In vivo chemical reprogramming of astrocytes into neurons

Abstract

In mammals, many organs lack robust regenerative abilities. Lost cells in impaired tissue could potentially be compensated by converting nearby cells in situ through in vivo reprogramming. Small molecule-induced cell reprogramming offers a temporally flexible and non-integrative strategy for altering cell fate, which is, in principle, favorable for in vivo reprogramming in organs with notoriously poor regenerative abilities, such as the brain. Here, we demonstrate that in the adult mouse brain, small molecules can reprogram astrocytes into neurons. The in situ chemically induced neurons resemble endogenous neurons in terms of neuron-specific marker expression, electrophysiological properties, and synaptic connectivity. Our study demonstrates the feasibility of in vivo chemical reprogramming in the adult mouse brain and provides a potential approach for developing neuronal replacement therapies.

Fig. 1. In vivo chemical induction of resident astrocytes into CiNs in conditional lineage-tracing system.

a Schematic diagram of chemical reprogramming in the striatum or cortex of the Aldh1l1-cre mice injected with AAV-FLEX-EGFP. IF, immunofluorescence. b Characterization of EGFP⁺ cells in the lineage-tracing mice (n = 3 mice). c Quantification of NEUN⁺/EGFP⁺ cells from 5 brain sections at 45-μm intervals per mouse in the striatum and cortex, respectively (n = 3 mice). Quantification of conversion efficiency of CiNs among EGFP-labeled cells in the striatum and the cortex, respectively (n = 3 mice). d Immunofluorescence analyses showing NEUN⁺/EGFP⁺ cells in the striatum after chemical treatment was conducted. Cc, corpus callosum. e Immunofluorescence analyses showing NEUN⁺/EGFP⁺ cells in the cortex following chemical treatment. f Schematic representation of the action potential traces and inward currents of CiNs induced in the striatum (n = 9). g Schematic representation of the action potential traces and inward currents of CiNs induced in the cortex (n = 3). Scale bars, 200 μm; 25 μm in high-magnification panels (d, e). Error bars represent SEM. **P < 0.01, ***P < 0.001 by two-way ANOVA with Sidak’s multiple comparisons test.

Fig. 2. Neuronal subtype characterizations of CiNs in the striatum and the cortex.

a Schematic diagram of chemical reprogramming in the striatum or the cortex of the Aldh1l1-cre mice injected with AAV-FLEX-EGFP and AAV-vGAT-mCherry or AAV-vGLUT2-mCherry sequentially. IF, immunofluorescence. b Conversion efficiency showing the generation of the VGAT-mCherry⁺/NEUN⁺/EGFP⁺ cells induced in the striatum and the VGLUT2-mCherry⁺/NEUN⁺/EGFP⁺ cells induced in the cortex (n = 3 mice). c Striatal neuronal subtype markers, including VGAT-mCherry, DARPP32, PVALB, and NPY were detected in CiNs generated in the striatum of adult mice. Arrowheads show the co-localization of EGFP and the corresponding markers. d Cortical neuronal subtype markers, including VGLUT2-mcherry, CTIP2, TBR1, and PVALB were detected in CiNs generated in the cortex of adult mice. Arrowheads indicate the co-localization of EGFP and the corresponding markers. Scale bars, 25 μm. Error bars represent SEM.

Fig. 3. Single-cell RNA sequencing analysis on CiNs in the striatum.

a Representative images of neuron-like EGFP⁺ cells in brain slices collected by patch pipette. Scale bar, 10 μm. b EGFP expression was confirmed in the 51 cells analyzed. c t-SNE analysis of highly variable genes that were detected in 51 EGFP⁺ cells. d Heatmap representation of 1256 differentially expressed genes cross the three clusters. e Enriched GO terms of 1256 differentially expressed genes cross the three clusters. f Gene expression profiles of the EGFP⁺ cells using the neuron score analysis. g Expression patterns of striatal neuronal subtype genes in the three clusters. h A heatmap showing expression levels of genes involved in mature neuronal functions in the three clusters. i A heatmap showing expression levels of GABAergic neuronal genes in the three clusters.

Fig. 4. Integration of in vivo reprogrammed CiNs into the mouse brain.

a Schematic representation illustrating the RABV-based retrograde transsynaptic tracing process. b Illustrative images of the initiating astrocytes (green), CiNs (yellow nucleus and red cytoplasm) and traced host neurons (red) in the chemically induced and vehicle groups at 8 wpi. Arrowheads point to the examples of the CiNs. High-magnification panels represent the co-localization of His-EGFP and DAPI. c Histogram representing the total number of His-EGFP⁺/DsRed⁺ cells (CiNs) in the chemically induced and vehicle groups (n = 5 mice). d Quantifications of His-EGFP⁺/DsRed⁺ CiNs and the connecting DsRed single-positive neurons across the injection area in the cortex (n = 4 mice). e Illustrative figure of the His-EGFP⁺/DsRed⁺ CiNs and the connecting DsRed single-positive neuron. White arrowhead points to a CiN, and the asterisk highlights a local neuron showing direct synaptic connections with the CiN. f The three-dimensional reconstruction of the image in e (left). The arrowhead and asterisk point to the same cells in e. Scale bars, 100 μm; 20 μm in high-magnification (b); 40 μm (e). Error bars represent SEM.