Ptbp1 deletion does not induce glia-to-neuron conversion in adult mouse retina and brain.
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA767059 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| Other | ||||
| SRR22141557.fastq.gz | Fastqsanger.gz | |||
| SRR22141558.fastq.gz | Fastqsanger.gz | |||
| SRR22141559.fastq.gz | Fastqsanger.gz | |||
| SRR22141560.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: scRNA-Seq was performed to profile changes in adult retina Muller glia cells and brain astrocytes after Ptbp1 deletion
2021-12-07 | GSE184933 | GEO
Project description:Direct reprogramming of glia into neurons is a potentially promising approach for the replacement of neurons lost to injury or neurodegenerative disorders. Knockdown of the polypyrimidine tract-binding protein Ptbp1 has been recently reported to induce efficient conversion of retinal Mϋller glia into functional neurons. Here, we use a combination of genetic lineage tracing, single-cell RNA sequencing (scRNA-seq), and electroretinogram analysis to show that selective induction of either heterozygous or homozygous loss-of-function mutants of Ptbp1 in adult retinal Mϋller glia does not lead to any detectable level of neuronal conversion. Only a few changes in gene expression are observed in Mϋller glia following Ptbp1 deletion, and glial identity is maintained. These findings highlight the importance of using genetic manipulation and lineage-tracing methods in studying cell-type conversion.
| S-EPMC9619396 | biostudies-literature
Project description:Reprogramming glial cells to convert them into neurons represents a potential therapeutic strategy that could repair damaged neural circuits and restore function. Recent studies show that downregulation of the RNA-binding protein PTBP1 leads to one-step conversion of Müller glia (MG) into retinal ganglion cells (RGCs) with a high efficiency. However, the original study did not perform fate-mapping experiments to confirm MG-to-RGC conversion after Ptbp1 downregulation. To address the fundamental question of whether Ptbp1 downregulation can convert MG into RGCs in the mouse retina, we perform fate-mapping experiments to lineage trace MG independent of the adeno-associated virus (AAV)-mediated labeling system. Here, we report that Ptbp1 downregulation by CRISPR-CasRx or small hairpin RNA is insufficient to convert MG to RGCs. The original conclusion of MG-to-RGC conversion is due to leaky labeling of endogenous RGCs. Our results emphasize the importance of using stringent fate mapping to determine glia-to-neuron conversion in cell reprogramming research.
| S-EPMC9371382 | biostudies-literature
Project description:The RNA-binding protein Ptbp1 has been proposed as a master regulator of neuronal fate, repressing neurogenesis through its effects on alternative splicing and miRNA maturation. While prior studies using RNA interference suggested that Ptbp1 loss promotes neurogenesis, recent genetic studies have failed to replicate glia-to-neuron conversion following Ptbp1 loss of function. To evaluate the role of Ptbp1 in developmental neurogenesis in vivo, we conditionally disrupted Ptbp1 in retinal progenitors. Ptbp1 was robustly expressed in both retinal progenitors and Müller glia but absent from postmitotic neurons, and efficient loss of function in mutant animals was confirmed using immunostaining for Ptbp1. Furthermore, bulk RNA-Seq at E16 revealed accelerated expression of late-stage progenitor and photoreceptor-specific genes and altered splicing patterns in Ptbp1 mutants, including increased inclusion of neuron- and retina-specific exons. However, we observed no defects in retinal lamination, progenitor proliferation, or cell fate specification in mature retina. ScRNA-Seq of mature mutant retinas revealed only modest transcriptional changes limited to Müller glia, recapitulating alterations seen following selective deletion of Ptbp1 in mature glia. Our findings demonstrate that Ptbp1 is fully dispensable for retinal development and suggest that its proposed role as a central repressor of neurogenesis should be reevaluated
2025-07-16 | GSE300607 | GEO
Project description:The RNA-binding protein Ptbp1 has been proposed as a master regulator of neuronal fate, repressing neurogenesis through its effects on alternative splicing and miRNA maturation. While prior studies using RNA interference suggested that Ptbp1 loss promotes neurogenesis, recent genetic studies have failed to replicate glia-to-neuron conversion following Ptbp1 loss of function. To evaluate the role of Ptbp1 in developmental neurogenesis in vivo, we conditionally disrupted Ptbp1 in retinal progenitors. Ptbp1 was robustly expressed in both retinal progenitors and Müller glia but absent from postmitotic neurons, and efficient loss of function in mutant animals was confirmed using immunostaining for Ptbp1. Furthermore, bulk RNA-Seq at E16 revealed accelerated expression of late-stage progenitor and photoreceptor-specific genes and altered splicing patterns in Ptbp1 mutants, including increased inclusion of neuron- and retina-specific exons. However, we observed no defects in retinal lamination, progenitor proliferation, or cell fate specification in mature retina. ScRNA-Seq of mature mutant retinas revealed only modest transcriptional changes limited to Müller glia, recapitulating alterations seen following selective deletion of Ptbp1 in mature glia. Our findings demonstrate that Ptbp1 is fully dispensable for retinal development and suggest that its proposed role as a central repressor of neurogenesis should be reevaluated
2025-07-16 | GSE300588 | GEO
Project description:Astrocyte-to-neuron conversion has developed into a promising avenue for neuronal replacement therapy. Neurons depend critically on mitochondria function and often die by ferroptosis during the conversion process. Here we examined the extent of adequate mitochondrial reprogramming by morphology and proteome analysis. While mitochondria profoundly changed their morphology during Neurogenin2 (Neurog2) – or Achaete-scute homolog 1 (Ascl1)-mediated astrocyte-to-neuron reprogramming, we found neuron-specific mitochondrial proteins, here identified in a comprehensive proteome analysis of isolated mitochondria from primary neurons and astrocytes, to be only partially and at late stages regulated during the process. To improve this, we used dCas9 technology to induce neuron-specific mitochondrial proteins early during reprogramming. This resulted not only in increased conversion efficiency, but also in faster neuronal generation. Taken together, reprogramming mitochondria in a cell type-specific manner has powerful effects on astrocyte-to-neuron conversion, suggesting mitochondria to be a driving force in this process.
2021-03-29 | PXD014886 | Pride
Project description:Neuronal reprogramming of astrocytes poses a promising therapeutic approach to replace degenerating cells with newly converted neurons. Recent studies reported depletion of polyprimidine tract binding protein PTBP1, a repressor of neuronal alternative splicing, could induce astrocyte to neuron conversion and rescue functional deficits in models of neurodegenerative disease, while others disputed the astrocytic origin of converted neurons. Mechanistic understanding of the conversion, or the lack thereof, requires investigating the transcriptomic effects of Ptbp1 loss of function in mature astrocytes on RNA splicing, in conjunction with lineage tracing, which have not yet been examined. Here, we used genetic depletion of Ptbp1 in adult Aldh1l1-Cre/ERT2 reporter mice to determine whether lineage traced Ptbp1 knockout astrocytes exhibited RNA splicing alterations underlying potential neuronal transdifferentiation. Our analysis employed an alternative genetic Ptbp1 knockout mouse model and an extended 12-week window of Ptbp1 knockout to match previous reports of positive conversion. We found no widespread induction of neuronal identity, despite detecting a minuscule fraction of Cre-positive cells with neuronal transcriptomic features following Ptbp1 deletion. Importantly, PTBP1 depletion in mature astrocytes induces splicing alternations unlike neuronal features of alternative splicing. These findings highlight the challenge of targeting astrocytes for neuronal conversion via Ptbp1 knockout and underscore the importance of cellular gene expression profiling and genetic lineage tracing for in vivo studies of cell type conversion.
2025-08-06 | GSE294763 | GEO
Project description:Neuronal reprogramming of astrocytes poses a promising therapeutic approach to replace degenerating cells with newly converted neurons. Recent studies reported depletion of polyprimidine tract binding protein PTBP1, a repressor of neuronal alternative splicing, could induce astrocyte to neuron conversion and rescue functional deficits in models of neurodegenerative disease, while others disputed the astrocytic origin of converted neurons. Mechanistic understanding of the conversion, or the lack thereof, requires investigating the transcriptomic effects of Ptbp1 loss of function in mature astrocytes on RNA splicing, in conjunction with lineage tracing, which have not yet been examined. Here, we used genetic depletion of Ptbp1 in adult Aldh1l1-Cre/ERT2 reporter mice to determine whether lineage traced Ptbp1 knockout astrocytes exhibited RNA splicing alterations underlying potential neuronal transdifferentiation. Our analysis employed an alternative genetic Ptbp1 knockout mouse model and an extended 12-week window of Ptbp1 knockout to match previous reports of positive conversion. We found no widespread induction of neuronal identity, despite detecting a minuscule fraction of Cre-positive cells with neuronal transcriptomic features following Ptbp1 deletion. Importantly, PTBP1 depletion in mature astrocytes induces splicing alternations unlike neuronal features of alternative splicing. These findings highlight the challenge of targeting astrocytes for neuronal conversion via Ptbp1 knockout and underscore the importance of cellular gene expression profiling and genetic lineage tracing for in vivo studies of cell type conversion.
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Project description:Loss of neurons in the neural retina is a leading cause of vision loss. Retinal regeneration in zebrafish is attributed to Müller glia, which are activated, adopt a stem cell-like state, proliferate, and generate new neurons. Despite the same endogenous Müller glia being present in all vertebrates including humans, Müller glia activation in mammals leads to glial scarring instead of neurogenesis. Therefore, understanding Müller glia cellular states and molecular processes during zebrafish regeneration may allow us to improve mammalian regeneration. Since only a subset of Müller glia activate following neuron loss, we asked whether a specialised subset of Müller glia might be genetically primed for activation. Single-cell RNA sequencing (sc-RNAseq) from integrated zebrafish Müller glia samples reveals heterogeneity of gene expression across the quiescent Müller glia pool with 4 main clusters emerging, two of which are genetically similar. Labelling for key differentially expressed markers identified that these main clusters correlated with glia spatially distributed across dorsal, central and ventral retina. Using a genetically driven, chemically induced nitroreductase approach, three distinct photoreceptor types were ablated: the long (Lws2), short wavelength-sensitive (Sws2) and rod (Xops) photoreceptors, which also differ in their abundance. As expected, histological analysis of the three injuries and sc-RNAseq data for the two cone photoreceptor ablations identified common genetic program involved in the transition of Müller glia quiescence to activation, and differential responses to injury related to either injury extent or subtype of photoreceptor targeted. Interestingly, despite differences in the distributed across the retina, biased activation of Müller glia was observed in dorsal and central regions. Gene ontology analysis revealed that these injury-responsive dorsal and central Müller glia express genes related to dorsal/ventral pattern formation, growth factor activity, and regulation of developmental process. Müller glia upregulated genes related to homeostasis as well as certain AP-1 and injury-responsive transcription factors, followed by expression of genes involved in cell cycle, chromatin remodeling, and microtubule organisation. Prior to cell cycle entry, Müller glia show a transient upregulation of genes involved in gliogenesis, and Notch signalling. These findings enhance our understanding of heterogeneous states of quiescent Müller glia, and how these differences relate to activation and regeneration potential following neural ablation. A comparison of the distinct quiescent Müller glia with glia states in mammals will reveal key molecular pathways that could be targeted for improved mammalian neural regeneration.
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