Repressing PTBP1 fails to convert reactive astrocytes to dopaminergic neurons in a 6-hydroxydopamine mouse model of Parkinson’s disease
- Guangdong Provincial Key Laboratory of Brain Function and Disease, Zhongshan School of Medicine, Sun Yat-sen University, China
- Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, China
Figures
Figure 1 with 1 supplement
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.
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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.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig1-data1-v2.zip
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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.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig1-data2-v2.zip
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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.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig1-data3-v2.zip
Figure 1—figure supplement 1
Adeno-associated virus (AAV)-shPtbp1 and AAV-shscramble predominantly infected astrocyte at early timepoint (7dpi).
(A) Representative images of brain slices co-stained with GFP (green) and AldoC (purple), NeuN (red), NG2(purple), or Iba-1(red) 7 days after AAV-shPtbp1 or AAV-shscramble delivery in the substantia nigra or striatum. Scale bar, 75 μm. (B) Ratio of GFP+ cells in the substantia nigra and striatum that shows positive staining for AldoC, NeuN, NG2, or Iba-1. n = 3 biological repeats per group. Data are presented as mean ± SEM. * indicates a significant difference (p<0.05). Two-way ANOVA (Turkey’s) is used. In the substantia nigra, F(3, 16) = 6.285. AAV-shscramble: AldoC vs NeuN, p<0.0001; AldoC vs NG2, p<0.0001; AldoC vs Iba-1, p<0.0001; AAV-shPtbp1: AldoC vs NeuN, p<0.0001; AldoC vs NG2, p<0.0001; AldoC vs Iba-1, p<0.0001. In the striatum, F(3, 16) = 0.000. AAV-shscramble: AldoC vs NeuN, p<0.0001; AldoC vs NG2, p<0.0001; AldoC vs Iba-1, p<0.0001; AAV-shPtbp1: AldoC vs NeuN, p<0.0001; AldoC vs NG2, p<0.0001; AldoC vs Iba-1, p<0.0001.
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Figure 1—figure supplement 1—source data 1
Brain slices co-stained with GFP (green) and AldoC (purple), NeuN (red), NG2 (purple), or Iba-1 (red) 7days after AAV-shPtbp1 or AAV-shscramble delivery in the substantia nigra or striatum for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig1-figsupp1-data1-v2.zip
Figure 2 with 1 supplement
No viral-reporter-labeled neuron including dopaminergic neuron is derived from quiescent astrocytes AAV-shPtbp1 delivery.
(A) Schematic of breeding strategy of Aldh1l1-CreERT2;Rpl22lsl-HA lineage-tracing mice. (B) Experimental design of Tamoxifen (TAM) induction and representative images of the substantia nigra or striatum of Aldh1l1-CreERT2;Rpl22lsl-HA mice co-stained hemagglutinin (HA) (red) with pan-astrocyte marker AldoC (green) and tyrosine hydroxylase (TH) (purple) 2 weeks after TAM administration. Scale bar, 100 μm. (C) Schematic of experimental design. Representative images of brain slices co-stained GFP (green), HA (red) with TH (purple) in the substantia nigra (D) or with NeuN (purple) in striatum (E) 3 months after AAV-shPtbp1 delivery. n = 3 biological repeats per group. Arrows indicate GFP/TH (D) or GFP/NeuN (E) double positive neurons that are HA negative. Scale bar, 75 μm.
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Figure 2—source data 1
Indicated brain regions of Aldh1l1-CreERT2;Rpl22lsl-HA mice co-stained hemagglutinin (red) with pan-astrocyte marker AldoC (green) and tyrosine hydroxylase (purple) 2 weeks after Tamoxifen administration for Figure 2B.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig2-data1-v2.zip
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Figure 2—source data 2
Brain slices co-stained GFP (green), HA (red) with TH (purple) in the substantia nigra 3 months after AAV-shPtbp1 delivery for Figure 2D.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig2-data2-v2.zip
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Figure 2—source data 3
Brain slices co-stained GFP (green), hemagglutinin (red) with NeuN (purple) in the striatum 3 months after adeno-associated virus-shPtbp1 delivery for Figure 2E.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig2-data3-v2.zip
Figure 2—figure supplement 1
Representative images of brain slices co-stained GFP (green), hemagglutinin (HA; red) with tyrosine hydroxylase (TH; purple) in the substantia nigra (A) or with NeuN (purple) in striatum (B) 3 months after adeno-associated virus (AAV)-shscramble delivery.
n = 2 biological repeats per group. Scale bar, 75 μm.
Figure 3
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.
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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.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig3-data1-v2.zip
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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.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig3-data2-v2.zip
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Figure 3—source data 3
Original data and statistical analysis of Figure 3D and E for Figure 3D&E.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig3-data3-v2.zip
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Figure 3—source data 4
Original data and statistical analysis of Figure 3F.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig3-data4-v2.zip
Figure 4 with 2 supplements
No neuron including dopaminergic neuron is derived from astrocytes with or without 6-hydroxydopamine (6-OHDA) lesion after antisense oligonucleotide (ASO)-mediated polypyrimidine tract binding protein 1 (PTBP1) repression.
(A) Schematic of experimental design. (B) Representative images of the brain slices of Aldh1l1-CreERT2;Rosa26lsl-YFP mice co-stained with YFP (green) and NeuN (red) or tyrosine hydroxylase (TH; purple) after ASO-Ptbp1 or ASO-Ctrl delivery in the substantia nigra. Scale bar, 75 μm. n = 6 mice for ASO-Ctrl group; n = 5 mice for ASO-Ptbp1 group. (C) Schematic of experimental design. (D) Representative images of brain slices of 6-OHDA lesioned Aldh1l1-CreERT2;Rpl22lsl-HA mice after ASO-Ptbp1 delivery in substantia nigra, co-stained with hemagglutinin (HA; green) and NeuN (red) or TH (purple). Scale bar, 75 μm. n = 2 mice for ASO-Ctrl group; n = 3 mice for ASO-Ptbp1 group. (E) Apomorphine-induced rotation test before and 2 months after ASO-Ptbp1 delivery in substantia nigra on 6-OHDA lesioned mice (n = 3 biological repeats). Data are presented as mean ± SEM. Paired t test is used in (E), p=0.1435, df = 2.
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Figure 4—source data 1
Brain slices of Aldh1l1-CreERT2;Rosa26lsl-YFP mice co-stained with YFP (green) and NeuN (red) or TH (purple) after ASO-Ptbp1 or ASO-Ctrl delivery in the substantia nigra for Figure 4B.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig4-data1-v2.zip
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Figure 4—source data 2
Brain slices of 6-OHDA lesioned Aldh1l1-CreERT2;Rpl22lsl-HA mice after ASO-Ptbp1 delivery in substantia nigra, co-stained with HA (green) and NeuN (red) or TH (purple) for Figure 4D.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig4-data2-v2.zip
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Figure 4—source data 3
Original data and statistical analysis of Figure 4G.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig4-data3-v2.zip
Figure 4—figure supplement 1
Knockdown efficiency of PTBP1 by ASO.
(A) In vivo knockdown efficiency of antisense oligonucleotide (ASO) by immunofluorescence. Representative images of the brain slices of Aldh1l1-CreERT2;Rosa26lsl-YFP mice co-stained with polypyrimidine tract binding protein 1 (PTBP1; purple), YFP (green), and Cy3 (red) after ASO-Ptbp1 or ASO-Ctrl delivery in the substantia nigra. n = 3 mice for ASO-Ctrl group; n = 5 mice for ASO-Ptbp1 group. Scale bar, 75 μm. (B) Quantitative data of relative PTBP1 intensity from (A) are shown. Unpaired t test is used. p<0.0001, F(2, 4) = 2.868. (C) In vivo knockdown efficiency of ASO by western blot, normalized against β-actin in mouse midbrain delivered with ASO-Ptbp1 or ASO-Ctrl (n = 3 biological repeats, respectively). (D) Quantitative data of relative PTBP1/β-actin ratio from (C) are shown. Unpaired t test is used. p=0.016, F(2, 2) = 6.268.
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Figure 4—figure supplement 1—source data 1
Brain slices of Aldh1l1-CreERT2;Rosa26lsl-YFP mice co-stained with PTBP1 (purple), YFP (green), and Cy3 (red) after ASO-Ptbp1 or ASO-Ctrl delivery in the substantia nigra for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig4-figsupp1-data1-v2.zip
Figure 4—figure supplement 2
Apomorphine-induced rotation test before and 2 months after antisense oligonucleotide (ASO)-Ctrl delivery in substantia nigra on 6-hydroxydopamine lesioned mice (n = 1 biological repeat).
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Figure 4—figure supplement 2—source data 1
Original data and statistical analysis of Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/75636/elife-75636-fig4-figsupp2-data1-v2.zip
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Repressing PTBP1 fails to convert reactive astrocytes to dopaminergic neurons in a 6-hydroxydopamine mouse model of Parkinson’s disease
eLife 11:e75636.
https://doi.org/10.7554/eLife.75636
