Abstract
BackgroundPurinoceptors have emerged as mediators of chronic inflammation and neurodegenerative processes. The ionotropic purinoceptor P2X7 (P2X7R) is known to modulate proinflammatory signaling and integrate neuronal-glial circuits. Evidence of P2X7R involvement in neurodegeneration, chronic pain, and chronic inflammation suggests that purinergic signaling plays a major role in microglial activation during neuroinflammation. In this study, we investigated the effects of methamphetamine (METH) on microglial P2X7R.MethodsESdMs were used to evaluate changes in METH-induced P2X7R gene expression via Taqman PCR and protein expression via western blot analysis. Migration and phagocytosis assays were used to evaluate functional changes in ESdMs in response to METH treatment. METH-induced proinflammatory cytokine production following siRNA silencing of P2X7R in ESdMs measured P2X7R-dependent functional changes. In vivo expression of P2X7R and tyrosine hydroxylase (TH) was visualized in an escalating METH dose mouse model via immunohistochemical analysis.ResultsStimulation of ESdMs with METH for 48 h significantly increased P2X7R mRNA (*p < 0.0336) and protein expression (*p < 0.022). Further analysis of P2X7R protein in cellular fractionations revealed increases in membrane P2X7R (*p < 0.05) but decreased cytoplasmic expression after 48 h METH treatment, suggesting protein mobilization from the cytoplasm to the membrane which occurs upon microglial stimulation with METH. Forty-eight hour METH treatment increased microglial migration towards Fractalkine (CX3CL1) compared to control (****p < 0.0001). Migration toward CX3CL1 was confirmed to be P2X7R-dependent through the use of A 438079, a P2X7R-competitive antagonist, which reversed the METH effects (****p < 0.0001). Similarly, 48 h METH treatment increased microglial phagocytosis compared to control (****p < 0.0001), and pretreatment of P2X7R antagonist reduced METH-induced phagocytosis (****p < 0.0001). Silencing the microglial P2X7R decreased TNF-α (*p < 0.0363) and IL-10 production after 48 h of METH treatment. Additionally, our studies demonstrate increased P2X7R and decreased TH expression in the striata of escalating dose METH animal model compared to controls.ConclusionsThis study sheds new light on the functional role of P2X7R in the regulation of microglial effector functions during substance abuse. Our findings suggest that P2X7R plays an important role in METH-induced microglial activation responses. P2X7R antagonists may thus constitute a novel target of therapeutic utility in neuroinflammatory conditions by regulating pathologically activated glial cells in stimulant abuse.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0553-3) contains supplementary material, which is available to authorized users.
Highlights
Purinoceptors have emerged as mediators of chronic inflammation and neurodegenerative processes
We suggest that P2X7 receptor (P2X7R) plays an important role in the underlying mechanism of METH-induced microglial activation
The P2X7R protein concentration in the cytoplasmic fraction (Fig. 1b, c) was reduced in comparison to the membrane fraction after METH treatment (48– 72 h). These results are consistent with previously published reports that whole cell lysates and membrane fractions of the P2X7R protein (75 kD) show a proteolytic product of the P2X7R around 60 kD [33]. These data suggest that is the P2X7R protein increased at 48 h and later time points but there may be trafficking of P2X7 from the cytosol to the membrane
Summary
Purinoceptors have emerged as mediators of chronic inflammation and neurodegenerative processes. Evidence of P2X7R involvement in neurodegeneration, chronic pain, and chronic inflammation suggests that purinergic signaling plays a major role in microglial activation during neuroinflammation. Microglial activation, known as microgliosis, may occur as a consequence of a single stimulus or multiple stimuli. Microgliosis is associated with the generation of neuroinflammatory mediators and cellular expansion [1, 3]. Chronic microgliosis is the result of hyperactive microglia creating a continuous, selfpropagating feedback loop that leads to sustained neuroinflammation. Sustained inflammation further promotes the generation and accumulation of neurotoxic inflammatory mediators that contribute to neural damage in neurodegenerative diseases and psychostimulant drug abuse [1, 3]
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