System-based proteomic and metabonomic analysis of the Df(16)A+/- mouse identifies potential miR-185 targets and molecular pathway alterations.

Deletions on chromosome 22q11.2 are a strong genetic risk factor for development of schizophrenia and cognitive dysfunction. We employed shotgun liquid chromatography-mass spectrometry (LC-MS) proteomic and metabonomic profiling approaches on prefrontal cortex (PFC) and hippocampal (HPC) tissue from Df(16)A+/- mice, a model of the 22q11.2 deletion syndrome. Proteomic results were compared with previous transcriptomic profiling studies of the same brain regions. The aim was to investigate how the combined effect of the 22q11.2 deletion and the corresponding miRNA dysregulation affects the cell biology at the systems level. The proteomic brain profiling analysis revealed PFC and HPC changes in various molecular pathways associated with chromatin remodelling and RNA transcription, indicative of an epigenetic component of the 22q11.2DS. Further, alterations in glycolysis/gluconeogenesis, mitochondrial function and lipid biosynthesis were identified. Metabonomic profiling substantiated the proteomic findings by identifying changes in 22q11.2 deletion syndrome (22q11.2DS)-related pathways, such as changes in ceramide phosphoethanolamines, sphingomyelin, carnitines, tyrosine derivates and panthothenic acid. The proteomic findings were confirmed using selected reaction monitoring mass spectrometry, validating decreased levels of several proteins encoded on 22q11.2, increased levels of the computationally predicted putative miR-185 targets UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110 kDa subunit (OGT1) and kinesin heavy chain isoform 5A and alterations in the non-miR-185 targets serine/threonine-protein phosphatase 2B catalytic subunit gamma isoform, neurofilament light chain and vesicular glutamate transporter 1. Furthermore, alterations in the proteins associated with mammalian target of rapamycin signalling were detected in the PFC and with glutamatergic signalling in the hippocampus. Based on the proteomic and metabonomic findings, we were able to develop a schematic model summarizing the most prominent molecular network findings in the Df(16)A+/- mouse. Interestingly, the implicated pathways can be linked to one of the most consistent and strongest proteomic candidates, (OGT1), which is a predicted miR-185 target. Our results provide novel insights into system-biological mechanisms associated with the 22q11DS, which may be linked to cognitive dysfunction and an increased risk to develop schizophrenia. Further investigation of these pathways could help to identify novel drug targets for the treatment of schizophrenia.

Deletion of Rapgef6, a candidate schizophrenia susceptibility gene, disrupts amygdala function in mice.

In human genetic studies of schizophrenia, we uncovered copy-number variants in RAPGEF6 and RAPGEF2 genes. To discern the effects of RAPGEF6 deletion in humans, we investigated the behavior and neural functions of a mouse lacking Rapgef6. Rapgef6 deletion resulted in impaired amygdala function measured as reduced fear conditioning and anxiolysis. Hippocampal-dependent spatial memory and prefrontal cortex-dependent working memory tasks were intact. Neural activation measured by cFOS phosphorylation demonstrated a reduction in hippocampal and amygdala activation after fear conditioning, while neural morphology assessment uncovered reduced spine density and primary dendrite number in pyramidal neurons of the CA3 hippocampal region of knockout mice. Electrophysiological analysis showed enhanced long-term potentiation at cortico-amygdala synapses. Rapgef6 deletion mice were most impaired in hippocampal and amygdalar function, brain regions implicated in schizophrenia pathophysiology. The results provide a deeper understanding of the role of the amygdala in schizophrenia and suggest that RAPGEF6 may be a novel therapeutic target in schizophrenia.

Cntnap4 differentially contributes to GABAergic and dopaminergic synaptic transmission.

Although considerable evidence suggests that the chemical synapse is a lynchpin underlying affective disorders, how molecular insults differentially affect specific synaptic connections remains poorly understood. For instance, Neurexin 1a and 2 (NRXN1 and NRXN2) and CNTNAP2 (also known as CASPR2), all members of the neurexin superfamily of transmembrane molecules, have been implicated in neuropsychiatric disorders. However, their loss leads to deficits that have been best characterized with regard to their effect on excitatory cells. Notably, other disease-associated genes such as BDNF and ERBB4 implicate specific interneuron synapses in psychiatric disorders. Consistent with this, cortical interneuron dysfunction has been linked to epilepsy, schizophrenia and autism. Using a microarray screen that focused upon synapse-associated molecules, we identified Cntnap4 (contactin associated protein-like 4, also known as Caspr4) as highly enriched in developing murine interneurons. In this study we show that Cntnap4 is localized presynaptically and its loss leads to a reduction in the output of cortical parvalbumin (PV)-positive GABAergic (γ-aminobutyric acid producing) basket cells. Paradoxically, the loss of Cntnap4 augments midbrain dopaminergic release in the nucleus accumbens. In Cntnap4 mutant mice, synaptic defects in these disease-relevant neuronal populations are mirrored by sensory-motor gating and grooming endophenotypes; these symptoms could be pharmacologically reversed, providing promise for therapeutic intervention in psychiatric disorders.

Neuroanatomical phenotypes in a mouse model of the 22q11.2 microdeletion.

Recurrent deletions at the 22q11.2 locus have been established as a strong genetic risk factor for the development of schizophrenia and cognitive dysfunction. Individuals with 22q11.2 deletions have a range of well-defined volumetric abnormalities in a number of critical brain structures. A mouse model of the 22q11.2 deletion (Df(16)A(+/-)) has previously been utilized to characterize disease-associated abnormalities on synaptic, cellular, neurocircuitry, and behavioral levels. We performed a high-resolution MRI analysis of mutant mice compared with wild-type littermates. Our analysis revealed a striking similarity in the specific volumetric changes of Df(16)A(+/-) mice compared with human 22q11.2 deletion carriers, including in cortico-cerebellar, cortico-striatal and cortico-limbic circuits. In addition, higher resolution magnetic resonance imaging compared with neuroimaging in human subjects allowed the detection of previously unknown subtle local differences. The cerebellar findings in Df(16)A(+/-) mice are particularly instructive as they are localized to specific areas within both the deep cerebellar nuclei and the cerebellar cortex. Our study indicates that the Df(16)A(+/-)mouse model recapitulates most of the hallmark neuroanatomical changes observed in 22q11.2 deletion carriers. Our findings will help guide the design and interpretation of additional complementary studies and thereby advance our understanding of the abnormal brain development underlying the emergence of 22q11.2 deletion-associated psychiatric and cognitive symptoms.

A Disc1 mutation differentially affects neurites and spines in hippocampal and cortical neurons.

A balanced chromosomal translocation segregating with schizophrenia and affective disorders in a large Scottish family disrupting DISC1 implicated this gene as a susceptibility gene for major mental illness. Here we study neurons derived from a genetically engineered mouse strain with a truncating lesion disrupting the endogenous Disc1 ortholog. We provide a detailed account of the consequences of this mutation on axonal and dendritic morphogenesis as well as dendritic spine development in cultured hippocampal and cortical neurons. We show that the mutation has distinct effects on these two types of neurons, supporting a cell-type specific role of Disc1 in establishing structural connections among neurons. Moreover, using a validated antibody we provide evidence indicating that Disc1 localizes primarily to Golgi apparatus-related vesicles. Our results support the notion that in vitro cultures derived from Disc1(Tm1Kara) mice provide a valuable model for future mechanistic analysis of the cellular and biochemical effects of this mutation, and can thus serve as a platform for drug discovery efforts.

Avoiding mouse traps in schizophrenia genetics: lessons and promises from current and emerging mouse models.

Schizophrenia is one of the most common psychiatric disorders, but despite progress in identifying the genetic factors implicated in its development, the mechanisms underlying its etiology and pathogenesis remain poorly understood. Development of mouse models is critical for expanding our understanding of the causes of schizophrenia. However, translation of disease pathology into mouse models has proven to be challenging, primarily due to the complex genetic architecture of schizophrenia and the difficulties in the re-creation of susceptibility alleles in the mouse genome. In this review we highlight current research on models of major susceptibility loci and the information accrued from their analysis. We describe and compare the different approaches that are necessitated by diverse susceptibility alleles, and discuss their advantages and drawbacks. Finally, we discuss emerging mouse models, such as second-generation pathophysiology models based on innovative approaches that are facilitated by the information gathered from the current genetic mouse models.

Disruption of thermal nociceptive behaviour in mice mutant for the schizophrenia-associated genes NRG1, COMT and DISC1.

Abnormalities in pain perception, especially altered warmth and heat pain sensitivity, have been reported in schizophrenia. Therefore, genes associated with schizophrenia, including neuregulin-1 (NRG1), catechol-O-methyltranferase (COMT) and disrupted-in-schizophrenia-1 (DISC1), may play a role in modulating the physiological and psychological effects of pain stimuli in such patients. Thermal pain sensitivity was assessed in NRG1, COMT and DISC1 mutant mice, and the anti-nociceptive effects of acute Delta(9)-tetrahydrocannabinol (THC) were compared in NRG1 and COMT mutants. At baseline, deletion of NRG1 and DISC1 each reduced thermal pain sensitivity, while deletion of COMT increased pain sensitivity. Neither NRG1 nor COMT deletion altered the anti-nociceptive effects of acute systemic THC (8.0mg/kg). These results indicate a differential contribution of NRG1 and DISC1 vis-à-vis COMT to the processing of thermal nociceptive stimuli and extend their phenotypic relationship to psychotic illness.

Importance of membrane-bound catechol-O-methyltransferase in L-DOPA metabolism: a pharmacokinetic study in two types of Comt gene modified mice.

BACKGROUND AND PURPOSE: Catechol-O-methyltransferase (COMT) metabolizes compounds containing catechol structures and has two forms: soluble (S-COMT) and membrane-bound (MB-COMT). Here we report the generation of a mouse line that expresses MB-COMT but not S-COMT. We compared the effects of deleting S-COMT only or both COMT forms on the pharmacokinetics of oral L-DOPA. EXPERIMENTAL APPROACH: L-DOPA (10 mg kg(-1)) and carbidopa (30 mg kg(-1)) were given to mice by gastric tube, and samples were taken at various times. HPLC was used to measure L-DOPA in plasma and tissue samples, and dopamine and its metabolites in brain. Immunohistochemistry and Western blotting were used to characterize the distribution of COMT protein isoforms. KEY RESULTS: Lack of S-COMT did not affect the levels of L-DOPA in plasma or peripheral tissues, whereas in the full COMT-knock-out mice, these levels were increased. The levels of 3-O-methyldopa were significantly decreased in the S-COMT-deficient mice. In the brain, L-DOPA levels were not significantly increased, and dopamine was increased only in females. The total COMT activity in the S-COMT-deficient mice was 22-47% of that in the wild-type mice. In peripheral tissues, female mice had lower COMT activity than the males. CONCLUSIONS AND IMPLICATIONS: In S-COMT-deficient mice, MB-COMT in the liver and the duodenum is able to O-methylate about one-half of exogenous L-DOPA. Sexual dimorphism and activity of the two COMT isoforms seems to be tissue specific and more prominent in peripheral tissues than in the brain.

Phenotypic characterization of cognition and social behavior in mice with heterozygous versus homozygous deletion of catechol-O-methyltransferase.

Catechol-O-methyltransferase is an important enzyme in the metabolism of dopamine and an important regulator of aspects of dopamine-dependent working memory in prefrontal cortex that are disturbed in schizophrenia. This study investigated the phenotype of mice with heterozygous deletion vs. homozygous knockout of the catechol-O-methyltransferase gene across paradigms that access processes relevant for psychotic illness. Homozygotes evidenced improved performance in spontaneous alternation, an index of immediate spatial working memory; this effect appeared more substantive in males and was reflected in performance in aspects of the Barnes maze, an index of spatial learning/memory. Heterozygotes evidenced impaired performance in object recognition, an index of recognition memory; this effect was evident for both sexes at a retention interval of 5 min but appeared more enduring in males. There were no material effects for either genotype in relation to sociability or social novelty preference. While homozygous catechol-O-methyltransferase deletion results in improvement in spatial learning/working memory with little effect on social behavior, heterozygous deletion results in impairment of recognition memory. We have reported recently, using similar methods, that mice with deletion of the schizophrenia risk gene neuregulin-1 evidence disruption to social behavior, with little effect on spatial learning/working memory. The data suggest that catechol-O-methyltransferase and neuregulin-1 may influence, respectively, primarily cognitive and social endophenotypes of the overall schizophrenia syndrome.

Evidence implicating the candidate schizophrenia/bipolar disorder susceptibility gene G72 in mitochondrial function.

G72 is a strong candidate susceptibility gene for schizophrenia and bipolar disorder, whose function remains enigmatic. Here we show that one splicing isoform of the gene (LG72) encodes for a mitochondrial protein. We also provide convergent lines of evidence that increase of endogenous or exogenous G72 levels promotes robust mitochondrial fragmentation in mammalian cell lines and primary neurons, which proceeds in a manner that does not depend on induction of apoptosis or alteration in mitochondrial transmembrane potential. Finally, we show that increase in G72 levels in immature primary neurons is accompanied by a marked increase in dendritic arborization. By contrast, we failed to confirm the originally proposed functional interaction between G72 and D-amino acid oxidase (DAO) in two tested cell lines. Our results suggest an alternative role for G72 in modulating mitochondrial function.

The contribution of three strong candidate schizophrenia susceptibility genes in demographically distinct populations.

Here we characterize and compare the contribution of three recently identified strong candidate schizophrenia susceptibility genes; G72, neuregulin 1 (NRG1) and dystrobrevin-binding protein 1 (DTNBP1) in two independent datasets of patients with distinct genetic backgrounds. On the basis of corrected P-values from single- and multilocus transmission distortion tests our analysis provides no support for a contribution of G72, NRG1 or DTNBP1 in the tested samples. When transmission of individual haplotypes was considered, a picture more consistent with the original studies emerged, where transmission distortions in the same direction as the original samples and involving the same core haplotypes were observed for G72 and NRG1. Interestingly, whereas the NRG1 gene analysis was dominated by the presence of over-transmitted haplotypes, the G72 gene analysis was consistently dominated in both datasets by under-transmissions. Negative transmissions involved a core haplotype complementary to the originally detected over-transmitted haplotype, suggesting the presence of a protective variant within the G72 locus.

Genome-wide scan in a large complex pedigree with predominantly male schizophrenics from the island of Kosrae: evidence for linkage to chromosome 2q.

It is widely accepted that founder populations hold promise for mapping loci for complex traits. However, the outcome of these mapping efforts will most likely depend on the individual demographic characteristics and historical circumstances surrounding the founding of a given genetic isolate. The 'ideal' features of a founder population are currently unknown. The Micronesian islandic population of Kosrae, one of the four islands comprising the Federated States of Micronesia (FSM), was founded by a small number of settlers and went through a secondary genetic 'bottleneck' in the mid-19th century. The potential for reduced etiological (genetic and environmental) heterogeneity, as well as the opportunity to ascertain extended and statistically powerful pedigrees makes the Kosraen population attractive for mapping schizophrenia susceptibility genes. Our exhaustive case ascertainment from this islandic population identified 32 patients who met DSM-IV criteria for schizophrenia or schizoaffective disorder. Three of these were siblings in one nuclear family, and 27 were from a single large and complex schizophrenia kindred that includes a total of 251 individuals. One of the most startling findings in our ascertained sample was the great difference in male and female disease rates. A genome-wide scan provided initial suggestive evidence for linkage to markers on chromosomes 1, 2, 3, 7, 13, 15, 19, and X. Follow-up multipoint analyses gave additional support for a region on 2q37 that includes a schizophrenia locus previously identified in another small genetic isolate, with a well-established recent genealogical history and a small number of founders, located on the eastern border of Finland. In addition to providing further support for a schizophrenia susceptibility locus at 2q37, our results highlight the analytic challenges associated with extremely large and complex pedigrees, as well as the limitations associated with genetic studies of complex traits in small islandic populations.

"Targeting" schizophrenia in mice.

The greatest challenge of schizophrenia research remains the identification of the multiple, common, interacting, and moderately penetrant mutations that interfere with the highly complex function of human brain and result to this devastating disease. The inaccessibility of the human central nervous system to experimental manipulations and the paramount difficulties in identifying genes for schizophrenia has led researchers to generate mouse models for candidate genes using gene-targeting approaches. Although such mouse models have proven very useful in deciphering the causes of several diseases of the central nervous system (such as neurodegenerative diseases), their use in dissecting the biology of schizophrenia is still in its infancy. We argue that progress in this direction depends highly on progress in human genetic studies and requires careful and critical interpretation of the accumulating data.

Genetic ablation and restoration of the olfactory topographic map.

In the olfactory sensory system, neurons expressing a given odorant receptor project with precision to two of 1800 spatially invariant glomeruli creating a topographic map within the olfactory bulb. Olfactory sensory neurons have a half-life of about 90 days and are continually renewing. This poses the problem of how this precise spatial map is maintained throughout the life of the organism. We have developed a genetic approach to effect the synchronous ablation of subpopulations of neurons expressing a given receptor. The axons of newly generated neurons can then be followed as they enter the brain and converge on glomerular targets during adult life. The observation that following neuronal cell killing, the spatial map is faithfully restored, demonstrates that the information necessary for the establishment of the sensory map persists throughout the life of the organism.

Family-based association studies support a sexually dimorphic effect of COMT and MAOA on genetic susceptibility to obsessive-compulsive disorder.

BACKGROUND: Obsessive-compulsive disorder (OCD) is a common and severe psychiatric illness that affects 1-3% of the population and presents a well-established co-morbidity with major depressive disorder (MDD). Twin and family studies have suggested a genetic component in the etiology of OCD, although the mode of inheritance is unknown. Pharmacotherapy of the disease implicates both serotonergic and dopaminergic pathways. Previously, guided by the 22q11 microdeletion-related psychiatric phenotype, we provided evidence for a sexually dimorphic association between OCD and the gene for catechol-O-methyltransferase (COMT). In this report, we use 110 nuclear OCD families to analyze the inheritance of variants of COMT and monoamine oxidase-A (MAOA), another gene modulating monoamine metabolism. METHODS: A sample of 110 nuclear OCD families was collected, and lifetime diagnoses were ascertained using the Diagnostic Interview for Genetic Studies (DIGS). DNA was genotyped for functional variants of the COMT and MAO genes, and allele inheritance was examined using the Transmission Disequilibrium Test (TDT) and Haplotype-based Haplotype Relative Risk (HHRR) test. RESULTS: We provide evidence supporting the previously reported sexually dimorphic association between low COMT enzymatic activity and OCD. We also provide evidence for a similar sexually dimorphic association between OCD and an allele of the MAOA gene, previously linked to high MAO-A enzymatic activity. In agreement with the well-established action of MAO-A inhibitors as antidepressants, this association is particularly marked among male OCD probands with co-morbid MDD, who represent more than 50% of our male OCD sample. CONCLUSIONS: Our analysis indicates that variants of two genes modulating monoamine metabolism contribute significantly to OCD susceptibility. Most importantly, an unexpected sexually dimorphic pattern of genetic susceptibility to OCD is revealed and suggests the possibility that profound gender differences in genetic predisposition may exist not only for other OCD susceptibility genes, but for an array of other psychiatric disorders as well.

The gene encoding proline dehydrogenase modulates sensorimotor gating in mice.

Hemizygous cryptic deletions of the q11 band of human chromosome 22 have been associated with a number of psychiatric and behavioural phenotypes, including schizophrenia. Here we report the isolation and characterization of PRODH, a human homologue of Drosophila melanogaster sluggish-A (slgA), which encodes proline dehydrogenase responsible for the behavioural phenotype of the slgA mutant. PRODH is localized at chromosome 22q11 in a region deleted in some psychiatric patients. We also isolated the mouse homologue of slgA (Prodh), identified a mutation in this gene in the Pro/Re hyperprolinaemic mouse strain and found that these mice have a deficit in sensorimotor gating accompanied by regional neurochemical alterations in the brain. Sensorimotor gating is a neural filtering process that allows attention to be focused on a given stimulus, and is affected in patients with neuropsychiatric disorders. Furthermore, several lines of evidence suggest that proline may serve as a modulator of synaptic transmission in the mammalian brain. Our observations, in conjunction with the chromosomal location of PRODH, suggest a potential involvement of this gene in the 22q11-associated psychiatric and behavioural phenotypes.

Catechol-O-methyltransferase-deficient mice exhibit sexually dimorphic changes in catecholamine levels and behavior.

Catechol-O-methyltransferase (COMT) is one of the major mammalian enzymes involved in the metabolic degradation of catecholamines and is considered a candidate for several psychiatric disorders and symptoms, including the psychopathology associated with the 22q11 microdeletion syndrome. By means of homologous recombination in embryonic stem cells, a strain of mice in which the gene encoding the COMT enzyme has been disrupted was produced. The basal concentrations of brain catecholamines were measured in the striatum, frontal cortex, and hypothalamus of adult male and female mutants. Locomotor activity, anxiety-like behaviors, sensorimotor gating, and aggressive behavior also were analyzed. Mutant mice demonstrated sexually dimorphic and region-specific changes of dopamine levels, notably in the frontal cortex. In addition, homozygous COMT-deficient female (but not male) mice displayed impairment in emotional reactivity in the dark/light exploratory model of anxiety. Furthermore, heterozygous COMT-deficient male mice exhibited increased aggressive behavior. Our results provide conclusive evidence for an important sex- and region-specific contribution of COMT in the maintenance of steady-state levels of catecholamines in the brain and suggest a role for COMT in some aspects of emotional and social behavior in mice.

Identification of sequence variants and analysis of the role of the catechol-O-methyl-transferase gene in schizophrenia susceptibility.

BACKGROUND: Deletions of 1.5-2 MB of chromosome 22q11 have been previously associated with schizophrenia. The deleted region includes proximally the region harboring genes involved in DiGeorge and velocardiofacial syndromes. Distally, it includes the gene for catechol-O-methyl-transferase (COMT), an enzyme that catalyzes the O-methylation of catecholamine neurotransmitters, including dopamine, and which therefore is considered a candidate gene for schizophrenia. METHODS: We address the issue of a direct involvement of the COMT gene in the development of schizophrenia by employing the first extensive mutational analysis of this gene in a sample of 157 schizophrenia patients and 129 healthy controls, using single-strand conformation polymorphism and chemical cleavage methodologies. RESULTS: No mutations were found, but several sequence variants were identified, including the genetic polymorphism that underlies the high/low activity of the enzyme (a Val158-->Met change, which results in the creation of an NlaIII restriction site in the low-activity allele). The distribution of the NlaIII genotypes among subsets of schizophrenia patients was analyzed. CONCLUSIONS: The results presented here argue against a major role of COMT in schizophrenia in general (although a minor effect could not be excluded) and represent a first step toward a more refined delineation of the phenotype/genotype relationship between 22q11 microdeletions and schizophrenia susceptibility.