01 Jul 2022

Nixing the expression of a single protein, PTBP1, converts astrocytes into fresh neurons within the brain. This landmark finding, reported in 2020, may have been too good to be true. A recent spate of papers report failed attempts at replicating the apparent conversion, which previously had been claimed for glial cells in the retina as well as in the substantia nigra and striatum.

Using different fate-mapping approaches to tag astrocytes prior to PTBP1 knockdown, the new studies found that nary a glial cell made the desired switch. The studies did find leaky expression of the knockdown construct via the GFAP promotor in neurons, which could explain why it had appeared that these neurons were astrocytic converts. The findings cast a pall on what some had considered a promising therapeutic strategy for Parkinson’s, vision disorders, and other neurodegenerative diseases.

In 2013, researchers led by Xiang-Dong Fu at the University of California, San Diego, reported that turning down expression of pyrimidine-tract-binding (PTB) triggered fibroblasts to transform into neurons (Jan 2013 news; Xue et al., 2013).

Scientists later extended those findings to astrocytes within the mouse brain. The UCSD group did so by injecting an adeno-associated virus (AAV) carrying a short hairpin RNA trained against PTBP1 directly into the mouse substantia nigra. To label infected astrocytes, the vector they used included a fluorescent tag that could only be activated by Cre recombinase, which, in this case, was expressed in the infected mice under control of the GFAP promoter. The researchers reported that fluorescent cells bearing neuronal markers formed connections with the nearby striatum, and reversed motor deficits in a PD model (Jul 2019 conference news; Jun 2020 news). Later, they replicated findings with an antisense oligonucleotide-based approach (Maimon et al., 2021).

Another group used a CRISPR-based approach, which also relied on an AAV vector and the GFAP promotor to tag infected glial cells—to reportedly convert Müller glia in the retina into retinal ganglion cells (Zhou et al., 2020). Other iterations of these approaches have also been reported (Fu et al., 2020). 

Alas, several recent studies suggest that the supposed converts may have been neurons all along. The studies used different fate-mapping approaches to tag astrocytes independently from the mouse GFAP promotor or AAV infection. Two of the studies, published in Cell Reports on June 14, reported that Müller glia in the retina did not convert to neurons, even when PTBP1 was efficiently knocked down (Xie et al., 2022; Hoang et al., 2022). Two others—one published in Life on May 10 and the other posted on bioRxiv on May 13—came to similar conclusions with astrocytes in the substantia nigra and striatum (Chen et al., 2022; Yang et al., 2022). Their findings jibe with a similar report published last year (Wang et al., 2021). Some also found leaky expression from the GFAP promotor in neurons, leading to the mistaken impression that they were former astrocytes.

In response to these and additional studies that question the astrocyte-to-neuron conversion data, Fu acknowledged that some leaky expression from the GFAP promotor occurs in neurons. Indeed, he reported that about 5 percent of cells expressing the AAV genes shortly after infection were neurons. However, Fu told Alzforum that lineage-tracing experiments performed in some studies may have preferentially labeled mature astrocytes, leaving open the possibility that conversion of more immature astrocytes to neurons may have been missed. A careful examination of astrocyte subtypes—and how their characteristics change following PTBP1 knockdown—could address this issue, he said.

Finally, PTBP1 knockdown restored dopamine levels and boosted motor function in a PD mouse model, Fu pointed out. If not by the creation of new neurons, what could explain these benefits? Fu said that in addition to producing fresh neurons, multiple possibilities remain on the table, including enhanced function of endogenous neurons and reduced neurotoxicity in astrocytes in response to PTBP1 downregulation. His group and others are investigating these additional mechanisms.—Jessica Shugart

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References

News Citations

1. Stem Cells: Simpler to Make, Easier on the Immune System 17 Jan 2013
2. Dopaminergic Neurons Conjured from Astrocytes Restore Motion 5 Jul 2019
3. Paper Alert: Astrocytes Convert to Neurons (of the Dopaminergic Persuasion) 24 Jun 2020

Paper Citations

1. Xue Y, Ouyang K, Huang J, Zhou Y, Ouyang H, Li H, Wang G, Wu Q, Wei C, Bi Y, Jiang L, Cai Z, Sun H, Zhang K, Zhang Y, Chen J, Fu XD. Direct Conversion of Fibroblasts to Neurons by Reprogramming PTB-Regulated MicroRNA Circuits. Cell. 2013 Jan 9; PubMed.
2. Maimon R, Chillon-Marinas C, Snethlage CE, Singhal SM, McAlonis-Downes M, Ling K, Rigo F, Bennett CF, Da Cruz S, Hnasko TS, Muotri AR, Cleveland DW. Therapeutically viable generation of neurons with antisense oligonucleotide suppression of PTB. Nat Neurosci. 2021 Aug;24(8):1089-1099. Epub 2021 Jun 3 PubMed.
3. Zhou H, Su J, Hu X, Zhou C, Li H, Chen Z, Xiao Q, Wang B, Wu W, Sun Y, Zhou Y, Tang C, Liu F, Wang L, Feng C, Liu M, Li S, Zhang Y, Xu H, Yao H, Shi L, Yang H. Glia-to-Neuron Conversion by CRISPR-CasRx Alleviates Symptoms of Neurological Disease in Mice. Cell. 2020 Apr 30;181(3):590-603.e16. Epub 2020 Apr 8 PubMed.
4. Fu X, Zhu J, Duan Y, Li G, Cai H, Zheng L, Qian H, Zhang C, Jin Z, Fu XD, Zhang K. Visual function restoration in genetically blind mice via endogenous cellular reprogramming. bioRxiv. April 08, 2020
5. Xie Y, Zhou J, Chen B. Critical examination of Ptbp1-mediated glia-to-neuron conversion in the mouse retina. Cell Rep. 2022 Jun 14;39(11):110960. PubMed.
6. Hoang T, Kim DW, Appel H, Pannullo NA, Leavey P, Ozawa M, Zheng S, Yu M, Peachey NS, Blackshaw S. Genetic loss of function of Ptbp1 does not induce glia-to-neuron conversion in retina. Cell Rep. 2022 Jun 14;39(11):110849. PubMed.
7. Chen W, Zheng Q, Huang Q, Ma S, Li M. Repressing PTBP1 fails to convert reactive astrocytes to dopaminergic neurons in a 6-hydroxydopamine mouse model of Parkinson's disease. Elife. 2022 May 10;11 PubMed.
8. Yang G, Yan Z, Wu X, Zhang M, Xu C, Shi L, Yang H, Fang K. Ptbp1 knockdown in mouse striatum did not induce astrocyte-to-neuron conversion using HA-tagged labeling system. BioRxiv, May 13, 2022.
9. Wang LL, Serrano C, Zhong X, Ma S, Zou Y, Zhang CL. Revisiting astrocyte to neuron conversion with lineage tracing in vivo. Cell. 2021 Oct 14;184(21):5465-5481.e16. Epub 2021 Sep 27 PubMed.

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