Additional file 1 of RGS5 augments astrocyte activation and facilitates neuroinflammation via TNF signaling
Creators: name: Yin, Shu
name: Ma, Xin-yue
name: Sun, Ying-feng
name: Yin, Yan-qing
name: Long, Ying
name: Zhao, Chun-lai
name: Ma, Jun-wei
name: Li, Sen
name: Hu, Yan
name: Li, Ming-tao
name: Hu, Gang
name: Zhou, Jia-wei
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. Selective ablation of Rgs5 in astrocytes inhibits LPS-induced inflammation in vivo. (A) Double immunofluorescence staining for GFAP and RGS5 on the SN. Arrowheads indicate the double-labeled cells. Scale bar, 10 μm. (B) Double immunofluorescence staining for GFAP and RGS5 on the spinal cord of A30P-mutant α-synuclein transgenic mice or littermate control. (C) Quantification of immunofluorescence integrated density for RGS5 shown in B. Scale bar, 10 μm. (D) Representative graph showing a reduction in Rgs5 mRNA expression in the SN and STR of Rgs5hGFAP cKO mice. n = 3–9. (E) Immunohistochemical staining of TH on the ventral mesencephalon of adult Rgs5mGFAP cKO and their littermate controls administered with a single intra-nigral injection of LPS. Scale bar, 100 μm. (F) Quantitative data of TH+ cell numbers shown in E (n = 8). (G) Representative graph showing a reduction in Rgs5 mRNA expression in the SN or GLAST+ astrocytes of Rgs5hGFAP−CreER cKO mice (2 months old). Rgs5hGFAP−CreER cKO mice were administered with tamoxifen (i.p.) and killed 3-week post-injection. n = 4–5. (H) Representative graph showing a reduction in Rgs5 mRNA expression in Rgs5mGFAP cKO astrocytes (RGS5-null). n = 5. Data are expressed as mean ± SEM. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.001. Figure S2. Selective ablation of Rgs5 in astrocytes inhibits 6-OHDA-induced DA neuron lose and glial activation in the SN. (A) Immunohistochemical staining for TH, GFAP and IBA1 on the striatum or ventral mesencephalon of adult Rgs5hGFAP−CreER cKO and their littermate controls received a single striatal injection of 6-OHDA (4 µg). Scale bars, 100 μm. (B) Quantification of TH positive cells shown in A, n = 5. (C, D) Quantification of immunoreactive cells or immunoreactivity for GFAP (C) or IBA1 (D) shown in A. n = 5. Data are expressed as mean ± SEM, two-tailed t- test. *P &lt; 0.05. Figure S3. Increased production of pro-inflammatory mediators induced by Rgs5 overexpression in vitro. (A) Representative fluorescent microphotographs of rat primary cultured astrocytes transfected with either Lenti-RGS5-HA-FLAG-2A-GFP (LV-RGS5) driven by CMV promoter or Lenti-control vector (LV-GFP) for 12 h and followed by treatment with LPS or vehicle. Cells were harvested 48 h later. Scale bars, 100 μm. (B) Representative graphs showing relative mRNA levels of indicated pro-inflammatory mediators in the transfected astrocytes with or without LPS challenge (500 ng/ml, 5 h). n = 4–8 per group. (C) NF-κB luciferase reporter assay in HEK293T cells co-expressing the NF-κB-Luc reporter and RGS5 or GFP shows the NF-κB luciferase reporter activities in cells incubated with TNF-α (20 ng/ml). The assay was performed in triplicates for 3 times. Data are expressed as mean ± SEM. Two-way ANOVA. *P &lt; 0.05; ****P &lt; 0.001. Figure S4. RGS5 interacts with TNFR1, TNFR2 or TNFR2-ICD, respectively. (A, B) Co-immunoprecipitation assay reveals that RGS5 interacts with TNFR1 and TNFR2. HEK293T cells were transiently transfected with HA-tagged TNFR1 or TNFR2 and empty vector (GFP), mouse TNF-α-3xFLAG or RGS5-3xFLAG. The assay is repeated at least three times. IB, immunoblot; IP, immunoprecipitation. (C-E) Co-immunoprecipitation assays show the interaction between RGS5 truncation mutants and the intracellular domain (ICD) of TNFR2. C2A-RGS5 refers to the cysteine-2-alanine mutant of RGS5 which slows down the degradation of the protein. RGS5 aa 1–108, but not RGS5 aa 109–181, interacts with TNFR2-ICD. Figure S5. Interrupting RGS5–TNFR interaction suppresses astrocytic TNF-α production. (A-D) Representative Western blots showing N-RGS5-induced attenuation of increased TNF-α expression in primary astrocytes following co-overexpression of RGS5 with TNFR1 (A, B) or TNFR2 (C, D). Primary astrocytes were transfected with lentivirus (∼106 infectious units per ml) followed by challenge with TNF-α (100 ng/ml). (n = 5). (E) Identification of RGS5 fragments that interact with TNFR2 or TNFR2(T377I). Experiments are repeated 4 times. (F) Reduced TNF-α expression in astrocytes transfected with TNFR2(T377I) compared to WT TNFR2 exposed to LPS. (G) Quantification of data shown in F (n = 5). (H) NF-κB luciferase reporter activities in HEK293T cells. Cells were pre-incubated with feshurin or butein for 2 h at indicated concentrations and challenged with TNF-α (20 ng/ml, 4 h). # indicates comparisons between DMSO and butein groups. * indicates comparisons between DMSO and feshurin groups.  (I, J) Reduced pro-IL-1β expression in astrocytes pre-incubated with feshurin or butein (50 µM) for 2 h and then challenged with PFFs (2 µg/ml, 4 h) (n = 4). Data are expressed as mean ± SEM. Two-way ANOVA followed with Bonferroni’s multiple comparisons test or two-tailed t- test. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001.