Regulator of G-Protein Signaling-10 Negatively Regulates NF-κB in Microglia and Neuroprotects Dopaminergic Neurons in Hemiparkinsonian Rats

RGS10 limits cytotoxicity of activated microglia on DA cells. Primary microglia from WT or Rgs10-null (P4) mice were isolated as described in Materials and Methods. A, Primary microglia were plated at a density of 50,000 cells/well in 24-well plates and treated with PBS or LPS (10 ng/ml or 1 μg/ml) for 24 h. Conditioned media were collected and transferred into differentiated MN9Ds (10,000 cells/well in 96-well plate). Target effector assay (MTS viability assay) was performed 48 h after incubation. Significant difference at ***p < 0.001 between vehicle and LPS within the genotype. Significant differences at ^##p < 0.01 or ^###p < 0.001, respectively, between WT and Rgs10-null group. Values shown are group means (n = 4) ± SEM from one experiment, representative of three independent experiments. B, Target effector assay using RGS10 siRNA or RGS4 siRNA transfected BV2 microglia as effector cells. BV2 cells were plated at a density of 150,000 cells/well in 12-well plates and transfected with control siRNA, RGS10 siRNA or RGS4 siRNA (final concentration at 60 nm) for 24 h. BV2 cells were treated with PBS or LPS (10 ng/ml or 1 μg/ml) for 24 h and conditioned media were collected and transferred into differentiated MN9D cells. Viability of MN9D cells was measured 48 h after addition of conditioned media. Significant differences between vehicle and LPS within the group at *p < 0.05 or ***p < 0.001, respectively. Significant differences between the groups (siCon and siRGS10 group, siRGS10 and siRGS4 group, or siCon and siRGS4 group) at ^#p < 0.05, ^##p < 0.01, or ^###p < 0.001, respectively. Values shown are group means (n = 4) ± SEM from one experiment, representative of two independent experiments. C, Western blot analysis of RGS10 and RGS4 in BV2 cells 24 h after transfection of siRNAs for control, RGS10, or RGS4. Expression levels were determined by densitometry and normalized to α-tubulin expression.

Effects of RGS10 deficiency on phagocytosis and chemotaxis. Primary microglia from Rgs10-null (P4) mice were isolated as described in Materials and Methods. Cells were plated in 96-well plates at a density of 50,000 cells/well and treated with LPS (10 ng/ml)/Aβ_1–42 (1 μm), M-CSF (50 ng/ml), or M-CSF (50 ng/ml)/Aβ_1–42 (1 μm) for 18 h. A, Phagocytic activity against E. coli particles was measured using the Vibrant Phagocytosis Assay using fluorescently labeled E. coli particles (Invitrogen) as described in Materials and Methods. B, Fc-receptor-mediated phagocytosis assay were performed using CytoSelect 96-Well Phagocytosis Assay (Cell Biolabs, Inc) as described in material and method. C, Fluorescently (Mitotracker Red) labeled primary microglia were plated at a density of 200,000 cells/insert. Inserts were transferred to a seeder plate which contains LPS (1 μg/ml), medium containing 10% FBS or LPS (1 μg/ml) plus medium containing 10% FBS for 18 h. Chemotaxis was measured by reading fluorescence at 585 nm excitation/620 nm emission. Significant differences between vehicle and treatments within the genotype at *p < 0.05, **p < 0.01, or ***p < 0.001 respectively. Significant differences between WT and Rgs10-null group at ^###p < 0.001. Values shown are group means (n = 4) ± SEM from one experiment, representative of three independent experiments. fAb, Fibrillar amyloid β.

RGS10 negatively regulates NF-κB activation in LPS and TNF-treated primary microglia and restoration of RGS10 is sufficient to reverse the NF-κB activation in LPS and TNF-treated BV2 microglia. Primary microglia from Rgs10-null (P4) mice were isolated as described in Materials and Methods. A, Cells were plated at the density of 200,000 cells/well in 12-well plates and treated with LPS (1 μg/ml) for indicated times. Western blot analysis were performed to measure expression of NF-κB signaling proteins (expressions were determined by densitometry and normalized to α-tubulin expression). Data are representative of two independent experiments. B, C, Primary microglia were plated at the density of 15,000 cells/well in 96-well plates. Cells were transfected with the inducible NF-κB-responsive firefly luciferase reporter and the Cignal lenti renilla as described in Materials and Methods. Cells were treated with LPS (1 μg/ml) (B) or TNF (10 ng/ml) (C) for 18 h. D, Primary microglia were isolated from WT and RGS10-null mice and plated at the density of 15,000 cells/well in 96-well plates. Cells were transfected with the inducible NF-κB-responsive firefly luciferase reporter and the Cignal lenti renilla as described in Materials and Methods and infected with lenti-GFP or lenti-RGS10 (∼7.5 × 10⁵ EU) for 24 h before LPS or TNF treatments. Cells were then treated with LPS (1 μg/ml) or TNF (10 ng/ml) for 18 h and luciferase assay was performed and values were normalized to renilla activity. E, BV2 cells were plated at the density of 5000 cells/well in 96-well plates. Cells were transfected with reporter as described above. Cells were then transfected with control siRNA, RGS10 siRNA or lenti-RGS10 virus for 24 h followed by LPS (1 μg/ml) or TNF (10 ng/ml) treatment for 18 h. Luciferase assay was performed values were normalized to renilla activity. Significant differences between vehicle and LPS within the genotype at *p < 0.05, **p < 0.01, or ***p < 0.001, respectively. Significant differences between WT and Rgs10-null group at ^#p < 0.05, ^##p < 0.01, or ^###p < 0.001 respectively. Values shown are group means (n = 4) ± SEM from one experiment representative of three independent experiments.

Restoration of RGS10 is sufficient to reverse the proinflammatory phenotype of Rgs10-null microglia. Primary microglia from Rgs10-null (P4) mice were isolated as described in Materials and Methods. Cells were plated at a density of 50,000 cells/well in 24-well plates and infected with lenti-RGS10 or lenti-GFP virus for 48 h. A, Immunofluorescent labeling of the GFP or RGS10 in primary RGS10-null microglia to confirm the expression of GFP and RGS10 after transduction. Scale bar, 100 μm. B, Western blot analysis of RGS10 in RGS10-null primary microglia after 24 h of viral transduction with lenti-RGS10 or lenti-GFP virus. Expression levels were determined by densitometry and normalized to α-tubulin expression. C, Primary microglia were plated at a density of 50,000 cells/well in 24-well plates and infected with lenti-RGS10 or lenti-GFP virus for 48 h. Cells were treated with PBS or LPS (1 μg/ml) for 24 h. Conditioned media were collected and inflammatory factor production was measured by multiplexed immunoassays (Meso-Scale Discovery). Significant differences between vehicle and LPS within the group at *p < 0.05 or ***p < 0.001 respectively. Significant differences between lenti-GFP and lenti-RGS group at ^#p < 0.05 or ^###p < 0.001, respectively. Values shown are group means (n = 4) ± SEM from one experiment, representative of three independent experiments.

Restoration of RGS10 is sufficient to reverse the enhanced cytotoxic phenotype of Rgs10-null microglia on dopaminergic cells. Primary microglia cells plated at a density of 50,000 cells/well in 24-well plates and infected with lenti-RGS10 or lenti-GFP for 48 h. Cells were treated with PBS or LPS (10 ng/ml or 1 μg/ml) for 24 h and conditioned media were collected for target-effector assays. Cytotoxic effects on differentiated MN9D dopaminergic cells were measured 48 h after incubation. Values shown are group means (n = 4) ± SEM from one experiment, representative of 3 independent experiments. Significant differences between vehicle and LPS within the group at **p < 0.01 or ***p < 0.001 respectively. Significant difference between lenti-GFP and lenti-RGS10 group at ^##p < 0.01.

RGS10-deficient ventral midbrain primary neuron-glia cultures display increased sensitivity to inflammatory and neurotoxic stimuli. Primary neuron/glia cultures from postnatal mouse ventral mesencephalon were prepared as described in Materials and Methods. Cells were plated in 24-well plates (one 75 μl-microisland per well at a density of 1 × 10⁶ cells/ml). Experiments were performed in cultures after 7 d in vitro. Cells were treated in quadruplicate with TNF (10 ng/ml), 6-OHDA (20 or 50 μm) for 72 h, or pretreated with 6-OHDA (20 μm) for 18 h followed by TNF (10 ng/ml) for 72 h. Cells were fixed, permeabilized, and blocked according to immunocytochemistry protocol. Cells were incubated for 24 h at 4°C with anti-TH (1:250; Millipore)- and Alexa-488-conjugated secondary antibody (1:1000) (Invitrogen). Significant differences between vehicle and treatments within the group at *p < 0.05 or **p < 0.01, respectively. Values shown are group means (n = 4) ± SEM from one experiment, representative of three independent experiments.

RGS10 gene transfer into the SNpc attenuates microgliosis in 6-OHDA-induced hemiparkinsonism in rats. Unilateral lesion was induced by injecting 6-OHDA into the striatum of rats as described in Materials and Methods. A, B, Animals were stereotaxically injected with 2 μl of lentivirus-expressing GFP (A) or RGS10 (B) into the SNpc. Animals were perfused 3 weeks after the lesion and brains were processed for immunohistology. Dual-labeling immunofluorescence for the microglial marker Iba1 (Alexa-594) and the dopaminergic neuron marker TH (Cy5) in the ventral midbrain of WT mice. Scale bar, 200 μm. Insets show high-magnification images of Iba1-immunoreactive microglia. C, Quantification of Iba1+ cells per field in midbrain of 6-OHDA/lenti-GFP or 6-OHDA/lenti-RGS10 rats (See Materials and Methods). Significant differences between contralateral (unlesioned) side and ipsilateral (lesioned) within the groups at ***p < 0.001. Significant differences between lenti-GFP and lenti-RGS10 groups at ^###p < 0.001.

RGS10 gene transfer in SNpc attenuates 6-OHDA-induced hemiparkinsonism in rats. Unilateral lesion was induced by stereotaxically injecting 20 μg of 6-OHDA into the striatum of rats and then animals were injected with lentivirus-expressing GFP (n = 7) or RGS10 (n = 8) into the SNpc as described in Materials and Methods. Animals were perfused 3 weeks after the lesion and brains were processed for immunohistology. A, Confocal immunofluorescence analyses of RGS10 (green), the microglial marker Iba1 (red) and the dopaminergic neuron marker TH (blue, pseudo color for Cy5) in the ventral midbrain. Scale bar, 10 μm. B, TH immunohistochemical analyses were performed after unilateral lesions were induced by a single injection of 6-OHDA (b, d) into the right striatum followed by a single injection of lenti-GFP (n = 7) (b) or lenti-RGS10 (n = 8) (d) into the right substantia nigra pars compacta (a, c; contralateral unlesioned side). Scale bar, 450 μm. C, Unbiased stereological estimate of DA neuron number (TH-positive cells) in ipsilateral (lesioned) SNpc or the contralateral (unlesioned) side. Significant differences between contralateral side and ipsilateral within the groups at **p < 0.01 or ***p < 0.001 respectively. Significant differences between lenti-GFP and lenti-RGS10 groups at ^##p < 0.01.