The emergence and rapid development of in vivo cell reprogramming technology, which converts deleterious astrocytes into functional neurons, holds great promise for neuroregenerative therapy (Torper and Götz, 2017). Various groups worldwide have successfully achieved astrocyte-to-neuron (AtoN) conversion by forced expression of different proneural transcription factors (TFs) such as Neurogenin2 (Ngn2) (Grande et al., 2013), Mash1 (Ascl1) (Liu et al., 2015), NeuroD1 (Guo et al., 2014), SOX2 (Niu et al., 2013), and various TF combinations (Rivetti di Val Cervo et al., 2017; Wu et al., 2020; Lentini et al., 2021). In contrast to the TF overexpression approach, by repressing an RNA-binding protein polypyrimidine tract binding protein 1 (PTBP1), two groups have reported that functional neurons including dopaminergic neurons (DAns) could be induced from astrocytes rapidly and efficiently in vivo, reconstructing the nigrostriatal circuit and improving motor deficits in a 6-hydroxydopamine (6-OHDA) mouse model of Parkinson’s disease (PD) (Qian et al., 2020; Zhou et al., 2020).

Nevertheless, without substantiating the exact origin of the nascent-induced DAns using a reliable lineage-tracing strategy, these two outstanding works soon arouse widespread debate and argument (Jiang et al., 2021; Arenas, 2020; Qian et al., 2021). Most recently, by adopting the stringent lineage-tracing method, two studies arguing against previous findings have been published. One group shows that adeno-associated virus (AAV)-shPtbp1-induced, presumed astrocyte-converted DAns are not truly converted from astrocytes but are merely AAV-infected endogenous neurons due to virus leakage (Wang et al., 2021). Another group reports that no astrocyte-derived neuron, including DAns are generated in multiple brain regions including the substantia nigra and striatum, in astrocyte-specific Ptbp1 deletion mice (Blackshaw et al., 2021). However, both studies only focus on quiescent astrocytes and whether reactive astrocytes could be converted to neurons more effectively after PTBP1 repression requires further verification.

During brain injury or neurodegeneration, astrocytes become activated and acquire certain characteristics of neural stem cells (NSCs) such as proliferation, Nestin- or Vimentin-immunoreactivity, and multipotency (Buffo et al., 2008; Shimada et al., 2012; Robel et al., 2011; Sirko et al., 2013). Some researchers have claimed that reactive astrocytes with stem cell hallmarks can be reprogrammed to neurons more easily and efficiently than quiescent astrocytes (Grande et al., 2013; Guo et al., 2014; Brulet et al., 2017; Wan et al., 2014; Mattugini et al., 2019). Therefore, we adopted the 6-OHDA PD model with lineage-tracing method to investigate whether reactive astrocytes could truly be converted to neurons including DAns.

In this study, through stringent and convincing lineage-tracing technology, we substantiated that neither AAV-shRNA- nor ASO-mediated astroglial PTBP1 repression could achieve AtoN or AtoDAn conversion either in the substantia nigra or in the striatum of a 6-OHDA mouse model of PD.

We first used AAV expressing EGFP followed by shPtbp1 under the full-length GFAP promoter to repress astroglial PTBP1 and AAV-shscramble as a control. Both AAV constructs were predominantly expressed in astrocytes 7 days post-infection. After 1–3 months of infection, AAV-shPtbp1 allowed for low levels of reporter protein (GFP) expression in neurons, whereas AAV-shscramble-mediated GFP expression was still restricted to astrocytes. Without stringent lineage-tracing, the result can be easily misinterpreted as PTBP1 repression-mediated AtoN conversion. The reason why AAV-shPtbp1 rather than AAV-shscramble leaked into neurons is currently unclear. According to a recent study, coding sequences of some proneural genes, such as Neurod1, could activate GFAP promoter elements in cis and drive the expression of the viral reporter gene in neurons (Wang et al., 2021). We presume that the shPtbp1 sequence might function similarly to Neurod1, activating GFAP promoter and driving GFP expression in neurons at a relatively low level.

It has been reported that AAV can exert toxic effects on doublecortin (DCX)⁺ neuroblasts or neural progenitor cells (Johnston et al., 2021), which might render the virus-infected astrocytes unable to convert into neurons. After reviewing previous studies of in vivo glia-to-neuron conversion, we found that DCX⁺ intermediate cells could hardly be detected using AAV for gene manipulation (Liu et al., 2015; Guo et al., 2014; Brulet et al., 2017; Mattugini et al., 2019; Chen et al., 2020; Lai et al., 2020; Leib et al., 2022; Liu et al., 2020; Xiang et al., 2021; Zheng et al., 2022; Torper et al., 2015; Pereira et al., 2017). In contrast, DCX⁺ intermediate cells can usually be seen in most lentivirus (LV)/retrovirus (RV)-mediated glia-to-neuron conversion studies in vivo (Grande et al., 2013; Guo et al., 2014; Niu et al., 2013; Buffo et al., 2005; Gascón et al., 2016; Torper et al., 2013; Rivetti di Val Cervo et al., 2017; Magnusson et al., 2014; Niu et al., 2015; Su et al., 2014; Wang et al., 2016; Zhang et al., 2022; Heinrich et al., 2014). This observation suggests that either AAV toxicity restrains the AtoN conversion process, or LV/RV boosts the AtoN conversion process in an unknown manner.

To exclude the potential toxicity of AAV, we adopted ASO as an alternative method for PTBP1 repression and found ASO-Ptbp1 also failed to convert astrocytes into neurons or DAns. This result is inconsistent with that of a previous study claiming that AtoN conversion occurred after ASO-Ptbp1 delivery (Qian et al., 2020). However, we believe that their result is not convincing since the reporter mice-Rosa-Tdtomato (Ai14) used for lineage-tracing by Qian et al., 2020 has been questioned for occasional leakage to neurons (Wang et al., 2021). Other possible reasons for the discrepancy may be different ASO-mediated PTBP1 repression efficiency, different lineage-tracing mouse types and genetic backgrounds, and different experimental time lengths.

The major weakness of the present study is that we only rule out the possibility of astrocyte conversion to neuron, including DAn using Aldh1l1 promoter-based lineage-tracing mice. Whether other latent neurogenic cell types such as NSC (Maimon et al., 2021), oligodendrocytes (Weinberg et al., 2017), or NG2 glia, could be converted to neurons upon PTBP1 repression requires further investigation. Our results showed that ASO had no cell selectivity and could non-specifically enter different cell types to repress PTBP1 expression. These neurogenic cell types could be converted into neurons after PTBP1 repression Therefore, a reliable lineage-tracing method targeting these neurogenic cell types is necessary for future studies to identify the genuine cell identity that might contribute to neuron restoration.

One important question of the present study is whether and to what extent the reactive state of astrocytes in the 6-OHDA model could truly reflect the real state of astrocytes in PD patients. Acute lesions induced by neurotoxins, such as 6-OHDA, usually results in substantial neuron loss, creating an inflammatory microenvironment characterized by the presence of both A1 (pro-inflammatory) and A2 (anti-inflammatory) astrocyte subtypes (Ryu et al., 2020). In contrast, as one of the most important risk factors for PD, normal aging induces pro-inflammatory A1 like astrocyte reactivity (Clarke et al., 2018), which may accurately reflect the real state of PD patients. In particular, during ischemia stroke, a classic model characterized by the presence of A2 astrocytes, astrocytes spontaneously become neurogenic and the Notch pathway is repressed (Magnusson et al., 2014). We therefore assume that pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), could be detrimental to the AtoN conversion process and the survival, maturation, and subsequent neurite outgrowth of the newborn neurons (Karimi-Abdolrezaee and Billakanti, 2012; Belenguer et al., 2021; Park and Bowers, 2010), whereas the anti-inflammatory cytokines and neurotrophic factors, such as brain-derived neurotrophic factor might be beneficial and even critical (Niu et al., 2013). Therefore, whether A2 astrocytes could be converted to neurons including DAns more efficiently, and more importantly, how to induce beneficial A2 astrocytes in the brains of PD patients for neural repair and regeneration needs further investigation.

All data generated or analysed during this study are included in the manuscript and supporting file; source data files have been provided for all the figures.

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Decision letter after peer review:

Thank you for submitting your article "Repressing PTBP1 is incapable to convert reactive astrocytes to dopaminergic neurons in a mouse model of Parkinson's disease" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Suzanne Pfeffer as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Benedikt Berninger (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

All three reviewers expressed a high level of enthusiasm for your work and made important comments/recommendations (see appended reviews) that are, for the most part, straightforward and aimed at strengthening your study. Thus, we would like to see these being addressed before your manuscript can be considered suitable for publication. Moreover, we would like you to also consider the following revisions:

(1) To tone-down your findings throughout including in the title as it was felt that you overstate the implications of this work given the fact that clearly, 6-OHDA does not capture the etiology of PD and that your data leave open the possibility that modulation of ptbp1 could facilitate dopaminergic neurogenesis if tested in other models, by other techniques, over longer time scales, or in greater Ns.

(2) To edit the presentation of the statistics as mentioned by Reviewer #1 throughout.

(3) to revise your discussion. Indeed, we concur with the Reviewers that a more formal discussion would increase the potential impact of your work. In particular, it would be important that the discussion better covers the limitations of the present work and stresses the outstanding questions.

Accordingly, we would recommend that you discuss at least the following two points:

(1) the acute nature of the 6-OHDA model and whether this may elicit cellular alterations, including in astrocytes, distinct from those that may arise in a chronic neurodegenerative process; and

(2) whether AAVs themselves could prevent neuronal conversion as suggested by Johnston et al., (eLife 2021). Although we recognize that many studies claiming successful glia-to-neuron conversion have used 6-OHDA and AAVs, we believe that you should discuss the possibility that other models of PD (as already mentioned above) and/or other approaches of knocking down Ptbp1, could potentially yield positive results. For instance, Maimon et al., (Nat Neurosci 2021) used antisense oligonucleotides against Ptbp1 to observe increased neurogenesis in the dentate gyrus, but not so markedly in other brain areas.

Reviewer #1 (Recommendations for the authors):

This is a very strong, elegant and straightforward study designed to test the question as to whether compared to quiescent astrocytes, reactive astrocytes may be more amendable to be converted into neurons, and specifically, dopaminergic neurons upon repression of PTB1. As a first step, the authors show that a number of neurons, including dopaminergic neurons, did express GFP in the substantia nigra of mice after injection of AAV-Gfap-EGFP-shPtb1. However, upon combining an elegant lineage tracing method, the authors found no evidence of GFP/NeuN or GFP/TH neurons derived from HA labeled astrocytes in both intact and 6-OHDA lesioned mice. In the latter the authors showed data in support of the reactive state of astrocytes. In light of this data, Chen and collaborators concluded that, in mice, there is no evidence of quiescent or reactive astrocyte conversion into dopaminergic neurons upon repression of PTB1.

This is a very convincing study, extremely well designed and conducted, and shows a set of striking data. The methods are excellent, and the results are well presented and discussed.

Reviewer #2 (Recommendations for the authors):

Considering the following points might improve the manuscript

1. It would be useful to show which cells were targeted by the AAV construct at earlier time points, e.g. 7days following infection. There is a general agreement that hGFAP promoter may be leaky and a tighter system would use AAVs with floxed transgenes. However, even if the system lacks in specificity, the authors do show convincingly that Ptbp1 is knocked down in astrocytes. This effect, illustrated in Figure 1B should be quantified.

2. In Figure 1C, the authors should provide 3D visualization of individual neurons to demonstrate co-localization of GFP with NeuN or TH more clearly.

3. The authors should quantitatively compare the number of NeuN and TH positive neurons in w/o 6-OHDA controls to those following scramble or Ptbp1 knockdown treatment in figure 3.

4. Given that the authors suspect that AAV-mediated GFP expression occurs in neurons despite being driven from hGAP promoter, would it make sense to show presence of GFP mRNA in neurons, even if very little, at an time point when GFP fluorescence is still undetectable in neurons but clearly seen in astrocytes? The authors could use RNA Scope towards this. I would expect to see clear signs of low-level expression compared to massive expression in astrocytes.

5. Given that hGFAP driven expression should eventually cease when astrocytes convert, would this have any impact on the interpretation? The authors should analyze whether there are any fate-mapped neurons that seem to lack AAV-mediated GFP expression. Or do the AAV constructs used in Figure 3 lack GFP to allow for TH IF?

6. The left panels in 3B must be controls, but this is not clearly indicated in the figure legend.

7. English should be revised.

8. Regarding the ethical endorsement of animal experiments, more clear information regarding the endorsing body should be provided. Only "university" is mentioned (Care and Use committee of university). Is there any legal number associated to this endorsement?

Reviewer #3 (Recommendations for the authors):

Chen et al., highlight a technical confound in previous publications that claim attenuation of PTBP1 results in trans-differentiation of astrocytes into dopamine neurons. While they clearly observe that previous use of recombinant AAV vectors lead to EGFP expression in dopaminergic neurons despite using an astrocyte "specific" promoter, the authors cannot conclude from the presented experiments that attenuation of PTBP1 does not achieve trans-differentiation. This is because the use of AAV could render astrocytes unable to differentiate into dopaminergic neurons in response to successful downregulation of PTBP1. Adding other, AAV independent, means of achieving PTBP1 downregulation in astrocytes in vivo would strengthen this study.

Essential revisions:

All three reviewers expressed a high level of enthusiasm for your work and made important comments/recommendations (see appended reviews) that are, for the most part, straightforward and aimed at strengthening your study. Thus, we would like to see these being addressed before your manuscript can be considered suitable for publication. Moreover, we would like you to also consider the following revisions:

(1) to tone-down your findings throughout including in the title as it was felt that you overstate the implications of this work given the fact that clearly, 6-OHDA does not capture the etiology of PD and that your data leave open the possibility that modulation of ptbp1 could facilitate dopaminergic neurogenesis if tested in other models, by other techniques, over longer time scales, or in greater Ns.

We appreciate the editor’s suggestion and have revised our title to ‘Repressing PTBP1 fails to convert reactive astrocytes to dopaminergic neurons in a mouse 6-hydroxydopamine model of Parkinson's disease’.

(2) To edit the presentation of the statistics as mentioned by Reviewer #1 throughout.

The detailed statistical information is provided in our revised manuscript.

(3) to revise your discussion. Indeed, we concur with the Reviewers that a more formal discussion would increase the potential impact of your work. In particular, it would be important that the discussion better covers the limitations of the present work and stresses the outstanding questions.

As suggested by the editor and reviewers, we have added a formal discussion in our revised manuscript that covers the limitations of the present work and the outstanding questions in this field.

Reviewer #2 (Recommendations for the authors):

Considering the following points might improve the manuscript

1. It would be useful to show which cells were targeted by the AAV construct at earlier time points, e.g. 7days following infection. There is a general agreement that hGFAP promoter may be leaky and a tighter system would use AAVs with floxed transgenes. However, even if the system lacks in specificity, the authors do show convincingly that Ptbp1 is knocked down in astrocytes. This effect, illustrated in Figure 1B should be quantified.

To identify the exact cell type targeted by our AAV construct seven days after AAV infection, we performed immunostaining and GFP⁺ cell number counting in the substantia nigra and striatum, respectively. The results showed that most of the infected cells were astrocytes (AldoC⁺), and a few other cells, such as neurons (NeuN⁺), NG2-glia (NG2⁺), and microglia (Iba-1⁺), were infected (Figure 1—figure supplement 1).

Moreover, as suggested by the reviewer, we have quantified the PTBP1 knockdown efficiency in Figure 1B, and the quantified data are included in the revised manuscript (Figure 1C).

2. In Figure 1C, the authors should provide 3D visualization of individual neurons to demonstrate co-localization of GFP with NeuN or TH more clearly.

To more clearly demonstrate the colocalization of GFP with NeuN or TH, we performed immunofluorescence experiments using anti-GFP, anti-TH, and anti-NeuN antibodies. Images were captured using a LSM780 confocal microscope (Zeiss). The enlarged 3D reconstruction of boxed individual neurons are shown in the lower panel of Figure 1D, F in the revised manuscript.

3. The authors should quantitatively compare the number of NeuN and TH positive neurons in w/o 6-OHDA controls to those following scramble or Ptbp1 knockdown treatment in figure 3.

As suggested by the reviewer, we have quantified the number of NeuN⁺ neurons and TH⁺ dopaminergic neurons in the substantia nigra before and after AAV-shPtbp1 or AAV-shscramble delivery in the 6-OHDA model and found no significant difference between the two groups, indicating that no neurons, including DA neurons, were regenerated in the substantia nigra after astroglial PTBP1 repression (Figure 3D, E).

4. Given that the authors suspect that AAV-mediated GFP expression occurs in neurons despite being driven from hGAP promoter, would it make sense to show presence of GFP mRNA in neurons, even if very little, at an time point when GFP fluorescence is still undetectable in neurons but clearly seen in astrocytes? The authors could use RNA Scope towards this. I would expect to see clear signs of low-level expression compared to massive expression in astrocytes.

We agree with the reviewer that low levels of GFP mRNA may appear in neurons at early time points, and we did observe low GFP protein levels in neurons 1 month after AAV-shPtbp1 delivery, but not AAV-shscramble (Figure 1 D, F). According to a recent study, the coding sequence of some proneural genes, such as Neurod1 could activate hGfap promoter via binding to cis-acting elements of the promoter in neurons (Wang et al., 2021). Therefore, we suppose that the shRNA sequence targeting Ptbp1 per se might function similar to Neurod1, activating the hGfap promoter to drive GFP expression in neurons at a relatively low level.

5. Given that hGFAP driven expression should eventually cease when astrocytes convert, would this have any impact on the interpretation? The authors should analyze whether there are any fate-mapped neurons that seem to lack AAV-mediated GFP expression. Or do the AAV constructs used in Figure 3 lack GFP to allow for TH IF?

We understand the reviewer’s concern. Because the hGfap promoter activity gradually ceased during AtoN conversion, we chose Rpl22^HA/HA reporter mice to permanently label astrocytes with HA-tag. If AtoN conversion occurrs, astrocyte-derived neurons or dopaminergic neurons should be marked with HA. However, we did not detect any HA⁺NeuN⁺ or HA⁺TH⁺ cells after PTBP1 repression, suggesting that no neurons or DAn were converted from astrocytes.

The two AAV constructs used in this study were attached to EGFP driven by the hGfap promoter as illustrated in Figure 1A. We apologize for the misleading comment because GFP images were not provided, which have been included in the revised manuscript (Figure 3C). The fluorescence images in the manuscript were captured on a gray scale and pseudocolored for overlay presentation.

6. The left panels in 3B must be controls, but this is not clearly indicated in the figure legend.

We appreciate the reviewer’s reminder and have revised according to reviewer’s suggestion.

7. English should be revised.

As suggested by the reviewer, we have sent our manuscript to the Editage (www.editage.cn) for English language editing.

8. Regarding the ethical endorsement of animal experiments, more clear information regarding the endorsing body should be provided. Only "university" is mentioned (Care and Use committee of university). Is there any legal number associated to this endorsement?

We supplied detailed ethical endorsement information of animal experiments including permission numbers in the Animal part of the Material and Methods section.

Reviewer #3 (Recommendations for the authors):

Chen et al., highlight a technical confound in previous publications that claim attenuation of PTBP1 results in trans-differentiation of astrocytes into dopamine neurons. While they clearly observe that previous use of recombinant AAV vectors lead to EGFP expression in dopaminergic neurons despite using an astrocyte "specific" promoter, the authors cannot conclude from the presented experiments that attenuation of PTBP1 does not achieve trans-differentiation. This is because the use of AAV could render astrocytes unable to differentiate into dopaminergic neurons in response to successful downregulation of PTBP1. Adding other, AAV independent, means of achieving PTBP1 downregulation in astrocytes in vivo would strengthen this study.

We deeply appreciate Reviewer 3’s constructive suggestions. We injected antisense oligonucleotide (ASO) against mouse Ptbp1 in the substantia nigra of lineage-tracing mice with or without 6-OHDA lesions for 2 months. While astroglial PTBP1 was efficiently repressed after ASO delivery for 2 months (Figure 4—figure supplement 1), no neurons, including DAn expressing astroglial tracing reporter, were detected, indicating that no neurons, including DAn, were converted from quiescent or reactive astrocytes after ASO-Ptbp1 delivery (Figure 4B, D). Moreover, motor deficits in 6-OHDA lesion mice did not improve 2 months after ASO-Ptbp1 delivery. These results have been added to the revised manuscript (Figure 4E).

References

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Author details

1. Weizhao Chen

   1. Guangdong Provincial Key Laboratory of Brain Function and Disease, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
   2. Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China

   Contribution

   Conceptualization, Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Qiongping Zheng

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6753-4854

2. Qiongping Zheng

   1. Guangdong Provincial Key Laboratory of Brain Function and Disease, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
   2. Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China

   Contribution

   Data curation, Methodology

   Contributed equally with

   Weizhao Chen

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1989-9234

3. Qiaoying Huang

   1. Guangdong Provincial Key Laboratory of Brain Function and Disease, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
   2. Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China

   Contribution

   Investigation, Validation, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

4. Shanshan Ma

   1. Guangdong Provincial Key Laboratory of Brain Function and Disease, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
   2. Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China

   Contribution

   Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – review and editing

   For correspondence

   mashsh3@mail.sysu.edu.cn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3004-9468

5. Mingtao Li

   1. Guangdong Provincial Key Laboratory of Brain Function and Disease, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
   2. Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China

   Contribution

   Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review and editing

   For correspondence

   limt@mail.sysu.edu.cn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5714-9322

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