P Onali

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Papers by P Onali

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

Journal of Neurochemistry, 2010

J. Neurochem. (2010) 115, 1421–1433.Type I interferons (IFNs) are known to cause neuropsychiatric... more J. Neurochem. (2010) 115, 1421–1433.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 ac...

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δ-Opioid Receptors Stimulate the Metabolic Sensor AMP-Activated Protein Kinase through Coincident Signaling with Gq/11-Coupled Receptors

Molecular Pharmacology, 2011

AMP-activated protein kinase (AMPK) and δ-opioid receptors (DOR) are both involved in controlling... more AMP-activated protein kinase (AMPK) and δ-opioid receptors (DOR) are both involved in controlling cell survival, energy metabolism and f ood intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DOR stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase t reatment, G q/11 antagonism and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca 2+ concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate accumulation. In SH-SY5Y neuroblastoma cells and primary olfactory bulb neurons, DOR activation failed to affect AMPK phosphorylation per se but potentiated the stimulation by either muscarinic agonists or 2-methyl-thio-ADP. Sequestration of G protein βγ subunits (Gβγ) blocked the DOR potentiation of AMPK phosphorylation induced by oxotremorine-M. In CHO cells, the AMPK activator 5-aminoimidazole-4-carboxamide1-β-D-ribonucleoside stimulated AMPK phosphorylation and glucose uptake, whereas pharmacological inhibition of AMPK, expression of a dom inant-negative mutant of AMPKα1 and P2Y receptor blockade reduced DOR-stimulated glucose uptake. The data indicate that in different cell systems DOR activation up-regulates AMPK through a Gβγ-dependent synergistic interaction with G q/11 -coupled receptors, potentiating Ca 2+ release and CaMKKβ-This article has not been copyedited and formatted. The final version may differ from this version.

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Mixed Agonist–Antagonist Properties of Clozapine at Different Human Cloned Muscarinic Receptor Subtypes Expressed in Chinese Hamster Ovary Cells

Neuropsychopharmacology, 1999

We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarini... more We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarinic receptor subtype. In the present study, we investigated whether the drug could elicit similar effects at the cloned human m1, m2, and m3 muscarinic receptor subtypes expressed in the Chinese hamster ovary (CHO) cells. Clozapine elicited a concentration-dependent stimulation of [ 3 H]inositol phosphates accumulation in CHO cells expressing either the m1 or the m3 receptor subtype. Moreover, clozapine inhibited forskolin-stimulated cyclic AMP accumulation and enhanced [ 35 S] GTP ␥ S binding to membrane G proteins in CHO cells expressing the m2 receptor. These agonist effects of clozapine were antagonized by atropine. The intrinsic activity of clozapine was lower than that of the full cholinergic agonist carbachol, and, when the compounds were combined, clozapine potently reduced the receptor responses to carbachol. These data indicate that clozapine behaves as a partial agonist at different muscarinic receptor subtypes and may provide new hints for understanding the receptor mechanisms underlying the antipsychotic efficacy of the drug.

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Expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) potentiates the effects of GnRH on gonadotropin subunit gene expression

Biochemical and Biophysical Research Communications, 2011

We examined the effect of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 r... more We examined the effect of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) on gonadotropin-releasing hormone (GnRH)-induced gonadotropin subunit promoter activities using the LβT2 gonadotroph cell line. In mock transfected cells, GnRH-increased LHβ and FSHβ promoters up to 2.74 ± 0.15-fold and 1.6 ± 0.05-fold respectively. When cells were transfected with PAC1R, both LHβ and FSHβ promoter activities were further increased up to 6.1 ± 0.87-fold and 2.22 ± 0.43-fold following GnRH stimulation. ERK phosphorylation, serum response element (SRE) promoters, and cAMP response element (CRE) promoters stimulated by GnRH were also potentiated in the presence of increasing amounts of PAC1R. The EC50 values for LHβ and FSHβ gene transcription by GnRH were significantly decreased by overexpression of PAC1R. PACAP 6-38, a PACAP receptor antagonist, failed to reduce the effect of GnRH on gonadotropin promoter activities in PAC1R overexpressing cells, suggesting that the potentiation of the effects of GnRH by PAC1R expression was not related to an autocrine mechanism of PACAP produced in the gonadotrophs. Our current results show that the action of GnRH in the regulation of gonadotropin subunit expression is enhanced by the presence of PAC1Rs.

The atypical antidepressant mianserin exhibits agonist activity at κ-opioid receptors

British Journal of Pharmacology, 2012

BACKGROUND AND PURPOSE Antidepressants are known to interact with the opioid system through mecha... more 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 [ 35 S]GTPgS binding and MAPK phosphorylation. KEY RESULTS Mianserin displayed 12-and 18-fold higher affinity for kthan mand d-opioid receptors respectively. In [ 35 S]GTPgS assays, mianserin selectively activated k-opioid receptors. The agonist activity was antagonized by the selective k-opioid blocker nor-binaltorphimine (nor-BNI). The mianserin analogue mirtazapine also displayed k-opioid agonist activity. Mianserin and mirtazapine increased ERK1/2 phosphorylation in CHO cells expressing k-opioid receptors and C6 cells, and these effects were antagonized by nor-BNI. In rat striatum and nucleus accumbens, mianserin stimulated [35S]GTPgS binding in a nor-BNI-sensitive manner with maximal effects lower than those of the full k-opioid agonists (-)-U50,488 and dynorphin A. When combined, mianserin antagonized the effects of the full k-opioid receptor agonists in [ 35 S]GTPgS assays and reduced the stimulation of p38 MAPK and ERK1/2 phosphorylation by dynorphin A.

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Protection from interferon-β-induced neuronal apoptosis through stimulation of muscarinic acetylcholine receptors coupled to ERK1/2 activation

British journal of pharmacology, Oct 30, 2016

Although clinically useful for their immunomodulatory, antiproliferative and antiviral properties... more 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. 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. In SH-SY5Y cells, CCh inhibited...

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The Receptor��Mediated Activation of Tyrosine Hydroxylation in the Superior Cervical Ganglion of the Rat

ABSTRACT The addition of carbachol to superior cervical ganglia causes a rapid increase in tyrosi... more ABSTRACT The addition of carbachol to superior cervical ganglia causes a rapid increase in tyrosine hydroxylation in situ. The increase occurs in ganglia from both newborn and adult animals, and in ganglia from animals pretreated with reserpine. The increase is not due to increased transport of the substrate. The increase is dependent upon the presence of calcium, and is additive to the stimulation produced by dibutyryl cyclic AMP. The stimulation seems specific for tyrosine hydroxylation; dopamine beta-hydroxylation is not increased. Preincubation experiments suggest that the carbachol-induced stimulation is due to a change in the availability of, or the affinity of the enzyme for, reduced pterin cofactor. The stimulation is inhibited by atropine and also by low concentrations of phenoxybenzamine or haloperidol, which suggests that it is caused by an action of carbachol on the interneurons in the ganglia.

Corticotropin-releasing hormone stimulates adenylyl cyclase activity in the retinas of different animal species

Regulatory peptides, Jan 3, 1993

In the present study we investigated the presence of corticotropin-releasing hormone (CRH)-stimul... more In the present study we investigated the presence of corticotropin-releasing hormone (CRH)-stimulated adenylyl cyclase activity in the retinas of different animal species. CRH significantly stimulated adenylyl cyclase activity in homogenates of calf, pig, rabbit and guinea pig retinas. The stimulatory effects were concentration-dependent with half-maximal responses occurring at 20-30 nM CRH. The enzyme activities increased by 37-80% at the maximal concentration of CRH (1 microM). On the other hand, adenylyl cyclase activities of chicken and pigeon retinas were poorly stimulated by CRH. In calf, pig and rabbit retinas, the CRH effect was completely antagonized by the CRH receptor antagonist alpha-helical CRH 9-41 and required the presence of GTP. The stimulatory response elicited by CRH was also found to be not additive with that produced by either vasoactive intestinal peptide or dopamine. These results provide evidence for the presence in retinas of different animal species of func...

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Beta-adrenergic receptor regulation of a cyclic AMP phosphodiesterase in C6 glioma cells

Advances in cyclic nucleotide and protein phosphorylation research, 1984

Involvement of store-operated Ca(2+) entry in activation of AMP-activated protein kinase and stimulation of glucose uptake by M3 muscarinic acetylcholine receptors in human neuroblastoma cells.

by P Onali, Simona Dedoni, and MC Olianas

Gq/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M1, M3 and M5 subtypes hav... more 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.

Stimulation of adenylate cyclase activity as a novel signal transaction mechanism for central muscarinic receptors

Interferon-β counter-regulates its own pro-apoptotic action by activating p38 MAPK signalling in human SH-SY5Y neuroblastoma cells.

by P Onali, Simona Dedoni, and MC Olianas

Type I interferons (IFNs) induce apoptosis of neuroblastoma cells, but the molecular mechanisms r... more 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 striatal muscarinic receptor inhibiting dopamine D1-stimulated adenylyl cyclase activity as a target for anticholinergic antiparkinson drugs

Life Sciences, 1997

receptors by ADP-ribosylation, and can be used to indicate participation of these subtypes it med... more receptors by ADP-ribosylation, and can be used to indicate participation of these subtypes it mediating physiological responses. PTX ADP-ribosyhttion of membranes in vitro is usuaII1 detent&d using ["PI NAD as substrate coupled with SDS-PAGE and autoradiography to identilj labeled GVO. This study describes an alternative method.

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Activation of Opioid and Muscarinic Receptors Stimulates Basal Adenylyl Cyclase but Inhibits Ca2+/Calmodulin- and Forskolin-Stimulated Enzyme Activities in Rat Olfactory Bulb

Journal of Neurochemistry, 2002

In rat olfactory bulb, muscarinic and opioid receptor agonists stimulate basal adenylyl cyclase a... more In rat olfactory bulb, muscarinic and opioid receptor agonists stimulate basal adenylyl cyclase activity in a GTP-dependent and pertussis toxin-sensitive manner. However, in the present study, we show that in the same brain area activation of these receptors causes inhibition of adenylyl cyclase activity stimulated by Ca2+ and calmodulin (CaM) and by forskolin (FSK), two direct activators of the catalytic unit of the enzyme. The opioid and muscarinic inhibitions consist of a decrease of the maximal stimulation elicited by either CaM or FSK, without a change in the potency of these agents. [Leu5]-Enkephalin and selective delta- and mu-, but not kappa-, opioid receptor agonists inhibit the FSK stimulation of adenylyl cyclase activity with the same potencies displayed in stimulating basal enzyme activity. Similarly, the muscarinic inhibition of FSK-stimulated adenylyl cyclase activity shows agonist and antagonist sensitivities similar to those characterizing the muscarinic stimulation of basal enzyme activity. Fluoride stimulation of adenylyl cyclase is not affected by either carbachol or [Leu5]enkephalin. In vivo treatment of olfactory bulb with pertussis toxin prevents both opioid and muscarinic inhibition of Ca2+/CaM- and FSK-stimulated enzyme activities. These results indicate that in rat olfactory bulb delta- and mu-opioid receptors and muscarinic receptors, likely of the M4 subtype, can exert a dual effect on cyclic AMP formation by interacting with pertussis toxin-sensitive GTP-binding protein(s) and possibly by affecting different molecular forms of adenylyl cyclase.

Expression of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Receptors and PACAP in Human Fetal Retina

by P Onali and Valeria Sogos

Journal of Neurochemistry, 2002

ABSTRACT

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Neuropeptide Y inhibits forskolin-stimulated adenylate cyclase activity in rat hippocampus

European Journal of Pharmacology, 1987

Neuropeptide Y inhibited the forskolin-stimulated adenylate cyclase activity in rat hippocampus w... more Neuropeptide Y inhibited the forskolin-stimulated adenylate cyclase activity in rat hippocampus with the half-maximal effect occurring at 73 nM. The maximal inhibition corresponded to a 17-22% decrease of the control level of enzyme activity. The effect of neuropeptide Y was mimicked by the peptide YY but not by the avian pancreatic polypeptide and required micromolar concentrations of GTP. These results indicate that, in the brain, the inhibition of adenylate cyclase activity constitutes a mechanism by which the receptor for neuropeptide Y transduces its signal.

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Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor

British Journal of Pharmacology, 1997

1 Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 ... more 1 Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 muscarinic receptor arti®cially expressed in Chinese hamster ovary (CHO) cells. In the present study we have investigated whether clozapine could activate the rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity, considered as pharmacologically equivalent to the m4 gene product. In addition, we have examined the eect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells.

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PACAP is a potent and highly effective stimulator of adenylyl cyclase activity in the retinas of different mammalian species

Brain Research, 1994

In rat, calf, pig and rabbit retinas the two forms of pituitary adenylate cyclase activating poly... more In rat, calf, pig and rabbit retinas the two forms of pituitary adenylate cyclase activating polypeptide with 38- and 27-amino acids (PACAP-38 and PACAP-27) produce a robust stimulation of adenylyl cyclase activity. PACAP-38 acts at picomolar concentrations and is generally more potent than PACAP-27. Both PACAPs are systematically more effective than the structurally homologous vasoactive intestinal peptide. Moreover, rat, calf and pig retinas contain significant amounts of PACAP-38 immunoreactivity. This study provides the first evidence for the action and occurrence of PACAP in mammalian retinas.

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.

by P Onali, Simona Dedoni, and MC Olianas

Neuroscience, 2007

Proteinase-activated receptors (PARs) are a family of four G protein–coupled receptors that are ... more 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.

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

by P Onali, Simona Dedoni, and MC Olianas

Type I interferons (IFNs) are known to cause neuropsychiatric side effects, which have been propo... more 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.

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