1,721,030 research outputs found
Neurotossicità degli interferoni alfa e beta: effetti diretti sulle cellule neuronali
No full textLa terapia con gli interferoni (IFN) di tipo I alfa e beta è frequentemente associata alla comparsa di disturbi neuropsichiatrici, ma i processi molecolari e cellulari coinvolti nell'azione della neurotossica degli IFN non sono ancora del tutto conosciuti. Precedenti studi hanno ipotizzato meccanismi indiretti mediati da effetti periferici sul metabolismo del triptofano o da un'azione centrale sulla glia. Nel nostro studio abbiamo esaminato la possibilità che gli IFN possano agire direttamente sulle cellule neuronali e ridurne la vitalità regolando le vie di segnalazione che controllano la sopravvivenza cellulare. In cellule di neuroblastoma umano SH-SY5Y e in colture primarie di neuroni corticali l'esposizione prolungata all' IFN beta causa la comparsa di apoptosi accompagnata da liberazione di citocromo C dai mitocondri, attivazione delle caspasi 9, 7, e 3, proteolisi della poli-(ADP ribosio)-polimerasi e frammentazione del DNA. Nelle cellule neuronali, l'attivazione dei recettori degli IFN di tipo I induce una rapida fosforilazione delle Janus tirosin-chinasi e dei fattori di trascrizione STAT1, 3, 5. L'inibizione delle KAK con conseguente blocco della fosforilazione e dell'attività trascrizionale di STAT, e l'inibizione della protein chinasi R, una proteina indotta da STAT1, prevengono l'apoptosi da IFN beta. Inoltre, l'esposizione prolungata ad IFN beta causa una ridotta attivazione della via di segnalazione PI3K-AKT/GSK-3beta in risposta ai fattori neurotrofici IGF-1 e BDNF. Questi risultati indicano che gli IFN di tipo I possono ridurre la sopravvivenza neuronale attraverso l'attivazione di meccanismi intracellulari distinti che co-operano nel promuovere l'apoptosi. L' effetto neurotossico diretto sui neuroni può contribuire allo sviluppo dei disturbi neuropsichiatrici indotti da queste citochine
Studio dell'attività biologica degli interferoni e di loro derivati mediante determinazione dell'attivazione della via JAK/STAT
No full textLPA1 mediates antidepressant-induced ERK1/2 signaling and protection from oxidative stress in glial cells
No full textAntidepressants have been shown to affect glial cell functions and intracellular signaling through mechanisms that are still not completely understood. In the present study, we provide evidence that in glial cells the lysophosphatidic acid (LPA) receptor LPA1 mediates antidepressant-induced growth factor receptor transactivation, ERK1/2 signaling, and protection from oxidative stress. Thus, in C6 glioma cells and rat cortical astrocytes, ERK1/2 activation induced by either amitriptyline or mianserin was antagonized by Ki16425 and VPC 12249 (S), which block LPA1 and LPA3 receptors, and by AM966, which selectively blocks LPA1 Cell depletion of LPA1 with siRNA treatment markedly reduced antidepressant- and LPA-induced ERK1/2 phosphorylation. LPA1 blockade prevented antidepressant-induced phosphorylation of the transcription factors CREB and Elk-1. Antidepressants and LPA signaling to ERK1/2 was abrogated by cell treatment with pertussis toxin and by the inhibition of fibroblast growth factor (FGF) receptor (FGF-R) and platelet-derived growth factor receptor (PDGF-R) tyrosine kinases. Both Ki16425 and AM966 suppressed antidepressant-induced phosphorylation of FGF-R. Moreover, blockade of LPA1 or inhibition of FGF-R and PDGF-R activities prevented antidepressant-stimulated Akt and GSK-3β phosphorylations. Mianserin protected C6 glioma cells and astrocytes from apoptotic cell death induced by H2O2, as indicated by increased cell viability, decreased expression of cleaved caspase 3, reduced cleavage of poly-ADP ribose polymerase and inhibition of DNA fragmentation. The protective effects of mianserin were antagonized by AM966. These data indicate that LPA1 constitutes a novel molecular target of the regulatory actions of tricyclic and tetracyclic antidepressants in glial cells
The GABAB positive allosteric modulators CGP7930 and GS39783 stimulate ERK1/2 signalling in cells lacking functional GABAB receptors
No full textThe 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
CCL5 activates a orphan G-protein coupled receptor 75 in human neuroblastoma SH-SY5Y cell line. 15-19 NOVEMBER, WASHINGTON DC
No full textThe chemokine CCL5 inhibits entry of M-tropic HIV strains into macrophages/microglia by affecting the binding of the envelop protein gp120 to the co-receptor CCR5. Interestingly, CCL5 also prevents neuronal cell death mediated by the T-tropic gp120 and the viral protein Tat, which have no affinity for CCR5. Thus, CCL5 could be used to reduce HIV-associated neurocognitive disorder (HAND). Nevertheless, the mechanism of action of CCL5 remains to be fully characterized. Recent studies have shown that CCL5 activates a G-protein coupled receptor 75 (GPR75) which encodes for a 540 amino-acid orphan receptor of the Gq α family. In the present study, we examined the interaction of CCL5 and GPR75 in neuroblastoma SH-SY5Y cells that do not express other receptors for CCL5, such as CCR5, CCR3, and CCR1. CCL5 then promoted GPR75 internalization within few minutes. In addition, CCL5 elicited a significant dose-dependent increase in pro-survival pathways, such as the phosphatidylinositol 3-kinase (PI3K) and the extracellular signal-regulated kinases (ERK1/2). Akt and ERK1/2 phosphorylation were blocked by the specific pathway inhibitors, Wortmannin and U73 122, respectively, but not by pertuxin toxin, suggesting that CCL5 activate a Gq-coupled receptor. In conclusion, we hypothesize that CCL5-GPR75 signaling could further activate a neuroprotective mechanism that could explain the multiple pro-survival roles of CCL5 in reducing gp120 and Tat cell death
Activation of μ- and ð-opioid receptors enhances dopamine D1-like receptor signalling in mouse medial prefrontal cortex.
No full textIn the present study we examined the interaction between opioid and dopamine (DA) D1 - like receptors in the regulation of adenylyl cyclase activity in membranes of microdissected mouse medial prefrontal cortex. We found that the μ-opiooid receptor agonist DAMGO and the ð-opioid receptor agonist DPDPE caused a concentration-dependent potentiation of cyclic AMP formation elicited by either DA or the selective DA D1 -like receptor agonist (+-)-chloro APB. Conversely, the k-opioid receptor agonist (-)U50,488 failed to affect the DA response. The facilitatory effects elicited by DAMGO and DPDPE were mimicked by the naturally occurring opioid agonist leu-enkephalin and completely blocked by the selective μ- and ð-opioid receptor antagonists CTAP and naltrindole, respectively. As prefrontal DA is involved in cognitive and emotional functions, the facilitatory effects on DA D1 –like receptor signaling may represent a novel molecular mechanism contributing to the opioid effects on mood and motivation
Expression and functional activity of proteinase-activated receptors 1 and 2 in the rat olfactory
No full textAlterations in the olfactory system and impairment of olfactory function occurr in neurodegenerative diseases, such as Alzheimer’ s and Parkinson’ s diseases. The participation of proteinase-activated receptors (PARs) in these conditions has been postulated, but little is known on the expression and activity of PARs in the olfactory system. We investigated the presence, the signaling properties and cellular actions of PAR1 and PAR2 in the rat main olfactory bulb and in primary cultures of olfactory bulb and olfactory sensory neurons. PAR1 and PAR2 activity was predominantly expressed in the olfactoty nerve-glomerular cell layer (ON-GL), where selective peptide agonists inhibited cyclic AMP formation and stimulated [35S ]GTPyS binding, phosphoinositide hydrolysis, CaMKII phosphorylation and Rho activation. Nanomolar concentrations of thrombin and trypsin mimicked the actions of the peptide agonists. Lesion of the olfactory mucosa caused a reduction of PAR1 and PAR2 activity in ON-GL, suggesting the possible localization of a receptor population on olfactory nerve terminals. In primary cultures of olfactory bulb cells and olfactory neuroepithelial cells, exposure to either serine proteinases or selective peptide agonists caused a rapid neurite retraction. Immunofluorescence analysis showed the presence of PAR1 and PAR2 in neurons and glial cells of olfactory bulb and in olfactory sensory neurons. These data provide the first evidence that PAR1 and PAR2 are expressed and functional in different structures of the olfactory system and suggest the possible involvement of the receptor in neurodegenerative processes affecting the olfactory function
Involvement of store-operated Ca2+ entry in AMP-activated protein kinase stimulation by Gq/11-coupled receptors
No full textAMP-activated protein kinase (AMPK) regulates cell energy balance under conditions of metabolic stress and may play a role in neuroprotection. Gq/11-coupled receptors have been reported to activate AMPK through a signaling pathway involving stimulation of phospholipase C, enhancement of intracellular Ca2+ and activation of Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ), which phosphorylates AMPK at Thr172. The enhancement of cytosolic Ca2+ is due to both Ca2+ release from intracellular stores and Ca2+ entry through plasma membrane channels, but the relative contribution of these two Ca2+ pathways to AMPK stimulation by Gq/11-coupled receptors has not been previously investigated. In the present study we report that in human neuroblastoma SH-SY5Y cells endogenously expressing M3 muscarinic receptors and CHO cells transfected with the human M1 muscarinic receptor carbachol (CCh) stimulation of AMPK phosphorylation was completely dependent on the presence of extracellular Ca2+. In CHO/K1 cells, the stimulation of AMPK phosphorylation by endogenous protease-activated receptor 1 also required the presence of extracellular Ca2+. In addition, both thapsigargin (Tg) and ionomycin, which mobilize Ca2+ from intracellular stores, failed to elicit AMPK phosphorylation when Ca2+ was omitted from the extracellular medium. Assay of cytosolic free Ca2+ with Fura-2 fluorescence indicated that either activation of Gq/11-coupled receptors or treatment with Ca2+ mobilizing agents was effective in increasing Ca2+ levels in the absence of extracellular Ca2+. To investigate the involvement of store-operated Ca2+ entry in AMPK activation, cells were pretreated with Tg plus CCh in the absence of extracellular Ca2+ to deplete intracellular stores and then exposed to Ca2+ in the presence of atropine. The addition of Ca2+ increased AMPK phosphorylation in cells pretreated with Tg and CCh, but not in cells pretreated with vehicle. These data indicate that Ca2+ release from intracellular stores elicited by either Gq/11-coupled receptors or other Ca2+ mobilizing agents is insufficient to elicit AMPK phosphorylation. Conversely, store-operated Ca2+ entry seems to be the major pathway triggering AMPK activation via CaMKK
Regulation of AMP-activated protein kinase by human delta opioid receptors
No full textAMP-activated protein kinase (AMPK) is a multisubstrate serine/threonine protein kinase that is activated under conditions of metabolic stress causing depletion of cellular ATP, such as glucose deprivation and hypoxia/ischemia. In the present study we investigated the regulation of AMPK by human delta opioid receptors stably expressed in Chinese hamster ovary cells. We found that exposure of cells to the delta opioid receptor agonists [D-Pen2,5]enkephalin and SNC 80 caused a rapid phosphorylation of AMPK on Thr172 of the α subunit, which is associated with enhanced enzyme activity. The stimulatory effect was concentration dependent, blocked by the selective delta opioid receptors antagonist naltrindole and cell treatment with pertussis toxin, indicating the involvement of G proteins of the Gi/Go family. The delta opioid receptor-induced AMPK phosphorylation was not prevented by cell treatment with inhibitors of extracellular signal-regulated protein kinases 1 and 2, p38 mitogen-activated protein kinase, c-Jun N-terminal protein kinase and protein kinase C. On the other hand, it was almost completely blocked by STO-609, a selective inhibitor of camodulin-dependent protein kinase kinase (CaMKK), which is a major upstream regulator of AMPK. These results indicate that human delta opioid receptor can up-regulate AMPK activity through a pathway involving increased intracellular Ca2+ and activation of CaMKK. This novel regulatory action may be relevant for the neuroprotective effects of delta opioid receptors under conditions of limited energy supply
Human delta opioid receptors stimulate AMP-activated protein kinase through coincident signaling with Gq/11-coupled receptors
No full textThe heterotrimeric AMP-activated protein kinase (AMPK) is activated under conditions of metabolic stress, such as glucose deprivation and hypoxia/ischemia, and it may play a neuroprotective role. We previously reported that activation of delta opioid receptors (DOR) stably expressed in CHO cells rapidly stimulated Thr172 phosphorylation of AMPK and increased the enzyme activity through a signaling pathway involving Gi/o proteins and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). In the present study we have further characterized this regulatory pathway by examining the possible occurrence of DOR interaction with Gq/11-coupled receptors. We found that in CHO cells DOR activation of AMPK was prevented by cell treatment with apyrase, which hydrolyzes extracellular ATP and ADP, and attenuated by either PPADS, a non-selective P2 purinergic receptor antagonist, or MRS 2179, a selective P2Y1 receptor antagonist. Apyrase treatment markedly depressed DOR enhancement of intracellular free Ca2+, whereas MRS 2179 blocked DOR stimulation of phospholipase C activity. Moreover, cell treatment with the Gq/11 antagonist YM-254890 inhibited DOR stimulation of AMPK phosphorylation. In human SH-SY5Y neuroblastoma cells, the DOR agonist SNC 80 failed to affect AMPK phosphorylation but significantly enhanced the stimulatory effect elicited by activation of M3 muscarinic receptors. These data indicate that in different cell systems DOR activation regulates AMPK through a synergistic interaction with Gq/11-coupled receptors, which leads to enhancement of intracellular free Ca2+ and AMPK phosphorylation by CaMKKβ. This mechanism of AMPK regulation may participate in DOR-induced neuroprotection against metabolic insults
- …
