Barbara Pelosi

Lack of brain serotonin affects postnatal development and serotonergic neuronal circuitry formation

Despite increasing evidence suggests that serotonin (5-HT) can influence neurogenesis, neuronal migration and circuitry formation, the precise role of 5-HT on central nervous system (CNS) development is only beginning to be elucidated. Moreover, how changes in serotonin homeostasis during critical developmental periods may have etiological relevance to human mental disorders, remains an unsolved question. In this study we address the consequences of 5-HT synthesis abrogation on CNS development using a knock-in mouse line in which the tryptophan hydroxylase 2 (Tph2) gene is replaced by the eGFP reporter. We report that lack of brain 5-HT results in a dramatic reduction of body growth rate and in 60% lethality within the first 3 weeks after birth, with no gross anatomical changes in the brain. Thanks to the specific expression of the eGFP, we could highlight the serotonergic system independently of 5-HT immunoreactivity. We found that lack of central serotonin produces severe abnormalities in the serotonergic circuitry formation with a brain region- and time- specific effect. Indeed, we observed a striking reduction of serotonergic innervation to the suprachiasmatic and thalamic paraventricular nuclei, while a marked serotonergic hyperinnervation was found in the nucleus accumbens and hippocampus of Tph2∷eGFP mutants. Finally, we demonstrated that BDNF expression is significantly up-regulated in the hippocampus of mice lacking brain 5-HT, mirroring the timing of the appearance of hyperinnervation and thus unmasking a possible regulatory feedback mechanism tuning the serotonergic neuronal circuitry formation. On the whole, these findings reveal that alterations of serotonin levels during CNS development affect the proper wiring of the brain that may produce long-lasting changes leading to neurodevelopmental disorders.

Rhes regulates dopamine D2 receptor transmission in striatal cholinergic interneurons

Ras homolog enriched in striatum (Rhes) is highly expressed in striatal medium spiny neurons (MSNs) of rodents. In the present study, we characterized the expression of Rhes mRNA across species, as well as its functional role in other striatal neuron subtypes. Double in situ hybridization analysis showed that Rhes transcript is selectively localized in striatal cholinergic interneurons (ChIs), but not in GABAergic parvalbumin- or in neuropeptide Y-positive cell populations. Rhes is closely linked to dopamine-dependent signaling. Therefore, we recorded ChIs activity in basal condition and following dopamine receptor activation. Surprisingly, instead of an expected dopamine D2 receptor (D2R)-mediated inhibition, we observed an aberrant excitatory response in ChIs from Rhes knockout mice. Conversely, the effect of D1R agonist on ChIs was less robust in Rhes mutants than in controls. Although Rhes deletion in mutants occurs throughout the striatum, we demonstrate that the D2R response is altered specifically in ChIs, since it was recorded in pharmacological isolation, and prevented either by intrapipette BAPTA or by GDP-β-S. Moreover, we show that blockade of Cav2.2 calcium channels prevented the abnormal D2R response. Finally, we found that the abnormal D2R activation in ChIs was rescued by selective PI3K inhibition thus suggesting that Rhes functionally modulates PI3K/Akt signaling pathway in these neurons. Our findings reveal that, besides its expression in MSNs, Rhes is localized also in striatal ChIs and, most importantly, lack of this G-protein, significantly alters D2R modulation of striatal cholinergic excitability.

Rhes influences striatal cAMP/PKA-dependent signaling and synaptic plasticity in a gender-sensitive fashion.

Mechanisms of gender-specific synaptic plasticity in the striatum, a brain region that controls motor, cognitive and psychiatric functions, remain unclear. Here we report that Rhes, a GTPase enriched in medium spiny neurons (MSNs) of striatum, alters the striatal cAMP/PKA signaling cascade in a gender-specific manner. While Rhes knockout (KO) male mice, compared to wild-type (WT) mice, had a significant basal increase of cAMP/PKA signaling pathway, the Rhes KO females exhibited a much stronger response of this pathway, selectively under the conditions of dopamine/adenosine-related drug challenge. Corticostriatal LTP defects are exclusively found in A2AR/D2R-expressing MSNs of KO females, compared to KO males, an effect that is abolished by PKA inhibitors but not by the removal of circulating estrogens. This suggests that the synaptic alterations found in KO females could be triggered by an aberrant A2AR/cAMP/PKA activity, but not due to estrogen-mediated effect. Consistent with increased cAMP signaling, D1R-mediated motor stimulation, haloperidol-induced catalepsy and caffeine-evoked hyper-activity are robustly enhanced in Rhes KO females compared to mutant males. Thus Rhes, a thyroid hormone-target gene, plays a relevant role in gender-specific synaptic and behavioral responses.

Rasd2 Modulates Prefronto-Striatal Phenotypes in Humans and 'Schizophrenia-Like Behaviors' in Mice.

Rasd2 is a thyroid hormone target gene, which encodes for a GTP-binding protein enriched in the striatum where, among other functions, it modulates dopaminergic neurotransmission. Here we report that human RASD2 mRNA is abundant in putamen, but it also occurs in the cerebral cortex, with a distinctive expression pattern that differs from that present in rodents. Consistent with its localization, we found that a genetic variation in RASD2 (rs6518956) affects postmortem prefrontal mRNA expression in healthy humans and is associated with phenotypes of relevance to schizophrenia, including prefrontal and striatal grey matter volume and physiology during working memory, as measured with magnetic resonance imaging. Interestingly, quantitative real-time PCR analysis indicated that RASD2 mRNA is slightly reduced in postmortem prefrontal cortex of patients with schizophrenia. In the attempt to uncover the neurobiological substrates associated with Rasd2 activity, we used knockout mice to analyze the in vivo influence of this G-protein on the prepulse inhibition of the startle response and psychotomimetic drug-related behavioral response. Data showed that Rasd2 mutants display deficits in basal prepulse inhibition that, in turn, exacerbate gating disruption under psychotomimetic drug challenge. Furthermore, we documented that lack of Rasd2 strikingly enhances the behavioral sensitivity to motor stimulation elicited by amphetamine and phencyclidine. Based on animal model data, along with the finding that RASD2 influences prefronto-striatal phenotypes in healthy humans, we suggest that genetic mutation or reduced levels of this G-protein might have a role in cerebral circuitry dysfunction underpinning exaggerated psychotomimetic drugs responses and development of specific biological phenotypes linked to schizophrenia.

Perturbation of Serotonin Homeostasis during Adulthood Affects Serotonergic Neuronal Circuitry

Visual Abstract Growing evidence shows that the neurotransmitter serotonin (5-HT) modulates the fine-tuning of neuron development and the establishment of wiring patterns in the brain. However, whether serotonin is involved in the maintenance of neuronal circuitry in the adult brain remains elusive. Here, we use a Tph2fl°x conditional knockout (cKO) mouse line to assess the impact of serotonin depletion during adulthood on serotonergic system organization. Data show that the density of serotonergic fibers is increased in the hippocampus and decreased in the thalamic paraventricular nucleus (PVN) as a consequence of brain serotonin depletion. Strikingly, these defects are rescued following reestablishment of brain 5-HT signaling via administration of the serotonin precursor 5-hydroxytryptophan (5-HTP). Finally, 3D reconstruction of serotonergic fibers reveals that changes in serotonin homeostasis affect axonal branching complexity. These data demonstrate that maintaining proper serotonin homeostasis in the adult brain is crucial to preserve the correct serotonergic axonal wiring.

Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin

Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2 flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2 null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2 null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84–178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2 null/null mice. Finally, we set up a tamoxifen administration protocol that allows an efficient, time-specific inactivation of brain serotonin synthesis. On the whole, we generated a suitable genetic tool to investigate how serotonin depletion impacts on time-specific events during central nervous system development and adulthood life.

A Tph2GFP Reporter Stem Cell Line To Model in Vitro and in Vivo Serotonergic Neuron Development and Function

Modeling biological systems in vitro has contributed to clarification of complex mechanisms in simplified and controlled experimental conditions. Mouse embryonic stem (mES) cells can be successfully differentiated toward specific neuronal cell fates, thus representing an attractive tool to dissect, in vitro, mechanisms that underlie complex neuronal features. In this study, we generated and characterized a reporter mES cell line, called Tph2GFP, in which the vital reporter GFP replaces the tryptophan hydroxylase 2 (Tph2) gene. Tph2GFP mES cells selectively express GFP upon in vitro differentiation toward the serotonergic fate, they synthesize serotonin, possess excitable membranes, and show the typical morphological, morphometrical, and molecular features of in vivo serotonergic neurons. Thanks to the vital reporter GFP, we highlighted by time-lapse video microscopy several dynamic processes such as cell migration and axonal outgrowth in living cultures. Finally, we demonstrated that predifferentiated Tph2GFP cells are able to terminally differentiate, integrate, and innervate the host brain when grafted in vivo. On the whole, the present study introduces the Tph2GFP mES cell line as a useful tool allowing accurate developmental and dynamic studies and representing a reliable platform for the study of serotonergic neurons in health and disease.

Automatic Tractography Analysis through Sparse Networks in Case-Control Studies

Magnetic Resonance Diffusion Tensor Imaging (DTI) has opened the way to a variety of white matter analysis approaches which leverage the axon networks in the brain. Even if tractography algorithms are widely used to reconstruct these networks, their topology is seldom employed to evaluate differences between patient and control groups, unless there is prior knowledge on the areas of interest. Using a sparse approach, we have developed a multivariate method to automatically identify the most significant connections bundles able to characterise differences between two groups. This will allow neuroscientist to explore inter-group differences in white-matter topology in an unbiased-fashion, and without the need of a priori knowledge. Here, we performed a preliminary test of the approach with serotonin dysfunctional mice and a control group. The results allowed us to identify inter-group differences in the density of white matter tracts originating from serotonergic areas, thus corroborating the predictive validity of the method.

Rasd2 Modulates Psychotomimetic Drug Effects in Mice and Schizophrenia-related Phenotypes in Humans

Background Rasd2 is a striatal GTP-binding protein that modulates Akt and mTOR signaling cascades, well known to be highly vulnerable pathways in psychiatric disorders. Aims We investigated the association of Rasd2 and its genetic variation with a series of prefronto-striatal phenotypes related to psychosis in rodents and humans. Objectives We want to provide evidence that Rasd2 controls the vulnerability to schizophrenia-related behavior induced by psychothomimetic drugs in mice. Moreover, we aim to find genetic variations within the Rasd2 gene that influence a series of brain schizophrenia-related phenotypes in human. Methods Rasd2 knockout mice were employed to evaluate schizophrenia-like behaviors induced by psychotomimetic drugs like amphetamine and phencyclidine. Furthermore, we investigated if RASD2 genetic variations in humans are associated with mRNA expression in post-mortem prefrontal cortex, as well as prefrontal and striatal grey matter volume and physiology during working memory as measured with MRI in healthy subjects. Finally, we assessed RASD2 mRNA expression levels in post-mortem brains of patients with schizophrenia and bipolar disorder. Results We found that both psychotomimetics triggered greater vulnerability to motor stimulation and to prepulse inhibition deficits in Rasd2 mutants. In humans, we found that a genetic variation (rs6518956) within RASD2 predicts prefrontal mRNA expression as well as prefrontal grey matter volume and prefronto-striatal activity during working memory. Finally, we reported that RASD2 mRNA expression is slightly reduced in post-mortem prefrontal cortex of patients with schizophrenia. Conclusions Collectively, our data suggests that RASD2 represents a gene of potential interest in psychiatric disorders for its ability to modulate prefronto-striatal phenotypes related to schizophrenia.