Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra (SN) and the reduction of dopamine levels in the striatum. Although details of the molecular mechanisms underlying dopaminergic neuronal death in PD remain unclear, neuroinflammation is also considered a potent mediator in the pathogenesis and progression of PD. In the present study, we present evidences that microglial NLRP3 inflammasome activation is critical for dopaminergic neuronal loss and the subsequent motor deficits in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Specifically, NLRP3 deficiency significantly reduces motor dysfunctions and dopaminergic neurodegeneration of MPTP-treated mice. Furthermore, NLRP3 deficiency abolishes MPTP-induced microglial recruitment, interleukin-1β production and caspase-1 activation in the SN of mouse brain. In primary microglia and mixed glial cell cultures, MPTP/ATP treatment promotes the robust assembly and activation of the NLRP3 inflammasome via producing mitochondrial reactive oxygen species. Consistently, 1-methyl-4-phenyl-pyridinium (MPP) induces NLRP3 inflammasome activation in the presence of ATP or nigericin treatment in mouse bone-marrow-derived macrophages. These findings reveal a novel priming role of neurotoxin MPTP or MPP for NLRP3 activation. Subsequently, NLRP3 inflammasome-active microglia induces profound neuronal death in a microglia-neuron co-culture model. Furthermore, Cx3Cr1-based microglia-specific expression of an active NLRP3 mutant greatly exacerbates motor deficits and dopaminergic neuronal loss of MPTP-treated mice. Taken together, our results indicate that microglial NLRP3 inflammasome activation plays a pivotal role in the MPTP-induced neurodegeneration in PD.

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A sensitive and rapid method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with rapid solid-phase extraction (SPE) has been developed and validated for the quantitative determination of enalapril and its active metabolite enalaprilat in human plasma. After addition of internal standard to human plasma, samples were extracted by 96-well SPE cartridge. The extracts were analyzed by HPLC with the detection of the analyte in the multiple reaction monitoring (MRM) mode. This method for the simultaneous determination of enalapril and enalaprilat was accurate and reproducible, with respective limits of quantitation of 0.2 and 1.0 ng/mL in plasma. The standard calibration curves for both enalapril and enalaprilat were linear (r(2) = 0.9978 and 0.9998) over the concentration ranges 0.2-200 and 1.0-100 ng/mL in human plasma, respectively. The intra- and inter-day precision over the concentration range for enalapril and enalaprilat were lower than 13.3 and 15.4% (relative standard deviation, %RSD), and accuracy was between 89.2-105.0 and 91.9-104.7%, respectively.

Recently, the glutathione S-transferase omega 1 (GSTO1) is suspected to be involved in certain cancers and neurodegenerative diseases. However, profound investigation on the pathological roles of GSTO1 has been hampered by the lack of specific methods to determine or modulate its activity in biological systems containing other isoforms with similar catalytic function. Here, we report a fluorescent compound that is able to inhibit and monitor the activity of GSTO1. We screened 43 fluorescent chemicals and found a compound (6) that binds specifically to the active site of GSTO1. We observed that compound 6 inhibits GSTO1 by covalent modification but spares other isoforms in HEK293 cells and demonstrated that compound 6 could report the activity of GSTO1 in NIH/3T3 or HEK293 cells by measuring the fluorescence intensity of the labeled amount of GSTO1 in SDS-PAGE. Compound 6 is a useful tool to study GSTO1, applicable as a specific inhibitor and an activity reporter.