Search

The pathophysiology of Anorexia Nervosa (AN) has not been fully elucidated. Anaplastic lymphoma kinase (ALK) receptor is a protein-tyrosine kinase mainly known as a key oncogenic driver. Recently, a genetic deletion of ALK in mice has been found to increase energy expenditure and confers resistance to obesity in these animals, suggesting its role in the regulation of thinness. Here, we investigated the expression of ALK and the downstream intracellular pathways in female rats subjected to the activity-based anorexia (ABA) model, which reproduces important features of human AN. In the hypothalamic lysates of ABA rats, we found a reduction in ALK receptor expression, a downregulation of Akt phosphorylation, and no change in the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. After the recovery from body weight loss, ALK receptor expression returned to the control baseline values, while it was again suppressed during a second cycle of ABA induction. Overall, this evidence suggests a possible involvement of the ALK receptor in the pathophysiology of AN, that may be implicated in its stabilization, resistance, and/or its exacerbation.

The antiepileptic and mood stabilizer agent valproic acid (VPA) has been shown to exert anti-tumour effects and to cause neuronal damage in the developing brain through mechanisms not completely understood. In the present study we show that prolonged exposure of SH-SY5Y and LAN-1 human neuroblastoma cells to clinically relevant concentrations of VPA caused a marked induction of the protein and transcript levels of the common neurotrophin receptor p75NTR and its co-receptor sortilin, two promoters of apoptotic cell death in response to proneurotrophins. VPA induction of p75NTR and sortilin was associated with an increase in plasma membrane expression of the receptor proteins and was mimicked by cell treatment with several histone deacetylase (HDAC) inhibitors. VPA and HDAC1 knockdown decreased the level of EZH2, a core component of the polycomb repressive complex 2, and upregulated the transcription factor CASZ1, a positive regulator of p75NTR. CASZ1 knockdown attenuated VPA-induced p75NTR overexpression. Cell treatment with VPA favoured proNGF-induced p75NTR/sortilin interaction and the exposure to proNGF enhanced JNK activation and apoptotic cell death elicited by VPA. Depletion of p75NTR or addition of the sortilin agonist neurotensin to block proNGF/sortilin interaction reduced the apoptotic response to VPA and proNGF. Exposure of mouse cerebellar granule cells to VPA upregulated p75NTR and sortilin and induced apoptosis which was enhanced by proNGF. These results indicate that VPA upregulates p75NTR apoptotic cell signalling through an epigenetic mechanism involving HDAC inhibition and suggest that this effect may contribute to the anti-neuroblastoma and neurotoxic effects of VPA. Electronic supplementary material The online version of this article (10.1007/s10495-020-01626-0) contains supplementary material, which is available to authorized users.

Neurotrophins and their receptors are relevant factors in controlling neuroblastoma growth and progression. The histone deacetylase (HDAC) inhibitor valproic acid (VPA) has been shown to downregulate TrkB and upregulate the p75NTR/sortilin receptor complex. In the present study, we investigated the VPA effect on the expression of the neurotrophin-3 (NT-3) receptor TrkC, a favorable prognostic marker of neuroblastoma. We found that VPA induced the expression of both full-length and truncated (TrkC-T1) isoforms of TrkC in human neuroblastoma cell lines without (SH-SY5Y) and with (Kelly, BE(2)-C and IMR 32) MYCN amplification. VPA enhanced cell surface expression of the receptor and increased Akt and ERK1/2 activation by NT-3. The HDAC inhibitors entinostat, romidepsin and vorinostat also increased TrkC in SH-SY5Y, Kelly and BE(2)-C but not IMR 32 cells. TrkC upregulation by VPA involved induction of RUNX3, stimulation of ERK1/2 and JNK, and ERK1/2-mediated Egr1 expression. In SH-SY5Y cell monolayers and spheroids the exposure to NT-3 enhanced the apoptotic cascade triggered by VPA. Gene silencing of both TrkC-T1 and p75NTR prevented the NT-3 proapoptotic effect. Moreover, NT-3 enhanced p75NTR/TrkC-T1 co-immunoprecipitation. The results indicate that VPA upregulates TrkC by activating epigenetic mechanisms and signaling pathways, and sensitizes neuroblastoma cells to NT-3-induced apoptosis.

Tumor necrosis factor-α (TNF-α), 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-α-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 LPA1. Mianserin failed to affect TNF-α-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 LPA1, mianserin also attenuated the enhanced pro-apoptotic response induced by the combination of TNF-α with other pro-inflammatory cytokines. TNF-α 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-2 at 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 LPA1 signalling, protect HT22 hippocampal cells from TNF-α-induced apoptosis through a mechanism involving, at least in part, the potentiation of the pro-survival pathways activated by the cytokine.

The aim of the study was to investigate the interaction between cannabinoid CBHT22 hippocampal cells were treated with different cannabinoid and LPA receptor agonists and antagonists. Western blot and immunofluorescence microscopy were used to study intracellular signaling and the expression of apoptotic markers. Cell viability was determined by a luminescence assay.Cannabinoid agonists induced activation of both ERK1/2 and p38 MAP kinases. The effects of the CBIn HT22 hippocampal cells CB

Aims The aim of the study was to investigate the interaction between cannabinoid CB1/CB2 and lysophosphatidic acid (LPA) receptors in controlling neuronal signaling and fate. Methods HT22 hippocampal cells were treated with different cannabinoid and LPA receptor agonists and antagonists. Western blot and immunofluorescence microscopy were used to study intracellular signaling and the expression of apoptotic markers. Cell viability was determined by a luminescence assay. Key findings Cannabinoid agonists induced activation of both ERK1/2 and p38 MAP kinases. The effects of the CB1/CB2 receptor agonist HU210 were antagonized by the CB1 antagonist rimonabant, whereas the responses to the CB2 agonist JWH133 were blocked by the CB2 antagonist SR144528. HU210 reduced the apoptotic cell death induced by the pro-inflammatory cytokine TNF-α, whereas JWH133 enhanced the cytokine cytotoxicity. Blockade of ERK1/2 and p38 MAPK activation abrogated the HU210 pro-survival and the JWH133 pro-apoptotic effects, respectively. HU210 and the endocannabinoid anandamide, but not JWH133, potentiated ERK1/2 stimulation by LPA and the tricyclic antidepressant amitriptyline acting through the LPA1 receptor. HU210 enhanced amitriptyline-stimulated CREB phosphorylation and protection against TNF-α-induced apoptosis, whereas JWH133 had no effect. ERK1/2 stimulation by either HU210 or amitriptyline was dependent on fibroblast growth factor receptor (FGF-R) kinase activity and the combination of the two stimulants induced FGF-R phosphorylation. Moreover, the CB1 receptor was found to co-immunoprecipitate with the LPA1 receptor. Conclusions In HT22 hippocampal cells CB1 and CB2 receptors differentially regulate TNF-α-induced apoptosis and CB1 receptors positively interact with amitriptyline-stimulated LPA1 in promoting FGF-R-mediated ERK1/2 signaling and neuroprotection.

Both lysophosphatidic acid (LPA) and antidepressants have been shown to affect neuronal survival and differentiation, but whether LPA signalling participates in the action of antidepressants is still unknown. In this study, we examined the role of LPA receptors in the regulation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activity and neuronal survival by the tetracyclic antidepressants, mianserin and mirtazapine in hippocampal neurons. In HT22 immortalized hippocampal cells, antidepressants and LPA induced a time- and concentration-dependent stimulation of ERK1/2 phosphorylation. This response was inhibited by either LPA1 and LPA1/3 selective antagonists or siRNA-induced LPA1 down-regulation, and enhanced by LPA1 over-expression. Conversely, the selective LPA2 antagonist H2L5186303 had no effect. Antidepressants induced cyclic AMP response element binding protein phosphorylation and this response was prevented by LPA1 blockade. ERK1/2 stimulation involved pertussis toxin-sensitive G proteins, Src tyrosine kinases and fibroblast growth factor receptor (FGF-R) activity. Tyrosine phosphorylation of FGF-R was enhanced by antidepressants through LPA1 . Serum withdrawal induced apoptotic death, as indicated by increased annexin V staining, caspase activation and cleavage of poly-ADP-ribose polymerase. Antidepressants inhibited the apoptotic cascade and this protective effect was curtailed by blockade of either LPA1 , ERK1/2 or FGF-R activity. Moreover, in primary mouse hippocampal neurons, mianserin acting through LPA1 increased phospho-ERK1/2 and protected from apoptosis induced by removal of growth supplement. These data indicate that in neurons endogenously expressed LPA1 receptors mediate intracellular signalling and neuroprotection by tetracyclic antidepressants.

The present study shows that the GABAB positive allosteric modulators (PAMs) CGP7930 and GS39783 stimulate extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signalling in cells that do not express functional GABAB receptors. In human SH-SY5Y neuroblastoma cells, CGP7930 and GS39783 induced a time- and concentration-dependent increase in ERK1/2 phosphorylation with potencies similar to those displayed as GABAB PAMs. Conversely, γ-aminobutyric acid and the GABAB receptor agonists (-)baclofen and SKF97541 were completely inactive. CGP7930 and GS39783 enhanced the nuclear localization of phospho-ERK1/2 and CGP7930 promoted the phosphorylation of the transcription factors Elk-1 and CREB. CGP7930-induced ERK1/2 stimulation was insensitive to pertussis toxin, the Gq/11 antagonist YM254890 and the phospholipase C-β inhibitor U-73122, but was completely blocked by the MEK1/2 inhibitor PD98059. Inhibition of insulin-like growth factor-1, platelet--derived growth factor, phosphoinositide 3-kinase and Akt activities potentiated CGP7930-induced ERK1/2 phosphorylation. CGP7930 enhanced the phosphorylation of myristoylated alanine-rich protein kinase C (PKC) substrate and inhibition of PKC attenuated the ERK1/2 stimulation. Over-expression of N17Ras, a dominant negative mutant of c-Ras, or inhibition of c-Raf by GW5074 partially antagonized CGP7930-induced ERK1/2 activation. CGP7930 enhanced the phosphorylation of transforming growth factor-β-activated kinase 1 (TAK-1) and TAK-1 inhibition by 5Z-7-oxozeaenol reduced CGP7930-induced ERK1/2 phosphorylation. CGP7930 activated ERK1/2 in CHO-K1 fibroblasts, which lack endogenous GABAB receptors, but not in HEK-293 cells, indicating that the response displayed cell type specificity. These data demonstrate that CGP7930 and GS39783 can trigger ERK1/2 signalling, a critical modulator of mood and drug addiction, independently of an action on GABAB receptors.

Preclinical 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 LPA

Valproic acid (VPA) has been shown to regulate the levels of brain-derived neurotrophic factor (BDNF), but it is not known whether this drug can affect the neuronal responses to BDNF. In the present study, we show that in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells, prolonged exposure to VPA reduces the expression of the BDNF receptor TrkB at the protein and mRNA levels and inhibits the intracellular signaling, neurotrophic activity, and prosurvival function of BDNF. VPA downregulates TrkB and curtails BDNF-induced signaling also in differentiated Kelly and LAN-1 neuroblastoma cells and primary mouse cortical neurons. The VPA effect is mimicked by several histone deacetylase (HDAC) inhibitors, including the class I HDAC inhibitors entinostat and romidepsin. Conversely, the class II HDAC inhibitor MC1568, the HDAC6 inhibitor tubacin, the HDAC8 inhibitor PCI-34051, and the VPA derivative valpromide have no effect. In neuroblastoma cells and primary neurons both VPA and entinostat increase the cellular levels of the transcription factor RUNX3, which negatively regulates TrkB gene expression. Treatment with RUNX3 siRNA attenuates VPA-induced RUNX3 elevation and TrkB downregulation. VPA, entinostat, HDAC1 depletion by siRNA, and 3-deazaneplanocin A (DZNep), an inhibitor of the polycomb repressor complex 2 (PRC2), decrease the PRC2 core component EZH2, a RUNX3 suppressor. Like VPA, HDAC1 depletion and DZNep increase RUNX3 and decrease TrkB expression. These results indicate that VPA downregulates TrkB through epigenetic mechanisms involving the EZH2/RUNX3 axis and provide evidence that this effect implicates relevant consequences with regard to BDNF efficacy in stimulating intracellular signaling and functional responses. SIGNIFICANCE STATEMENT: The tropomyosin-related kinase receptor B (TrkB) mediates the stimulatory effects of brain-derived neurotrophic factor (BDNF) on neuronal growth, differentiation, and survival and is highly expressed in aggressive neuroblastoma and other tumors. Here we show that exposure to valproic acid (VPA) downregulates TrkB expression and functional activity in retinoic acid-differentiated human neuroblastoma cell lines and primary mouse cortical neurons. The effects of VPA are mimicked by other histone deacetylase (HDAC) inhibitors and HDAC1 knockdown and appear to be mediated by an epigenetic mechanism involving the upregulation of RUNX3, a suppressor of TrkB gene expression. TrkB downregulation may have relevance for the use of VPA as a potential therapeutic agent in neuroblastoma and other pathologies characterized by an excessive BDNF/TrkB signaling.

Background and purpose Although clinically useful for their immunomodulatory, antiproliferative and antiviral properties, type I interferons (IFNs) are involved in the pathogenesis of several neurodegenerative/neuroinflammatory diseases. In the present study, we investigated the ability of cholinergic stimulation to protect from IFN-β-induced neuronal apoptosis. Experimental approach The effects of the cholinergic agonist carbachol (CCh) on IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells were examined by using Western blot, immunofluorescence and cytofluorimetry. The involvement of muscarinic acetylcholine receptors (mAChRs) was assessed by using selective antagonists and siRNA transfection. Pharmacological inhibitors and over-expression of ERK2 and an ERK2 constitutively active form (ERK2-CA) were employed to study ERK1/2 signalling. The effects of oxotremorine-M (Oxo-M) on IFN-β-induced apoptosis of mouse hippocampal neurons were examined by measuring cleaved caspase 3 expression. Key results In SH-SY5Y cells, CCh inhibited IFN-β-induced mitochondrial cytochrome c release, activation of caspases 9, 7 and 3, PARP cleavage and DNA fragmentation. The anti-apoptotic effect of CCh was mediated by M3 mAChRs, blocked by the Gq/11 antagonist YM254890 and the protein kinase C inhibitor Go 6983, impaired by inhibition of ERK1/2 pathway, potentiated by over-expression of ERK2, and mimicked by ERK2-CA. Blockade of JNK activation enhanced the CCh anti-apoptotic response. IFN-β inhibited JNK activation and up-regulated CCh-induced ERK1/2 signalling. In hippocampal neurons, IFN-β-induced apoptosis was reduced by Oxo-M and the effect was antagonized by blockade of M1/M3 mAChRs and ERK1/2. Conclusions and implications Stimulation of mAChRs counteracted IFN-β-induced neuronal apoptosis through the activation of ERK1/2 signalling. The data indicate that activation of ERK1/2-coupled mAChRs may be an effective strategy for preventing IFNs neurotoxicity. This article is protected by copyright. All rights reserved.

Neurotoxicity of human immunodeficiency virus-1 (HIV) includes synaptic simplification and neuronal apoptosis. However, the mechanisms of HIV-associated neurotoxicity remain unclear, thus precluding an effective treatment of the neurological complications. The present study was undertaken to characterize novel mechanisms of HIV neurotoxicity that may explain how HIV subjects develop neuronal degeneration. Several neurodegenerative disorders are characterized by mitochondrial dysfunction; therefore, we hypothesized that HIV promotes mitochondrial damage. We first analyzed brains from HIV encephalitis (HIVE) by electron microscopy. Several sections of HIVE subjects contained enlarged and damaged mitochondria compared to brains from HIV subjects with no neurological complications. Similar pathologies were observed in mice overexpressing the HIV protein gp120, suggesting that this viral protein may be responsible for mitochondrial pathology found in HIVE. To gain more information about the cellular mechanisms of gp120 neurotoxicity, we exposed rat cortical neurons to gp120 and we determined cellular oxygen consumption rate, mitochondrial distribution, and trafficking. Our data show that gp120 evokes impairment in mitochondrial function and distribution. These data suggest that one of the mechanisms of HIV neurotoxicity includes altered mitochondrial dynamics in neurons.

Although clinically useful for the treatment of various diseases, type I interferons (IFNs) have been implicated as causative factors of a number of neuroinflammatory disorders characterized by neuronal damage and altered CNS functions. As neurotrophin 3 (NT3) plays a critical role in neuroprotection, we examined the effects of IFN-β on the signalling and functional activity of the NT3/TrkC system. We found that prolonged exposure of differentiated human SH-SY5Y neuroblastoma cells to IFN-β impaired the ability of NT3 to induce transphosphorylation of the full-length TrkC receptor (TrkC-FL) and the phosphorylation of downstream signalling molecules, including PLCγ1, Akt, GSK-3β and ERK1/2. NT3 was effective in protecting the cells against apoptosis triggered by serum withdrawal or thapsigargin but not IFN-β. Prolonged exposure to the cytokine had little effects on TrkC-FL levels but markedly enhanced the messenger RNA (mRNA) and protein levels of the truncated isoform TrkC-T1, a dominant-negative receptor that inhibits TrkC-FL activity. Cell depletion of TrkC-T1 by small interfering RNA (siRNA) treatment enhanced NT3 signalling through TrkC-FL and allowed the neurotrophin to counteract IFN-β-induced apoptosis. Furthermore, the upregulation of TrkC-T1 by IFN-β was associated with the inhibition of NT3-induced recruitment of the scaffold protein tamalin to TrkC-T1 and tamalin tyrosine phosphorylation. These data indicate that IFN-β exerts a negative control on NT3 pro-survival signalling through a novel mechanism involving the upregulation of TrkC-T1.

Preclinical 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.

Different lines of evidence indicate that the lysophosphatidic acid (LPA) receptor LPA1 is involved in neurogenesis, synaptic plasticity and anxiety-related behavior, but little is known on whether this receptor can be targeted by neuropsychopharmacological agents. The present study investigated the effects of different antidepressants on LPA1 signaling. We found that in Chinese hamster ovary (CHO)-K1 fibroblasts expressing endogenous LPA1 tricyclic and tetracyclic antidepressants and fluoxetine induced the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and CREB. This response was antagonized by either LPA1 blockade with Ki16425 and AM966 or knocking down LPA1 with siRNA. Antidepressants induced ERK1/2 phosphorylation in human embryonic kidney (HEK)-293 cells overexpressing LPA1, but not in wild-type cells. In PathHunter™ assay measuring receptor-β-arrestin interaction, amitriptyline, mianserin and fluoxetine failed to induce activation of LPA2 and LPA3 stably expressed in CHO-K1 cells. ERK1/2 stimulation by antidepressants and LPA was suppressed by pertussis toxin and inhibition of Src, phosphatidylinositol-3 kinase and insulin-like growth factor-I receptor (IGF-IR) activities. Antidepressants and LPA induced tyrosine phosphorylation of IGF-IR and insulin receptor-substrate-1 through LPA1 and Src. Prolonged exposure of CHO-K1 fibroblasts to either mianserin, mirtazapine or LPA enhanced cell proliferation as indicated by increased [3H]-thymidine incorporation and Ki-67 immunofluorescence. This effect was inhibited by blockade of LPA1- and ERK1/2 activity. These data provide evidence that different antidepressants induce LPA1 activation, leading to receptor tyrosine kinase transactivation, stimulation of ERK1/2 signaling and enhanced cell proliferation.

The chemokine CCL5 prevents neuronal cell death mediated both by amyloid β, as well as the human immunodeficiency virus viral proteins gp120 and Tat. Because CCL5 binds to CCR5, CCR3 and/or CCR1 receptors, it remains unclear which of these receptors plays a role in neuroprotection. Indeed, CCL5 also has neuroprotective activity in cells lacking these receptors. CCL5 may bind to a G-protein-coupled receptor 75 (GPR75), which encodes for a 540 amino-acid orphan receptor of the Gqα family. In this study, we have used SH-SY5Y human neuroblastoma cells to characterize whether CCL5 could activate a Gq signaling through GPR75. Both qPCR and flow cytometry show that these cells express GPR75 but do not express CCR5, CCR3 or CCR1 receptors. SY-SY5Y cells were then used to examine CCL5-mediated signaling. We report that CCL5 promotes a time- and concentration-dependent phosphorylation of protein kinase B (AKT), glycogen synthase kinase 3β, and extracellular signal-regulated kinase (ERK) 1/2. Specific antagonists of CCR5, CCR3, and CCR1 did not prevent CCL5 from increasing phosphorylated AKT or ERK. Moreover, CCL5 promotes a time-dependent internalization of GPR75. Lastly, knocking down GPR75 expression by a CRISPR-Cas9 approach inhibited the ability of CCL5 to activate pERK in SH-SY5Y cells. Therefore, we propose that GPR75 is a novel receptor for CCL5 that could explain some of the pharmacological action of this chemokine. These findings may help in the development of small molecule GPR75 agonists that mimic CCL5. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of the cellular energy metabolism and may induce either cell survival or death. We previously reported that in SH-SY5Y human neuroblastoma cells stimulation of muscarinic acetylcholine receptors (mAChRs) activate AMPK by triggering store-operated Ca

Both lysophosphatidic acid (LPA) and antidepressants have been shown to affect neuronal survival and differentiation, but whether LPA signalling participates in the action of antidepressants is still unknown. In this study, we examined the role of LPA receptors in the regulation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activity and neuronal survival by the tetracyclic antidepressants, mianserin and mirtazapine in hippocampal neurons. In HT22 immortalized hippocampal cells, antidepressants and LPA induced a time- and concentration-dependent stimulation of ERK1/2 phosphorylation. This response was inhibited by either LPA

Gq/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M1, M3 and M5 subtypes have been shown to activate the metabolic sensor AMP-activated protein kinase (AMPK) through Ca(2+)/calmodulin-dependent protein kinase kinase-β (CaMKKβ)-mediated phosphorylation at Thr172. However, the source of Ca(2+) required for this response has not been yet elucidated. Here, we investigated the involvement of store-operated Ca(2+) entry (SOCE) in AMPK activation by pharmacologically defined M3 mAChRs in human SH-SY5Y neuroblastoma cells. In Ca(2+)-free medium the cholinergic agonist carbachol (CCh) caused a transient increase of phospho-Thr172 AMPK that rapidly ceased within 2min. Conversely, in the presence of extracellular Ca(2+) CCh-induced AMPK phosphorylation lasted for at least 180min. The SOCE modulator 2-aminoethoxydiphephenyl borate (2-APB), at a concentration (50μM) that suppressed CCh-induced intracellular Ca(2+) ([Ca(2+)]i) plateau, inhibited CCh-induced AMPK phosphorylation. CCh triggered the activation of the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1, as indicated by redistribution of STIM1 immunofluorescence into puncta, and promoted the association of STIM1 with the SOCE channel component Orai1. Cell depletion of STIM1 by siRNA treatment reduced both CCh-induced [Ca(2+)]i plateau and AMPK activation. M3 mAChRs increased glucose uptake and this response required extracellular Ca(2+) and was inhibited by 2-APB, STIM1 knockdown, CaMKKβ and AMPK inhibitors, and adenovirus infection with dominant negative AMPK. Thus, the study provides evidence that SOCE is required for sustained activation of AMPK and stimulation of downstream glucose uptake by M3 mAChRs and suggests that SOCE is a critical process connecting M3 mAChRs to the control of neuronal energy metabolism.

Type I interferons (IFNs) induce apoptosis of neuroblastoma cells, but the molecular mechanisms regulating this event have not been completely elucidated. Here, we investigated the role of p38 mitogen activated protein kinase (MAPK) activity, a key regulator of apoptosis and a known modulator of IFN-induced responses in non-neuronal cells. We show that in SH-SY5Y human neuroblastoma cells IFN-β induced a delayed and sustained increase of p38 MAPK activity through a novel mechanism involving the sequential activation of Janus kinase-signal transducer and activator of transcription-1 signalling, enhanced expression of the NADPH oxidase catalytic subunit gp91(phox), increased reactive oxygen species production and stimulation of the MAPK kinase kinase transforming growth factor-β-activated kinase 1. Either blockade of p38 MAPK by the second generation inhibitors BIRB0796 and VX745 or siRNA knockdown of p38α MAPK enhanced IFN-β-induced apoptosis of neuroblastoma cells. Exposure to IFN-β increased the phosphorylation of the small heat shock protein HSP27 at Ser15, Ser78 and Ser82 with a time course similar to p38 MAPK activation and this response was suppressed by either p38α MAPK depletion or pharmacological inhibition of p38 MAPK and MAPK-activated protein kinase 2 (MK2). Either silencing of HSP27 expression by siRNA or MK2 inhibition potentiated IFN-β-induced apoptotic death. These results indicate that IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells is associated with a long-lasting up-regulation of p38 MAPK activity, stimulation of MK2 and phosphorylation of the pro-survival protein HSP27. Moreover, the data show that inhibition of p38 MAPK signalling potentiates the anti-neuroblastoma activity of the cytokine, indicating that this pathway mediates a counter-regulatory response.

The present study shows that the GABA

Antidepressants 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.

Opioid receptors located in the ventral tegmental area are known to regulate dopamine (DA) release from mesocortical afferents to medial prefrontal cortex (mPFC) but little is known on whether in this cortical region activation of opioid receptors affect DA receptor signaling. In the present study we show that in mouse mPFC concomitant activation of either δ- or μ-opioid receptors, but not κ-opioid receptors, potentiated DA D1-like receptor-induced stimulation of adenylyl cyclase activity through a G protein βγ subunit-dependent mechanism. In tissue slices of mPFC, the combined addition of the opioid agonist leu-enkephalin and the DA D1-like receptor agonist SKF 81297 produced more than additive increase in the phosphorylation state of AMPA and NMDA receptor subunits GluR1 and NR1, respectively. Moreover, in primary cultures of mouse frontal cortex neurons, DA D1-like receptor-induced Ser133 phosphorylation of the transcription factor cyclic AMP responsive element binding protein was potentiated by concurrent stimulation of opioid receptors. Double immunofluorescence analysis of cultured cortical cells indicated that a large percentage of DA D1 receptor positive cells expressed either δ- or μ-opioid receptor immunoreactivity. These data indicate that in mouse mPFC activation of μ- and δ-opioid receptors enhances DA D1-like receptor signaling likely through converging regulatory inputs on βγ-stimulated adenylyl cyclase isoforms. This previously unrecognized synergistic interaction may selectively affect DA D1 transmission at specific postsynaptic sites where the receptors are co-localized and may play a role in prefrontal DA D1 regulation of opioid addiction.

BACKGROUND AND PURPOSE Antidepressants are known to interact with the opioid system through mechanisms not completely understood. We previously reported that tricyclic antidepressants act as agonists at distinct opioid receptors. Here, we investigated the effect of the atypical antidepressant mianserin at cloned and native opioid receptors. EXPERIMENTAL APPROACH Effects of mianserin were examined in CHO cells transfected with human opioid receptors, C6 glioma cells and rat brain membranes by the use of radioligand binding and functional assays including the stimulation of [35S]GTPγS binding and MAPK phosphorylation. KEY RESULTS Mianserin displayed 12- and 18-fold higher affinity for κ- than µ- and δ-opioid receptors respectively. In [35S]GTPγS assays, mianserin selectively activated κ-opioid receptors. The agonist activity was antagonized by the selective κ-opioid blocker nor-binaltorphimine (nor-BNI). The mianserin analogue mirtazapine also displayed κ-opioid agonist activity. Mianserin and mirtazapine increased ERK1/2 phosphorylation in CHO cells expressing κ-opioid receptors and C6 cells, and these effects were antagonized by nor-BNI. In rat striatum and nucleus accumbens, mianserin stimulated [35S]GTPγS binding in a nor-BNI-sensitive manner with maximal effects lower than those of the full κ-opioid agonists (–)-U50,488 and dynorphin A. When combined, mianserin antagonized the effects of the full κ-opioid receptor agonists in [35S]GTPγS assays and reduced the stimulation of p38 MAPK and ERK1/2 phosphorylation by dynorphin A. CONCLUSIONS AND IMPLICATIONS In different cell systems, mianserin directly activates κ-opioid receptors, displaying partial agonist activity at brain receptors. Thus, this property appears to be a common feature of different classes of antidepressants.

Type I interferons (IFNs) have been shown to act on neurons and to cause neuronal damage through mechanisms not completely defined. Here, we investigated the effects of type I IFNs on brain-derived neurotrophic factor (BDNF)-induced TrkB receptor signaling and neurotrophic activity. In retinoic acid-treated human SH-SY5Y neuroblastoma cells and mouse primary cortical neurons, long-term exposure to IFNs curtailed BDNF-induced activation of phosphatidylinositol 3-kinase, phospholipase Cγ and extracellular-regulated kinases 1 and 2 signaling. Moreover, IFN-β inhibited BDNF-induced cell survival, neurite outgrowth, and expression of neuronal markers, such as neurofilament proteins, growth-associated protein-43 and glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluR1. The IFN inhibitory effects were associated with down-regulation of TrkB and inhibition of TrkB autophosphorylation. In SH-SY5Y cells, blockade of either Janus kinase with pyridone 6 or signal transducer and activator of transcription (STAT) 1 with siRNA transfection attenuated IFN-β-induced TrkB down-regulation. Quantitative real time RT-PCR indicated that IFN-β significantly reduced TrkB mRNA levels. Moreover, blockade of protein kinase R counteracted IFN-β-induced inhibition of TrkB expression and signaling. These data indicate that in neuronal cells IFNs negatively regulate BDNF signaling and neurotrophic activity through inhibition of TrkB activation and Janus kinase/Signal transducer and activator of transcription-dependent down-regulation of TrkB.

BACKGROUND AND PURPOSE Although opioids have been reported to affect glucose homeostasis, relatively little is known on the role of δ-opioid receptors. We have investigated the regulation of glucose transport by human δ-opioid receptors expressed in Chinese hamster ovary cells. EXPERIMENTAL APPROACH The uptake of [3H]-2-deoxy-D-glucose and 3-O-[methyl-[3H]]-D-glucose in response to δ-opioid receptor ligands and the expression of GLUT1, GLUT3 and GLUT4 glucose transporters were examined. Moreover, the effects of intracellular signal transduction inhibitors on δ-opioid receptor-regulated [3H]-2-deoxy-D-glucose uptake and protein phosphorylation were investigated. KEY RESULTS Activation of δ-opioid receptors rapidly stimulated [3H]-2-deoxy-D-glucose and 3-O-[methyl-[3H]]-D-glucose uptakes, which were blocked by the GLUT inhibitors cytochalasin B and phloretin. The stimulation of [3H]-2-deoxy-D-glucose uptake that occurred without a change in plasma membrane GLUT1 – required the coupling to Gi/Go proteins – was independent of cAMP and extracellular signal-regulated protein kinases, and was suppressed by blockade of Src and insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinases. Inhibition of phosphatidylinositol 3-kinase (PI3K) by wortmannin or LY294002 and by PI3Kα, but not γ, isoform-selective inhibitors greatly reduced the δ-opioid receptor stimulation of glucose uptake. Moreover, the response was attenuated by overexpressing a dominant-negative kinase-deficient Akt form and by chemical inhibition of Akt. Stimulation of δ-opioid receptors increased protein kinase Cζ/λ (PKCζ/λ) phosphorylation and a selective PKCζ/λ inhibitor slightly reduced opioid stimulation of glucose uptake. CONCLUSIONS AND IMPLICATIONS δ-Opioid receptors stimulated glucose transport probably by enhancing GLUT1 intrinsic activity through a signalling cascade involving Gi/Go, Src, IGF-1R, PI3Kα, Akt and, to a minor extent, PKCζ/λ. This effect may contribute to the opioid regulation of glucose homeostasis in physio-pathological conditions.

Tricyclic 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.

The clozapine metabolite N-desmethylclozapine (NDMC) has been recently shown to act at different neurotransmitter receptors and to display both antagonist and agonist activities. We have previously reported that in cells over-expressing the recombinant delta-opioid receptor NDMC behaved as partial agonist with high intrinsic activity, but its action at the receptors naturally expressed in human brain remained to be investigated. In the present study, we examined whether NDMC was able to bind to and activate delta-opioid receptors in membranes of post-mortem human frontal cortex. In radioligand binding assays, NDMC competition curves displayed high- (K(i)=26 nM) and low-affinity (K(i)=3 microM) components, whose proportion was regulated by guanine nucleotides in an agonist-like fashion. In functional assays, NDMC stimulated [(35)S]GTPgammaS binding (EC(50)=905 nM) and inhibited cyclic AMP formation (EC(50)=590 nM) as effectively as delta-opioid agonists, whereas clozapine was much less potent and efficacious and clozapine N-oxide was completely inactive. The NDMC agonist activity was potently antagonized by the delta-opioid antagonist naltrindole, but not by the micro-opioid receptor antagonist CTAP (D-phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)) or the kappa-opioid antagonist nor-binaltorphimine. Moreover, blockade of either acetylcholine muscarinic, dopamine D(2) or serotonin 5HT(1A) receptors failed to affect NDMC agonist activity. These data demonstrate that at clinically relevant concentrations NDMC behaves as an efficacious agonist at delta-opioid receptors of human frontal cortex.

Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit dopamine (DA) release in basal ganglia mainly by acting on NOP receptors in substantia nigra and ventral tegmental area. We investigated whether N/OFQ could affect DA transmission by acting at either DA nerve endings or DA-targeted post-synaptic neurons. In synaptosomes of rat nucleus accumbens and striatum N/OFQ inhibited DA synthesis and tyrosine hydroxylase (TH) phosphorylation at Ser40 via NOP receptors coupled to inhibition of the cAMP/protein kinase A pathway. Immunofluorescence studies showed that N/OFQ preferentially inhibited phospho-Ser40-TH in nucleus accumbens shell and that in this subregion NOP receptors partly colocalized with either TH or DA D1 receptor positive structures. In accumbens and striatum N/OFQ inhibited DA D1 receptor-stimulated cAMP formation, but failed to affect either adenosine A2A or DA D2 receptor regulation of cAMP. In accumbens slices, N/OFQ inhibited DA D1-induced phosphorylation of NMDA and α-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate receptors, whereas in primary cultures of accumbal cells, which were found to coexpress NOP and DA D1 receptors, N/OFQ curtailed DA D1 receptor-induced cAMP-response element-binding protein phosphorylation. Thus, in accumbens and striatum N/OFQ exerts an inhibitory constraint on DA transmission by acting on either pre-synaptic NOP receptors inhibiting TH phosphorylation and DA synthesis or post-synaptic NOP receptors selectively down-regulating DA D1 receptor signaling.

Proteinase-activated receptors (PARs) are a family of four G protein-coupled receptors that are widely distributed in the CNS and involved in neural cell proliferation, differentiation and survival. The olfactory system undergoes continuous neurogenesis throughout life and may represent a critical target of PAR cellular actions. In the present study we investigated the functional activity of PAR1 and PAR2 in microdissected tissue preparations of olfactory nerve-glomerular layer (ON-GL), external plexiform layer (EPL) and granule cell layer (GRL) of the rat main olfactory bulb and in primary cultures of olfactory neuroepithelial cells. Activation of either PAR1 or PAR2 regulated multiple signaling pathways, including activation of pertussis-toxin sensitive Gi/o proteins, inhibition of cyclic AMP formation, stimulation of Gq/11-mediated phosphoinositide (PI) hydrolysis, phosphorylation of Ca2+/calmodulin-dependent protein kinase II and activation of the monomeric G protein Rho, predominantly in ON-GL, whereas only activation of Rho was detected in the deeper layers. Olfactory nerve lesion by nasal irrigation with ZnSO4 induced a marked decrease of PAR signaling in ON-GL. In primary cultures of olfactory neurons, double immunofluorescence analysis showed the localization of PAR1 and PAR2 in cells positive for olfactory-marker protein and neuron-specific enolase. Cell exposure to either nanomolar concentrations of thrombin and trypsin or PAR-activating peptides caused rapid neurite retraction. This study provides the first characterization of the laminar distribution of PAR1 and PAR2 signaling in rat olfactory bulb, demonstrates the presence of the receptors in olfactory sensory neurons and suggests a role of PARs in olfactory sensory neuron neuritogenesis.

Both type I interferons (IFNs) and neurotrophins regulate neuroadaptive responses, but relatively little is known on the interaction between these two classes of regulatory proteins. Here we investigated the effect of IFN-β on the expression and functional activity of the common neurotrophin receptor p75NTR and the nerve growth factor (NGF) receptor TrkA. In differentiated human SH-SY5Y neuroblastoma cells prolonged exposure to IFN-β up-regulated p75NTR and TrkA levels, failed to affect the content of sortilin, a p75NTR co-receptor, and, consistent with our previous finding, down-regulated the brain-derived neurotrophic factor receptor TrkB. Quantitative real time RT-PCR indicated that IFN-β increased p75NTR and TrkA mRNA levels. In control and IFN-β treated cells proNGF failed to induce c-Jun N-terminal kinase and nuclear factor/kB activation, two p75NTR/sortilin signalling pathways mediating neuronal death. On the other hand, IFN-β treatment enhanced TrkA autophosphorylation and signalling induced by NGF and proNGF. Knockdown of p75NTR by siRNA reduced TrkA activation by proNGF and a subnanomolar concentration of NGF, whereas co-immunoprecipitation indicated close association of p75NTR and TrkA. Co-treatment with either NGF or proNGF reduced IFN-β pro-apoptotic and anti-neurotrophic effects. Similarly, in primary mouse hippocampal neurons IFN-β increased p75NTR and TrkA expression, down-regulated TrkB and enhanced NGF-induced phosphorylation of the pro-survival protein kinase Akt. The data demonstrate that in neuronal cells IFN-β differentially affects the expression and signalling of neurotrophin receptors and suggest that the up-regulation of the p75NTR/TrkA signalling complex may constitute a novel mechanism by which this cytokine selectively attenuates its pro-apoptotic effect in NGF-responsive cells.

Human glucagon-like peptide-1 (GLP-1) is a physiological gastrointestinal peptide with glucose-dependent insulinotropic effects which is therefore considered an interesting antidiabetic agent. However, after in vivo administration, exogenous GLP-1 does not exert its physiological action due to the combination of rapid proteolytic degradation by ubiquitous dipeptidyldipeptidase IV (DPP IV) enzyme and renal clearance resulting in an extremely short circulating half-life. In this work we describe the conjugation of GLP-1-(7-36)-amide derivatives with polyethylene glycol (PEG) by enzymatic site-specific transglutamination reaction as an approach to reduce both the proteolysis and the renal clearance rates. The compound GLP-1-(7-36)-amide-Q23-PEG 20 kDa monopegylated on the single glutamine residue naturally present in position 23 maintained the ability to activate the GLP-1 receptor expressed in the rat β-cell line RIN-m5F with nanomolar potency along with an increased in vitro resistance to DDP IV and a circulating half-life of about 12 h after subcutaneous administration in rats. These properties enabled GLP-(7-36)-amide-Q23-PEG 20 kDa to exert a glucose-stabilizing effect for a period as long as 8 h, as demonstrated by a single subcutaneous injection to diabetic mice concomitantly challenged with an oral glucose load. The results reported in this work indicate that GLP-(7-36)-amide-Q23-PEG 20 kDa could be a lead compound for the development of long-lasting anti-diabetic agents useful in the treatment of type 2 diabetes affected patients.

Type I interferons (IFNs) have been shown to act on neurons and to cause neuronal damage through mechanisms not completely defined. Here, we investigated the effects of type I IFNs on brain-derived neurotrophic factor (BDNF)-induced TrkB receptor signaling and neurotrophic activity. In retinoic acid-treated human SH-SY5Y neuroblastoma cells and mouse primary cortical neurons, long-term exposure to IFNs curtailed BDNF-induced activation of phosphatidylinositol 3-kinase, phospholipase Cγ and extracellular-regulated kinases 1 and 2 signaling. Moreover, IFN-β inhibited BDNF-induced cell survival, neurite outgrowth, and expression of neuronal markers, such as neurofilament proteins, growth-associated protein-43 and glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluR1. The IFN inhibitory effects were associated with down-regulation of TrkB and inhibition of TrkB autophosphorylation. In SH-SY5Y cells, blockade of either Janus kinase with pyridone 6 or signal transducer and activator of transcription (STAT) 1 with siRNA transfection attenuated IFN-β-induced TrkB down-regulation. Quantitative real time RT-PCR indicated that IFN-β significantly reduced TrkB mRNA levels. Moreover, blockade of protein kinase R counteracted IFN-β-induced inhibition of TrkB expression and signaling. These data indicate that in neuronal cells IFNs negatively regulate BDNF signaling and neurotrophic activity through inhibition of TrkB activation and Janus kinase/Signal transducer and activator of transcription-dependent down-regulation of TrkB.

Type I interferons (IFNs) are known to cause neuropsychiatric side effects, which have been proposed to be mediated by either peripheral actions or activation of glial cells. In the present study, we have investigated whether these cytokines could act directly on neuronal cells and regulate signaling pathways involved in cell death. In human SH-SY5Y neuroblastoma cells, type I IFNs rapidly stimulated tyrosine phosphorylation of Janus kinase and signal transducer and activator of transcription (STAT) through type I IFN receptor. Prolonged exposure to IFN-β induced apoptotic cell death accompanied by cytochrome C release, cleavage of caspases 9, 7, 3 and poly-(ADP ribose) polymerase and DNA fragmentation. Janus kinase inhibition reduced IFN-β-stimulated TyK2 and STAT1 phosphorylation, STAT1 transcriptional activity, induction of double-stranded RNA-activated protein kinase (PKR) and caspase cleavage. PKR induction was associated with enhanced PKR activity and chemical inhibition of PKR reduced IFN-stimulated caspase activation. Moreover, long-term IFN-β treatment led to down-regulation of phosphatidylinositol 3-kinase/Akt signaling and IFN-β-induced apoptosis was attenuated in cells expressing constitutively active Akt. Similarly, in mouse primary neurons IFN-β induced STAT phosphorylation, caspase 3 cleavage and inhibition of Akt signaling. Thus, type I IFNs can directly impair neuronal survival by regulating multiple signaling molecules promoting the intrinsic apoptotic pathway. This effect may contribute to the cytokine neurotoxicity.

Besides being involved in analgesia, δ-opioid receptors have recently been shown to exert antidepressant-like and neuroprotective effects. Glycogen synthase kinase-3β (GSK-3β), a key enzyme involved in cellular apoptosis and in mood disorders, may constitute a molecular target of δ-opioid receptors. However, relatively little is known on how δ-opioid receptors affect the multiple signaling pathways regulating GSK-3β. In the present study, we show that activation of human δ-opioid receptors stably expressed in Chinese hamster ovary (CHO) cells induced a rapid GSK-3β phosphorylation on Ser9 and a significant inhibition of the kinase activity. This effect was dependent on G proteins Gi/Go, unaffected by cell transfection with the Gβγ scavenger transducin, required the Src non-receptor tyrosine kinase and the specific involvement of the α isoform of phosphatidylinositol 3-kinase. δ-Opioid agonists activated the protein kinase Akt in a Src-dependent manner and chemical inhibition of Akt or stable expression of a dominant negative Akt1 mutant reduced the stimulation of GSK-3β phosphorylation. Moreover, δ-opioid receptor regulation of Akt and GSK-3β was dependent on transphosphorylation and transactivation of platelet-derived growth factor and insulin-like growth factor-1 receptor tyrosine kinases. AMP-activated protein kinase (AMPK) activity was also required, as δ-opioid effects on Akt and GSK-3β were mimicked by the AMPK activator A-769662 and reduced by the AMPK inhibitor Compound C. Conversely, inhibition of protein kinase C isoforms, extracellular signal-regulated protein kinases 1/2 and mammalian target of rapamycin was without effect, although the latter two kinases were activated by δ-opioid agonists. The results identify Src-dependent transactivation of receptor tyrosine kinases as a key process in δ-opioid receptor inhibitory control of GSK-3β and reveal a novel δ-opioid regulatory mechanism mediated by AMPK. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit dopamine (DA) release in basal ganglia mainly by acting on NOP receptors in substantia nigra and ventral tegmental area. We investigated whether N/OFQ could affect DA transmission by acting at either DA nerve endings or DA-targeted post-synaptic neurons. In synaptosomes of rat nucleus accumbens and striatum N/OFQ inhibited DA synthesis and tyrosine hydroxylase (TH) phosphorylation at Ser40 via NOP receptors coupled to inhibition of the cAMP/protein kinase A pathway. Immunofluorescence studies showed that N/OFQ preferentially inhibited phospho-Ser40-TH in nucleus accumbens shell and that in this subregion NOP receptors partly colocalized with either TH or DA D(1) receptor positive structures. In accumbens and striatum N/OFQ inhibited DA D(1) receptor-stimulated cAMP formation, but failed to affect either adenosine A(2A) or DA D(2) receptor regulation of cAMP. In accumbens slices, N/OFQ inhibited DA D(1)-induced phosphorylation of NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate receptors, whereas in primary cultures of accumbal cells, which were found to coexpress NOP and DA D(1) receptors, N/OFQ curtailed DA D(1) receptor-induced cAMP-response element-binding protein phosphorylation. Thus, in accumbens and striatum N/OFQ exerts an inhibitory constraint on DA transmission by acting on either pre-synaptic NOP receptors inhibiting TH phosphorylation and DA synthesis or post-synaptic NOP receptors selectively down-regulating DA D(1) receptor signaling.

Filgrastim or methionyl-granulocyte colony-stimulating factor (Met-G-CSF), is a recombinant therapeutic protein widely used to treat severe neutropenia caused by myelosuppressive drugs in patients with nonmyeloid malignancies. In addition to its role in the regulation of granulopoiesis, treatment with G-CSF is considered the standard approach to mobilize CD34 positive (CD34+) mononuclear cells for reconstituting hemopoietic ability for bone marrow transplantation. An intended biosimilar filgrastim (coded BK0023) was produced in GMP conditions by E.coli fermentation according to an original recombinant process and showed physico-chemical properties and purity profile similar to Neupogen®, a commercial preparation of filgrastim. The aim of the present study was to demonstrate the comparability of BK0023 to Neupogen® in terms of both in vitro biological activities and in vivo toxicology, pharmacokinetics and pharmacodynamics. Cell proliferation and radioligand binding assays were conducted in NFS-60 cells to compare the biological activity and functional interaction with the G-CSF receptor in vitro, while preclinical in vivo studies, including pharmacokinetics and pharmacodynamics after repeated dose were performed in normal and neutropenic rats. A phase I study was carried out in healthy male volunteers treated by multiple-dose subcutaneous administration of BK0023 and Neupogen® to evaluate their pharmacodynamic effects as well as their pharmacokinetic and safety profile and to demonstrate their pharmacodynamic equivalence and pharmacokinetic bioequivalence. The results reported in this work demonstrate that BK0023 is comparable in terms of biological activity, efficacy and safety to Neupogen®. BK0023 has the same pharmacokinetic profile, efficacy and safety as the reference commercial filgrastim Neupogen® and therefore could be further developed to become a convenient option to treat neutropenia in oncological patients. Trial registration number (TRN): NCT01933971 . Date of registration: Sept 6th 2013.