JIP1 binding to RBP-Jk mediates cross-talk between the Notch1 and JIP1-JNK signaling pathway.

Notch1 signaling has a critical function in maintaining a balance among cell proliferation, differentiation, and apoptosis. Our earlier work showed that the Notch1 intracellular domain interferes with the scaffolding function of c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1), yet the effect of JIP1 for Notch1-recombining binding protein suppressor of hairless (RBP-Jk) signaling remains unknown. Here, we show that JIP1 suppresses Notch1 activity. JIP1 was found to physically associate with either intracellular domain of Notch1 or RBP-Jk and interfere with the interaction between them. Furthermore, we ascertained that JIP1 caused the cytoplasmic retention of RBP-Jk through an interaction between the C-terminal region of JIP1 including Src homology 3 domain and the proline-rich domain of RBP-Jk. We also found that RBP-Jk inhibits JIP1-mediated activation of the JNK1 signaling cascade and cell death. Our results suggest that direct protein-protein interactions coordinate cross-talk between the Notch1-RBP-Jk and JIP1-JNK pathways.

Neuroprotection by nicotine against hypoxia-induced apoptosis in cortical cultures involves activation of multiple nicotinic acetylcholine receptor subtypes.

Activation of neuronal nicotinic acetylcholine receptors (nAChR) by nicotine has been suggested to protect neurons against a hypoxic insult. The objective of this study was to examine the nature of cell death induced by acute hypoxia in rat primary cortical cultures and the neuroprotective potential of nicotine in ameliorating these processes. Neuronal cell death induced by a 4-h exposure to hypoxia (0.1% O(2)) was apoptotic, as shown by TUNEL staining and assays monitoring DNA strand breaks and caspase-3/7 activity. The presence of nicotine (10 microM) during the hypoxic insult protected a subpopulation of susceptible neurones against DNA damage and apoptosis induced by oxygen deprivation. This protective effect of nicotine was prevented by a 30-min pre-incubation with either 100 nM alpha-bungarotoxin or 1 microM dihydro-beta-erythroidine, but not 1 microM atropine, suggesting that activation of at least two subtypes of nAChR, alpha7 and beta2* nAChR, is involved in mediating nicotine neuroprotection.

Nicotine activates the extracellular signal-regulated kinase 1/2 via the alpha7 nicotinic acetylcholine receptor and protein kinase A, in SH-SY5Y cells and hippocampal neurones.

Neuronal nicotinic acetylcholine receptors (nAChR) can modulate many cellular mechanisms, such as cell survival and memory processing, which are also influenced by the serine/threonine protein kinases ERK1/2. In SH-SY5Y cells and hippocampal neurones, nicotine (100 microM) increased the activity of ERK1/2. This effect was Ca2+ dependent, and prevented by the alpha7 nAChR antagonist alpha-bungarotoxin (alpha-Bgt) and an inhibitor (PD98059) of the upstream kinase MEK. To determine the intervening steps linking Ca2+ entry to MEK-ERK1/2 activation, inhibitors of Ca2+-dependent kinases were deployed. In SH-SY5Y cells, selective blockers for PKC (Ro 31-8220), CaM kinase II (KN-62) or PI3 kinase (LY 294002) failed to inhibit the nicotine-evoked increase in ERK1/2 activity. In contrast, two structurally different inhibitors of PKA (KT 5720 and H-89) completely prevented the nicotine-dependent increase in ERK1/2 activity. Inhibition of the nicotine-evoked increase in ERK1/2 activity by H-89 was also observed in hippocampal cultures. Down stream of PKA, the activity of B-Raf was significantly decreased by nicotine in SH-SY5Y cells, as determined by direct measurement of MEK1 phosphorylation or in vitro kinase assays, whereas the modulation of MEK1 phosphorylation by Raf-1 tended to increase. Thus, this study provides evidence for a novel signalling route coupling the stimulation of alpha7 nAChR to the activation of ERK1/2, in a Ca2+ and PKA dependent manner.

The alpha7 nicotinic acetylcholine receptor subtype mediates nicotine protection against NMDA excitotoxicity in primary hippocampal cultures through a Ca(2+) dependent mechanism.

Neuronal nicotinic acetylcholine receptors (nAChR) have been suggested to play a role in a variety of modulatory and regulatory processes, including neuroprotection. Here we have characterized the neuroprotective effects of nicotine against an excitotoxic insult in primary hippocampal cultures. Exposure of hippocampal neurons to 200 microM NMDA for 1 h decreased cell viability by 25+/-5%, an effect blocked by NMDA receptor antagonists. Nicotine (10 microM) counteracted the NMDA-induced cell death when co-incubated with NMDA or when present subsequent to the NMDA treatment. Nicotine protection was prevented by 1 microM MLA, confirming that it was mediated by nAChR, and by 1 microM alpha-bungarotoxin, demonstrating that the alpha7 nAChR subtype was responsible. Both the NMDA evoked neurotoxicity and nicotine neuroprotection were Ca(2+)-dependent. In Fura-2-loaded hippocampal neurons, nicotine (10 microM) and NMDA (200 microM) acutely increased intracellular resting Ca(2+) from 70 nM to 200 and 500 nM, respectively. Responses to NMDA were unaffected by the presence of nicotine. (45)Ca(2+) uptake after a 1 h exposure to nicotine or NMDA also demonstrated quantitative differences between the two drugs. This study demonstrates that the alpha7 subtype of nAChR can support neuronal survival after an excitotoxic stimulus, through a Ca(2+) dependent mechanism that operates downstream of NMDA receptor activation.

Dopaminergic pharmacology and antioxidant properties of pukateine, a natural product lead for the design of agents increasing dopamine neurotransmission.

The dopaminergic and antioxidant properties of pukateine [(R)-11-hydroxy-1,2-methylenedioxyaporphine, PUK], a natural aporphine derivative, were analyzed in the rat central nervous system. At dopamine (DA) D1 ([3H]-SCH 23390) and D2 ([3H]-raclopride) binding sites, PUK showed IC50 values in the submicromolar range (0.4 and 0.6 microM, respectively). When the uptake of tritiated dopamine was assayed by using a synaptosomal preparation, PUK showed an IC50 = 46 microM. In 6-hydroxydopamine unilaterally denervated rats, PUK (8 mg/kg but not 4 mg/kg) elicited a significant contralateral circling, a behavior classically associated with a dopaminergic agonist action. When perfused through a microdialysis probe inserted into the striatum, PUK (340 microM) induced a significant increase in dopamine levels. In vitro experiments with a crude rat brain mitochondrial suspension showed that PUK did not affect monoamine oxidase activities, at concentrations as high as 100 microM. PUK potently (IC50 = 15 microM) and dose-dependently inhibited the basal lipid peroxidation of a rat brain membrane preparation. As a whole, PUK showed a unique profile of action, comprising an increase in extracellular DA, an agonist-like interaction with DA receptors, and antioxidant activity. Thus, PUK may be taken as a lead compound for the development of novel therapeutic strategies for Parkinson disease.

Effects of alpha-erabutoxin, alpha-bungarotoxin, alpha-cobratoxin and fasciculin on the nicotine-evoked release of dopamine in the rat striatum in vivo.

Snake neurotoxins (NTX) have proven to be valuable tools for the characterisation of muscular nicotinic acetylcholine receptor structure and function. It is very likely that they could also be utilised to identify subtypes of neuronal nicotinic receptors controlling specific functions within the central nervous system. In this study we examined the effects of long alpha NTX (alpha-bungarotoxin, alpha-Bgt, and alpha-cobratoxin, alpha-Cbt) and short alpha NTX (alpha-erabutoxin a, alpha-Ebt) as well as the anticholinesterase toxin fasciculin-2 (FAS), on the nicotine-evoked release of dopamine (DA) in the striatum, using the in vivo push-pull technique. The short toxins alpha-Ebt and FAS blocked the extracellular increase of DA evoked by nicotine at 4.2 microM concentrations and alpha-Ebt was more potent, as reflected by the blockade at the lower dose of 0.42 microM. In contrast, the long toxins showed a different profile of action. Alpha-Cbt did not show any blockade of the nicotine-evoked release of DA at the doses studied while alpha-Bgt did block it only at the higher dose (4.2 microM) These results indicate that short neurotoxins show a stronger interaction with striatal nicotinic receptors subtypes controlling DA release when compared to the long ones. This interaction of short neurotoxin polypeptides and presynaptic receptors may permit the further elucidation of the particular nicotinic receptor populations responsible for the modulation of striatal DA release.

Hydroxyl radical production in the substantia nigra after 6-hydroxydopamine and hypoxia-reoxygenation.

To study the involvement of oxidative stress in 6-OHDA neurotoxicity, we investigated the production of the hydroxyl free radical (OH.) in the substantia nigra (SN) and the striatum (CS) several moments after intranigral injection of the neurotoxin, with or without an added episode of hypoxia (30 min, 95% N2, 5% O2). We utilized the hydroxylation of salicylate to 2,3 dihydroxybenzoic acid (2,3 DHBA) as indication of OH. production. When 2.3 DHBA levels were not modified, the levels of 2,5 DHBA were taken as an indication of cytochrome P450 (CYP 450) metabolism. 6-OHDA alone did not increase the production of 2,3 DHBA in the SN. 2,5 DHBA increased significantly after 120 min and was high up to 24 h. An episode of hypoxia (60 min after 6-OHDA injection) significantly worsened the decrease of dopamine (DA) in the striatum assessed 8 days after injection of 6-OHDA in the SN. Hypoxia performed 60 min and 24 h before or 24 h after 6-OHDA did not show any additional effect on striatal DA levels. Contrary to results obtained after 6-OHDA alone, 2,3 DHBA increased significantly 120 min after the injection, when the hypoxia-reoxygenation was added to the 6-OHDA treatment. Our data are showing a relationship between the increase in OH. production and a concomitant worsening of neuronal degeneration. As a whole, the results support the idea that neurons undergoing 6-OHDA neurotoxicity have their antioxidant defences affected and that oxidative stress is actually an important eliciting factor in 6-OHDA dependant neurodegeneration. However, OH. may not be the main radical species involved in this process. Additionally, 6-OHDA also appeared to provoke a long-term increase in CYP 450 activity.

Acetylcholinesterase inhibitors block acetylcholine-evoked release of dopamine in rat striatum, in vivo.

In the rat striatum, acetylcholine (ACh) increases dopamine (DA) release. The role of increased cholinergic activity provoked by acetylcholinesterase inhibitors (AChEi) on DA release is currently under revision after recent papers have shown a blockade of nicotinic transmission by AChEi in vitro. To study the effects of AChEi in vivo, Fasciculin2 (FAS), a peptidergic AChEi, and physostigmine (PHY), a classical carbamate AChEi, were applied through push-pull or microdialysis cannulae respectively, to the striatum of rats, alone or with ACh. Extracellular concentrations of DA were assessed by HPLC with electrochemical detection. Alone, the AChEi studied did not provoke changes in basal extracellular levels of DA, in the different doses studied. ACh (100 microM, 1 and 5 mM) applied through the push-pull cannulae in basal conditions provoked a dose-dependent increase of extracellular DA. This effect was not observed with ACh in concentrations of 100 microM and 1 mM if FAS (0.4 and 4.2 microM) was applied first. Higher concentrations of ACh (5 mM) evoked a partial response after FAS 0.42 microM, an effect still blocked by FAS at 4.2 microM. PHY 50 microM applied through microdialysis completely blocked the increase in DA release provoked by ACh 10, 20 mM, while at ACh 30 mM, PHY 50 microM only partially blocked the evoked increase. A partial blockade was also observed with PHY 20 microM, on the three different concentrations of ACh. On the other hand PHY 10 microM did not block any of the ACh doses perfused. These results showed that AChEi like FAS and PHY interfere with the ACh-evoked DA release in the striatum.