17-ß-estradiol potentiates the neurotrophic and neuroprotective effects mediated by the dopamine D3/acetylcholine nicotinic receptor heteromer in dopaminergic neurons.

Dopaminergic neurons express a heteromer composed of the dopamine D3 receptor and the α4ß2 nicotinic acetylcholine receptor, the D3R-nAChR heteromer, activated by both nicotine and dopamine D2 and D3 receptors agonists, such as quinpirole, and crucial for dopaminergic neuron homeostasis. We now report that D3R-nAChR heteromer activity is potentiated by 17-ß-estradiol which acts as a positive allosteric modulator by binding a specific domain on the α4 subunit of the nicotinic receptor protomer. In mouse dopaminergic neurons, in fact, 17-ß-estradiol significantly increased the ability of nicotine and quinpirole in promoting neuron dendritic remodeling and in protecting neurons against the accumulation of α-synuclein induced by deprivation of glucose, with a mechanism that does not involve the classical estrogen receptors. The potentiation induced by 17-ß-estradiol required the D3R-nAChR heteromer since either nicotinic receptor or dopamine D3 receptor antagonists and interfering TAT-peptides, but not the estrogen receptor antagonist fulvestrant, specifically prevented 17-ß-estradiol effects. Evidence of estrogens neuroprotection, mainly mediated by genomic mechanisms, have been provided, which is in line with epidemiological data reporting that females are less likely to develop Parkinson's Disease than males. Therefore, potentiation of D3R-nAChR heteromer activity may represent a further mechanism by which 17-ß-estradiol reduces dopaminergic neuron vulnerability.

Generation of human induced pluripotent stem cell lines derived from three Noonan syndrome patients from a single family carrying the heterozygous PTPN11 c.188 A > G (p.Y63C) mutation.

We have established Noonan syndrome (NS)-derived induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) of a family cohort carrying the heterozygous PTPN11 c.188 A > G (p.Y63C) mutation. The new iPSC lines were validated by confirming the normal karyotype and targeted mutation, the pluripotent gene expression, and the differentiation capacity into three germ layers.

G Protein-Dependent Activation of the PKA-Erk1/2 Pathway by the Striatal Dopamine D1/D3 Receptor Heteromer Involves Beta-Arrestin and the Tyrosine Phosphatase Shp-2.

The heteromer composed of dopamine D1 and D3 receptors (D1R-D3R) has been defined as a structure able to trigger Erk1/2 and Akt signaling in a G protein-independent, beta-arrestin 1-dependent way that is physiologically expressed in the ventral striatum and is likely involved in the control of locomotor activity. Indeed, abnormal levels of D1R-D3R heteromer in the dorsal striatum have been correlated with the development of L-DOPA-induced dyskinesia (LID) in Parkinson's disease patients, a motor complication associated with striatal D1R signaling, thus requiring Gs protein and PKA activity to activate Erk1/2. Therefore, to clarify the role of the D1R/D3R heteromer in LID, we investigated the signaling pathway induced by the heteromer using transfected cells and primary mouse striatal neurons. Collectively, we found that in both the cell models, D1R/D3R heteromer-induced activation of Erk1/2 exclusively required the D1R molecular effectors, such as Gs protein and PKA, with the contribution of the phosphatase Shp-2 and beta-arrestins, indicating that heterodimerization with the D3R abolishes the specific D3R-mediated signaling but strongly allows D1R signals. Therefore, while in physiological conditions the D1R/D3R heteromer could represent a mechanism that strengthens the D1R activity, its pathological expression may contribute to the abnormal PKA-Shp-2-Erk1/2 pathway connected with LID.

Central nervous system interaction and crosstalk between nAChRs and other ionotropic and metabotropic neurotransmitter receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in both the peripheral and the central nervous systems. nAChRs exert a crucial modulatory influence on several brain biological processes; they are involved in a variety of neuronal diseases including Parkinson's disease, Alzheimer's disease, epilepsy, and nicotine addiction. The influence of nAChRs on brain function depends on the activity of other neurotransmitter receptors that co-exist with nAChRs on neurons. In fact, the crosstalk between receptors is an important mechanism of neurotransmission modulation and plasticity. This may be due to converging intracellular pathways but also occurs at the membrane level, because of direct physical interactions between receptors. In this line, this review is dedicated to summarizing how nAChRs and other ionotropic and metabotropic receptors interact and the relevance of nAChRs cross-talks in modulating various neuronal processes ranging from the classical modulation of neurotransmitter release to neuron plasticity and neuroprotection.

Recent Advances in Dopamine D3 Receptor Heterodimers: Focus on Dopamine D3 and D1 Receptor-Receptor Interaction and Striatal Function.

G protein-coupled receptors (GPCR) heterodimers represent new entities with unique pharmacological, signalling, and trafficking properties, with specific distribution restricted to those cells where the two interacting receptors are co-expressed. Like other GPCR, dopamine D3 receptors (D3R) directly interact with various receptors to form heterodimers: data showing the D3R physical interaction with both GPCR and non-GPCR receptors have been provided including D3R interaction with other dopamine receptors. The aim of this chapter is to summarize current knowledge of the distinct roles of heterodimers involving D3R, focusing on the D3R interaction with the dopamine D1 receptor (D1R): the D1R-D3R heteromer, in fact, has been postulated in both ventral and motor striatum. Interestingly, since both D1R and D3R have been implicated in several pathological conditions, including schizophrenia, motor dysfunctions, and substance use disorders, the D1R-D3R heteromer may represent a potential drug target for the treatment of these diseases.

Tryptophan Hydroxylase 2 Knockout Male Rats Exhibit a Strengthened Oxytocin System, Are Aggressive, and Are Less Anxious.

The central serotoninergic system is critical for stress responsivity and social behavior, and its dysregulations have been centrally implicated in virtually all neuropsychiatric disorders. Genetic serotonin depletion animal models could provide a tool to elucidate the causes and mechanisms of diseases and to develop new treatment approaches. Previously, mice lacking tryptophan hydroxylase 2 (Tph2) have been developed, showing altered behaviors and neurotransmission. However, the effect of congenital serotonin deficiency on emotional and social behavior in rats is still largely unknown, as are the underlying mechanisms. In this study, we used a Tph2 knockout (Tph2-/-) male rat model to study how the lack of serotonin in the rat brain affects anxiety-like and social behaviors. Since oxytocin is centrally implicated in these behaviors, we furthermore explored whether the effects of Tph2 knockout on behavior would relate to changes in the oxytocin system. We show that Tph2-/- rats display reduced anxiety-like behavior and a high level of aggression in social interactions. In addition, oxytocin receptor expression was increased in the infralimbic and prelimbic cortices, paraventricular nucleus, dorsal raphe nucleus, and some subregions of the hippocampus, which was paralleled by increased levels of oxytocin in the medial frontal cortex and paraventricular nucleus but not the dorsal raphe nucleus, central amygdala, and hippocampus. In conclusion, our study demonstrated reduced anxiety but exaggerated aggression in Tph2-/- male rats and reveals for the first time a potential involvement of altered oxytocin system function. Meanwhile, the research of oxytocin could be distinguished in almost any psychiatric disorder including anxiety and mental disorders. This research potentially proposes a new target for the treatment of such disorders, from a genetic serotonin deficiency aspect.

Repeated testing modulates chronic unpredictable mild stress effects in male rats.

Depression is a highly prevalent, debilitating mental disorder. Chronic unpredictable mild stress (CUMS) is the most widely applied model to study this affliction in rodents. While studies incorporating CUMS prior to an intervention often require long-lasting stress effects that persist after exposure is ceased, the longevity of these effects is rarely studied. Additionally, it is unclear whether behavioural assessments can be performed before and after interventions without repeated testing effects. In rats, we investigated CUMS effects on components of depressive-like behaviour both acutely after stress cessation and after a recovery period, as well as effects of repeated testing. We observed acute disruptions of the circadian locomotor rhythm and a reduced sucrose preference immediately after CUMS exposure. While circadian locomotor rhythm effects persisted up until four weeks after stress cessation, independently of repeated testing, sucrose preference effects did not. Interestingly, CUMS animals tested once after a recovery period of four weeks showed reduced anxiety-like behaviour in the open field and elevated plus maze compared to their control group and repeatedly-tested CUMS animals. These findings suggest that distinct CUMS-induced components of depressive-like behaviour are affected differentially by recovery time and repeated testing; these aspects should be considered carefully in future study designs.

Peripheral Serotonin Deficiency Affects Anxiety-like Behavior and the Molecular Response to an Acute Challenge in Rats.

Serotonin is synthetized through the action of tryptophan hydroxylase (TPH) enzymes. While the TPH2 isoform is responsible for the production of serotonin in the brain, TPH1 is expressed in peripheral organs. Interestingly, despite its peripheral localization, alterations of the gene coding for TPH1 have been related to stress sensitivity and an increased susceptibility for psychiatric pathologies. On these bases, we took advantage of newly generated TPH1-/- rats, and we evaluated the impact of the lack of peripheral serotonin on the behavior and expression of brain plasticity-related genes under basal conditions and in response to stress. At a behavioral level, TPH1-/- rats displayed reduced anxiety-like behavior. Moreover, we found that neuronal activation, quantified by the expression of Bdnf and the immediate early gene Arc and transcription of glucocorticoid responsive genes after 1 h of acute restraint stress, was blunted in TPH1-/- rats in comparison to TPH1+/+ animals. Overall, we provided evidence for the influence of peripheral serotonin levels in modulating brain functions under basal and dynamic situations.

Stress Modifies the Expression of Glucocorticoid-Responsive Genes by Acting at Epigenetic Levels in the Rat Prefrontal Cortex: Modulatory Activity of Lurasidone.

Epigenetics is one of the mechanisms by which environmental factors can alter brain function and may contribute to central nervous system disorders. Alterations of DNA methylation and miRNA expression can induce long-lasting changes in neurobiological processes. Hence, we investigated the effect of chronic stress, by employing the chronic mild stress (CMS) and the chronic restraint stress protocol, in adult male rats, on the glucocorticoid receptor (GR) function. We focused on DNA methylation specifically in the proximity of the glucocorticoid responsive element (GRE) of the GR responsive genes Gadd45ß, Sgk1, and Gilz and on selected miRNA targeting these genes. Moreover, we assessed the role of the antipsychotic lurasidone in modulating these alterations. Chronic stress downregulated Gadd45ß and Gilz gene expression and lurasidone normalized the Gadd45ß modification. At the epigenetic level, CMS induced hypermethylation of the GRE of Gadd45ß gene, an effect prevented by lurasidone treatment. These stress-induced alterations were still present even after a period of rest from stress, indicating the enduring nature of such changes. However, the contribution of miRNA to the alterations in gene expression was moderate in our experimental conditions. Our results demonstrated that chronic stress mainly affects Gadd45ß expression and methylation, effects that are prolonged over time, suggesting that stress leads to changes in DNA methylation that last also after the cessation of stress procedure, and that lurasidone is a modifier of such mechanisms.

Chronic Treatment with a Phytosomal Preparation Containing Centella asiatica L. and Curcuma longa L. Affects Local Protein Synthesis by Modulating the BDNF-mTOR-S6 Pathway.

Brain derived neurotrophic factor (Bdnf) is the most diffuse neurotrophin in the central nervous system and it is crucial for the proper brain development and maintenance. Indeed, through the binding to its high affinity receptor TRKB and the activation of different intracellular cascades, it boosts cell survival, neurite growth and spine maturations mechanisms. Here, we evaluated if the chronic oral treatment for 10 days with a phytosomal preparation containing Centella asiatica L. and Curcuma longa L. could improve Bdnf levels in the prefrontal cortex of adult rats. Interestingly we found an increased expression of Bdnf with main effect of the treatment on the mTOR-S6 downstream signaling pathway. Accordingly, we found an increase in the expression of eukaryotic elongation factor (eEF2) with a shift towards the phosphorylated form thus increasing the transcription of Oligophrenin-1, a protein carrying the upstream Open Reading Frame (uORF) which reduction is paralleled by memory dysfunctions. These results show the ability of the phytosome to enhance mTOR-S6 regulated transcription and suggest the possibility to use this preparation in subjects with impairments in neuroplastic mechanisms, memory and cognitive abilities.

Enrichment Environment Positively Influences Depression- and Anxiety-Like Behavior in Serotonin Transporter Knockout Rats through the Modulation of Neuroplasticity, Spine, and GABAergic Markers.

The serotonin transporter (5-HTT in humans, SERT in rodents) is the main regulator of serotonergic transmission in the brain. The short allelic variant of the 5-HTT gene is in humans associated with psychopathologies and may enhance the vulnerability to develop depression after exposure to stressful events. Interestingly, the short allele also increases the sensitivity to a positive environment, which may buffer the vulnerability to depression. Since this polymorphism does not exist in rodents, male SERT knockout (SERT-/-) rats were tested to explore the molecular mechanisms based on this increased predisposition. This article investigates the influences of a positive manipulation, namely, enriched environment (EE), on the depressive-like behavior observed in SERT-/- rats. We found that one month of EE exposure normalized the anhedonic and anxious-like phenotype characteristics of this animal model. Moreover, we observed that EE exposure also restored the molecular alterations in the prefrontal cortex by positively modulating the expression of the neurotrophin Bdnf, and of spines and gamma-aminobutyric acid (GABA)ergic markers. Overall, our data confirm the depression-like phenotype of SERT-/- rats and highlight the ability of EE to restore behavioral and molecular alterations, thus promoting the opportunity to use EE as a supporting non-pharmacological approach to treat mood disorders.

Chronic Restraint Stress Inhibits the Response to a Second Hit in Adult Male Rats: A Role for BDNF Signaling.

Depression is a recurrent disorder, with about 50% of patients experiencing relapse. Exposure to stressful events may have an adverse impact on the long-term course of the disorder and may alter the response to a subsequent stressor. Indeed, not all the systems impaired by stress may normalize during symptoms remission, facilitating the relapse to the pathology. Hence, we investigated the long-lasting effects of chronic restraint stress (CRS) and its influence on the modifications induced by the exposure to a second hit on brain-derived neurotrophic factor (BDNF) signaling in the prefrontal cortex (PFC). We exposed adult male Sprague Dawley rats to 4 weeks of CRS, we left them undisturbed for the subsequent 3 weeks, and then we exposed animals to one hour of acute restraint stress (ARS). We found that CRS influenced the release of corticosterone induced by ARS and inhibited the ability of ARS to activate mature BDNF, its receptor Tropomyosin receptor kinase B (TRKB), and their associated intracellular cascades: the TRKB-PI3K-AKT), the MEK-MAPK/ERK, and the Phospholipase C γ (PLCγ) pathways, positively modulated by ARS in non-stressed animals. These results suggest that CRS induces protracted and detrimental consequences that interfere with the ability of PFC to cope with a challenging situation.

The Absence of Serotonin in the Brain Alters Acute Stress Responsiveness by Interfering With the Genomic Function of the Glucocorticoid Receptors.

Alterations in serotonergic transmission have been related to a major predisposition to develop psychiatric pathologies, such as depression. We took advantage of tryptophan hydroxylase (TPH) 2 deficient rats, characterized by a complete absence of serotonin in the brain, to evaluate whether a vulnerable genotype may influence the reaction to an acute stressor. In this context, we investigated if the glucocorticoid receptor (GR) genomic pathway activation was altered by the lack of serotonin in the central nervous system. Moreover, we analyzed the transcription pattern of the clock genes that can be affected by acute stressors. Adult wild type (TPH2+/+) and TPH2-deficient (TPH2-/-) male rats were sacrificed after exposure to one single session of acute restraint stress. Protein and gene expression analyses were conducted in the prefrontal cortex (PFC). The acute stress enhanced the translocation of GRs in the nucleus of TPH2+/+ animals. This effect was blunted in TPH2-/- rats, suggesting an impairment of the GR genomic mechanism. This alteration was mirrored in the expression of GR-responsive genes: acute stress led to the up-regulation of GR-target gene expression in TPH2+/+, but not in TPH2-/- animals. Finally, clock genes were differently modulated in the two genotypes after the acute restraint stress. Overall our findings suggest that the absence of serotonin within the brain interferes with the ability of the HPA axis to correctly modulate the response to acute stress, by altering the nuclear mechanisms of the GR and modulation of clock genes expression.

Centella asiatica L. Phytosome Improves Cognitive Performance by Promoting Bdnf Expression in Rat Prefrontal Cortex.

A wide range of people in the world use natural remedies as primary approaches against illnesses. Accordingly, understanding the mechanisms of action of phytochemicals has become of great interest. In this context, Centella asiatica L. is extensively used, not only as anti-inflammatory or antioxidant agent but also as brain tonic. On this basis, the purpose of this study was to evaluate whether the chronic administration of C. asiatica L. to adult male rats was able to improve the expression of Bdnf, one of the main mediators of brain plasticity. Moreover, we assessed whether the treatment could affect the cognitive performance in the novel object recognition (NOR) test. We confirmed the presence of the main compounds in the plasma. Furthermore, C. asiatica L. administration induced an increase of Bdnf in the prefrontal cortex, and the administration of the higher dose of the extract was able to improve cognitive performance. Finally, the increase in the preference index in the NOR test was paralleled by a further increase in Bdnf expression. Overall, we highlight the ability of C. asiatica L. to affect brain functions by increasing Bdnf expression and by enhancing the cognitive performance.

Effect of lurasidone treatment on chronic mild stress-induced behavioural deficits in male rats: The potential role for glucocorticoid receptor signalling.

BACKGROUND: Stress represents one of the main precipitating factors for psychiatric diseases, characterised by an altered function of glucocorticoid receptors (GR), known to play a role in mood and cognitive function. We investigated the ability of the antipsychotic lurasidone to modulate the involvement of genomic and non-genomic GR signalling in the behavioural alterations due to chronic stress exposure. METHODS: Male Wistar rats were exposed to seven weeks of chronic mild stress (CMS) and treated with lurasidone (3 mg/kg/day) starting from the second week of stress for more five weeks. Gene expression and protein analyses were conducted in dorsal hippocampus. RESULTS: Seven weeks of CMS induced anhedonia and cognitive impairment, which were normalised by lurasidone. At molecular level, CMS rats showed an increase of GR protein levels by 60% (p<0.001 vs. CTRL/VEH) in the membrane compartment, which was paralleled by an up-regulation of phosphoSINAPSYN Ia/b by 88% (p<0.01 vs. CTRL/VEH) and of the mitochondrial marker Cox3 by 21% (p<0.05 vs. CTRL/VEH). Moreover, while exposure to the novel object recognition test increased the nuclear translocation of GRs by 96% (p<0.01 vs. CTRL/VEH/Naïve) and their transcriptional activity in non-stressed rats, such mechanisms were impaired in CMS rats. Interestingly, the genomic and non-genomic alterations of GR, induced by CMS, were normalised by lurasidone. CONCLUSION: Our results further support the role of glucocorticoid signalling in the dysfunction associated with stress exposure. We provide novel insights on the mechanism of lurasidone, suggesting its effectiveness on different domains associated with psychiatric disorders.

Acute Stress Induces Cognitive Improvement in the Novel Object Recognition Task by Transiently Modulating Bdnf in the Prefrontal Cortex of Male Rats.

Stress response involves several mechanisms and mediators that allow individuals to adapt to a changing environment. The effects of stress may be adaptive or maladaptive, based on the timing and intensity of exposure as well as on the individual vulnerability. In particular, exposure to mild and brief stressors provides beneficial advantages in a short-term period, by activating protective functions to react with the external demands. On these bases, the purpose of our study was to establish the time-dependent effects of acute stress exposure on neuroplastic mechanisms in adult male rats. Moreover, we aim at establishing the consequences of the acute challenge on memory processes by testing rats in the Novel Object Recognition (NOR) test. We found that acute restraint stress up-regulated total Bdnf expression 1 h post stress specifically in rat prefrontal cortex, an effect that was sustained by the increase of Bdnf isoform IV as well as by the pool of Bdnf transcripts with long 3'UTR. Furthermore, in the same brain region, the acute stress modulated in a time-specific manner the expression of different activity-dependent genes, namely Arc, Gadd45ß and Nr4a1. At behavioral level, the challenge was able to improve the performance in the NOR test specifically 1 h post stress, an effect that positively correlated with the expression of the neurotrophic factors. Taken together, our results suggest that a single session of acute stress enhances memory and learning functions with a specific temporal profile, by improving neuroplastic mechanisms within the prefrontal cortex.

TPH2 Deficiency Influences Neuroplastic Mechanisms and Alters the Response to an Acute Stress in a Sex Specific Manner.

Dysregulations of the central serotoninergic system have been implicated in several psychopathologies, characterized by different susceptibility between males and females. We took advantage of tryptophan hydroxylase 2 (TPH2) deficient rats, lacking serotonin specifically in the brain, to investigate whether a vulnerable genotype can be associated with alterations of neuronal plasticity from the early stage of maturation of the brain until adulthood. We found a significant increase, in both gene and protein expression, of the neurotrophin brain-derived neurotrophic factor (BDNF), in the prefrontal cortex (PFC) of adult TPH2-deficient (TPH2-/-) male and female rats in comparison to wild type (TPH2+/+) counterparts. Interestingly, a development-specific pattern was observed during early postnatal life: whereas the increase in Bdnf expression, mainly driven by the modulation of Bdnf isoform IV was clearly visible after weaning at postnatal day (pnd) 30 in both sexes of TPH2-/- in comparison to TPH2+/+ rats, at early stages (pnd1 and pnd10) Bdnf expression levels did not differ between the genotypes, or even were downregulated in male TPH2-/- animals at pnd10. Moreover, to establish if hyposerotonergia may influence the response to a challenging situation, we exposed adult rats to an acute stress. Although the pattern of corticosterone release was similar between the genotypes, neuronal activation in response to stress, quantified by the expression of the immediate early genes activity regulated cytoskeleton associated protein (Arc) and Fos Proto-Oncogene (cFos), was blunted in both sexes of animals lacking brain serotonin. Interestingly, although upregulation of Bdnf mRNA levels after stress was observed in both genotypes, it was less pronounced in TPH2-/- in comparison to TPH2+/+ rats. In summary, our results demonstrated that serotonin deficiency affects neuroplastic mechanisms following a specific temporal pattern and influences the response to an acute stress.