Limited astrocyte-to-neuron conversion in the mouse brain using NeuroD1 overexpression

Abstract

The central nervous system (CNS) is largely unable to generate new neurons to compensate for the loss of neurons caused by disease or injury. One potentially promising approach to replace CNS neurons is to convert resident astrocytes into neurons in situ by gene therapy. Conversion of astrocytes to neurons in vitro by overexpression of the transcription factor Pax6 was first reported nearly 20 years ago.1Heins N. Malatesta P. Cecconi F. Nakafuku M. Tucker K.L. Hack M.A. Chapouton P. Barde Y.A. Götz M. Glial cells generate neurons: the role of the transcription factor Pax6.Nat. Neurosci. 2002; 5: 308-315https://doi.org/10.1038/nn828Google Scholar Since then, multiple groups have reported astrocyte-to-neuron conversion in vitro and in vivo following various genetic manipulations.2Buffo A. Vosko M.R. Ertürk D. Hamann G.F. Jucker M. Rowitch D. Götz M. Expression pattern of the transcription factor Olig2 in response to brain injuries: implications for neuronal repair.Proc. Natl. Acad. Sci. U S A. 2005; 102: 18183-18188https://doi.org/10.1073/pnas.0506535102Google Scholar, 3Berninger B. Costa M.R. Koch U. Schroeder T. Sutor B. Grothe B. Götz M. Functional properties of neurons derived from in vitro reprogrammed postnatal astroglia.J. Neurosci. 2007; 27: 8654-8664https://doi.org/10.1523/JNEUROSCI.1615-07.2007Google Scholar, 4Heinrich C. Blum R. Gascón S. Masserdotti G. Tripathi P. Sánchez R. Tiedt S. Schroeder T. Götz M. Berninger B. Directing astroglia from the cerebral cortex into subtype specific functional neurons.PLoS Biol. 2010; 8: e1000373https://doi.org/10.1371/journal.pbio.1000373Google Scholar, 5Grande A. Sumiyoshi K. López-Juárez A. Howard J. Sakthivel B. Aronow B. Campbell K. Nakafuku M. Environmental impact on direct neuronal reprogramming in vivo in the adult brain.Nat. Commun. 2013; 4: 2373https://doi.org/10.1038/ncomms3373Google Scholar, 6Niu W. Zang T. Zou Y. Fang S. Smith D.K. Bachoo R. Zhang C.L. In vivo reprogramming of astrocytes to neuroblasts in the adult brain.Nat. Cell Biol. 2013; 15: 1164-1175https://doi.org/10.1038/ncb2843Google Scholar, 7Torper O. Pfisterer U. Wolf D.A. Pereira M. Lau S. Jakobsson J. Björklund A. Grealish S. Parmar M. Generation of induced neurons via direct conversion in vivo.Proc. Natl. Acad. Sci. U S A. 2013; 110: 7038-7043https://doi.org/10.1073/pnas.1303829110Google Scholar, 8Liu Y. Miao Q. Yuan J. Han S. Zhang P. Li S. Rao Z. Zhao W. Ye Q. Geng J. Zhang X. Cheng L. Ascl1 converts dorsal midbrain astrocytes into functional neurons in vivo.J. Neurosci. 2015; 35: 9336-9355https://doi.org/10.1523/JNEUROSCI.3975-14.2015Google Scholar, 9Aravantinou-Fatorou K. Ortega F. Chroni-Tzartou D. Antoniou N. Poulopoulou C. Politis P.K. Berninger B. Matsas R. Thomaidou D. CEND1 and NEUROGENIN2 reprogram mouse astrocytes and embryonic fibroblasts to induced neural precursors and differentiated neurons.Stem Cell Rep. 2015; 5: 405-418https://doi.org/10.1016/j.stemcr.2015.07.012Google Scholar, 10Berninger B. Jessberger S. Engineering of adult neurogenesis and Gliogenesis.Cold Spring Harb. Perspect. Biol. 2016; 8https://doi.org/10.1101/cshperspect.a018861Google Scholar, 11Lei W. Li W. Ge L. Chen G. Non-engineered and engineered adult neurogenesis in mammalian brains.Front. Neurosci. 2019; 13: 131https://doi.org/10.3389/fnins.2019.00131Google Scholar Recently, overexpression of NeuroD112Guo Z. Zhang L. Wu Z. Chen Y. Wang F. Chen G. In vivo direct reprogramming of reactive glial cells into functional neurons after brain injury and in an Alzheimer's disease model.Cell Stem Cell. 2014; 14: 188-202https://doi.org/10.1016/j.stem.2013.12.001Google Scholar, 13Chen Y.C. Ma N.X. Pei Z.F. Wu Z. Do-Monte F.H. Keefe S. Yellin E. Chen M.S. Yin J.C. Lee G. et al.A NeuroD1 AAV-based gene therapy for functional brain repair after ischemic injury through in vivo astrocyte-to-neuron conversion.Mol. Ther. 2020; 28: 217-234https://doi.org/10.1016/j.ymthe.2019.09.003Google Scholar, 14Wu Z. Parry M. Hou X.Y. Liu M.H. Wang H. Cain R. Pei Z.F. Chen Y.C. Guo Z.Y. et al.Gene therapy conversion of striatal astrocytes into GABAergic neurons in mouse models of Huntington's disease.Nat. Commun. 2020; 11: 1105https://doi.org/10.1038/s41467-020-14855-3Google Scholar, 15Ge L.J. Yang F.H. Li W. Wang T. Lin Y. Feng J. Chen N.H. Jiang M. Wang J.H. Hu X.T. et al.In vivo neuroregeneration to treat ischemic Stroke through NeuroD1 AAV-based gene therapy in adult non-human primates.Front. Cell Dev. Biol. 2020; 8: 590008https://doi.org/10.3389/fcell.2020.590008Google Scholar, 16Puls B. Ding Y. Zhang F. Pan M. Lei Z. Pei Z. Jiang M. Bai Y. Forsyth C. Metzger M. et al.Regeneration of functional neurons after spinal cord injury via in situ NeuroD1-mediated astrocyte-to-neuron conversion.Front. Cell Dev. Biol. 2020; 8: 591883https://doi.org/10.3389/fcell.2020.591883Google Scholar, 17Tang Y. Wu Q. Gao M. Ryu E. Pei Z. Kissinger S.T. Chen Y. Rao A.K. Xiang Z. Wang T. et al.Restoration of visual function and cortical connectivity after ischemic injury through NeuroD1-mediated gene therapy.Front. Cell Dev. Biol. 2021; 9: 720078https://doi.org/10.3389/fcell.2021.720078Google Scholar and knockdown of the RNA splicing factor Ptbp118Qian H. Kang X. Hu J. Zhang D. Liang Z. Meng F. Zhang X. Xue Y. Maimon R. Dowdy S.F. et al.Reversing a model of Parkinson's disease with in situ converted nigral neurons.Nature. 2020; 582: 550-556https://doi.org/10.1038/s41586-020-2388-4Google Scholar,19Zhou H. Su J. Hu X. Zhou C. Li H. Chen Z. Xiao Q. Wang B. Wu W. Sun Y. et al.Glia-to-Neuron conversion by CRISPR-CasRx alleviates symptoms of neurological disease in mice.Cell. 2020; 181: 590-603.e16https://doi.org/10.1016/j.cell.2020.03.024Google Scholar were assessed for their ability to convert astrocytes to neurons in vivo in multiple CNS areas, with therapeutic effects in mouse models of disease, ischemia, and injury. However, key findings from this work were not readily reproduced,20Wang L.L. Serrano C. Zhong X. Ma S. Zou Y. Zhang C.L. Revisiting astrocyte to neuron conversion with lineage tracing in vivo.Cell. 2021; 184: 5465-5481.e16https://doi.org/10.1016/j.cell.2021.09.005Google Scholar prompting responses from the original authors21Chen G. In vivo confusion over in vivo conversion.Mol. Ther. 2021; 29: 3097-3098https://doi.org/10.1016/j.ymthe.2021.10.017Google Scholar,22Xiang Z. Xu L. Liu M. Wang Q. Li W. Lei W. Chen G. Lineage tracing of direct astrocyte-to-neuron conversion in the mouse cortex.Neural Regen. Res. 2021; 16: 750-756https://doi.org/10.4103/1673-5374.295925Google Scholar and generating controversy around astrocyte-to-neuron conversion. Here we report limited, brain-area-specific astrocyte-to-neuron conversion in mice and compare our findings with recent reports. We initially set out to test astrocyte-to-neuron conversion across the mouse CNS using systemic delivery of the blood-brain-barrier-penetrating AAV-PHP.eB capsid23Chan K.Y. Jang M.J. Yoo B.B. Greenbaum A. Ravi N. Wu W.L. Sánchez-Guardado L. Lois C. Mazmanian S.K. Deverman B.E. et al.Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.Nat. Neurosci. 2017; 20: 1172-1179https://doi.org/10.1038/nn.4593Google Scholar and a shortened human GFAP promoter (hGFAP)24Lee Y. Messing A. Su M. Brenner M. GFAP promoter elements required for region-specific and astrocyte-specific expression.Glia. 2008; 56: 481-493https://doi.org/10.1002/glia.20622Google Scholar to restrict expression to astrocytes. We achieved improved broad expression in astrocytes with self-complementary AAV genomes25McCarty D.M. Fu H. Monahan P.E. Toulson C.E. Naik P. Samulski R.J. Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo.Gene Ther. 2003; 10: 2112-2118https://doi.org/10.1038/sj.gt.3302134Google Scholar (Figures 1A–1C ; Figure S1B) and confirmed that miR-124 targeting sites (miR-124-TSs)26Taschenberger G. Tereshchenko J. Kügler S. A MicroRNA124 target sequence restores astrocyte specificity of gfaABC1D-driven transgene expression in AAV-mediated gene transfer.Mol. Ther. Nucleic Acids. 2017; 8: 13-25https://doi.org/10.1016/j.omtn.2017.03.009Google Scholar in the 3′ untranslated region (UTR) helped restrict expression to astrocytes (Figures S1A and S1B). However, the expression level was generally low, and we noted trace levels of off-target expression in neurons (Figures 1C and S1B) as well as scattered expression in the liver (data not shown). These results underscore the difficulty of targeting astrocytes specifically by systemic AAV injection. We instead decided to permanently label astrocytes with tdTomato (tdTom) in Aldh1l1CreERT2; Rosa26LSL-tdTom mice27Srinivasan R. Lu T.Y. Chai H. Xu J. Huang B.S. Golshani P. Coppola G. Khakh B.S. New transgenic mouse lines for selectively targeting astrocytes and studying calcium signals in astrocyte processes in situ and in vivo.Neuron. 2016; 92: 1181-1195https://doi.org/10.1016/j.neuron.2016.11.030Google Scholar and deliver AAV1 vectors expressing conversion factors directly into the brain parenchyma. Tamoxifen administration in these mice led to tdTom expression in the vast majority of astrocytes, with only rare expression in neurons (data not shown). To test NeuroD1-mediated conversion, we designed expression cassettes with hGFAP promoters driving NeuroD1.P2A.EGFP or EGFP alone. Both cassettes included the 3′UTR miR-124-TS. We also designed an artificial microRNA28McBride J.L. Boudreau R.L. Harper S.Q. Staber P.D. Monteys A.M. Martins I. Gilmore B.L. Burstein H. Peluso R.W. Polisky B. et al.Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi.Proc. Natl. Acad. Sci. U S A. 2008; 105: 5868-5873https://doi.org/10.1073/pnas.0801775105Google Scholar targeting Ptbp1 (miPtbp1) and confirmed its activity in vitro (Figure S1C). We then cloned miPtbp1 or a control microRNA29Boudreau R.L. Spengler R.M. Davidson B.L. Rational design of therapeutic siRNAs: minimizing off-targeting potential to improve the safety of RNAi therapy for Huntington's disease.Mol. Ther. 2011; 19: 2169-2177https://doi.org/10.1038/mt.2011.185Google Scholar (miControl) into the 3′UTR of a hGFAP-EGFP transgene. Four weeks after tamoxifen administration, we infused AAV1 vectors with each of these four transgenes unilaterally into the striatum, hippocampus, and cerebellum at 1 × 109 vector genomes (vg) per injection site (Figure 1D). We perfused the mice after 4 weeks and collected their brains for histology. We next performed immunofluorescence for the neuronal marker NeuN to identify fate-mapped tdTom-positive cells that had converted to neuron-like cells. In mice treated with NeuroD1, we observed extensive tdTom/NeuN overlap in the hippocampus and cerebellar cortex but not in the striatum (Figures 1E and 1F). In the hippocampus, viral expression was largely confined to the molecular layers of the dentate gyrus (DG) and cornu ammonis 1 (CA1), and apparent conversion was entirely confined to these regions (Figure S1D). Concerningly, conversion in the cerebellar cortex was localized to regions with a damaged granule cell layer (Figure S1D). In the hippocampus and cerebellar cortex, conversion was always within a few hundred micrometers of the needle track. However, despite the proximity to the needle track, NeuN staining in the far-red channel in converted cells was not due to autofluorescence, which was punctate and faint (data not shown). In addition, the hGFAP promoter remained active in induced NeuN-positive cells, unlike in mature neurons, because we noted continued EGFP expression (Figures 1E and 1F). In contrast to NeuroD1, we observed no evidence of astrocyte-to-neuron conversion in mice treated with the EGFP control virus (Figure S1E), miPtbp1 virus (Figure 1G; Figure S1D), or miControl virus (data not shown). Although the NeuroD1 results were encouraging, apparent astrocyte-to-neuron conversion occurred in a narrow region close to the injection site. It has been proposed that reactive astrocytes may be more readily converted into neurons than nonreactive astrocytes.30Brulet R. Matsuda T. Zhang L. Miranda C. Giacca M. Kaspar B.K. Nakashima K. Hsieh J. NEUROD1 Instructs neuronal conversion in non-reactive astrocytes.Stem Cell Rep. 2017; 8: 1506-1515https://doi.org/10.1016/j.stemcr.2017.04.013Google Scholar Therefore, we decided to test astrocyte-to-neuron conversion in Tpp1−/− mice,31Sleat D.E. Wiseman J.A. El-Banna M. Kim K.H. Mao Q. Price S. Macauley S.L. Sidman R.L. Shen M.M. Zhao Q. et al.A mouse model of classical late-infantile neuronal ceroid lipofuscinosis based on targeted disruption of the CLN2 gene results in a loss of tripeptidyl-peptidase I activity and progressive neurodegeneration.J. Neurosci. 2004; 24: 9117-9126https://doi.org/10.1523/JNEUROSCI.2729-04.2004Google Scholar a model of late infantile neuronal ceroid lipofuscinosis. These mice develop progressive astrogliosis beginning by 9 weeks of age and some neurodegeneration by the end stage at 21 weeks.32Chang M. Cooper J.D. Sleat D.E. Cheng S.H. Dodge J.C. Passini M.A. Lobel P. Davidson B.L. Intraventricular enzyme replacement improves disease phenotypes in a mouse model of late infantile neuronal ceroid lipofuscinosis.Mol. Ther. 2008; 16: 649-656https://doi.org/10.1038/mt.2008.9Google Scholar As expected, we observed elevated levels of the reactive astrocyte marker Gfap by 11 weeks of age that increased by 14 weeks (Figure S1F). We next infused the cerebral cortex, striatum, hippocampus, and cerebellum of 10-week-old Tpp1−/− mice with 1 × 109 vg of the same viruses as above (Figure 1H). To identify converted neurons, we performed NeuN immunofluorescence and identified transduced astrocytes by EGFP expression. In NeuroD1-treated Tpp1−/− mice, we detected no EGFP/NeuN overlap in the cerebral cortex (Figure 1I). In brain areas tested previously in Aldh1l1CreERT2; Rosa26LSL-tdTom mice, results were comparable in Tpp1−/− mice; we observed no NeuN/EGFP overlap in the striatum but significant overlap in the molecular layers of the hippocampus and in the cerebellar cortex (Figure 1I). As above, cerebellar regions with apparent conversion were marked by a damaged granule cell layer, and we noted particularly strong Gfap expression in these areas (Figures 1I). We again saw no evidence of astrocyte-to-neuron conversion in Tpp1−/− mice injected with EGFP control (Figure S1G) or miPtbp1 viruses (data not shown). Therefore, the astrogliosis in Tpp1−/− mice did not enhance astrocyte-to-neuron conversion compared with wild-type mice. In summary, we observed evidence of NeuroD1-mediated astrocyte-to-neuron conversion in the mouse hippocampus and cerebellar cortex but not in the cerebral cortex or striatum. Where conversion was observed, it was limited spatially to within a few hundred micrometers of the injection site. This may be due to a localized dose effect or damage and inflammation along the needle track. In addition, converted cells were likely not fully mature neurons by 4 weeks after injection because the hGFAP promoter remained highly active in these cells. We did not follow converted cells for a longer time period because of the limited scope of conversion. In contrast to NeuroD1 overexpression, delivery of an artificial microRNA targeting Ptbp1 did not lead to astrocyte-to-neuron conversion in any brain area, although we did not quantify Ptbp1 knockdown in vivo. One caveat regarding our results is the small scale of this pilot study. However, the results were consistent across multiple animals in two mouse lines, including Aldh1l1CreERT2 mice, which enable robust fate mapping of astrocytes. We also note that Aldh1l1 and Gfap, the genes on which our Cre and AAV reporter systems are based, are expressed in neural stem cells in the subgranular zone (SGZ), which normally generate granule cells in the DG granule cell layer. Thus, it is possible that neural stem cells, rather than astrocytes, gave rise to the apparent converted neurons in the hippocampus. This possibility is made less likely by the distance between the SGZ and the converted neurons, the farthest of which were across the hippocampal fissure in CA1, and the relatively short time frame of the experiments. Published results on astrocyte-to-neuron conversion using NeuroD1 overexpression vary widely.12Guo Z. Zhang L. Wu Z. Chen Y. Wang F. Chen G. In vivo direct reprogramming of reactive glial cells into functional neurons after brain injury and in an Alzheimer's disease model.Cell Stem Cell. 2014; 14: 188-202https://doi.org/10.1016/j.stem.2013.12.001Google Scholar, 13Chen Y.C. Ma N.X. Pei Z.F. Wu Z. Do-Monte F.H. Keefe S. Yellin E. Chen M.S. Yin J.C. Lee G. et al.A NeuroD1 AAV-based gene therapy for functional brain repair after ischemic injury through in vivo astrocyte-to-neuron conversion.Mol. Ther. 2020; 28: 217-234https://doi.org/10.1016/j.ymthe.2019.09.003Google Scholar, 14Wu Z. Parry M. Hou X.Y. Liu M.H. Wang H. Cain R. Pei Z.F. Chen Y.C. Guo Z.Y. et al.Gene therapy conversion of striatal astrocytes into GABAergic neurons in mouse models of Huntington's disease.Nat. Commun. 2020; 11: 1105https://doi.org/10.1038/s41467-020-14855-3Google Scholar,16Puls B. Ding Y. Zhang F. Pan M. Lei Z. Pei Z. Jiang M. Bai Y. Forsyth C. Metzger M. et al.Regeneration of functional neurons after spinal cord injury via in situ NeuroD1-mediated astrocyte-to-neuron conversion.Front. Cell Dev. Biol. 2020; 8: 591883https://doi.org/10.3389/fcell.2020.591883Google Scholar,17Tang Y. Wu Q. Gao M. Ryu E. Pei Z. Kissinger S.T. Chen Y. Rao A.K. Xiang Z. Wang T. et al.Restoration of visual function and cortical connectivity after ischemic injury through NeuroD1-mediated gene therapy.Front. Cell Dev. Biol. 2021; 9: 720078https://doi.org/10.3389/fcell.2021.720078Google Scholar,30Brulet R. Matsuda T. Zhang L. Miranda C. Giacca M. Kaspar B.K. Nakashima K. Hsieh J. NEUROD1 Instructs neuronal conversion in non-reactive astrocytes.Stem Cell Rep. 2017; 8: 1506-1515https://doi.org/10.1016/j.stemcr.2017.04.013Google Scholar In a recent publication, Wang et al.20Wang L.L. Serrano C. Zhong X. Ma S. Zou Y. Zhang C.L. Revisiting astrocyte to neuron conversion with lineage tracing in vivo.Cell. 2021; 184: 5465-5481.e16https://doi.org/10.1016/j.cell.2021.09.005Google Scholar propose that the Neurod1 DNA sequence upregulates GFAP promoter activity in neurons and that this can lead to spurious reports of astrocyte-to-neuron conversion. When they instead fate mapped astrocytes in Aldh1l1CreERT2; Rosa26LSL-tdTom mice, as we did above, they reported no NeuroD1-mediated conversion in the cerebral cortex and striatum. This is consistent with our results showing a lack of conversion in these brain areas but some conversion in the hippocampus and cerebellar cortex. However, Xian et al.22Xiang Z. Xu L. Liu M. Wang Q. Li W. Lei W. Chen G. Lineage tracing of direct astrocyte-to-neuron conversion in the mouse cortex.Neural Regen. Res. 2021; 16: 750-756https://doi.org/10.4103/1673-5374.295925Google Scholar do report conversion in the cerebral cortex and striatum in these mice. We cannot rule out the possibility of efficient conversion in the cerebral cortex and striatum with alternative viral vectors, a higher dose, or a longer time period after injection. Similar to NeuroD1-mediated conversion, widely varying efficiencies of astrocyte-to-neuron conversion by Ptbp1 knockdown have been reported.18Qian H. Kang X. Hu J. Zhang D. Liang Z. Meng F. Zhang X. Xue Y. Maimon R. Dowdy S.F. et al.Reversing a model of Parkinson's disease with in situ converted nigral neurons.Nature. 2020; 582: 550-556https://doi.org/10.1038/s41586-020-2388-4Google Scholar, 19Zhou H. Su J. Hu X. Zhou C. Li H. Chen Z. Xiao Q. Wang B. Wu W. Sun Y. et al.Glia-to-Neuron conversion by CRISPR-CasRx alleviates symptoms of neurological disease in mice.Cell. 2020; 181: 590-603.e16https://doi.org/10.1016/j.cell.2020.03.024Google Scholar, 20Wang L.L. Serrano C. Zhong X. Ma S. Zou Y. Zhang C.L. Revisiting astrocyte to neuron conversion with lineage tracing in vivo.Cell. 2021; 184: 5465-5481.e16https://doi.org/10.1016/j.cell.2021.09.005Google Scholar,33Maimon R. Chillon-Marinas C. Snethlage C.E. Singhal S.M. McAlonis-Downes M. Ling K. Rigo F. Bennett C.F. Da Cruz S. et of neurons with of Neurosci. 2021; 24: Scholar The of in vivo knockdown of Ptbp1 is to compare between these and may be to efficient conversion. we are by it is to efficient astrocyte-to-neuron conversion. work is to the results of in vivo conversion and the that conversion. Although astrocyte-to-neuron conversion may be regarding its therapeutic NeuN expression was only induced in astrocytes close to the injection site. Conversion on this scale is to have a therapeutic effect in the mouse brain and less likely in the human Therefore, the of administration and dose have to be to conversion minimizing delivery of AAVs may be an for conversion, but this likely a dose of AAVs and lead to expression in the as noted above for AAV-PHP.eB in mice. more conversion is using be to induced NeuN-positive cells develop into mature neurons that can into neural and disease Although these are it may be to astrocyte-to-neuron conversion We and for AAV vector The was by from the of and Stroke The is the of the and not the of the of the of designed the performed and and designed the artificial microRNA targeting and designed the designed and the and the with from is on the for the from for and with and to In vivo confusion over in vivo et with of our Cell in recent to the

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