Francesco Papaleo

Genetic dissection of the role of catechol-O-methyltransferase in cognition and stress reactivity in mice

The COMT (catechol-O-methyltransferase) gene has been linked to a spectrum of human phenotypes, including cognition, anxiety, pain sensitivity and psychosis. Doubts about its clinical impact exist, however, because of the complexity of human COMT polymorphism and clinical variability. We generated transgenic mice overexpressing a human COMT-Val polymorphism (Val-tg), and compared them with mice containing a null COMT mutation. Increased COMT enzyme activity in Val-tg mice resulted in disrupted attentional set-shifting abilities, and impaired working and recognition memory, but blunted stress responses and pain sensitivity. Conversely, COMT disruption improved working memory, but increased stress responses and pain sensitivity. Amphetamine ameliorated recognition memory deficits in COMT-Val-tg mice but disrupted it in wild types, illustrating COMT modulation of the inverted-U relationship between cognition and dopamine. COMT-Val-tg mice showed increased prefrontal cortex (PFC) calcium/calmodulin-dependent protein kinase II (CaMKII) levels, whereas COMT deficiency decreased PFC CaMKII but increased PFC CaMKKβ and CaMKIV levels, suggesting the involvement of PFC CaMK pathways in COMT-regulated cognitive function and adaptive stress responses. Our data indicate a critical role for the COMT gene in an apparent evolutionary trade-off between cognitive and affective functions.

Role of dysbindin in dopamine receptor trafficking and cortical GABA function

Dysbindin has been implicated in the pathogenesis of schizophrenia, but little is known about how dysbindin affects neuronal function in the circuitry underlying psychosis and related behaviors. Using a dysbindin knockout line (dys−/−) derived from the natural dysbindin mutant Sandy mice, we have explored the role of dysbindin in dopamine signaling and neuronal function in the prefrontal cortex (PFC). Combined cell imaging and biochemical experiments revealed a robust increase in the dopamine receptor D2, but not D1, on cell surface of neurons from dys−/− cortex. This was due to an enhanced recycling and insertion, rather than reduced endocytosis, of D2. Disruption of dysbindin gene resulted in a marked decrease in the excitability of fast-spiking (FS) GABAergic interneurons in both PFC and striatum. Dys−/− mice also exhibited a decreased inhibitory input to pyramidal neurons in layer V of PFC. The increased D2 signaling in dys−/− FS interneurons was associated with a more pronounced increase in neuronal firing in response to D2 agonist, compared to that in wild-type interneurons. Taken together, these results suggest that dysbindin regulates PFC function by facilitating D2-mediated modulation of GABAergic function.

Chronic and Acute Intranasal Oxytocin Produce Divergent Social Effects in Mice

Intranasal administration of oxytocin (OXT) might be a promising new adjunctive therapy for mental disorders characterized by social behavioral alterations such as autism and schizophrenia. Despite promising initial studies in humans, it is not yet clear the specificity of the behavioral effects induced by chronic intranasal OXT and if chronic intranasal OXT could have different effects compared with single administration. This is critical for the aforementioned chronic mental disorders that might potentially involve life-long treatments. As a first step to address these issues, here we report that chronic intranasal OXT treatment in wild-type C57BL/6J adult mice produced a selective reduction of social behaviors concomitant to a reduction of the OXT receptors throughout the brain. Conversely, acute intranasal OXT treatment produced partial increases in social behaviors towards opposite-sex novel-stimulus female mice, while on the other hand, it decreased social exploration of same-sex novel stimulus male mice, without affecting social behavior towards familiar stimulus male mice. Finally, prolonged exposure to intranasal OXT treatments did not alter, in wild-type animals, parameters of general health such as body weight, locomotor activity, olfactory and auditory functions, nor parameters of memory and sensorimotor gating abilities. These results indicate that a prolonged over-stimulation of a ‘healthy’ oxytocinergic brain system, with no inherent deficits in social interaction and normal endogenous levels of OXT, results in specific detrimental effects in social behaviors.

Mouse models of genetic effects on cognition: Relevance to schizophrenia

Cognitive dysfunction is a core feature of schizophrenia. Growing evidence indicates that a wide variety of genetic mutations and polymorphisms impact cognition and may thus be implicated in various aspects of this mental disorder. Despite differences between human and rodent brain structure and function, genetic mouse models have contributed critical information about brain mechanisms involved in cognitive processes. Here, we summarize discoveries of genetic modifications in mice that impact cognition. Based on functional hypotheses, gene modifications within five model systems are described: 1) dopamine (D1, D2, D3, D4, D5, DAT, COMT, MAO); 2) glutamate (GluR-A, NR1, NR2A, NR2B, GRM2, GRM3, GLAST); 3) GABA (α(5), γ(2), α(4), δGABA(A), GABA(B(1)), GAT1); 4) acetylcholine (nAChRβ2, α7, CHRM1); and 5) calcium (CaMKII-α, neurogranin, CaMKKβ, CaMKIV). We also consider other risk-associated genes for schizophrenia such as dysbindin (DTNBP1), neuregulin (NRG1), disrupted-in-schizophrenia1 (DISC1), reelin and proline dehydrogenase (PRODH). Because of the presumed importance of environmental factors, we further consider genetic modifications within the stress-sensitive systems of corticotropin-releasing factor (CRF), brain-derived neurotrophic factor (BDNF) and the endocannabinoid systems. We highlight the missing information and limitations of cognitive assays in genetically modified mice models relevant to schizophrenia pathology.

Dysbindin-1 modulates prefrontal cortical activity and schizophrenia-like behaviors via dopamine/D2 pathways

Dysbindin-1 regulates D2-receptor trafficking and is implicated in schizophrenia and related cognitive abnormalities, but whether this molecular effect mediates the clinical manifestations of the disorder is unknown. We explored in dysbindin-1-deficient mice (dys−/−) (1) schizophrenia-related behaviors, (2) molecular and electrophysiological changes in medial prefrontal cortex (mPFC) and (3) the dependence of these on D2-receptor stimulation. Dysbindin-1 disruption altered dopamine-related behaviors and impaired working memory under challenging/stressful conditions. Dys−/− pyramidal neurons in mPFC layers II/III were hyperexcitable at baseline but hypoexcitable following D2 stimulation. Dys−/− were also respectively more and less sensitive to D2 agonist- and antagonist-induced behavioral effects. Dys−/− had reduced expression of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and CaMKKβ in mPFC. Chronic D2 agonist treatment reproduced these changes in protein expression, and some of the dys−/− behavioral effects. These results elucidate dysbindins modulation of D2-related behavior, cortical activity and mPFC CaMK components, implicating cellular and molecular mechanisms of the association of dysbindin with psychosis.

Neuregulin 1-ErbB4-PI3K signaling in schizophrenia and phosphoinositide 3-kinase-p110δ inhibition as a potential therapeutic strategy

Neuregulin 1 (NRG1) and ErbB4, critical neurodevelopmental genes, are implicated in schizophrenia, but the mediating mechanisms are unknown. Here we identify a genetically regulated, pharmacologically targetable, risk pathway associated with schizophrenia and with ErbB4 genetic variation involving increased expression of a PI3K-linked ErbB4 receptor (CYT-1) and the phosphoinositide 3-kinase subunit, p110δ (PIK3CD). In human lymphoblasts, NRG1-mediated phosphatidyl-inositol,3,4,5 triphosphate [PI(3,4,5)P3] signaling is predicted by schizophrenia-associated ErbB4 genotype and PIK3CD levels and is impaired in patients with schizophrenia. In human brain, the same ErbB4 genotype again predicts increased PIK3CD expression. Pharmacological inhibition of p110δ using the small molecule inhibitor, IC87114, blocks the effects of amphetamine in a mouse pharmacological model of psychosis and reverses schizophrenia-related phenotypes in a rat neonatal ventral hippocampal lesion model. Consistent with these antipsychotic-like properties, IC87114 increases AKT phosphorylation in brains of treated mice, implicating a mechanism of action. Finally, in two family-based genetic studies, PIK3CD shows evidence of association with schizophrenia. Our data provide insight into a mechanism of ErbB4 association with schizophrenia; reveal a previously unidentified biological and disease link between NRG1-ErbB4, p110δ, and AKT; and suggest that p110δ is a previously undescribed therapeutic target for the treatment of psychiatric disorders.

Working memory deficits, increased anxiety-like traits, and seizure susceptibility in BDNF overexpressing mice

BDNF regulates components of cognitive processes and has been implicated in psychiatric disorders. Here we report that genetic overexpression of the BDNF mature isoform (BDNF-tg) in female mice impaired working memory functions while sparing components of fear conditioning. BDNF-tg mice also displayed reduced breeding efficiency, higher anxiety-like scores, high self-grooming, impaired prepulse inhibition, and higher susceptibility to seizures when placed in a new empty cage, as compared with wild-type (WT) littermate controls. Control measures of general health, locomotor activity, motor coordination, depression-related behaviors, and sociability did not differ between genotypes. The present findings, indicating detrimental effects of life-long increased BDNF in mice, may inform human studies evaluating the role of BDNF functional genetic variations on cognitive abilities and vulnerability to psychiatric disorders.

Decreased motivation to eat in µ-opioid receptor-deficient mice

Altered motivational processes might participate to the physiopathology of eating‐related disorders. The endogenous opioid system is thought to mediate the hedonic properties of food intake. To assess the role for the µ‐opioid receptor (MOR) pathway in the motivational properties of food intake, in the present study we tested wild‐type and MOR‐deficient mice (MOR–/–) in a nose‐poke operant paradigm for chow or sucrose pellets. To avoid confounding factors linked to food restriction/deprivation experience, mice were always provided with food ad libitum. Although less MOR–/– than wild‐type mice initiated operant behaviour, under a fixed ratio‐1 (FR‐1) reinforcement schedule the two genotypes showed similar patterns of food‐driven nose‐poking, indicating preserved cognitive abilities in MOR‐deficient mice. However, during FR‐3 and progressive ratio (PR) reinforcement experiments, MOR–/– mice showed lower levels of nose‐poking for either chow or sucrose pellets than wild‐type mice, indicating a crucial role for the MOR pathway in the motivational properties of food intake. Moreover, under the PR reinforcement schedule mice nose‐poking for sucrose pellets showed higher genotype‐independent breakpoint levels than mice working for chow pellets, indicating that the MOR pathway is not essential for hedonic processing of palatable food intake. Finally, MOR–/– mice did not differ from wild‐type mice in the rate of operant responding extinction, further supporting the notion of unaltered cognitive abilities in the MOR‐deficient mice. The present findings strongly indicate that the MOR pathway mediates the motivational properties of food intake, but it is not essential for hedonic processing of ingestive behaviour.

Effects of sex and COMT genotype on environmentally modulated cognitive control in mice

Cognitive functioning differs between males and females, likely in part related to genetic dimorphisms. An example of a common genetic variation reported to have sexually dimorphic effects on cognition and temperament in humans is the Val/Met polymorphism in catechol-O-methyltransferase (COMT). We tested male and female wild-type mice (+/+) and their COMT knockout littermates (+/− and −/−) in the five-choice serial reaction time task (5CSRTT) to investigate the effects of sex, COMT genotype, and their interactions with environmental manipulations of cognitive functions such as attention, impulsivity, compulsivity, motivation, and rule-reversal learning. No sex- or COMT-dependent differences were present in the basic acquisition of the five-choice serial reaction time task. In contrast, specific environmental manipulations revealed a variety of sex- and COMT-dependent effects. Following an experimental change to trigger impulsive responding, the sexes showed similar increases in impulsiveness, but males eventually habituated whereas females did not. Moreover, COMT knockout mice were more impulsive compared with wild-type littermates. Manipulations involving mild stress adversely affected cognitive performance in males, and particularly COMT knockout males, but not in females. In contrast, following amphetamine treatment, subtle sex by genotype and sex by treatment interactions emerged primarily limited to compulsive behavior. After repeated testing, female mice showed improved performance, working harder and eventually outperforming males. Finally, removing the food-restriction condition enhanced sex and COMT differences, revealing that overall, females outperform males and COMT knockout males outperform their wild-type littermates. These findings illuminate complex sex- and COMT-related effects and their interactions with environmental factors to influence specific executive cognitive domains.

Epistatic interaction between COMT and DTNBP1 modulates prefrontal function in mice and in humans

Cognitive functions are highly heritable and the impact of complex genetic interactions, though undoubtedly important, has received little investigation. Here we show in an animal model and in a human neuroimaging experiment a consistent non-linear interaction between two genes—catechol-O-methyl transferase (COMT) and dysbindin (dys; dystrobrevin-binding protein 1 (DTNBP1))—implicated through different mechanisms in cortical dopamine signaling and prefrontal cognitive function. In mice, we found that a single genetic mutation reducing expression of either COMT or DTNBP1 alone produced working memory advantages, while, in dramatic contrast, genetic reduction of both in the same mouse produced working memory deficits. We found evidence of the same non-linear genetic interaction in prefrontal cortical function in humans. In healthy volunteers (N=176) studied with functional magnetic resonance imaging during a working memory paradigm, individuals homozygous for the COMT rs4680 Met allele that reduces COMT enzyme activity showed a relatively more efficient prefrontal engagement. In contrast, we found that the same genotype was less efficient on the background of a dys haplotype associated with decreased DTNBP1 expression. These results illustrate that epistasis can be functionally multi-directional and non-linear and that a putatively beneficial allele in one epistastic context is a relatively deleterious one in another. These data also have important implications for single-locus association analyses of complex traits.