Astrocytes are reprogrammed to express neuronal genes via perturbation of REST/NRSF function. (A) Total RNAs were extracted from astrocytes transduced with control or DN-REST/NRSF–expressing vector, and from primary cultured neurons. These RNAs were then subjected to quantitative RT-PCR with primers specific for individual REST/NRSF target genes, GAD1, BDNF, Syt4, and Cal. (B) Expression of a neuronal marker in astrocytes after perturbation of REST/NRSF activity. Introduction of a DN-REST/NRSF–expressing construct into astrocytes resulted in ectopic expression of βIII-tubulin (red) in GFAP-positive (blue) astrocytes. Insets: Hoechst nuclear staining of each field. Bar, 50 µm. The percentage of βIII-tubulin–positive cells in GFP- and GFAP-positive populations was determined (right graph); the effect of REST/NRSF-VP16 in astrocytes is also indicated. Mean ± SD (n = 3). Statistical significance was examined by Student’s t test (*, P < 0.05). (C) Expression of Map2ab in DN-REST/NRSF-expressing astrocytes. Astrocytes were cultured as in B and stained with anti-GFP and -Map2ab antibodies. (D) Ectopic expression of neuronal markers in S100β-positive astrocytes in vivo after introduction of REST/NRSF-VP16. Lentiviruses were injected stereotactically into the striatum of adult mouse brain, followed by immunohistochemical analysis 3 wk after the surgery. Insets: higher magnification image of boxed region in each field. Quantification is shown in the table (Control, 164 S100β+/EGFP+ cells from three animals; REST/NRSF-VP16, 67 S100β+/EGFP+ cells from three animals).