94 research outputs found
Enhancement of human neuroblastoma cell apoptosis by combined treatment with valproic acid and interferonβ
No full textType I interferons (IFNs), including IFNα and IFNβ, possess antitumoral activity and have been proposed to be potentially useful in the treatment of
neuroblastoma. We have previously shown that IFNβ causes cell death of human SHSY5Y neuroblastoma cells by inducing apoptosis through the
intrinsic mitochondrial pathway (Dedoni et al. J. Neurochem. 115,14211433, 2010). As valproic acid, an histone deacetylase inhibitor (HDACi) has been reported to enhance the antitumoral effects of type I IFNs, in the present study we have examined the effects of this drug on IFNβinduced apoptosis of neuroblastoma cells. We found that prolonged exposure (24 h)
of SHSY5Y cells to therapeutic concentrations of valproic acid (0.61 mM) had no effect on cell viability. However, when combined with IFNβ,
valproic acid markedly enhanced cell apoptosis induced by the cytokine, as demonstrated by the increase in annexin V staining, caspase activation and
poly( ADP ribose) polymerase (PARP) cleavage. Acetylation of histone 2B at Lys5 was markedly enhanced by valproic acid, but this response was not
affected by cotreatment with IFNβ. Conversely, valproic acid enhanced the IFNβstimulated Tyrphosphorylation of STAT1, which is crucial for cytokineinduced
apoptosis. Sodium butyrate (1 mM), another HDACi,
elicited similar potentiating effects on IFNβinduced apoptosis and STAT1 phosphorylation, whereas valpromide (1 mM), which did not enhance
histone acetylation, had no effects. MYCNamplified human LAN1 neuroblastoma cells displayed no sensitivity to the proapoptotic activity of IFNβ, but underwent apoptosis when treated with either valproic acid or sodium butyrate. Cotreatment of LAN1 cells with IFNβ and HDACi, but not valpromide, resulted in a significant increase in cell apoptosis. These data show a synergistic interaction of IFNβ and valproic acid in promoting
neuroblastoma cells apoptosis, which appears to be related to enhancementof histone acetylation
Antidepressants induce profibrotic responses via the lysophosphatidic acid receptor LPA1
No full textPreclinical and clinical studies have indicated that antidepressants can promote inflammation and fibrogenesis, particularly in the lung, by mechanisms not fully elucidated. We have previously shown that different classes of antidepressants can activate the lysophosphatidic acid (LPA) receptor LPA1, a major pathogenetic mediator of tissue fibrosis. The aim of the present study was to investigate whether in cultured human dermal and lung fibroblasts antidepressants could trigger LPA1-mediated profibrotic responses. In both cell types amitriptyline, clomipramine and mianserin mimicked the ability of LPA to induce the phosphorylation/activation of extracellular signal –regulated kinases 1 and 2 (ERK1/2), which was blocked by the selective LPA1 receptor antagonist AM966 and the LPA1/3 antagonist Ki16425. Antidepressant-induced ERK1/2 stimulation was absent in fibroblasts stably depleted of LPA1 by short hairpin RNA transfection and was prevented by pertussis toxin, an uncoupler of receptors from Gi/o proteins. Like LPA, antidepressants stimulated fibroblasts proliferation and this effect was blocked by either AM966 or the MEK1/2 inhibitor PD98059. Moreover, by acting through LPA1 antidepressants induced the expression of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, and caused an ERK1/2-dependent increase in the cellular levels of transforming growth factor-β (TGF-β)1, a potent fibrogenic cytokine. Pharmacological blockade of TGF-β receptor type 1 prevented antidepressant- and LPA-induced α-SMA expression. These data indicate that in human dermal and lung fibroblasts different antidepressants can induce proliferative and differentiating responses by activating the LPA1 receptor coupled to ERK1/2 signalling and suggest that this property may contribute to the promotion of tissue fibrosis by these drugs
Inhibition of TNF-α-induced neuronal apoptosis by antidepressants acting through the lysophosphatidic acid receptor LPA1
No full textTumor necrosis factor- (TNF-), a pro-inflammatory cytokine considered to be implicated in the pathogenesis of major depressive disorder, is a critical regulator of neuronal cell fate. In the present study we found that TNF--induced apoptosis of HT22 hippocampal cells, a neuroblast-like cell line, was markedly attenuated by the antidepressants mianserin, mirtazapine and amitriptyline. The anti-apoptotic effect of the antidepressants was blocked by either pharmacological inhibition or gene silencing of the lysophosphatidic acid receptor LPA(1). Mianserin failed to affect TNF--induced caspase 8 activation, but inhibited the loss of mitochondrial membrane potential, the release of cytochrome c from mitochondria, procaspase 9 cleavage and downstream activation of caspase 3 in response to the cytokine. By acting through LPA(1), mianserin also attenuated the enhanced pro-apoptotic response induced by the combination of TNF- with other pro-inflammatory cytokines. TNF- appeared to counterbalance its own pro-apoptotic response by activating NF-kB, ERK1/2 and JNK. Antidepressants had no significant effects on NF-kB activation, but potentiated the TAK-1-dependent phosphorylation of ERK1/2 and JNK elicited by the cytokine. This synergistic interaction was associated with enhanced JNK-mediated phosphorylation of Bcl-2at Ser70 and increased ERK1/2-dependent mitochondrial accumulation of Mcl-1, two anti-apoptotic proteins that promote mitochondrial outer membrane stability. These results indicate that certain antidepressants, by activating LPA(1) signalling, protect HT22 hippocampal cells from TNF--induced apoptosis through a mechanism involving, at least in part, the potentiation of the pro-survival pathways activated by the cytokine
Evidence that the GABAB positive allosteric modulator CGP7930 activates MAPK cascade independently of GABAB receptor
No full textThe GABAB positive allosteric modulator (PAM) CGP7930 potentiates GABAB receptor signaling in transfected cells (Urwyler et al. Mol. Pharmacol. 60, 963971, 2001) and rat and human brain (Onali et al., Eur. J. Pharmacol. 471, 7784, 2003; Olianas et al., Neurochem. Int. 46, 149158, 2005). A number of behavioral studies have also shown that CGP7930 exerts anxiolytic effects and reduces selfadministration of drugs of abuse (Adams and Lawrence, CNS Drug Rev. 13, 308316, 2007). However, little is known on whether this drug can affect neuronal signaling independently of GABAB receptor activity. In the present study we report that in human SHSY5Y neuroblastoma cells, CGP7930 (30 μM) induced a rapid increase of dual phosphorylation (activation) of ERK1/2, which reached a maximum at 15 min and lasted for at least 60 min. CGP7930 also triggered CREB phosphorylation at Ser133 with a similar kinetic profile. Under the same experimental conditions, the GABAB receptor agonist () baclofen (100 μM) failed to affect ERK1/2 phosphorylation, and when combined with CGP7930, it did not elicit any further ERK1/2 stimulation. CGP7930induced ERK1/2 phosphorylation was not prevented by cell pretreatment with either the GABAB receptor antagonists CGP55845A and CGP54626, the Gi/oreceptor uncoupler pertussis toxin, the Gq/11 antagonist YM254890, or the tyrosine kinase inhibitor genistein. Conversely, it was completely blocked by the MEK inhibitor PD98059 and significantly attenuated by the protein kinase C inhibitors Go 6983 and bisindolylmaleimide I. CGP7930 (30 μM) was also found to stimulate ERK1/2 phosphorylation in CHOK1 cells, which do not express GABAB receptors. However, CGP7930 (1100 μM) had no effect in HEK293 cells, indicating that ERK1/2 activation was not a generalized cellular response to the PAM. These data indicate that CGP7930 can affect ERK1/2 signaling, which is known to be involved in the control of mood and drug addiction, independently of increased GABAB receptor activity
LPA1 mediates antidepressant-induced ERK1/2 signaling and protection from oxidative stress in glial cells
No full textAntidepressants have been shown to affect glial cell functions and intracellular signaling through mechanisms that are still not completely understood. In the present study, we provide evidence that in glial cells the lysophosphatidic acid (LPA) receptor LPA1 mediates antidepressant-induced growth factor receptor transactivation, ERK1/2 signaling, and protection from oxidative stress. Thus, in C6 glioma cells and rat cortical astrocytes, ERK1/2 activation induced by either amitriptyline or mianserin was antagonized by Ki16425 and VPC 12249 (S), which block LPA1 and LPA3 receptors, and by AM966, which selectively blocks LPA1 Cell depletion of LPA1 with siRNA treatment markedly reduced antidepressant- and LPA-induced ERK1/2 phosphorylation. LPA1 blockade prevented antidepressant-induced phosphorylation of the transcription factors CREB and Elk-1. Antidepressants and LPA signaling to ERK1/2 was abrogated by cell treatment with pertussis toxin and by the inhibition of fibroblast growth factor (FGF) receptor (FGF-R) and platelet-derived growth factor receptor (PDGF-R) tyrosine kinases. Both Ki16425 and AM966 suppressed antidepressant-induced phosphorylation of FGF-R. Moreover, blockade of LPA1 or inhibition of FGF-R and PDGF-R activities prevented antidepressant-stimulated Akt and GSK-3β phosphorylations. Mianserin protected C6 glioma cells and astrocytes from apoptotic cell death induced by H2O2, as indicated by increased cell viability, decreased expression of cleaved caspase 3, reduced cleavage of poly-ADP ribose polymerase and inhibition of DNA fragmentation. The protective effects of mianserin were antagonized by AM966. These data indicate that LPA1 constitutes a novel molecular target of the regulatory actions of tricyclic and tetracyclic antidepressants in glial cells
The atypical antidepressants mianserin and mirtazapine induce ERK1/2 signaling and antiapoptotic effects through activation of the lysophosphatidic acid LPA1 receptor in mouse hippocampal HT22 cells
No full textA large body of evidence indicate that the bioactive phospholipid lysophosphatidic acid (LPA) affects neurogenesis, synaptic plasticity and
anxietyrelated behaviour by activating specific Gprotein coupled receptors, termed LPA16, which are expressed in the developing and adult brain. However, relatively little is known on whether the LPA receptor system can be targeted by drugs used to treat neuropsychiatric diseases. We
have previously reported that different antidepressants promote growth factor receptor transactivation and cell proliferation in CHOk1 fibroblasts
and glia cells. In the present study, we show that in immortalized mouse hippocampal HT22 cells the atypical antidepressants mianserin and mirtazapine trigger ERK1/2 phosphorylation and protect against apoptosis through LPA1 receptors. ERK1/ 2 phosphorylation induced by acute exposure to either mianserin, mirtazapine or LPA was inhibited by cell pretreatment with pertussis toxin, indicating the involvement of G proteins of the Gi/o family. LPA, mianserin and mirtazapine also promoted the phosphorylation of the transcription factor CREB, a downstream effector of ERK1/2 signaling. Cell treatment with either AM966, a selective LPA1 receptor antagonist, or Ki16425, which preferentially blocks LPA1 and LPA3, significantly inhibited ERK1/2 and CREB phosphorylation elicited by mianserin and mirtazapine. Serum withdrawalinduced apoptosis of HT22 cells was markedly attenuated by either LPA, mianserin or mirtazapine, as indicated by the decreased cell death, decreased percentage of annexin Vpositive cells, inhibition of procaspase activation and poly( ADP ribose) polymerase cleavage. AM966 reduced the protective effects of mianserin, mirtazapine and LPA against HT22 cell apoptosis. Moreover, cell treatment with the MEK inhibitor PD98059 prevented the antiapoptotic effect of LPA and mianserin. These data provide further evidence
for the involvement of the LPA1 receptor in the pharmacological action of antidepressants
Expression and functional activity of proteinase-activated receptors 1 and 2 in the rat olfactory
No full textAlterations in the olfactory system and impairment of olfactory function occurr in neurodegenerative diseases, such as Alzheimer’ s and Parkinson’ s diseases. The participation of proteinase-activated receptors (PARs) in these conditions has been postulated, but little is known on the expression and activity of PARs in the olfactory system. We investigated the presence, the signaling properties and cellular actions of PAR1 and PAR2 in the rat main olfactory bulb and in primary cultures of olfactory bulb and olfactory sensory neurons. PAR1 and PAR2 activity was predominantly expressed in the olfactoty nerve-glomerular cell layer (ON-GL), where selective peptide agonists inhibited cyclic AMP formation and stimulated [35S ]GTPyS binding, phosphoinositide hydrolysis, CaMKII phosphorylation and Rho activation. Nanomolar concentrations of thrombin and trypsin mimicked the actions of the peptide agonists. Lesion of the olfactory mucosa caused a reduction of PAR1 and PAR2 activity in ON-GL, suggesting the possible localization of a receptor population on olfactory nerve terminals. In primary cultures of olfactory bulb cells and olfactory neuroepithelial cells, exposure to either serine proteinases or selective peptide agonists caused a rapid neurite retraction. Immunofluorescence analysis showed the presence of PAR1 and PAR2 in neurons and glial cells of olfactory bulb and in olfactory sensory neurons. These data provide the first evidence that PAR1 and PAR2 are expressed and functional in different structures of the olfactory system and suggest the possible involvement of the receptor in neurodegenerative processes affecting the olfactory function
Coincidence signaling of dopamine D1-like and M1 muscarinic receptors in the regulation of cyclic AMP formation and CREB phosphorylation in mouse prefrontal cortex
No full textIn the prefrontal cortex, dopamine D(1)-like and M(1) muscarinic receptors are both involved in the regulation of attentional, cognitive and emotional processes but so far no information has been provided on their functional interaction. In the present study we show that in mouse medial prefrontal cortex, concomitant activation of M(1) muscarinic receptors potentiated D(1)-like receptor-induced cyclic AMP formation through a mechanism involving activation of G(q/11) and the release of G protein βγ subunits. Immunohistochemical studies indicated that the adenylyl cyclase isoforms AC2 and AC4 are expressed in mouse prefrontal cortex and that they colocalize with D(1)-like receptors with a greater association for AC4. In primary cultures of frontal cortex neurons, D(1)-like receptor-induced Ser133 phosphorylation of the transcription factor cyclic AMP-responsive element binding protein (CREB) was potentiated by concurrent stimulation of M(1) receptors. Suppression of AC4 expression with small interfering RNA transfection reduced D(1) stimulation of cyclic AMP formation and CREB phosphorylation and abolished the M(1) potentiation, whereas knockdown of AC2 had no significant effects. These data indicate that in mouse prefrontal cortex G(q/11)-coupled M(1) receptor and G(s)-coupled D(1)-like receptor inputs converge on AC4 with a consequent enhancement of cyclic AMP formation and signaling to the nucleus
The induction of the activity-based anorexia is associated to the modulation of neuroinflammatory and oxidative balance mediators in the rat prefrontal cortex
Get PDFAnorexia nervosa (AN) is a life-threatening and complex psychiatric disorder characterized by altered body perception and the adoption of disturbed eating behavior and excessively exercising to reach the desired thinness. Reiteration of these habits leads to severe consequences for general and mental health, and the presence of psychiatric comorbidities makes this disorder one of those with the highest mortality rate. Although the exact molecular mechanisms underlying AN development have not been fully elucidated, clinical and preclinical data suggest the involvement of the immune system as well as of mediators of the redox balance, systems with an established role also in other psychiatric disorders. On these bases, aim of our study was to assess cerebral levels of specific mediators of neuroinflammation and redox balance by using the activity-based anorexia (ABA) model.
After a period of acclimation and adaptation, adolescent Sprague-Dawley female rats were divided in four experimental groups to be exposed for 6 days to (1) a combination of food restriction and wheel activity (ABA group), (2) voluntary running wheel activity (EXE group), (3) food restriction (RESTR group), (4) normal maintenance procedure (CTRL group). During the whole experiments, running wheel activity and animal body-weight were constantly monitored. The animals were then euthanized, the prefrontal cortex rapidly dissected and frozen in dry ice for the subsequent molecular analyses. Specifically, by using real time RT-PCR, we measured the gene expression of cardinal markers of neuroinflammation including pro-inflammatory and anti-inflammatory cytokines as well as markers of microglia activation. Moreover, we assessed the mRNA levels of specific mediators of the antioxidant component of the redox machinery.
We observed an overall reduction of neuroinflammation in the ABA group, as indicated by a significant decrease of the pro-inflammatory cytokines TNF-α and IL-1β, the inflammasome NLRP3, and the marker of microglia activation CD11b. Conversely, we found a significant increase of the mRNA levels for IL-6, whose dual role as pro- and anti-inflammatory cytokine is well-established. These effects were paralleled by the upregulation of the antioxidant enzymes GPX1, SRXN1 and MT1α.
Our data clearly indicate that the induction of activity-based anorexia in adolescent female rats is associated with alteration of both mediators of inflammation and oxidative balance in the prefrontal cortex. In particular we found that animals in the ABA group were characterized by lower neuroinflammation and increased antioxidant response. Since our experimental paradigm mimics the early stage of this complex pathology, we may hypothesize that the observed changes are the result of an adaptive response to the ABA induction Accordingly, further studies aimed to evaluate long-term effects will be crucial to better understand the molecular impact of ABA in the brain
Direct agonist activity of tricyclic antidepressants at distinct opioid receptor subtypes
No full textTricyclic antidepressants (TCAs) have been reported to interact with the opioid system, but their pharmacological activity at opioid receptors has not yet been elucidated. In the present study, we investigated the actions of amoxapine, amitriptyline, nortriptyline, desipramine, and imipramine at distinct cloned and native opioid receptors. In Chinese hamster ovary (CHO) cells expressing delta-opioid receptors (CHO/DOR), TCAs displaced [3H]naltrindole binding and stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding at micromolar concentrations with amoxapine displaying the highest potency and efficacy. Amoxapine and amitriptyline inhibited cyclic AMP formation and induced the phosphorylation of signaling molecules along the extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol-3 kinase pathways. Amoxapine also activated delta-opioid receptors in rat dorsal striatum and nucleus accumbens and human frontal cortex. In CHO cells expressing kappa-opioid receptors (CHO/KOR), TCAs, but not amoxapine, exhibited higher receptor affinity and more potent stimulation of [(35)S]GTPgammaS binding than in CHO/DOR and effectively inhibited cyclic AMP accumulation. Amitriptyline regulated ERK1/2 phosphorylation and activity in CHO/KOR and C6 glioma cells endogenously expressing kappa-opioid receptors, and this effect was attenuated by the kappa-opioid antagonist nor-binaltorphimine. In rat nucleus accumbens, amitriptyline slightly inhibited adenylyl cyclase activity and counteracted the inhibitory effect of the full kappa agonist trans-(-)-3,4dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488). At the cloned mu-opioid receptor, TCAs showed low affinity and no significant agonist activity. These results show that TCAs differentially regulate opioid receptors with a preferential agonist activity on either delta or kappa subtypes and suggest that this property may contribute to their therapeutic and/or side effects
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