Repressing PTBP1 fails to convert reactive astrocytes to dopaminergic neurons in a 6-hydroxydopamine mouse model of Parkinson’s disease

  1. Weizhao Chen
  2. Qiongping Zheng
  3. Qiaoying Huang
  4. Shanshan Ma
  5. Mingtao Li

  • Curated by eLife

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    Evaluation Summary:

    This new work from Chen et al. reports on a critical question that is whether astrocytes can be converted in situ into dopaminergic neurons in response to the targeting of specific factors using, for example, gene therapy. This is a very strong, elegant and straightforward study. It is of broad interest and of high translational relevance.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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Abstract

Lineage reprogramming of resident glial cells to dopaminergic neurons (DAns) is an attractive prospect of the cell-replacement therapy for Parkinson’s disease (PD). However, it is unclear whether repressing polypyrimidine tract binding protein 1 (PTBP1) could efficiently convert astrocyte to DAns in the substantia nigra and striatum. Although reporter-positive DAns were observed in both groups after delivering the adeno-associated virus (AAV) expressing a reporter with shRNA or CRISPR-CasRx to repress astroglial PTBP1, the possibility of AAV leaking into endogenous DAns could not be excluded without using a reliable lineage-tracing method. By adopting stringent lineage-tracing strategy, two other studies show that either knockdown or genetic deletion of quiescent astroglial PTBP1 fails to obtain induced DAns under physiological condition. However, the role of reactive astrocytes might be underestimated because upon brain injury, reactive astrocyte can acquire certain stem cell hallmarks that may facilitate the lineage conversion process. Therefore, whether reactive astrocytes could be genuinely converted to DAns after PTBP1 repression in a PD model needs further validation. In this study, we used Aldh1l1-CreERT2 -mediated specific astrocyte-lineage-tracing method to investigate whether reactive astrocytes could be converted to DAns in a 6-hydroxydopamine (6-OHDA) mouse model of PD. However, we found that no astrocyte-originated DAn was generated after effective and persistent knockdown of astroglial PTBP1 either in the substantia nigra or in striatum, while AAV ‘leakage’ to nearby neurons was easily observed. Our results confirm that repressing PTBP1 does not convert astrocytes to DAns, regardless of physiological or PD-related pathological conditions.

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  1. Version published to 10.7554/elife.75636 on eLife
  2. eLife

    Evaluation Summary:

    This new work from Chen et al. reports on a critical question that is whether astrocytes can be converted in situ into dopaminergic neurons in response to the targeting of specific factors using, for example, gene therapy. This is a very strong, elegant and straightforward study. It is of broad interest and of high translational relevance.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    This new work from Chen et al. reports on a critical question that is whether astrocytes can be converted in situ into dopaminergic neurons in response to the targeting of specific factors using, for example, gene therapy. This concept is enormously significant because, if correct, it may open powerful new repair strategies for disorders of the nervous system, such as Parkinson disease (PD). Relevant to this view is the remarkable demonstration by two groups that repressing the expression of the RNA-binding protein PTBP1 promotes the conversation of astrocytes into dopaminergic neurons in rodents associated with behavioral and neuropathological improvements in a model of PD. However, soon after the publication of this amazing data, independent research teams were unable to confirm these findings and rather attributed them to reported apparent conversion of technical issues, including leakage of viral vector. The lack of evidence of astrocyte conversion into dopaminergic neurons upon repression of PTB1 was done in a context where astrocytes were quiescent, a point that caught the attention of Chen and collaborators, who sought in the present study to revisit the same question of astrocyte conversion to dopaminergic neurons in a context where astrocytes are reactive. This difference is highly significant because studying reactive astrocytes would not only model PD more closely but would also examine whether particular intrinsic properties of reactive vs quiescent astrocytes may lead to very different responses to PTB1 repression. Therefore, to address this important point of cell biology, Chen and collaborators embarked on a very elegant and straightforward study using the 6-OHDA model of PD in mice to show that despite the presence of reactive astrocytes, there was no evidence of astrocytes conversion into neurons, including into dopaminergic neurons, neither in the striatum, nor in the substantia nigra upon repression of PTB1. This study is extremely solid and elegant and while perhaps beyond the scope of the study, it would have been valuable to also obtain behavioral and neuropathological data in these mice. Lastly, it would also be nice to test the authors' hypothesis in a more chronic model of PD, as produced by gene mutations which may cause a more protracted response of astrocytes. Having said this, it is true, however, that genetic models of PD are problematic and overt, and reproducible neurodegeneration is often questionable, shown in initial reports where conversion was done in 6-OHDA models.

  4. eLife

    Reviewer #2 (Public Review):

    This brief manuscript by Chen et al. revisits the important question whether knock-down of the RNA binding protein Ptbp1 can convert quiescent or reactive astrocytes residing in the striatum or the substantia nigra into induced dopaminergic neurons. Previous work in the literature suggests that this might occur and could contribute to functional repair in a mouse model of Parkinsonism. However, in these earlier studies there was no unambiguous proof of the astrocyte origin of the allegedly induced dopaminergic neurons.

    Thus, in the present study, the authors subjected this notion to scrutiny by combining genetic fate mapping to ascertain astrocyte origin with or without 6-hyrodxydopamine induced death of endogenous dopamine neurons, followed by adeno-associated virus-mediated Ptbp1 knockdown. The authors illustrate effective Ptbp1 knockdown in astrocytes by an AAV encoding a short-hairpin RNA against Ptbp1. A (possibly minor) weakness is that hGFAP regulatory sequences are used to drive transgene expression, known to be less specific than other approaches using AAVs. In any case, the authors report then that they do indeed observe a progressive increase in virus-labelled neurons over the time course of 3 months, most prominently in the substantia nigra, and less so in the striatum. No increase is observed when using an AAV encoding a scramble shRNA for control. However, in sharp contrast to expectation, they then find that none of the virus-labelled neurons could be traced back to fate-mapped astrocytes. To test whether Ptbp1 knockdown would be more effective in reactive astrocytes, they induce a 6-OHDA lesion which results in massive loss of TH immunoreactivity. The authors state that Ptbp1 knockdown does not improve TH levels, and none of the TH-positive neurons remaining in the substantia nigra can be traced back to astrocytes.

    This study strongly suggested that knock-down of Ptbp1 is not sufficient to induce the conversion of quiescent (shown for striatum or substantia nigra) or reactive astrocytes (substantia nigra) into dopaminergic neurons.

  5. eLife

    Reviewer #3 (Public Review):

    This paper re-evaluates whether repression of the expression of the RNA binding protein PTBP1 in astrocytes results in the trans-differentiation of astrocytes into mesencephalic dopamine neurons which are lost in Parkinson's Disease. The authors conclude that repression of PTBP1 in astrocytes does not induce the formation of dopamine neurons in contrast to the findings published earlier by two independent groups. By extension, the authors speculate that previous observations mis-identified neurogenesis due to compromised tissue specificity of an astrocyte specific promoter when expressed from an AAV vector. Clarifying the effects of PTBP1 repression on dopaminergic neurogenesis is of significance in order to guide efforts to develop strategies for neuronal replacement from endogenous sources in Parkinson's Disease.

    Strengths:
    The authors applied a stringent in vivo cell fate tracing technique to determine whether astrocytes can contribute to dopaminergic neuron replacement. This strategy revealed the significance of an underappreciated confound in previous published work, namely tissue specificity of the utilized promotors in the context of AAV.

    Weaknesses:
    The authors observed a qualitative downregulation of PTBP1 expression by immunohistochemistry assessment. They also show that AAV infected astrocytes reveal altered morphology. It is not clear whether the observed down-regulation of PTBP1 is due to the expression of the silencer RNA or secondary to AAV induced physiological stress in infected astrocytes. Thus, ruling out attenuation of PTBP1 as a technique of inducing dopaminergic neurons appears premature in absence of AAV independent techniques to modulate PTBP1 selectively in astrocytes.

  6. Version published to 10.1101/2021.11.12.468309v2 on bioRxiv
  7. Version published to 10.1101/2021.11.12.468309v1 on bioRxiv