of glutamatergic neurotransmission. In mice, the constitutive knockdown of NMDA receptors leads to social deficits, repetitive behavior, and self-injurious responses that reflect aspects of the autism clinical profile. However, social phenotypes differ with age: mice with reduced NMDA-receptor function exhibit hypersociability in adolescence, but markedly deficient sociability in adulthood. The present studies determined whether acute disruption of NMDA neurotransmission leads to exaggerated social approach, similar to that observed with constitutive disruption, in adolescent C57BL/6J mice. The effects of MK-801, an NMDA receptor antagonist, were compared with amphetamine, a dopamine agonist, and fluoxetine, a selective serotonin reuptake inhibitor, on performance in a three-chamber choice task. Results showed that acute treatment with MK-801 led to social approach deficits at doses without effects on entry numbers. Amphetamine also decreased social preference, but increased number of entries at every dose. Fluoxetine (10 mg/kg) had selective effects on social novelty preference. Withdrawal from a chronic ethanol regimen decreased activity, but did not attenuate sociability. Low doses of MK-801 and amphetamine were also evaluated in a marble-burying assay for repetitive behavior. MK-801, at a dose that did not disrupt sociability or alter entries, led to a profound reduction in marble-burying. Overall, these findings demonstrate that moderate alteration of NMDA, dopamine, or serotonin function can attenuate social preference in wild type mice.

Preweaning Sensorimotor Deficits and Adolescent Hypersociability in Grin1 Knockdown Mice

Mice with knockdown of the N-methyl-d-aspartate (NMDA) receptor NR1 subunit, encoded by the gene Grin1, have been investigated as a model for the intrinsic NMDA hypofunction hypothesized for schizophrenia. Previous work has shown that adult Grin1 mutant mice have overt deficits in habituation and sensorimotor gating, exaggerated reactivity to environmental stimuli, reduced social approach, and other alterations that reflect behavioral manifestations of schizophrenia. In humans, the emergence of overt symptoms of the disorder typically occurs in adolescence or early adulthood, suggesting a role for aberrant maturation of NMDA receptor signaling in symptom onset. The following study evaluated Grin1 mutant mice for abnormal behavioral phenotypes during the preweaning, adolescent, and adult periods. Measures included open field activity, prepulse inhibition of acoustic startle responses, and social preference in a three-chamber choice task. Mice from the C57BL/6J inbred strain, one of the parental strains for the Grin1 line, were also tested. The results showed that developmental reduction of NMDA receptor function led to significant alterations in behavior during the second and third weeks of life, including exaggerated startle responses and sensorimotor gating deficits on postnatal day 13, and pronounced hypersociability in adolescence. Male Grin1 mutant mice were more susceptible than female mice to the detrimental effects of decreased NMDA signaling. Overall, these findings provide evidence that reduced Grin1 function leads to abnormal phenotypes in the preweaning period, and that deficient NMDA signaling can lead to both overt hypersociability or marked asociality, dependent upon sex and age.

Disruption of the MicroRNA 137 Primary Transcript Results in Early Embryonic Lethality in Mice

To the editors: Multiple lines of evidence support a role for microRNA 137 (miR-137) in the etiology of schizophrenia (1–4) and fundamental neuronal processes (5–7). In the largest genome-wide association meta-analysis for schizophrenia,(8) the second most significant association is in MIR137HG, the gene encoding miR-137 (rs1702294, P=3.4×10−19). Prior reports indicated that genes with predicted miR-137 target sites were enriched for smaller GWAS P-values (2), raising the possibility that miR-137 regulates a gene network involved in schizophrenia. To understand more about the role of miR-137, we describe here our characterization of embryonic development, behavior, and gene expression in mice with targeted disruption of the Mir137 transcript (supplementary detailed information can be found at: http://crowley.web.unc.edu/files/2014/03/mir137.ko_.supplement.pdf). Mir137tm1Mtm mice were created in a project to generate conditional, reporter-tagged targeted mutations in 162 miRNAs (9). These mice were created using a “knockout-first” strategy (10) to produce a knockout at the RNA processing level. We purchased heterozygous (+/−) breeder mice (www.mmrrc.org/catalog/sds.php?mmrrc_id=36301). We confirmed genetic background (~75% C57BL/6J) and backcrossed twice to C57BL/6J to create animals of >95% C57BL/6J ancestry. To our knowledge, these Mir137 mutant mice have not been characterized previously. Seven heterozygous × heterozygous matings produced 35 living offspring, none of which were homozygous (−/−) for the targeted Mir137 allele (Figure 1A, P < 0.001), suggesting complete embryonic lethality in the presence of knockout of Mir137. Figure 1 Mir137 knockout mice (−/−) are embryonic lethal. (A) Heterozygous × heterozygous matings failed to yield a live born (−/−) mouse or embryo, and timed matings revealed an excess of resorbed embryos at E11.5. Heterozygous ... To confirm embryonic lethality and to determine when lethality occurred, we conducted timed matings (six heterozygous × heterozygous and four heterozygous × wildtype). On embryonic day 11.5 (E11.5), all embryos were collected (Figure 1A). Again, no homozygous (−/−) embryos were identified (P < 0.001). We observed significantly more resorbed embryos from heterozygous × heterozygous matings (P < 0.0001), indicating that embryonic lethality must occur after implantation (~E4.5) but before E11.5. Efforts to identify a Mir137 −/− mouse by genotyping resorbed embryos at E11.5 or collecting flushed embryos at E3.5 were unsuccessful, likely due to insufficient and/or impure DNA preparations from small amounts of tissue. We did not observe reduced viability of heterozygous mice (+/−) after genotyping 256 offspring (P=0.14) collected from birth to three weeks of age, indicating that one copy of Mir137 is sufficient for survival (Figure 1A). We subjected wild-type and heterozygous animals to a battery of behavioral tests (e.g., basic sensorium, activity, social behavior, learning/memory, etc.), and observed no consistent and interpretable differences. These results also suggest compensation from the remaining allele. We next examined the expression of Mir137 and the levels of mature miR-137 in the brains of heterozygous and wildtype animals (−/− embryos died too early for collection of sufficient tissue). Using allele-specific reverse transcriptase PCR, we confirmed that the targeted allele leads to complete loss of Mir137 expression downstream of the polyA sequence (Figure 1B). However, mature miR-137 levels were not significantly different between heterozygous and wild-type animals (Figure 1C), suggesting that heterozygous mice have compensatory upregulation or decreased degradation of miR-137. These expression data are consistent with the lack of a viability or behavioral phenotype in the heterozygous mice. To circumvent the embryonic lethality of Mir137 −/− mice, we attempted to make conditional knockouts (9), such that ablation of Mir137 could be limited to a particular tissue. The construct contains a combination of FRT and loxP sites intended to allow conditional mutagenesis by crossing to germline deleter Flp mice (expected to restore the wild-type allele) and then crossing to tissue-specific Cre transgenic mice (expected to generate adult brain-specific knockouts). Crosses between Mir137 +/− mice and an efficient Flp line led to several successfully recombined mice, heterozygous for the rescued allele. Repeated intercrosses of these recombined mice, however, failed to yield any homozygous offspring. Therefore, we were unable to rescue embryonic lethality. We reasoned that there were two major possibilities for the rescue failure. First, the targeting construct could have inserted into the wrong genomic position or could have sequence errors. We have likely excluded this possibility by confirming the location of the integration site and re-sequencing >95% of the construct without identifying an error. Second, Flp may have been unable to restore proper gene function due to issues inherent with the gene construct (e.g., FRT or LoxP sites in critical part of the gene). We favor this possibility because we were unable to detect any reporter gene (beta-galactosidase) activity in Mir137 +/− embryos or adult tissues, suggesting that the endogenous Mir137 promoter was inadvertently inactivated. Furthermore, bioinformatic analysis of the region (using additional data that became available after we were well into this project), suggests that the exogenous gene targeting elements may indeed interfere with endogenous functional elements, and act to prevent conditional mutagenesis (Figure 1D). The LoxP site upstream of Mir137 is in a putative splice donor sequence and the FRT site lies between highly conserved DNase hypersensitivity sites that are active in embryonic brain. In conclusion, these results suggest that at least one functional copy of Mir137 is essential for embryonic development. These data are consistent with miR-137 playing important roles in development and perhaps also in neurodevelopmental disorders like schizophrenia. It appears that miR-137’s biological pathway is capable of homeostatic compensation and, while we do not yet understand the functional impact of Mir137 schizophrenia risk variants, it is conceivable that this homeostatic capacity could be affected in some way. It is clear, however, that this genomic region is more complex than previously thought, and the design of targeted mutations in this region should incorporate all available genomic data.

The Temporal Dynamics of Arc Expression Regulate Cognitive Flexibility

Summary Neuronal activity regulates the transcription and translation of the immediate-early gene Arc/Arg3.1, a key mediator of synaptic plasticity. Proteasome-dependent degradation of Arc tightly limits its temporal expression, yet the significance of this regulation remains unknown. We disrupted the temporal control of Arc degradation by creating an Arc knockin mouse (ArcKR) where the predominant Arc ubiquitination sites were mutated. ArcKR mice had intact spatial learning but showed specific deficits in selecting an optimal strategy during reversal learning. This cognitive inflexibility was coupled to changes in Arc mRNA and protein expression resulting in a reduced threshold to induce mGluR-LTD and enhanced mGluR-LTD amplitude. These findings show that the abnormal persistence of Arc protein limits the dynamic range of Arc signaling pathways specifically during reversal learning. Our work illuminates how the precise temporal control of activity-dependent molecules, such as Arc, regulates synaptic plasticity and is crucial for cognition.

Enhanced Operant Extinction and Prefrontal Excitability in a Mouse Model of Angelman Syndrome

Angelman syndrome (AS), a neurodevelopmental disorder associated with intellectual disability, is caused by loss of maternal allele expression of UBE3A in neurons. Mouse models of AS faithfully recapitulate disease phenotypes across multiple domains, including behavior. Yet in AS, there has been only limited study of behaviors encoded by the prefrontal cortex, a region broadly involved in executive function and cognition. Because cognitive impairment is a core feature of AS, it is critical to develop behavioral readouts of prefrontal circuit function in AS mouse models. One such readout is behavioral extinction, which has been well described mechanistically and relies upon prefrontal circuits in rodents. Here we report exaggerated operant extinction in male AS model mice, concomitant with enhanced excitability in medial prefrontal neurons from male and female AS model mice. Abnormal behavior was specific to operant extinction, as two other prefrontally dependent tasks (cued fear extinction and visuospatial discrimination) were largely normal in AS model mice. Inducible deletion of Ube3a during adulthood was not sufficient to drive abnormal extinction, supporting the hypothesis that there is an early critical period for development of cognitive phenotypes in AS. This work represents the first formal experimental analysis of prefrontal circuit function in AS, and identifies operant extinction as a useful experimental paradigm for modeling cognitive aspects of AS in mice. SIGNIFICANCE STATEMENT Prefrontal cortex encodes “high-level” cognitive processes. Thus, understanding prefrontal function is critical in neurodevelopmental disorders where cognitive impairment is highly penetrant. Angelman syndrome is a neurodevelopmental disorder associated with speech and motor impairments, an outwardly happy demeanor, and intellectual disability. We describe a behavioral phenotype in a mouse model of Angelman syndrome and related abnormalities in prefrontal cortex function. We hypothesize that robust and reliable prefrontally encoded behavior may be used to model cognitive impairments in Angelman syndrome.

Amelioration of Muscle and Nerve Pathology in LAMA2 Muscular Dystrophy by AAV9-Mini-Agrin

LAMA2-related muscular dystrophy (LAMA2 MD) is the most common and fatal form of early-onset congenital muscular dystrophies. Due to the large size of the laminin α2 cDNA and heterotrimeric structure of the protein, it is challenging to develop a gene-replacement therapy. Our group has developed a novel adeno-associated viral (AAV) vector carrying the mini-agrin, which is a non-homologous functional substitute for the mutated laminin α2. A significant therapeutic effect in skeletal muscle was observed in our previous study using AAV serotype 1 (AAV1). In this investigation, we examined AAV9 vector, which has more widespread transduction than AAV1, to determine if the therapeutic effects could be further improved. As expected, AAV9-mini-agrin treatment offered enhanced therapeutic effects over the previously used AAV1-mini-agrin in extending mouse lifespan and improvement of muscle pathology. Additionally, overexpression of mini-agrin in peripheral nerves of dyw/dyw mice partially amended nerve pathology as evidenced by improved motor function and sensorimotor processing, partial restoration of myelination, partial restoration of basement membrane via EM examination, as well as decreased regeneration of Schwann cells. In conclusion, our studies indicate that overexpression of mini-agrin into dyw/dyw mice offers profound therapeutic effects in both skeletal muscle and nervous system.