Neuroscience

Lineage tracing and live-cell imaging reveal that NeuroD1 does not reprogram microglia into neurons

Xiaoyu Li
Yuxin Li
Yue Cao
Niewen Hu
Baozhi Yang
Pei Ouyang
Yuxiao Jin
Shuai Gao
Bo Peng author has email address
Yanxia Rao author has email address

National Children’s Medical Center, Children’s Hospital, Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, MOE Innovative Center for New Drug Development of Immune Inflammatory Diseases, Shanghai Key Laboratory of Gene Editing and Cell Therapy for Rare Diseases, Fudan University, Shanghai, China
Department of Neurology, Zhongshan Hospital, Laboratory Animal Center, MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China
Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China

https://doi.org/10.7554/eLife.112083.1

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Figures and data

Genetic gain of function of NeuroD1 does not result in microglia-to-neuron conversion in the adult mouse brain.
(A) Schematic of the specific expression of NeuroD1 and lineage tracing of microglia. (B) Experimental timeline. (C) Representative confocal images at D18 showing that NeuroD1 is robustly co-expressed in GFP-positive cells after Cre recombination. (D) Quantification of the percentage of NeuroD1⁺GFP⁺ cells among GFP⁺ cells and NeuroD1⁺GFP^- cells among NeuroD1⁺ at D18, revealing NeuroD1 is highly co-expressed with GFP. N = 5 mice for each group. (E) Representative confocal images at D18 showing that GFP-positive cells do not colocalize with neuronal marker NeuN. (F) Quantification of NEUN⁺GFP⁺ double-positive cells in NEUN⁺ cells at D18. N = 6 mice for each group. (G) Representative confocal images at D18 showing that GFP-positive cells do colocalize with microglial marker IBA1. (H) Quantification of IBA1⁺GFP⁺ double-positive cells in GFP⁺ cells at D18. N = 5 mice for each group. Data are presented as mean ± SD.

Ectopic NeuroD1 expression in microglia does not induce microglia-to-neuron conversion across time.
(A) Experimental timeline. (B) Representative confocal images at D4, D10, D30 and D45 showing that GFP is co-labeled with IBA1. (C) Representative confocal images at D4, D10, D30 and D45 showing that GFP is not co-labeled with NeuN. (D) Quantification of IBA1⁺GFP⁺ double-positive cells among GFP⁺ cells at D4, D10, D30 and D45. N = 4 or 5 mice for each group. (D) Quantification of NeuN⁺GFP⁺ double-positive cells among GFP⁺ cells at D4, D10, D30 and D45. N = 4 or 5 mice for each group.

NeuroD1-expressing microglia retain microglial morphology and decline in number over time.
(A) Experimental timeline. (B) Representative images showing GFP-positive cells with highly ramified morphologies characteristic of microglia rather than neurons. (C) Represent images illutrating changes in the number of GFP-postive cells over time (green dots indicate GFP-positive cell). (D) Quantification of GFP-positive cells density.N = 4 - 6 mice per time point. Data are presented as mean ± SD. One Way ANOVA with Turkey’s multiple comparison test. (E) Represent confocal image shows the LC3 and P62 level in control and NeuroD1MG mice. Scale bar = 50μm. (F) Quantification of P62⁺ area per cell. Unpaired t test. (G) Quantification of LC3⁺ area per cell. Unpaired t test. (H) Data are presented as mean ± SD.

NeuroD1 does not induce microglia-to-neuron conversion even in TBI model.
(A) Experiment design. (B) Quantification of grip strength showing no improvement in NeuroD1-expressing TBI mice. Two-way ANOVA with multiple comparisons. (C) Quantification of rotarod performance showing no improvement in NeuroD1-expressing mice. Two-way ANOVA with multiple comparisons. (D) Representative confocal images of DAPI, GFP and NeuN co-staining results in NeuroD1^MG TBI mice, showing no co-localization of GFP with NeuN in either the lesion core or distal regions. Scale bar, 50 μm. (E) Quantification of the percentage of GFP⁺NeuN⁺ cells among total NeuN⁺ cells in both the lesion area and distal area. n.d., not detected. (F) Representative confocal images of DAPI, GFP, and MAP2 in NeuroD1^MG mice, showing no co-localization of GFP with MAP2 in either the lesion core or distal regions. Scale bar, 50 μm. (G) Quantification of the percentage of GFP⁺MAP2⁺ cells among total MAP2⁺ cells in the lesion area and distal region. n.d., not detected. (H) UMAP reduction shows the cell types of the FACS-sorted reporter⁺ cells in each group; note that there are no neurons. (I) Feature plot showing that the microglia-specific marker P2ry12 was highly expressed even under TBI conditions, but the neuron-specific marker Rbfox3 was barely expressed. (J) Violin plot showing the expression of microglia-specific genes (Hexb and Tmem119) and neuron-specific genes (Tubb3 and Map2) in the collected cells.

Lentivirus- or AAV-based infection caused leaky neuronal expression in vivo.
(A) The study design shows the different viruses used here. (B) Representative confocal images showing the expression of GFP, IBA1 and NEUN in the brains of the lentivirus- or AAV-infected mice. (C) The efficacy of infection was quantified, and the data are presented as the ratio of IBA1⁺GFP⁺/GFP⁺ cells. N = 3 mice for each group.

NeuroD1 is robustly co-expressed in GFP-positive cells after Cre recombination.
(A) Experimental timeline. (B) Representative confocal images at D4, D10, D30 and D45 showing that NeuroD1 is robustly co-expressed in GFP-positive cells after Cre recombination. (C) Quantification of NeuroD1⁺GFP⁺ double-positive cells in GFP⁺ cells and NeuroD1⁺GFP^- in NeuroD1⁺ cells at D4, D10, D30 and D45. N = 5 mice for each group.

Ectopic NeuroD1 expression in microglia does not induce microglia-to-neuron conversion, as GFP does not co-localize with TUJ1 and MAP2.
(A) Experimental timeline. (B) Representative confocal images at D4, D10, D30 and D45 showing that GFP is not co-labeled with TUJ1 in the brain. (C) Representative confocal images at D4, D10, D30 and D45 showing that GFP is not co-labeled with MAP2 in the brain. (D) Quantification of TUJ1⁺GFP⁺ double-positive cells among TUJ1⁺ cells at D4, D10, D30 and D45. N = 4 or 5 mice for each group. (E) Quantification of MAP2⁺GFP⁺ double-positive cells among MAP2⁺ cells at D4, D10, D30 and D45. N = 4 or 5 mice for each group.

TUNEL staining shows NeuroD1expression triggers microglia apoptosis.
(A) Experiment timeline. (B) Represent confocal image shows TUNEL⁺reporter⁺ cells are observed in NeuroD1^MG mice at D4. Scale bar = 50μm. (C) Represent confocal image shows TUNEL⁺reporter⁺ cells are observed in NeuroD1^MG mice at D18. Scale bar = 50μm. (D) Quantification result shows TUNEL⁺reporter⁺ cells are higher than control group.

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