Apocynin prevents mitochondrial burdens, microglial activation, and pro-apoptosis induced by a toxic dose of methamphetamine in the striatum of mice via inhibition of p47phox activation by ERK

Abstract

Background: Activation of NADPH oxidase (PHOX) plays a critical role in mediating dopaminergic neuroinflammation. In the present study, we investigated the role of PHOX in methamphetamine (MA)-induced neurotoxic and inflammatory changes in mice.

Methods: We examined changes in mitogen-activated protein kinases (MAPKs), mitochondrial function [i.e., mitochondrial membrane potential, intramitochondrial Ca(2+) accumulation, mitochondrial oxidative burdens, mitochondrial superoxide dismutase expression, and mitochondrial translocation of the cleaved form of protein kinase C delta type (cleaved PKCδ)], microglial activity, and pro-apoptotic changes [i.e., cytosolic cytochrome c release, cleaved caspase 3, and terminal deoxynucleotidyl transferase dUDP nick-end labeling (TUNEL) positive populations] after a neurotoxic dose of MA in the striatum of mice to achieve a better understanding of the effects of apocynin, a non-specific PHOX inhibitor, or genetic inhibition of p47phox (by using p47phox knockout mice or p47phox antisense oligonucleotide) against MA-induced dopaminergic neurotoxicity.

Results: Phosphorylation of extracellular signal-regulated kinases (ERK1/2) was most pronounced out of MAPKs after MA. We observed MA-induced phosphorylation and membrane translocation of p47phox in the striatum of mice. The activation of p47phox promoted mitochondrial stresses followed by microglial activation into the M1 phenotype, and pro-apoptotic changes, and led to dopaminergic impairments. ERK activated these signaling pathways. Apocynin or genetic inhibition of p47phox significantly protected these signaling processes induced by MA. ERK inhibitor U0126 did not exhibit any additional positive effects against protective activity mediated by apocynin or p47phox genetic inhibition, suggesting that ERK regulates p47phox activation, and ERK constitutes the crucial target for apocynin-mediated inhibition of PHOX activation.

Conclusions: Our results indicate that the neuroprotective mechanism of apocynin against MA insult is via preventing mitochondrial burdens, microglial activation, and pro-apoptotic signaling process by the ERK-dependent activation of p47phox.

Fig. 1

MA-induced activation of p47phox and MAPKs. Phosphorylation (a) and membrane translocation (b) of p47phox and phosphorylations of ERK (c), p38 (d), and JNK (e) after MA treatment (35 mg/kg, i.p.). Sal saline. Each value is the mean ± S.E.M. of six animals. ^* P < 0.05, ^** P < 0.01, and ^& P < 0.001 vs. saline (one-way ANOVA was followed by Fisher’s LSD pairwise comparisons)

Fig. 2

Effects of U0126 and apocynin or p47phox knockout against MA-induced activations in ERK and p47phox. Phosphorylation of ERK (a) and phosphorylation (b) and membrane translocation (c) of p47phox after MA (35 mg/kg, i.p.). WT wild-type mice, p47 KO p47phox knockout mice, Sal saline, U U0126 (2 μg, i.c.v.), Apo apocynin (50 mg/kg, i.p.), V or Veh vehicle [10 % (v/v) DMSO] for U0126 or apocynin. Each value is the mean ± SEM of six animals. ^** P < 0.01, ^& P < 0.001 vs. vehicle/WT with saline. ^# P < 0.05 or ^## P < 0.01 vs. vehicle/WT with MA (three-way ANOVA was followed by Fisher’s LSD pairwise comparisons)

Fig. 3

Effects of U0126, apocynin, or p47phox knockout on mitochondrial dysfunction after MA treatment. MA-induced changes in mitochondrial membrane potential (a) and intramitochondrial Ca²⁺ level (c), and effects of ERK1/2 inhibitor U0126, apocynin, or p47phox gene knockout on mitochondrial membrane potential (b) and intramitochondrial Ca²⁺ level (d). WT wild-type mice, p47 KO p47phox knockout mice, Sal saline, U U0126 (2 μg, i.c.v.), Apo apocynin (50 mg/kg, i.p.), V or Veh vehicle [10 % (v/v) DMSO] for U0126 or apocynin. Each value is the mean ± S.E.M. of six animals. ^* P < 0.05 vs. saline. ^** P < 0.01 vs. saline or vehicle/WT with saline. ^# P < 0.05 vs. vehicle/WT with MA [one-way ANOVA (a and c) or three-way ANOVA (b and d) was followed by Fisher’s LSD pairwise comparisons]

Fig. 4

Effects of U0126, apocynin, or p47phox knockout on cytosolic and mitochondrial oxidative burdens after MA. Changes in cytosolic and mitochondrial reactive oxygen species (ROS) formation (a) and mitochondrial MnSOD expression (c) after MA treatment and effects of U0126, apocynin, or p47phox knockout on ROS (b) and MnSOD expression (d) 2 h after MA (35 mg/kg, i.p.). WT wild-type mice. p47 KO p47phox knockout mice, Sal saline, U U0126 (2 μg, i.c.v.), Apo apocynin (50 mg/kg, i.p.), V or Veh vehicle [10 % (v/v) DMSO] for U0126 or apocynin. Each value is the mean ± S.E.M. of six animals. ^* P < 0.05, ^** P < 0.01 vs. saline or vehicle/WT with saline. ^& P < 0.001 vs. saline. ^# P < 0.05, ^## P < 0.01 vs. vehicle/WT with MA [one-way ANOVA (a and c) or three-way ANOVA (b and d) was followed by Fisher’s LSD pairwise comparisons]

Fig. 5

Effects of U0126, apocynin, or p47phox knockout on mitochondrial translocation of cleaved PKCδ after MA. Mitochondrial translocation of cleaved PKCδ after MA (35 mg/kg, i.p.) treatment (a) and effects of U0126, apocynin, or p47phox knockout on mitochondrial translocation of cleaved PKCδ (b). WT wild-type mice, p47 KO p47phox knockout mice, Sal saline, U U0126 (2 μg, i.c.v.), Apo apocynin (50 mg/kg, i.p.), V or Veh vehicle [10 % (v/v) DMSO] for U0126 or apocynin. Each value is the mean ± S.E.M. of six animals. ^** P < 0.01 vs. saline. ^& P < 0.001 vs. vehicle/WT with saline. ^## P < 0.01 vs. vehicle/WT with MA [one-way ANOVA (a) or three-way ANOVA (b) was followed by Fisher’s LSD pairwise comparisons]

Fig. 6

Effects of U0126, apocynin, or p47phox knockout on Iba-1 expression after MA. Changes in Iba-1 expression after MA treatment (a) and effects of U0126, apocynin, or p47phox knockout on Iba-1 expression (b) and microglial activation as labeled by Iba-1 (c, d) 1 day after MA (35 mg/kg, i.p.). WT wild-type mice, p47 KO p47phox knockout mice, Sal saline, U or U0126 U0126 (2 μg, i.c.v.), Apo apocynin (50 mg/kg, i.p.), V or Veh vehicle [10 % (v/v) DMSO] for U0126 or apocynin. Each value is the mean ± S.E.M. of six animals. ^* P < 0.05, ^** P < 0.01 vs. saline or vehicle/WT with saline. ^# P < 0.05, ^## P < 0.01 vs. vehicle/WT with MA [one-way ANOVA (a) or three-way-ANOVA (b, d) was followed by Fisher’s LSD pairwise comparisons]. Scale bar = 100 μm

Fig. 7

Effects of U0126, apocynin, or p47phox knockout on microglial differentiation 1 day after MA. Microglial differentiation into M1 type (a–c) and into M2 type (d–e). Gene primer sequences for RT-PCR analysis were shown in Table 1. WT wild-type mice, p47 KO p47phox knockout mice, Sal saline, U U0126 (2 μg, i.c.v.), Apo apocynin (50 mg/kg, i.p.), V or Veh vehicle [10 % (v/v) DMSO] for U0126 or apocynin. Each value is the mean ± S.E.M. of six animals. ^** P < 0.01 vs. vehicle/WT with saline. ^# P < 0.05 vs. vehicle/WT with MA (three-way ANOVA was followed by Fisher’s LSD pairwise comparisons)

Fig. 8

Effects of U0126, apocynin, or p47phox knockout on pro-apoptotic changes after MA. Effects of U0126, apocynin, or p47phox gene knockout on TUNEL-positive cells (a), cytosolic release of cytochrome c (b), and cleaved caspase 3 expression (c) 1 day after MA (35 mg/kg, i.p.) in the Taconic ICR mice. p47 SO or p47phox SO p47phox sense oligonucleotide, p47 ASO or p47phox ASO p47phox antisense oligonucleotide, Sal saline, U or U0126 U0126 (2 μg, i.c.v.), Apo apocynin (50 mg/kg, i.p.), V or Veh vehicle [10 % (v/v) DMSO] for U0126 or apocynin. Each value is the mean ± SEM of six animals. ^** P < 0.01 vs. respective saline-group. ^# P < 0.05, ^## P < 0.01 vs. vehicle/p47phox SO with MA or vehicle/vehicle with MA (three-way ANOVA was followed by Fisher’s LSD pairwise comparisons). Scale bar = 100 μm

Fig. 9

Effects of U0126, apocynin, or p47phox knockout on dopaminergic impairments after MA. Changes in TH expression (a), dopamine level (d), and dopamine turnover rate (f) after MA treatment and effects of U0126, apocynin, or p47phox gene knockout on TH expression (b), TH-immunoreactivity (c), dopamine level (e), and dopamine turnover rate (g). WT wild-type mice, p47 KO p47phox knockout mice, Sal saline, U or U0126 U0126 (2 μg, i.c.v.), Apo apocynin (50 mg/kg, i.p.), V or Veh vehicle [10 % (v/v) DMSO] for U0126 or apocynin. Each value is the mean ± S.E.M. of six animals. ^* P < 0.05, ^** P < 0.01 vs. saline or vehicle/WT with saline. ^# P < 0.05 vs. vehicle/WT with MA [one-way ANOVA (a, d, and f) or three-way ANOVA (b, c, e, and g) was followed by Fisher’s LSD pairwise comparisons]. Scale bar = 1 mm

Fig. 10

A schematic depiction of the role of PHOX in MA-induced ERK-dependent dopaminergic neurotoxicity. Treatment with a toxic dose of MA resulted in significant phosphorylations in ERK1/2 (> p38 and JNK), and p47phox followed by membrane translocation of p47phox in the striatum of mice. The activation of p47phox promotes mitochondrial stress followed by microglial activation into M1 phenotype, and pro-apoptotic process, and leading to dopaminergic impairments. These signalings were promoted by ERK1/2. ERK inhibitor U0126 did not exhibit any additional positive effect in response to the protection offered by apocynin or p47phox genetic inhibition, suggesting that ERK regulates p47phox activation, and thus ERK is the crucial target for apocynin-mediated inhibition of PHOX activation