Chronic Trazodone and Citalopram Treatments Increase Trophic Factor and Circadian Rhythm Gene Expression in Rat Brain Regions Relevant for Antidepressant Efficacy.

Trazodone is an efficacious atypical antidepressant acting both as an SSRI and a 5HT2A and 5HT2C antagonist. Antagonism to H1-histaminergic and alpha1-adrenergic receptors is responsible for a sleep-promoting action. We studied long-term gene expression modulations induced by chronic trazodone to investigate the molecular underpinning of trazodone efficacy. Rats received acute or chronic treatment with trazodone or citalopram. mRNA expression of growth factor and circadian rhythm genes was evaluated by qPCR in the prefrontal cortex (PFCx), hippocampus, Nucleus Accumbens (NAc), amygdala, and hypothalamus. CREB levels and phosphorylation state were evaluated using Western blotting. BDNF levels were significantly increased in PFCx and hippocampus by trazodone and in the NAc and hypothalamus by citalopram. Likewise, TrkB receptor levels augmented in the PFCx after trazodone and in the amygdala after citalopram. FGF-2 and FGFR2 levels were higher after trazodone in the PFCx. The CREB phosphorylation state was increased by chronic trazodone in the PFCx, hippocampus, and hypothalamus. Bmal1 and Per1 were increased by both antidepressants after acute and chronic treatments, while Per2 levels were specifically augmented by chronic trazodone in the PFCx and NAc, and by citalopram in the PFCx, amygdala, and NAc. These findings show that trazodone affects the expression of neurotrophic factors involved in antidepressant responses and alters circadian rhythm genes implicated in the pathophysiology of depression, thus shedding light on trazodone's molecular mechanism of action.

Early-life nicotine or cotinine exposure produces long-lasting sleep alterations and downregulation of hippocampal corticosteroid receptors in adult mice.

Early-life exposure to environmental toxins like tobacco can permanently re-program body structure and function. Here, we investigated the long-term effects on mouse adult sleep phenotype exerted by early-life exposure to nicotine or to its principal metabolite, cotinine. Moreover, we investigated whether these effects occurred together with a reprogramming of the activity of the hippocampus, a key structure to coordinate the hormonal stress response. Adult male mice born from dams subjected to nicotine (NIC), cotinine (COT) or vehicle (CTRL) treatment in drinking water were implanted with electrodes for sleep recordings. NIC and COT mice spent significantly more time awake than CTRL mice at the transition between the rest (light) and the activity (dark) period. NIC and COT mice showed hippocampal glucocorticoid receptor (GR) downregulation compared to CTRL mice, and NIC mice also showed hippocampal mineralocorticoid receptor downregulation. Hippocampal GR expression significantly and inversely correlated with the amount of wakefulness at the light-to-dark transition, while no changes in DNA methylation were found. We demonstrated that early-life exposure to nicotine (and cotinine) concomitantly entails long-lasting reprogramming of hippocampal activity and sleep phenotype suggesting that the adult sleep phenotype may be modulated by events that occurred during that critical period of life.

Activation of Antioxidant and Proteolytic Pathways in the Nigrostriatal Dopaminergic System After 3,4-Methylenedioxymethamphetamine Administration: Sex-Related Differences.

3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") is an amphetamine-related drug that may damage the dopaminergic nigrostriatal system. To investigate the mechanisms that sustain this toxic effect and ascertain their sex-dependence, we evaluated in the nigrostriatal system of MDMA-treated (4 × 20 mg/kg, 2 h apart) male and female mice the activity of superoxide dismutase (SOD), the gene expression of SOD type 1 and 2, together with SOD1/2 co-localization with tyrosine hydroxylase (TH)-positive neurons. In the same mice and brain areas, activity of glutathione peroxidase (GPx) and of ß2/ß5 subunits of the ubiquitin-proteasome system (UPS) were also evaluated. After MDMA, SOD1 increased in striatal TH-positive terminals, but not nigral neurons, of males and females, while SOD2 increased in striatal TH-positive terminals and nigral neurons of males only. Moreover, after MDMA, SOD1 gene expression increased in the midbrain of males and females, whereas SOD2 increased only in males. Finally, MDMA increased the SOD activity in the midbrain of females, without affecting GPx activity, decreased the ß2/ß5 activities in the striatum of males and the ß2 activity in the midbrain of females. These results suggest that the mechanisms of MDMA-induced neurotoxic effects are sex-dependent and dopaminergic neurons of males could be more sensitive to SOD2- and UPS-mediated toxic effects.

Nociceptive behavior and central neuropeptidergic dysregulations in male and female mice of a Fabry disease animal model.

Fabry disease (FD) is an X-linked inherited disorder characterized by glycosphingolipid accumulation due to deficiency of α-galactosidase A (α-Gal A) enzyme. Chronic pain and mood disorders frequently coexist in FD clinical setting, however underlying pathophysiologic mechanisms are still unclear. Here we investigated the mechanical and thermal sensitivity in α-Gal A (-/0) hemizygous male and the α-Gal A (-/-) homozygous female mice. We also characterized the gene expression of dynorphinergic, nociceptinergic and CRFergic systems, known to be involved in pain control and mood disorders, in the prefrontal cortex, amygdala and thalamus of α-Gal A (-/0) hemizygous male and the α-Gal A (-/-) homozygous female mice. Moreover, KOP receptor protein levels were evaluated in the same areas. Fabry knock-out male, but not female, mice displayed a decreased pain threshold in both mechanical and thermal tests compared to their wild type littermates. In the amygdala and prefrontal cortex, we observed a decrease of pDYN mRNA levels in males, whereas an increase was assessed in females, thus suggesting sex-related dysregulation of stress coping and pain mechanisms. Elevated mRNA levels for pDYN/KOP and CRF/CRFR1 systems were observed in male and female thalamus, a critical crossroad for both painful signals and cognitive/emotional processes. KOP receptor protein level changes assessed in the investigated areas, appeared mostly in agreement with KOP gene expression alterations. Our data suggest that α-Gal A enzyme deficiency in male and female mice is associated with distinct neuropeptide gene and protein expression dysregulations of investigated systems, possibly related to the neuroplasticity underlying the neurological features of FD.

Deregulated FASN Expression in BRAF Inhibitor-Resistant Melanoma Cells Unveils New Targets for Drug Combinations.

Metabolic changes promoting cell survival are involved in metastatic melanoma progression and in the development of drug resistance. In BRAF-inhibitor resistant melanoma cells, we explored the role of FASN, an enzyme involved in lipogenesis overexpressed in metastatic melanoma. Resistant melanoma cells displaying enhanced migratory and pro-invasive abilities increased sensitivity to the BRAF inhibitor PLX4032 upon the molecular targeting of FASN and upon treatment with the FASN inhibitor orlistat. This behavior was associated with a marked apoptosis and caspase 3/7 activation observed for the drug combination. The expression of FASN was found to be inversely associated with drug resistance in BRAF-mutant cell lines, both in a set of six resistant/sensitive matched lines and in the Cancer Cell Line Encyclopedia. A favorable drug interaction in resistant cells was also observed with U18666 A inhibiting DHCR24, which increased upon FASN targeting. The simultaneous combination of the two inhibitors showed a synergistic interaction with PLX4032 in resistant cells. In conclusion, FASN plays a role in BRAF-mutated melanoma progression, thereby creating novel therapeutic opportunities for the treatment of melanoma.

Targeting the JAK/STAT Pathway: A Combined Ligand- and Target-Based Approach.

Janus kinases (JAKs) are a family of proinflammatory enzymes able to mediate the immune responses and the inflammatory cascade by modulating multiple cytokine expressions as well as various growth factors. In the present study, the inhibition of the JAK-signal transducer and activator of transcription (STAT) signaling pathway is explored as a potential strategy for treating autoimmune and inflammatory disorders. A computationally driven approach aimed at identifying novel JAK inhibitors based on molecular topology, docking, and molecular dynamics simulations was carried out. For the best candidates selected, the inhibitory activity against JAK2 was evaluated in vitro. Two hit compounds with a novel chemical scaffold, 4 (IC50 = 0.81 µM) and 7 (IC50 = 0.64 µM), showed promising results when compared with the reference drug Tofacitinib (IC50 = 0.031 µM).

The active second-generation proteasome inhibitor oprozomib reverts the oxaliplatin-induced neuropathy symptoms.

Oxaliplatin-induced neuropathy (OXAIN) is a major adverse effect of this antineoplastic drug, widely used in the treatment of colorectal cancer. Although its molecular mechanisms remain poorly understood, recent evidence suggest that maladaptive neuroplasticity and oxidative stress may participate to the development of this neuropathy. Given the role played on protein remodeling by ubiquitin-proteasome system (UPS) in response to oxidative stress and in neuropathic pain, we investigated whether oxaliplatin might cause alterations in the UPS-mediated degradation pathway, in order to identify new pharmacological tools useful in OXAIN. In a rat model of OXAIN (2.4 mg kg-1 i.p., daily for 10 days), a significant increase in chymotrypsin-(ß5) like activity of the constitutive proteasome 26S was observed in the thalamus (TH) and somatosensory cortex (SSCx). In addition, the selective up-regulation of ß5 and LMP7 (ß5i) subunit gene expression was assessed in the SSCx. Furthermore, this study revealed that oprozomib, a selective ß5 subunit proteasome inhibitor, is able to normalize the spinal prodynorphin gene expression upregulation induced by oxaliplatin, as well as to revert mechanical allodynia and thermal hyperalgesia observed in oxaliplatin-treated rats. These results underline the relevant role of UPS in the OXAIN and suggest new pharmacological targets to counteract this severe adverse effect. This preclinical study reveals the involvement of the proteasome in the oxaliplatin-induced neuropathy and adds useful information to better understand the molecular mechanism underlying this pain condition. Moreover, although further evidence is required, these findings suggest that oprozomib could be a therapeutic option to counteract chemotherapy-induced neuropathy.

Modulation of the Negative Affective Dimension of Pain: Focus on Selected Neuropeptidergic System Contributions.

It is well known that emotions can interfere with the perception of physical pain, as well as with the development and maintenance of painful conditions. On the other hand, somatic pain can have significant consequences on an individual's affective behavior. Indeed, pain is defined as a complex and multidimensional experience, which includes both sensory and emotional components, thus exhibiting the features of a highly subjective experience. Over the years, neural pathways involved in the modulation of the different components of pain have been identified, indicating the existence of medial and lateral pain systems, which, respectively, project from medial or lateral thalamic nuclei to reach distinct cortex regions relating to specific functions. However, owing to the limited information concerning how mood state and painful input affect each other, pain treatment is frequently unsatisfactory. Different neuromodulators, including endogenous neuropeptides, appear to be involved in pain-related emotion and in its affective influence on pain perception, thus playing key roles in vulnerability and clinical outcome. Hence, this review article focuses on evidence concerning the modulation of the sensory and affective dimensions of pain, with particular attention given to some selected neuropeptidergic system contributions.

Interplay between the Endogenous Opioid System and Proteasome Complex: Beyond Signaling.

Intracellular signaling mechanisms underlying the opioid system regulation of nociception, neurotransmitters release, stress responses, depression, and the modulation of reward circuitry have been investigated from different points of view. The presence of the ubiquitin proteasome system (UPS) in the synaptic terminations suggest a potential role of ubiquitin-dependent mechanisms in the control of the membrane occupancy by G protein-coupled receptors (GPCRs), including those belonging to the opioid family. In this review, we focused our attention on the role played by the ubiquitination processes and by UPS in the modulation of opioid receptor signaling and in pathological conditions involving the endogenous opioid system. The collective evidence here reported highlights the potential usefulness of proteasome inhibitors in neuropathic pain, addictive behavior, and analgesia since these molecules can reduce pain behavioral signs, heroin self-administration, and the development of morphine analgesic tolerance. Moreover, the complex mechanisms involved in the effects induced by opioid agonists binding to their receptors include the ubiquitination process as a post-translational modification which plays a relevant role in receptor trafficking and degradation. Hence, UPS modulation may offer novel opportunities to control the balance between therapeutic versus adverse effects evoked by opioid receptor activation, thus, representing a promising druggable target.

FoxO-1 contributes to the efficacy of the combination of the XPO1 inhibitor selinexor and cisplatin in ovarian carcinoma preclinical models.

The XPO1/CRM1 inhibitor selinexor (KPT-330), is currently being evaluated in multiple clinical trials as an anticancer agent. XPO1 participates in the nuclear export of FoxO-1, which we previously found to be decreased in platinum-resistant ovarian carcinoma. The aim of this study was to determine whether enriching FoxO-1 nuclear localization using selinexor would increase ovarian cancer cell sensitivity to cisplatin. Selinexor, as a single agent, displayed a striking antiproliferative effect in different ovarian carcinoma cell lines. A schedule-dependent synergistic effect of selinexor in combination with cisplatin was found in cisplatin-sensitive IGROV-1, the combination efficacy being more evident in sensitive than in the resistant cells. In IGROV-1 cells, the combination was more effective when selinexor followed cisplatin exposure. A modulation of proteins involved in apoptosis (p53, Bax) and in cell cycle progression (p21WAF1) was found by Western blotting. Selinexor-treated cells exhibited enriched FoxO-1 nuclear staining. Knock-down experiments with RNA interference indicated that FOXO1-silenced cells displayed a reduced sensitivity to selinexor. FOXO1 silencing also tended to reduce the efficacy of the drug combination at selected cisplatin concentrations. Selinexor significantly inhibited tumor growth, induced FoxO-1 nuclear localization and improved the efficacy of cisplatin in IGROV-1 xenografts. Taken together, our results support FoxO-1 as one of the key factors promoting sensitivity towards selinexor and the synergistic interaction between cisplatin and selinexor in ovarian carcinoma cells with selected molecular backgrounds, highlighting the need for treatment regimens tailored to the molecular tumor features.