Fig 2: No neuron including dopaminergic neuron (DAn) is derived from reactive astrocytes in 6-hydroxydopamine (6-OHDA) model after polypyrimidine tract binding protein 1 (PTBP1) repression.(A) Schematic of experimental design. (B) Representative images of the substantia nigra or striatum after 6-OHDA lesion co-stained with tyrosine hydroxylase (TH; green) and GFAP (red). Scale bar, 200 µm for the substantia nigra and 50 µm for the striatum. (C) Representative images of brain slices of Aldh1l1-CreERT2;Rpl22lsl-HA mice subjected to 6-OHDA lesion and adeno-associated virus (AAV)-shPtbp1 or AAV-shscramble injection in substantia (upper panel) and striatum (lower panel), co-stained with GFP (cyan), hemagglutinin (HA; red) and NeuN (green) or TH(green). Scale bar, 50 µm. Number of NeuN+ neurons (D) and TH+ DAns (E) in the substantia nigra 3 months after treatment with AAV-shPtbp1 or AAV-shscramble on 6-OHDA lesioned mice. n = 5 mice for AAV-shscramble group; n = 8 mice for AAV-shPtbp1 group. (F) Apomorphine-induced rotation test before and 3 months after AAV-shPtbp1 or AAV-shscramble delivery on 6-OHDA lesioned mice. n = 8 mice for AAV-shPtbp1 group; n = 5 mice for AAV-shscramble group. Data are presented as mean ± SEM. Unpaired t test is used in (D) F(4, 7) = 3.266, p=0.4627 and (E) F(4, 7) = 3.856 p=0.5534. Unpaired t test is used in (F) 0 M AAV-shPtbp1 vs AAV-shscramble F(7, 4) = 3.59, p=0.3669; 3 M AAV-shPtbp1 vs AAV-shscramble F(7, 4) = 2.157, p=0.7915. Paired t test is used in (F), AAV-shscramble 3 M vs 0 M: p=0.0489, df = 4; AAV-shPtbp1 3 M vs 0 M: p=0.068, df = 7. Figure 3—source data 1.Brain slices of the substantia nigra or striatum after 6-OHDA lesion, co-stained with TH (green) and GFAP (red) for Figure 3B. Figure 3—source data 2.Brain slices of Aldh1l1-CreERT2;Rpl22lsl-HA mice subjected to 6-OHDA lesion and AAV-shPtbp1 or AAV-shscramble injection in substantia or striatum, co-stained with GFP (green) and HA (red), or NeuN (green) and HA (red), or TH(green) and HA (red) for Figure 3C. Figure 3—source data 3.Original data and statistical analysis of Figure 3D and E for Figure 3D&E. Figure 3—source data 4.Original data and statistical analysis of Figure 3F.

Fig 3: Viral-reporter-labeled neurons including dopaminergic neurons are induced in the substantia nigra and striatum after adeno-associated virus (AAV)-shPtbp1 delivery.(A) Schematic of AAV-shPtbp1 and AAV-shscramble vector design and the experimental design. (B) Representative images of brain slices co-stained with polypyrimidine tract binding protein 1 (PTBP1) (red) and GFP (green) at indicated timepoints after AAV-shPtbp1 or AAV-shscramble delivery in the substantia nigra. Scale bar, 50 µm. (C) Quantitative data of GFP+PTBP1+ cells ratio from (B) are shown. Representative images of brain slices co-stained GFP (green) with tyrosine hydroxylase (TH; purple) or NeuN (red) at indicated timepoints after AAV-shPtbp1 or AAV-shscramble delivery in the substantia nigra (D) and striatum (F), the enlarged 3D reconstruction of boxed individual neurons are shown in the lower panel (counterstained with Hoechst–blue). Scale bars: low magnification, 75 µm; high magnification, 10 µm. Quantitative data of GFP+NeuN+ or GFP+NeuN+ cells ratio from the substantia nigra (D) and striatum (F) are shown in (E) and (G). n = 3 biological repeats per group. Data are presented as mean ± SEM. * indicates a significant difference between AAV-shPtbp1 and AAV-shscramble (p<0.05). Two-way ANOVA followed by Tukey’s multiple comparisons test is used. (C) F(2,12) = 5.297, 1 M: p<0.0001; 2 M: p<0.0001; 3 M: p<0.0001. (E) F(2,12) = 5.321, 1 M: p=0.5220; 2 M: p=0.0016; 3 M: p=0.0007. (G) F(2,12) = 1.132, 1 M: p=0.0004; 2 M: p=0.0006; 3 M: p=0.0085. Figure 1—source data 1.Brain slices co-stained with PTBP1 (red) and GFP (green) at indicated timepoints after AAV-shPtbp1 or AAV-shscramble delivery in the substantia nigra for Figure 1B, C. Figure 1—source data 2.Brain slices co-stained GFP (green) with TH (purple) or NeuN (red) at indicated timepoints after AAV-shPtbp1 or AAV-shscramble delivery in the substantia nigra for Figure 1D, E. Figure 1—source data 3.Brain slices co-stained GFP (green) with TH (purple) or NeuN (red) at indicated timepoints after AAV-shPtbp1 or AAV-shscramble delivery in the striatum for Figure 1F, G.