DHED treatment increases brain estradiol and markers of estrogenic effects. a, Mice treatment and analysis paradigm. For studying therapeutic effects, 7-month-old (symptomatic) 3K males were injected subcutaneously for 90 consecutive days with 100 μg DHED, a brain-selective estrogen-prodrug (Prokai et al., 2015). Motor performance was recorded at baseline, after 30 d, and finally assessed after 90 d. b, High-sensitive ELISA measurement of estradiol level in brain extracts after DHED treatment shows significant increase in male brains. Note the general higher 17β-estradiol level in female mice. c, Confocal microscopy of brain sections from (vehicle-treated) male 3K-Sham and 3K-DHED mice, and 3K female mice and WT female controls labeled for ERα in cells displaying pSer129-αS (red) aggregates as indicated. d, Expression data for representative (selected) estrogen-responsive and autophagy-relevant genes. Data are mean ± SEM. **p < 0.01 vs. DHED (b, middle, unpaired two-tailed t test; b, right, one-way ANOVA post Tukey). **p < 0.01; ***p < 0.001; ^#p < 0.05; vs. DHED (d, two-way ANOVA post Tukey). Scale bar: c, 20 μm.