LRRK2 G2019S promotes astrocytic inflammation induced by oligomeric α-synuclein through NF-κB pathway

Abstract

Parkinson's disease (PD) is characterized by the irreversible loss of dopaminergic neurons and the accumulation of α-synuclein in Lewy bodies. The oligomeric α-synuclein (O-αS) is the most toxic form of α-synuclein species, and it has been reported to be a robust inflammatory mediator. Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are also genetically linked to PD and neuroinflammation. However, how O-αS and LRRK2 interact in glial cells remains unclear. Here, we reported that LRRK2 G2019S mutation, which is one of the most frequent causes of familial PD, enhanced the effects of O-αS on astrocytes both in vivo and in vitro. Meanwhile, inhibition of LRRK2 kinase activity could relieve the inflammatory effects of both LRRK2 G2019S and O-αS. We also demonstrated that nuclear factor κB (NF-κB) pathway might be involved in the neuroinflammatory responses. These findings revealed that inhibition of LRRK2 kinase activity may be a viable strategy for suppressing neuroinflammation in PD.

Keywords: Biological sciences; Immunology; Molecular biology; Neuroscience.

Graphical abstract

Figure 1

LRRK2 G2019S promoted early parkinsonism-like behaviors and dopaminergic neuronal loss in O-αS-treated mouse model (A) Identification of α-syn protein (monomer and oligomer) with Coomassie blue-stained native-PAGE. (B) Representative electron microscopy image of α-syn oligomers (scale bar = 50 nm). (C and D) Stance time of control (CTR), G2019S, O-αS, and G2019S+O-αS mice in gait performance. Two-way ANOVA followed by Turkey’s post-hoc test, n = 6. ∗p < 0.05; ns, not significant. (E) Latency to fall off the accelerating Rota-rod during the days training of the between G2019S group and G2019S+O-αS group. Two-way ANOVA followed by Turkey’s post-hoc test, n = 6. ^#p < 0.05. (F) Representative images of IHC staining of TH-positive neurons in the substantia nigra pars compacta (SNpc) of mice in the control, G2019S, O-αS, and G2019S+O-αS groups. (scale bar = 1 mm). (G) Statistical comparison of TH-positive neurons of control, G2019S, O-αS, and G2019S+O-αS groups in the substantia nigra pars compacta. Two-way ANOVA followed by Sidak’s post-hoc test, n = 3. ∗p < 0.05; ns, not significant. Data are represented as mean ± SEM.

Figure 2

LRRK2 G2019S enhanced activation and morphological changes of glia induced by O-αS (A) Immunostaining for astrocyte and microglia on the substantia nigra of mice with anti-IBA1 antibody and anti-GFAP antibody in the control, G2019S, O-αS, and G2019S+O-αS groups. (Scale bar = 50 μm , n = 3 per group). (B) and (C) Statistical comparison of relative fluorescence intensity of control, G2019S, O-αS, and G2019S+O-αS groups reflected activation of astrocytes and microglia. Two-way ANOVA followed by Sidak’s post-hoc test, n = 3. ∗∗p < 0.01; ∗∗∗p < 0.001. (D) Representative images of Sholl analysis for detecting glial morphology. (Scale bar = 10 μm). (E) Morphological (branching index) changes of astrocytes in control, G2019S, O-αS, and G2019S+O-αS groups by Sholl analysis. Two-way ANOVA followed by Sidak’s post-hoc test, n = 3. ∗∗∗p < 0.001. (F) Morphological (ending radius) changes in astrocytes in control, G2019S, O-αS, and G2019S+O-αS groups by Sholl analysis. Two-way ANOVA followed by Sidak’s post-hoc test, n = 3. ∗∗p < 0.01; ns, not significant. (G) Morphological (branching index) changes in microglia in control, G2019S, O-αS, and G2019S+O-αS groups by Sholl analysis. Two-way ANOVA followed by Sidak’s post-hoc test, n = 3. ∗p < 0.05; ns, not significant. (H) Morphological (ending radius) changes in microglia in control, G2019S, O-αS, and G2019S+O-αS groups by Sholl analysis. Two-way ANOVA followed by Sidak’s post-hoc test, n = 3. ∗p < 0.05; ns, not significant. Data are represented as mean ± SEM.

Figure 3

LRRK2 G2019S mutation exacerbated inflammatory response in O-αS-treated astrocytes, and IN-1 reduced the effects of LRRK2 G2019S and O-αS (A) Flow-process diagram of experimental treatment of primary astrocytes. (B and C) Western blotting assessment of NLRP3 protein expression in astrocytes in the control, G2019S, O-αS, and G2019S+O-αS groups. Two-way ANOVA followed by Sidak’s post-hoc test, n = 3. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (D‒F) The levels of IL-1β, IL-10, and TNF-α in supernatants of different treatments were determined by ELISA in the control, G2019S, O-αS, and G2019S+O-αS groups. Two-way ANOVA followed by Sidak’s post-hoc test, n = 3. ∗∗∗p < 0.001. Data are represented as mean ± SEM.

Figure 4

LRRK2 G2019S regulated inflammatory activity through NF-κB pathway (A) Western blotting assessment of P-IKK, P-P65, and IKB protein expression in astrocytes in the control, G2019S, O-αS, and G2019S+O-αS groups. (B‒D) Quantitative expression of P-IKK, P-P65, and IKB in astrocytes in the control, G2019S, O-αS, and G2019S+O-αS groups. Two-way ANOVA followed by Sidak’s post-hoc test, n = 3. ∗p < 0.05; ns, not significant. (E) Immunostaining for primary astrocyte on the substantia nigra of mice with anti-P65 antibody in the control, G2019S, O-αS, and G2019S+O-αS groups. Nuclei are stained with DAPI. (Scale bar = 50 μm， n = 3 per group). Data are represented as mean ± SEM.

Figure 5

Schematic diagram for the mechanism of LRRK2 regulating inflammation in astrocytes induced by O-αS LRRK2 G2019S enhanced O-αS induced inflammatory levels in astrocytes through NF-κB pathway. Treatment with LRRK2 kinase inhibitor, IN-1, reduced NLRP3 inflammasome activation and the level of IL-1β and TNF-α and increased the level of IL-10.