Peer review process
Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, public reviews, and a provisional response from the authors.
Read more about eLife’s peer review process.Editors
- Reviewing EditorXin DuanUniversity of California, San Francisco, San Francisco, United States of America
- Senior EditorSacha NelsonBrandeis University, Waltham, United States of America
Reviewer #1 (Public review):
Summary:
Zhang et al. used a conditional knockout mouse model to re-examine the role of the RNA-binding protein PTBP1 in the transdifferentiation of astroglial cells into neurons. Several earlier studies reported that PTBP1 knockdown can efficiently induce the transdifferentiation of rodent glial cells into neurons, suggesting potential therapeutic applications for neurodegenerative diseases. However, these findings have been contested by subsequent studies, which in turn have been challenged by more recent publications. In their current work, Zhang et al. deleted exon 2 of the Ptbp1 gene using an astrocyte-specific, tamoxifen-inducible Cre line and investigated, using fluorescence imaging and bulk and single-cell RNA-sequencing, whether this manipulation promotes the transdifferentiation of astrocytes into neurons across various brain regions. The data strongly indicate that genetic ablation of PTBP1 is not sufficient to drive efficient conversion of astrocytes into neurons. Interestingly, while PTBP1 loss alters splicing patterns in numerous genes, these changes do not shift the astroglial transcriptome toward a neuronal profile.
Strengths:
Although this is not the first report of PTBP1 ablation in mouse astrocytes in vivo, this study utilizes a distinct knockout strategy and provides novel insights into PTBP1-regulated splicing events in astrocytes. The manuscript is well written, and the experiments are technically sound and properly controlled. I believe this study will be of considerable interest to a broad readership.
Weaknesses:
(1) The primary point that needs to be addressed is a better understanding of the effect of exon 2 deletion on PTBP1 expression. Figure 4D shows successful deletion of exon 2 in knockout astrocytes. However, assuming that the coverage plots are CPM-normalized, the overall PTBP1 mRNA expression level appears unchanged. Figure 6A further supports this observation. This is surprising, as one would expect that the loss of exon 2 would shift the open reading frame and trigger nonsense-mediated decay of the PTBP1 transcript. Given this uncertainty, the authors should confirm the successful elimination of PTBP1 protein in cKO astrocytes using an orthogonal approach, such as Western blotting, in addition to immunofluorescence. They should also discuss possible reasons why PTBP1 mRNA abundance is not detectably affected by the frameshift.
(2) The authors should analyze PTBP1 expression in WT and cKO substantia nigra samples shown in Figure 3 or justify why this analysis is not necessary.
(3) Lines 236-238 and Figure 4E: The authors report an enrichment of CU-rich sequences near PTBP1-regulated exons. To better compare this with previous studies on position-specific splicing regulation by PTBP1, it would be helpful to assess whether the position of such motifs differs between PTBP1-activated and PTBP1-repressed exons.
(4) The analyses in Figure 5 and its supplement strongly suggest that the splicing changes in PTBP1-depleted astrocytes are distinct from those occurring during neuronal differentiation. However, the authors should ensure that these comparisons are not confounded by transcriptome-wide differences in gene expression levels between astrocytes and developing neurons. One way to address this concern would be to compare the new PTBP1 cKO data with publicly available RNA-seq datasets of astrocytes induced to transdifferentiate into neurons using proneural transcription factors (e.g., PMID: 38956165).
Reviewer #2 (Public review):
Summary:
The manuscript by Zhang and colleagues describes a study that investigated whether the deletion of PTBP1 in adult astrocytes in mice led to an astrocyte-to-neuron conversion. The study revisited the hypothesis that reduced PTBP1 expression reprogrammed astrocytes to neurons. More than 10 studies have been published on this subject, with contradicting results. Half of the studies supported the hypothesis while the other half did not. The question being addressed is an important one because if the hypothesis is correct, it can lead to exciting therapeutic applications for treating neurodegenerative diseases such as Parkinson's disease.
In this study, Zhang and colleagues conducted a conditional mouse knockout study to address the question. They used the Cre-LoxP system to specifically delete PTBP1 in adult astrocytes. Through a series of carefully controlled experiments, including cell lineage tracing, the authors found no evidence for the astrocyte-to-neuron conversion.
The authors then carried out a key experiment that none of the previous studies on the subject did: investigating alternative splicing pattern changes in PTBP1-depleted cells using RNA-seq analysis. The idea is to compare the splicing pattern change caused by PTBP1 deletion in astrocytes to what occurs during neurodevelopment. This is an important experiment that will help illuminate whether the astrocyte-to-neuron transition occurred in the system. The result was consistent with that of the cell staining experiments: no significant transition was detected.
These experiments demonstrate that, in this experimental setting, PTBT1 deletion in adult astrocytes did not convert the cells to neurons.
Strengths:
This is a well-designed, elegantly conducted, and clearly described study that addresses an important question. The conclusions provide important information to the field.
To this reviewer, this study provided convincing and solid experimental evidence to support the authors' conclusions.
Weaknesses:
The Discussion in this manuscript is short and can be expanded. Can the authors speculate what led to the contradictory results in the published studies? The current study, in combination with the study published in Cell in 2021 by Wang and colleagues, suggests that observed difference is not caused by the difference of knockdown vs. knockout. Is it possible that other glial cell types are responsible for the transition? If so, what cells? Oligodendrocytes?