Abnormal behavior in a chromosome-engineered mouse model for human 15q11-13 duplication seen in autism.

Substantial evidence suggests that chromosomal abnormalities contribute to the risk of autism. The duplication of human chromosome 15q11-13 is known to be the most frequent cytogenetic abnormality in autism. We have modeled this genetic change in mice by using chromosome engineering to generate a 6.3 Mb duplication of the conserved linkage group on mouse chromosome 7. Mice with a paternal duplication display poor social interaction, behavioral inflexibility, abnormal ultrasonic vocalizations, and correlates of anxiety. An increased MBII52 snoRNA within the duplicated region, affecting the serotonin 2c receptor (5-HT2cR), correlates with altered intracellular Ca(2+) responses elicited by a 5-HT2cR agonist in neurons of mice with a paternal duplication. This chromosome-engineered mouse model for autism seems to replicate various aspects of human autistic phenotypes and validates the relevance of the human chromosome abnormality. This model will facilitate forward genetics of developmental brain disorders and serve as an invaluable tool for therapeutic development.

Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice.

Qing Dai/Indigo Naturalis (QD) has been shown to ameliorate ulcerative colitis (UC) in clinical trials; however, its mechanism remains elusive. This study investigates the effects of QD on murine dextran sulfate sodium salt-induced colitis. Oral administration of QD protected the animals from colitis as manifested by weight loss, diarrhea, and rectal bleeding. QD was distinguishingly more effective than 5-aminosalicylate. Focused microarray analysis of genes expressed in the distal colon suggested that QD influences the inflammatory pathway. Anti-inflammatory activity of QD was confirmed by the suppression of nitric oxide (NO) production in response to interleukin-1ß in cultured hepatocytes. Some of the constituents in QD, such as tryptanthrin (TRYP) and indigo, suppressed NO production. TRYP maintained body weight but did not inhibit bleeding. Indigo, on the other hand, partially ameliorated bleeding, but did not maintain body weight. The combination of TRYP and indigo did not show additive ameliorating activity. The methanol extract of QD showed an anti-colitis activity like that of TRYP. In contrast, the methanol-insoluble QD fraction moderately ameliorated diarrhea and bleeding. Combining these two fractions resulted in full anti-colitis activity. Further clarification of the active constituents will help in the discovery of a safe and potent prescription for UC.

Model mice for 15q11-13 duplication syndrome exhibit late-onset obesity and altered lipid metabolism.

Copy number variations on human chromosome 15q11-q13 have been implicated in several neurodevelopmental disorders. A paternal loss or duplication of the Prader-Willi syndrome/Angelman syndrome (PWS/AS) region confers a risk of obesity, although the mechanism remains a mystery due to a lack of an animal model that accurately recreates the obesity phenotype. We performed detailed analyses of mice with duplication of PWS/AS locus (6 Mb) generated by chromosome engineering and found that animals with a paternal duplication of this region (patDp/+) show late-onset obesity, high sensitivity for high-fat diet, high levels of blood leptin and insulin without an increase in food intake. We show that prior to becoming obese, young patDp/+ mice already had enlarged white adipocytes. Transcriptome analysis of adipose tissue revealed an up-regulation of Secreted frizzled-related protein 5 (Sfrp5), known to promote adipogenesis. We additionally generated a new mouse model of paternal duplication focusing on a 3 Mb region (3 Mb patDp/+) within the PWS/AS locus. These mice recapitulate the obese phenotypes including expansion of visceral adipose tissue. Our results suggest paternally expressed genes in PWS/AS locus play a significant role for the obesity and identify new potential targets for future research and treatment of obesity.

Mechanosensory Genes Pkd1 and Pkd2 Contribute to the Planar Polarization of Brain Ventricular Epithelium.

Directional beating of ependymal (E) cells' cilia in the walls of the ventricles in the brain is essential for proper CSF flow. E cells display two forms of planar cell polarity (PCP): rotational polarity of individual cilium and translational polarity (asymmetric positioning of cilia in the apical area). The orientation of individual E cells varies according to their location in the ventricular wall (location-specific PCP). It has been hypothesized that hydrodynamic forces on the apical surface of radial glia cells (RGCs), the embryonic precursors of E cells, could guide location-specific PCP in the ventricular epithelium. However, the detection mechanisms for these hydrodynamic forces have not been identified. Here, we show that the mechanosensory proteins polycystic kidney disease 1 (Pkd1) and Pkd2 are present in primary cilia of RGCs. Ablation of Pkd1 or Pkd2 in Nestin-Cre;Pkd1(flox/flox) or Nestin-Cre;Pkd2(flox/flox) mice, affected PCP development in RGCs and E cells. Early shear forces on the ventricular epithelium may activate Pkd1 and Pkd2 in primary cilia of RGCs to properly polarize RGCs and E cells. Consistently, Pkd1, Pkd2, or primary cilia on RGCs were required for the proper asymmetric localization of the PCP protein Vangl2 in E cells' apical area. Analyses of single- and double-heterozygous mutants for Pkd1 and/or Vangl2 suggest that these genes function in the same pathway to establish E cells' PCP. We conclude that Pkd1 and Pkd2 mechanosensory proteins contribute to the development of brain PCP and prevention of hydrocephalus. SIGNIFICANCE STATEMENT: This study identifies key molecules in the development of planar cell polarity (PCP) in the brain and prevention of hydrocephalus. Multiciliated ependymal (E) cells within the brain ventricular epithelium generate CSF flow through ciliary beating. E cells display location-specific PCP in the orientation and asymmetric positioning of their cilia. Defects in this PCP can result in hydrocephalus. Hydrodynamic forces on radial glial cells (RGCs), the embryonic progenitors of E cells, have been suggested to guide PCP. We show that the mechanosensory proteins Pkd1 and Pkd2 localize to primary cilia in RGCs, and their ablation disrupts the development of PCP in E cells. Early shear forces on RGCs may activate Pkd1 and Pkd2 in RGCs' primary cilia to properly orient E cells. This study identifies key molecules in the development of brain PCP and prevention of hydrocephalus.

Voluntary running exercise modifies astrocytic population and features in the peri-infarct cortex.

Rehabilitative exercise following a brain stroke has beneficial effects on the morphological plasticity of neurons. Particularly, voluntary running exercise after focal cerebral ischemia promotes functional recovery and ameliorates ischemia-induced dendritic spine loss in the peri-infarct motor cortex layer 5. Moreover, neuronal morphology is affected by changes in the perineuronal environment. Glial cells, whose phenotypes may be altered by exercise, are known to play a pivotal role in the formation of this perineuronal environment. Herein, we investigated the effects of voluntary running exercise on glial cells after middle cerebral artery occlusion. Voluntary running exercise increased the population of glial fibrillary acidic protein-positive astrocytes born between post-operative days (POD) 0 and 3 on POD15 in the peri-infarct cortex. After exercise, transcriptomic analysis of post-ischemic astrocytes revealed 10 upregulated and 70 downregulated genes. Furthermore, gene ontology analysis showed that the 70 downregulated genes were significantly associated with neuronal morphology. In addition, exercise reduced the number of astrocytes expressing lipocalin 2, a regulator of dendritic spine density, on POD15. Our results suggest that exercise modifies the composition of astrocytic population and their phenotype.

Ependymoma associated protein Zfta is expressed in immature ependymal cells but is not essential for ependymal development in mice.

The fusion protein of uncharacterised zinc finger translocation associated (ZFTA) and effector transcription factor of tumorigenic NF-κB signalling, RELA (ZFTA-RELA), is expressed in more than two-thirds of supratentorial ependymoma (ST-EPN-RELA), but ZFTA's expression profile and functional analysis in multiciliated ependymal (E1) cells have not been examined. Here, we showed the mRNA expression of mouse Zfta peaks on embryonic day (E) 17.5 in the wholemount of the lateral walls of the lateral ventricle. Zfta was expressed in the nuclei of FoxJ1-positive immature E1 (pre-E1) cells in E18.5 mouse embryonic brain. Interestingly, the transcription factors promoting ciliogenesis (ciliary TFs) (e.g., multicilin) and ZFTA-RELA upregulated luciferase activity using a 5' upstream sequence of ZFTA in cultured cells. Zftatm1/tm1 knock-in mice did not show developmental defects or abnormal fertility. In the Zftatm1/tm1 E1 cells, morphology, gene expression, ciliary beating frequency and ependymal flow were unaffected. These results suggest that Zfta is expressed in pre-E1 cells, possibly under the control of ciliary TFs, but is not essential for ependymal development or flow. This study sheds light on the mechanism of the ZFTA-RELA expression in the pathogenesis of ST-EPN-RELA: Ciliary TFs initiate ZFTA-RELA expression in pre-E1 cells, and ZFTA-RELA enhances its own expression using positive feedback.

Comparative distribution of Arcadlin/Protocadherin-8 mRNA in the intact and ischemic brains of adult mice.

The structural plasticity of dendritic spines serves as the adaptive capabilities of the central nervous system to various stimuli. Among these stimuli, cerebral ischemia induces dynamic alterations in neuronal network activity. Arcadlin/Paraxial protocadherin/Protocadherin-8 (Acad), a regulator of dendritic spine density, is strongly induced by activating stimuli to the neurons. However, the detailed distribution of Acad in normal and ischemic adult brains remains unclear. We comprehensively described Acad expression patterns in normal and ischemic adult brains by in situ hybridization histochemistry. We found that intact adult brains expressed Acad in the piriform cortex, dentate gyrus, hippocampal CA3, entorhinal cortex, amygdala, and hypothalamus. Acad expression was dramatically upregulated in the piriform cortex, olfactory area, dentate gyrus, entorhinal cortex, prefrontal cortex, insular cortex, amygdala, and septohippocampal nucleus 4 h after cerebral ischemia. Cerebral ischemia induced widespread neuronal activation, which was required for Acad upregulation. Our data suggested the involvement of Acad in the adaptive plasticity and remodeling of the neuronal network in the limbic and paralimbic systems.

Indigo enhances wound healing activity of Caco-2 cells via activation of the aryl hydrocarbon receptor.

Indigo Naturalis, also known as Qing Dai (QD) is a compound obtained from Indigofera tinctoria, Isatis tinctoria, and Polygonum tinctoria and is known to ameliorate refractory ulcerative colitis (UC) by an unknown mechanism. QD maintains both homeostasis and the integrity of colon epithelia in mice that have experimentally induced colitis. The primary component of QD, indigo, comprises 42.4% of the compound. Indigo efficiently suppresses rectal bleeding and reduces the erosion of the colon epithelium, whereas it does not reduce weight loss or increase survival in a certain condition. Indigo is a ligand of the aryl hydrocarbon receptor (AhR), which is involved in the anti-colitis activity of QD. Here we investigate the effects of indigo on wound (erosion) closure in colon epithelial cells. Oral administration of indigo induced expression of Cytochrome P450 1A1 (Cyp1a1) in the colon but not in the liver, suggesting that indigo stimulates AhR from the luminal side of the colon. The erosion-closure activity tested in the scratch assays using Caco-2 cells was accelerated by addition of QD and indigo to the culture medium. QD and indigo also induced nuclear localization of AhR and expression of CYP1A1 in the Caco-2 cells. Acceleration of scratch wound closure was abolished by addition of the AhR-antagonist CH223191. Cell proliferation and actin polymerization were also shown to contribute to erosion closure. The results suggest that indigo exerts its erosion-healing effects by increasing proliferation and migration of colon epithelial cells via activation of AhR in intestinal epithelia.

Voluntary running exercise after focal cerebral ischemia ameliorates dendritic spine loss and promotes functional recovery.

Cerebral infarction causes motor, sensory, and cognitive impairments. Although rehabilitation enhances recovery of activities of daily living after cerebral infarction, its mechanism remains elusive due to the lack of reproducibility and low survival rate of brain ischemic model animals. Here, to investigate the relationship between rehabilitative intervention, motor function, and pathophysiological remodeling of the tissue in the ipsilateral hemisphere after cerebral infarction, we took advantage of a highly reproducible model of cerebral infarction using C.B-17/Icr-+/+Jcl mice. In this model, we confirmed that voluntary running exercise improved functional recovery after ischemia. Exercise did not alter the volume of infarction or survived cortex, or the number of NeuN-labeled cells in the peri-infarct cortex. In mice who did not exercise, the number of basal dendritic spines of layer 5 pyramidal cells decreased in the peri-infarct motor cortex, whereas in mice who exercised it remained at the normal level. The voluntary exercise intervention maintained basal dendritic spine density within the peri-infarct area, which may reflect an adaptive remodeling of the surviving neural circuitry that might contribute to promoting the recovery of activities of daily living.

Serotonin induces Arcadlin in hippocampal neurons.

The monoamine hypothesis does not fully explain the delayed onset of recovery after antidepressant treatment or the mechanisms of recovery after electroconvulsive therapy (ECT). The common mechanism that operates both in ECT and monoaminergic treatment presumably involves molecules induced in both of these conditions. A spine density modulator, Arcadlin (Acad), the rat orthologue of human Protocadherin-8 (PCDH8) and of Xenopus and zebrafish Paraxial protocadherin (PAPC), is induced by both electroconvulsive seizure (ECS) and antidepressants; however, its cellular mechanism remains elusive. Here we confirm induction of Arcadlin upon stimulation of an N-methyl-d-aspartate (NMDA) receptor in cultured hippocampal neurons. Stimulation of an NMDA receptor also induced acute (20 min) and delayed (2 h) phosphorylation of the p38 mitogen-activated protein (MAP) kinase; the delayed phosphorylation was not obvious in Acad-/- neurons, suggesting that it depends on Arcadlin induction. Exposure of highly mature cultured hippocampal neurons to 1-10 µM serotonin for 4 h resulted in Arcadlin induction and p38 MAP kinase phosphorylation. Co-application of the NMDA receptor antagonist d-(-)-2-amino-5-phosphonopentanoic acid (APV) completely blocked Arcadlin induction and p38 MAP kinase phosphorylation. Finally, administration of antidepressant fluoxetine in mice for 16 days induced Arcadlin expression in the hippocampus. Our data indicate that the Arcadlin-p38 MAP kinase pathway is a candidate neural network modulator that is activated in hippocampal neurons under the dual regulation of serotonin and glutamate and, hence, may play a role in antidepressant therapies.

Dendritic Spine Density is Increased in Arcadlin-deleted Mouse Hippocampus.

The neural network undergoes remodeling in response to neural activity and interventions, such as antidepressants. Cell adhesion molecules that link pre- and post-synaptic membranes are responsible not only for the establishment of the neural circuitry, but also for the modulation of the strength of each synaptic connection. Among the various classes of synaptic cell adhesion molecules, a non-clustered protocadherin, Arcadlin/Paraxial protocadherin/Protocadherin-8 (Acad), is unique in that it is induced quickly in response to neural activity. Although the primary structure of Arcadlin implies its cell adhesion activity, it weakens the adhesion of N-cadherin. Furthermore, Arcadlin reduces the dendritic spine density in cultured hippocampal neurons. In order to gain an insight into the function of Arcadlin in the brain, we examined the dendritic morphologies of the hippocampal neurons in Acad-/- mice. Acad-/- mice showed a higher spine density than wild-type mice. Following an electroconvulsive seizure (ECS), which strongly induces Arcadlin in the hippocampus, the spine density gradually decreased for 8 h. ECS did not reduce the spine density of CA1 apical dendrites in Acad-/- mice. Daily intraperitoneal injection of the antidepressant fluoxetine (25 mg/kg/day) for 18 days resulted in the induction of Arcadlin in the hippocampus. This treatment reduced spine density in the dentate gyrus and CA1. Chronic fluoxetine treatment did not suppress spine density in Acad-/- mice, suggesting that fluoxetine-induced decrease in spine density is largely due to Arcadlin. The present findings confirm the spine-repulsing activity of Arcadlin and its involvement in the remodeling of hippocampal neurons in response to antidepressants.

Serotonin rebalances cortical tuning and behavior linked to autism symptoms in 15q11-13 CNV mice.

Serotonin is a critical modulator of cortical function, and its metabolism is defective in autism spectrum disorder (ASD) brain. How serotonin metabolism regulates cortical physiology and contributes to the pathological and behavioral symptoms of ASD remains unknown. We show that normal serotonin levels are essential for the maintenance of neocortical excitation/inhibition balance, correct sensory stimulus tuning, and social behavior. Conversely, low serotonin levels in 15q dup mice (a model for ASD with the human 15q11-13 duplication) result in impairment of the same phenotypes. Restoration of normal serotonin levels in 15q dup mice revealed the reversibility of a subset of ASD-related symptoms in the adult. These findings suggest that serotonin may have therapeutic potential for discrete ASD symptoms.

Neuroanatomical Phenotypes Are Consistent With Autism-Like Behavioral Phenotypes in the 15q11-13 Duplication Mouse Model.

Paternally and maternally inherited deletions and duplications of human chromosome 15q11-13 are relatively common in the human population. Furthermore, duplications in the 15q region are often associated with autism. Both maternal and paternal interstitial 15q11-13 duplication mouse models have been previously created, where several behavioral differences were found in the paternal duplication (patDp/+) mouse but not in the maternal duplication (matDp/+). These included decreased sociability, behavioral inflexibility, abnormal ultrasonic vocalizations, decreased spontaneous activity, and increased anxiety. Similarly, in the current study, we found several anatomical differences in the patDp/+ mice that were not seen in the matDp/+ mice. Regional differences that are evident only in the paternal duplication are a smaller dentate gyrus and smaller medial striatum. These differences may be responsible for the behavioral inflexibility. Furthermore, a smaller dorsal raphe nucleus could be responsible for the reported serotonin defects. This study highlights consistency that can be found between behavioral and anatomical phenotyping.

Transcriptome profiling of white adipose tissue in a mouse model for 15q duplication syndrome.

Obesity is not only associated with unhealthy lifestyles, but also linked to genetic predisposition. Previously, we generated an autism mouse model (patDp/+) that carries a 6.3 Mb paternal duplication homologous to the human 15q11-q13 locus. Chromosomal abnormalities in this region are known to cause autism spectrum disorder, Prader-Willi syndrome, and Angelman syndrome in humans. We found that, in addition to autistic-like behaviors, patDp/+ mice display late-onset obesity and hypersensitivity to a high-fat diet. These phenotypes are likely to be the results of genetic perturbations since the energy expenditures and food intakes of patDp/+ mice do not significantly differ from those of wild-type mice. Intriguingly, we found that an enlargement of adipose cells precedes the onset of obesity in patDp/+ mice. To understand the underlying molecular networks responsible for this pre-obese phenotype, we performed transcriptome profiling of white adipose tissue from patDp/+ and wild-type mice using microarray. We identified 230 genes as differentially expressed genes. Sfrp5 - a gene whose expression is positively correlated with adipocyte size, was found to be up-regulated, and Fndc5, a potent inducer of brown adipogenesis was identified to be the top down-regulated gene. Subsequent pathway analysis highlighted a set of 35 molecules involved in energy production, lipid metabolism, and small molecule biochemistry as the top candidate biological network responsible for the pre-obese phenotype of patDp/+. The microarray data were deposited in NCBI Gene Expression Omnibus database with accession number GSE58191. Ultimately, our dataset provides novel insights into the molecular mechanism of obesity and demonstrated that patDp/+ is a valuable mouse model for obesity research.

Loss of Dishevelleds disrupts planar polarity in ependymal motile cilia and results in hydrocephalus.

Defects in ependymal (E) cells, which line the ventricle and generate cerebrospinal fluid flow through ciliary beating, can cause hydrocephalus. Dishevelled genes (Dvls) are essential for Wnt signaling, and Dvl2 has been shown to localize to the rootlet of motile cilia. Using the hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) mouse, we show that compound genetic ablation of Dvls causes hydrocephalus. In hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) mutants, E cells differentiated normally, but the intracellular and intercellular rotational alignments of ependymal motile cilia were disrupted. As a consequence, the fluid flow generated by the hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) E cells was significantly slower than that observed in control mice. Dvls were also required for the proper positioning of motile cilia on the apical surface. Tamoxifen-induced conditional removal of Dvls in adult mice also resulted in defects in intracellular rotational alignment and positioning of ependymal motile cilia. These results suggest that Dvls are continuously required for E cell planar polarity and may prevent hydrocephalus.

Cerebellar plasticity and motor learning deficits in a copy-number variation mouse model of autism.

A common feature of autism spectrum disorder (ASD) is the impairment of motor control and learning, occurring in a majority of children with autism, consistent with perturbation in cerebellar function. Here we report alterations in motor behaviour and cerebellar synaptic plasticity in a mouse model (patDp/+) for the human 15q11-13 duplication, one of the most frequently observed genetic aberrations in autism. These mice show ASD-resembling social behaviour deficits. We find that in patDp/+ mice delay eyeblink conditioning--a form of cerebellum-dependent motor learning--is impaired, and observe deregulation of a putative cellular mechanism for motor learning, long-term depression (LTD) at parallel fibre-Purkinje cell synapses. Moreover, developmental elimination of surplus climbing fibres--a model for activity-dependent synaptic pruning--is impaired. These findings point to deficits in synaptic plasticity and pruning as potential causes for motor problems and abnormal circuit development in autism.

Decreased exploratory activity in a mouse model of 15q duplication syndrome; implications for disturbance of serotonin signaling.

Autism spectrum disorders (ASDs) have garnered significant attention as an important grouping of developmental brain disorders. Recent genomic studies have revealed that inherited or de novo copy number variations (CNVs) are significantly involved in the pathophysiology of ASDs. In a previous report from our laboratory, we generated mice with CNVs as a model of ASDs, with a duplicated mouse chromosome 7C that is orthologous to human chromosome 15q11-13. Behavioral analyses revealed paternally duplicated (patDp/+) mice displayed abnormal behaviors resembling the symptoms of ASDs. In the present study, we extended these findings by performing various behavioral tests with C57BL/6J patDp/+ mice, and comprehensively measuring brain monoamine levels with ex vivo high performance liquid chromatography. Compared with wild-type controls, patDp/+ mice exhibited decreased locomotor and exploratory activities in the open field test, Y-maze test, and fear-conditioning test. Furthermore, their decreased activity levels overcame increased appetite induced by 24 hours of food deprivation in the novelty suppressed feeding test. Serotonin levels in several brain regions of adult patDp/+ mice were lower than those of wild-type control, with no concurrent changes in brain levels of dopamine or norepinephrine. Moreover, analysis of monoamines in postnatal developmental stages demonstrated reduced brain levels of serotonin in young patDp/+ mice. These findings suggest that a disrupted brain serotonergic system, especially during postnatal development, may generate the phenotypes of patDp/+ mice.

Fezf1 is required for penetration of the basal lamina by olfactory axons to promote olfactory development.

In the development of the olfactory system, olfactory receptor neurons (ORNs) project their axons from the olfactory epithelium (OE) to the olfactory bulb (OB). The surface of the OB is covered by the central nervous system (CNS) basal lamina. To establish this connection, pioneer axons of the ORNs penetrate the CNS basal lamina at embryonic day 12.5 in mice. The importance of this penetration is highlighted by the Kallmann syndrome. However, little has been known about the molecular mechanism underlying this penetration process. Fezf1 (also called as Fez, Zfp312-like, and 3110069A13Rik) is a C2H2-type zinc-finger gene expressed in the OE and hypothalamic region in mice. In Fezf1-deficient mice, ORN axons (olfactory axons) do not reach the OB. Here we demonstrate that Fezf1-deficient olfactory axons do not penetrate the CNS basal lamina in vivo, and the penetration activity of the axons in Matrigel is impaired in vitro. Coculture experiments using the OE and OB reveal that axonal projection of ORNs is rescued in Fezf1-deficient mice in which the meninges including the CNS basal lamina are removed from the mutant OB. These data indicate that Fezf1 is required for the penetration of olfactory axons through the CNS basal lamina before they innervate the OB.