Fig 2: The expression of mutant ataxin-2 leads to cell death and neuroinflammation.Cleaved caspase-3 immunostaining of mouse striatum sections injected with Atx2WT at 12 weeks (A) and Atx2MUT at 4-, 8- and 12- weeks postinjection (B–D). Quantification of cleaved caspase-3 puncta relative to Atx2 positive cells showed that expression of Atx2MUT in the mouse striatum leads to a significant increase of cleaved caspase-3 over the different timepoints, suggesting an increase in apoptosis (Q) (values are expressed as mean ± SEM n = 3 per timepoint and normalized to Atx2WT; *P < 0.05, ***P < 0.001, ****P < 0.0001; one-way ANOVA followed by post hoc Tukey’s multiple comparison test). E–H Cresyl violet staining revealed that expression of Atx2MUT resulted in increased presence of pycnotic nuclei structures over time, suggesting cell lesion and striatal degeneration (R). Immunohistochemistry for GFAP (astrocytes marker) and Iba-1 (microglia marker) revealed low levels of astrogliosis (I) and microgliosis (M) upon expression of Atx2WT. In contrast, the overexpression of Atx2MUT induced an inflammatory response, highlighted by increased staining of GFAP (J–L) and Iba-1 (N–P) at 4-, 8- and 12-weeks after disease beginning. These neuropathological alterations are higher at 12-weeks of experiment (R).

Fig 3: Astrocyte-specific expression of ChR2(H134R)-mCherry and Opto-a1AR-EYFP constructs. (A) Confocal images of fixed slices of mouse brain tissue three weeks after unilateral administration of AAV2/5 GfaABC1D_ChR2(H134R)–mCherry (red). GFAP staining (green) was used to label astrocytes. Scale bar is 100 µm. (B) Confocal images of fixed slices of mouse brain tissue three weeks after unilateral administration of AAV2/5 GfaABC1D_Opto-a1AR_EYFP (green). GFAP staining (red) was used to label astrocytes. Scale bar is 100 µm. (C) Confocal image of fixed slice of mouse brain tissue three weeks after unilateral an administration of AAV2/5 GfaABC1D_Opto-a1AR_EYFP (green), nuclei are labeled with DAPI (blue), 4× magnification.

Fig 4: BC168687 siRNA downregulates the expression of P2X7 and GFAP induced by HGHF in SGCs. (A) Representative western blot analysis and the relative expression levels of P2X7 and (B) GFAP. ß-actin from the same sample was used as an internal control. The expression of P2X7 protein and GFAP in the HGHF group was significantly increased compared with the control group. The expression levels of P2X7 protein and GFAP in the BC168687si group significantly decreased compared with the HGHF group. **P<0.05, ***P<0.001 vs. Control group. ##P<0.05, ###P<0.001 vs. HGHF group. siRNA, small interfering RNA; P2X7, P2X purinoceptor 7; GFAP, glial fibrillary acidic protein; HGHF, high glucose and high free fatty acids; SGC, satellite glial cell; NCsi, negative control siRNA; IOD, integrated optical density; VD, empty vector control.

Fig 5: Aged animals displayed a significantly higher number of activated astrocytes. (A–H) Representative immunohistochemistry of striatal sections stained with GFAP. Green: astrocytes; blue: DAPI. Scale bar: 500 µm. (I) Quantitative analysis of the number of astrocytes in the 3 m (n = 5) and 18 m (n = 3) animal groups. Values are expressed ± SEM (standard error of the mean) (unpaired t-test with Welch’s correction).