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Cited by 159Neurogenesis and neuroplasticity mechanismsPluripotent Stem Cells ResearchNuclear Receptors and Signaling

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Inducing Different Neuronal Subtypes from Astrocytes in the Injured Mouse Cerebral Cortex

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Mattugini, Nicola, Bocchi, Riccardo, Scheuss, Volker, Russo, Gianluca Luigi, Torper, Olof, Lao, Chu Lan, Götz, Magdalena·2019·Neuron
DOI 10.1016/j.neuron.2019.08.009PMC PMC6859713
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Abstract

Astrocytes are particularly promising candidates for reprogramming into neurons, as they maintain some of the original patterning information from their radial glial ancestors. However, to which extent the position of astrocytes influences the fate of reprogrammed neurons remains unknown. To elucidate this, we performed stab wound injury covering an entire neocortical column, including the gray matter (GM) and white matter (WM), and targeted local reactive astrocytes via injecting FLEx switch (Cre-On) adeno-associated viral (AAV) vectors into mGFAP-Cre mice. Single proneural factors were not sufficient for adequate reprogramming, although their combination with the nuclear receptor-related 1 protein (Nurr1) improved reprogramming efficiency. Nurr1 and Neurogenin 2 (Ngn2) resulted in high-efficiency reprogramming of targeted astrocytes into neurons that develop lamina-specific hallmarks, including the appropriate long-distance axonal projections. Surprisingly, in the WM, we did not observe any reprogrammed neurons, thereby unveiling a crucial role of region- and layer-specific differences in astrocyte reprogramming.

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Graphical abstract CC BY-NC-ND · PubMed Central
Figure 1 Neurogenic Factors Reprogram Astrocytes into Neurons after Traumatic Brain Injury (A and B) Scheme of the AAV-FLEx constructs (A) and experimental design (B). (C) Photomicrographs showing an overview with GFP+ cells at 24 dpi of AAV encoding for GFP, GFP/Ngn2/Nurr1, and GFP/Ascl1/Nurr1. (D–I) Photomicrographs showing GFP+/NeuN+ neurons (full arrowheads) and GFP+/GFAP+ astrocytes (empty arrowheads) at 24 (D) and 72 (G) dpi of GFP, Ngn2/Nurr1, and Ascl1/Nurr1. Example of Z-projection (E and H) of GFP/Ngn2/Nurr1 neurons (dashed square) used for the co-localization analysis (F and I). n = 3, 4, and 4 for GFP, Ngn2/Nurr1, and Ascl1/Nurr1, respectively. Data are shown as median ± interquartile range (IQR). Student’s t test. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗∗p ≤ 0.0001. AAV, adeno-associated virus; dpi, days post-injection. Scale bars: 100 μm (C, left); 50 μm (C, right); 20 μm (D and G). See also Figures S1 and S2.
Figure 1 Neurogenic Factors Reprogram Astrocytes into Neurons after Traumatic Brain Injury (A and B) Scheme of the AAV-FLEx constructs (A) and experimental design (B). (C) Photomicrographs showing an overview with GFP+ cells at 24 dpi of AAV encoding for GFP, GFP/Ngn2/Nurr1, and GFP/Ascl1/Nurr1. (D–I) Photomicrographs showing GFP+/NeuN+ neurons (full arrowheads) and GFP+/GFAP+ astrocytes (empty arrowheads) at 24 (D) and 72 (G) dpi of GFP, Ngn2/Nurr1, and Ascl1/Nurr1. Example of Z-projection (E and H) of GFP/Ngn2/Nurr1 neurons (dashed square) used for the co-localization analysis (F and I). n = 3, 4, and 4 for GFP, Ngn2/Nurr1, and Ascl1/Nurr1, respectively. Data are shown as median ± interquartile range (IQR). Student’s t test. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗∗p ≤ 0.0001. AAV, adeno-associated virus; dpi, days post-injection. Scale bars: 100 μm (C, left); 50 μm (C, right); 20 μm (D and G). See also Figures S1 and S2.CC BY-NC-ND · PubMed Central
Figure 2 Neurons Induced by Ngn2/Nurr1 Acquire Adequate Laminar Identity and Pyramidal Morphology (A) Photomicrographs and quantitative analysis of endogenous and induced neurons at 72 dpi of AAV-mScarlet-I and AAV-FLEx-mScarlet-I/Ngn2/Nurr1. The vertical dashed line in the right panel represents the intensity threshold (intensity of 0.2) defining positive cells. The heatmap shows the cell distribution in ten bins. The color-coded columns represent the percentage of mScarlet-I+/CUX1+ or mScarlet-I+/CTIP2+ cells per bin of the total number of mScarlet-I+/CUX1+ or mScarlet-I+/CTIP2+, respectively, in an entire column. Histograms illustrate the average of percentage for each bin; n = 5 animals. (B) Photomicrographs showing pyramidal morphology of mScarlet-I+ endogenous and induced neurons at 24 and 72 dpi. (C–G) Histograms depicting the morphological analyses of endogenous and induced neurons at 24 and 72 dpi for the following parameters: percentage of cell with an apical dendrite (C), thickness of the apical dendrite (D), circularity of the cell soma (E), number of basal dendrites (F), and their orientation (G), demonstrating the gradual maturation process of iNs; n = 4 animals. Data are shown as median ± IQR. One-way ANOVA. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. L, layer. Scale bars: 100 μm (A, overview); 20 μm (A, close up); 10 μm (B); 5 μm (G). See also Figures S3 and S4.
Figure 2 Neurons Induced by Ngn2/Nurr1 Acquire Adequate Laminar Identity and Pyramidal Morphology (A) Photomicrographs and quantitative analysis of endogenous and induced neurons at 72 dpi of AAV-mScarlet-I and AAV-FLEx-mScarlet-I/Ngn2/Nurr1. The vertical dashed line in the right panel represents the intensity threshold (intensity of 0.2) defining positive cells. The heatmap shows the cell distribution in ten bins. The color-coded columns represent the percentage of mScarlet-I+/CUX1+ or mScarlet-I+/CTIP2+ cells per bin of the total number of mScarlet-I+/CUX1+ or mScarlet-I+/CTIP2+, respectively, in an entire column. Histograms illustrate the average of percentage for each bin; n = 5 animals. (B) Photomicrographs showing pyramidal morphology of mScarlet-I+ endogenous and induced neurons at 24 and 72 dpi. (C–G) Histograms depicting the morphological analyses of endogenous and induced neurons at 24 and 72 dpi for the following parameters: percentage of cell with an apical dendrite (C), thickness of the apical dendrite (D), circularity of the cell soma (E), number of basal dendrites (F), and their orientation (G), demonstrating the gradual maturation process of iNs; n = 4 animals. Data are shown as median ± IQR. One-way ANOVA. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. L, layer. Scale bars: 100 μm (A, overview); 20 μm (A, close up); 10 μm (B); 5 μm (G). See also Figures S3 and S4.CC BY-NC-ND · PubMed Central
Figure 3 Neurons Induced by Ngn2/Nurr1 Develop Spines and Project to the Appropriate Brain Regions (A) Photomicrographs and quantification of spines on secondary dendrite segments of endogenous (top) and induced (bottom) neurons at 24 and 72 dpi of mScarlet-I/Ngn2/Nurr1; n = 4 animals. (B) Patch-clamp recording of endogenous or induced neurons in acute slice preparations of the cortex at 72 dpi as shown in the laser-Dodt-contrast and fluorescence images of mScarlet-I+ cell, before (top) and after filling the recorded cell with Alexa 488 (bottom). (C) Left: action potential in response to 100-ms current injection in 72 dpi iN shown in (B) (timescale expanded). Right: current-voltage responses recorded during 1-s current pulses from −300 to +400 pA are shown (of the same cell). (D) Excitatory spontaneous synaptic currents recorded in voltage clamp at −70 mV in mScarlet-I+ iNs (top trace) are blocked by NBQX (2nd trace). Inhibitory spontaneous synaptic currents recorded at −30 mV (3rd trace from top) are blocked by bicuculline (bottom trace). (E and F) Photomicrographs of mScarlet-I+ iN axons at 72 dpi in the ipsilateral (E) and contralateral (F) cortical hemisphere. (G) Left: schematic drawing indicating the experimental schedule and positions of callosal neurons with a low-power overview photomicrograph depicting the Fluorogold injection site. Middle: retrogradely traced iNs at 72 dpi are mainly located in L2/3 and L5. Right: histograms illustrate the percentage of Fluorogold+ endogenous or induced neurons in L2/3 that are SATB2+/CTIP2− and in L5 that are SATB2+/CTIP2− or SATB2−/CTIP2+ neurons; n = 3 animals. Data are shown as median ± IQR. One-way ANOVA. ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. cc, corpus callosum; Crb, cerebellum; Ctx, cortex; ic, internal capsule; OB, olfactory bulb; SC: spinal cord; Str, striatum; Th, thalamus; VL, lateral ventricle. Scale bars: 100 μm (E, left; F, left; and G, overview); 50 μm (E, right and F, middle); 20 μm (B and G, close up); 10 μm (A and F, right). See also Figure S5.
Figure 3 Neurons Induced by Ngn2/Nurr1 Develop Spines and Project to the Appropriate Brain Regions (A) Photomicrographs and quantification of spines on secondary dendrite segments of endogenous (top) and induced (bottom) neurons at 24 and 72 dpi of mScarlet-I/Ngn2/Nurr1; n = 4 animals. (B) Patch-clamp recording of endogenous or induced neurons in acute slice preparations of the cortex at 72 dpi as shown in the laser-Dodt-contrast and fluorescence images of mScarlet-I+ cell, before (top) and after filling the recorded cell with Alexa 488 (bottom). (C) Left: action potential in response to 100-ms current injection in 72 dpi iN shown in (B) (timescale expanded). Right: current-voltage responses recorded during 1-s current pulses from −300 to +400 pA are shown (of the same cell). (D) Excitatory spontaneous synaptic currents recorded in voltage clamp at −70 mV in mScarlet-I+ iNs (top trace) are blocked by NBQX (2nd trace). Inhibitory spontaneous synaptic currents recorded at −30 mV (3rd trace from top) are blocked by bicuculline (bottom trace). (E and F) Photomicrographs of mScarlet-I+ iN axons at 72 dpi in the ipsilateral (E) and contralateral (F) cortical hemisphere. (G) Left: schematic drawing indicating the experimental schedule and positions of callosal neurons with a low-power overview photomicrograph depicting the Fluorogold injection site. Middle: retrogradely traced iNs at 72 dpi are mainly located in L2/3 and L5. Right: histograms illustrate the percentage of Fluorogold+ endogenous or induced neurons in L2/3 that are SATB2+/CTIP2− and in L5 that are SATB2+/CTIP2− or SATB2−/CTIP2+ neurons; n = 3 animals. Data are shown as median ± IQR. One-way ANOVA. ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. cc, corpus callosum; Crb, cerebellum; Ctx, cortex; ic, internal capsule; OB, olfactory bulb; SC: spinal cord; Str, striatum; Th, thalamus; VL, lateral ventricle. Scale bars: 100 μm (E, left; F, left; and G, overview); 50 μm (E, right and F, middle); 20 μm (B and G, close up); 10 μm (A and F, right). See also Figure S5.CC BY-NC-ND · PubMed Central
Figure 4 Absence of Induced Neurons after Transduction of White Matter Astrocytes with Ngn2/Nurr1 after Traumatic Brain Injury (A and B) Photomicrographs showing GFP+ cells at 10, 24, and 72 dpi of GFP (A) and GFP/Ngn2/Nurr1 (B). Histograms show the percentage of marker+/− cells among the GFP+ cells for each experimental animal (3 or 4 per condition). Note that GFP+ cells were always detected in the control (all were astrocytes) but either not detectable or none were NeuN+ after Ngn2/Nurr1 injection. Data are shown as median ± IQR of different technical replicates, i.e., immunostainings of different sections. Scale bars: 20 μm.
Figure 4 Absence of Induced Neurons after Transduction of White Matter Astrocytes with Ngn2/Nurr1 after Traumatic Brain Injury (A and B) Photomicrographs showing GFP+ cells at 10, 24, and 72 dpi of GFP (A) and GFP/Ngn2/Nurr1 (B). Histograms show the percentage of marker+/− cells among the GFP+ cells for each experimental animal (3 or 4 per condition). Note that GFP+ cells were always detected in the control (all were astrocytes) but either not detectable or none were NeuN+ after Ngn2/Nurr1 injection. Data are shown as median ± IQR of different technical replicates, i.e., immunostainings of different sections. Scale bars: 20 μm.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Mattugini, N., Bocchi, R., Scheuss, V., Russo, G. L., Torper, O., Lao, C. L., & Götz, M.
Affiliations
Helmholtz Munich (DE)
Corresponding author
Götz, Magdalena
ORCID
Venue
Neuron
Publisher
Elsevier BV
Published
2019-09
Volume
103
Issue
6
Pages
1086-1095.e5
ISSN
0896-6273 (ISSN-L)
DOI
10.1016/j.neuron.2019.08.009
PMC
PMC6859713
OpenAlex
W2971841027
Cited by
159
References
42
Citations by year
2026: 12 · 2025: 26 · 2024: 28 · 2023: 19 · 2022: 26 · 2021: 35 · 2020: 11 · 2019: 2
FWCI
6.97
Type
journal article
Language
en
Open access
Licence
Locations
  • Neuron (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Open access LMU (Ludwid Maxmilian's Universitat Munchen) (submittedVersion, cc-by-nc-nd)
  • Site cant be reached (submittedVersion, cc-by-nc-nd)
  • PuSH - Publication Server of Helmholtz Zentrum München (submittedVersion, cc-by-nc-nd)
Funders
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (P400PB_183826, P2GEP3_174900)
  • European Research Council (340793)
  • Fondation Roger de Spoelberch
  • ERA-LEARN 2020
  • Deutsche Forschungsgemeinschaft (01EW1705B, SFB 870, A06)
Topics
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Nuclear Receptors and Signaling
Keywords
neuronal subtype specification, stab wounds, cerebral cortex, Nurr1, Neurogenin2, white matter, gray matter, adeno-associated virus vectors
MeSH terms
Cellular Reprogramming Techniques, Brain Injuries, Traumatic, Dependovirus, Animals, Astrocytes, Cerebral Cortex, Genetic Vectors, Gliosis, Nerve Tissue Proteins, Neurons, Wounds, Stab, Pyramidal Cells, Mice, Basic Helix-Loop-Helix Transcription Factors, Basic Helix-Loop-Helix Proteins, Nuclear Receptor Subfamily 4, Group A, Member 2, White Matter, Gray Matter

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