​​​​"Contending" "glia-to-neuron" research needs to be cautious, lineage tracing is indispensable-denying that knocking out Ptbp1 can induce glial cells to transdifferentiate into neurons

Written | There are many types of neurodegenerative diseases, but they have the same feature-functional neuron loss
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In recent years, cell reprogramming has been booming, and the transformation of resident glial cells into functional neurons (glia-to-neuron) in the body has become a very promising strategy for nerve regeneration
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Therefore, scientists have spent tremendous efforts to explore how to transdifferentiate endogenous glial cells into functional neurons in order to compensate for those damaged or lost neuron functions in the disease
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There are many strategies to realize glia-to-neuron: overexpression of some genes that can promote the conversion of glial cells into neurons by viral vectors in glial cells, including some transcription factors and miRNAs [1-2], or through genes or small Molecular-mediated methods modify the extracellular signaling pathway [3-4]
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The success rates of these methods are uneven: some only work in vitro or in immature glia, and some can only achieve partial reprogramming or cannot induce the required neuron types
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These methods also have an obvious flaw, that is, there is no direct evidence showing the lineage (lineage) relationship between reprogrammed glial cells and newborn neurons
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The major progress in the field of Glia-to-neuron is the reliable and efficient realization of in vivo glial reprogramming by manipulating a single factor
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The most representative of these is to achieve "in vivo glia-to-neuron transdifferentiation" by overexpression of NeuroD1 or knockdown of Ptbp1 by AAV vector
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These induced neurons are very similar to endogenous neurons in terms of morphology, electrophysiological characteristics, and tissue arrangement
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Using this strategy in mice can effectively improve the behavioral or pathological defects in diseases such as stroke, Parkinson's syndrome, and Huntington's disease, and is expected to be applied to the treatment of human neurological diseases
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In the face of such a major research breakthrough, scientists have not been dizzy, and there are still teams thinking about whether these near-perfect neurons are really derived from glial cell transdifferentiation
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They believe that this simple and elegant method of loss of function can indeed solve many of the difficulties associated with previous directed glial reprogramming, such as low reprogramming efficiency and over-expression vectors that are too complex
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However, there are still several major problems to be solved: First, the current data cannot directly prove that the expression of Ptbp1 in situ in glial cells is reduced; second, the previous studies mainly inferred from the AAV vector or transgene of the GFAP promoter The lineage relationship between glial cells and neurons, but this method is not rigorous, because studies have shown that in some cases expression vectors based on the GFAP promoter will also be expressed in neurons [5]; Third, there is a lack of reliable genetic lineage analysis and/or scRNA-Seq-based trajectory analysis to directly prove the transdifferentiation of glial to neuron
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Therefore, these issues must be clearly explained before applying the method of Ptbp1 knockdown to induce neurons in the clinic
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However, tomorrow and accident, you never know which one will come first
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When everyone is eagerly looking forward to the application of Ptbp1 related reprogramming strategies to the treatment of human neurological diseases, Zhang Chunli's team from the University of Texas Southwestern Medical Center and Seth Blackshaw's team from the Johns Hopkins University School of Medicine in the United States have successively published articles on NeuroD1 or Ptbp1 Related glia-to-neuron transdifferentiation studies have raised questions
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Zhang Chunli’s team published a research paper titled Revising astrocyte to neuron conversion with lineage tracing in vivo on September 27, 2021 (see Bioart report: CellChallenge the entire field! Zhang Chunli’s team uses the pedigree tracing method to re-examine the small Transdifferentiation of glial cells into neurons in mice)
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The study systematically proved that overexpression of NeuroD1 or knockdown of Ptbp1 did not transform astrocytes into neurons in vivo using lineage tracing methods
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Those so-called "newborn neurons" are just in situ neurons in the brain itself
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Subsequently, on October 5, 2021, the Seth Blackshaw team published an online article titled Ptbp1 deletion does not induce glia-to-neuron conversion in adult mouse retina and brain on BioRxiv (this report will interpret this article in detail)
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The researchers used a strict lineage tracing system combined with scRNA-seq and electrophysiological findings to specifically knock down or knock out Ptbp1 in retinal Mϋller glia or brain astrocytes.
The phenomenon of glia-to-neuron transdifferentiation cannot be observed
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First, the authors evaluated the function of Ptbp1 knockout in retinal cells
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In order to simultaneously interfere with Ptbp1 and visually label these cells in Mϋller glia in the retina, the authors used three transgenic mouse strains: GlastCreERT2, treated with tamoxifen, and selectively induced Cre-dependent recombination in Mϋller glia; Sun1-GFPlox/ After activation of lox, Cre, GFP targeted to the nuclear membrane is expressed under the drive of the CAG promoter; Ptbp1 lox/lox, loxP sites are flanked by the Ptbp1 promoter and the first exon coding sequence.
After Cre is activated, it will disturb Its transcription
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Three genotype mice were produced by mating for subsequent experiments (Figure 1A): wild type (GlastCreERT2; Sun1-GFPlox/lox; Ptbp1+/+), Ptbp1 heterozygous knockout (GlastCreERT2; Sun1-GFPlox/lox; Ptbp1lox/ +), Ptbp1 homozygous knockout (GlastCreERT2; Sun1-GFPlox/lox; Ptbp1lox/lox)
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In the retinal cells of adult wild-type mice, PTBP1 protein is mainly enriched in Mϋller glia
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In Ptbp1 homozygous knockout mice, the number of PTBP1-positive Müller glia was reduced by about 90% (Figure 1B-C), but the number of GFP-positive Müller glia did not change significantly, indicating that they did not generate retinal ganglion cells or light.
Receptor
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To confirm this result, the authors tested the ganglion cell-specific markers RBPMS and BRN3B
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After Ptbp1 was knocked out, RBPMS and BRN3B did not have any co-localization with GFP (Figure 1D)
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Similarly, there is no co-localization phenomenon between GFP and cone neuron marker arrestin or photoreceptor marker OTX2 (Figure 1E)
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Previous studies have also shown that knocking out Ptbp1 will make Mϋller glia transdifferentiate into retinal ganglion cells, which can effectively alleviate visual defects caused by NMDA excitotoxic injury [6]
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The authors found that although the loss of RBPMS-positive retinal ganglion cells increased after NMDA treatment, no GFP/RBPMS or GFP/BRN3B-positive cells or recovery of retinal ganglion cells were observed
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These results indicate that after Ptbp1 deletion, Mϋller glia cannot be converted into retinal neurons in both normal and damaged retinas
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Figure 1.
Knockout of Ptbp1 cannot induce the transdifferentiation of Mϋller glia into neurons in the retina.
Subsequently, the author conducted a related investigation in the brain astrocytes (Astrocytes)
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Three mouse strains were used in the experiment: tamoxifen inducible Aldh1l1CreERT2 mice, Sun1-GFPlox/lox and Ptbp1lox/lox mice
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Through mating, wild-type, Ptbp1 knockout mice that are heterozygous or homozygous in astrocytes were obtained
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Previous studies have found that knocking down Ptbp1 in the cortex, striatum, and substantia nigra can effectively convert Astrocytes into neurons [6-7], so the author mainly focused on these three brain regions for follow-up analysis
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No co-localization of GFP with neuronal markers NeuN and HuC/D was observed in Ptbp1 deletion mice
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Through whole-cell patch clamp experiments, the authors found that in the cortex of Ptbp1 heterozygous or homozygous knockout mice, GFP-positive Astrocytes do not generate action potentials, on the contrary, adjacent GFP-negative wild-type neurons have the ability to generate action potentials.
It shows that the physiological characteristics of Astrocytes have not changed after the deletion of Ptbp1
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These experimental results indicate that the knockout of Ptbp1 in Astrocytes cannot cause any specific neuron-like electrophysiological changes
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Finally, because previous studies have not analyzed the gene expression profile after Ptbp1 knockdown in glial cells
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In order to fully analyze the cell phenotype caused by knocking out Ptbp1 in Müller glia and Astrocytes, the authors used scRNA-Seq to analyze the retina, cortex, striatum and substantia nigra of wild-type, heterozygous and homozygous mice.
Gene expression profile
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In all tissues, the expression level of Ptbp1 decreased, but no significant changes in gene expression were observed in the glial cell population, and no significant decrease in Müller glia-specific marker genes was observed (including Sox9, Glul, Rlbp1, Slc1a3, Apoe, Aqp4, Mlc1, Kcnj10 and Tcf7l2), or induce any specific marker genes for neural cells or mature neurons
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Immunostaining proved that Müller glia lacking Ptbp1 still expresses the glial cell marker gene SOX9
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Similar to Müller glia, after knocking out Ptbp1 in Astrocytes, only very subtle changes in gene expression profiles were observed
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Up-regulated genes include mt-Nd4, Son, Hes5, and Mt3; down-regulated genes include mt-Nd3, Lars2, lvd
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Most of the marker genes of Astrocytes did not change significantly after Ptbp1 was knocked out
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Immunostaining confirmed that GFP-positive Astrocytes lacking Ptbp1 still express the glial cell marker gene SOX9, and no expression of any neuronal cell or mature neuron-specific genes was detected.

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In general, the study used genetic deletion and cell lineage analysis, combined with scRNA-Seq, and the authors did not obtain any data in the retina and brain tissue to confirm that partial or complete knockout of Ptbp1 can induce glial cells to transdifferentiate into nerves.
Yuan
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These data are completely contrary to the previous research conclusions of using ASO, shRNA and/or CasRx to knock down Ptbp1 [5-7], and are consistent with the research conclusions of Zhang Chunli's team
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Once again, it is proved that the previous report on glia-to-neuron transdifferentiation is unlikely to be caused by the loss of Ptbp1 function in glial cells
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So, what is the reason for the restoration of visual function after NMDA excitotoxicity causes damage? What is the reason for the recovery of behavioral defects in a mouse model of Parkinson's disease? The author believes that one possibility is the ectopic expression of GFAP-based agents in endogenous neurons; the other possibility is the off-target effect of agents against Ptbp1 in previous studies
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The author emphasizes that no matter what the situation is, strict pedigree tracking should be used to verify relevant conclusions in future glia-to-neuron studies
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Original link: https://doi.
org/10.
1101/2021.
10.
04.
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[6] Zhou, Haibo, Jinlin Su, Xinde Hu, et al.
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[7] Qian, Hao, Xinjiang Kang, Jing Hu, et al.
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” Nature 582(7813):550-556.
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" Nature 582(7813):550-556.
Instructions for reprinting [Original Articles] BioArt original articles, personal forwarding and sharing are welcome, reprinting without permission is prohibited, all published The copyright of the work is owned by BioArt"Reversing a Model of Parkinson's Disease with in Situ Converted Nigral Neurons.
" Nature 582(7813):550-556.
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