2,5-Hexanedione induces dopaminergic neurodegeneration through integrin αMβ2/NADPH oxidase axis-mediated microglial activation

Abstract

Recent study demonstrated that chronic exposure to solvents increases the risk of Parkinson's disease (PD), the second most common neurodegenerative disorder characterized by progressive dopaminergic neurodegeneration in the substantia nigra (SN). n-Hexane, a widely used organic solvent, displays central-peripheral neurotoxicity, which is mainly mediated by its active metabolite, 2,5-hexanedione (HD). However, whether HD exposure contributes to PD remains unclear. In this study, we found that rats exposed to HD displayed progressive dopaminergic neurodegeneration in the nigrostriatal system. Microglial activation was also detected in HD-treated rats, which occurred prior to degeneration of dopaminergic neurons. Moreover, depletion of microglia markedly reduced HD-induced dopaminergic neurotoxicity. Mechanistic study revealed an essential role of microglial integrin α_Mβ₂-NADPH oxidase (NOX2) axis in HD-elicited neurotoxicity. HD activated NOX2 by inducing membrane translocation of NOX2 cytosolic subunit, p47^phox. Integrin α_Mβ₂ was critical for HD-induced NOX2 activation since inhibition or genetic deletion of α_Mβ₂ attenuated NOX2-generated superoxide and p47^phox membrane translocation in response to HD. Src and Erk, two downstream signals of α_Mβ₂, were recognized to bridge HD/α_Mβ₂-mediated NOX2 activation. Finally, pharmacological inhibition of α_Mβ₂-NOX2 axis attenuated HD-induced microglial activation and dopaminergic neurodegeneration. Our findings revealed that HD exposure damaged nigrostriatal dopaminergic system through α_Mβ₂-NOX2 axis-mediated microglial activation, providing, for the first time, experimental evidence for n-hexane exposure contributing to the etiology of PD.

Fig. 1. HD exposure induces progressive dopaminergic neurodegeneration

a After 1, 3, and 5 weeks of HD treatment, dopaminergic neurons in the SN and striatum were immunostained with antibody against TH and the representative images were shown. b The number of TH⁺ neurons in the SNpc was counted. c Striatal dopaminergic neuron fibers were quantified by TH density. *p < 0.05, **p < 0.01; n = 4–6; Scale bar = 200 μm

Fig. 2. HD exposure induces microglial activation in the SN of rats

a After 1, 3, and 5 weeks of HD treatment, microglia in the SN were stained with antibody against Iba-1 and the representative images were shown. Activated microglia display larger cell body size and intensified Iba-1 staining. b Microglial activation was quantified by calculating the density Iba-1 immunostaining. c The gene expressions of TNF-α and iNOS were measured in the midbrain of HD-treated rats by using RT-PCR. *p < 0.05, **p < 0.01; n = 4–6; Scale bar = 50 μm

Fig. 3. Microglia are essential for HD-induced dopaminergic neurodegeneration

a Rat primary midbrain neuron–glia or microglia-deleted cultures were stained with antibody against Iba-1 or TH after 2 days of HD treatment and the representative images were shown. b The density of Iba-1 staining was quantified. c The number of TH⁺ neurons was counted in both neuron–glia and microglia-deleted cultures. Results were expressed as a percentage of controls from three experiments performed in duplicate.*p < 0.05, **p < 0.01; Scale bar = 50 μm

Fig. 4. HD activates NOX2

a BV2 microglial cells were treated with 1, 4, 8, and 16 mM HD with or without apocynin (APO) pre-treatment. The production of superoxide was assessed by DHE and the representative images of DHE oxidation were shown. b The density of red fluorescence of DHE oxidation was quantified. c Cell viability of BV2 microglia treated with different concentrations of HD was detected using LDH assay. d The effects of apocynin on HD-induced superoxide production was quantified. e The membrane translocation of NOX2 cytosolic subunit, p47^phox in HD-treated BV2 microglial cells was detected after 30 min of HD stimulation by using Western blot and the density of blots was quantified. Gp91phox, an abundant membrane protein, and GAPDH were used as an internal membrane and cytosolic control, respectively. Previous reports indicated that gp91phox is a reliable internal membrane control^,. f HD-induced superoxide production was examined in primary mixed-glia cells by DHE and the representative images of DHE oxidation were shown. g The density of red fluorescence of DHE oxidation was quantified. Results were expressed as a percentage of controls from three experiments performed in duplicate. h The membrane translocation p47^phox was detected in midbrain tissues of HD-treated rats by Western blot and the density of blots was quantified. i The levels of 4-HNE were determined in midbrains of HD-treated rats by Western blot and the density of blots was quantified. *p < 0.05, **p < 0.01; n = 3–6; Scale bar = 100 μm

Fig. 5. SRs inhibitor fucoidan fails to block HD-induced NOX2 activation

a The production of superoxide induced by HD was assessed in BV2 microglial cells with or without fucoidan pre-treatment. b The density of red fluorescence of DHE oxidation was quantified. c The effects of fucoidan on HD-induced p47^phox membrane translocation was detected using Western blot and the density of blots was quantified. Results were expressed as a percentage of controls from three experiments performed in duplicate. N.S. = not significant. Bar = 100 μm

Fig. 6. The integrin α_Mβ₂ is essential for NOX2 activation in response to HD

a BV2 microglial cells were pre-treated with RGD or anti-α_M antibody and then HD-induced superoxide production was measured. c-IgG: control IgG. b The density of red fluorescence of DHE oxidation was quantified. c The effects of RGD or anti-α_M antibody on HD-induced membrane translocation of p47^phox was detected using Western blot and the density of blots was quantified. d, e HD-induced superoxide production was measured in primary mixed-glia cultures prepared from wild type and α_M KO mice and the density of red fluorescence of DHE oxidation was quantified. Results were expressed as a percentage of controls from three experiments performed in duplicate. *p < 0.05, **p < 0.01; n = 3–6; Scale bar = 100 μm

Fig. 7. The activation of Src-Erk signals contributes to HD-induced NOX2 activation in vitro and in vivo

a BV2 microglia were pre-treated with saracatinib (Src inhibitor) and U0126 (Erk inhibitor) prior to HD and then the production of superoxide was measured using DHE. b The density of red fluorescence of DHE oxidation was quantified. c The effects of saracatinib and U0126 on HD-induced p47^phox membrane translocation was detected using Western blot and the density of blots was quantified. Results were expressed as a percentage of controls from three experiments performed in duplicate. d The levels of α_M, phosphorylated and total Src and Erk were determined in the midbrain tissues of HD-treated rats by Western blot and the representative blots were shown. e–g The density of blots was quantified. *p < 0.05, **p < 0.01; n = 3–6; Scale bar = 100 μm

Fig. 8. Pharmacological inhibition of α_Mβ₂-NOX2 axis attenuates HD-induced microglial activation and dopaminergic neurodegeneration

a Rat midbrain neuron–glia cultures were pretreated with anti-α_M antibody or apocynin (APO) and then stimulated by HD. Microglial cells were stained with antibody against Iba-1 and the representative images were shown. c-IgG: control IgG. b The density of Iba-1 immunostaining was quantified. c Dopaminergic neurons were stained with antibody against TH and the representative images were shown. d The effects of anti-α_M antibody or apocynin on HD-induced dopaminergic neurodegeneration were assessed by THir neuron counts. Results were expressed as a percentage of controls from three experiments performed in duplicate. *p < 0.05, **p < 0.01; Scale bar = 50 μm