Fig 2: Localization of aromatase in astrocytes in adult rat brain sections. Nuclear (nucleus: blue) appearance of Aro enzyme (red; c,g,k,o; yellow arrowheads) in GFAP-labelled (green) astrocytes (a,b,e,f,i,j,m,n; white arrowheads) is also detectable in frozen cortical tissue sections from male (a–h) and female (i–p) rats. Interestingly, the Aro immunopositive signal in the adult cortical astrocytes is abundant in the processes (c,g,k,o; green arrowheads), while astrocytes derived from newborn rats do not show an Aro signal in the branches. Quantitative analysis of total GFAP-positive astrocytes and nuclear Aro immunopositive astrocytes (q) in 10 randomly selected microscopic fields of different frozen sections from both sexes showed that the abundance of nuclear Aro immunopositive GFAP-labelled astrocytes is about 20% in both males (19.79%; n = 197) and females (20.13%; n = 154). Scale bar: 100 µm.

Fig 3: The levels of cellular senescence markers such as LaminB1 and HMGB1 were significantly lower in a-syn PFF-injected mouse midbrain (MB) and striatum (STR) than PBS-treated brains, whereas the levels of p21, GFAP and Iba-1 were enhanced by 5–6 months after PFF treatment (n = 6/group). In Western blots, the examples of cellular senescence markers are displayed in triplicates per group ((A) MB, (J) STR). The quantified levels of Lamin B1 (B,K), HMGB1 (C,L), SATB1 (D,M), p16 (E,N), p21 (F,O), GFAP (G,P), Iba-1 (H,Q) and ß-III-tubulin (I,R) are demonstrated. The level of ß-III-tubulin decreased with a-syn PFF injection due to the neuronal loss. In quantification, the band intensity was normalized by a loading control, GAPDH and displayed in mean ± SEM in relativity (100% for age-matched controls, n = 6/group). The data analysis was applied to unpaired Student’s t-test for statistical significance. *: p < 0.05, **: p < 0.01, ***: p < 0.001 and ****: p < 0.0001. ns: not significant.

Fig 4: The levels of cellular senescence markers, such as LaminB1, HMGB1, SATB1 and p16, gradually decrease with a-syn PFF exposure, while the levels of p21 and GFAP increase in the isolated astrocyte culture over 3 days with a-syn PFF. In Western blots, cellular senescence markers were quantified in primary astrocytes culture at 12, 24, 48 and 72 h after a-syn PFF treatment to detect expression patterns over time (A). For assessing the expression patterns over 3 days of a-syn PFF exposure, we quantified the levels of Lamin B1 (B), HMGB1 (C), SATB1 (D), p16 (E), p21 (F) and GFAP (G). GAPDH was used as a loading control. The relative band intensities (100% for no PFF treatment) are displayed in mean ± SEM and applied to one-way ANOVA, Dunnett’s post-hoc test (#: compared with T = 0 point) for statistical significance. *: p <0.05, **: p < 0.01 and ***: p < 0.001.

Fig 5: Astrogliogenesis in the SGZ following Ethanol Consumption(A–C) Representative images of standard chow, liquid diet control, and long-term ethanol male and female mouse brains stained with stem cell marker (GFP green), astrocyte marker (GFAP red), and merged with nuclear maker DAPI (blue). The region in the red box is shown enlarged on the right.(D–F) Quantification in SGZ of average GFAP+ (D) GFAP+GFP+ (E), and percentage of GFAP+GFP+ over total GFP+ cells (F).Scale bars, 45 µm, and 15 µm in enlarged images. Values are shown as mean ± SEM, n = 3 mice per sex per group. **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA with Tukey's test compared with controls. See also Figure S3.