Base editing of Ptbp1 in neurons alleviates symptoms in a mouse model for Parkinson’s disease

Reviewer #1 (Public Review):

Summary:

Recent years have seen spectacular and controversial claims that loss of function of the RNA splicing factor Ptbp1 can efficiently reprogram astrocytes into functional neurons that can rescue motor defects seen in 6-hydroxydopamine (6-OHDA)-induced mouse models of Parkinson's disease (PD). This latest study is one of a series that fails to reproduce these observations, but remarkably also reports that neuronal-specific loss of function of Ptbp1 both induces expression of dopaminergic neuronal markers in striatal neurons and rescues motor defects seen in 6-OHDA-treated mice. The claims, if replicated, are remarkable and identify a straightforward and potentially translationally relevant mechanism for treating motor defects seen in PD models. However, while the reported behavioral effects are strong and were collected without sample exclusion, other claims made here are less convincing. In particular, no evidence that Ptbp1 loss of function actually occurs in striatal neurons is provided, and the immunostaining data used to claim that dopaminergic markers are induced in striatal neurons is not convincing. Furthermore, no characterization of the molecular identity of Ptbp1-deficient striatal neurons is provided using single-cell RNA-Seq or spatial transcriptomics, making it difficult to conclude that these cells are indeed adopting a dopaminergic phenotype.

Overall, while the claims of behavioral rescue of 6-OHDA-treated mice appear compelling, it is essential that these be independently replicated as soon as possible before further studies on this topic are carried out. Insights into the molecular mechanisms by which neuronal-specific loss of function of Ptbp1 induces behavioral rescue are lacking, however. Moreover, the claims of induction of neuronal identity in striatal neurons by Ptbp1 require considerable additional work to be convincing.

Strengths of the study:

(1) The effect size of the behavioral rescue in the stepping and cylinder tests is strong and significant, essentially restoring 6-OHDA-lesioned mice to control levels.

(2) Since the neurotoxic effects of 6-OHDA treatment are highly variable, the fact that all behavioral data was collected blinded and that no samples were excluded from analysis increases confidence in the accuracy of the results reported here.

Weaknesses of the study:

(1) Neurons express relatively little Ptbp1. Indeed, cellular expression levels as measured by scRNA-Seq are substantially below those of astrocytes and other non-neuronal cell types, and Ptbp1 immunoreactivity has not been observed in either striatal or midbrain neurons (e.g. Hoang, et al. Nature 2023). This raises the question of whether any recovery of Th expression is indeed mediated by the loss of function of Ptbp1 rather than by off-target effects. AAV-mediated rescue of Ptbp1 expression could help clarify this.

(2) It is not clear why dopaminergic neurons, which are not normally found in the striatum, are observed following Ptbp1 knockout. This is very similar to the now-debunked claims made in Zhou, et al. Cell 2020, but here performed using the hSyn rather than GFAP mini promoter to control AAV expression. While this is the most dramatic and potentially translationally relevant claim of the study, this claim is extremely surprising and lacks any clear mechanistic explanation for why it might happen in the first place. This observation is even more surprising in light of reports that antisense oligonucleotide-mediated knockdown of Ptbp1, which should have affected both neuronal and glial Ptbp1 expression, failed to induce expression of dopaminergic neuronal markers in the striatum (Chen, et al. eLife 2022). Selective loss of function of Ptbp1 in striatal and midbrain astrocytes likewise results in only modest changes in gene expression It is critically important that this claim be independently replicated, and that additional data be provided to conclusively show that striatal neurons are indeed expressing dopaminergic markers.

(3) More generally, since multiple spectacular and irreproducible claims of single-step glial-to-neuron reprogramming have appeared in high-profile journals in recent years, a consensus has emerged that it is essential to comprehensively characterize the identity of "transformed" cells using either single-cell RNA-Seq or spatial transcriptomics (e.g. Qian, et al. FEBS J 2021; Wang and Zhang, Dev Neurobiol 2022). These concerns apply equally to claims of neuronal subtype conversion such as those advanced here, and it is essential to provide these same datasets.

(4) Low-power images are generally lacking for immunohistochemical data shown in Figures 3 and 4, which makes interpretation difficult. DAPI images in Figure 3C do not appear nuclear. Immunostaining for Th, DAT, and Dcx in Figure 4 shows a high background and is difficult to interpret.

(5) Insights into the mechanism by which neuronal-specific loss of Ptbp1 function induces either functional recovery, or dopaminergic markers in striatal neurons, is lacking.

Reviewer #2 (Public Review):

Summary:

The manuscript by Bock and colleagues describes the generation of an AAV-delivered adenine base editing strategy to knockdown PTBP1 and the behavioral and neurorestorative effects of specifically knocking down striatal or nigral PTBP1 in astrocytes or neurons in a mouse model of Parkinson's disease. The authors found that knocking down PTBP1 in neurons, but not astrocytes, and in striatum, but not nigra, results in the phenotypic reorganization of neurons to TH+ cells sufficient to rescue motor phenotypes, though insufficient to normalize responses to dopaminomimetic drugs.

Strengths:

The manuscript is generally well-written and adds to the growing literature challenging previous findings by Qian et al., 2020 and Zhou et al., 2020 indicating that astrocytic downregulation of PTBP1 can induce conversion to dopaminergic neurons in the midbrain and improve parkinsonian symptoms. The base editing approach is interesting and potentially more therapeutically relevant than previous approaches.

Weaknesses:

The manuscript has several weaknesses in approach and interpretation. In terms of approach, the animal model utilized, the 6-OHDA model, though useful to examine dopaminergic cell loss, exhibits accelerated neurodegeneration and none of the typical pathological hallmarks (synucleinopathy, Lewy bodies, etc.) compared to the typical etiology of Parkinson's disease, limiting its translational interpretation. In addition, there is no confirmation of a neuronal or astrocytic knockdown of PTBP1 in vivo; all base editing validation experiments were completed in cell lines. Finally, it is unclear why the base editing approach was used to induce loss-of-function rather than a cell-type specific knockout, if the goal is to assess the effects of PTBP1 loss in specific neurons. In terms of interpretation, the conclusion by the authors that PTBP1 knockdown has little likelihood to be therapeutically relevant seems overstated, particularly since they did observe a beneficial effect on motor behavior. We know that in PD, patients often display negligible symptoms until 50-70% of dopaminergic input to the striatum is lost, due to compensatory activity of remaining dopaminergic cells. Presumably, a small recovery of dopaminergic neurons would have an outsized effect on motor ability and may improve the efficacy of dopaminergic drugs, particularly levodopa, at lower doses, averting many problematic side effects. Since striatal dopamine was assessed by whole-tissue analysis, which is not necessarily reflective of synaptic dopamine availability, it is difficult to assess whether the ~10% increase in TH+ cells in the striatum was sufficient to improve dopamine function. However, the improvement in motor activity suggests that it was.

Reviewer #3 (Public Review):

This study explores the use of an adenine base editing strategy to knock down PTBP1 in astrocytes and neurons of a Parkinson's disease mouse model, as a potential AAV-BE therapy. The results indicate that editing Ptbp1 in neurons, but not astrocytes, leads to the formation of tyrosine hydroxylase (TH)+ cells, rescuing some motor symptoms.

Several aspects of the manuscript stand out positively. Firstly, the clarity of the presentation. The authors communicate their ideas and findings in a clear and understandable manner, making it easier for readers to follow.

The Materials and methods section is well-elaborated, providing sufficient detail for reproducibility.

The logical flow of the manuscript makes sense, with each section building upon the previous one coherently.

The ABE strategy employed by the authors appears sound, and the manuscript presents a coherent and well-supported argument.

Positively, some of the data in this study effectively counteracts previous work in line with more recent publications, demonstrating the authors' ability to contribute to the ongoing conversation in the field.

However, while the in vitro data yields promising results, it may have been overly optimistic to assume that the efficiencies observed in dividing cells will directly translate to in vivo conditions. This consideration is important given the added complexities of vector optimization, different cell types targeted in vitro versus in vivo, as well as unknown intrinsic limitations of the base editing technology.

In addition, certain aspects of the manuscript would benefit from a more in-depth and comprehensive discussion rather than being only briefly touched upon. Such a discussion would enhance the relevance of the obtained results and provide the foundation for improvement when using similar approaches.