Methamphetamine alters microglial immune function through P2X7R signaling

Abstract

Background: Purinoceptors 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.

Methods: ESdMs 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.

Results: Stimulation 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.

Conclusions: This 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.

Keywords: Methamphetamine; Microglia; Purinergic receptor X7.

Fig. 1

METH increases P2X7 purinergic receptor mRNA and protein expression in microglia. a The expression level of P2X7R mRNA in ESdM cells was modestly increased by treatment with 100 μM METH for 48 h (P < 0.0336). b Representative immunoblots of P2X7R in whole cell lysate, protein fraction, and Ponceau S staining (Po-S) as loading control. The ratios of P2X7R to protein loading control are shown in the histogram. Error bars represent mean ± SE of four independent experiments (p < 0.0003). (METH-treated versus control)

Fig. 2

METH increases migratory capacity of microglia that is P2X7R-dependent. In vitro migration was performed using the transwell migration assay. ESdM cells (5 × 10⁴) were loaded with 5 mM Calcein-AM. In the lower chamber of the transwell plate, 10 ng/mL of fractalkine (CX3CL1) was added to determine the number of migrating cells and was visualized under ×40 magnification. a shows representative images of control (a), 10 μM P2X7R-antagonist A 438079 (b), 100 μM METH (c), and 100 μM METH and 10 μM P2X7R-antagonist A 438079 (d) treated ESdM cells. b Quantification of total cells migrated in response to 100 μM METH and/or A 438079 represents a statistically significant increase in migration towards CX3CL1 in METH-treated cells as compared to control (****p < 0.0001) that is reversed by pretreatment with A 438079 (****p < 0.0001) (ANOVA)

Fig. 3

METH increases phagocytic capacity of microglia that is P2X7-dependent. Graphical representation of microglial phagocytosis in response to treatment with Cyto D (5 μM), A 438079 (10 μM), METH (100 μM), or METH (100 μM), and A 438079 (10 μM) was quantified using flow cytometry. METH (100 μM) significantly increases microglia phagocytosis (****p < 0.001). Pretreatment with antagonist decreases phagocytosis as compared to METH-treated cells. (****p < 0.0001) (ANOVA). Data consist of means ± SEM of three independent experiments

Fig. 4

METH-induced TNF-α and IL-10 secretion is P2X7R dependent. P2X7R expression was silenced around 80% compared to scrambled siRNA in ESdM cells (a). Cytokines TNF-α and IL-10 showed trend toward increased expression after treatment with METH (100 μM) (data not shown). Following P2X7R silencing, there was a significant (*p < 0.0363) decrease in TNF-α in response to METH when compared to scrambled siRNA (b). Evaluation of IL-10 release showed increased trend with METH treatment followed by a decreased trend with P2X7R silencing, which was not statistically significant (b)

Fig. 5

METH decreases tyrosine hydroxylase expression in vivo. Mice were subjected to an escalating METH dose schedule (s.c; twice daily) for 6 days, followed by a once daily administration of 10 mg/kg METH up to 56 days (a). Representative images of mouse coronal sections after 56 days are shown (b): Control (n = 3) and METH (n = 3) were stained for tyrosine hydroxylase. Five images at ×20 were taken for each mouse, and subsequent immunohistochemical analysis and quantification of tissue showed significant decreases (****p < 0.0001) in tyrosine hydroxylase expression in the striata of mice exposed to chronic METH treatment (c)

Fig. 6

METH increases P2X7R expression in murine microglia in vivo. Representative images of immunohistochemical analysis of coronal sections from control and METH mice are shown: purinergic receptors (green), Iba-1 (red), and DAPI (blue). Images show the presence of P2X7R in neurons and an increase in P2X7R expression following escalating doses (to 10 mg/kg) of METH in microglia. Yellow arrows point to P2X7R co-staining in Iba-1-positive microglia, and blue arrows point to neuronal P2X7R staining. To highlight the co-staining, the larger images were taken under ×20 objective magnification (original scale bars, 50 μm) to show Iba-1 (red) and P2X7R (green) without DAPI. The inset images of co-staining in a single cell were taken under ×40 objective magnification. The small images on the left were taken under ×20 objective magnification (original scale bars, 50 μm)