Fig. 2

Selective Rgs5 overexpression in astrocytes induces expression of pro-inflammatory mediators and promotes α-synuclein aggregation. A Schematic drawing showing the stereotaxic injection of AAV2/5 virus into mouse substantia nigra. Mice received unilateral viral injection of AAV-RGS5-FLAG-2A-GFP driven by GFAP promoter (AAV-RGS5) on one side and AAV-GFP on the other side, and were harvested 3 weeks post-injection. B Immunofluorescence staining for GFAP on the SN of mice injected with AAV-GFAP-RGS5 or AAV-GFAP-GFP. The right panel is an enlarged view of the rectangular area of the corresponding photo. Arrows indicate double-labeled cells. Scale bars, 100 μm. C–E Effect of Rgs5 overexpression on the production of pro-inflammatory mediators in the SN upon LPS stimulation as revealed by qPCR (C) or Western blot analysis (D and E). LPS, 2.5 mg/kg/day, i.p., 2 consecutive days, n = 4 per group. F Diagram indicating the experimental design of lentivirus-mediated Rgs5 overexpression targeting astrocytes in A30P-mutant α-synuclein transgenic mice. Lenti-GFP (LV-GFP) or Lenti-RGS5-HA-3xFLAG-2A-GFP (LV-RGS5) driven by GFAP promoter were stereotaxically injected into the lateral ventricle of the brain. G Immunofluorescence staining for GFAP on the spinal cord of A30P-mutant a-synuclein transgenic mice injected with LV-RGS5 or LV-GFP. Arrows indicate double-labeled cells. Scale bars, 100 μm. H Behavioral changes of A30P-mutant α-synuclein transgenic mice injected with LV-RGS5 or LV-GFP, as assessed by the pole test, wire hang test and open field test. n = 8–11. I Kaplan–Meier survival rates in wild-type mice and A30P mutant α-synuclein transgenic mice receiving LV-RGS5, LV-GFP, respectively. Log-rank test, n = 6–8 per group. J Increased TNF-α and/or IL-1β transcription in RGS5-overexpressing A30P-mutant a-synuclein transgenic mouse spinal cord as revealed by qPCR analysis. n = 8–13, two-side t test. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001