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. 2016 Apr 16:11:28.
doi: 10.1186/s13024-016-0094-3.

MicroRNA-7 targets Nod-like receptor protein 3 inflammasome to modulate neuroinflammation in the pathogenesis of Parkinson's disease

Yan Zhou  1 , Ming Lu  1 , Ren-Hong Du  1 , Chen Qiao  1 , Chun-Yi Jiang  1 , Ke-Zhong Zhang  2 , Jian-Hua Ding  1 , Gang Hu  3   4   5
Affiliations

MicroRNA-7 targets Nod-like receptor protein 3 inflammasome to modulate neuroinflammation in the pathogenesis of Parkinson's disease

Yan Zhou et al. Mol Neurodegener. .

Abstract

Background: α-Synuclein (α-Syn), a pathological hallmark of Parkinson's disease (PD), has been recognized to induce the production of interleukin-1β in a process that depends, at least in vitro, on nod-like receptor protein 3 (NLRP3) inflammasome in monocytes. However, the role of NLRP3 inflammasome activation in the onset of PD has not yet been fully established.

Results: In this study, we showed that NLRP3 inflammasomes were activated in the serum of PD patients and the midbrain of PD model mice. We further clarified that α-syn activated the NLRP3 inflammasome through microglial endocytosis and subsequent lysosomal cathepsin B release. Deficiency of caspase-1, an important component of NLRP3 inflammasome, significantly inhibited α-syn-induced microglia activation and interleukin-1β production, which in turn alleviated the reduction of mesencephalic dopaminergic neurons treated by microglia medium. Specifically, we demonstrated for the first time that Nlrp3 is a target gene of microRNA-7 (miR-7). Transfection of miR-7 inhibited microglial NLRP3 inflammasome activation whereas anti-miR-7 aggravated inflammasome activation in vitro. Notably, stereotactical injection of miR-7 mimics into mouse striatum attenuated dopaminergic neuron degeneration accompanied by the amelioration of microglial activation in MPTP-induced PD model mice.

Conclusions: Our study provides a direct link between miR-7 and NLRP3 inflammasome-mediated neuroinflammation in the pathogenesis of PD. These findings will give us an insight into the potential of miR-7 and NLRP3 inflammasome in terms of opening up novel therapeutic avenues for PD.

Keywords: NLRP3 inflammasome; Neuroinflammation; Parkinson’s disease; microRNA-7; α-Synuclein.

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Figures

Fig. 1
Fig. 1
α-Synuclein activates the NLRP3 inflammasome in BV2 cells. a Wide-type and A53T mutants showing α-Syn induces the up-regulation of NLRP3 and Caspase-1 in BV2 cells. Data are presented as the mean ± S.E.M from four independent experiments. b Caspase-1-specific inhibitor (z-YVAD, 10 μM) suppresses IL-1β formation induced by both A53T mutant and wide-type α-Syn. LPS + ATP were used for a positive control drug. Data are presented as the mean ± S.E.M from four independent experiments. c Caspase-1 knockout abolishes WT or A53T α-Syn (10 μg/ml) induced elevation of IL-1β release in primary cultured microglia. d Midbrain TH neurons showed an attenuated impairment and a resistance to condition medium (CM) that was obtained from Caspase-/- microglia treated with WT or A53T α-Syn. Data are presented as the mean ± S.E.M from four independent experiments. * p < 0.05, ** p < 0.01 vs. control group, # p < 0.05, ## p < 0.01 vs. α-Syn treatment group
Fig. 2
Fig. 2
α-Syn activates NLRP3 inflammasome via endocytosis and lysosomal impairment. a Immunoblot analysis of α-Syn in BV2 cells stimulated with A53T mutant or wide-type α-Syn, cytochalasin D (3 μM) inhibits endocytosis of α-Syn into BV2 cells. Data are presented as the mean ± S.E.M from four independent experiments. b Confocal microscopy of immortalized microglia BV2 cells incubated for 4 h with FITC-labeled α-Syn (10 μM) and then processed for immunocytochemistry, cell nuclei were visualized with hoechst. Data are presented as the mean ± S.E.M from three independent experiments. c Immunoblot analysis of caspase-1 in BV2 cells stimulated with A53T mutant or wide-type α-Syn, cytochalasin D (3 μM) inhibits activation of caspase-1 in a concentration-dependent manner in BV2 cells. Data are presented as the mean ± S.E.M from three independent experiments. d ELISA of the release of IL-1β into supernatants of BV2 cells treated with cytochalasin D during stimulation with A53T mutant or wide-type α-Syn or ATP. Data are presented as the mean ± S.E.M from four independent experiments. e α-Syn-containing lysosomes adopt a swollen morphology and underwent structural damage once α-Syn was phagocytosed by BV2 cells. Data are presented as the mean ± S.E.M from three independent experiments. f WT or A53T α-Syn in turn triggers the release of lysosomal protease cathepsin B into the cytoplasm. g-h Cathepsin B inhibitor significantly suppresses α-Syn evoked increase of caspase-1 expression and IL-1β production. Data are presented as the mean ± S.E.M from four independent experiments. * p < 0.05, ** p < 0.01 vs. control group, # p < 0.05, ## p < 0.01 vs. α-Syn treatment group
Fig. 3
Fig. 3
Inflammasome is activated in serum of PD patients and in the midbrain of α-Syn-overexpressed mice. a Serum levels of IL-1β and b caspase-1 activity are upregulated in PD patients. * p < 0.05, ** p < 0.01 vs. normal group. Data are presented as the mean ± S.E.M, n = 12. c Immunofluorescence exhibits enhanced α-Syn accumulation and aggravated Iba-1+ microglial immunofluorescence intensity in the SNc of A53T transgenic mice. Green: Iba-1, red: α-Syn, blue: DAPI, n = 5–6. d-e A53T mutant mice exhibit aggravated activation of NLRP3 inflammasome and the lower levels of TH expression in the midbrain in the presence or absence of MPTP injections, n = 4. * p < 0.05 vs. wild type group, # p < 0.05 vs. A53Ttg/tg mice
Fig. 4
Fig. 4
Caspase-1 deficiency attenuates α-Syn-induced microglial activation in mouse SNc. a NeuN immunostaining exhibits a decrease of total neurons in the SNc of MPTP/p mice. Stereological counts reveal that MPTP treatment for 5-week results in a loss of TH+ DA neurons in SNc (b) and a dramatic increase of α-Syn-ir cells accumulation (c). d Concomitantly, MPTP induces the overactivation of microglia which was characterized by the increased Iba-1-ir cells in SNc. Scale bar: 150 μm, data are presented as the mean ± S.E.M, n = 5. e IBA-1+ microglial activation and f IBA-1 expressions in WT, A53T alone transgenic mice and A53Ttg/tg;Caspase-1-/- double transgenic mice. Scale bar: 40 μm, data are presented as the mean ± S.E.M, n = 4–6. * p < 0.05 vs. saline group
Fig. 5
Fig. 5
Nlrp3 is a target gene of microRNA-7. a Luciferase reporter assays confirm that Nlrp3 is a direct target gene of miR-7. Data are presented as the mean ± S.E.M from three independent experiments. b Transfection of miR-7 into BV2 cells significantly reduces NLRP3 expression but fails to affect procaspase-1 and caspase-1 expressions. c Anti-miR-7 upregulates NLRP3 expression but has no effect on caspase-1 maturation. Data are presented as the mean ± S.E.M from four independent experiments. d Transfection of miR-7 into BV2 cells markedly suppresses ATP or MSU triggerd upregulation of NLRP3 and caspase-1 as well as inhibits IL-1β production. e In contrast, miR-7 inhibitor exacerbates inflammasome activation characterized by aggravated NLRP3, caspase-1 expression and IL-1β release upon the stimulus of ATP or MSU. f Transfection of miR-7 suppresses WT or A53T α-Syn-induced upregulation of NLRP3 and caspase-1 as well as reduces IL-1β production. Data are presented as the mean ± S.E.M from four independent experiments. * p < 0.05, ** p < 0.01 vs. control or NC group, # p < 0.05, ## p < 0.01 vs. 30 nM of miR-7, anti-miR-7 treatment or corresponding NC plus ATP, MSU or α-Syn group
Fig. 6
Fig. 6
miR-7 inhibits NLRP3 inflammasome activation and attenuates DA neuronal loss in vivo. Relative miR-7 levels in the serum samples of PD patients (a, n = 12), the midbrain of MPTP-treated mice (b) and A53Ttg/tg mice (c) as well as α-Syn-treated BV2 cells (d). Data are presented as the mean ± S.E.M from four independent experiments. e miR-7 dramatically decreases the activation of IBA-1+ microglia in WT mice treated with MPTP. Scale bar: 150 μm (f-g) miR-7 mimics remarkedly increase the number of TH+ neurons in the SNc of WT mice treated with MPTP. Data are presented as the mean ± S.E.M, n = 6. h-i Injection of miR-7 mimics obviously inhibited NLRP3 inflammasome activation in MPTP-treated mice. j-m Stereotactic injection of miR-7 mimics into the striatum of A53T mice significantly downregulates NLRP3 expression and inhibits production of caspase-1 and IL-1β in vivo. n-o immunostaining showed miR-7 mimics had no significant effect on TH+ cell number in A53Ttg/tg mice at basal state. Data are presented as the mean ± S.E.M, n = 4. * p < 0.05, ** p < 0.01 vs. NC or vehicle group, # p < 0.05, ## p < 0.01 vs MPTP-NC group
Fig. 7
Fig. 7
Schematic illustration demonstrates that microglial NLRP3 inflammasome mediates α-Syn-induced neuroinflammation and DA neuron degeneration in MPTP/p PD mouse model. miR-7 targets Nlrp3 expression and modulates neuroinflammation in the pathogenesis of PD
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