Region-Restrict Astrocytes Exhibit Heterogeneous Susceptibility to Neuronal Reprogramming

Abstract

The adult CNS has poor ability to replace degenerated neurons following injury or disease. Recently, direct reprogramming of astrocytes into induced neurons has been proposed as an innovative strategy toward CNS repair. As a cell population that shows high diversity on physiological properties and functions depending on their spatiotemporal distribution, however, whether the astrocyte heterogeneity affect neuronal reprogramming is not clear. Here, we show that astrocytes derived from cortex, cerebellum, and spinal cord exhibit biological heterogeneity and possess distinct susceptibility to transcription factor-induced neuronal reprogramming. The heterogeneous expression level of NOTCH1 signaling in the different CNS regions-derived astrocytes is shown to be responsible for the neuronal reprogramming diversity. Taken together, our findings demonstrate that region-restricted astrocytes reveal different intrinsic limitation of the response to neuronal reprogramming.

Keywords: ASCL1; NGN2; astrocytes; heterogeneity; neurons; reprogramming.

Figure 1

Astrocyte Heterogeneity in Gene Expression and Proliferation (A) Immunohistochemical analysis of GFAP and Aldoc in astrocytes from adult mouse cortex, cerebellum, and spinal cord (n = 3; cells = 1,500–2,000 for each condition). (B) The heterogeneous gene expression in cortex-, cerebellum-, and spinal cord-derived cultured postnatal or adult astrocytes determined by real-time PCR (a representative result from three independent experiments). (C) The heterogeneous cell proliferation of regional postnatal astrocytes (n = 3; cells = 1,000–1,500 for each condition). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 by ANOVA Tukey's post hoc test. Scale bars, 100 μm (A) and 50 μm (C).

Figure 2

NGN2-Induced Neuronal Reprogramming Efficiency of Postnatal and Adult Astrocytes (A) Representative pictures of NGN2-induced neurons from postnatal astrocytes by staining with Tuj1 at 12 dpi. (B) NGN2-induced neuronal reprogramming efficiency of postnatal astrocytes (n = 3; cells = 900–1,500 for each condition). (C) Representative pictures of NGN2-induced neurons from adult astrocytes by staining with Tuj1 at 12 dpi. (D) Comparing NGN2-induced neuronal reprogramming efficiency of adult astrocytes compared with that of postnatal astrocytes (n = 3; cells = 700–1,500 for each condition). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 by ANOVA Tukey's post hoc test. Scale bar, 100 μm.

Figure 3

Maturation of NGN2-Induced Neurons (A) Representative micrographs of mature NGN2-induced neurons from postnatal astrocytes by staining with mature neuronal marker MAP2 and NeuN at 18 dpi. (B) Quantification of mature neurons induced from different region-derived postnatal astrocytes (n = 3; cells = 500–1,000 for each condition). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 by ANOVA Tukey's post hoc test. Scale bar, 50 μm.

Figure 4

NGN2-Induced Neurons Mainly Matured into Glutamatergic Neurons (A) Representative pictures of mature GABAergic and glutamatergic neurons induced from different region-derived postnatal astrocytes at 18 dpi. (B and C) Quantification of GABAergic and glutamatergic neurons reprogrammed from different region-derived postnatal (B) or adult (C) astrocytes (n = 3; cells = 300–600 for each condition). ^∗∗∗p < 0.001 by ANOVA Tukey's post hoc test. Scale bar, 50 μm.

Figure 5

Expression of Notch1 Signaling Molecules in Heterogeneous Astrocytes (A) Heterogeneous expression of molecules associated with neuronal differentiation and specialization in postnatal astrocytes (n = 3 for each group, ^∗p < 0.05 by ANOVA Tukey's post hoc test). (B) Notch1 expression in regional postnatal astrocytes determined by real-time PCR (n = 3 for each group, ^∗p < 0.05 by ANOVA Tukey's post hoc test). (C) Comparison of Notch1 expression between postnatal and adult astrocytes by real-time PCR (n = 3 for each group, ^∗p < 0.05 by Student's t test). (D) NOTCH1 expression in regional postnatal astrocytes determined by western blotting. (E) Notch1 signaling molecules Hes1 and Nrarp expression in postnatal astrocytes determined by real-time PCR (n = 3 for each group; ^∗p < 0.05, ^∗∗∗p < 0.001 by ANOVA Tukey's post hoc test).

Figure 6

DAPT Treatment Increased the Efficiency of NGN2-Induced Neuronal Reprogramming from Postnatal Astrocytes (A) The knockdown efficiency of DATP in postnatal astrocytes was determined by western blotting. Quantitative analysis showed no significant difference in the knockdown efficiency of DAPT between cortex (CTX), cerebellum (CE), and spinal cord (SC) astrocytes. n = 3 for each group. ^∗∗∗p < 0.001 (Student's t test); p ≥ 0.05, no statistically significant difference (n.s.) (ANOVA Tukey's post hoc test). (B) Experimental scheme of blocking NOTCH1 signaling in astrocytes by DAPT during neuronal reprogramming. AM, astrocyte culture medium; NM, neuronal induction medium. (C) Representative pictures of NGN2-induced neurons with or without DAPT treatment by staining with Tuj1 at 12 dpi. (D) The efficiency of NGN2-induced neuronal reprogramming in the DAPT group compared with control group (n = 3; cells = 1,000–1,700 for each condition). ^∗p < 0.05 by ANOVA Tukey's post hoc test. Scale bar, 100 μm.

Figure 7

Interference of NOTCH1 Signaling Increased the Efficiency of NGN2-Induced Neuronal Reprogramming from Postnatal Astrocytes (A) Western blotting analysis of the knockdown efficiency of Notch1 shRNA in postnatal cortex astrocytes. n = 3 for each group. ^∗∗p < 0.01, ^∗∗∗p < 0.001 by ANOVA Tukey's post hoc test. (B) The knockdown efficiency of Notch1 shRNA in postnatal astrocytes was determined by western blotting. Quantitative analysis showed no significant difference in the knockdown efficiency of Notch1 shRNA between cortex (CTX), cerebellum (CE), and spinal cord (SC) astrocytes. n = 3 for each group. ^∗∗∗p < 0.001 (Student's t test); p ≥ 0.05, no statistically significant difference (n.s.) (ANOVA Tukey's post hoc test). (C) Experimental scheme of Notch1 knockdown in astrocytes by lentivirus-mediated shRNA during neuronal reprogramming. (D) Representative pictures of NGN2-induced neurons with or without shRNA interference by staining with Tuj1 at 12 dpi. (E) The efficiency of NGN2-induced neuronal reprogramming in Notch1 shRNA group compared with control group (n = 3; cells = 1,000–1,700 for each condition). ^∗p < 0.05 by ANOVA Tukey's post hoc test. Scale bar, 100 μm.