Olig2 acts as an inducible barrier to in vivo astrocyte-to-neuron conversion

Domain: neuroscience
Published: 2026-01-26
Editorial outcome: major_revision

Abstract
In vivo astrocyte-to-neuron (AtN) conversion suffers low efficiency due to pre-existing intrinsic barriers. However, it is unclear whether astrocytes have inducible barriers to reprogramming. Here, we identify Olig2, a basic helix-loop-helix (bHLH) transcription factor, as an inducible barrier to Ngn2-mediated AtN conversion. Olig2 is strongly upregulated in cortical astrocytes following the ectopic expression of bHLH neuronal reprogramming factors such as Ngn2, NeuroD1, and Ascl1, but is barely expressed in normal astrocytes. Knocking down Olig2 in Ngn2-transduced astrocytes reduces astrocyte-specific gene expression, enhances neurogenesis-related gene expression, and increases AtN conversion efficiency by approximately threefold. Further multi-omics analysis shows that astrocytic Olig2 directly binds to regulatory regions of pro-neurogenic genes, including Ngn2, inhibiting their expression and impeding the expression of neural progenitor genes. Collectively, our findings reveal Olig2 as an inducible barrier to AtN conversion, providing insights into the regulation of neuronal reprogramming.

Introduction
The adult mammalian central nervous system has limited neurogenic capacity after neuronal loss, while glial cells can proliferate and typically produce glial scars throughout life1. Among these glia, astrocytes exhibit neural stem cell-like properties in certain diseased conditions2,3, and are thus considered potential endogenous candidate cells for in vivo AtN conversion4,5. In general, the bHLH proneural transcription factors (TFs, e.g., Ngn2, Ascl1 and NeuroD1) are frequently selected as the key pioneer factors for driving AtN conversion because of their essential contribution to neurogenesis during development6–8. Despite significant progress in AtN conversion in animal studies, the barriers that inhibit AtN conversion are still not fully understood. Therefore, the low efficiency of AtN conversion will inevitably hinder its future clinical application.

Terminal differentiated cells maintain their identity through a complex interplay of genetic and epigenetic mechanisms that hinder cellular reprogramming9. In general, to achieve efficient cell reprogramming, we have to overcome these resistances existing within the cells10,11. For instance, the RE1 silencing transcription factor (REST) complex, known for its competition with Ngn2 for binding to the NeuroD4 promoter, acts as a barrier to AtN conversion12; the Notch signaling pathway, a highly conserved signaling system that is critical for regulating neurogenesis and promoting astrocytogenesis, is identified as another obstacle to AtN conversion3,13,14; unfolded protein response caused by gene mutation is recently identified as a novel hurdle for astrocyte reprogramming15; one study used a transcriptionally activated form of Olig2-VP16 to antagonize the function of Olig2 in dividing glial cells, promoting the generation of immature neurons, imply that Olig2 acts as a suppressor of neurogenesis in these dividing glial cells16. In addition, DNA methylation and microRNA (miRNA) inside cells also exert an important influence on AtN conversion at epigenetic level4,8,17. Apparently, the aforementioned obstacles are intrinsic and pre-existed in astrocytes regardless of AtN initiation, acting as the “innate” barriers to AtN conversion. Whether there is any obstacle that is absent in the normal astrocytes but induced after the ectopic expression of proneural TFs to induce neuronal reprogramming remains largely unknown.

In this study, we discovered the increase of Olig2+ cells during the in vivo TFs-mediated AtN conversion in the adult mouse cortex. These Olig2+ cells did not originate from the proliferation of oligodendrocyte precursor cells, but rather from astrocytes that ectopically expressed bHLH proneural TFs (Ngn2, Ascl1, or NeuroD1). Knockdown of Olig2 expression in astrocytes significantly enhanced the efficiency of Ngn2-mediated AtN conversion. ScRNA-seq study demonstrated that Olig2 knockdown in Ngn2-transduced astrocytes significantly altered their transcriptome, including further suppression of astrocyte-related genes and upregulation of neurogenesis-related genes. CUT&Tag-seq analysis revealed that astrocytic Olig2 directly bound to pro-neurogenesis genes and repressed their expression. Collectively, our study identifies a distinct inhibitory mechanism for AtN conversion, where the induced Olig2 upregulation in astrocytes counteracts the effect of proneural TFs, revealing a concept of “inducible” cellular defense against fate switching.

Results
bHLH pioneer transcription factors induce the expression of Olig2 in astrocytes
The brain parenchyma is predominantly composed of neurons and glial cells, encompassing astrocytes, oligodendrocytes, and microglia, which exhibit relatively stable population densities under normal conditions. In vivo AtN conversion utilizes endogenous astrocytes to generate neurons, which may affect the cell composition and microenvironments in the conversion regions. Therefore, to illuminate how AtN conversion affects the local cell populations, we examined the density of different cell types within the conversion regions. AAV9-based gene delivery system was used to target astrocytes via a synthetic GFAP promoter (hGFAP). The specificity of AAV9-hGFAP system was verified by immunostaining at 7 days post injection (dpi) of AAV9-hGFAP::tdTomato (tdT, control, Fig. 1a). Approximately 100% of tdT+ cells were co-labeled with the pan-astrocytic marker S100β (Fig. 1b, c). tdT reporter gene was not expressed in oligodendrocyte lineage cells (Sox10), microglia (Iba1), and neurons (NeuN, Fig. 1b). When AAV9-hGFAP::Ngn2-tdT, AAV9-hGFAP::Ascl1-tdT, or AAV9-hGFAP::NeuroD1-tdT was respectively injected in the mouse cortex (AAV9-hGFAP::TF-tdT, Fig. 1a), they were specifically expressed in the transduced astrocytes, as demonstrated by immunostaining (tdT+, Supplementary Fig. 1a–c). These results indicate that AAV9 combined with the hGFAP promoter is a highly specific gene delivery system for targeting astrocytes.

Then we examined the density of local cells in the AAV-TF transduced regions at 7 dpi and found no significant difference in the density of neurons (NeuN), astrocytes (S100β), microglia (Iba1), mature oligodendrocytes (CC1), and NG2 cells in the cortex of Ngn2-transduced mice compared with mice that transduced with tdT (Supplementary Fig. 1d). Furthermore, Ngn2 overexpression did not affect the morphology of microglia, that are the primary immune cell inside the brain (Supplementary Fig. 2). However, the density of Olig2+ cells increased 1.5-fold in AAV9-Ngn2 infected regions compared to the control group at 7 dpi (Fig. 1d, e). This phenomenon was also observed in the cortex of mice injected with AAV9-Ascl1 and AAV9-NeuroD1 (Fig. 1d, e; 7 dpi). Olig2+ oligodendrocyte progenitor cells (OPCs) are the major proliferating cells in the adult brain, so we wondered whether the increased density of Olig2+ cells was caused by OPC proliferation. To test this hypothesis, Sox10-CreERT2 mice were crossed with Rosa-tdTomato reporter mice (Ai14) to label oligodendrocytes and OPCs in the mouse brain18. As expected, Sox10 and Olig2 immunosignals were specifically observed in tdT+ cells in the cortex, indicating that these tdT+ cells are derived from the oligodendrocyte lineage (Supplementary Fig. 3a–e). Then, the lineage-tracing transgenic mice were administrated with AAV9-hGFAP::GFP (control) or AAV9-hGFAP::Ngn2-P2A-GFP in cortical regions at 2–5 months of age 10 days after tamoxifen induction, and mice were sacrificed for immunohistochemistry (IHC) at 7 days after AAV9 injection (Fig. 1f). Consistently, the reporter gene GFP was not expressed in the oligodendrocyte lineage cells (tdT+, Fig. 1f left) and the Olig2+ cells were dramatically increased in AAV9-Ngn2 injected cortical regions compared to the control (Fig. 1f right, 1g). In the control group, Olig2 was essentially detected in all oligodendrocyte lineage cells (Ai14-tdT+), but not in AAV-infected cells (GFP+, Fig. 1h), showing that AAV-GFP didn’t change the Olig2 expression pattern and that Olig2 was restricted to oligodendrocyte lineage cells in the adult mouse cortex. In contrast, when AAV9-Ngn2-GFP was transduced into the astrocytes, Olig2 immunoreactivity was not only detected in oligodendrocyte lineage cells (Ai14-tdT+) but also in the Ngn2-transduced cells (GFP+, Fig. 1f right, 1h). Quantitatively, about 30% of Olig2+ cells were not co-labeled with tdTomato but with GFP in AAV9-Ngn2 injected areas (Fig. 1i). However, the number of oligodendrocyte lineage cells (Ai14-tdT+) was comparable in both groups (Fig. 1j). In addition, there was no significant difference in the Olig2 expression intensity between the two groups in oligodendrocyte lineage cells (Supplementary Fig. 3f, g). Together, these results suggest that the increase of Olig2+ cells after the overexpression of Ngn2 in cortical astrocytes is not derived from the proliferation of OPCs.

Olig2 is primarily expressed in oligodendrocytes and their progenitors in the normal adult brain. However, Olig2 transient expression in the astrocytes has been reported after acute cortical injury19. Since AAV9 combined with the hGFAP promoter specifically targets astrocytes (Fig. 1b, c and Supplementary Fig. 1a–c), we wanted to know the relationship between the upregulation of Olig2 and the ectopic expression of proneural TFs in the astrocytes. In the control group, there were few Olig2 immunostaining signals in tdT+ cells (Fig. 1k left and Supplementary Fig. 1e left), whereas almost all tdT+ cells that expressed Ngn2, Ascl1, or NeuroD1 were Olig2 positive (Supplementary Fig. 1f–h). To further investigate that Olig2 was upregulated in cortical astrocytes, immunostaining for three typical astrocyte markers (GFAP, S100β, and Aldh1l1) was performed to identify astrocytes. About 5% of astrocytes were observed with Olig2 in the control group, however, this co-labeling rate was dramatically increased to nearly 100% after overexpression of Ngn2 at 7 dpi (Fig. 1k right, 1l and Supplementary Fig. 1e right). Notably, astrocytic Olig2 was induced as early as 3 dpi following AAV9-Ngn2 administration and remained at a high level until 90 dpi in our examined time window (Fig. 1m, n). Collectively, these results indicate that Olig2 is significantly upregulated in cortical astrocytes following the ectopic expression of bHLH proneural transcriptional factors.

Non-bHLH transcription factors such as Sox2 and Dlx2 are also commonly selected to drive AtN conversion in mouse brain20–22. We then asked whether non-bHLH transcription factors could induce the expression of Olig2 in the astrocytes. In contrast to the bHLH proneural TFs, neither Sox2 (AAV9-hGFAP::Sox2-GFP) nor Dlx2 (AAV9-hGFAP::Dlx2-GFP) was able to induce the expression of Olig2 in the transduced cells (Supplementary Fig. 4). Thus, these data suggest that the ectopic expression of the bHLH pioneer TFs (Ngn2, Ascl1, and NeuroD1) in cortical astrocytes results in upregulation of Olig2 in the transduced astrocytes, whereas other non-bHLH transcription factors do not exhibit this effect.

Inhibiting Olig2 expression promotes AtN conversion
Olig2 is a member of the bHLH family and plays a critical role during the differentiation and maturation of oligodendrocyte lineage cells in the central nervous system23–25, but the impact of Olig2 on AtN conversion needs further elucidation. While the molecular mechanisms of Ngn2-mediated AtN conversion are well understood, most studies have been in vitro rather than in vivo, and none have used the AAV-GFAP system employed here8,12,26. Therefore, we selected AAV-GFAP::Ngn2-mediated AtN conversion as a model to elucidate the role of Olig2 in the AtN conversion. For this purpose, we designed shRNAs to downregulate Olig2 (shOlig2) in astrocytes. We used EGFP for visualization (Supplementary Fig. 5a) and observed that the reporter gene (GFP) was primarily expressed in astrocytes and neurons, with minimal expression in NG2+ OPCs, CC1+ oligodendrocytes, or Iba1+ microglia (Supplementary Fig. 5b, c). Therefore, there was almost no impact on the endogenous Olig2 expression in OPCs and oligodendrocytes with AAV9-shOlig2 injection, as indicated by the similar proportion and expression level of Olig2 in OPCs and oligodendrocytes between the control shRNA (shNC) group and the shOlig2 group (Supplementary Fig. 5d–g). To utilize more channels for immunofluorescence staining, we excluded the EGFP sequence from the knockdown vector in subsequent experiments. When we combined Olig2 knockdown with Ngn2 overexpression, we observed a significant decrease in the mRNA level of Olig2 in fluorescence-activated cell sorting (FACS) purified tdT+ cells in the Ngn2+shOlig2 group (Fig. 2a, b). Further immunostaining studies showed that at 7 dpi, Olig2 was sharply decreased in Ngn2+shOlig2 treated mice (0.1% in tdT+ cells) compared to the Ngn2+shNC group (94.2%; Fig. 2c, d), indicating that the upregulation of Olig2 in Ngn2-overexpressing astrocytes was effectively inhibited by shOlig2.

Next, we wanted to investigate the effect of Olig2 knockdown on Ngn2-mediated neuronal reprogramming, and four groups (tdT, Ngn2, Ngn2+shNC, Ngn2+shOlig2) were included for comparison (Fig. 2e). At 7 dpi, essentially all virus-infected cells were S100β+ astrocytes among the four groups (Supplementary Fig. 6a). However, at 45 dpi, more tdT+ cells showed NeuN signal in the Ngn2+shOlig2 injected mouse brain (Fig. 2f). Quantitative analyses revealed that approximately 30% of tdT+ cells became neurons (indicated by NeuN signal, Fig. 2g). Parallel to this trend, the percentage of tdT+ cells co-localized with S100β decreased from 98.1% at 7 dpi to 33.3% at 45 dpi in the Ngn2+shOlig2 group (Fig. 2h). Conversely, among the other three groups (tdT, Ngn2, and Ngn2+shNC), the number of tdT+ cells acquiring neuronal identity (NeuN+) was significantly lower than that of the Ngn2+shOlig2 group at 45 dpi (0.1% for tdT, 9.9% for Ngn2, 11.0% for Ngn2+shNC), and the remaining tdT+ cells were immunopositive for S100β (Fig. 2g, h). Notably, most of the converted neurons in the Ngn2+shOlig2 group were labeled by the glutamatergic neuron marker CamKII (Supplementary Fig. 6c, d), which is consistent with previous findings that Ngn2 directs the reprogramming of astrocytes into glutamatergic neurons12,27.

To elucidate the dynamic process of AtN conversion, we examined the morphological changes of tdT+ cells in the Ngn2+shNC and Ngn2+shOlig2 groups from 7 to 45 dpi. In the Ngn2+shOlig2 group, tdT+ cells initially displayed a typical astrocytic morphology during the first 14 days, co-localizing with S100β but not with the neuronal marker NeuN (Fig. 2g, h, j). At 21 dpi, round-shaped tdT+ cells emerged, which were double-negative for both S100β and NeuN, though their proportion was still small (Fig. 2i, j). At this stage, the neural stem cell markers Vimentin and Nestin were expressed in the tdT+ cells in Ngn2+shOlig2 group, while DCX expression was absent (Supplementary Fig. 7). By 30 dpi, the percentage of these double-negative cells increased, and a small number of tdT+ cells began to express NeuN (Fig. 2g, i, j). By 45 dpi, the proportion of tdT+ cells expressing NeuN had further increased, while the proportion of double-negative cells began to decline (Fig. 2g–j). In contrast, the Ngn2+shNC group exhibited minimal morphological changes, with most tdT+ cells retaining a typical astrocytic morphology throughout the time course (Supplementary Fig. 6b). Only at 45 dpi did a small fraction of tdT+ cells (approximately 10%) express NeuN (Fig. 2g–i). These findings indicate that Olig2 knockdown in astrocytes significantly enhances Ngn2-mediated AtN reprogramming and suggest the existence of a transient double-negative intermediate state during the conversion of astrocytes to neurons.

It was worth noting that during the conversion process, some tdT+ cells in the Ngn2+shOlig2 group were double negative for astrocytic marker S100β and neuronal marker NeuN (28.6% at 21 dpi, 44.8% at 30 dpi, 37.9% at 45 dpi), which was rare in other 3 groups (tdT, Ngn2 and Ngn2+shNC; Fig. 2i). We next wondered whether these double-negative cells had lost typical astrocyte features and used two other astrocyte markers, GFAP and Sox9, co-stained with NeuN at 30 dpi, respectively. The three populations of tdT+ cells were detected in the brains of mice treated with Ngn2+shOlig2. First, GFAP and Sox9 were positive in tdT+ cells without NeuN expression, and these cells represented astrocytes (Fig. 2k, left; quantified in 2l). Second, some tdT+ cells showed neither GFAP, Sox9, nor NeuN signals (Fig. 2k, middle; quantified in 2l), which may represent intermediate-stage cells as reported by a previous study21. These intermediate cells likely represent a transitional stage during AtN reprogramming, where the cells have lost their astrocytic identity, but neuronal gene expression has not yet been fully upregulated, resulting in the observation of these double-negative intermediate cells. Finally, when NeuN signals became detectable, tdT+ cells showed neuronal morphology and were negative for GFAP and Sox9 (Fig. 2k, right; quantified in 2l), and these cells represented converted neurons. Together, these data indicate that Olig2 knockdown promotes AtN conversion, which may go through an intermediate stage between astrocytes and neurons.

To further confirm the inhibitory effect of Olig2 on Ngn2-mediated AtN conversion, we used a dominant active form of Olig2 (Olig2-VP16) to antagonize the inhibitory function of endogenous Olig2 in astrocytes16,28. Olig2-VP16 was driven by the hGFAP promoter and co-overexpressed with tdT or Ngn2-tdT (Supplementary Fig. 8a). At 7 dpi, we confirmed that control tdT had high GFAP+ astrocytes targeting rate, Olig2 and VP16 were also strongly expressed in tdT+ cells (Supplementary Fig. 8b–e). Multi-timepoint analysis revealed that Olig2-VP16 alone failed to convert astrocytes into neurons in the absence of Ngn2 from 7 dpi to 45 dpi, with almost all tdT+ cells still expressing S100β but not NeuN. However, when Olig2-VP16 worked with Ngn2, the AtN conversion efficiency reached up to 40% at 45 dpi, as indicated by a decrease in the proportion of tdT+S100β+ cells and an increase in tdT+NeuN+ cells (Supplementary Fig. 8f–h). Taken together, these findings further demonstrate that Olig2 plays a negative regulatory role in Ngn2-induced AtN conversion, and removing the inhibition of Olig2 could promote cell fate conversion.

Functional analysis of converted neurons
To test whether the converted neurons were functional, we performed whole-cell patch-clamp recordings in acute cortical slices at 40–45 dpi, with the infected cells identified by the expression of tdTomato (Fig. 3a). We first compared the Na+-K+ currents between Ngn2+shNC (control) and Ngn2+shOlig2 (conversion) treated mice and found larger K+ currents in the control group cells, without a significant fast Na+ current, exhibiting typical astrocyte electrophysiological properties (Fig. 3b, top). However, in the conversion group cells, we not only recorded outward K+ currents but also inward fast Na+ currents (Fig. 3b, bottom). Of note, the K+ currents were significantly reduced while Na+ currents were dramatically increased in conversion group cells compared to the control group (Fig. 3c). Next, for testing the probability of action potential firing, step currents were injected into the AAV-infected cells under the current patch-clamp model. Consistently, no evoked action potentials were recorded in the control group (Fig. 3d top), while 95.5% of conversion group cells were able to generate action potentials (18.2% with single spike and 77.3% with regular spikes) upon the step current injection (Fig. 3d bottom and quantified in 3e). Thus, Ngn2+shOlig2 can convert cortical astrocytes from non-excitatory glial cells to excitable neurons.

In addition, when comparing the tdT+ cells between the control and conversion group, tdT+ cells with Olig2 knockdown had lower membrane capacitance (Fig. 3f) and higher membrane input resistance (Fig. 3g), implying a reduction in gap junctions when astrocytes were converted into neurons. We also found that tdT+ cells in the control group displayed a more negative resting membrane potential (RMP) than those in the conversion group (Fig. 3h). Moreover, we further examined whether the converted neurons could integrate into the local circuit and form functional synaptic connections. To this end, both spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in tdT+ cells. As expected, none of the tdT+ cells exhibited any spontaneous synaptic events in control group cells, whereas both sEPSCs and sIPSCs were detected in half of the conversion group cells (Fig. 3i–n). Notably, approximately half of the converted neurons (47.6%) acquired the ability to generate action potentials but no detectable postsynaptic currents (PSCs, Fig. 3o), implying that these newly converted cells require a longer period of maturation to establish functional synaptic connections. Taken together, our electrophysiological results demonstrate that cortical astrocytes can be converted into functional neurons by Ngn2+shOlig2 and integrate into local synaptic circuits.

Knockdown of Olig2 enhances AtN conversion efficiency in astrocyte lineage-tracing mice and retro-labeling study
To further validate the enhanced conversion efficiency induced by Olig2 knockdown, we employed astrocytic lineage-tracing mice generated by crossing Aldh1l1-CreERT2 mice with Ai14 mice (Fig. 4a). After tamoxifen treatment, around 98.9±0.7% and 98.1±0.8% tdT+ cells expressed astrocyte marker S100β and Sox9 in cortex, respectively (Figs. 4b, c), indicating highly efficient to pre-label endogenous astrocytes. Next, we found that under normal conditions, around 6.7±2.2% tdT+S100β+ astrocytes expressed Olig2 in the adult mouse cortex, consistent with previous reports in wild-type mice. Seven days after Ngn2 injection, Olig2 was markedly upregulated in tdT+ astrocytes (94.2±1.7% in tdT+ Ngn2+ cells, Figs. 4d, e). Thus, these results indicate that overexpression of Ngn2 in lineage-tracing mice also significantly increases Olig2 expression in pre-labeling astrocytes.

Given that Olig2 knockdown increased conversion efficiency about three-fold in wild-type mice at 45 dpi (Fig. 2g), we extended this analysis to the lineage-traced mice. First, we confirmed the knockdown efficiency at 7 dpi. Quantitative analysis revealed that Ngn2 co-expression with shOlig2, compared to shNC controls, significantly reduced Olig2 protein levels (Fig. 4d-e). In the absence of AAV injection, the lineage-traced mice exhibited over 98% labeling specificity for astrocytes in the cortical area (Fig. 4f top, g left), with tdT+ neurons comprising less than 2% (Fig. 4f top, g right). At 45 dpi, we observed that around 5.0% tdT+ cells became NeuN+ in the Ngn2+shNC group, accompanied by a corresponding decrease in tdT+S100β+ astrocytes compared to the normal conditions. As expect, shOlig2 significantly promoted the generation of tdT+NeuN+ cells, reaching up to 12.2% (Fig. 4f, g), further supporting the inhibitory role of Olig2 in Ngn2-mediated AtN conversion.

To further validate AtN reprogramming in the mouse cortex, we pre-labeled endogenous neurons with GFP by injecting engineered AAV2 variants. Adult mice were first injected with rAAV2-retro-CAG::GFP (Retro-GFP) into the T6 corticospinal tract (CST), followed by intracerebral injections of AAV9-Ngn2-tdT+shNC or AAV9-Ngn2-tdT+shOlig2 viruses (Supplementary Fig. 9a). Forty-five days after the AAV9 injection, we observed 7.6% and 22.3% tdT+ neurons in the brains injected with Ngn2-tdT+shNC and Ngn2-tdT+shOlig2 viruses, respectively (Supplementary Fig. 9b, c). Specifically, in the Ngn2+shNC group, 416 tdT+ AAV-transduced cells were analyzed, of which 34 were neurons. Among these, 5 tdT+ neurons were pre-labeled with GFP, representing approximately 15% of the neuronal population analyzed (Supplementary Fig. 9b, d). In the Ngn2+shOlig2 group, 968 tdT+ AAV-transduced cells were analyzed, with 224 tdT+ neurons identified. Notably, the majority of these tdT+ neurons (96%) were not labeled by GFP (Supplementary Fig. 9b, d). These results suggest that Olig2 knockdown in astrocytes significantly enhances the efficiency of Ngn2-mediated AtN reprogramming, rather than causing nonspecific tdT reporter expression in endogenous neurons.

ScRNA-seq reveals the underlying mechanism of reprogramming after knocking down Olig2 in astrocytes
To gain mechanistic insight into how Olig2 impedes AtN conversion, we performed single-cell RNA sequencing (scRNA-seq) of freshly pooled cortical samples infected with Ngn2 either in the presence or absence of shOlig2 vectors at 18, 20 and 22 dpi (n=2 mice for each time-point, Fig. 5a), when astrocytes infected with Ngn2 and shOlig2 began to lose astrocytic features (Fig. 2h). After data-quality filtering, we obtained 32,095 cells from the Ngn2+shNC group (median gene count: 928) and 32,439 cells from the Ngn2+shOlig2 group (median gene count: 894). Uniform manifold approximation and projection (UMAP) analysis identified 11 cell types based on the respective representative marker genes (Supplementary Fig. 10a, b). The overall cell composition of the Ngn2+shNC group and the Ngn2+shOlig2 group was similar (Supplementary Fig. 10c, d). The tdTomato transcript was mainly distributed in astrocytes, whereas tdT reporter gene was barely detected in neurons (Supplementary Fig. 10e, f). This might be due to the limited number of reprogrammed neurons at these time points (Fig. 2g, j), the decreased activity of the GFAP promoter in converted neurons, and neuronal damage caused by tissue dissociation. Additionally, tdT transcripts were detected in a small subset of oligodendrocytes (Supplementary Fig. 10e, f). However, analysis of the distribution and relative expression levels of tdT transcripts revealed that both were much higher in astrocytes than in oligodendrocytes. (Supplementary Fig. 10g). To compare the transcriptomic changes in AAV-infected astrocytes between the two groups, we first separated tdT+ astrocytes from the total cell population for further analysis (Fig. 5b, c). We confirmed the downregulation of Olig2 transcript and protein expression level in AAV-shOlig2 infected cells at 21 dpi (Fig. 5d, e), similar to the immunostaining results at 7 dpi (Fig. 2c, d). We then performed differential expression analysis between Olig2 knockdown tdT+ cells (Ngn2+shOlig2) and the controls (Ngn2+shNC), identifying 1281 upregulated genes including those associated with neurogenesis (e.g., Nes, Sox4, and Pax6) and 597 downregulated genes embracing those related to mature astrocyte function (e.g., Slc1a2, S100β, and Aqp4, Fig. 5f). Gene Ontology (GO) enrichment analysis revealed that the upregulated genes after Olig2 knockdown were related to mRNA processing, translation and neurogenesis, whereas metabolism and gliogenesis-related pathways were significantly downregulated (Fig. 5g, h). Real-time PCR further demonstrated that the mRNA of Nestin, Vimentin and Olig1 were significantly upregulated in Ngn2+shOlig2 infected astrocytes (Fig. 5i). Furthermore, we confirmed that Nestin and Vimentin, known to be expressed in neural progenitor cells, were slightly expressed in Ngn2-transduced astrocytes and then significantly increased after Olig2 knockdown, implying that astrocytic Olig2 represses neurogenesis gene expression (Fig. 5j, k).

To provide detailed insights into the changes in cellular feature of the astrocytes after the knockdown of Olig2 during the Ngn2-mediated AtN conversion, we performed an unbiased astrocyte sub-clustering analysis and further divided the astrocyte population into five sub-clusters (A1-A5, Fig. 5l) according to the differentially expressed markers (Supplementary Fig. 10h). Gene set variation analysis (GSVA) showed that A1 astrocyte was enriched for genes related to synapse regulations. A2 and A5 astrocytes shared similar functions related to metabolism and energy production. A3 and A4 astrocytes were featured by active gene transcription and translation (Supplementary Fig. 10i). Interestingly, the percentages of A1 and A2 were reduced following Olig2 knockdown, whereas A3 (green) and A4 (red) showed significant increases (Fig. 5m). In addition, tdT+ astrocytes in both groups were detected in the A3 and A4 subpopulations (Fig. 5c, l and supplementary Fig. 12g), indicating that Ngn2 alone is sufficient to change the profile of astrocytes and push them to enter a transcriptionally and translationally active state. But Olig2 knockdown further increased the proportion of A3 and A4 astrocyte populations in the Ngn2-transduced astrocytes (Fig. 5n). Additionally, we performed a supplementary analysis on the Ngn2+shNC group without AAV infection in astrocytes (tdT-). We found that only 9.3% of these astrocytes were distributed in the A3 and A4 subpopulations, while about 90% were observed in the A1 and A2 subpopulations (Supplementary Fig. 10j). Together, these data suggest that the induced astrocytic Olig2 may inhibit the Ngn2-transduced astrocytes to enter a transcriptionally and translationally active state.

We next analyzed detailed features of the A3 and A4 subpopulations. Genes related to mature astrocyte functions, such as Aqp4 for BBB maintenance, Slc1a2 for glutamate transport, Apoe for lipid transport, and Glul for glutamine synthesis, were downregulated in these subpopulations (Fig. 5o). Meanwhile, genes related to cell dedifferentiation, chromatin remodeling, neurogenesis and axonogenesis were significantly upregulated (Fig. 5o). We then selected four representative genes associated with astrocyte dedifferentiation (Sox4, Nestin, Olig1 and Vimentin) for scatter plot visualization. Our analysis revealed that these genes were predominantly concentrated within the A3 and A4 subpopulations and the frequency of these astrocyte populations was significantly elevated in the Olig2 knockdown group (Fig. 5p). Moreover, A3 and A4 astrocytes also upregulated genes involved in cell cycle regulation (e.g., Ccnd2, Fgfr1 and Akt1), chromatin remodeling (e.g., H2aj, Hdgf and Hat1), RNA processing and splicing (e.g., Dhx9, Ddx5 and Akt1), translation (e.g., Rpls and Rpss), and actin cytoskeleton organization (e.g., Ezr, Coro1c and Actg1; Fig. 5o). Importantly, genes associated with neurogenesis and axonogenesis, such as Pax6, Stmn1, Smn1, Robo2, and Carpin1, were also upregulated in A3 and A4 (Fig. 5o). Collectively, these data reveal that A3 and A4 astrocytes undergo a phenotypic transition that exhibits characteristics of reprogramming toward neural progenitor cells.

CytoTRACE is a computational method to predict the potential of cell reprogramming and differentiation potential based on scRNA-seq data, with higher scores suggesting better differentiation potential29. Analysis and quantification showed that knocking down Olig2 in Ngn2-transduced astrocytes led to a significant increase in the CytoTRACE score (Supplementary Fig. 11a). Further analysis of astrocyte subpopulations revealed that A3 and A4 subclusters showed higher CytoTRACE scores compared with A1 and A2 (Supplementary Fig. 11b), suggesting that A3 and A4 astrocytes have stronger reprogramming potential. Next, to investigate the AtN conversion at the single-cell level, we employed cell fate trajectory and pseudotime analysis using Monocle 2, incorporating neural progenitor cells (NPCs) into this analysis. Of note, NPCs were mainly found in Ngn2+shOlig2 treated mouse cortex (Supplementary Fig. 12a–c), which were highly expressed Nnat, Tubb2b, Igfbpl1, and Marcksl1 genes (Supplementary Fig. 12b). These genes are mainly related to axon growth, neuronal differentiation, and regeneration, and are highly expressed in immature neurons. Our analysis revealed a cellular continuum with two distinct branch points that originated from astrocytes with mature astrocyte function (A1 and A2) and differentiated into astrocytes with features of immature astrocytes and neurogenesis (A3 and A4). A branch point emerged from A1 and A2, diverging into two distinct paths: one predominantly leading to A3 (cell fate 1) and the other to A4 (cell fate 2). In control cells (Ngn2+shNC), A1 and A2 were predominantly located in the pre-branch region (Fig. 5q, left panel). However, upon knocking down Olig2, A1, A2, and A3 cells moved beyond the branch point, adopting characteristics of cell fates 1 and 2 (Fig. 4q, right panel). In addition, most A3 cells and some A4 cells were located near NPCs (Fig. 5q), indicating intrinsic similarity, and this distribution pattern was more pronounced after knockdown of Olig2 (Supplementary Fig. 12d–g). Along the cellular trajectory, mature astrocyte markers (e.g., Glul and Aqp4) were downregulated, while neural progenitor marker Vimentin was gradually upregulated (Fig. 5r), indicating a transition towards a neural progenitor fate. Olig1, another bHLH transcription factor reported to have the ability to promote axonogenesis30, also showed a gradual increase and then was maintained at a certain level in cellular trajectory (Fig. 5r). Together, knocking down Olig2 in Ngn2-transduced astrocytes leads to significant transcriptomic changes and upregulates neural stem cell signature genes.

Olig2 inhibits Ngn2-mediated AtN conversion in multiple ways
To further illuminate how Olig2 knockdown in Ngn2-transduced astrocytes facilitates AtN conversion, we investigated the additional alterations in the transcriptome of Ngn2-overexpressing astrocytes resulting from the knockdown of Olig2. We first compared the transcriptome of Ngn2-transduced astrocytes (tdT+) and that of non Ngn2-transduced astrocytes (tdT-) in the Ngn2+shNC group to figure out the changes induced by Ngn2 (Fig. 6a left, DEGs-1). Ngn2 alone is sufficient to change the gene expression profiles of astrocytes (Supplementary Fig. 13a–c). Among these genes, we identified Vim, Sox4, Nes, and Olig1 genes have been upregulated, while Aldoc, Glul and Gjb6 genes have been downregulated in tdT+ astrocytes (Supplementary Fig. 13a). GO analysis revealed that genes associated with biosynthesis and membrane transport were significantly downregulated, while genes related to cellular respiration and translation were markedly upregulated (Supplementary Fig. 13b, c). Notably, genes involved in neurogenesis and axonogenesis, which were enriched in the Ngn2+shOlig2 group (Fig. 5h), were conspicuously absent from the upregulated pathways induced by Ngn2 alone, suggesting that this may underlie the limited efficiency of Ngn2-mediated AtN conversion.

Then, we compared the two groups of DEGs that were generated from the comparisons between either tdT+ and tdT- astrocytes in Ngn2+shNC group (defined as DEGs-1), or tdT+ astrocytes in Ngn2+shOlig2 and those in Ngn2+shNC (defined as DEGs-2; Fig. 6a). Venn diagram displayed the common and unique DEGs in the 2 datasets (Fig. 6a). There were 367 common DEGs shared between the two datasets, while 542 and 1308 unique DEGs were found in DEGs-1 and DEGs-2, respectively (Fig. 6a). DEGs unique to DEGs-1 represent the profile change induced by the Ngn2 that contained Olig2 in astrocytes, which were associated with cytoplasmic translation and cellular respiration (Fig. 6b). DEGs unique to DEGs-2 represent the profile change induced by knocking down Olig2 upon Ngn2 overexpression in astrocytes, which included specific genes relevant for RNA splicing, processing and protein catabolic process (Fig. 6c). Furthermore, upregulated DEGs related to neurogenesis, such as Pax6, Robo2, and Stmn1, as well as downregulated DEGs associated with mature astrocyte functions, such as Aqp4 and Slc1a2, were also unique to Olig2 knockdown in Ngn2-transduced astrocytes (Fig. 6a, bottom Venn diagram). Thus, knocking down Olig2 in Ngn2-transduced astrocytes further substantially alters the astrocyte transcriptome and promotes neurogenic related genes expression.

Next, we focused on common DEGs shared by DEGs-1 and DEGs-2 that may reflect the patterns of how Olig2 impacts Ngn2-mediated AtN conversion, and identified four distinct patterns (Fig. 6d). Pattern 1 included genes that were upregulated by Ngn2 and further upregulated by Olig2 knockdown (Fig. 6d, left), which were mainly associated with ribosome biogenesis and cytoplasmic translation (Fig. 6e). These results suggest that knocking down Olig2 may alter the expression of genes involved in protein synthesis, which could be linked to the cellular reprogramming process and the need for new functional proteins. Pattern 2 contained genes that were initially downregulated by Ngn2 and further downregulated by Olig2 knockdown (Fig. 6d, middle left). The functions of these genes were mainly associated with membrane and cell junction adjustment, as well as synapse regulation (Fig. 6f), which are typical functions of astrocytes. These results indicate that knockdown of Olig2 further inhibited the expression of astrocyte signature genes, suggesting that knockdown of Olig2 may facilitate Ngn2-mediated astrocyte dedifferentiation. Moreover, our analysis revealed not only a synergistic relationship between Ngn2 and Olig2, but also two distinct patterns of astrocytic Olig2 functionality that exhibit opposing effects. In Pattern 3, genes were upregulated by Ngn2 but downregulated by Ngn2+shOlig2 (Fig. 6d, middle right). We observed several pathways related to metabolism (Fig. 6g), consistent with previous findings that cellular reprogramming is accompanied by many metabolic changes7,31. Notably, among these genes, we also identified Hes5, which has been reported as a repressor for neurogenesis32, and it was significantly upregulated by Ngn2 overexpression but downregulated by Olig2 knockdown (Fig. 6d, middle right). Finally, Pattern 4 exhibited genes that were downregulated by Ngn2 but upregulated upon the knockdown of Olig2 (Fig. 6d, right), which are primarily related to receptor regulation (Fig. 6h). Cellular receptor remodeling represents a pivotal event in the process of reprogramming, which involves the reorganization and functional changes of cell receptors to support the cell fate switch. Collectively, knockdown of Olig2 further alters the gene expression signature of astrocytes and then promotes Ngn2-mediated astrocyte reprogramming in multiple aspects.

Olig2 knockdown alters astrocyte metabolic gene expression
Given the common occurrence of metabolic shifts during AtN conversion7,31, we compared changes in gene expression associated with metabolic preference of tdT+ astrocytes in Ngn2+shNC or Ngn2+shOlig2 treated animals. The results indicated that cells with Olig2 knockdown exhibited an overall low metabolic score (Fig. 7a). Classical metabolic pathways preferred in astrocytes, including canonical glycolysis, glucose metabolism, fatty acid metabolism, β-oxidation, glutamate and pyruvate metabolism were notably downregulated in the Ngn2+shOlig2 group (Fig. 7b–h). Oxidative phosphorylation (OXPHOS), a pathway typically favored in neurons, did not show a significant difference (Fig. 7i), primarily due to the impact of Ngn2 alone on the elevation of OXPHOS levels (Fig. 7j). We then performed a metabolic pathway activity scoring analysis on subclusters of astrocytes and neurons, observing that A3 and A4 exhibited lower metabolic value, a trend also detected in neurons (Supplementary Fig. 14a). ScMetabolism pathway analysis revealed that astrocytes had distinct metabolic transcriptome profiles compared to neurons and neural stem cells. Specifically, metabolic pathways that were highly active in A2 and A5 astrocytes, but relatively quiescent in neurons and NPCs (glycolysis/gluconeogenesis, pentose phosphate pathway, steroid biosynthesis, etc.), were found to be downregulated in A3 and A4 astrocytes. Interestingly, a metabolic pathway (N-glycan biosynthesis) that had high value in excitatory neurons (ENs) but less value in A1, A2 and A5 astrocytes, was upregulated in A3 and A4 (Supplementary Fig. 14b), consistent with Ngn2 primarily inducing ENs (Supplementary Fig. 6c, d).

Mitochondria have been reported to play a crucial role in metabolic shifts, and specific mitochondrial proteins are essential for implementing the AtN conversion7. Studies indicate that astrocytes and neurons exhibit distinct mitochondrial proteomes, and cells lacking the upregulation of neuron-enriched mitochondrial proteins often fail to achieve successful reprogramming7,15. In our data, we identified 31 mitochondrial protein-related genes enriched in astrocytes and 21 enriched in neurons that underwent transcriptional changes following Olig2 knockdown (Fig. 7k). Among the astrocyte-enriched genes, 74.2% (23 out of 31, e.g., Sfxn5, Prdx6, and Cox20) were downregulated in the Ngn2+shOlig2 group. In contrast, 85.7% (18 out of 21, e.g., Prdx2, Bax, and Slc25a22) of neuron-enriched genes were upregulated with knockdown of Olig2 (Fig. 7l). Sfxn5 is a mitochondrial transporter responsible for citrate transport and is enriched in astrocyte-derived mitochondria7. Immunostaining results revealed that the expression of Sfxn5 in astrocytes was significantly decreased in the Ngn2+shOlig2 group (Fig. 7m, n). These findings suggest that the knockdown of astrocytic Olig2 promotes the shift from astrocytic mitochondria transcriptome profile to that of neurons, which may facilitate Ngn2-mediated AtN reprogramming.

CUT&Tag genome binding profiling of astrocytic Olig2
Olig2 belongs to the bHLH family, which forms heterodimers or homodimers to bind to the DNA and serves as a transcription regulator24. To uncover the targets of Olig2, which impede the Ngn2-mediated AtN conversion, we conducted Olig2 CUT&Tag sequencing to analyze the genomic binding loci of Olig2 in the Ngn2-transduced cells. We collected tdT+ astrocytes infected with AAV9-Ngn2 through FACS and then subjected them to Olig2-CUT&Tag sequencing (Fig. 8a). A total of approximately 60,000 sorted cells from 12 mice were pooled for a single experiment, and there two replicates were conducted in this study (Fig. 8b). We identified 8372 Olig2-binding sites within 3kb upstream or downstream of the annotated transcription start site (TSS, Fig. 8c). Among these Olig2-occupied loci, most of them were in the regulatory regions including intergenic regions (44.3%), introns (35.0%) and promoters (16.4%, Fig. 8d). The closest annotated gene to each Olig2-binding locus was considered as a presumed target, and GO analysis revealed that Olig2-target genes were primarily associated with cell communication, signaling regulation, and development (Fig. 8e). We then performed motif analysis to investigate whether the DNA motifs of Olig2 binding sites in cortical astrocytes were similar to those in OPCs. We did not detect canonical E-box motif (CANNTG) in AAV-Ngn2 transduced astrocytes in two replicate experiments. Instead, Olig2-occupied loci in these astrocytes displayed GC-rich, TC-rich, and AG-rich repeat motifs (Supplementary Fig. 15). To determine whether the motifs enriched in astrocytes are unique, we conducted additional experiments using the same CUT&Tag methodology to investigate Olig2 binding sites in oligodendrocyte lineage cells, a glial cell type that naturally expresses Olig2 in the normal adult mouse brain. We collected tdT+ cells from adult Sox10-CreERT2; Ai14 transgenic mice via FACS, and identified peaks along with their binding distribution after sequencing (Supplementary Fig. 16a–c). We detected a GC-enriched motif similar to that in astrocytes (such as Sp and Klf genes), as well as the previously reported canonical E-box CAGCTG motif (Supplementary Fig. 16d, e). Together, these results suggested that Olig2 may have some unique regulatory mechanisms in astrocytes undergoing Ngn2-mediated reprogramming.

To further explore the unique features of Olig2-targeting regions in Ngn2-transduced astrocytes, we compared the Olig2 occupancy profiles in these astrocytes with those in mouse embryonic stem cells (mESCs)33, OPCs23 and Sox10-tdT+ cells, respectively (Supplementary Fig. 17a). Our analysis revealed that there was only a small portion of overlapping genes between astrocytes and ESCs (1180 shared vs 7192 unique), which were primarily associated with functions related to axonogenesis, synapse organization, and forebrain development (Supplementary Fig. 17b). In contrast, the genes unique to Ngn2-transduced astrocytes were more enriched in the regulation of apoptotic signaling pathway, immune cell migration and tissue development, although the enrichment was less significant (Supplementary Fig. 17c). Additionally, the ratio of the shared genes between astrocytes and OPCs was also small (1376 shared vs 6996 unique), with the top GO biological process terms including axonogenesis, synapse organization and regulation of membrane potential (Supplementary Fig. 17d). Genes unique to astrocytes compared to OPCs were notably enriched in response to cell-substrate adhesion, stem cell population maintenance and maintenance of cell number (Supplementary Fig. 17e). Finally, we found that more genes shared between adult astrocytes and Sox10-tdT+ oligodendrocyte lineage cells (2526 shared vs 5846 unique). The overlapping genes were primarily enriched in pathways related to synaptic organization and signal transduction (Supplementary Fig. 17f), whereas the genes unique to astrocytes showed highly close association with axonogenesis and regulation of membrane potentials (Supplementary Fig. 17g). Together, these results reveal that Olig2 may play an important gene regulatory role during astrocyte reprogramming.

To further investigate whether the genes targeted by Olig2 undergo transcriptional changes, we conducted the integrative analysis of the scRNA-seq transcriptome data and CUT&Tag-seq gene binding loci data of Olig2. We discovered that 90 Olig2-targeted genes exhibited downregulated expression after Olig2 knockdown, while 201 Olig2-targeted genes upregulated after reducing the expression of Olig2 (Fig. 8f, g). For the upregulated targets, there was a group of genes related to neuronal development, including Nes, Vim, Gria3, and Pou3f2. Notably, Hes5, a transcriptional repressor of genes that require bHLH proteins for transcription and plays a negative regulator of neurogenesis32, was downregulated following Olig2 knockdown (Fig. 8g). Stub1 (also known as CHIP) acts as an E3 ubiquitin ligase that regulates the ubiquitination and degradation of specific proteins (such as GPX4), promoting ferroptosis34. In Ngn2-transduced astrocytes, Olig2 knockdown reduced Stub1 expression (Fig. 8g), supporting the idea that AtN reprogramming is regulated by programmed cell death31. GO analysis indicated that the downregulated genes were enriched in processes related to steroid metabolism, synaptic maintenance and organization, and actin binding (Supplementary Fig. 18a). Meanwhile, the upregulated genes were mainly related to actin filament organization, intrinsic component of organelle membrane and transcription coregulatory activity (Supplementary Fig. 18b). Overall, the integrated analysis of CUT&Tag and scRNA-seq suggests that Olig2 plays a key regulatory role in the expression of genes involved in Ngn2-mediated astrocyte reprogramming.

Ngn2 acts as a pioneer transcription factor that reprograms astrocytes into functional neurons. Interestingly, we found that Olig2 in astrocytes can directly target the Ngn2 gene (Fig. 8h), potentially revealing a regulatory link. The prominent genomic binding peak of Olig2 within the Ngn2 exon region motivated us to explore the possibility of the direct regulation of Olig2 to the expression of Ngn2 (Fig. 8h), given that Olig2 typically functions as a transcription repressor25,35. qRT-PCR data revealed that Ngn2 mRNAs were significantly increased after knocking down Olig2 in AAV9 transduced cortical regions (Fig. 8i). Next, the expression of Ngn2 was examined through immunostaining in mice injected with AAV-Ngn2, Ngn2+shNC, and Ngn2+shOlig2 at 7 dpi. As expected, the Ngn2 protein was specifically expressed in cortical astrocytes (tdT+) in all groups (Fig. 8j). Noteworthy, the fluorescence intensity of Ngn2 almost doubled after Olig2 knockdown (Fig. 8k), suggesting that removing Olig2 increases Ngn2 expression in astrocytes. Together, these results suggest a negative feedback of the ectopic expression of Ngn2 in the astrocytes, which induces the Olig2 expression to inhibit Ngn2 expression and neuronal reprogramming (Fig. 8i).

Methods
Animals
Experiments were conducted on adult (age 2–3 months) wild-type C57BL/6J mice (Guangdong Yaokang Biotechnology, China) and transgenic mice (The Jackson laboratory). The transgenic mouse strains used in this study include: Sox10-CreERT2 [CBA;B6-Tg(Sox10-icre/ERT2)388Wdr/J, stock number: 027651], Aldh1l1-CreERT2 [B6N.FVB-Tg(Aldh1l1-cre/ERT2)1Khakh/J, stock number: 031008], and Ai14 [B6.Cg Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, stock number: 007914]. Sox10-CreERT2 mice or Aldh1l1-CreERT2 mice were crossed with the Ai14 mice for cell type-specific lineage tracing. Both male and female mice were used in this study. All mice were housed under a controlled temperature and a 12-h light/dark cycle with free access to water and food in the animal facility. For all surgical experiments, mice were anesthetized using isoflurane. Anesthesia was initiated at 5% and then reduced to 2% to maintain a stable deep anesthetic state. Animal procedures and protocols were approved by the Laboratory Animal Ethics Committee of Jinan University, China (approval no. IACUC-20220613-05 and 20230703-12).

Virus preparation and injection
AAV was used to deliver transcription factors into the mouse cortex. tdTomato was inserted into the AAV vector to generate AAV-tdTomato, and Neurogenin2/Ascl1/NeuroD1 was cloned into the AAV-tdTomato to generate AAV-Ngn2/Ascl1/NeuroD1-tdTomato. Sox2 or Dlx2 was cloned into the AAV-GFP to generate AAV-Sox2/Dlx2-GFP. The transactivation domain of herpes simplex VP16 was fused to bHLH domain of Olig2 to generate AAV-Olig2VP16. Three designed Olig2 shRNA were cloned into one AAV backbone (Olig2 shRNA1: gcacttgtggaatggaatattggatccaatattccattccacaagtgc; Olig2 shRNA2: ggattatggggagtcgaatttggatccaaattcgactccccataatcc; Olig2 shRNA3: gcgagcacctcaaatctaattggatccaattagatttgaggtgctcgc). All transcription factors were under the control of the human GFAP promoter, and shRNA was driven by U6 promoter. Human GFAP promoter (hGFAP) is a synthetic promoter that is composed of GfaABCD1 derived of human GFAP promoter gfa2 enhancer element, derived from the human glial fibrillary acidic protein gene. All recombinant AAVs were produced by PackGene® Biotech, purified through iodixanol gradient ultracentrifuge and subsequent concentration. Purified AAV viruses were tittered using a quantitative PCR-based method. AAV (1 μL) was injected stereotactically into both sides of the mouse cortex with titers of 0.5–2×1011 GC mL−1 (transcription factors) and 2×1010 GC mL−1 (shRNA). Equal ratios of viruses were mixed for multifactor injections. Injection coordinates are as follows: anterior/posterior, 1.3 mm; medial/lateral, ±1.5 mm; and dorsal/ventral, −0.7 mm. Brain slices with tdT expression within approximately 400 μm surrounding the injection site were included for analysis. For AAV9-Ngn2 (lacking a reporter gene) analysis, we performed serial sectioning and immunofluorescence staining for Ngn2 on every fifth section to confirm Ngn2 expression, and the intervening brain sections were included for conversion validation.

Retrograde labeling and intracerebral injection
The use of AAV to pre-label endogenous neurons was based on a previous study38. Briefly, AAV2-retro-CAG-GFP virus (2 µL per mouse) was injected into the T6 level of the dorsal spinal cord to target the corticospinal tract. Ten days later, we stereotaxically injected AAV9-Ngn2 with shNC or shOlig2 into the cortex at these coordinates: −0.11 mm AP, ±1.5 mm ML, and 0.7 mm DV. Analysis was conducted 45 days after the cortical injection.

Tissue dissociation
Mice were sacrificed, perfused with aCSF, and the virus-infected brain tissue was collected. The brain was dissociated into single-cell suspension using the Papain Dissociation System (Worthington, LK003150) according to the manufacturer’s protocol. Myelin was removed using a debris removal solution (Miltenyi Biotec, 130-109-398) according to the manufacturer’s instructions. For fluorescent-assisted cell sorting (FACS, BD FACS Aria SORP), cells were resuspended in 1× PBS supplemented with 1% BSA and 2 mM EDTA, and kept at 4°C until sorting. For scRNA-seq, cells were resuspended in 1× PBS.

CUT&Tag and data analysis
For astrocytic Olig2 profiling, tdTomato+ cells were sorted from the cortical tissues of animals (two samples, 12 mice per sample) 7 days after AAV9-GFAP::Ngn2-tdTomato injection using FACS (BD FACS Aria SORP). For Olig2 profiling in oligodendrocyte lineage cells, tdTomato+ cells were sorted from the brains of 3-month-old Sox10-CreERT2; Ai14 transgenic mice (two samples, 2 mice per sample) using FACS. Cells were first gated for singlets based on Forward Scatter–Area (FSC-A) versus Forward Scatter–Height (FSC-H), then live cells were selected by excluding DAPI-positive dead cells, and finally, the brightest tdT+ cells were gated (10.0% of total tdT+ cells). The sorted cells were performed with CUT&Tag-IT™ Assay Kit (Active Motif, catalog No. 53160) to get the final CUT&Tag libraries, followed by Illumina NovaSeq. Rabbit anti-Olig2 (Millipore, catalog No. AB9610) was used as the primary antibody. Before read mapping, clean reads were obtained from the raw reads by removing the adaptor sequences. The sequencing data were mapped to the mm10 genome assembly, and peak calling was performed using MACS2 software with a cutoff value < 0.05. The deeptool was used for showing read distributions. A unified peak atlas was generated through the integration of aligned reads (BAM files) derived from two biological samples. Peaks were annotated by the bedtools, and the HOMER’s findMotifsGenome.pl tool was used for Motif analysis.

ScRNA-seq and data analysis
Eighteen, twenty, and twenty-two days after virus injection (n=2 for each timepoint), the single-cell suspensions were directly loaded onto a microfluidic chip (GEXSCOPE™ Single Cell RNA-seq Kit, Singleron Biotechnologies) and processed for scRNA-seq library preparation according to the manufacturer’s protocol. Raw reads were processed to generate gene expression profiles using CeleScope v1.5.2. Scanpy v1.8.1 was used for quality control, dimensionality reduction, and clustering under Python 3.7. Low quality and duplet cells were removed before further analysis. Cells with less than 200 or with top 2% gene counts were removed. Cells with top 2% UMIs were excluded. Cells with mitochondrial content > 20% were filtered. Cells with genes detected in less than 5 cells were removed. Total 64,534 cells were retained for the downstream analyses, with an average of 972 genes and 1836 UMIs per cell. The raw count matrix was normalized by total counts per cell and logarithmically transformed into normalized data matrix. The top 2000 variable genes were chosen for principal component analysis (PCA), and top 20 principle components were used for clustering and dimensional reduction. Cell clusters were visualized by using Uniform Manifold Approximation and Projection (UMAP). All single-cell analysis results in this study do not incorporate data from other publications.

CytoTRACE analysis
CytoTRACE (Cellular Trajectory Reconstruction Analysis using gene Counts and Expression) is a computational framework designed to infer cellular differentiation potential and reconstruct developmental trajectories from scRNA-seq data29. CytoTRACE first calculates the number of detectably expressed genes per cell. It then identifies genes whose expression patterns correlate with gene counts to derive the Gene Count Signature (GCS). Finally, it employs a local similarity algorithm between cells and applies an iterative two-step smoothing procedure to refine the GCS. By leveraging gene expression heterogeneity, CytoTRACE predicts the relative “stemness” or differentiation status of individual cells, enabling the identification of progenitor-like cells and lineage hierarchies within heterogeneous populations.

Quantitative real-time PCR
Seven days or twenty days after virus injection, brain tissue was dissociated into single-cell suspension, and tdTomato positive cells were collected using FACS. RNA samples were harvested using Flysis Amp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, China) according to the manufacturer’s instructions. QPCR was performed using CFX Duet Real-Time PCR System (Bio-Rad). Relative expression was calculated as 2(−ΔΔCT). The primers for targeted genes were as follows: Olig2 (F: TTACAGACCGAGCCAACACC, R: TGGCCCCAGGGATGATCTAA), Nestin (F: CCTTCTCTAGTGCTCCACGTC, R: CGTCGATTGAGCTCCCACA), Vimentin (F: GCGAGAGAAATTGCAGGAGG, R: CCGTTCAAGGTCAA-GACGTG), Olig1 (F: CTCGCCCAGGTGTTTTGTTG, R: TATAAGCCTGCGCTACGACG) and Gapdh (F: AGGTCGGTGTGAACGGATTTG, R: TGT AGACCATGTAGTTGAGGTCA).

Electrophysiology
Brain slices were prepared 40-45 days after virus injection and cut to 300 μm thick coronal sections with a Leica vibratome (VT-1200S) in ice-cold cutting solution (containing 75 mM sucrose, 85 mM NaCl, 2.5 mM KCl, 0.5 mM CaCl2, 4 mM MgCl2, 24 mM NaHCO3, 1.25 mM NaH2PO4, and 25 mM glucose). Slices were transferred to holding solution (containing 93 mM NMDG, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM sodium ascorbate, 2 mM Thiourea, 3 mM sodium pyruvate, 10 mM MgSO4·7H2O, 0.5 mM CaCl2, pH 7.3 adjusted with HCl, 300–310 mOsm L−1), with continuous 95% O2 and 5% CO2 bubbling, first at 34°C for 30 min, and then at room temperature. Whole-cell recordings were performed using Multiclamp 700B patch-clamp amplifier (Molecular Devices, Palo Alto, CA), and the slices were maintained in the artificial cerebral spinal fluid (aCSF) containing 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 2 mM MgCl2, 2 mM CaCl2, and 10 mM glucose. The pH of bath solution was adjusted to 7.3 with HCl, and osmolarity at 300–310 mOsm L−1. Patch pipettes were pulled from borosilicate glass (~5–8 MΩ) and filled with a pipette solution consisting of 126 mM K-Gluconate, 4 mM KCl, 10 mM HEPES, 4 mM Mg ATP, 0.3 mM Na2 GTP, 10 mM Phospho-Creatnine (pH 7.3 adjusted with KOH, 290 mOsm L−1). Data were collected using pClamp 10 and Clampex 10.4 software (Molecular Devices, Palo Alto, CA), sampled at 10 kHz and filtered at 3 kHz, analyzed with Clampfit 10.4.

Immunofluorescence
Mice were deeply anesthetized with 2.5% Avertin and perfused transcardially with ice-cold artificial cerebrospinal fluid (aCSF), followed by post-fixation in 4% PFA overnight at 4°C in darkness. Then, the samples were cut into 30-μm sections by a vibratome (Leica, VTS1200). Brain slices were washed three times in 1× PBS for 10 min each. Blocking was performed for 2 h in 0.3% triton PBS + 5% donkey serum. Primary antibody was diluted in 0.05% triton PBS + 5% donkey serum and incubated at 4°C for two nights. After washing three times with PBS, the samples were incubated with secondary antibodies conjugated to Alexa Flour 488, 555, or 647 (1:500, Jackson ImmunoResearch) for 2 h at room temperature, followed by extensive washing in PBS. The secondary antibody was diluted in 0.05% triton PBS + 5% donkey serum. Sections were counterstained with DAPI and mounted in fluorescent mounting media (DAKO). Images were taken with a Zeiss LSM880 confocal microscope.
Primary antibodies used were listed as follows: rabbit anti-GFAP (1:1000, Cat# AB9610, Millipore), guinea pig anti-Olig2 (1:1000, Cat# OB-PGP040-01, Oasis Biofarm), rat anti-RFP (1:1000, Cat# 5F8, Chromotek), rat anti-GFAP (1:1000, Cat# 13-0300, Invitrogen), rabbit anti-S100β (1:1000, Cat# ab52642, Abcam), rabbit anti-Ascl1 (1:1000, Cat# ab211327, Abcam), rabbit anti-ND1 (1:1000, Cat# AB205300, Millipore), mouse anti-Aldh1l1 (1:500, Cat# AB10673448, NeuroMab), guinea pig anti-NeuN (1:1000, Cat# ABN90, Millipore), rabbit anti-Ngn2 (1:1000, Cat# PA5-78556, Invitrogen), guinea pig anti-Sox10 (1:1000, Cat# OB-PGP001-01, Oasis Biofarm), rabbit anti-Iba1 (1:1000, Cat# 019-19741, Wako), rabbit anti-Sox9 (1:1000, Cat# AB5535, Millipore), rabbit anti-Sox2 (1:1000, Cat# AB5603, Millipore), rabbit anti-Dlx2 (1:1000, Cat# ab135620, Abcam), rabbit anti-NG2 (1:500, Cat# AB5320, Millipore), guinea pig anti-CC1 (1:1000, Cat# OB-PRT039-01, Oasis Biofarm), mouse anti-Nestin (1:500, Cat# Rat-401, DHSB), rabbit anti-Vimentin (1:1000, Cat# ab92527-1002, Abcam), chicken anti-GFP (1:2000, Cat# ab13970, Abcam), mouse anti-VP16 (1:500, Cat# sc-7545, Santa Cruz), rabbit anti-CamkII (1:500, Cat# ab5683, Abcam), rabbit anti-GABA (1:500, Cat# A2052, Sigma), rabbit anti Sfxn5 (1:1000, Cat# ab172971, Abcam), guinea pig anti-DCX (1:1000, Cat# AB2253, Sigma).

Statistics
Statistical analysis was performed with GraphPad Prism 8 and Origin 2021. Statistical significance was tested with the unpaired two-sided Student’s t-test and One-Way ANOVA test followed with the Bonferroni’s post hoc test. Significance was determined at p value ≤ 0.05, and data were presented as mean ± SD.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors investigated whether the reprogramming TFs turn on downstream genes that serve as a non-pre-existing barrier to reprogramming. They found that three reprogramming TFs, Ngn2, Ascl1 and Neurod1, all turn on Olig2 very rapidly post transduction. They then go on to demonstrate that Olig2 is turned on in astrocytes, and that by knocking down Olig2, more neurons are labeled by their tdTomato reporter (see major issue below). The tdTomato+ neurons are electrically excitable, especially those in which Olig2 is first knocked down. Using scRNA-seq, the authors demonstrate that Ngn2 and Ngn2+shOlig2 have distinct effects on gene expression within astrocytes, which can be broken down into 5 clusters. They further go onto to perform CUT&Tag to identify the Olig2 binding sites, and demonstrate that Olig2 binds to and represses neurogenic genes, including Ngn2 itself.\nTaken together, this is a nice study, and the identification of Olig2 as a barrier to reprogramming is a novel finding. However, there are several issues that need to be addressed prior to publication:\nMajor issue:\nThe authors have not acknowledged the issue with the AAV-GFAP vector, which is that when coupled to a neurogenic TF, such as the bHLH TFs used herein, the GFAP promoter loses its astrocytic specificity and transgene expression is observed in endogenous neurons (Revisiting astrocyte to neuron conversion with lineage tracing in vivo. Wang et al. 2021. Cell. 184, 5465–5481). Thus, for all the analyses of tdtomato+ cells in this study, especially after 7 days post-transduction, the majority of the labeled cells will be endogenous neurons in which transgene expression has been activated.\nThis caveat affects the following analyses and conclusions:\n1. To trace converted cells, it is essential to use the pre-labeling of glia, which was not done in this study. Thus, for Figure 2e-i, without assessing pre-labeled astrocytes, it is unclear to which extent the tdT+ cells were endogenous neurons that expressed tdTomato, versus astrocytes that had converted into neurons. The loss of astrocytic marker co-expression with tdTomato reflects at least in part a change in the promoter activity from driving astrocytic to neuronal expression.\n2. The electrophysiological studies in Figure 3 that targets tdtomato+ cells. For these analyses, while the shOlig2 improves the electrophysiology of labeled neurons, it is not clear whether it has also affected the extent of endogenous neuronal activity. These studies could be performed in a pre-glial labeling model, with tdTomato and the pre-labeled glia (e.g., GFP+) targeted for recordings.\nFinally, it is essential that the Wang et al. study above is highlighted, and the review by Magdalena Gotz that highlights a Call for Obligatory Controls and Standards.\nBocchi R, Masserdotti G, Götz M. Direct neuronal reprogramming: Fast forward from new concepts toward therapeutic approaches. Neuron. 2022 Feb 2;110(3):366-393. doi: 10.1016/j.neuron.2021.11.023. Epub 2021 Dec 18. PMID: 34921778.\nGeneral comments:\n1. Typos/Grammar:\n• line 92 should be TdT reporter, not TdT report\n• line 97, should be immunostaininig, not immunostainging;\n• line 111, “in great contrast” should just be “in contrast”;\n• line 119 “detected all in” should be detected in all.\n• line 200-201 reads “Then three populations of tdT cells… and should read “The three populations of tdT cells..”\n• line 332 Molocle 2, should be Monocle 2.\n• line 651 “Methds” should be “Methods”\n2. Actual p values should be written on the figures rather than using the asterisks.\n3. There is a lack of detail of stages of analysis post AAV transduction throughout, which makes it difficult to evaluate the data.\n• Figure 1d-k, no where in text or figure legend is it indicated at how many days post transduction the cells were quantified. Only by a careful inspection of the figure is it clear that it is at 7 days post-transduction. In general, the authors should indicate time of analysis throughout the text.\n• Figure 1f schematic, it appears that the tamoxifen injections were between 14 and 10 days post viral transduction, but in the text, the only information on line 115 is that the AAVs were introduced in cortical regions at 2-5 months of age after tamoxifen induction. This description is too vague and does not highlight important information, including time after tamoxifen injection and how much time post AAV transduction the cells were counted.\n• Suppl Figure 1 – all panels- how many days post transduction were these counts performed?\n• Suppl Figure 2- how many days post transduction were these counts performed?\n• Suppl Figure 3 – how many days post transduction were these images collected? Counts should be performed.\n4. There is some vagueness to the scRNA-seq description, along with a lack of consideration of alternative interpretations of the data.\n• Lines 256-259 “To gain mechanistic insight into how Olig2 impedes AtN conversion, we performed single-cell RNA sequencing (scRNA-seq) of freshly pooled cortical samples infected with Ngn2 either in the presence or absence of shOlig2 vectors from18 to 22 dpi (n = 2 mice for each timepoint, Fig. 4a).”\n-This is a vague description, in the methods, it says 18, 20 and 22 dpi, which is what should be stated here. Otherwise, it is not clear which days between 18 and 22 the analyses were performed.\n-In addition, there should be a control transduction with a GFAP-tdTomato vector alone to know which gene expression changes are related to the reprogramming TF.\n• Lines 266-270 “The tdTomato transcript was mainly distributed in astrocytes, whereas tdTomato reporter gene was barely detected in neurons (Supplementary Fig. 7e, f). This might be due to the limited number of reprogrammed neurons at these time points (Fig. 2h) and neuronal damage caused by tissue dissociation.”\nSince tdTomato expression is driven by a GFAP promoter, once astrocytes convert to neurons, or GFAP promoter activity changes to driving expression in endogenous neurons when coupled to a neurogenic bHLH TF, the GFAP promoter will no longer be active to drive tdTomato expression. Thus, the only cells that one would expect tdTomato to be expressed in are astrocytes. The rationale in lines 266-270 is not the most obvious.\n• Lines 272-274 “We confirmed the downregulation of Olig2 transcript and protein expression level in AAV-shOlig2 infected cells at 21 dpi (Fig. 4d,e), similar to the immunostaining results at 7 dpi (Fig. 2c, d).”\nWhat about Ngn2 expression?\n5. The identification of 5 astrocyte clusters is of interest, but it is not clear whether these astrocytic clusters would already exist in a control brain, or their appearance would be associated with viral transduction in general (an AAV-GFAP-tdTomato control is missing). Further comparisons should be made to these additional controls to fully understand the significance of these clusters. As it stands, it is not clear how this analysis helps to demonstrate that there is neuronal conversion.\n• Lines 298-301 “In addition, tdT+ astrocytes in both groups were mainly enriched in the A3 and A4 subpopulations (Fig. 4c, l), indicating that Ngn2 alone is sufficient to change the profile of astrocytes and push them to enter a transcriptionally and translationally active state.” As mentioned above, if tdT turns off in astrocytes that have already converted to neurons, it is unclear whether the tdT+ astrocytes are the most representative of cells at a later stage in lineage conversion. Furthermore, a comparison to a tdt control vector would make it much easier to distinguish changes associated with the reprogramming TF. (The following section of the manuscript “Olig2 inhibits Ngn2-mediated AtN conversion in multiple ways” could be better integrated into this section.\n• Lines 323-330. What it is the criteria for Cytotrace and what makes it a predictive tool for differentiation potential? The tool should be further described. It is rather vague as it stands.\n• Lines 350-352 “Olig1, another bHLH transcription factor reported to have the ability to promote axonogenesis, also showed a significant increase in cellular trajectory (Fig. 4r).” It is unclear what a “significant increase in cellular trajectory” means?\n• Many of the conclusions from the scRNA-seq data analysis in the results section are overreaching. For instance: (1) line 394, a change in ribosomal gene expression does not necessarily mean that protein synthesis is altered, (2) line 400-401, indicated that Olig2 promotes Ngn2-mediated astrocyte dedifferentiation, and (3) metabolic changes in the subsequent section should be referred to as changes in gene expression and not a metabolic shift. There are not metabolic assays conducted to support that contention. In summary, most of the GO and DEG description in the results should be more succinct, and possible interpretations mainly moved to the discussion\nOther minor comments:\n1. Suppl. Figure 1a should show lower magnification images to reveal the full extent of the transduced area.\n2.Without showing DAPI, it is hard to confirm the signal is a colocalization or its nearby cells. I suggest including the Dapi.\n3. Suppl. Fig. 1d does not show Olig2 expression in the control brain. Perhaps a lower magnification image could be shown to demonstrate that Olig2 is expressed.\n4. -Line 137, it is very important to know the percentage of tdT+ cells expressing the TF from the total number of tdT+ cells, as your Sup 1a shows 100% and Sup 1f shows a lower percentage.\n5. -Line 151, Please make it clear that Sox2 and Dlx2 were incorporated in AAV-GFAP.\n6. -Line 161, highlights that the role of Ngn2 in AtN conversion is relatively well characterized but mainly in vitro and not in vivo and not with the AAV-GFAP system used herein. References should be provided.\n7. Why is “neuronal conversion” between 30 (10% NeuN+) and 45 days (30% NeuN+) so different if you had complete Olig2 knockdown since day 7? What causes this gradual increase? Figure 2G.\n8. Line 198, what is the characteristics of the so-called intermediate cells? Do they express Olig2? Do they express immature neuronal markers or both? Further marker analyses would be informative.\n9. Line 215. The experiments in suppl. Fig 6 are not fully described.\n10. Line 266, How can you explain the Td+ transcripts or gene reporter in the Oligodendrocyte cluster and few other cell clusters, if it was Astrocyte specific? Since you stated that 100% of Td+ cells expressed S100b, Aldh1/1, and GFAP? A feature plot of Olig2 expression for both conditions as in Fig7 d and e would be helpful.\n11. -Line 362, Vim, Sox4, Nes, and Olig1 were upregulated, is this upregulation is in the TdT+ or TdT- cells? If it is in the positive cells, same genes were upregulated in the Ngn2 shOlig2 and considered to be dedifferentiation genes, how this can be explained?\n12. Line 519, to conclude that Olig2 inhibited the expression of Ngn2 specially in Fig 7k and l, it would be important to perform a quantitative analysis, such as qPCR on FACS enriched cells.", "reviewer_2": "Review of Lai et al. on Olig2 as a Barrier to Ngn2-Mediated Astrocyte-to-Neuron Conversion\nSummary:\nLai et al. present a study investigating the role of Olig2 in astrocyte-to-neuron (AtN) conversion mediated by proneural transcription factors (TFs). Olig2 has previously been highlighted as a potential barrier to neuronal fate conversion following cortical stab wound injury (Buffo A, et al. 2005. PNAS). Here Lai et al. make a very interesting observation that Olig2 is upregulated in astrocytes following forced AAV-mediated expression of bLHL proneural TFs (Ngn2, Ascl1, Nd1) in the absence of injury and propose that here too, Olig2 may act as a inducible barrier to neuronal conversion. While this is an intriguing idea, we find significant issues in the experimental evidence and interpretations. Without addressing these concerns, the central claims of the paper cannot be substantiated.\nMajor Concerns:\n1. Lack of Robust Evidence for Astrocyte-to-Neuron Conversion\nThe central claim of the paper is that downregulation of Olig2, combined with ectopic expression of proneural bHLH TFs (Ngn2, Ascl1, NeuroD1), induces AtN conversion in adult mouse cortical astrocytes. However, the evidence presented is insufficient to establish true lineage conversion for the following reasons:\n• Contextual Regulation of hGFAP Promoter: The hGFAP mini-promoter in the AAV vector system is now well appreciated to show variable and highly contextual expression in endogenous neurons (Wang et al., 2021, Cell; Le et al., 2022, Front. Cell. Dev. Biol.; Xie et al., 2023, eBioMedicine; Enbar et al, 2024, Sci. Rep.). Thus, the observation of viral reporter protein (tdTomato) fluorescence in NeuN+ cells is not sufficient evidence to demonstrate the cells arise from astrocytes.\n• Alternative Hypotheses Not Addressed: The authors argue that the increased percentage of tdT+S100B-NeuN+ cells (neurons) and reduced percentage of tdT+S100B+NeuN- (astrocytes) observed following Olig2 downregulation (but not in scrambled shRNA control) is evidence for astrocyte to neuron conversion. However, the authors do not consider any other alternative hypotheses. For example, these data would also result from the death of astrocytes following the intervention with leaky expression of tdTomato in endogenous neurons.\n• Absence of Robust Fate-Mapping Experiments: To validate the claim of AtN conversion, it is imperative that - at a minimum - robust, viral vector independent fate-mapping are performed. Ideally, these should use well validated tools such as Aldh1l1-CreERT2 mice crossed with Cre-reporter lines. The current experiments do not exclude the possibility of fluorophore expression in endogenous neurons or other non-astrocytic cells. Given the importance of the claim the paper is making, we would also recommend that efforts are made to label endogenous neurons prior to AAV injections to confirm that tdT is not being expressed in endogenous neurons. There are several ways researchers could go about this (e.g. using transgenic excitatory or pan-neuronal cre-driver lines with a GFP/YFP reporter, EdU/BrDU labelling in utero, or using additional viral tools).\n• No Evidence for Intermediate Stages: In figure 2L, authors present a schematic of astrocytes passing via an intermediate stage to neurons, however, this is highly misleading as no substantive evidence has been presented for intermediate stages. Whilst authors note that there is an increased number of tdT+S100B-NeuN- cells when Olig2 is downregulated, the absence of S100B is not of itself evidence that these transduced cells will go on to acquire neuronal properties and there is no positive identification of immature neuronal genes (e.g. Dcx) in the histology or scRNA dataset provided. As such, it the tdT+S100B-NeuN- cells have not been identified and should not be presented alone as an intermediate step of a conversion process as there may be other explanations (e.g. we note that in the scRNA dataset many Oligodendrocyte lineage cells express the viral transcript. Alternatively, astrocyte toxicity may result in absence of detection of S100B downregulation, etc). Finally, there is no morphological characterisation or clear imaging data presented that would corroborate an intermediate or immature neuronal stage.\n• scRNA-seq Analysis: Authors self-report that they were unable to collect reprogrammed neurons that express viral transcripts. They speculate this may be for technical reasons or due to limited cell numbers converted. Either way, this data is not capable of supporting the central claim of AtN conversion. Additionally, the scRNA-seq analysis revealed the presence of multiple cell types, including mural cells, endothelial cells, microglia, and oligodendroglia (which express viral transcripts at broadly levels similar to astrocytes). This could demonstrate a lack of specificity in the AAV targeting astrocytes or issues around cell sorting. Together these technical issues somewhat limits what can be concluded from these data but there are still some interesting observations. Namely, downregulation of Olig2 along with AAV-mediated expression of Ngn2 seems to result in a more neural stem-cell like transcriptome, however, this should not be over-interpreted this as a step towards to neuronal conversion, given that currently no evidence is provided for a trajectory towards lineage converted or immature neurons.\n2. Issues with Olig2’s Role and Mechanistic Insight\n• CuT&Tag Analysis of Olig2 Binding: The authors attempt to investigate Olig2’s function as a reprogramming barrier using CuT&Tag experiments. Surprisingly, the canonical Olig2 binding site is not enriched in the genomic regions identified as bound by Olig2. Whilst we accept that this could suggest Olig2 binds non-canonical sites, the data are more plausibly explained by a failure of the CuT&Tag experiment. Promoters are enriched in GC regions, and the 27% of enriched regions containing Gs or Cs likely reflect promoter capture rather than specific Olig2 binding. To support the authors claim of Olig2 binding non-canonical sites, the authors should include additional replicates of the CuT&Tag experiment and validate binding through ChIP-qPCR or band shift assays for targets lacking canonical E-box sites.\n4. Additional concerns/observations:\n• Marker Expression Over Time: The authors claim that the hGFAP in AAV9 is \"highly specific\" for targeting astrocytes. However, the authors find that even following conversion (and at late, 45dpi, timepoints) there is expression of tdTomato in putative converted NeuN+ cells. It is unclear how the authors rationalise ongoing tdTomato expression in reprogrammed neurons if the hGFAP promoter is specific for astrocytes? How can authors distinguish between genuinely converted cells and mislabelled (i.e. leaky tdTomato expression in) endogenous neurons in experiments (e.g. in electrophysiology)?\nRecommendations:\nTo address these concerns, the authors should:\n1. Include Fate-Mapping Experiments: Use transgenic mouse lines (e.g., Aldh1l1-CreERT2) with Cre-reporter systems to ensure the origin of induced neurons is astrocytic and to exclude targeting of endogenous neurons.\n2. Demonstrate that tdTomato is not expressed in endogenous neurons: (or characterise the extent of this and show how authors distinguish between these and astrocyte-derived cells). E.g. label endogenous neurons prior to AAV injections (e.g., using pan-neuronal Cre-driver lines or BrdU/EdU labeling) to confirm tdT is not expressed in pre-existing neurons.\n3. Show morphology: Microscopy should demonstrate the morphology of putative lineage converted cells, ideally with some intermediate/immature morphological stages.\n4. Use Intermediate Markers: Demonstrate the progression of reprogramming using markers (e.g. DCX) for intermediate/immature neuronal stages.\n5. Replicate & validate CuT&Tag result: Authors should do further work on this aspect as the current claims are strong considering that failure to identify canonical Olig2 binding sites may be consistent with experimental failure.\nConclusion:\nLai et al. provide an interesting observation regarding Olig2 upregulation during ectopic expression of proneural TFs in astrocytes. However, the evidence presented does not support the claim of astrocyte-to-neuron conversion or establish Olig2’s role as a barrier to reprogramming. Since this is the central claim of the paper, more experimental work is needed before this could be published. If the claims made were confirmed to an appropriate standard, the findings would be of considerable interest.", "reviewer_3": "In the present manuscript, C. Lai, K. Hou and colleagues identified Olig2 as barrier during in vivo direct reprogramming of astrocytes into neurons. First, the authors showed that Olig2 is upregulated in astrocytes following the expression of bHLH TFs. Then, they investigated the effect of Olig2 knock-down in Ngn2-mediated direct conversion, unravelling an increased reprogramming efficiency and maturation of Ngn2-shOlig2-iNs; last, they performed single cell RNAseq analysis to investigate the mechanisms regulated by (sh)Olig2 in the conversion process.\nOverall the manuscript is interesting and of interest for the in vivo direct reprogramming field.\nHowever, there are several points that need to addressed before considering for publication;\n_ the identification of Olig2 as barrier for in vivo direct reprogramming was postulated and partially revealed years ago (doi/10.1073/pnas.0506535102). While the present manuscript is a substantial step forward towards understanding the mechanisms underlying the conversion process, the role of Olig2 in direct neuronal reprogramming is not “unexplored” (line 160). The authors are well-aware of the paper, as they used the same approach to manipulate Olig2 (using Olig2-VP16). Therefore, it is important to reshape the introduction to include previous work on Olig2;\n_ Previous work from the same group showed that the proportion of astrocytes expressing Olig2 in vivo is region-specific, spanning from 8% to 13% in the cortex (doi.org/10.1186/s13041-021-00747-0). However, in their control-injected mice (Fig.1f, h), the proportion of GFP+ cells expressing Olig2 is very low. Can the authors comment on the discrepancy of the results? Is it possible that their AAVs or Gfap promoter have a preference for a subset of astrocytes?\n_ Can the authors quantify the intensity of Olig2 in OPCs following control and Ngn2 transduction (figure 1f)?. According to the figure, it seems that in Ngn2-injection condition, tdT+GFP- cells have a stronger signal than tdT+GFP- cells in control condition; that would suggest a cell non-autonomous effect;\n_ the authors quantified the number of Iba1+ cells and did not find a change in different conditions; can the authors comment and discuss the morphology of Iba1+ cells, to better understand whether the presence of the reprogramming factor leads to an inflammatory state?\n_ Please specify in the text and figures when transgenics mice and when viruses are used. For instance, in figure 1k it is not clear that the authors used tdT comes from the AAV injection and not from the Sox10-CreERT2::Ai14 transgenic mouse used in experiment in panel 1f;\n_ can the authors include as well GFP channel in Figure 2C, to better understand the proportion of infected cells with the sole AAV-shNC/Olig2? Can the authors quantify the proportion of double-infected cells (tdT+GFP+). See comment in the scRNASeq;\n_ Supplementary figure 2 is missing, at least in the downloaded pdf file;\n_ the observation that cells at intermediate stage are increased in Ngn2-shOlig2 condition is very interesting, but is not followed up;\n- can the authors test whether these cells are still expressing low levels of Olig2? Or do they express higher levels of neural stem cells markers (e.g., Sox2, Pax6)? Can the authors find them in their scRNAseq? what is their signature?\n- please specify that the quantification of the intensity in suppl. Fig. 4e refers to supply. Fig. 4d and suppl. Fig.4g refers to Suppl. Fig.4f;\n_ scRNAseq data are interesting and raise some important questions:\n- Supplementary figure 7e shows the distribution of TdT+ cells in various clusters; if correctly interpreted, suppl. Fig. 7s shows the proportion of TdT+ cells in various cluster as well, providing quantitative information to Supplement. Fig. 7e. From these 2 panels, it is evident that at least 25% of TdT+ cells are located in the oligodendrocyte lineage. However, in the initial characterisation of the vector system, the authors claim that the system is very tight and specific for astrocytes. Can the authors explain this discrepancy? Which of the data is true? Can the authors analyze as well these “oligodendrocyte” cluster and compare it to the “astrocyte” cluster of infected cells to get an insight whether a neuronal cascade is being induced in oligodendrocytes or not?\n_ As the authors use 2 different AAV vectors, one carrying TF-TdT and one shControl/Olig2-GFP, it is very likely the presence of 3 groups of infected cells: single one (TdT+, GFP+) and double positive (tdT+GFP+). Can the authors detect these 3 groups in the scRNAseq?\n_ If so, can the authors show in which clusters the various subgroups are localized? Do Ngn2-tdT+ cells coming from Ngn2-shControl localize together with Ngn2-shControl and Ngn2 only from Ngn2-shOlig2?\n_ Are Ngn2-shOlig2 double positive cells (and not just tdT+ cells) enriched in astrocyte cluster 4 (Fig.4n)? It is not clear whether the authors subset only the double positive astrocytes, or they consider all astrocytes coming from the injection, irrespective of whether they are single, double or not transduced at all. A clarification in the text and figure will greatly improve the interpretation of the data;\n_ the inclusion of NPCs into the data allows for a better interpretation of the data; however, it is not clear the source of the NPCs. Did the author sequence as well NPCs? Did they merge the data from another publication? This information could not be found in the material and method;\n_ can the authors confirm via IF the change in the expression of at least 1 gene coding for mitochondrial protein? This would substantiate the interesting finding.\n_ The interpretation that the increase in Ngn2 depends on the derepression of the endogenous Ngn2 promoter in shOlig 2 conditions is difficult to prove. As the authors stain for Ngn2, can the authors discriminate between the endogenous Ngn2 and the over-expressed? It has previously been shown that Olig2 can regulate Gfap expression (doi:10.1242/dev.02847). A possibility is that Olig2 regulates the Gfap promoter used to induce the expression of Ngn2 in AAV. Can the authors exclude this hypothesis by testing it? For instance by using a luciferase assay?\n_ In the discussion, the authors suggest that Olig2 activation might be an “second line of defence against bHLH proneural transcription factor mediate cell reprogramming” (lines 623-624) based on the the fact that Sox2 or Dlx2 did not induce Olig2. This sounds a kind of deterministic hypothesis, where cells have “implemented” the possibility that bHLH-mediate cell reprogramming occurs and it needs to be prevented. However, it is well known that upon injury or neurodegeneration, astrocytes become Olig2 positive. Therefore, it seems more likely that Olig2 induction reflects some sort of reactive state. Can the authors check if shOlig2+ cells show lower level of reactivity in scRNAseq and via IF? Either case, the cited sentence needs to be changed.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point responses:\nReviewer #1 (Remarks to the Author):\nIn this study, the authors investigated whether the reprogramming TFs turn on downstream genes that serve as a non-pre-existing barrier to reprogramming. They found that three reprogramming TFs, Ngn2, Ascl1 and Neurod1, all turn on Olig2 very rapidly post transduction. They then go on to demonstrate that Olig2 is turned on in astrocytes, and that by knocking down Olig2, more neurons are labeled by their tdTomato reporter (see major issue below). The tdTomato+ neurons are electrically excitable, especially those in which Olig2 is first knocked down. Using scRNA-seq, the authors demonstrate that Ngn2 and Ngn2+shOlig2 have distinct effects on gene expression within astrocytes, which can be broken down into 5 clusters. They further go onto to perform CUT&Tag to identify the Olig2 binding sites, and demonstrate that Olig2 binds to and represses neurogenic genes, including Ngn2 itself.\nTaken together, this is a nice study, and the identification of Olig2 as a barrier to reprogramming is a novel finding. However, there are several issues that need to be addressed prior to publication:\nResponse: We would like to sincerely thank the reviewer for your thoughtful comments and for recognizing the novelty of our study. We greatly appreciate the acknowledgment of our identification of Olig2 as a barrier to reprogramming, which we believe represents an important advancement in understanding the molecular mechanisms of AtN reprogramming. We are encouraged that the novelty of this finding was recognized, and we are grateful for the constructive feedback provided.\nMajor issue:\nThe authors have not acknowledged the issue with the AAV-GFAP vector, which is that when coupled to a neurogenic TF, such as the bHLH TFs used herein, the GFAP promoter loses its astrocytic specificity and transgene expression is observed in endogenous neurons (Revisiting astrocyte to neuron conversion with lineage tracing in vivo. Wang et al. 2021. Cell. 184, 5465–5481). Thus, for all the analyses of tdtomato+ cells in this study, especially after 7 days post-transduction, the majority of the labeled cells will be endogenous neurons in which transgene expression has been activated.\nResponse: Thank you for your important feedback. We have carefully reviewed the article you referenced (Wang et al., 2021, Cell) and considered the potential issue of AAV-GFAP vector leakage into neurons when combined with bHLH neurogenic transcription factors. In fact, the Cell paper also shows that Ngn2 does not induce significant leakage expression, and the probability of leakage is relatively low compared to other neurogenic transcription factors (Wang et al., 2021, Cell, Fig. 5F, I)1. In our own experiments, we observed that by 7 days post-transduction (7 dpi), there was almost no expression of the reporter fluorescent protein (tdTomato) in endogenous neurons (Fig. 2g; Supplementary Fig. 6a, b). This suggests that, despite the presence of the neurogenic transcription factor Ngn2, the GFAP promoter largely retains its specificity for astrocytes, with minimal leakage into endogenous neurons.\nThis caveat affects the following analyses and conclusions:\n1. To trace converted cells, it is essential to use the pre-labeling of glia, which was not done in this study. Thus, for Figure 2e-i, without assessing pre-labeled astrocytes, it is unclear to which extent the tdT+ cells were endogenous neurons that expressed tdTomato, versus astrocytes that had converted into neurons. The loss of astrocytic marker co-expression with tdTomato reflects at least in part a change in the promoter activity from driving astrocytic to neuronal expression.\nResponse: We sincerely appreciate the reviewer’s critical point regarding the potential loss of GFAP promoter specificity when co-expressing neurogenic TFs. We acknowledge the caveat raised by Wang et al. (2021) and agree that pre-labeling astrocytes is essential for conclusive lineage tracing. To address this, we employed an astrocyte lineage tracing transgenic mice (Aldh1l1CreERT2 x Ai14), enabling tamoxifen-dependent astrocyte pre-labeling prior to Ngn2 induction. Post-transduction analyses (45 dpi) confirmed that tdTomato+ cells exhibited neuronal morphology, expressed mature neuronal markers (NeuN), and lacked astrocytic markers (S100β), strongly supporting bona fide AtN conversion. These new findings have been added in our revised manuscript (lines 295-317, new Fig. 4). Moreover, we have revised the text to explicitly discuss this limitation (lines 631-659; lines 779-785).\n2. The electrophysiological studies in Figure 3 that targets tdtomato+ cells. For these analyses, while the shOlig2 improves the electrophysiology of labeled neurons, it is not clear whether it has also affected the extent of endogenous neuronal activity. These studies could be performed in a pre-glial labeling model, with tdTomato and the pre-labeled glia (e.g., GFP+) targeted for recordings.\nFinally, it is essential that the Wang et al. study above is highlighted, and the review by Magdalena Gotz that highlights a Call for Obligatory Controls and Standards.\nBocchi R, Masserdotti G, Götz M. Direct neuronal reprogramming: Fast forward from new concepts toward therapeutic approaches. Neuron. 2022 Feb 2;110(3):366-393. doi: 10.1016/j.neuron.2021.11.023. Epub 2021 Dec 18. PMID: 34921778.\nResponse: We thank the reviewer for raising this critical concern and for recommending the key references. While Wang et al. (2021) demonstrated that GFAP-driven bHLH neurogenic TFs may lead to neuronal leakage, we note that Ngn2 exhibits significantly lower off-target expression compared to Pax6, ASCL1 and NeuroD1 (Wang et al., 2021, Cell, Fig. 5F, I)1. Consistent with this, Mattugini et al. (2019, Neuron) reported minimal leakage when Ngn2 alone was expressed in adult cortical astrocytes (Supplementary Fig. 2A)2. In our study, Ngn2 or Ngn2+NCshRNA expression resulted in only ~10% tdTomato+ neurons, whereas Ngn2+shOlig2 increased this to ~30% at 45 dpi (Fig. 2f, g), suggesting Olig2 knockdown enhances conversion efficiency rather than amplifying leakage.\nCrucially, electrophysiological analyses revealed that 18.2% of tdTomato+ neurons fired only single action potentials, and nearly half lacked detectable synaptic activity (Fig. 3e and 3o), resembling immature neurons rather than endogenous mature neurons in adult mouse cortex. While we cannot entirely rule out minor leaky expression of tdTomato in endogenous neurons, our astrocyte lineage tracing mice minimized confounding effects. We have modified the text to meet the criteria proposed by Götz and colleagues (Bocchi et al., 2022), including an explicit discussion of the limitations of the GFAP promoter (lines 631-640)\nGeneral comments:\n1. Typos/Grammar:\n• line 92 should be TdT reporter, not TdT report\n• line 97, should be immunostaininig, not immunostainging;\n• line 111, “in great contrast” should just be “in contrast”;\n• line 119 “detected all in” should be detected in all.\n• line 200-201 reads “Then three populations of tdT cells… and should read “The three populations of tdT cells..”\n• line 332 Molocle 2, should be Monocle 2.\n• line 651 “Methds” should be “Methods”\nResponse: Thank you, we have corrected these typos/grammar and highlight them in the revised manuscript. TdT reporter (line 95); immunostaining (line 100); in contrast (line 128); detected in all (line 125); The three populations of tdT cells (line 231); Monocle 2 (line 406); Methods (line 793).\n2. Actual p values should be written on the figures rather than using the asterisks.\nResponse: Thanks for your suggestion, we have added actual p values in the figures.\n3. There is a lack of detail of stages of analysis post AAV transduction throughout, which makes it difficult to evaluate the data.\n• Figure 1d-k, no where in text or figure legend is it indicated at how many days post transduction the cells were quantified. Only by a careful inspection of the figure is it clear that it is at 7 days post-transduction. In general, the authors should indicate time of analysis throughout the text.\n• Figure 1f schematic, it appears that the tamoxifen injections were between 14 and 10 days post viral transduction, but in the text, the only information on line 115 is that the AAVs were introduced in cortical regions at 2-5 months of age after tamoxifen induction. This description is too vague and does not highlight important information, including time after tamoxifen injection and how much time post AAV transduction the cells were counted.\n• Suppl Figure 1 – all panels- how many days post transduction were these counts performed?\n• Suppl Figure 2- how many days post transduction were these counts performed?\n• Suppl Figure 3 – how many days post transduction were these images collected? Counts should be performed.\nResponse: I sincerely apologize for making it difficult to evaluate the data due to unclear labeling. Based on your suggestions, we've added this information to the images and captions. (line 1125 and lines 1133-1134 in main text; all changes in supplementary materials are highlighted with yellow)\n4. There is some vagueness to the scRNA-seq description, along with a lack of consideration of alternative interpretations of the data.\n• Lines 256-259 “To gain mechanistic insight into how Olig2 impedes AtN conversion, we performed single-cell RNA sequencing (scRNA-seq) of freshly pooled cortical samples infected with Ngn2 either in the presence or absence of shOlig2 vectors from18 to 22 dpi (n = 2 mice for each timepoint, Fig. 4a).”\n-This is a vague description, in the methods, it says 18, 20 and 22 dpi, which is what should be stated here. Otherwise, it is not clear which days between 18 and 22 the analyses were performed.\nResponse: Thanks for your suggestion, we have clarified this information (line 323).\n-In addition, there should be a control transduction with a GFAP-tdTomato vector alone to know which gene expression changes are related to the reprogramming TF.\nResponse: Thank you for your thorough evaluation of our manuscript and your insightful suggestion regarding the inclusion of a GFAP-tdTomato control to distinguish transcriptional changes specific to the reprogramming TF, but this will require sacrificing more experimental mice. Here, we are confident that using astrocytes from the existing sc-RNAseq database that have not been transduced with AAV as a control provides a sufficient basis for demonstrating the transcriptional changes resulting from Ngn2 overexpression in astrocytes. In conclusion, our failure to add GFAP-tdTomato as a control for the sc-RNAseq data is one of the limitations of this study, which we have added to the Discussion section (lines 772-776).\n• Lines 266-270 “The tdTomato transcript was mainly distributed in astrocytes, whereas tdTomato reporter gene was barely detected in neurons (Supplementary Fig. 7e, f). This might be due to the limited number of reprogrammed neurons at these time points (Fig. 2h) and neuronal damage caused by tissue dissociation.”\nSince tdTomato expression is driven by a GFAP promoter, once astrocytes convert to neurons, or GFAP promoter activity changes to driving expression in endogenous neurons when coupled to a neurogenic bHLH TF, the GFAP promoter will no longer be active to drive tdTomato expression. Thus, the only cells that one would expect tdTomato to be expressed in are astrocytes. The rationale in lines 266-270 is not the most obvious.\nResponse: Thank you for your suggestion. We fully agree with your suggestion and have added this possibility into our revised manuscript (lines 334-335).\n• Lines 272-274 “We confirmed the downregulation of Olig2 transcript and protein expression level in AAV-shOlig2 infected cells at 21 dpi (Fig. 4d,e), similar to the immunostaining results at 7 dpi (Fig. 2c, d).”\nWhat about Ngn2 expression?\nResponse: Thank you for raising this point. We observed that at 7 days post AAV9 injection (dpi), Ngn2 expression was significantly higher in the Ngn2+shOlig2 group compared to the Ngn2+shNC group (Fig. 8k, l). However, by 21 dpi, the expression of Ngn2 in both groups showed no significant difference. This lack of difference at 21 dpi may be attributed to a combination of factors, including the potential decrease in GFAP promoter activity during AtN reprogramming, as noted by the reviewer. At 7 dpi, astrocytes are in the early stages of reprogramming, and GFAP activity in both groups is similar. Knockdown of Olig2 at this stage likely reduces Olig2-mediated inhibition of Ngn2 expression. By 21 dpi, the knockdown of Olig2 promotes AtN reprogramming, which may lead to a decrease in GFAP promoter activity. These two factors—reduced Olig2 inhibition and decreased GFAP promoter activity—may contribute to the lack of significant difference in Ngn2 expression between the two groups at 21 dpi. Please check this new data below:\nFigure legend: a, TdT and Ngn2 double staining at 21 dpi. Scale bar, 20 μm. b, Quantified data showing no significant different in Ngn2 intensity between two groups; n = 5 per group, unpaired Student’s t-test, mean ± SD.\n5. The identification of 5 astrocyte clusters is of interest, but it is not clear whether these astrocytic clusters would already exist in a control brain, or their appearance would be associated with viral transduction in general (an AAV-GFAP-tdTomato control is missing). Further comparisons should be made to these additional controls to fully understand the significance of these clusters. As it stands, it is not clear how this analysis helps to demonstrate that there is neuronal conversion.\nResponse: Thank you for your insightful comment. We apologize for not including an AAV-GFAP::tdTomato control. To address this, we performed a supplementary analysis on the Ngn2+shNC group without AAV infection (tdT-). We found that only 9.3% of these astrocytes were in the A3 and A4 subclusters, while 90% were in the A1 and A2 subclusters. This suggests that while normal astrocytes may contain a small proportion of transcriptionally active A3 and A4 cells, the majority belong to the A1 and A2 subclusters. This new analysis has been added into our revised manuscript (lines 373-376; supplementary Fig. 9j).\n• Lines 298-301 “In addition, tdT+ astrocytes in both groups were mainly enriched in the A3 and A4 subpopulations (Fig. 4c, l), indicating that Ngn2 alone is sufficient to change the profile of astrocytes and push them to enter a transcriptionally and translationally active state.” As mentioned above, if tdT turns off in astrocytes that have already converted to neurons, it is unclear whether the tdT+ astrocytes are the most representative of cells at a later stage in lineage conversion. Furthermore, a comparison to a tdt control vector would make it much easier to distinguish changes associated with the reprogramming TF. (The following section of the manuscript “Olig2 inhibits Ngn2-mediated AtN conversion in multiple ways” could be better integrated into this section.\nResponse: We appreciate your constructive suggestion. Considering the possibility that tdT is turned off in astrocytes that have already converted into neurons, we specifically analyzed mouse cerebral cortex samples about 3 weeks after AAV injection (Figure 5a). This early time point was chosen because the reprogramming efficiency was still relatively low (Fig. 2g, h and j) and most astrocytes still maintained detectable tdT signals (Fig. 5e). Therefore, our results mainly reflect the effect of Olig2 on transcriptome regulation in the early to mid-stage of AtN reprogramming. We acknowledge that scRNA-seq analysis at the later stage of reprogramming will undoubtedly reveal more obvious transcriptional changes, which is also a direction worthy of future research.\nTo assess the effect of the Ngn2 transcription factor on the astrocyte transcriptome, we compared Td+ and Td- astrocytes within the same set of scRNA-seq datasets (Fig.6a DEGs-1 and Fig. S12). This intra-dataset comparison helps minimize systematic errors and reduces the need for experimental animals. However, whether viral transduction (AAV-GFAP-tdTomato) itself affects the astrocyte transcriptome and influences cell cluster analysis results is worth investigating. A review of the literature reveals no scRNA-seq studies on the impact of AAV-GFAP vector-mediated tdTomato labeling on the astrocyte transcriptome. However, our previous bulk RNA-seq study showed that when less than 109 GC of AAV9-GFAP::GFP was injected into the cerebral cortex of adult mice, only 13 DEGs were identified at 10 dpi, compared to PBS injection3. In this study, the AAV dose was between 0.2-2 x 108 GC. Therefore, we hypothesize that AAV-GFAP::tdTomato will not significantly affect the astrocyte transcriptome. Based on these findings, we believe that using the same scRNA-seq datasets for comparison of Td+ and Td- astrocytes is a valid and reasonable approach that will not impact our study's conclusions. Please check these data below:\nFigure legend: a, Low-magnification images show the transfection range of AAV9-GFAP::GFP virus in the cerebral cortex of adult mice. Scale bar, 200 μm. b, High-magnification images show that GFP is highly co-labeled with the astrocyte marker s100β. Scale bar, 20 μm. c, Bulk-RNAseq experimental flow chart. d, Only 13 DEGs were found. e, The 13 DEGs were plotted on the volcano map. Data shown as mean ± SD.\nIn the section titled “Olig2 inhibits Ngn2-mediated AtN conversion in multiple ways”, our goal is to explore the new effects of the Ngn2 transcription factor on astrocytes after Olig2 knockdown. To prevent the section from becoming too lengthy, we believe it would be better to present this as an independent part.\nWe again thank the reviewers for their insightful comments and look forward to these improvements effectively responding to your concerns.\n• Lines 323-330. What it is the criteria for Cytotrace and what makes it a predictive tool for differentiation potential? The tool should be further described. It is rather vague as it stands.\nResponse: Thank you for your suggestion, we have added more information in the Methods section (lines 873-883).\n• Lines 350-352 “Olig1, another bHLH transcription factor reported to have the ability to promote axonogenesis, also showed a significant increase in cellular trajectory (Fig. 4r).” It is unclear what a “significant increase in cellular trajectory” means?\nResponse: Thank you for your correction, we have modified the description of the results in this section (lines 425-426).\n• Many of the conclusions from the scRNA-seq data analysis in the results section are overreaching. For instance: (1) line 394, a change in ribosomal gene expression does not necessarily mean that protein synthesis is altered, (2) line 400-401, indicated that Olig2 promotes Ngn2-mediated astrocyte dedifferentiation, and (3) metabolic changes in the subsequent section should be referred to as changes in gene expression and not a metabolic shift. There are not metabolic assays conducted to support that contention. In summary, most of the GO and DEG description in the results should be more succinct, and possible interpretations mainly moved to the discussion.\nResponse: Thank you very much for your suggestion. We understand your concerns regarding the interpretation of the scRNA-seq data and would like to address each point in turn.\n(1) Ribosomal gene expression and protein synthesis: We acknowledge your comment regarding line 394, where we suggested that changes in ribosomal gene expression might reflect alterations in protein synthesis. We agree that this does not necessarily imply a direct change in protein synthesis, and we have revised the text to clarify this point (lines 469-471).\n(2) Olig2's role in Ngn2-mediated astrocyte dedifferentiation: In response to your concern about the statement on lines 400-401, we have revised the text to more cautiously describe the role of Olig2 in Ngn2-mediated astrocyte dedifferentiation. We have updated the language to avoid overstating the conclusions and have made it clear that these results are suggestive rather than definitive (lines 475-477).\n(3) Metabolic changes and gene expression: We appreciate your comment regarding the interpretation of metabolic changes. Since no direct metabolic assays were conducted, we have revised the section to more accurately reflect these changes as alterations in gene expression, rather than a metabolic shift. This adjustment ensures that our conclusions are more consistent with the available data (line 494; 496; 504; 506; 507; 508; 512-513; 530).\nAdditionally, we have revised the descriptions of the GO and DEG results to make them more concise and focused. We hope these revisions address your concerns.\nOther minor comments:\n1. Suppl. Figure 1a should show lower magnification images to reveal the full extent of the transduced area.\nResponse: Thank you for your valuable suggestion. We agree that including lower magnification images in Supplementary Figure 1a would provide a clearer overview of the transduced area. We have revised the figure to include additional low-magnification images, which now better illustrate the full extent of the transduction. The updated figure (new supplementary figure 1) has been uploaded in the revised manuscript for your consideration.\n2.Without showing DAPI, it is hard to confirm the signal is a colocalization or its nearby cells. I suggest including the Dapi.\nResponse: Thank you for your constructive feedback. We have revised the figure 1 to include DAPI staining in 1b and 1m, which is now shown in the updated version of the manuscript. We apologize for not adding DAPI fluorescent dye when performing immunofluorescence staining in Figure 1f. This addition should improve the clarity of the colocalization analysis. The revised figure has been uploaded for your review.\n3. Suppl. Fig. 1d does not show Olig2 expression in the control brain. Perhaps a lower magnification image could be shown to demonstrate that Olig2 is expressed.\nResponse: Thank you for pointing this out. Higher magnification images help to visualize fine structures and the localization of the fluorescence signal. However, the region we initially selected did not contain any Olig2-positive cells. Following your suggestion, we have now selected a new high-magnification image that includes a region with Olig2+ cells (new supplementary fig. 1e). The updated figure has been revised accordingly in the manuscript for your review.\n4. -Line 137, it is very important to know the percentage of tdT+ cells expressing the TF from the total number of tdT+ cells, as your Sup 1a shows 100% and Sup 1f shows a lower percentage.\nResponse: Thank you for your valuable comment. We agree that it is important to quantify the percentage of tdT+ cells expressing the transcription factor (TF) relative to the total number of tdT+ cells. Upon revisiting the data, we have calculated this percentage and included it in the revised manuscript (new supplementary Fig. 1h). The revised data and figures have been updated in the manuscript for your review.\n5. -Line 151, Please make it clear that Sox2 and Dlx2 were incorporated in AAV-GFAP.\nResponse: Thanks, we have added this information in the revised manuscript (lines 160-161).\n6. -Line 161, highlights that the role of Ngn2 in AtN conversion is relatively well characterized but mainly in vitro and not in vivo and not with the AAV-GFAP system used herein. References should be provided.\nResponse: Thank you, we have added your suggestion in the revised manuscript (lines 170-173).\n7. Why is “neuronal conversion” between 30 (10% NeuN+) and 45 days (30% NeuN+) so different if you had complete Olig2 knockdown since day 7? What causes this gradual increase? Figure 2G.\nResponse: Thank you for your thoughtful question. As you correctly noted, the process of AtN reprogramming is a dynamic biological process that takes time. While we initiated complete Olig2 knockdown from day 7, Ngn2-mediated neuronal conversion is not an instantaneous event and involves a complex series of molecular and cellular changes. This temporal progression can be attributed to several factors, including the time required for the expression of neuronal markers, synaptic integration, and functional maturation. The gradual increase in the percentage of NeuN+ cells between 30 and 45 days reflects the ongoing maturation of reprogrammed cells over time.\n8. Line 198, what is the characteristics of the so-called intermediate cells? Do they express Olig2? Do they express immature neuronal markers or both? Further marker analyses would be informative.\nResponse: Thank you for your insightful comment. The intermediate cells identified in our study exhibited distinct characteristics, including the lack of expression of both astrocyte and neuronal markers. These intermediate cells likely represent a transitional stage in astrocyte-to-neuron reprogramming, where the progenitor cells lose their astrocytic identity, but neuronal gene expression is not yet fully upregulated, resulting in the observation of these double-negative intermediate cells. These cells were only observed in the Ngn2+shOlig2 group, suggesting that they may represent a transitional cell type. Additionally, we tracked the proportion of these double-negative cells in AAV9-transduced samples and found that they first appeared at 14 dpi, peaked at 30 dpi, and subsequently declined (Fig. 2i). Based on these observations, we hypothesize that these cells are indeed transitional, and we have provided a brief description of these intermediate cells in the revised manuscript (line 230; lines 236-240).\nWhile we did not perform extensive marker analysis in this study, we can confirm that these cells do not express Olig2, as we have previously shown in our knockdown experiments.\n9. Line 215. The experiments in suppl. Fig 6 are not fully described.\nResponse: Thank you for pointing this out. We apologize for the lack of detail in the description of the experiments in Supplementary Figure 6. In the revised manuscript, we have added a more comprehensive description of the experimental procedures and data analysis related to this figure (lines 247-256).\n10. Line 266, How can you explain the Td+ transcripts or gene reporter in the Oligodendrocyte cluster and few other cell clusters, if it was Astrocyte specific? Since you stated that 100% of Td+ cells expressed S100b, Aldh1/1, and GFAP? A feature plot of Olig2 expression for both conditions as in Fig7 d and e would be helpful.\nResponse: Thank you for your insightful question. To clarify, the Td+ transcripts or gene reporter expression in the oligodendrocyte cluster and other cell clusters can be attributed to the high sensitivity of single-cell RNA sequencing. As we are detecting TdTomato mRNA expression in this analysis, it is possible that the TdTomato signal, while being specific to astrocytes at the protein level (as confirmed by immunofluorescence staining showing 100% of Td+ cells expressing S100b, Aldh1l1, and GFAP), may also be detected in other cell types at the transcriptional level due to potential cross-expression or residual mRNA in neighboring cells. This does not necessarily imply that these other cell clusters, such as oligodendrocytes, are expressing TdTomato protein but rather reflects the sensitivity and potential overlap in mRNA expression in the single-cell RNA-sequencing data. Similar findings have been reported in previous studies4-6. In addition, we also directly compared the tdT expression proportion and level between astrocytes and oligodendrocytes and found the proportion and level are much higher in astrocyte (new supplementary Fig. 9g).\n11. -Line 362, Vim, Sox4, Nes, and Olig1 were upregulated, is this upregulation is in the TdT+ or TdT- cells? If it is in the positive cells, same genes were upregulated in the Ngn2 shOlig2 and considered to be dedifferentiation genes, how this can be explained?\nResponse: The upregulation of Vim, Sox4, Nes, and Olig1 in line 362 occurs specifically in the TdT+ cells, which we have clarified in the manuscript (line 439).\nRegarding the reviewer’s concern about the upregulation of these genes in the Ngn2 shOlig2 condition, we would like to highlight that Ngn2 is a pioneer transcription factor for AtN reprogramming and can initiate the expression of genes related to neurogenesis7, 8. On the other hand, Olig2, a bHLH family transcription factor, is often considered a transcriptional repressor that represses neurogenesis in response to brain injury9. Therefore, the combined condition of Ngn2+shOlig2 may enhance the expression of neurogenesis-related genes, as the inhibition of Olig2 can lift its repressive effect, allowing Ngn2 to drive gene expression more effectively. In conclusion, overexpression of Ngn2 alone in astrocytes can increase the expression of Vim, Sox4, Nes and Olig1. On this basis, knocking down Olig2 can further upregulate the expression of the above genes (Fig. 6d).This could explain the observed differences in gene expression between TdT+ cells in Ngn2+shNC group and TdT+ cells in Ngn2+shOlig2 group.\n12. Line 519, to conclude that Olig2 inhibited the expression of Ngn2 specially in Fig 7k and l, it would be important to perform a quantitative analysis, such as qPCR on FACS enriched cells.\nResponse: We thank the reviewer for this valuable suggestion. Since Ngn2 expression is extremely low in adult mouse cortical tissue (Supplementary Fig. 1b), we did not need to isolate TdT+ cells by FACS. To analyze the expression of Ngn2 and its potential regulation by Olig2, we directly extracted RNA from AAV-infected cortical regions and performed qPCR analysis. We found that Ngn2 mRNAs were significantly increased after knocking down of Olig2. The new data have been added into the revised manuscript (new Fig. 8k; lines 605-607).\nReviewer #2 (Remarks to the Author):\nReview of Lai et al. on Olig2 as a Barrier to Ngn2-Mediated Astrocyte-to-Neuron Conversion\nSummary:\nLai et al. present a study investigating the role of Olig2 in astrocyte-to-neuron (AtN) conversion mediated by proneural transcription factors (TFs). Olig2 has previously been highlighted as a potential barrier to neuronal fate conversion following cortical stab wound injury (Buffo A, et al. 2005. PNAS). Here Lai et al. make a very interesting observation that Olig2 is upregulated in astrocytes following forced AAV-mediated expression of bLHL proneural TFs (Ngn2, Ascl1, Nd1) in the absence of injury and propose that here too, Olig2 may act as a inducible barrier to neuronal conversion. While this is an intriguing idea, we find significant issues in the experimental evidence and interpretations. Without addressing these concerns, the central claims of the paper cannot be substantiated.\nResponse: We would like to sincerely thank the reviewer for their thoughtful comments and for recognizing the intriguing nature of our study. We are grateful for the positive acknowledgment of our observation that Olig2 is upregulated in astrocytes following forced AAV-mediated expression of neurogenic TFs, and the proposal that Olig2 may act as an inducible barrier to neuronal reprogramming. We greatly appreciate the reviewer’s recognition of the significance of this idea and its potential implications for understanding the molecular mechanisms underlying AtN reprogramming.\nMajor Concerns:\n1. Lack of Robust Evidence for Astrocyte-to-Neuron Conversion\nThe central claim of the paper is that downregulation of Olig2, combined with ectopic expression of proneural bHLH TFs (Ngn2, Ascl1, NeuroD1), induces AtN conversion in adult mouse cortical astrocytes. However, the evidence presented is insufficient to establish true lineage conversion for the following reasons:\n• Contextual Regulation of hGFAP Promoter: The hGFAP mini-promoter in the AAV vector system is now well appreciated to show variable and highly contextual expression in endogenous neurons (Wang et al., 2021, Cell; Le et al., 2022, Front. Cell. Dev. Biol.; Xie et al., 2023, eBioMedicine; Enbar et al, 2024, Sci. Rep.). Thus, the observation of viral reporter protein (tdTomato) fluorescence in NeuN+ cells is not sufficient evidence to demonstrate the cells arise from astrocytes.\nResponse: We sincerely appreciate the reviewer’s critical point regarding the potential loss of GFAP promoter specificity when co-expressing neurogenic transcription factors. We fully acknowledge the caveat raised by Wang et al. (2021) regarding the variability in the expression of the hGFAP mini-promoter, and we agree that pre-labeling astrocytes is crucial for conclusive lineage tracing in such studies.\nTo address this concern, we employed astrocyte lineage tracing transgenic mice (Aldh1l1CreERT2 x Ai14), which allowed for tamoxifen-dependent pre-labeling of astrocytes prior to the induction of Ngn2. Post-transduction analyses (45 days) revealed that the tdTomato+ cells exhibited characteristic neuronal morphology, expressed mature neuronal markers such as NeuN, which strongly supports the conclusion of bona fide AtN reprogramming (new Fig. 4; lines 295-317).\nAdditionally, we conducted electrophysiological analyses that demonstrated that 18.2% of tdTomato+ cells fired only single action potentials, and nearly half of them lacked detectable synaptic activity (Fig. 3e and 3o). These findings suggest that the converted cells resemble immature neurons rather than fully mature endogenous neurons in the adult mouse cortex. While we cannot entirely rule out the possibility of minor leaky expression of tdTomato in endogenous neurons, the use of our astrocyte-specific lineage tracing mice significantly minimized these confounding effects.\nWe have also revised the manuscript to include a detailed discussion of the limitations of the GFAP promoter and its potential impact on our results (line 631-659). We hope these additional data and clarifications adequately address the reviewer’s concerns. We are grateful for the reviewer’s insightful comments, which have helped to enhance the robustness of our study.\n• Alternative Hypotheses Not Addressed: The authors argue that the increased percentage of tdT+S100B-NeuN+ cells (neurons) and reduced percentage of tdT+S100B+NeuN- (astrocytes) observed following Olig2 downregulation (but not in scrambled shRNA control) is evidence for astrocyte to neuron conversion. However, the authors do not consider any other alternative hypotheses. For example, these data would also result from the death of astrocytes following the intervention with leaky expression of tdTomato in endogenous neurons.\nResponse: Thank you very much for your valuable feedback. The point you raised about the lack of consideration for alternative hypotheses is indeed an important perspective, and we appreciate the opportunity to address it in detail.\nWe agree with your observation that simply attributing the increase in tdT+S100B-NeuN+ cells and the decrease in tdT+S100B+NeuN- cells to astrocyte-to-neuron conversion could be limited without ruling out other potential factors. As you pointed out, one possible explanation could be that the downregulation of Olig2 leads to the death of some astrocytes, which may indirectly trigger tdTomato expression in endogenous neurons, leading to potential false-positive results. This is a potential confounding factor we considered when designing the experiment, but since our current data do not conclusively exclude this possibility, we did not provide a detailed discussion of it in the paper.\nTo mitigate this issue, we used an astrocyte lineage tracing mouse model (Aldh1l1CreERT2 x Ai14), where astrocytes are pre-labeled with tamoxifen before Ngn2 induction (new Fig. 4; lines 295-317). This approach ensures that the tdTomato+ cells observed originate from astrocytes rather than endogenous neurons. However, we acknowledge that we cannot completely rule out the possibility that, in some cases, tdTomato might label endogenous neurons via non-specific pathways.\nAdditionally, we performed electrophysiological analysis, which showed that about 18.2% of tdTomato+ cells only evoked a single action potential, and nearly half of the tdTomato+ cells lacked detectable synaptic activity (see Figure 3e and 3o). This suggests that these cells are more similar to immature neurons rather than mature endogenous neurons. While this finding supports the hypothesis of astrocyte-to-neuron conversion, we also acknowledge that the data do not entirely exclude other potential explanations.\nIn summary, we have highlighted these experimental limitations in our discussion and provided a detailed explanation of these potential issues (lines 631-659). We hope to further verify these hypotheses through future experiments and clarify any potential confounding factors. Thank you again for your valuable suggestions, which have been instrumental in improving the design and analysis of our experiments.\n• Absence of Robust Fate-Mapping Experiments: To validate the claim of AtN conversion, it is imperative that - at a minimum - robust, viral vector independent fate-mapping are performed. Ideally, these should use well validated tools such as Aldh1l1-CreERT2 mice crossed with Cre-reporter lines. The current experiments do not exclude the possibility of fluorophore expression in endogenous neurons or other non-astrocytic cells. Given the importance of the claim the paper is making, we would also recommend that efforts are made to label endogenous neurons prior to AAV injections to confirm that tdT is not being expressed in endogenous neurons. There are several ways researchers could go about this (e.g. using transgenic excitatory or pan-neuronal cre-driver lines with a GFP/YFP reporter, EdU/BrDU labelling in utero, or using additional viral tools).\nResponse: Thank you for your valuable feedback. We understand and appreciate your concern regarding the lack of lineage tracing experiments to validate the fate of astrocyte-derived neurons. We recognize that this aspect could significantly strengthen our conclusions.\nBased on the resources and technology of our laboratory, in order to confirm the existence of AtN as soon as possible, we chose the astrocyte lineage tracing mice that we already had in our hands to pre-label astrocytes. We used the Aldh1l1-CreERT2 x Ai14 lineage tracing mouse model to specifically label astrocytes before inducing Ngn2, ensuring that the tdTomato-positive cells are derived from astrocytes. This method has provided us with compelling data confirming the fate of the AtN reprogrammed cells. Given the specificity of Aldh1l1-CreERT2 expression in astrocytes and its reliable control in adult mice1, 10, we are confident in the validity of our findings based on these lineage tracing experiments (new Fig.4 lines 295-317). In addition, the BrdU pre-labeling of endogenous neurons you mentioned is also a very good idea. In the future, we will learn this technology and apply it to our future study. We sincerely appreciate your suggestion and believe that the lineage tracing data we have presented are robust and conclusive for validating our hypothesis.\n• No Evidence for Intermediate Stages: In figure 2L, authors present a schematic of astrocytes passing via an intermediate stage to neurons, however, this is highly misleading as no substantive evidence has been presented for intermediate stages. Whilst authors note that there is an increased number of tdT+S100B-NeuN- cells when Olig2 is downregulated, the absence of S100B is not of itself evidence that these transduced cells will go on to acquire neuronal properties and there is no positive identification of immature neuronal genes (e.g. Dcx) in the histology or scRNA dataset provided. As such, it the tdT+S100B-NeuN- cells have not been identified and should not be presented alone as an intermediate step of a conversion process as there may be other explanations (e.g. we note that in the scRNA dataset many Oligodendrocyte lineage cells express the viral transcript. Alternatively, astrocyte toxicity may result in absence of detection of S100B downregulation, etc). Finally, there is no morphological characterisation or clear imaging data presented that would corroborate an intermediate or immature neuronal stage.\nResponse: We sincerely thank the reviewers for their valuable feedback and for giving us the opportunity to further elaborate on the theoretical basis for proposing tdT+S100B⁻NeuN⁻ (double negative) cells as a potential intermediate state in the process of astrocyte-to-neuron conversion. Below, we will systematically elaborate on these issues and analyze the limitations of our existing data in combination with existing experimental evidence：\n(1) The tdT+S100B-NeuN- population only appeared in the Ngn2+shOlig2 group (Figure 2i). This population specificity suggests that knockdown of astrocyte Olig2 promotes the emergence of the tdT+S100B-NeuN- cell population, rather than the nonspecificity of the AAV viral expression system. (2) We tracked the changes in tdT+S100B-NeuN- cells over time and found that they first appeared at 14 dpi, reached a peak at 30 dpi, and then declined (Fig. 2i). This dynamic change feature is difficult to explain by cell death; (3) ScRNA-seq data (18-22 dpi) also revealed downregulation of signature genes of astrocytes, which has also been reported in other related studies8, 11; (4) In addition to S100B, we also detected three other typical astrocyte markers (GFAP, SOX9 and glutamine synthesis) at 30 dpi and found 30-40% double-negative cells (tdT+GFAP-NeuN- and tdT+Sox9-NeuN-; Fig. 2k, l). These results indicate that the expression of some identity-signature genes is lost during astrocyte reprogramming.\nWe thank the reviewer for highlighting the importance of caution in interpreting transitional states. While our data collectively suggest that Olig2 knockdown enables astrocytes to enter a reprogramming-permissive state (marked by loss of astrocytic markers and upregulation of progenitor genes), we emphasize that this model remains provisional. Ongoing studies using live imaging and inducible lineage tracing will provide definitive evidence for or against this hypothesis. In summary, based on current data, we cannot 100% guarantee that these tdT+S100B-NeuN- cells will definitely be reprogrammed into neurons. Therefore, we have removed the cell fate map from the figure 2 and toned down the affirmative tone in the revised manuscript (lines 236-240; lines 242-244).\n• scRNA-seq Analysis: Authors self-report that they were unable to collect reprogrammed neurons that express viral transcripts. They speculate this may be for technical reasons or due to limited cell numbers converted. Either way, this data is not capable of supporting the central claim of AtN conversion. Additionally, the scRNA-seq analysis revealed the presence of multiple cell types, including mural cells, endothelial cells, microglia, and oligodendroglia (which express viral transcripts at broadly levels similar to astrocytes). This could demonstrate a lack of specificity in the AAV targeting astrocytes or issues around cell sorting. Together these technical issues somewhat limits what can be concluded from these data but there are still some interesting observations. Namely, downregulation of Olig2 along with AAV-mediated expression of Ngn2 seems to result in a more neural stem-cell like transcriptome, however, this should not be over-interpreted this as a step towards to neuronal conversion, given that currently no evidence is provided for a trajectory towards lineage converted or immature neurons.\nResponse: Thank you for your thorough review and valuable feedback on our study. We fully understand your concerns and would like to clarify our experimental design and data analysis.\nRegarding the insufficient capture of converted neurons expressing viral transcripts in our scRNA-seq analysis, we acknowledge that this is due to several technical limitations. As stated in the manuscript, the primary goal of our study was to investigate the molecular mechanisms of Olig2 in astrocyte-to-neuron reprogramming. Therefore, we collected samples at an early mid-stage time point when reprogramming efficiency was low (around 10%, as shown in Fig. 2G). Moreover, previous studies have shown that neurons derived from astrocyte reprogramming are fragile12, which could lead to their loss during single-cell suspension preparation. Additionally, our choice of scRNA-seq over snRNA-seq was intended to capture mRNA directly from the cells, but it also resulted in some loss of neurons during cell dissociation, as reported in other studies4-6. Nevertheless, we believe the inhibitory role of Olig2 in astrocyte reprogramming remains robust and reliable.\nRegarding the detection of viral transcripts in other cell types, this is likely due to the high sensitivity of scRNA-seq. Notably, while tdT transcripts were detected in some oligodendrocytes, analysis of the distribution and relative expression levels of tdT transcripts revealed that both were much higher in astrocytes than in oligodendrocytes. (Supplementary Fig. 9g, lines 335-339). In addition, while scRNA-seq detects tdTomato mRNA within cells, our immunofluorescence staining and flow cytometry sorting detect tdTomato protein. Immunofluorescence staining showed that viral-encoded TdTomato protein is primarily expressed in astrocytes, which co-express astrocyte markers such as S100b, Aldh1l1, and GFAP (Fig. 1b, 1k; Fig. 2f; Supplementary Fig. 1b, 1e; Supplementary Fig. 6a). This indicates that while scRNA-seq data may detect viral transcripts in other cell types, protein expression remains highly specific. Since flow cytometry sorting is based on tdTomato protein fluorescence, the sorting results are reliable.\nRegarding the role of Olig2 downregulation and AAV-mediated Ngn2 overexpression, we agree with your point that these findings require cautious interpretation. While our data suggest that downregulation of Olig2 and Ngn2 overexpression leads to a transcriptome resembling neural stem cells, this does not imply the cells have committed to a neuronal fate. We will clarify this in the revised manuscript, ensuring that the data are not over-interpreted, and stress that further experiments are needed to validate the formation and maturation of converted neurons.\nThank you again for your careful review and constructive comments. We have revised the manuscript accordingly in section of Abstract (lines 33-34) and Results (line 396; 428; 463; 492; 580-581) and also added further discussions (lines 777-7779) to ensure our conclusions are more precise and rigorous.\n2. Issues with Olig2’s Role and Mechanistic Insight\n• CuT&Tag Analysis of Olig2 Binding: The authors attempt to investigate Olig2’s function as a reprogramming barrier using CuT&Tag experiments. Surprisingly, the canonical Olig2 binding site is not enriched in the genomic regions identified as bound by Olig2. Whilst we accept that this could suggest Olig2 binds non-canonical sites, the data are more plausibly explained by a failure of the CuT&Tag experiment. Promoters are enriched in GC regions, and the 27% of enriched regions containing Gs or Cs likely reflect promoter capture rather than specific Olig2 binding. To support the authors claim of Olig2 binding non-canonical sites, the authors should include additional replicates of the CuT&Tag experiment and validate binding through ChIP-qPCR or band shift assays for targets lacking canonical E-box sites.\nResponse: Thank you for your valuable feedback. We fully understand your concerns regarding the absence of canonical E-box motifs in our CUT&Tag results for Olig2 binding.\nThe choice of the CUT&Tag technique was driven by the limited number of tdT+ cells available after AAV injection in the adult mouse cortex, with only about 62,000 tdT+ cells isolated from 12 mice by FACS. Given the insufficient cell number for ChIP-seq, we opted for CUT&Tag, which is ideal for low cell input and offers significant advantages over traditional ChIP-seq, including shorter experimental time and simpler procedures.\nAlthough we expected to detect E-box motifs in adult mouse cortical astrocytes, they were not enriched in the CUT&Tag analysis. We agree that this may indicate a potential technical issue. Moreover, Olig2 expression in adult cortical astrocytes is minimal and studies of Olig2 binding to this specific cell type are limited. Therefore, there are no published articles available to support the fact that Olig2 in astrocytes does not bind E-box motifs. Therefore, to validate our approach, we performed a similar CUT&Tag experiment with oligodendrocyte lineage reporter mice (Sox10-CreERT2 x Ai14), isolating about 130,000 tdT+ cells from 2 mice (3-month old). In this case, we successfully detected the canonical E-box binding sites for Olig2 (new Supplementary Fig. 14), confirming the reliability of the CUT&Tag technique. We have added these new data in our revised manuscript (lines 550-558; 574-579).\nIn conclusion, the lack of E-box enrichment in astrocytes appears to be cell-type specific rather than a limitation of the CUT&Tag method itself. We have updated our manuscript to reflect this clarification and included our additional validation experiment. Thank you again for your insightful comments.\n3. Additional concerns/observations:\n• Marker Expression Over Time: The authors claim that the hGFAP in AAV9 is \"highly specific\" for targeting astrocytes. However, the authors find that even following conversion (and at late, 45dpi, timepoints) there is expression of tdTomato in putative converted NeuN+ cells. It is unclear how the authors rationalise ongoing tdTomato expression in reprogrammed neurons if the hGFAP promoter is specific for astrocytes? How can authors distinguish between genuinely converted cells and mislabelled (i.e. leaky tdTomato expression in) endogenous neurons in experiments (e.g. in electrophysiology)?\nResponse: Thank you very much for your thoughtful comments and for bringing up this important issue regarding the specificity of the hGFAP promoter and the expression of tdTomato in converted neurons. The hGFAP promoter is commonly used to drive transgene expression specifically in astrocytes, but its activity may decrease after astrocytes are reprogrammed into neurons, limiting long-term tracking. The detection of tdTomato fluorescence in reprogrammed neurons could be explained by several mechanisms:\n(1) Gradual Transition of Astrocytes to Neurons: The conversion of astrocytes into neurons takes time. As a result, the GFAP promoter may not immediately lose its activity, and thus the expression of tdTomato may continue during the early stages of reprogramming.\n(2) Persistence of mRNA and Protein Products: In the early stages of reprogramming, the GFAP promoter activates downstream genes such as tdTomato, leading to the production of mRNA, which is then translated into proteins by ribosomes. Even if the activity of the GFAP promoter diminishes later, mRNA and protein that have already been synthesized will not immediately degrade. Studies have shown that certain proteins can remain in cells for several days to months13-15.\n(3) Signal Amplification by Immunohistochemistry: Immunohistochemistry was used to detect tdTomato fluorescence protein, and the antigen-antibody reaction involved in this technique itself serves to amplify the signal. This amplification could contribute to the continued detection of tdTomato despite the potential loss of promoter activity.\nRegarding the electrophysiological results, tdTomato fluorescence protein was again used to identify the reprogrammed neurons. While Wang et al. (2021) demonstrated that GFAP-driven bHLH neurogenic transcription factors may lead to neuronal leakage, we noted that the off-target expression of Ngn2 was significantly lower than that of Pax6, Ascl1 and NeuroD1 (Wang et al., 2021, Cell, Fig. 5F, I)1. Consistent with this, Mattugini et al. (2019, Neuron) reported minimal leakage expression of Ngn2 when expressed alone in adult cortical astrocytes (Supplementary Fig. 2A)2. Importantly, our electrophysiological analysis showed that 18.2% of tdTomato+ neurons elicited only a single action potential, and nearly half lacked detectable synaptic activity (Fig. 3e and 3o), resembling immature neurons rather than endogenous mature neurons in adult mouse cortex. While we cannot completely rule out slight leakage expression of tdTomato in endogenous neurons, we have discussed the limitations of the GFAP promoter in the discussion section (lines 631-659).\nRecommendations:\nTo address these concerns, the authors should:\n1. Include Fate-Mapping Experiments: Use transgenic mouse lines (e.g., Aldh1l1-CreERT2) with Cre-reporter systems to ensure the origin of induced neurons is astrocytic and to exclude targeting of endogenous neurons.\nResponse: We appreciate this recommendation, and we are pleased to inform you that we have already conducted fate-mapping experiments using astrocyte-specific lineage tracing mice. In these experiments, we utilized the Aldh1l1-CreERT2 system combined with a Cre-reporter (Ai14) system to confirm that the induced neurons originate from astrocytes. We have included these additional data in the revised manuscript and believe they directly address your concern (new Fig.4; lines 295-317). Thank you again for your insightful suggestion, which has undoubtedly strengthened the robustness of our findings.\n2. Demonstrate that tdTomato is not expressed in endogenous neurons: (or characterise the extent of this and show how authors distinguish between these and astrocyte-derived cells). E.g. label endogenous neurons prior to AAV injections (e.g., using pan-neuronal Cre-driver lines or BrdU/EdU labeling) to confirm tdT is not expressed in pre-existing neurons.\nResponse: Thank you for your thoughtful suggestion regarding the need to label endogenous neurons prior to AAV injections using pan-neuronal Cre-driver lines or BrdU/EdU labeling. While we fully understand the importance of confirming that tdTomato is not expressed in pre-existing neurons, we regret to inform you that we do not have access to the relevant transgenic mouse lines for pan-neuronal Cre-drivers, nor do we have experience with BrdU/EdU labeling to mark endogenous neurons. However, we have performed extensive lineage tracing using the Aldh1l1-CreERT2 system, which strongly supports our conclusion that tdTomato+ neurons are derived from astrocytes. We hope these findings address your concern and clarify the specificity of our results. We truly appreciate your valuable feedback, which has helped improve the manuscript.\n3. Show morphology: Microscopy should demonstrate the morphology of putative lineage converted cells, ideally with some intermediate/immature morphological stages.\nResponse: We greatly appreciate your valuable input, which has greatly contributed to improving the manuscript. In response to your suggestion, we will incorporate high-resolution microscopy images to show the morphology of the converted cells, highlighting both mature and intermediate stages (Revised Fig.2j; lines 207-225). We believe this additional data will provide a clearer understanding of the cellular reprogramming process.\n4. Use Intermediate Markers: Demonstrate the progression of reprogramming using markers (e.g. DCX) for intermediate/immature neuronal stages.\nResponse: Thank you very much for your thoughtful suggestion to use intermediate markers, such as DCX, to demonstrate the progression of reprogramming. In response to your recommendation, we did perform DCX staining on the lineage-converted cells, but unfortunately, we did not observe any expression of DCX in the Td+ cells. However, under the same experimental conditions, we observed that tdT+ cells expressed other neurogenic stem cell markers, such as Vimentin and Nestin, suggesting that the reprogramming process might be at an early stage. Furthermore, in our single-cell sequencing analysis, we found that knockdown of Olig2 led to a significant increase in the transcription of Vimentin and Nestin (Fig.5i). We further validated this result using immunofluorescence staining, which confirmed that Olig2 knockdown significantly enhanced the expression of both Vimentin and Nestin in tdT+ cells (Fig.5j, k).\nIn summary, although we did not detect DCX expression, the significant upregulation of Vimentin and Nestin following Olig2 knockdown provides strong evidence that knocking down Olig2 promotes Ngn2-overexpressing astrocyte reprogrammed into neural stem-like cells. These findings support the conclusion that Olig2 may play a crucial role in repressing the astrocyte-to-neuron reprogramming. We have added these new data in our revised manuscript (Supplementary Fig. 7; lines 213-215).\n5. Replicate & validate CuT&Tag result: Authors should do further work on this aspect as the current claims are strong considering that failure to identify canonical Olig2 binding sites may be consistent with experimental failure.\nResponse: Thank you for your insightful feedback regarding the CUT&Tag results. We greatly appreciate your suggestion to replicate and validate our findings, especially given the concerns about the failure to identify canonical Olig2 binding sites in astrocytes.\nIn response to your comment, we conducted additional experiments using the same CUT&Tag methodology to investigate Olig2 binding sites in oligodendrocyte lineage cells, a glial cell type that naturally expresses Olig2 in the adult mouse brain. Interestingly, we observed some motifs, such as Sp1 and Klf3, which were similar to those seen in astrocytes. More importantly, we successfully detected the canonical E-box motif, which is known to be a key Olig2 binding site, in the oligodendrocyte lineage cells. This suggests that the lack of E-box detection in astrocytes might be specific to this cell type and not a result of experimental failure. We have added these new data in our revised manuscript (Supplementary Fig. 14; lines 550-558).\nConclusion:\nLai et al. provide an interesting observation regarding Olig2 upregulation during ectopic expression of proneural TFs in astrocytes. However, the evidence presented does not support the claim of astrocyte-to-neuron conversion or establish Olig2’s role as a barrier to reprogramming. Since this is the central claim of the paper, more experimental work is needed before this could be published. If the claims made were confirmed to an appropriate standard, the findings would be of considerable interest.\nResponse: Thank you for your insightful feedback on our manuscript. We greatly appreciate your positive comments regarding our observation of Olig2 upregulation during astrocyte reprogramming and your constructive suggestions for improving the manuscript.\nWe agree that the molecular mechanisms underlying astrocyte-to-neuron (AtN) reprogramming are complex. While the precise role of Olig2 remains to be fully elucidated, our data strongly support the hypothesis that Olig2 acts as a critical barrier in this process. We have revised the manuscript to clarify that, although the exact mechanisms are not yet fully understood, our current findings suggest that Olig2 significantly impedes AtN reprogramming. We believe that our study provides a solid foundation for future investigations into the molecular barriers and mechanisms involved in AtN reprogramming. We hope that our findings will stimulate further research in this area, contributing to the broader goal of advancing neural regeneration.\nPublishing in a high-impact journal such as Nature Communications would facilitate the dissemination of these results to a broader audience and promote continued progress in the field.\nThank you again for your constructive feedback. We believe the revisions have strengthened the manuscript, and we look forward to the opportunity to share our findings with the research community.\nReviewer #3 (Remarks to the Author):\nIn the present manuscript, C. Lai, K. Hou and colleagues identified Olig2 as barrier during in vivo direct reprogramming of astrocytes into neurons. First, the authors showed that Olig2 is upregulated in astrocytes following the expression of bHLH TFs. Then, they investigated the effect of Olig2 knock-down in Ngn2-mediated direct conversion, unravelling an increased reprogramming efficiency and maturation of Ngn2-shOlig2-iNs; last, they performed single cell RNAseq analysis to investigate the mechanisms regulated by (sh)Olig2 in the conversion process.\nOverall the manuscript is interesting and of interest for the in vivo direct reprogramming field.\nHowever, there are several points that need to addressed before considering for publication;\nResponse: Thank you for your kind and encouraging comments regarding our manuscript. We greatly appreciate your recognition of our work, particularly the identification of Olig2 as a barrier in the in vivo direct reprogramming of astrocytes into neurons. We are pleased that you found our study interesting and relevant to the field of direct reprogramming. Your positive evaluation of our research motivates us to continue exploring the molecular mechanisms involved in reprogramming and to further advance this area of study. We sincerely appreciate the time and effort you invested in reviewing our manuscript.\n_ the identification of Olig2 as barrier for in vivo direct reprogramming was postulated and partially revealed years ago (doi/10.1073/pnas.0506535102). While the present manuscript is a substantial step forward towards understanding the mechanisms underlying the conversion process, the role of Olig2 in direct neuronal reprogramming is not “unexplored” (line 160). The authors are well-aware of the paper, as they used the same approach to manipulate Olig2 (using Olig2-VP16). Therefore, it is important to reshape the introduction to include previous work on Olig2;\nResponse: Thank you for your valuable and insightful comments. We acknowledge your point regarding the role of Olig2 in in vivo direct reprogramming. Indeed, as you correctly noted, the identification of Olig2 as a barrier was postulated and partially revealed in the paper you referenced (doi/10.1073/pnas.0506535102). We apologize for the imprecise use of the term \"unexplored\" in our manuscript. In response to your comment, we have revised the Introduction to reflect the prior work on Olig2 (lines 58-61), including the study you mentioned, and we have adjusted our phrasing to accurately contextualize our findings within the existing literature (line 170).\n_ Previous work from the same group showed that the proportion of astrocytes expressing Olig2 in vivo is region-specific, spanning from 8% to 13% in the cortex (doi.org/10.1186/s13041-021-00747-0). However, in their control-injected mice (Fig.1f, h), the proportion of GFP+ cells expressing Olig2 is very low. Can the authors comment on the discrepancy of the results? Is it possible that their AAVs or Gfap promoter have a preference for a subset of astrocytes?\nResponse: Thank you for your valuable feedback. We agree that the discrepancy may be related to differences in experimental conditions, such as antibody selection, antibody concentrations, fluorescence imaging parameters, and the specific brain regions studied. In our previous work, we used S100β as a marker for astrocytes, and found that 8% to 13% of cortical astrocytes expressed Olig2 (Adult C57BL/6J mice). In the current study, we also performed immunostaining for Tdtomato, S100β, and Olig2 (Supplementary Fig. 1e left) on five adult mice (C57BL/6J). Our data show that 5.5% of AAV-transduced astrocytes (tdT+ and S100β+) are Olig2-positive (Fig. 1l left bar). Given that AAV infection does not target all astrocytes, we believe that the differences between our data and the previous study are not fundamentally different. We hope this clarification addresses your concern and thank you again for your thoughtful comments.\n_ Can the authors quantify the intensity of Olig2 in OPCs following control and Ngn2 transduction (figure 1f)?. According to the figure, it seems that in Ngn2-injection condition, tdT+GFP- cells have a stronger signal than tdT+GFP- cells in control condition; that would suggest a cell non-autonomous effect;\nResponse: Thank you for your helpful suggestion. In response, we have now quantified the Olig2 intensity in oligodendrocyte lineage cells (tdT+ GFP-) following both control and Ngn2 transduction. Our analysis shows that there is no significant difference in Olig2 intensity between the two conditions. We believe the visual difference you observed, where tdT+GFP- cells in the Ngn2-injected group seem to have stronger fluorescence, is due to the large size of astrocyte nuclei and the strong induction of Olig2 in this condition. This could give the illusion of increased Olig2 expression in the Ngn2-injected group, but our data shows there is no actual difference at the cellular level. We have added the new statistical analysis in the revised manuscript (lines 134-136; Supplementary Fig. 3f, g).\n_ the authors quantified the number of Iba1+ cells and did not find a change in different conditions; can the authors comment and discuss the morphology of Iba1+ cells, to better understand whether the presence of the reprogramming factor leads to an inflammatory state?\nResponse: Thank you for your insightful comment. We apologize for not including the Iba1 immunostaining results in the original manuscript, which could have addressed your concern more directly. To address your question, we conducted a more detailed morphological analysis of Iba1+ microglia cells following the overexpression of the reprogramming factor Ngn2 (7 dpi). Our updated analysis revealed that both the expression intensity of Iba1 and the morphology of the Iba1+ microglia were comparable to those observed in the control group (line 107-108; Supplementary Fig. 2).\nBased on these findings, we conclude that overexpression of the reprogramming factor Ngn2 does not appear to cause significant changes in the number, morphology, or inflammatory state of microglia within the first week post AAV-Ngn2 injection. This suggests that, at least within the timeframe examined, Ngn2-induced astrocyte reprogramming does not provoke a marked inflammatory response in the region of interest. However, we acknowledge that this is an important and intriguing point. As you suggested, a more comprehensive study exploring the potential long-term effects of reprogramming factors on local inflammation and immune responses would be a valuable direction for future research.\n_ Please specify in the text and figures when transgenics mice and when viruses are used. For instance, in figure 1k it is not clear that the authors used tdT comes from the AAV injection and not from the Sox10-CreERT2::Ai14 transgenic mouse used in experiment in panel 1f;\nResponse: Thank you for your helpful comment. To avoid confusion, we have updated the manuscript to clearly distinguish between the fluorescent markers used in the transgenic mice and the AAV viral vector. Specifically, we now refer to the fluorescent protein expressed in the Ai14 transgenic mice as Ai14-tdT, while the red fluorescent protein expressed from the AAV vector is labeled as tdT. Regarding Figure 1k, we have clarified in the figure legend (lines1133-1134) and the main text in our revised manuscript (line 126; 130; 133)\n_ can the authors include as well GFP channel in Figure 2C, to better understand the proportion of infected cells with the sole AAV-shNC/Olig2? Can the authors quantify the proportion of double-infected cells (tdT+GFP+). See comment in the scRNASeq;\n_ Supplementary figure 2 is missing, at least in the downloaded pdf file;\nResponse: We apologize for not providing a clear explanation in the original manuscript. In the initial knockdown vector, we included the EGFP sequence to study the targeting of AAV-shOlig2, which allowed us to track the infected cells. However, in subsequent experiments, we used a knockdown system without the EGFP marker. We apologize for the confusion caused by this omission. To avoid misunderstanding, we added the information in the revised manuscript (lines 182-184).\nWe have now ensured that all supplementary materials, including figure 2, are correctly included in the revised manuscript and accompanying files.\n_ the observation that cells at intermediate stage are increased in Ngn2-shOlig2 condition is very interesting, but is not followed up;\nResponse: We completely agree with your observation and also find the intermediate stage cells in the Ngn2-shOlig2 condition to be very interesting. This is indeed a promising area for further investigation, and we acknowledge that it warrants deeper exploration in future studies. Thank you for highlighting this point.\n- can the authors test whether these cells are still expressing low levels of Olig2? Or do they express higher levels of neural stem cells markers (e.g., Sox2, Pax6)? Can the authors find them in their scRNAseq? what is their signature?\nResponse: Thank you for your insightful questions. After AAV virus injection, we performed Olig2 immunostaining at 21 days (21 dpi), which showed consistent results with our 7-day data (7 dpi). In the Ngn2+shOlig2 group, tdT+ cells did not express Olig2 (Fig. 5e), suggesting that the intermediate stage cells in this condition do not express Olig2.\nRegarding neural stem cell markers, we did additional immunostaining for Sox2 and Pax6 as per your suggestion. We observed widespread expression of Sox2 in tdT+ cells. This may be due to the fact that cortical astrocytes widely express Sox2 and are not related to the overexpression of Ngn2, which has been reported in some previous studies16-18. We noticed that the intensity of Sox2 was significantly decreased in Ngn2+shOlig2 group compared to Ngn2+shNC group. Interestingly, studies have reported that depletion of Sox2 in cortical astrocytes is beneficial to the repair and functional recovery of damaged brain tissue16, 17. As for Pax6, no positive cells were detected in either group. Interestingly, two other neural stem cell markers, Nestin and Vimentin, were significantly upregulated in the Ngn2+shOlig2 group (Fig. 5j, k), suggesting the presence of neural stem cell-like features in these cells. Please check our Sox2 and Pax6 immunostaining results below:\nFigure legend: a,Immunostaining shows that transcription factor Sox2 is expressed in tdT+ cells in both groups. b, Statistical data shows that the Sox2 intensity in the Ngn2+shOlig2 group is significantly lower than that in the Ngn2+shNC group. Data shown as mean ± SD.\nFigure legend: a,Immunostaining shows that transcription factor Pax6 is not detected in both groups.\n- please specify that the quantification of the intensity in suppl. Fig. 4e refers to supply. Fig. 4d and suppl. Fig.4g refers to Suppl. Fig.4f;\nResponse: Thank you for your suggestion. We have clarified that the quantification of the intensity shown in Supplementary Fig. 5e has referred to the data presented in Supplementary Fig. 5d, and that Supplementary Fig. 5g has corresponded to the data in Supplementary Fig. 5f. This distinction has been made clear in the revised manuscript (Supplementary Fig.5, highlighted with yellow in legend). Thank you for pointing this out.\n_ scRNAseq data are interesting and raise some important questions:\n- Supplementary figure 7e shows the distribution of TdT+ cells in various clusters; if correctly interpreted, suppl. Fig. 7s shows the proportion of TdT+ cells in various cluster as well, providing quantitative information to Supplement. Fig. 7e. From these 2 panels, it is evident that at least 25% of TdT+ cells are located in the oligodendrocyte lineage. However, in the initial characterisation of the vector system, the authors claim that the system is very tight and specific for astrocytes. Can the authors explain this discrepancy? Which of the data is true? Can the authors analyze as well these “oligodendrocyte” cluster and compare it to the “astrocyte” cluster of infected cells to get an insight whether a neuronal cascade is being induced in oligodendrocytes or not?\nResponse: Thank you for your question and valuable feedback. We would like to clarify the apparent discrepancy regarding the distribution of tdTomato (TdT+) cells in the oligodendrocyte lineage as shown in Supplementary Figure 7e and 7f (now is supplementary figure 9e and 9f in the revised manuscript).\nThe key point here is that the tdTomato signal in our study is based on mRNA expression, which is detected by single-cell RNA sequencing (scRNA-Seq). While immunofluorescence staining confirmed that tdTomato protein expression is highly specific to astrocytes (with 100% of TdT+ cells co-expressing astrocyte markers such as S100b, Aldh1l1, and GFAP), scRNA-Seq has a much higher sensitivity, which enables the detection of extremely low-abundance mRNA molecules in single cells, but this may also increase the risk of detecting residual or cross-expressed mRNAs in neighboring cells. In addition, we directly compared the distribution ratio and relative expression abundance of tdT transcripts in astrocytes and oligodendrocytes and found that both indicators were much greater in astrocytes than in oligodendrocytes (Supplementary Fig. 9g; line 335-339). This explains why tdTomato mRNA is also detected in other cell types, including oligodendrocytes, even though they do not express tdTomato protein. This phenomenon has also been observed in other related studies as well4-6.\nFurthermore, the AAV9 virus used in our study has very low tropism for oligodendrocytes (less than 0.4%)19, reinforcing the idea that the tdTomato signal in oligodendrocyte clusters reflects mRNA detection rather than protein expression. Regarding the potential induction of a neuronal cascade in oligodendrocytes, this is an interesting question. However, our study focused on astrocytes and the role of Olig2 in their reprogramming. Thus, we have not specifically analyzed the oligodendrocyte clusters for this purpose. This will be a valuable area for future research.\nIn summary, the discrepancy arises from the high sensitivity of scRNA-Seq, which can detect mRNA with extremely low-abundance, even in the absence of protein expression. The data in Supplementary Figure 7e and 7f (now is supplementary figure 9e and 9f in the revised manuscript) are consistent with this explanation, and further experiments will be needed to explore potential reprogramming in the oligodendrocyte lineage.\n_ As the authors use 2 different AAV vectors, one carrying TF-TdT and one shControl/Olig2-GFP, it is very likely the presence of 3 groups of infected cells: single one (TdT+, GFP+) and double positive (tdT+GFP+). Can the authors detect these 3 groups in the scRNAseq?\nResponse: Thank you for your question. We would like to clarify the shOlig2 vector used in the scRNA-Seq experiments does not carry the EGFP expression sequence. Therefore, there is no potential for detecting a double-positive group (tdT+GFP+) in the scRNA-Seq data, as the shOlig2 vector lacks GFP expression. We have added this information in the revised manuscript (lines 182-184).\n_ If so, can the authors show in which clusters the various subgroups are localized? Do Ngn2-tdT+ cells coming from Ngn2-shControl localize together with Ngn2-shControl and Ngn2 only from Ngn2-shOlig2?\nResponse: Thank you for your follow-up question. In response to your query regarding the localization of the various subgroups, we would like to clarify that in our scRNA-Seq analysis, we did not observe the presence of a double-positive group (tdT+GFP+) due to the absence of the EGFP expression sequence in both shNC and shOlig2 vector, as previously mentioned in the revised manuscript (lines 182-184). Therefore, the subgroups we detect are limited to tdTomato (tdT+) cells.\n_ Are Ngn2-shOlig2 double positive cells (and not just tdT+ cells) enriched in astrocyte cluster 4 (Fig.4n)? It is not clear whether the authors subset only the double positive astrocytes, or they consider all astrocytes coming from the injection, irrespective of whether they are single, double or not transduced at all. A clarification in the text and figure will greatly improve the interpretation of the data;\nResponse: Thank you for your question. To clarify, while our shOlig2 vector does not carry the EGFP expression sequence and we cannot directly identify double-positive cells (Ngn2+shOlig2), we have used tdT as a surrogate marker to indicate cells that have been transduced by both AAV-Ngn2 and AAV-shOlig2. This is supported by our flow cytometry enrichment of tdT+ cells, RT-qPCR data showing a significant downregulation of Olig2 mRNA in tdT+ cells in the Ngn2+shOlig2 group (Fig. 5d), and immunofluorescence staining confirming that tdT+ cells in this group do not express Olig2 (Fig. 5e). Moreover, Olig2 was sharply decreased in Ngn2+shOlig2 treated mice (0.1% in tdT+ cells) compared to the Ngn2+shNC group (94.2%; Fig. 2c, d) at 7 dpi. These data suggest that when the two AAV vectors were mixed together and injected into the mouse cortex, the probability of the two AAV vectors infecting the same astrocyte was very high. Meanwhile, even if a small number of astrocytes are only infected with AAV9-shOlig2, it will not have a substantial impact on the experiment because the vast majority of astrocytes do not express Olig2 in the absence of Ngn2 overexpression.\nWe hope this clarifies the methodology and interpretation. Thank you again for your feedback!\n_ the inclusion of NPCs into the data allows for a better interpretation of the data; however, it is not clear the source of the NPCs. Did the author sequence as well NPCs? Did they merge the data from another publication? This information could not be found in the material and method;\nResponse: Thank you for your thoughtful question. The Neural Progenitor Cells (NPCs) included in our dataset were derived from our own experiments, and they were part of the single-cell sequencing results we generated. Specifically, we found that the number of NPCs in the Ngn2+shOlig2 group (n = 43) was four times greater than in the Ngn2+shNC group (n = 11). We apologize for any confusion, and we will update the Materials and Methods section to make this clearer (lines 870-871).\n_ can the authors confirm via IF the change in the expression of at least 1 gene coding for mitochondrial protein? This would substantiate the interesting finding.\nResponse: Thank you for your question. Yes, we can confirm the change in the expression of a mitochondrial protein using immunofluorescence (IF). Specifically, we have observed a significant decrease in the expression of Sfxn5, a mitochondrial transport protein responsible for citrate transport and enriched in mitochondria derived from astrocytes20. Our recent immunostaining results show that Sfxn5 expression is notably reduced in astrocytes from the Ngn2+shOlig2 treatment group. This data has already been included in the revised manuscript to support our findings (lines 526-529; Fig. 7m, n).\n_ The interpretation that the increase in Ngn2 depends on the derepression of the endogenous Ngn2 promoter in shOlig 2 conditions is difficult to prove. As the authors stain for Ngn2, can the authors discriminate between the endogenous Ngn2 and the over-expressed? It has previously been shown that Olig2 can regulate Gfap expression (doi:10.1242/dev.02847). A possibility is that Olig2 regulates the Gfap promoter used to induce the expression of Ngn2 in AAV. Can the authors exclude this hypothesis by testing it? For instance by using a luciferase assay?\nResponse: Thank you for your thoughtful comment. We acknowledge that the interpretation of the increased Ngn2 expression as a result of derepression of the endogenous Ngn2 promoter in the shOlig2 conditions is challenging to prove definitively. Our immunostaining results cannot differentiate between endogenous Ngn2 and the overexpressed Ngn2 from the AAV vector, as both forms would appear similar. However, our CUT&Tag data show strong binding signals of Olig2 to the exon region of the Ngn2 gene, suggesting a potential regulatory role of Olig2 in modulating the expression of endogenous Ngn2. Given that Olig2 typically functions as a transcription repressor21, 22, we hypothesize that Olig2 may inhibit the endogenous Ngn2 expression in our experimental conditions. We have revised our description in the Results section of the manuscript (lines 605-607).\nWe also fully agree with the reviewer’s suggestion that Olig2 could regulate the GFAP promoter, which is used to drive the expression of Ngn2 in the AAV vector. This is a plausible alternative mechanism that could affect Ngn2 expression. We have now included this point in the discussion section, as we believe it is an interesting avenue for future research (lines 711-7143).\n_ In the discussion, the authors suggest that Olig2 activation might be an “second line of defence against bHLH proneural transcription factor mediate cell reprogramming” (lines 623-624) based on the the fact that Sox2 or Dlx2 did not induce Olig2. This sounds a kind of deterministic hypothesis, where cells have “implemented” the possibility that bHLH-mediate cell reprogramming occurs and it needs to be prevented. However, it is well known that upon injury or neurodegeneration, astrocytes become Olig2 positive. Therefore, it seems more likely that Olig2 induction reflects some sort of reactive state. Can the authors check if shOlig2+ cells show lower level of reactivity in scRNAseq and via IF? Either case, the cited sentence needs to be changed.\nResponse: Upon further analysis of our scRNA-seq data, we observed that knockdown of Olig2 in astrocytes leads to the differential regulation of reactive-related genes, most of which show upregulation, suggesting an enhanced reactive state. This supports the idea that Olig2 activation may not simply be a \"second line of defense,\" but rather part of a reactive response to injury. However, we do not yet have definitive mechanistic studies to explain how the overexpression of reprogramming transcription factors leads to the upregulation of Olig2 expression. The mechanisms involved in Olig2 upregulation could share similarities with those seen in injury or neurodegenerative processes, but they may also differ. Regardless of the exact mechanism, our current data strongly support the conclusion that the upregulation of Olig2 in astrocytes inhibits Ngn2-mediated neuronal reprogramming, which remains the key finding of our study.\nPlease check our scRNA-seq analysis results below:\nFigure legend: a, Heatmap of typical DEGs in different phenotypes of astrocytes. For analysis parameters, refer to this article: Nature. 2017 Jan 26;541(7638):481-48723.\nIn light of this, we have revised the sentence in the discussion to reflect this nuanced view: \"Olig2 activation might represent a reactive response in astrocytes to injury, potentially modulating the reprogramming process mediated by bHLH transcription factors, though the precise mechanisms governing Olig2 upregulation remain to be fully elucidated.\"\nOverall, you have raised a very interesting scientific question, which is worth exploring further in our future study. Thank you again for your thoughtful suggestions, which have helped to further refine our manuscript. We have updated the manuscript accordingly (lines 750-754).\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: Thank you for your thoughtful comments and for your involvement in co-reviewing our manuscript as part of the Nature Communications initiative. We truly appreciate the time and effort you have dedicated to providing valuable feedback, and we are grateful for the opportunity to have our work reviewed in such a constructive manner. We also appreciate the initiative to support Early Career Researchers, and we are encouraged by the collaborative and supportive nature of the peer review process.\nReferences:\n1. Wang, L.L. et al. Revisiting astrocyte to neuron conversion with lineage tracing in vivo. Cell 184, 5465-5481 e5416 (2021).\n2. Mattugini, N. et al. Inducing Different Neuronal Subtypes from Astrocytes in the Injured Mouse Cerebral Cortex. Neuron103, 1086-1095 e1085 (2019).\n3. Guo, Y. et al. High-titer AAV disrupts cerebrovascular integrity and induces lymphocyte infiltration in adult mouse brain. Mol Ther Methods Clin Dev31, 101102 (2023).\n4. Giehrl-Schwab, J. et al. Parkinson's disease motor symptoms rescue by CRISPRa-reprogramming astrocytes into GABAergic neurons. Embo Mol Med14 (2022).\n5. Wu, Z. et al. Brain-Wide Neuroregenerative Gene Therapy Improves Cognition in a Mouse Model of Alzheimer's Disease. Adv Sci (2025).\n6. Zhang, Y. et al. A single factor elicits multilineage reprogramming of astrocytes in the adult mouse striatum. Proc Natl Acad Sci U S A119, e2107339119 (2022).\n7. Masserdotti, G. et al. Transcriptional Mechanisms of Proneural Factors and REST in Regulating Neuronal Reprogramming of Astrocytes. Cell Stem Cell17, 74-88 (2015).\n8. Pereira, A. et al. Direct neuronal reprogramming of mouse astrocytes is associated with multiscale epigenome remodeling and requires Yy1. Nat Neurosci (2024).\n9. Buffo, A. et al. Expression pattern of the transcription factor Olig2 in response to brain injuries: implications for neuronal repair. Proc Natl Acad Sci U S A102, 18183-18188 (2005).\n10. Srinivasan, R. et al. New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo. Neuron92, 1181-1195 (2016).\n11. Ma, N.X., Puls, B. & Chen, G. Transcriptomic analyses of NeuroD1-mediated astrocyte-to-neuron conversion. Dev Neurobiol82, 375-391 (2022).\n12. Gascón, S. et al. Identification and Successful Negotiation of a Metabolic Checkpoint in Direct Neuronal Reprogramming. Cell Stem Cell18, 396-409 (2016).\n13. Bomba-Warczak, E., Edassery, S.L., Hark, T.J. & Savas, J.N. Long-lived mitochondrial cristae proteins in mouse heart and brain. J Cell Biol220 (2021).\n14. Mohar, B. et al. DELTA: a method for brain-wide measurement of synaptic protein turnover reveals localized plasticity during learning. Nat Neurosci (2025).\n15. Savas, J.N., Toyama, B.H., Xu, T., Yates, J.R. & Hetzer, M.W. Extremely Long-Lived Nuclear Pore Proteins in the Rat Brain. Science335, 942-942 (2012).\n16. Chen, C. et al. Astrocyte-Specific Deletion of Sox2 Promotes Functional Recovery After Traumatic Brain Injury. Cereb Cortex29, 54-69 (2019).\n17. Liu, Y. et al. Specific knockout of Sox2 in astrocytes reduces reactive astrocyte formation and promotes recovery after early postnatal traumatic brain injury in mouse cortex. Glia 71, 602-615 (2023).\n18. Wang, Y. et al. SOX2 is essential for astrocyte maturation and its deletion leads to hyperactive behavior in mice. Cell Rep41 (2022).\n19. Giannelli, S.G. et al. New AAV9 engineered variants with enhanced neurotropism and reduced liver off-targeting in mice and marmosets. Iscience27 (2024).\n20. Russo, G.L. et al. CRISPR-Mediated Induction of Neuron-Enriched Mitochondrial Proteins Boosts Direct Glia-to-Neuron Conversion. Cell Stem Cell28, 524-534 e527 (2021).\n21. Meijer, D.H. et al. Separated at birth? The functional and molecular divergence of OLIG1 and OLIG2. Nat Rev Neurosci13, 819-831 (2012).\n22. Novitch, B.G., Chen, A.I. & Jessell, T.M. Coordinate regulation of motor neuron subtype identity and pan-neuronal properties by the bHLH repressor Olig2. Neuron31, 773-789 (2001).\n23. Liddelow, S.A. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature541, 481-487 (2017).\nWe greatly appreciate the constructive feedback provided by the reviewers. We have carefully considered all comments and made appropriate revisions to improve the manuscript, with the changes highlighted in yellow. Below, we will address each issue in detail, incorporating new data, clarifications, and modifications to enhance the rigor and clarity of the research. Our responses are presented in black text.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have systematically addressed all of my concerns. I am not convinced by the following statement in the rebuttal\" “In fact, the Cell paper also shows that Ngn2 does not induce significant leakage expression, and the probability of leakage is relatively low compared to other neurogenic transcription factors (Wang et al., 2021, Cell, Fig. 5F, I)”\nIn fact, from the Wang et al. paper, the authors stated “ASCL1 and NEUROG2 also induced approximately 52.7% ± 5.4% and 27.3% ± 8.5% of mCherry+ neurons (Figures 5I and 5K). Importantly, many of these mCherry+ neurons were also labeled with GFP, indicating an origin of endogenous neurons (Figures 5I and 5K). “\nHowever, the way this information is conveyed in the manuscript is acceptable \"Notably, while some neural transcription factors, such as Ascl1, NeuroD1, and Pax6, have been shown to cause non-specific expression in endogenous neurons, the leakage of expression from Ngn2 is relatively low36, 37.\"\nThe revised manuscript is suitable for publication.\nResponse: We are grateful for your positive evaluation of the revised manuscript, and we are pleased to hear that it is now suitable for publication. Thank you once again for your time and thoughtful feedback.\nReviewer #2 (Remarks to the Author):\nOur primary concern with the manuscript revolved around unsatisfactory scientific evidence for the central claim of the paper, namely that AAV-mediated expression of Neurogenin2 and Olig2 knockdown converts adult cortical astrocytes into neurons. Due to widespread acknowledgement that many molecular tools to genetically label astrocytes suffer from “leaky” expression in neurons, robust scientific evidence is required to ensure labelled neurons are derived from astrocytes. Unfortunately, whilst the authors have acknowledged these concerns to some extent, we find the quality and nature of evidence and rebuttal provided in response to our concerns continue to fall short of providing sufficiently robust evidence to support the central claims of the paper.\nResponse: Thank you for your thorough and thoughtful review of our manuscript. We greatly appreciate your feedback and the opportunity to address the concerns you have raised regarding the central claim of our study—that Olig2 knockdown enhances the efficiency of Ngn2-mediated AtN conversion.\nWe fully acknowledge your concern about the potential “leaky” expression of fluorescent reporter proteins in neurons, which is indeed a well-recognized issue in astrocyte-specific expression systems. To address this, we wish to emphasize that Ngn2, which is the key focus of our study, exhibits significantly lower leakage in neuronal expression compared to other pioneer neurogenic transcription factors such as PAX6, NeuroD1, and Ascl11-3. Furthermore, to minimize potential side effects associated with high-titer AAV viral vectors4, we carefully adjusted the viral titer, which also helped reduce the occurrence of unwanted “leaky” expression in neurons. In our study, mouse cortical areas were injected with a total volume of 1 μL of AAV9 with titers of 0.5-2 × 1011 GC/mL (transcription factors) and 2 × 1010 GC/mL (shRNA),which was more than 100-fold lower than in previous studies1, 2. We have emphasized this important information in the Methods section of the revised manuscript (lines 837-839).\nAdditionally, to further validate the astrocyte-to-neuron conversion process, we employed an established transgenic mouse model for astrocyte lineage tracing, which has been widely recognized in the field. Using this model, we observed that knockdown of Olig2 in astrocytes significantly enhanced the efficiency of Ngn2-mediated astrocyte-to-neuron conversion. We believe that this provides robust evidence supporting our central claim that Olig2 acts as an inducible barrier to astrocyte-to-neuron conversion.\nWe understand that the issue of “leaky” expression remains a key concern, and we have taken extensive steps to ensure that our results are scientifically sound. We hope the additional experiments and clarifications provided in our response help to strengthen the evidence for our conclusions and address the concerns regarding the specificity of the conversion process. Thank you once again for your valuable feedback and for contributing to the improvement of our work.\n1- Lack of Robust Evidence for Astrocyte-to-Neuron Conversion\nSome of our concerns involving neuronal expression from the hGFAP promoter have received attention in the discussion. However, authors were unable to provide additional evidence to either demonstrate that reporter transgene labeled neurons do not arise from endogenous neurons (see recommendation 2) or quantify the extent of this.\nAuthors did attempt to follow Recommendation 1, to perform fate-mapping of putative reprogrammed neurons. However, the experimental design chosen raises additional concerns and ultimately fails to provide compelling evidence of lineage conversion. We outline these concerns below:\nResponse: Thank you once again for your thoughtful and constructive feedback. We appreciate your attention to detail and your helpful suggestions. We have carefully reviewed your concerns and would like to provide further clarification and address the issues raised regarding the evidence for astrocyte-to-neuron conversion.\nWe fully acknowledge the challenges associated with demonstrating that reporter transgene-labeled neurons do not arise from endogenous neurons, and we understand your concern regarding the robustness of the evidence provided. In response to your recommendation, we have made extensive efforts to improve the specificity of our labeling strategy, though certain experimental limitations have affected the overall outcome.\n1. Pan-Neuronal Cre-Driver Lines\nAs per your suggestion, we explored the use of pan-neuronal Cre-driver lines to validate the specificity of our reporter protein, Tdt. We investigated the hsyn-bgi-cre:ERT2-bGHpA transgenic mouse (JAX Stock #009585), which showed low labeling efficiency in this mouse strain (Crossed with Ai4 mice) by checking the data in the Allen Institute Brain Science Database (http://connectivity.brain-map.org/transgenic/experiment/175707256). While this strategy was suggested as a potential solution, the low efficiency of labeling endogenous neurons made it unsuitable for robustly distinguishing between reporter expression in endogenous neurons and the converted neurons.\n2.BrdU/EdU Labeling Strategy\nWe also considered the use of BrdU/EdU labeling during embryonic neurogenesis to pre-label endogenous neurons1. However, we realized that this strategy could inadvertently label astrocytes as well, making it difficult to distinguish between leakage expression and actual astrocyte-to-neuron conversion. Given the risk of misinterpretation, we decided that this approach would not provide the clarity required to address your concerns effectively.\n3. AAV-Based Pre-labeling of Endogenous Neurons\nAs an alternative, we attempted to use an AAV retro viral vector to pre-label endogenous neurons2. Specifically, we injected AAV2 retro-hSyn::GFP into downstream projection areas of the somatomotor region, such as the thalamus, to mark a subset of endogenous neurons (RFig.1a). While we were able to successfully label a portion of the endogenous neurons in this region, we observed a low labeling efficiency in the AAV9-Ngn2+shOlig2 injected regions (RFig.1b). This low efficiency may be due to immune responses, but unfortunately, this further complicates the interpretation of the results. Importantly, we did observe that the neurons undergoing conversion (tdT+NeuN+) were not labeled by GFP, suggesting that these converted neurons may not derived from the endogenous neurons (RFig.1c). However, due to the low pre-labeling efficiency, we acknowledge that the lack of GFP labeling in some of these neurons could be due to the limitations of this technique. Please check these results below:\nFigure legend: a, Timeline of the experimental design. b, Low-magnification images of pre-labeling neurons (Green), Ngn2+shOlig2 AAV9 transduced area (Red) and merged figure. Scale bar = 200 μm. c, High- magnification images showing converted neurons (arrowheads) are not pre-labeled by GFP. Scale bar = 20 μm.\n4. Transgenic Mouse Model for Astrocyte Lineage Tracing\nTo directly address the lineage of the reprogrammed neurons, we followed your recommendation to use an astrocyte-specific lineage tracing approach. We adopted a well-established transgenic mouse model for astrocyte lineage tracing and observed that knockdown of Olig2 in astrocytes significantly enhanced the efficiency of Ngn2-mediated astrocyte-to-neuron conversion. This approach has been widely recognized in the field, and we believe it provides the most reliable evidence to support our central claim that Olig2 acts as an inducible barrier to astrocyte-to-neuron conversion.\nIn conclusion, while we encountered challenges with some of the pre-labeling techniques, we believe that the use of the astrocyte lineage tracing model provides the most compelling evidence for our conclusions. Thank you again for your insightful comments. We sincerely hope that this explanation adequately addresses your concerns.\n- In Figure 4 in the amended manuscript, authors remove the fluorescent reporter from the viral constructs. They use a Ai14 transgenic reporter cre line with the Aldh1l1-CreERT2. This is a highly surprising choice of experimental design, since it would have been far more intuitive to use the same viral vector used elsewhere in the paper with a YFP/GFP cre-reporter line. The rationale has not been discussed.\nResponse: To address the origin of the converted neurons, we crossed Aldh1l1-CreERT2 transgenic mice (JAX stock #031008) with fluorescent reporter mice to trace the in vivo reprogramming of astrocytes. The Jackson Lab offers several fluorescent reporter lines, including YFP/GFP and tdTomato lines. Our lab had the red fluorescent reporter mouse R26R-tdTomato (Ai14, JAX stock #007914), so we chose the Ai14 line for this study. This transgenic mouse has been used in other studies tracking astrocyte fate reprogramming in vivo.\nSince the astrocyte lineage tracing mouse already carries the red fluorescent reporter gene (tdT), we could not continue using the same AAV9-GFAP::Ngn2-P2A-Tdt vector and had to repackage a new AAV vector for Ngn2 overexpression. Additionally, to facilitate multi-channel immunofluorescence staining—especially for detecting whether Ngn2-overexpressing astrocytes also express Olig2—we decided to remove the fluorescent reporter gene from the Ngn2 overexpression vector.\nOur immunofluorescence results show that in control mice (without AAV virus injection), only 6.7% of astrocytes in the cortex express Olig2 (baseline). In the Ngn2 overexpression region, approximately 95% of astrocytes co-express both Ngn2 and Olig2. Furthermore, when AAV9 hGFAP::Ngn2 + shOlig2 was injected, nearly 95% of the astrocytes only expressed Ngn2. Immunofluorescence results show that AAV9 vectors without fluorescent reporter genes can overexpress Ngn2 in astrocytes in the cortex of lineage-traced mice, leading to Olig2 upregulation.\nIn conclusion, while the use of the Ai14 reporter line and the modifications to the viral constructs might seem surprising at first glance, we believe this strategy provided the most precise and robust means to track astrocyte reprogramming and to investigate the role of Olig2 in this process. We hope this explanation clarifies our rationale and demonstrates the thoughtful consideration behind our experimental design.\n- Authors point out that there is a modest increase (from around 5 to 12% of tdTomato+ cells that express NeuN in Olig2 knockdown condition compared to Ng2 alone. This is the primary basis for the claim that these cells are astrocyte derived.\nResponse: Thank you for your insightful comments. We appreciate your attention to the details of our data regarding the increase in Ngn2-mediated AtN conversion efficiency under Olig2 knockdown conditions. To clarify, our study shows that in the Olig2 knockdown condition, the efficiency of Ngn2-induced AtN conversion increased from 5.0% to 12.2%, which represents a 2.4-fold improvement. This increase is statistically significant, and we believe it highlights the role of Olig2 in inhibiting the reprogramming of astrocytes into neurons. However, while we consider this increase significant, we also recognize that the 12.2% efficiency still leaves room for further improvement. This suggests that there are additional factors within astrocytes that may prevent a more complete conversion into neurons. We have acknowledged this in the discussion section of our manuscript, emphasizing that further investigation into these other potential barriers is warranted (lines 791-797). We hope this explanation addresses your concerns and clearly supports our claim that Olig2 is barrier for AtN conversion.\n- However, one consequence of this experimental approach is that there is now no way from these data presented to identify whether neurons analyzed were even transduced by the AAV. As such the authors are not fate-mapping viral transduced cells per se, or at least no evidence is provided to demonstrate these were viral transduced cells. No discussion or evidence is provided that even explains how authors knew that the regions analyzed were from the viral injection site, nor how many cells have been analyzed.\nResponse: Thank you for your valuable comments. We apologize for not providing enough clarity regarding how we identified the AAV-transduced regions in our study. We understand your concern about the lack of evidence confirming that the neurons analyzed were transduced by the AAV.\nTo address this, we determined the AAV infection area (the target region for analysis) based on the following criteria:\n1. We used a stereotaxic apparatus for intracranial injection into the mouse brain, which allowed us to target specific coordinates for injection. This helped us collect brain samples from a ±400 μm region surrounding the injection site.\n2. We performed serial sectioning and immunofluorescence staining for Ngn2 on every fifth sample, which enabled us to verify that the samples we collected were indeed from the AAV-infected regions. Other intermediate sections were used for additional immunofluorescence staining and analysis.\nFrom the staining results, we observed efficient Ngn2 expression in lineage traced astrocytes (Main Fig. 4d). Furthermore, similar studies have used virus vectors without fluorescent reporter genes, as we did, for fate-mapping5-7.\nWe have now added a more detailed explanation of how we identified the AAV-infected regions in the methods section of the revised manuscript (lines 841-845). Additionally, the number of cells analyzed is now specified in the figure legend (lines 1237-1239). We hope this addresses your concerns, and we appreciate your feedback to improve the clarity of our study.\n- Ai14 is generally appreciated to be more easily recombined that some other cre-reporter lines (e.g. Álvarez-Aznar, et al. Transgenic Res. 2020) and can lead to some neurons being labelled with tdTomato+. While authors have shown the vast majority of tdTomato+ cells stain for astrocyte markers, they have not quantified the percentage of tdTomato+ cells in this line that express NeuN+ in control AAV or no virus conditions.\nResponse: Thank you for your thoughtful comment. Prior to exposure to Cre recombinase, Ai14 may exhibit low levels of tdTomato expression, which could potentially lead to non-specific labeling. To this end, we examined brain samples from three lineage-traced mice that were not injected with tamoxifen and found very few cells expressing tdT locally. Even if there were a few sporadic positive cells, their morphology was typical astrocyte-like. Please check these data below:\nFigure legend: a, Fluorescent images from 3 mice before tamoxifen injection.\nAfter tamoxifen injection, we analyzed 1252 neurons from the cortices of five control mice (without AAV injection) and found that only 22 neurons expressed tdTomato, which is nearly 1.7%. This result suggests that our Aldh1l1-CreERT2 x Ai14 mice exhibit very high specificity for labeling astrocytes in the cortex (>98%), whereas the labeling efficiency of endogenous neurons is very limited under control conditions (<2%). We have now included these new data in the revised manuscript (lines 312-314, Fig. 4f, g) to further support the specificity of our astrocyte lineage tracing system.\n- Finally, if there were rare recombination events in endogenous neurons (resulting in small fraction of endogenous neurons expressing tdTomato) then an alternative hypothesis for a reasonably small increase in the percentage of tdTomato+NeuN+ cells reported could be a modest increase in astrocyte cell death due to Ng2 expression and Olig2 knockdown.\nResponse: Thank you for your insightful suggestion. To address the hypothesis regarding astrocyte cell death potentially contributing to the increase in percentage of converted neurons (tdTomato+NeuN+), we directly quantified the number of tdTomato+NeuN+ cells in the target regions. Our results show that knockdown of Olig2 in Ngn2 overexpressed astrocytes significantly increased the number of converted neurons, suggesting that the observed increase in percentage of converted neurons (conversion efficiency) is due to enhanced AtN reprogramming. Please check this data in this table:\n- A robust approach would present clear evidence of co-expression of viral reporter and a genetic label of astrocytes and improved quantification. At minimum, this would include the proportion of neurons expressing AAVs that co-express lineage tracing reporter and compare this to the overall recombination efficiency of the line across all conditions (control, Ng2+shNC and Ng2+shOlig2). Currently, the evidence presented is ambiguous and difficult to interpret although potentially promising.\nResponse: We agree that a robust approach would involve clear evidence of co-expression between the viral reporter and the genetic label for astrocytes, along with improved quantification. To address this, we acknowledge that in our current study, we removed the fluorescent reporter gene from the AAV vector to enable multiplex immunofluorescence staining. We agree that future experiments could benefit from using AAV vectors carrying a fluorescent reporter gene in combination with astrocyte lineage tracing, which would allow for better visualization and quantification of co-expression. We thank you for your valuable suggestions and plan to use this approach in our future studies.\nAside from concerns over demonstrating the astrocyte origins of NeuN+tdT+ neurons (both in the original manuscript and in the revised lineage tracing figure), we had several concerns around absence of compelling evidence for immature or intermediate neuronal phenotypes, the presence of which would both support lineage conversion as well as fill mechanistic gaps. We discuss the authors response to these below.\nResponse: Thank you for your thoughtful comments and for raising important concerns regarding the demonstration of immature or intermediate neuronal phenotypes in our study. We have now provided compelling evidence for the existence of such phenotypes across multiple levels of analysis. Our immunofluorescence results show a significant presence of double-negative AAV-infected cells in the Ngn2+shOlig2 group, along with upregulation of neural stem cell markers, Nestin and Vimentin. Single-cell RNA sequencing further supports this, revealing a marked downregulation of astrocyte-specific genes (e.g., Slc1a2, S100β, Aqp4. Fig.5f, 5o) and a significant upregulation of genes associated with neurogenesis (e.g., Nes, Sox4, Pax6. Fig.5f, 5o). Additionally, the transcriptomic analysis of mitochondrial energy metabolism indicates a transition from astrocyte to neuronal characteristics (Fig.7). Electrophysiological data also provide functional evidence of intermediate neuronal states, as we observed some tdT+ cells firing single action potentials (Fig.3d), while some tdT+ cells could fire action potentials but lacked spontaneous postsynaptic currents (Fig.3o), consistent with newly generated or immature neurons. These findings, taken together, provide robust support for the existence of intermediate cell states and help fill the mechanistic gaps in understanding AtN lineage conversion. We hope this additional evidence clarifies the issue and strengthens our conclusions. Thank you again for your suggestion, we have added the above information to the discussion section of the revised manuscript (lines 642-659).\nWhilst the high-resolution confocal images are somewhat useful (recommendation 3), the crop and lack of morphological analysis do not greatly advance the underlying point that motivated this recommendation. In other words, these imaging data could still be consistent with gradual accumulation of expression of viral reporter transgenes in pre-existing endogenous neurons. However, since the authors do not rely on morphological analysis to make their central claim, this remains a minor problem – although had they provided more solid morphological characterization it could have given the central claim considerably more weight.\nResponse: We understand your concern about the potential for viral reporter genes to gradually accumulate in pre-existing endogenous neurons, which might account for the observed tdTomato expression. In our study, we used the tdTomato fluorescent protein driven by the GFAP promoter, which may result in a gradual reduction of tdTomato signal during the AtN reprogramming process (Discussed, lines 660-671). This reduction could hinder a more detailed morphological analysis. To address this limitation, we plan to use brighter fluorescent proteins, such as mScarletI1, 8, in future studies to label both endogenous and reprogrammed neurons. This would allow for more detailed morphological analysis and comparisons at different time points between reprogrammed and mature endogenous neurons. We appreciate your suggestion and will implement these improvements in future work to enhance the clarity and strength of our findings.\nMore pressingly, we raised concerns about the absence of robust evidence of immature or transitional molecular neuronal phenotypes which must presumably exist for a bona fide conversion process. The timepoint of scRNA sequencing corresponds to the peak of the so-called \"intermediate stage\" (as defined by 2i), when neuronal commitment should already be underway. Absence of early neuronal genes (e.g., Dcx, Sox11, Neurod1) in the scRNA data is disappointing. Authors acknowledge failure to capture reprogrammed neurons expressing viral transcripts in scRNA-seq, citing technical limitations, early sampling (~10% conversion efficiency), and fragility of induced neurons during dissociation. However, authors also failed to detect Dcx at the protein level – which would not be affected by this technical limitation. Instead, authors found evidence of Vimentin and Nestin expression in both scRNA-seq and validated at the protein level. Both Vimentin and Nestin are broad progenitor markers, insufficient to support neuronal lineage commitment or maturation. These markers may well indicate a stem-like or reactive astrocyte state but do not demonstrate transition toward a neuronal fate. Overall, the new immunohistochemistry data match very well with the scRNA-seq data and could support a hypothesis that Ng2 + shOlig2 may induce astrocyte reactivity or stem-cell like progenitor state, but don’t contribute further evidence of neuronal fate commitment or conversion.\nResponse: Thank you for your insightful comments and your concerns about the molecular evidence of immature or transitional neuronal phenotypes in our study. Although early neuronal markers such as Dcx, Sox11, and Neurod1 are lacking in our scRNA-seq data, upregulation of genes such as Vim, Nes, Nfix, Stmn1, Sox4, and Pax6 was found (Fig.5f, 5i, 5o and 5p), which play crucial roles in neurogenesis and neural stem cell function. For example, Vimentin and Nestin are typical neural stem cell markers, which are widely expressed in neural stem cells and neural progenitor cells9, 10. Nfix is a transcription factor that plays a key role in neural development, especially in the maturation and differentiation of neurons11, 12. Stmn1 is a protein associated with microtubule dynamic regulation, which plays an important role in neuronal development and axon growth13. Similar to DCX, a latest study has shown that Stmn1 has a high expression level in immature neurons14. Sox4 is a key transcription factor involved in the fate determination of neural stem cells and the differentiation of neurons15, 16. Pax6 is a key factor in maintaining the stemness state and initiating differentiation in neural stem cells, and plays an important role in the development of the cerebral cortex17, 18. Secondly, the transcriptome characteristics of mitochondrial energy metabolism were also found to change from astrocyte characteristics to neurons (Fig.7a-l). Further immunostaining results showed that in the Ngn2+shOlig2 group, the expression of Sfxn5 enriched in astrocytes was significantly reduced (Fig.7m, n), a similar finding with the previous study19. Finally, we combined CUT&Tag with single-cell sequencing analysis and found that after knocking down Olig2, the expression of some Olig2-binding genes was upregulated, including some genes closely related to neurogenesis, such as Sox5, Pou3f2, etc (Fig.8i). Sox5 plays an important role in the development of the cerebral cortex, especially in the differentiation of cortical neurons20, 21. Pou3f2 is a transcription factor of the POU family and plays a key role in neurogenesis22. The absence or abnormal function of Pou3f2 can lead to neurodevelopmental defects and affect the generation and differentiation of neurons in human brain organoids23. The above results suggest that the AtN conversion requires upregulation of functional genes related to neurogenesis, but it is may not necessary to upregulate specific types of functional genes. We have already added some of the above points to the discussion (lines 637-652).\nReactive astrocytes and neural stem cells may share some markers in some cases, such as Vimentin and Nestin. S100β is a marker of astrocytes and is upregulated in reactive astrocytes24, while our single-cell sequencing results showed that it was downregulated. Based on this, we speculate that Ngn2 + shOlig2 may induce astrocytes to enter a neural stem cell-like cell state rather than a reactive astrocyte state. Combined with our astrocyte lineage tracing results, we conclude that knocking down Olig2 can significantly enhance the conversion efficiency of Ngn2-mediated AtN.\nWe do acknowledge that the authors comment that observations of some single spiking neurons constitute evidence of neuronal conversion. It would have been useful to present these traces in the main figure to assess the quality of that claim. However, we don’t believe that this supersedes the need for additional evidence to support claims that observed neurons derive from astrocytes.\nResponse: Thank you for your constructive comments. To better demonstrate the quality of our observations and substantiate the claim of neuronal conversion, we now add a typical trace of single spiking neuron in the main figure 3 (Fig. 3d). Combined with the immunohistochemical results, we found that four groups (tdT, Ngn2-tdT, Ngn2-tdT+shNC and Ngn2-tdT+shOlig2) of immunofluorescent reporter proteins were highly specifically expressed in astrocytes at early time points (Fig.2h and Supplementary figure 6a), indicating that transdifferentiated neurons are derived from astrocytes. In addition, we used astrocyte lineage tracing mice to further demonstrate that the origin of converted neurons is astrocytes (Fig. 4).\n2 - Issues with Olig2’s Role and Mechanistic Insight\nWhile we acknowledge that CUT&Tag was an appropriate method given the limited number of isolated cells, the authors do not provide sufficient evidence to demonstrate that the results reflect genuine Olig2 binding in astrocytes, rather than technical artifacts. As such, any conclusions drawn from this experiment are not adequately supported. Although the Olig2 CUT&Tag data in oligodendrocytes may be valid and potentially informative for understanding the differential role of Olig2 in oligodendrocytes versus astrocytes, no definitive conclusions can be drawn from the astrocyte data.\nConducting CUT&Tag in a different model system does not constitute strong evidence that the original CUT&Tag experiment in astrocytes was successful. Although motif enrichment analysis identifies the closest matching motifs, the reported enrichment of Sp1 and Klf—both of which are G/C-rich and commonly located in promoter regions—is not sufficient to validate the experiment. Promoter regions are typically accessible and may be overrepresented in assays such as ChIP-seq, CUT&RUN, or CUT&Tag when the target protein does not directly bind DNA or when antibody specificity is compromised. Thus, the presence of these motifs alone cannot be taken as evidence of successful Olig2 binding in astrocytes. A second biological replicate of the same experiment, or complementary binding assays as previously suggested, would be required to substantiate these findings.\nResponse: Thank you for your thoughtful comments. I would like to address your concerns regarding the validity of the Olig2 CUT&Tag data in astrocytes.\nFirst, the Olig2 antibody (Millipore, catalog No. AB9610) we used is IP grade and widely recognized in the field (Check Olig2 antibody information at: https://www.merckmillipore.com/HK/en/product/Anti-Olig-2-Antibody,MM_NF-AB9610?ReferrerURL=https%3A%2F%2Fwww.google.com%2F). Now, let me briefly explain the CUT&Tag process. The pA-Tn5 enzyme, guided by the antibody, fragments DNA only in chromatin regions bound by histone modifications, transcription factors, or chromatin regulators. Tn5 transposase then directly adds sequencing adapters to the fragmented DNA, and the product is released from the cell. This technique significantly enhances the signal-to-noise ratio, as most non-specific chromatin regions remain inside the cell nucleus25, 26. For CUT&Tag to work, the cells must remain viable throughout the experiment. This technology has been widely validated and used, including for detecting Olig2 binding sites27, 28.\nDue to technical limitations, we did not perform repeated CUT&Tag sequencing in this study. To isolate Olig2-bound DNA in astrocytes, we first isolate AAV-infected areas under a fluorescent stereomicroscope, then dissociate the tissue to obtain a single-cell suspension. We use flow cytometry to purify the astrocytes before constructing the CUT&Tag sequencing library, which takes about 12 hours. In this study, we sacrificed 12 mice to obtain approximately 62,000 tdT+ astrocytes, which was sufficient for one sequencing run. However, if we increase the number of mice, the viability of cells collected from previous samples significantly decreases. Given these technical constraints, repeated CUT&Tag sequencing on low-abundance cell samples is currently not feasible. Single-cell CUT&Tag sequencing may be a more viable solution in the future.\nAlthough we didn’t repeat the sequencing, we believe the data's reliability and authenticity can still be justified. Initially, we did not find the classic Olig2 binding motif (E-box) in astrocytes, which we thought might be a limitation of the CUT&Tag technology. To validate this method, we replicated the CUT&Tag process for Olig2 in oligodendrocyte lineage cells (Sox10+), where we successfully identified the E-box motif (Supplementary figure 14e), confirming the validity of our sequencing process. Furthermore, our immunofluorescence staining results showed that Ngn2 overexpression in astrocytes induced Olig2 expression comparable to that in oligodendrocyte lineage cells (Fig. 1f). Thus, it is difficult to dismiss the data obtained from astrocytes using the same CUT&Tag protocol, especially when it has been validated in another cell type.\nAdditionally, we integrated our CUT&Tag data for astrocyte Olig2 with published or self-generated data from oligodendrocyte lineage cells (Fig.8g). GO analysis revealed similar enriched biological pathways, such as neural development, axon regeneration, and synaptic integration (Supplementary figure 6a, 6c and 6e). Notably, the astrocyte-specific Olig2 binding genes were enriched in pathways related to ion channel activity, particularly voltage-dependent ion channels (Supplementary figure 6f), which are crucial for neuronal action potential firing and closely related to the AtN process. If the CUT&Tag data in astrocytes were a random technical artifact, we would not expect the joint analysis with different databases to yield consistent biological signals.\nLastly, we conducted additional validation experiments on the key target gene Ngn2 using qRT-PCR and immunofluorescence (Fig.8k-m). The results showed that astrocyte Olig2 inhibits Ngn2 expression, consistent with the known transcriptional repression function of Olig2.\nIn conclusion, while we were unable to perform repeated CUT&Tag sequencing due to technical limitations, the integration and validation of key target genes, along with the high signal-to-noise ratio of CUT&Tag sequencing, support the reliability and scientific rigor of our data.\nReferences\n1. Mattugini, N. et al. Inducing Different Neuronal Subtypes from Astrocytes in the Injured Mouse Cerebral Cortex. Neuron103, 1086-1095 e1085 (2019).\n2. Wang, L.L. et al. Revisiting astrocyte to neuron conversion with lineage tracing in vivo. Cell 184, 5465-5481 e5416 (2021).\n3. Chen, S. et al. Ngn2 failed to induce astrocyte-to-neuron reprogramming after intracerebral hemorrhage. Brain Hemorrhages (2025).\n4. Guo, Y. et al. High-titer AAV disrupts cerebrovascular integrity and induces lymphocyte infiltration in adult mouse brain. Mol Ther Methods Clin Dev31, 101102 (2023).\n5. Niu, W. et al. In vivo reprogramming of astrocytes to neuroblasts in the adult brain. Nat Cell Biol15, 1164-1175 (2013).\n6. Tai, W. et al. In vivo reprogramming of NG2 glia enables adult neurogenesis and functional recovery following spinal cord injury. Cell Stem Cell28, 923-937 e924 (2021).\n7. Zhang, Y. et al. A single factor elicits multilineage reprogramming of astrocytes in the adult mouse striatum. Proc Natl Acad Sci U S A119, e2107339119 (2022).\n8. Bindels, D.S. et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat Methods14, 53-56 (2017).\n9. Bernal, A. & Arranz, L. Nestin-expressing progenitor cells: function, identity and therapeutic implications. Cell Mol Life Sci75, 2177-2195 (2018).\n10. Morrow, C.S. et al. Vimentin Coordinates Protein Turnover at the Aggresome during Neural Stem Cell Quiescence Exit. Cell Stem Cell26, 558-+ (2020).\n11. Harris, L. et al. Neurogenic differentiation by hippocampal neural stem and progenitor cells is biased by NFIX expression. Development145 (2018).\n12. Heng, Y.H.E. et al. NFIX Regulates Neural Progenitor Cell Differentiation During Hippocampal Morphogenesis. Cereb Cortex24, 261-279 (2014).\n13. Ogushi, K. et al. High Tumoral STMN1 Expression Is Associated with Malignant Potential and Poor Prognosis in Patients with Neuroblastoma. Cancers15 (2022).\n14. Zhou, Y. et al. Molecular landscapes of human hippocampal immature neurons across lifespan. Nature607, 527-533 (2022).\n15. Stevanovic, M. et al. SOX Transcription Factors as Important Regulators of Neuronal and Glial Differentiation During Nervous System Development and Adult Neurogenesis. Front Mol Neurosci14 (2021).\n16. Mu, L.F. et al. SoxC Transcription Factors Are Required for Neuronal Differentiation in Adult Hippocampal Neurogenesis. J Neurosci32, 3067-3080 (2012).\n17. Sansom, S.N. et al. The Level of the Transcription Factor Pax6 Is Essential for Controlling the Balance between Neural Stem Cell Self-Renewal and Neurogenesis. Plos Genet5 (2009).\n18. Xu, Y. et al. PAUPAR and PAX6 sequentially regulate human embryonic stem cell cortical differentiation. Nucleic Acids Res49, 1935-1950 (2021).\n19. Russo, G.L. et al. CRISPR-Mediated Induction of Neuron-Enriched Mitochondrial Proteins Boosts Direct Glia-to-Neuron Conversion. Cell Stem Cell28, 524-534 e527 (2021).\n20. Lai, T. et al. SOX5 controls the sequential generation of distinct corticofugal neuron subtypes. Neuron57, 232-247 (2008).\n21. Leone, D.P., Srinivasan, K., Chen, B., Alcamo, E. & McConnell, S.K. The determination of projection neuron identity in the developing cerebral cortex. Curr Opin Neurobiol 18, 28-35 (2008).\n22. Hashizume, K., Yamanaka, M. & Ueda, S. POU3F2 participates in cognitive function and adult hippocampal neurogenesis via mammalian-characteristic amino acid repeats. Genes Brain Behav17, 118-125 (2018).\n23. Notaras, M. et al. Schizophrenia is defined by cell-specific neuropathology and multiple neurodevelopmental mechanisms in patient-derived cerebral organoids. Mol Psychiatr 27, 1416-1434 (2022).\n24. Li, K.Y., Li, J.T., Zheng, J.L. & Qin, S. Reactive Astrocytes in Neurodegenerative Diseases. Aging Dis10, 664-675 (2019).\n25. Kaya-Okur, H.S. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun10, 1930 (2019).\n26. Kaya-Okur, H.S., Janssens, D.H., Henikoff, J.G., Ahmad, K. & Henikoff, S. Efficient low-cost chromatin profiling with CUT&Tag. Nat Protoc15, 3264-3283 (2020).\n27. Tian, Y. et al. Drive Astrocytic Glioblastoma Proliferation Through Transcriptional Co-Regulation of Various Cyclins. Genes-Basel16 (2025).\n28. Wang, Z. et al. Coordinated regulation of cortical astrocyte maturation by OLIG1 and OLIG2 through BMP7 signaling modulation. J Genet Genomics (2025).\nReviewer #3 (Remarks to the Author):\nIn the revised manuscript, the authors have addressed most of the concerns raised in the initial submission. Notably, they have moderated some of their conclusions and incorporated a discussion of alternative interpretations of their findings.\nThe use of Aldh1l1:CreERT2 mice to label astrocytes prior to direct reprogramming is a significant addition that reinforces the astrocytic origin of the targeted cells. Overall, the manuscript presents compelling findings and will be of interest to the readership.\nOne minor point:\n_ in line 238 the authors use the term: “progenitor cells” to define intermediate state of cells negative for both glial and neuronal markers. However, the authors cannot claim they are “progenitor cells” as evidences are lacking that these cells are indeed progenitors cells(in their scRNASeq they did not detect an increase in Sox2 or Pax6). The sentence should be rephrase to just mention the existence of such intermediate state.\nResponse: Thank you for your thoughtful and constructive feedback on our revised manuscript. We truly appreciate your positive evaluation and are pleased to hear that the manuscript presents compelling findings that will be of interest to the readership.\nWe have made the suggested revision regarding the use of the term \"progenitor cells\" in the revised manuscript (lines 236-240). We have rephrased the sentence to avoid overstating the characterization of these cells as progenitors, given the lack of supporting evidence. Thank you once again for your time and consideration.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: Thank you very much for your involvement in reviewing our manuscript. Your participation is invaluable, and we are grateful for your time and thoughtful consideration of our work.\nAfter three rounds of peer review, Reviewer 2 still had some remaining concerns. In response to these suggestions, we have conducted two additional key experiments, and the related results are included in the revised manuscript. All new modifications are highlighted with a yellow background for clarity. We sincerely appreciate the constructive feedback from the reviewers, which has been crucial in enhancing the quality of our research. We hope that the revised manuscript can be considered for publication soon, as we believe our latest findings contribute valuable insights to the field.\nReviewers' comments:\nReviewer #2 (Remarks to the Author):\nThe authors failed to address our key concerns, i.e., proper fate mapping using state-of-the-art approaches and proper validation of the Cut&Tag results.\nWhile the findings of Olig2 induction in astrocytes in response to forced proneural factor expression are of interest, without the necessary scientific rigor in resolving those above issues, the paper will not sufficiently advance the field.\nResponse: Thank you for your valuable feedback and for highlighting the key concerns regarding our study. We have carefully addressed these issues in the revised manuscript and would like to provide a detailed response.\nRegarding the fate mapping of neurons, we acknowledge the need for additional evidence to demonstrate that the reporter-labeled neurons are not derived from endogenous neurons. In response to your suggestion, we followed the approach from a previously published study and used rAAV2-CAG::EGFP to pre-label cortical neurons in mice (Wang et al., 2021, Cell). This allowed us to specifically track AtN reprogramming in the pre-labeled region. Our results confirm that the reprogramming efficiency with Ngn2 alone is low, and the leakage expression of the reporter protein in endogenous neurons is minimal, consistent with prior findings (Wang et al., 2021, Cell). Importantly, we observed that knockdown of Olig2 in astrocytes significantly enhanced Ngn2-mediated reprogramming, with about 96% of the reprogrammed neurons (tdT+/NeuN+) not being endogenous neurons (GFP-). These findings are now included in the revised manuscript (lines 320-333 and Supplementary Figure 9).\nIn response to your concern about the single Cut&Tag result for Olig2, we have conducted an additional round of Cut&Tag experiments to validate our findings. We integrated the results from both experiments and re-analyzed all data. The updated results, where changes occurred, are highlighted in yellow. Both experiments consistently showed strong binding peaks of Olig2 to the Ngn2 gene in astrocytes (Figure 8h). Furthermore, our RT-qPCR and immunofluorescence results confirm that Olig2 binding to the Ngn2 gene significantly inhibits its expression, thus hindering AtN reprogramming. These new findings have been added to the manuscript (lines 549-636, new Figure 8, new Supplementary Figures 17 and 18).\nWe appreciate your constructive suggestions, which have greatly improved the rigor of our study. We hope the revised manuscript now addresses all of your concerns.\nReviewer #3 (Remarks to the Author):\nFrom the first submission, the manuscript has undergone substantial improvement.\nThe addition of Supplementary Figure 9, which includes data on pre-labelling endogenous neurons, is an important enhancement and strengthens the study.\nIn the newly added paragraph, the authors report that in the Ngn2 + shNC condition, 5 out of 34 tdT⁺ neurons were also GFP⁺. This corresponds to approximately 15% of the neuronal population analyzed, a small but not negligible fraction of transduced cells. I recommend that the authors explicitly include this percentage in the text and add a corresponding graph to Supplementary Figure 9, similar to what was presented for the Ngn2 + shOlig2 condition.\nResponse: We thank the reviewer for this helpful suggestion. As recommended, we have added the corresponding statistical graph to Supplementary Figure 9d, consistent with the presentation used for the Ngn2 + shOlig2 condition."}]

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