Simona Dedoni

Interferon‐β induces apoptosis in human SH‐SY5Y neuroblastoma cells through activation of JAK–STAT signaling and down‐regulation of PI3K/Akt pathway

J. Neurochem. (2010) 115, 1421–1433.

Direct Agonist Activity of Tricyclic Antidepressants at Distinct Opioid Receptor Subtypes

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 δ-opioid receptors (CHO/DOR), TCAs displaced [3H]naltrindole binding and stimulated guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) 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 δ-opioid receptors in rat dorsal striatum and nucleus accumbens and human frontal cortex. In CHO cells expressing κ-opioid receptors (CHO/KOR), TCAs, but not amoxapine, exhibited higher receptor affinity and more potent stimulation of [35S]GTPγS 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 κ-opioid receptors, and this effect was attenuated by the κ-opioid antagonist nor-binaltorphimine. In rat nucleus accumbens, amitriptyline slightly inhibited adenylyl cyclase activity and counteracted the inhibitory effect of the full κ agonist trans-(−)-3,4dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488). At the cloned μ-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 δ or κ subtypes and suggest that this property may contribute to their therapeutic and/or side effects.

Activation of nociceptin/orphanin FQ‐NOP receptor system inhibits tyrosine hydroxylase phosphorylation, dopamine synthesis, and dopamine D1 receptor signaling in rat nucleus accumbens and dorsal striatum

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 1 and 2 in rat olfactory system: Layer-specific regulation of multiple signaling pathways in the main olfactory bulb and induction of neurite retraction in olfactory sensory neurons

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.

Type I interferons impair BDNF-induced cell signaling and neurotrophic activity in differentiated human SH-SY5Y neuroblastoma cells and mouse primary cortical neurons

J. Neurochem. (2012) 122, 58–71.

δ-Opioid receptors stimulate GLUT1-mediated glucose uptake through Src- and IGF-1 receptor-dependent activation of PI3-kinase signalling in CHO cells

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.

Agonist activity of N-desmethylclozapine at δ-opioid receptors of human frontal cortex

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.

Preclinical and clinical phase I studies of a new recombinant Filgrastim (BK0023) in comparison with Neupogen

BackgroundFilgrastim 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.MethodsCell 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.ResultsThe results reported in this work demonstrate that BK0023 is comparable in terms of biological activity, efficacy and safety to Neupogen®.ConclusionsBK0023 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 registrationTrial registration number (TRN): NCT01933971. Date of registration: Sept 6th 2013.

Regulation of PI3K/Akt signaling by N-desmethylclozapine through activation of δ-opioid receptor.

We have previously reported that N-desmethylclozapine (NDMC), a major clozapine metabolite, acts as a δ-opioid receptor agonist. Here, we show that in different cellular systems NDMC regulates protein kinase B/Akt (Akt) signaling through the activation of δ-opioid receptors. In Chinese hamster ovary cells transfected with the human δ-opioid receptor (CHO/DOR), NDMC induced a time- and concentration-dependent phosphorylation of Akt at Thr308 and glycogen synthase kinase-3β (GSK-3β) at Ser9 and these effects were fully blocked by the δ-opioid receptor antagonist naltrindole. NDMC-induced Akt and GSK-3β phosphorylations were completely prevented by pertussis toxin, the Src tyrosine kinase inhibitor PP2 and the selective insulin-like growth factor-I (IGF-I) receptor tyrosine kinase inhibitor tyrphostin AG 1024. NDMC stimulated IGF-I receptor β subunit tyrosine phosphorylation and this effect was prevented by either naltrindole or PP2. Blockade of phosphatidylinositol 3-kinase (PI3K) α, but not PI3Kγ, suppressed NDMC-induced Akt and GSK-3β phosphorylation, whereas inhibition of Akt curtailed the stimulation of GSK-3β phosphorylation. In rat nucleus accumbens, NDMC induced Akt and GSK-3β phosphorylation either in vitro or in vivo and these effects were prevented by naltrindole. NDMC also regulated Akt and GSK-3β phosphorylation through δ-opioid receptors in NG108-15 cells. In these cells NDMC counteracted oxidative stress-induced apoptosis and the effect was lost following PI3K inhibition. These data demonstrate that in different cell systems NDMC can stimulate Akt signaling by activating Gi/Go-coupled δ-opioid receptors, which, at least in CHO/DOR cells, regulate PI3Kα through Src-dependent transactivation of the IGF-I receptor, and indicate that through this mechanism NDMC can exert neuroprotective effects.

Antidepressants activate the lysophosphatidic acid receptor LPA1 to induce insulin-like growth factor-I receptor transactivation, stimulation of ERK1/2 signaling and cell proliferation in CHO-K1 fibroblasts

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 [(3)H]-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.