Mitochondrial dysfunction is involved in P2X7 receptor-mediated neuronal cell death.

P2X7 receptor (P2X7R) is known to be a 'death receptor' in immune cells, but its functional expression in non-immune cells such as neurons is controversial. Here, we examined the involvement of P2X7R activation and mitochondrial dysfunction in ATP-induced neuronal death in cultured cortical neurons. In P2X7R- and pannexin-1-expressing neuron cultures, 5 or more mM ATP or 0.1 or more mM BzATP induced neuronal death including apoptosis, and cell death was prevented by oxATP, P2X7R-selective antagonists. ATP-treated neurons exhibited Ca(2+) entry and YO-PRO-1 uptake, the former being inhibited by oxATP and A438079, and the latter by oxATP and carbenoxolone, while P2X7R antagonism with oxATP, but not pannexin-1 blocking with carbenoxolone, prevented the ATP-induced neuronal death. The ATP treatment induced reactive oxygen species generation through activation of NADPH oxidase and activated poly(ADP-ribose) polymerase, but both of them made no or negligible contribution to the neuronal death. Rhodamine123 efflux from neuronal mitochondria was increased by the ATP-treatment and was inhibited by oxATP, and a mitochondrial permeability transition pore inhibitor, cyclosporine A, significantly decreased the ATP-induced neuronal death. In ATP-treated neurons, the cleavage of pro-caspase-3 was increased, and caspase inhibitors, Q-VD-OPh and Z-DEVD-FMK, inhibited the neuronal death. The cleavage of apoptosis-inducing factor was increased, and calpain inhibitors, MDL28170 and PD151746, inhibited the neuronal death. These findings suggested that P2X7R was functionally expressed by cortical neuron cultures, and its activation-triggered Ca(2+) entry and mitochondrial dysfunction played important roles in the ATP-induced neuronal death.

Microglial zinc uptake via zinc transporters induces ATP release and the activation of microglia.

Previously, we demonstrated that extracellular zinc plays a key role in transient global ischemia-induced microglial activation through sequential activation of NADPH oxidase and poly(ADP-ribose) polymerase (PARP)-1. However, it remains unclear how zinc causes the sequential activation of microglia. Here, we examined whether transporter-mediated zinc uptake is necessary for microglial activation. Administration of zinc to microglia activated them through reactive oxygen species (ROS) generation and poly(ADP-ribose) (PAR) formation, which were suppressed by intracellular zinc chelation with 25 µM TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine) or 2 µM BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). The (65)Zn uptake by microglia was temperature- and dose-dependent, and it was blocked by metal cations, but not by L-type calcium channel blockers nifedipine and nimodipine. Expression of Zrt-Irt-like protein (ZIP)1, a plasma membrane-type zinc transporter, was detected in microglia, and nickel, a relatively sensitive substrate/inhibitor of ZIP1, showed cis- and trans-inhibitory effects on the (65)Zn uptake. Exposure of microglia to zinc increased the extracellular ATP concentration, which was suppressed by intracellular zinc chelation and inhibition of hemichannels. mRNA expression of several types of P2 receptors was detected in microglia, and periodate-oxidized ATP, a selective P2×7 receptor antagonist, attenuated the zinc-induced microglial activation via NADPH oxidase and PARP-1. Exogenous ATP and 2'(3')-O-(4-benzoyl-benzoyl) ATP also caused microglial activation through ROS generation and PAR formation. These findings demonstrate that ZIP1-mediated uptake of zinc induces ATP release and autocrine/paracrine activation of P2X(7) receptors, and then activates microglia, suggesting that zinc transporter-mediated uptake of zinc is a trigger for microglial activation via the NADPH oxidase and PARP-1 pathway. © 2011 Wiley-Liss, Inc.

Haloperidol, spiperone, pimozide and aripiprazole reduce intracellular dopamine content in PC12 cells and rat mesencephalic cultures: Implication of inhibition of vesicular transport.

Accumulating evidence suggests that antipsychotics affect dopamine release from dopaminergic neurons, but the precise mechanisms are not fully understood. Besides, there are few studies on the effects of antipsychotics on intracellular dopamine content. In this study, the effects of 8 antipsychotics on dopamine release and intracellular dopamine content in PC12 cells were investigated. Pretreatment with haloperidol, spiperone, pimozide, aripiprazole and risperidone markedly inhibited high potassium-evoked dopamine release. By contrast, pretreatment with chlorpromazine slightly increased high potassium-evoked dopamine release, while pretreatment with sulpiride and olanzapine had no effect. Haloperidol, spiperone, pimozide, chlorpromazine, aripiprazole and olanzapine evoked dopamine release, while sulpiride and risperidone had no effect. In addition, haloperidol, spiperone, pimozide, aripiprazole and risperidone reduced intracellular dopamine content in a concentration-dependent manner. These results suggest that the reduction in high potassium-evoked dopamine release by pretreatment with antipsychotics results from the reduction in vesicular dopamine content. Treatment with the 8 antipsychotics did not affect the expression of total or phosphorylated tyrosine hydroxylase. Instead, haloperidol, spiperone, pimozide and aripiprazole as well as reserpine transiently increased extracellular levels of dopamine metabolites. In addition, haloperidol, spiperone, pimozide, aripiprazole and risperidone reduced vesicular [3H]dopamine transport. These results suggest that the inhibition of vesicular dopamine transport by haloperidol, spiperone, pimozide and aripiprazole results in a reduction in vesicular dopamine content.

Elevation of heme oxygenase-1 by proteasome inhibition affords dopaminergic neuroprotection.

Postmortem studies have shown that heme oxygenase-1 (HO-1) immunoreactivity is increased in patients with Parkinson disease. HO-1 expression is highly upregulated by a variety of stress. Since the proteasome activity is decreased in patients with Parkinson disease, we investigated whether proteasome activity regulates HO-1 content. MG-132, a proteasome inhibitor, increased the amount of HO-1 protein mainly in astrocytes of primary mesencephalic cultures. Quantitative RT-PCR analysis revealed that lactacystin upregulated HO-1 mRNA expression. Proteasome inhibition with MG132 also increased the cytomegalovirus promoter-driven expression of Flag-HO-1 protein and resulted in an accumulation of ubiquitinated Flag-HO-1 in Flag-HO-1-overexpressing PC12 cells. In addition, a cycloheximide chase assay demonstrated that the degradation of Flag-HO-1 protein was slowed by MG-132. Next, the function of HO-1 which was upregulated by proteasome inhibitors was examined. Proteasome inhibitors protected dopaminergic neurons from 6-hydroxydopamine (6-OHDA)-induced toxicity and this neuroprotection was abrogated by co-treatment with zinc protoporphyrin IX, a HO-1 inhibitor. Furthermore, 6-OHDA-induced toxicity was blocked by bilirubin and carbon monoxide, products of the HO-1-catalyzed degradation of heme. These results suggest that mesencephalic HO-1 protein level is regulated by proteasome activity and the elevation by proteasome inhibition affords neuroprotection.