Manuela Di Benedetto

Selective DNA Methylation of BDNF Promoter in Bipolar Disorder: Differences Among Patients with BDI and BDII

The etiology of bipolar disorder (BD) is still poorly understood, involving genetic and epigenetic mechanisms as well as environmental contributions. This study aimed to investigate the degree of DNA methylation at the promoter region of the brain-derived neurotrophic factor (BDNF) gene, as one of the candidate genes associated with major psychoses, in peripheral blood mononuclear cells isolated from 94 patients with BD (BD I=49, BD II=45) and 52 healthy controls. A significant BDNF gene expression downregulation was observed in BD II 0.53±0.11%; P<0.05), but not in BD I (1.13±0.19%) patients compared with controls (CONT: 1±0.2%). Consistently, an hypermethylation of the BDNF promoter region was specifically found in BD II patients (CONT: 24.0±2.1%; BDI: 20.4±1.7%; BDII: 33.3±3.5%, P<0.05). Of note, higher levels of DNA methylation were observed in BD subjects on pharmacological treatment with mood stabilizers plus antidepressants (34.6±4.2%, predominantly BD II) compared with those exclusively on mood-stabilizing agents (21.7±1.8%; P<0.01, predominantly BD I). Moreover, among the different pharmacological therapies, lithium (20.1±3.8%, P<0.05) and valproate (23.6±2.9%, P<0.05) were associated with a significant reduction of DNA methylation compared with other drugs (35.6±4.6%). Present findings suggest selective changes in DNA methylation of BDNF promoter in subjects with BD type II and highlight the importance of epigenetic factors in mediating the onset and/or susceptibility to BD, providing new insight into the mechanisms of gene expression. Moreover, they shed light on possible mechanisms of action of mood-stabilizing compounds vs antidepressants in the treatment of BD, pointing out that BDNF regulation might be a key target for their effects.

Kainate seizures increase nociceptin/orphanin FQ release in the rat hippocampus and thalamus: a microdialysis study

The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been suggested to play a facilitatory role in kainate seizure expression. Furthermore, mRNA levels for the N/OFQ precursor are increased following kainate seizures, while its receptor (NOP) density is decreased. These data suggest increased N/OFQ release. To obtain direct evidence that this is the case, we have developed a microdialysis technique, coupled with a sensitive radioimmunoassay, that allows measurement of N/OFQ release from the hippocampus and thalamus of awake, freely moving animals. In both these brain areas, the spontaneous N/OFQ efflux decreased by approximately 50% and 65% when Ca2+ was omitted and when tetrodotoxin was added to the perfusion medium, respectively. Perfusion of the dialysis probe with high K+ increased N/OFQ release (approximately threefold) in a Ca2+‐dependent and tetrodotoxin‐sensitive manner. Kainate seizures caused a twofold increase in N/OFQ release followed, within 3 h, by a return to baseline levels. Approximately 5 h after kainate, a late increase in N/OFQ release was observed. On the following day, when animals were having only low grade seizures, N/OFQ release was not significantly different from normal. These phenomena were observed with similar patterns in the hippocampus and in the thalamus. The present data indicate that acute limbic seizures are associated with increased N/OFQ release, which may prime the molecular changes described above, i.e. cause down‐regulation of NOP receptors and activation of N/OFQ biosynthesis.

Alterations of N/OFQ and NOP receptor gene expression in the substantia nigra and caudate putamen of MPP+ and 6-OHDA lesioned rats.

It has been suggested that the opioid-like neuropeptide nociceptin/orphanin FQ(N/OFQ) and its receptor (NOPr) may contribute to Parkinsons disease. Based on this idea, the aim of our study was to investigate the involvement of the N/OFQ-NOPr system in an animal model of Parkinsons disease and to evaluate if this neuropeptidergic system is acting through mechanisms involving glutamate and/or GABA. We injected the neurotoxins MPP+ or 6-OHDA into the cerebral ventricles and 10 days later measured N/OFQ and NOPr gene expression in caudate putamen (CP) and substantia nigra (SN), by RT-PCR. A large reduction in N/OFQ and NOPr mRNAs was observed in the CP of rat treated with either MPP+ or 6-OHDA, MPP+ being more effective than 6-OHDA. Both the neurotoxins induced an increase in N/OFQ gene expression in the SN, but only MPP+ evoked a significant down-regulation of NOPr in this area, showing a slight trend of reduction in 6-OHDA treated rats. Moreover, a reduction in the levels of glutamic acid decarboxylase (GAD65/67), an enzyme that converts the excitatory neurotransmitter glutamate to the inhibitory neurotransmitter y-aminobutyric acid (GABA), was also observed in the SN following 6-OHDA. These data suggest that DA modulates N/OFQ-NOPr system gene expression in SN and CP, strengthening the hypothesis that this neuropeptidergic system could be implicated in the mechanisms underlying Parkinsons disease. Our data might also suggest that the GABAergic system plays a role in the regulation of nigral function, although further studies are necessary to confirm this hypothesis. In agreement with previous studies, we also support the hypothesis of a potential value for NOP receptor antagonists to attenuate symptoms related to the degeneration of nigrostriatal dopaminergic pathway.

Dynorphin/KOP and nociceptin/NOP gene expression and epigenetic changes by cocaine in rat striatum and nucleus accumbens.

Cocaine induces neurochemical changes of endogenous prodynorphin-kappa opioid receptor (pDYN-KOP) and pronociceptin/orphaninFQ-nociceptin receptor (pN/OFQ-NOP) systems. Both systems play an important role in rewarding mechanisms and addictive stimulus processing by modulating drug-induced dopaminergic activation in the mesocortico-limbic brain areas. They are also involved in regulating stress mechanisms related to addiction. The aim of this study was to investigate possible changes of gene expression of the dynorphinergic and nociceptinergic system components in the nucleus accumbens (NA) and in medial and lateral caudate putamen (mCPu and lCPu, respectively) of rats, following chronic subcutaneous infusion of cocaine. In addition, the epigenetic histone modifications H3K4me3 and H3K27me3 (an activating and a repressive marker, respectively) at the promoter level of the pDYN, KOP, pN/OFQ and NOP genes were investigated. Results showed that cocaine induced pDYN gene expression up-regulation in the NA and lCPu, and its down-regulation in the mCPu, whereas KOP mRNA levels were unchanged. Moreover, cocaine exposure decreased pN/OFQ gene expression in the NA and lCPu, while NOP mRNA levels appeared significantly increased in the NA and decreased in the lCPu. Specific changes of the H3K4me3 and H3K27me3 levels were found at pDYN, pN/OFQ, and NOP gene promoter, consistent with the observed gene expression alterations. The present findings contribute to better define the role of endogenous pDYN-KOP and pN/OFQ-NOP systems in neuroplasticity mechanisms following chronic cocaine treatment. The epigenetic histone modifications underlying the gene expression changes likely mediate the effects of cocaine on transcriptional regulation of specific gene promoters that result in long-lasting drug-induced plasticity.

Role of serotonin on cocaine-mediated effects on prodynorphin gene expression in the rat brain.

The effect of the selective serotonin uptake inhibitor fluoxetine was examined on prodynorphin gene expression. Fluoxetine or vehicle was infused continuously for 7 d via osmotic minipumps into male rats. Northern blot analysis showed significant increases in prodynorphin gene expression in the hypothalamus (171% of controls) and significant decreases in the caudate putamen and nucleus accumbens (62% and 70% of controls, respectively). There were no significant changes in the hippocampus. Thus, chronic inhibition of serotonin uptake can regulate prodynorphin gene expression in the hypothalamus, caudate putamen, and nucleus accumbens. Fluoxetine effects were also evaluated in rats treated with p-chloroamphetamine (PCA), a neurotoxin that depletes serotonin. Because we previously reported that continuous infusion of cocaine for 7 d (which inhibits dopamine, serotonin, and norepinephrine uptake), or GBR 12909 (a selective dopamine uptake inhibitor), produced significant decreases in the hypothalamus and cocaine also produced a significant increase in prodynorphin gene expression in caudate putamen, regulation of prodynorphin gene expression by fluoxetine is suggested to be different from that by cocaine. Because neither a selective dopamine uptake inhibitor nor a selective serotonin uptake inhibitor produced the same effect as cocaine in the caudate putamen, this effect is likely regulated by the inhibition of norepinephrine uptake, by a combination of effects on two or three neurotransmitter transporters, or by a mechanism unrelated to transporter inhibition.

Chronic cocaine produces decreases in N/OFQ peptide levels in select rat brain regions

The interaction of opioids and stimulants is well established; however, the mechanisms that underlie the role that opioid receptors play in psychostimulant action are not. Nociceptin/orphaninFQ (N/OFQ), the endogenous agonist at NOP receptors, attenuates the behavioral effects of cocaine. The effects of cocaine on N/OFQ were examined in rats using immunoautoradiographic and RIA techniques. Chronic administration of cocaine decreased N/OFQ in medial regions of the caudate putamen, the nucleus accumbens shell, and the substantia nigra. These studies show that N/OFQ levels are altered by treatment with cocaine. Furthermore the changes in N/OFQ parallel those seen for K-opioid receptors, suggesting that the interactions between cocaine and these systems might be similar.

Combined exposure to agriculture pesticides, paraquat and maneb, induces alterations in the N/OFQ—NOPr and PDYN/KOPr systems in rats: Relevance to sporadic Parkinson's disease

Despite several years of research, the aetiology of Parkinsons disease (PD) is quite far from being solved. In PD, as well as in other neurodegenerative disorders, it has been proposed that the combination of multiple factors might contribute to the onset of the disease. Indeed, several authors have suggested that environmental factors, such as pollutants and chemicals, might be associated with the onset of several neurodegenerative disorders. On the other hand, several studies have described that the nociceptin/orphanin‐NOP and prodynorphin‐KOP opioid systems are implicated in the pathology of Parkinsons disease. Considering the nonrestricted commercial availability and common use of several pesticides, such as paraquat and maneb, in agriculture of less developed countries, the aim of our study was to investigate the involvement of nociceptin/orphanin‐NOP and prodynorphin‐KOP systems in a chronic paraquat and maneb animal model of Parkinsons disease. Our results showed that after paraquat/maneb (5/15 mg kg−1) treatment, a significant reduction in tyrosine hydroxylase (TH) levels, the rate‐limiting enzyme for dopamine synthesis, was observed. Also, the association of paraquat and maneb (5/15 mg kg−1) induced an increase in nociceptin/orphanin and a decrease of prodynorphin gene expression levels in the substantia nigra with a down‐regulation of NOP and KOP receptors after both treatments in the substantia nigra and caudate putamen. These data further confirm that paraquat and maneb toxicity can modulate gene expression of the nociceptin/orphanin‐NOP receptor and prodynorphin‐KOP receptor systems in the substantia nigra and caudate putamen, offering further support to the hypothesis that chronic exposure to these agrochemicals might be implicated in the mechanisms underlying sporadic Parkinsons disease.

The κ-opioid receptor agonist U-69593 prevents cocaine-induced phosphorylation of DARPP-32 at Thr34 in the rat brain

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein) is a potent endogenous inhibitor of protein phosphatase-1, which plays an important role in dopaminergic transmission. A large body of evidence supports the key role of DARPP-32-dependent signalling in mediating the actions of multiple drugs of abuse, including cocaine, which, when acutely administered, increases the Thr(34) phosphorylation of DARPP-32 in the striatal and cortical areas. In this study, we have examined the contribution of the kappa opioid system to the regulation of DARPP-32 phosphorylation at Thr(34), following acute cocaine administration, in selected rat brain areas. Results showed that a single injection of cocaine induces a significant increase in DARPP-32 phosphorylation at Thr(34) in the hippocampus, caudate putamen and prefrontal cortex. In addition, pretreatment with the kappa opioid receptor agonist U-69593 prevented cocaine effects in all the investigated areas. These data could be considered consistent with the ability of kappa opioid agonists to attenuate many behavioural and neurochemical effects of cocaine.

Alterations of CREB and DARPP-32 phosphorylation following cocaine and monoaminergic uptake inhibitors.

The cAMP response element-binding protein (CREB) is a transcription factor that can contribute to drug-induced changes in gene expression. It is well known that the dopamine and cAMP-regulated phosphoprotein (DARPP-32), via activation, is converted into a potent inhibitor of protein phosphatase-1 (PP-1), which regulates the activity of CREB. We previously reported that the continuous infusion of cocaine for 7 days produced a significant increase in prodynorphin mRNA in the rat caudate putamen and we also studied the role of the different monoamines in these cocaine effects. Since multiple cAMP response element (CRE) sequences are present on the prodynorphin gene promoter, the aim of our study was to investigate the effects of cocaine and monoaminergic uptake inhibitors on CREB and DARPP-32 phosphorylation and moreover the possible correlation with the changes already observed on prodynorphin gene expression. Here we investigated the alterations on phospho-Ser133 CREB, phospho-Thr34 DARPP-32 and phospho-Thr75 DARPP-32 induced by continuous infusions of cocaine, GBR12909, fluoxetine and nisoxetine. A significant decrease in both phospho-CREB at Ser133 and phospho-DARPP-32 at Thr34 in the rat caudate putamen was produced by cocaine, GBR 12909, fluoxetine or nisoxetine. No alterations were observed on phospho-Thr75 DARPP-32 levels. We hypothesize that the decrease in phospho-Thr34 DARPP-32 could evoke an increase in PP-1 activity which is responsible for the reduction of CREB activation. These effects could in turn elicit the reduction in the transcriptional cascade of the prodynorphin gene in the caudate putamen, observed following chronic fluoxetine and nisoxetine. On the other hand, these mechanisms do not seem to be involved in cocaine- or GBR 12909-induced effects.