Methamphetamine alters microglial immune function through P2X7R signaling.
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.
Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine at Temple University, MERB 845A, 3500 N. Broad Street, Philadelphia, 19140, PA, USA.
Nicole C
NC
Fernandes
Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine at Temple University, MERB 845A, 3500 N. Broad Street, Philadelphia, 19140, PA, USA.
Uma
U
Sriram
Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine at Temple University, MERB 845A, 3500 N. Broad Street, Philadelphia, 19140, PA, USA.
Larisa
L
Gofman
Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine at Temple University, MERB 845A, 3500 N. Broad Street, Philadelphia, 19140, PA, USA.
Jonathan M
JM
Cenna
Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine at Temple University, MERB 845A, 3500 N. Broad Street, Philadelphia, 19140, PA, USA.|Center for Substance Abuse Research, Lewis Katz School of Medicine, Philadelphia, PA, USA.
Servio H
SH
Ramirez
Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine at Temple University, MERB 845A, 3500 N. Broad Street, Philadelphia, 19140, PA, USA. raghava.potula@tuhs.temple.edu.|Center for Substance Abuse Research, Lewis Katz School of Medicine, Philadelphia, PA, USA. raghava.potula@tuhs.temple.edu.
Raghava
R
Potula
D019969:Amphetamine-Related Disorders / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D000697:Central Nervous System Stimulants / Q000494:pharmacology*; D004195:Disease Models, Animal; D055785:Gene Knockdown Techniques; D007150:Immunohistochemistry; D008297:Male; D008694:Methamphetamine / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*
D000697:Central Nervous System Stimulants; D058486:Receptors, Purinergic P2X7; D008694:Methamphetamine
Methamphetamine; Microglia; Purinergic receptor X7