Marta Busnelli

PHARMACOLOGIC RESCUE OF IMPAIRED COGNITIVE FLEXIBILITY, SOCIAL DEFICITS, INCREASED AGGRESSION, AND SEIZURE SUSCEPTIBILITY IN OXYTOCIN RECEPTOR NULL MICE: A NEUROBEHAVIORAL MODEL OF AUTISM

BACKGROUND Oxytocin (OT) has been suggested as a treatment to improve social behavior in autistic patients. Accordingly, the OT (Oxt(-/-)) and the OT receptor null mice (Oxtr(-/-)) display autistic-like deficits in social behavior, increased aggression, and reduced ultrasonic vocalization. METHODS Oxtr(-/-) mice were characterized for general health, sociability, social novelty, cognitive flexibility, aggression, and seizure susceptibility. Because vasopressin (AVP) and OT cooperate in controlling social behavior, learning, and aggression, they were tested for possible rescue of the impaired behaviors. Primary hyppocampal cultures from Oxtr(+/+) and Oxtr(-/-) mouse embryos were established to investigate the balance between gamma-aminobutyric acid (GABA) and glutamate synapses and the expression levels of OT and AVP (V1a) receptors were determined by autoradiography. RESULTS Oxtr(-/-) mice display two additional, highly relevant, phenotypic characteristics: 1) a resistance to change in a learned pattern of behavior, comparable to restricted interests and repetitive behavior in autism, and 2) an increased susceptibility to seizures, a frequent and clinically relevant symptom of autism. We also show that intracerebral administration of both OT and AVP lowers aggression and fully reverts social and learning defects by acting on V1a receptors and that seizure susceptibility is antagonized by peripherally administered OT. Finally, we detect a decreased ratio of GABA-ergic versus total presynapses in hippocampal neurons of Oxtr(-/-) mice. CONCLUSIONS Autistic-like symptoms are rescued on administration of AVP and OT to young Oxtr(-/-) adult animals. The Oxtr(-/-) mouse is thus instrumental to investigate the neurochemical and synaptic abnormalities underlying autistic-like disturbances and to test new strategies of pharmacologic intervention.

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.

A Family with Complete Resistance to Thyrotropin-Releasing Hormone

To the Editor: We report on a family with recessive resistance to the action of thyrotropin-releasing hormone (TRH) due to a nonsense homozygous mutation in the TRH receptor (TRHR) gene (p.R17X). W...

Functional Selective Oxytocin-derived Agonists Discriminate between Individual G Protein Family Subtypes

Background: The oxytocin receptor couples to multiple G proteins, leading to different physiological responses. Results: We screened for functional selective oxytocin receptor agonists and identified two analogs that activate individual Gi subunits. Conclusion: Functional selective analogs discriminate among different receptor conformations coupled to Gi proteins. Significance: These compounds will contribute to the development of selective drugs with new selectivity and therapeutic profiles. We used a bioluminescence resonance energy transfer biosensor to screen for functional selective ligands of the human oxytocin (OT) receptor. We demonstrated that OT promoted the direct engagement and activation of Gq and all the Gi/o subtypes at the OT receptor. Other peptidic analogues, chosen because of specific substitutions in key OT structural/functional residues, all showed biased activation of G protein subtypes. No ligand, except OT, activated GoA or GoB, and, with only one exception, all of the peptides that activated Gq also activated Gi2 and Gi3 but not Gi1, GoA, or GoB, indicating a strong bias toward these subunits. Two peptides (DNalOVT and atosiban) activated only Gi1 or Gi3, failed to recruit β-arrestins, and did not induce receptor internalization, providing the first clear examples of ligands differentiating individual Gi/o family members. Both analogs inhibited cell proliferation, showing that a single Gi subtype-mediated pathway is sufficient to prompt this physiological response. These analogs represent unique tools for examining the contribution of Gi/o members in complex biological responses and open the way to the development of drugs with peculiar selectivity profiles. This is of particular relevance because OT has been shown to improve symptoms in neurodevelopmental and psychiatric disorders characterized by abnormal social behaviors, such as autism. Functional selective ligands, activating a specific G protein signaling pathway, may possess a higher efficacy and specificity on OT-based therapeutics.

Mice heterozygous for the oxytocin receptor gene (Oxtr(+/-) ) show impaired social behaviour but not increased aggression or cognitive inflexibility : evidence of a selective haploinsufficiency gene effect

We characterised the behavioural phenotype of mice heterozygous (Oxtr+/−) for the oxytocin receptor gene (Oxtr) and compared it with that of Oxtr null mice (Oxtr−/−), which display autistic‐like behaviours, including impaired sociability and preference for social novelty, impaired cognitive flexibility, and increased aggression. Similar to Oxtr−/− mice, the Oxtr+/− showed impaired sociability and preference for social novelty but, unlike the null genotype, their cognitive flexibility and aggression were normal. By autoradiography, Oxtr+/− mice were found to have approximately 50% fewer oxytocin receptors (OXTRs) in all of the examined brain regions. Thus, because a partial reduction in Oxtr gene expression is sufficient to compromise social behaviour, the Oxtr acts as a haploinsufficient gene. Furthermore, the inactivation of the Oxtr gene affects specific behaviours in a dose‐dependent manner: social behaviour is sensitive to even a partial reduction in Oxtr gene expression, whereas defects in aggression and cognitive flexibility require the complete inactivation of the Oxtr gene to emerge. We then investigated the rescue of the Oxtr+/− social deficits by oxytocin (OT) and Thr4Gly7OT (TGOT) administered i.c.v. at different doses. TGOT was more potent than OT in rescuing sociability and social novelty in both genotypes. Furthermore, the TGOT doses that reverted impaired sociability and preference for social novelty in Oxtr+/− were lower than those required in Oxtr−/−, thus suggesting that the rescue effect is mediated by OXTR in Oxtr+/− and by other receptors (presumably vasopressin V1a receptors) in Oxtr−/−. In line with this, a low dose of the selective oxytocin antagonist desGlyDTyrOVT blocks the rescue effect of TGOT only in the Oxtr+/− genotype, whereas the less selective antagonist SR49059 blocks rescue in both genotypes. In conclusion, the Oxtr+/− mouse is a unique animal model for investigating how partial loss of the Oxtr gene impair social interactions, and for designing pharmacological rescue strategies.

Selective and Potent Agonists and Antagonists for Investigating the Role of Mouse Oxytocin Receptors

The neuropeptides oxytocin (OT) and vasopressin (AVP) have been shown to play a central role in social behaviors; as a consequence, they have been recognized as potential drugs to treat neurodevelopmental and psychiatric disorders characterized by impaired social interactions. However, despite the basic and preclinical relevance of mouse strains carrying genetic alterations in the OT/AVP systems to basic and preclinical translational neuroscience, the pharmacological profile of mouse OT/AVP receptor subtypes has not been fully characterized. To fill in this gap, we have characterized a number of OT and AVP agonists and antagonists at three murine OT/AVP receptors expressed in the nervous system as follows: the oxytocin (mOTR) and vasopressin V1a (mV1aR) and V1b (mV1bR) subtypes. These three receptors were transiently expressed in vitro for binding and intracellular signaling assays, and then a homology model of the mOTR structure was constructed to investigate how its molecular features compare with human and rat OTR orthologs. Our data indicate that the selectivity profile of the natural ligands, OT and AVP, is conserved in humans, rats, and mice. Furthermore, we found that the synthetic peptide [Thr4Gly7]OT (TGOT) is remarkably selective for the mOTR and, like the endogenous OT ligand, activates Gq and Gi and recruits β-arrestins. Finally, we report three antagonists that exhibit remarkably high affinities and selectivities at mOTRs. These highly selective pharmacological tools will contribute to the investigation of the specific physiologic and pathologic roles of mOTR for the development of selective OT-based therapeutics.

Dual modulation of inward rectifier potassium currents in olfactory neuronal cells by promiscuous G protein coupling of the oxytocin receptor

J. Neurochem. (2010) 114, 1424–1435.

The Timing of the Excitatory-to-Inhibitory GABA Switch Is Regulated by the Oxytocin Receptor via KCC2.

Summary Oxytocin and its receptor (Oxtr) play a crucial role in the postnatal transition of neuronal GABA neurotransmission from excitatory to inhibitory, a developmental process known as the GABA switch. Using hippocampal neurons from Oxtr-null mice, we show that (1) Oxtr is necessary for the correct timing of the GABA switch by upregulating activity of the chloride cotransporter KCC2, (2) Oxtr, in a very early and narrow time window, directly modulates the functional activity of KCC2 by promoting its phosphorylation and insertion/stabilization at the neuronal surface, and (3) in the absence of Oxtr, electrophysiological alterations are recorded in mature neurons, a finding consistent with a reduced level of KCC2 and increased susceptibility to seizures observed in adult Oxtr-null mice. These data identify KCC2 as a key target of oxytocin in postnatal events that may be linked to pathogenesis of neurodevelopmental disorders.

Blood cell mitochondrial DNA content and premature ovarian aging

Primary ovarian insufficiency (POI) is a critical fertility defect characterized by an anticipated and silent impairment of the follicular reserve, but its pathogenesis is largely unexplained. The frequent maternal inheritance of POI together with a remarkable dependence of ovarian folliculogenesis upon mitochondrial biogenesis and bioenergetics suggested the possible involvement of a generalized mitochondrial defect. Here, we verified the existence of a significant correlation between blood and ovarian mitochondrial DNA (mtDNA) content in a group of women undergoing ovarian hyperstimulation (OH), and then aimed to verify whether mtDNA content was significantly altered in the blood cells of POI women. We recruited 101 women with an impaired ovarian reserve: 59 women with premature ovarian failure (POF) and 42 poor responders (PR) to OH. A Taqman copy number assay revealed a significant mtDNA depletion (P<0.001) in both POF and PR women in comparison with 43 women of similar age and intact ovarian reserve, or 53 very old women with a previous physiological menopause. No pathogenic variations in the mitochondrial DNA polymerase γ (POLG) gene were detected in 57 POF or PR women with low blood mtDNA content. In conclusion, blood cell mtDNA depletion is a frequent finding among women with premature ovarian aging, suggesting that a still undetermined but generalized mitochondrial defect may frequently predispose to POI which could then be considered a form of anticipated aging in which the ovarian defect may represent the first manifestation. The determination of mtDNA content in blood may become an useful tool for the POI risk prediction.

Specific roles of Gi protein family members revealed by dissecting SST5 coupling in human pituitary cells

Summary Despite intensive investigation over the past 20 years, the specific role played by individual Gi protein family members in mediating complex cellular effects is still largely unclear. Therefore, we investigated the role of specific Gi proteins in mediating somatostatin (SS) effects in somatotroph cells. Because our previous data showed that SS receptor type 5 (SST5) carrying a spontaneous R240W mutation in the third intracellular loop had a similar ability to inhibit intracellular cAMP levels to the wild-type protein but failed to mediate inhibition of growth hormone (GH) release and cell proliferation, we used this model to check specific receptor–G-protein coupling by a bioluminescent resonance energy transfer analysis. In HEK293 cells, wild-type SST5 stimulated the activation of G&agr;i1–3 and G&agr;oA, B, whereas R240W SST5 maintained the ability to activate G&agr;i1–3 and G&agr;oB, but failed to activate the splicing variant G&agr;oA. To investigate the role of the selective deficit in G&agr;oA coupling, we co-transfected human adenomatous somatotrophs with SST5 and a pertussis toxin (PTX)-resistant G&agr;oA (G&agr;oA(PTX-r)) protein. In PTX-treated cells, G&agr;oA(PTX-r) rescued the ability of the selective SST5 analog BIM23206 to inhibit extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation, GH secretion and intracellular cAMP levels. Moreover, we demonstrated that silencing of G&agr;oA completely abolished SST5-mediated inhibitory effects on GH secretion and ERK1/2 phosphorylation, but not on cAMP levels. In conclusion, by analysing the coupling specificity of human SST5 to individual G&agr;i and G&agr;o subunits, we identified a crucial role for G&agr;oA signalling in human pituitary cells.