Abstract
BackgroundPrevious studies indicate a role of P2X7 receptors in processes that lead to neuronal death. The main objective of our study was to examine whether genetic deletion or pharmacological blockade of P2X7 receptors influenced dopaminergic cell death in various models of Parkinson's disease (PD).ResultsmRNA encoding P2X7 and P2X4 receptors was up-regulated after treatment of PC12 cells with 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). P2X7 antagonists protected against MPTP and rotenone induced toxicity in the LDH assay, but failed to protect after rotenone treatment in the MTT assay in PC12 cells and in primary midbrain culture. In vivo MPTP and in vitro rotenone pretreatments increased the mRNA expression of P2X7 receptors in the striatum and substantia nigra of wild-type mice. Basal mRNA expression of P2X4 receptors was higher in P2X7 knockout mice and was further up-regulated by MPTP treatment. Genetic deletion or pharmacological inhibition of P2X7 receptors did not change survival rate or depletion of striatal endogenous dopamine (DA) content after in vivo MPTP or in vitro rotenone treatment. However, depletion of norepinephrine was significant after MPTP treatment only in P2X7 knockout mice. The basal ATP content was higher in the substantia nigra of wild-type mice, but the ADP level was lower. Rotenone treatment elicited a similar reduction in ATP content in the substantia nigra of both genotypes, whereas reduction of ATP was more pronounced after rotenone treatment in striatal slices of P2X7 deficient mice. Although the endogenous amino acid content remained unchanged, the level of the endocannabinoid, 2-AG, was elevated by rotenone in the striatum of wild-type mice, an effect that was absent in mice deficient in P2X7 receptors.ConclusionsWe conclude that P2X7 receptor deficiency or inhibition does not support the survival of dopaminergic neurons in an in vivo or in vitro models of PD.
Highlights
Previous studies indicate a role of P2X7 receptors in processes that lead to neuronal death
Effect of P2X7 receptor antagonists on rotenone and MPTP-induced inhibition of cell viability in PC12 cells In conjunction with our previous study [33], treatment of PC12 cells with the mitochondrial complex I inhibitor, rotenone, elicited a concentration-dependent (0.0130 μM) decline in cell viability, measured by the MTT assay, that resulted in a significant decrease to 79.2 ± 3.7% of the control after a 20 h treatment with a 1 μM concentration (n = 4, P < 0.01, Figures 1C, E)
Pretreatment with the selective P2X7 receptor antagonists, brilliant blue G (BBG) (10 and 100 nM) and AZ10606120 (10 and 100 nM) had no effect on cell viability after rotenone treatment when measured with the MTT assay (Figure 1E)
Summary
Previous studies indicate a role of P2X7 receptors in processes that lead to neuronal death. The activation of P2X7 receptors elicits Ca2+ influx, which is followed by increased glutamate and subsequent GABA release [8,9,10] that could reinforce glutamate-mediated excitotoxicity under pathological conditions. P2X7 receptors play a governing role in the activation and proliferation of microglia following pathological signals [11,12] and directly contribute to neurodegeneration by eliciting microglia-mediated neuronal death [13]. P2X7 receptors are involved in the processing and release of other key mediators in neurodegeneration, such as interleukin-1b, [2,14], and in the production of endocannabinoids, which are neuroprotective. P2X7 receptor activation is one of the most powerful stimuli that lead to the synthesis and subsequent release of endocannabinoids from activated microglia [15] and astrocytes [16]
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