Revisiting astrocyte to neuron conversion with lineage tracing in vivo

Abstract

In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.

Keywords: AAV; CRISPR-CasRx; DLX2; NEUROD1; PAX6; PTBP1; astrocyte-to-neuron conversion; in vivo reprogramming; lineage tracing; shRNA.

Figure 1.. NEUROD1-induced reporter+ neurons neither pass through an immature stage nor come from reactive astrocytes.

(A) Study design to examine AtN conversion. IHC, immunohistochemistry; dpv, days post virus-injection. (B) Confocal images of the indicated markers. Scales, 50 μm. (C) Efficient induction of mCherry⁺ neurons by NEUROD1 (n = 3-4 mice per group per time-point; mean ± SEM; ***p<0.001; n.s., not significant). (D) Cell numbers in virus-injected cortex (n = 3-4 mice per group per time-point; mean ± SEM; n.s., not significant). (E) Study design to examine immature neurons. (F) Confocal images showing a lack of DCX⁺ cells in virus-injected cortex. Cells in the lateral ventricle (LV) are used as controls. Scales, 50 μm. (G) Study design to trace reactive cells. BrdU was delivered through drinking water. CCI, controlled cortical impact. (H) Robust BrdU-labeling of reactive astrocytes (n = 4 mice per group; mean ± SEM; n.s., not significant). (I) Confocal images of BrdU-labeled cells. Scales, 50 μm. (J) Quantification of mCherry⁺ cells (n = 4 mice per group; mean ± SEM; ****p < 0.0001; n.s., not significant). (K) Quantification of neurons in virus-injected cortex (n = 4 mice per group; mean ± SEM; n.s., not significant). (L) Confocal images of the indicated markers. A BrdU⁺ astrocyte-like NeuN⁺ cell is shown in ND1-mCh group. Scales, 50 μm. See also Figure S1 and Table S1.

Figure 2.. Genetically traced astrocytes are not an origin for NEUROD1-induced reporter⁺ neurons.

(A) Study design to trace resident astrocytes with the YFP reporter. Tam, tamoxifen. (B) Confocal images of YFP-traced cells. Scales, 50 μm. (C) Quantifications of mCherry⁺ cells (n = 3-4 mice per group; mean ± SEM; ***p = 0.0003; n.s., not significant). (D) Study design to trace resident astrocytes with the tdTomato reporter. (E) Confocal images of tdTomato-traced cells. Scales, 50 μm. (F) Quantifications of GFP⁺ cells (n = 4 mice per group; mean ± SEM; ****p < 0.0001; n.s., not significant). (G) Study design to examine the effect of brain injury on AtN conversion. (H) Confocal images of YFP-traced cells. Scales, 50 μm. (I) Quantifications of mCherry⁺ cells after CCI (n = 4 mice per group; mean ± SEM; **p = 0.0022; n.s., not significant). See also Figure S2.

Figure 3.. Astrocyte-restricted NEUROD1 cannot rapidly induce reporter+ neurons.

(A) A schematic illustration of the Cre-FLEX system. (B) Study design to examine AtN conversion through the Cre-FLEX system. (C) Confocal images of the indicated markers. Scale, 50 μm. (D) Quantifications showing robust NEUROD1 expression in astrocytes (n = 4 mice per group; mean ± SEM). (E) Confocal images of the indicated markers. Scales, 50 μm. (F) Quantifications showing a basal number of mCherry⁺ neurons (n = 4 mice per group; mean ± SEM; n.s., not significant). (G) Study design to examine AtN conversion from reactive cells. (H) Confocal images of the indicated markers. Scales, 50 μm. (I) Quantifications showing a basal number of mCherry⁺ neurons (n = 4 mice per group; mean ± SEM; n.s., not significant). (J) Quantification of neurons in virus-injected cortex (n = 3-4 mice per group; mean ± SEM; n.s., not significant). (K) Study design to examine the effect of tamoxifen on induction of mCherry⁺ neurons. (L) Confocal images of the indicated markers. Scales, 50 μm. (M) Quantifications of mCherry⁺ cells (n = 4 mice per group; mean ± SEM; ****p < 0.0001; n.s., not significant). See also Figures S3 and S4.

Figure 4.. A combination of NEUROD1 and DLX2 fails to convert striatal astrocytes.

(A) A schematic illustration of the Cre-FLEX system. (B) Study design to examine AtN conversion in the striatum. Astrocytes were traced with YFP after Tamoxifen treatments. (C) Confocal images of the indicated markers. Scales, 50 μm. (D) Quantifications showing a lack of YFP-traced mCherry⁺ neurons (n = 4 mice per group; mean ± SEM; ****p < 0.0001). (E) Study design to examine AtN conversion in the striatum. Astrocytes were constitutively traced with YFP. (F) Confocal images of the indicated markers. Scales, 50 μm. (G) Quantifications showing a lack of YFP-traced mCherry⁺ neurons (n = 4 mice per group; mean ± SEM; ****p < 0.0001). (H) Study design to examine NEUROD1 and DLX2 expression. (I) Confocal images of the indicated markers. DLX2 expression is indicated by the HA staining. Scales, 50 μm. (J) Quantifications showing robust expression of NEUROD1 and DLX2 (indicated by HA) in mCherry⁺ cells (n = 3 mice per group; mean ± SEM). See also Figure S5.

Figure 5.. Virus-induced reporter⁺ neurons are endogenous neurons.

(A) Study design to label corticomotor neurons. T6, the 6^th thoracic spinal cord level. (B) Series of brain sections showing retrograde labeling of the motor cortex. Ventricles are outlined. Scale, 1 mm. (C) Confocal images of the motor cortex showing GFP-traced neurons. Scales, 50 μm. (D) Confocal images showing a complete lack of GFP-traced astrocytes in the motor cortex. Scale, 50 μm. (E) Study design to determine the contribution of endogenous neurons to virus-induced mCherry⁺ neurons. (F) Confocal images of the motor cortex showing reporter-labeled neurons. Arrowheads show examples of neurons with dual reporters. Scales, 50 μm. (G) Quantifications showing robust induction of mCherry⁺ neurons by NEUROD1 in the cortex (n = 4-5 mice per group; mean ± SEM; ****p < 0.0001). (H) Quantifications showing endogenous neurons as the cell source for NEUROD1-induced mCherry⁺ neurons (n = 4-5 mice per group; mean ± SEM; ***p = 0.0002). (I) Confocal images showing reporter-labeled cortical neurons using study designs as shown in panel E. Arrowheads show examples of neurons with dual reporters. Scales, 50 μm. (J) Confocal images showing examples of factors with minimal induction of mCherry⁺ neurons. Scales, 50 μm. (K) Quantifications of the indicated factors for their ability to induce mCherry⁺ neurons (n = 4 mice per group; mean ± SEM).

Figure 6.. NeuroD1 cis-regulates the viral promoter and does not require its neurogenic activity to induce reporter⁺ neurons.

(A) Study design to examine NEUROD1 on viral promoter activity. (B) Study design to examine a mixture of individually packaged AAV5 viruses. (C) Confocal images showing cell type-specificity of the indicated viral reporters. Scales, 50 μm. (D) Quantifications showing neuronal expression of the cis-expressed mCherry but not the trans-expressed GFP (n = 3 mice per group; mean ± SEM). (E) Study design to examine co-packaged AAV5 viruses. (F) Confocal images showing cell type-specificity of the indicated viral reporters. Scales, 50 μm. (G) Quantifications showing neuronal expression of the cis-expressed mCherry but not the trans-expressed GFP (n = 3 mice per group; mean ± SEM). (H) A schematic of NEUROD1 and its mutants. bHLH, basic helix-loop-helix; AD1, activation domain 1; AD2, activation domain 2. (I) Study design to examine neurogenic activity in U251 cells. ICC, immunocytochemistry. (J) Confocal images showing a loss of neurogenic ability of NEUROD1 point-mutants (n = 4 biological replicates). DCX and MAP2 were used as markers of neurons. Scales, 50 μm. (K) Confocal images of the indicated markers in the cortex at 17 dpv. Scales, 50 μm. (L) Quantifications showing robust induction of mCherry⁺ neurons by NEUROD1 point-mutants (n = 3-4 mice per group; mean ± SEM).

Figure 7.. PTBP1 knockdown fails to convert striatal astrocytes in vivo.

(A) Study design. Striatal astrocytes were constitutively traced with YFP. (B) Confocal images of the indicated markers. Scales, 50 μm. (C, D) Quantifications showing robust and efficient PTBP1 knockdown (n = 4 mice per group; mean ± SEM; ****p < 0.0001). (E) A schematic showing the striatal region for analysis (F) Confocal images of the indicated markers. Scales, 50 μm. (G) Quantifications showing a lack of YFP-traced RFP⁺ neurons (n = 4 mice per group; mean ± SEM; ****p < 0.0001). (H) Study design. Striatal astrocytes were constitutively traced with YFP. (I) Confocal images of the indicated markers. Scales, 50 μm. (J) Quantifications showing a lack of YFP-traced RFP⁺ neurons (n= 4 mice per group; mean ± SEM; **p = 0.0056). (K) Study design. AAV2 viruses were from Fu laboratory. Striatal astrocytes were constitutively traced with YFP. (L) Confocal images of the indicated markers. Scales, 50 μm. (M) Quantifications showing a lack of YFP-traced RFP⁺ neurons (n = 4 mice per group; mean ± SEM; n.s., not significant). See also Figures S6 and S7.