Parkinson’s disease is characterized by loss of dopamine neurons in the substantia nigra1. Similar to other major neurodegenerative disorders, there are no disease-modifying treatments for Parkinson’s disease. While most treatment strategies aim to prevent neuronal loss or protect vulnerable neuronal circuits, a potential alternative is to replace lost neurons to reconstruct disrupted circuits2. Here we report an efficient one-step conversion of isolated mouse and human astrocytes to functional neurons by depleting the RNA-binding protein PTB (also known as PTBP1). Applying this approach to the mouse brain, we demonstrate progressive conversion of astrocytes to new neurons that innervate into and repopulate endogenous neural circuits. Astrocytes from different brain regions are converted to different neuronal subtypes. Using a chemically induced model of Parkinson’s disease in mouse, we show conversion of midbrain astrocytes to dopaminergic neurons, which provide axons to reconstruct the nigrostriatal circuit. Notably, re-innervation of striatum is accompanied by restoration of dopamine levels and rescue of motor deficits. A similar reversal of disease phenotype is also accomplished by converting astrocytes to neurons using antisense oligonucleotides to transiently suppress PTB. These findings identify a potentially powerful and clinically feasible approach to treating neurodegeneration by replacing lost neurons.
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RNA-seq data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE142250. Independently generated data are available upon request. Methods have been converted into stepwise protocols and deposited in Protocol Exchange (doi: 10.21203/rs.3.pex-902/v1). Repeats of individual experiments are summarized in Supplementary Table 2, which has been independently verified. All data generated or analysed in this study are included in this published article (and its Supplementary Information files).
Poewe, W. et al. Parkinson disease. Nat. Rev. Dis. Primers 3, 17013 (2017).
Barker, R. A., Götz, M. & Parmar, M. New approaches for brain repair—from rescue to reprogramming. Nature 557, 329–334 (2018).
Sonntag, K. C. et al. Pluripotent stem cell-based therapy for Parkinson’s disease: current status and future prospects. Prog. Neurobiol. 168, 1–20 (2018).
Cohen, D. E. & Melton, D. Turning straw into gold: directing cell fate for regenerative medicine. Nat. Rev. Genet. 12, 243–252 (2011).
Yu, X., Nagai, J. & Khakh, B. S. Improved tools to study astrocytes. Nat. Rev. Neurosci. 21, 121–138 (2020).
Rivetti di Val Cervo, P. et al. Induction of functional dopamine neurons from human astrocytes in vitro and mouse astrocytes in a Parkinson’s disease model. Nat. Biotechnol. 35, 444–452 (2017).
Wu, Z. et al. Gene therapy conversion of striatal astrocytes into GABAergic neurons in mouse models of Huntington’s disease. Nat. Commun. 11, 1105 (2020).
Gascón, S., Masserdotti, G., Russo, G. L. & Götz, M. Direct Neuronal Reprogramming: Achievements, Hurdles, and New Roads to Success. Cell Stem Cell 21, 18–34 (2017).
Xue, Y. et al. Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits. Cell 152, 82–96 (2013).
Xue, Y. et al. Sequential regulatory loops as key gatekeepers for neuronal reprogramming in human cells. Nat. Neurosci. 19, 807–815 (2016).
Hu, J., Qian, H., Xue, Y. & Fu, X. D. PTB/nPTB: master regulators of neuronal fate in mammals. Biophys. Rep. 4, 204–214 (2018).
Bennett, C. F., Krainer, A. R. & Cleveland, D. W. Antisense Diseases. Annu. Rev. Neurosci. 42, 385–406 (2019).
Guo, Z. et al. In vivo direct reprogramming of reactive glial cells into functional neurons after brain injury and in an Alzheimer’s disease model. Cell Stem Cell 14, 188–202 (2014).
Lu, T. et al. REST and stress resistance in ageing and Alzheimer’s disease. Nature 507, 448–454 (2014).
Li, Q. et al. The splicing regulator PTBP2 controls a program of embryonic splicing required for neuronal maturation. eLife 3, e01201 (2014).
Laywell, E. D., Rakic, P., Kukekov, V. G., Holland, E. C. & Steindler, D. A. Identification of a multipotent astrocytic stem cell in the immature and adult mouse brain. Proc. Natl Acad. Sci. USA 97, 13883–13888 (2000).
Sofroniew, M. V. Transgenic techniques for cell ablation or molecular deletion to investigate functions of astrocytes and other GFAP-expressing cell types. Methods Mol. Biol. 814, 531–544 (2012).
Tateno, T. & Robinson, H. P. The mechanism of ethanol action on midbrain dopaminergic neuron firing: a dynamic-clamp study of the role of I(h) and GABAergic synaptic integration. J. Neurophysiol. 106, 1901–1922 (2011).
Kimm, T., Khaliq, Z. M. & Bean, B. P. Differential regulation of action potential shape and burst-frequency firing by BK and Kv2 Channels in substantia nigra dopaminergic neurons. J. Neurosci. 35, 16404–16417 (2015).
Boisvert, M. M., Erikson, G. A., Shokhirev, M. N. & Allen, N. J. The aging astrocyte transcriptome from multiple regions of the mouse brain. Cell Rep. 22, 269–285 (2018).
Nott, A. et al. Brain cell type-specific enhancer-promoter interactome maps and disease-risk association. Science 366, 1134–1139 (2019).
Grealish, S. et al. Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson’s disease. Cell Stem Cell 15, 653–665 (2014).
Thiele, S. L., Warre, R. & Nash, J. E. Development of a unilaterally-lesioned 6-OHDA mouse model of Parkinson’s disease. J. Vis. Exp. 60, 3234 (2012).
Beal, M. F. Parkinson’s disease: a model dilemma. Nature 466, S8–S10 (2010).
Stott, S. R. & Barker, R. A. Time course of dopamine neuron loss and glial response in the 6-OHDA striatal mouse model of Parkinson’s disease. Eur. J. Neurosci. 39, 1042–1056 (2014).
Boix, J., Padel, T. & Paul, G. A partial lesion model of Parkinson’s disease in mice—characterization of a 6-OHDA-induced medial forebrain bundle lesion. Behav. Brain Res. 284, 196–206 (2015).
Zhu, H. & Roth, B. L. DREADD: a chemogenetic GPCR signaling platform. Int. J. Neuropsychopharmacol. 18, pyu007 (2015).
Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl Acad. Sci. USA 104, 5163–5168 (2007).
Chen, Y. et al. Chemical control of grafted human PSC-derived neurons in a mouse model of Parkinson’s disease. Cell Stem Cell 18, 817–826 (2016).
Zhou, H. et al. Glia-to-neuron conversion by CRISPR-CasRx alleviates symptoms of neurological disease in mice. Cell 181, 590-603 (2020).
Ouyang, H. et al. WNT7A and PAX6 define corneal epithelium homeostasis and pathogenesis. Nature 511, 358–361 (2014).
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12 (2011).
Srivastava, A., Malik, L., Smith, T., Sudbery, I. & Patro, R. Alevin efficiently estimates accurate gene abundances from dscRNA-seq data. Genome Biol. 20, 65 (2019).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Abercrombie, M. Estimation of nuclear population from microtome sections. Anat. Rec. 94, 239–247 (1946).
Falk, T. et al. Vascular endothelial growth factor-B is neuroprotective in an in vivo rat model of Parkinson’s disease. Neurosci. Lett. 496, 43–47 (2011).
Baker, H., Joh, T. H. & Reis, D. J. Genetic control of number of midbrain dopaminergic neurons in inbred strains of mice: relationship to size and neuronal density of the striatum. Proc. Natl Acad. Sci. USA 77, 4369–4373 (1980).
Kordower, J. H. et al. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson’s disease. Science 290, 767–773 (2000).
Bahat-Stroomza, M. et al. Induction of adult human bone marrow mesenchymal stromal cells into functional astrocyte-like cells: potential for restorative treatment in Parkinson’s disease. J. Mol. Neurosci. 39, 199–210 (2009).
Liu, G., Chen, J. & Ma, Y. Simultaneous determination of catecholamines and polyamines in PC-12 cell extracts by micellar electrokinetic capillary chromatography with ultraviolet absorbance detection. J. Chromatogr. B 805, 281–288 (2004).
De Benedetto, G. E. et al. A rapid and simple method for the determination of 3,4-dihydroxyphenylacetic acid, norepinephrine, dopamine, and serotonin in mouse brain homogenate by HPLC with fluorimetric detection. J. Pharm. Biomed. Anal. 98, 266–270 (2014).
Tareke, E., Bowyer, J. F. & Doerge, D. R. Quantification of rat brain neurotransmitters and metabolites using liquid chromatography/electrospray tandem mass spectrometry and comparison with liquid chromatography/electrochemical detection. Rapid Commun. Mass Sp. 21, 3898–3904 (2007).
Wang, S. R. et al. Role of vesicle pools in action potential pattern-dependent dopamine overflow in rat striatum in vivo. J. Neurochem. 119, 342–353 (2011).
Xu, H. et al. Striatal dopamine release in a schizophrenia mouse model measured by electrochemical amperometry in vivo. Analyst 140, 3840–3845 (2015).
Wang, C. et al. Synaptotagmin-11 is a critical mediator of parkin-linked neurotoxicity and Parkinson’s disease-like pathology. Nat. Commun. 9, 81 (2018).
Wang, L. et al. Modulation of dopamine release in the striatum by physiologically relevant levels of nicotine. Nat. Commun. 5, 3925 (2014).
Caiazzo, M. et al. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476, 224–227 (2011).
Grealish, S., Mattsson, B., Draxler, P. & Björklund, A. Characterisation of behavioural and neurodegenerative changes induced by intranigral 6-hydroxydopamine lesions in a mouse model of Parkinson’s disease. Eur. J. Neurosci. 31, 2266–2278 (2010).
Piallat, B., Benazzouz, A. & Benabid, A. L. Subthalamic nucleus lesion in rats prevents dopaminergic nigral neuron degeneration after striatal 6-OHDA injection: behavioural and immunohistochemical studies. Eur. J. Neurosci. 8, 1408–1414 (1996).
Dunnett, S. B., Björklund, A., Stenevi, U. & Iversen, S. D. Behavioural recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal pathway. I. Unilateral lesions. Brain Res. 215, 147–161 (1981).
Iancu, R., Mohapel, P., Brundin, P. & Paul, G. Behavioral characterization of a unilateral 6-OHDA-lesion model of Parkinson’s disease in mice. Behav. Brain Res. 162, 1–10 (2005).
Cohen, J. Statistical Power Analysis for the Behavioral Sciences (Academic Press, 1988).
Cohen, J. Eta-squared and partial eta-squared in fixed factor ANOVA designs. Educ. Psychol. Meas. 33, 107–112 (1973).
We thank members of the Fu laboratory for cooperation, reagent sharing and insightful discussion during the course of this investigation and A. Muotri for the gift of the human embryonic stem cell-derived neural progenitors. D.W.C. received a salary from the Ludwig Institute for Cancer Research and is a Nomis Foundation Distinguished Scientist. Z.Z. and X.K. were supported by NSFC grants (31930061, 31761133016, 21790394 and 81974203). W.C.M. and X.-D.F. were supported by a grant from the Larry Hillblom Foundation (2019-A-006-NET). This work was supported by NIH grants (GM049369 and GM052872) to X.-D.F.
X.-D.F. is a founder of CurePharma. The University of California, San Diego has filed a patent application on neuronal reprogramming induced by inactivating PTB by any means for treatment of neurological disorders.
Peer review information Nature thanks Ernest Arenas, Anders Bjorklund, Aaron D. Gitler, Malin Parmar and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Relative purity of mouse and human astrocytes. Astrocytes isolated from mouse cortex and midbrain or obtained from human embryonic brain (gestational age 19 weeks) were probed with a panel of markers for neurons and common non-neuronal cell types in the central nervous system, including those for astrocytes: GFAP (green) and ALDH1L1 (red); for neurons: TUJ1, NSE, NeuN, GAD67, VGlut1 and TH; for oligodendrocytes: OLIG2; for microglia: CD11b; for NG2 cells: NG2; for neural progenitors: nestin; for pluripotent stem cells: NANOG; and for fibroblasts: fibronectin. Scale bar, 30 μm. These results demonstrated that isolated astrocytes are largely free of neurons and common non-neuronal cells. The experiment was independently repeated twice with similar results. b, c, Levels of key components in the regulatory loops controlled by PTB and nPTB in mouse midbrain. Levels of miR-124 (b, top) and miR-9 (b, bottom) were quantified by RT–qPCR in human astrocytes (hAstrocytes), human dermal fibroblasts (HDFs), and human neurons (hNeurons) differentiated from human neuronal progenitor cells. Data were normalized against U6 snRNA and the levels in human dermal fibroblasts were set to 1 for comparative analysis. Levels of BRN2 were determined by western blotting and normalized against β-actin (c). Results show low miR-124, but high miR-9 and BRN2 in human astrocytes, suggesting that the PTB-regulated loop is inactive and components of the nPTB-regulated loop are active in human astrocytes. d, Levels of PTB, nPTB, BRN2 and REST in mouse midbrain. Cell types in mouse midbrain were marked by GFAP for astrocytes, TH for DA neurons, and fibronectin for adjacent meningeal fibroblasts and double-stained for BRN2, PTB, nPTB and REST. Scale bar, 20 μm. Relative immunofluorescence intensities in different cell types were quantified (right). n = 3 mice with a total of 54 cells counted in each. Note that REST is decreased, but not eliminated, in endogenous DA neurons, which is in agreement with the documented requirement for REST for viability of mature neurons. e, f, Dynamic nPTB expression in response to PTB knockdown. nPTB expression was monitored by western blotting after PTB knockdown in human dermal fibroblasts (e, left), mouse cortical astrocytes (e, middle) and human astrocytes (e, right). f, Data from 3 biological repeats were quantified. Results show that nPTB remains stably expressed in human dermal fibroblasts, but undergoes transient expression in astrocytes from both mice and humans. In b–d, ANOVA with post hoc Tukey test; mean ± s.e.m. (n = 3 biological repeats). P-values are indicated. All except those pairwise comparisons indicated as NS (not significant) in panels b and d are considered statistically significant.
Extended Data Fig. 2 Global evidence for programmed switch of gene expression from astrocytes to neurons in response to PTB depletion.
a, Clustering analysis. RNA-seq data (available under GSE142250) were generated on independent isolates of astrocytes from mouse cortex or midbrain before and after conversion to neurons by depleting PTB for 2 or 4 weeks. By clustering analysis, the global gene expression profiles were compared with the public datasets for astrocytes or neurons as indicated by the colour key and the data sources on the right. The selection of these public data for comparison was based on astrocytes without further culture and on neurons directly isolated from mouse brain or differentiated from embryonic stem cells (ESCs). b, c, Comparison of gene expression profiles between independent libraries prepared from mouse cortical (b) or midbrain (c) astrocytes before and after PTB depletion for 2 or 4 weeks. Selective astrocyte-specific (blue) and neuron-specific (red) genes are highlighted. Results show a degree of heterogeneity between independent isolates of astrocytes, but notably, their converted neurons became more homogeneous. d, Comparison between induced gene expression upon PTB depletion in cortical versus midbrain astrocytes. Several commonly induced DA neuron-specific genes (that is, Otx2, En1 and Aldh1a1) are highlighted when comparing between neurons derived from cortical versus midbrain astrocytes (right). Significantly induced DA neuron-associated genes are listed in Supplementary Table 1. Note that most genes are enriched, but not uniquely expressed, in DA neurons (thus, they are not specific markers for DA neurons), as evidenced by their induction to different degrees in shPTB-treated cortical astrocytes.
a, b, Conversion of mouse and human astrocytes to neurons. Cells were immunostained with the indicated markers after conversion from mouse cortical astrocytes (a) or human astrocytes (b). Converted glutamatergic (marked by VGlut1) and GABAergic (marked by GAD67) neurons constituted approximately 90% and 80% of total TUJ1-marked neurons from mouse and human astrocytes, respectively. Data were based on 4 (a) or 5 (b) biological repeats and represented as mean ± s.e.m. Scale bars, 30 μm (a); 40 μm (b). c, d, Efficient conversion from human astrocytes to neurons. Converted neurons were characterized by immunostaining with TUJ1 and MAP2 (c). Scale bar, 80 μm. n = 4 biological repeats. d, These neurons are functional as indicated by repetitive action potentials (top left), large currents of voltage-dependent sodium and potassium channels (top right) and spontaneous postsynaptic currents after co-culture with rat astrocytes (bottom). Indicated in each panel is the number of cells that showed the recorded activity versus the number of cells examined. e–h, Electrophysiological characterization of neurons converted from mouse (e) and human (f) astrocytes, showing spontaneous excitatory and inhibitory postsynaptic currents that could be sequentially blocked with the inhibitors against the excitatory (NBQX and APV) and inhibitory (PiTX) receptors, indicative of their secretion of glutamine and GABA neurotransmitters. g, h, Control shRNA (shCtrl)-treated mouse (g) and human (h) astrocytes failed to show action potentials (top), currents of voltage-dependent channels (middle) or postsynaptic events (bottom). The number of cells that showed the recorded activity versus the total number of cells examined is indicated on the top right of each panel.
a, Schematic of the substantia nigral region (white box) for AAV injection and immunochemical analysis. b, Cre-dependent RFP expression. RFP+ cells were not detected in midbrain of wild-type mice injected with either AAV-empty or AAV-shPTB (left). In comparison, both viruses generated abundant RFP signals in Gfap-cre transgenic mice. Scale bar, 150 μm. c, d, Co-staining of RFP+ cells with the astrocyte markers S100b and ALDH1L1 10 weeks after injecting AAV-empty (c), indicating that most RFP+ cells in AAV-empty-transduced midbrain were astrocytes. Scale bar, 25 μm. d, No RFP expression was detectable in NG2-labelled cells. Scale bar, 15 μm. Experiments in b–d were independently repeated three times with similar results. e, Reprogramming-dependent conversion from astrocytes to neurons. Immunostaining with the astrocyte marker GFAP and the pan-neuronal marker NeuN was performed 10 weeks after injection of AAV-empty or AAV-shPTB in the midbrain. Scale bar, 30 μm. Quantified results show that cells transduced with AAV-empty were all GFAP+ astrocytes, whereas cells transduced with AAV-shPTB were mostly NeuN+ neurons. Quantified data were based on three mice as shown on the right. Two-sided Student’s t-test. Data are mean ± s.e.m. f, g, Further characterization of AAV-shPTB-induced neurons in midbrain with additional neuronal markers, including pan-neuronal specific markers TUJ1, MAP2, NSE and PSD95 (f; scale bar, 10 μm) and specific markers for glutamatergic (VGlut2) and GABAergic (GAD65) neurons (g; scale bar, 20 μm).
Extended Data Fig. 5 Progressive conversion of AAV-shPTB treated astrocytes to DA neurons within the dopamine domain.
a, b, Time-dependent appearance of RFP+DDC+ DA neurons. AAV-shPTB-transduced midbrain was characterized for time-dependent appearance of DA neurons with the DA neuron marker DDC (a; scale bar, 50 μm). Few initial RFP+ cells were co-stained with DDC 3 weeks after AAV-shPTB transduction, and the fraction of RFP+DDC+ cells progressively increased 8 and 12 weeks after AAV-shPTB injection. Images from substantia nigra 12 weeks after AAV-shPTB transduction are enlarged to highlight RFP+DDC+ neurons (b; scale bar, 25 μm). c–e, Conversion of midbrain astroyctes to DA neurons within the dopamine domain. AAV-shPTB-induced neuronal reprogramming was determined relative to the site of injection. c, A low-magnification view of a substantia nigra section. Circles mark brain areas with progressively larger diameters from the centre of the injection site. Scale bar, 100 μm. d, Enlarged views show the representative proximal and distal sites from the injection site 12 weeks after AAV-shPTB transduction, positively stained for TH (green) over RFP-labelled cells. Scale bar, 10 μm. Note the presence of RFP+TH+ cells in the proximal site, but only RFP+TH− cells in the distal site. e, The percentages of TH+ cells among total RFP+ cells in the three different areas defined in (c) were quantified based on 3 mice with at least 100 cells counted in each. Data are mean ± s.e.m. These data show the generation of TH+ neurons within the dopamine domain of midbrain. f, g, Further characterization of converted DA neurons with additional DA neuron-specific markers DAT, VMAT2, EN1, LMX1A, PITX3 and DDC, all showing positive signals (f). RFP+TH+ cell bodies are highlighted by orthogonal views of z-stacked images, attached on right and bottom of the main image (f; scale bar, 10 μm). Cell body diameters were compared between newly converted RFP+TH+ neurons and endogenous RFP−TH+ DA neurons (g, left; scale bar, 5 μm). The size distribution of both populations of neurons shown on the right suggests that converted TH+ cells have a similar cell size to endogenous TH+RFP– DA neurons (g, right). Quantification based on 62 RFP+ cells and 64 RFP−TH+ cells from 3 mice. Two-sided Student’s t-test. h, Schematic depiction for further analysis of converted neurons in substantia nigra and ventral tegmental area. i, j, Representative immunostaining of SOX6, OTX2 and ALDH1A1, showing that SOX6-marked RFP+ cells were confined to the substantia nigra, whereas OTX2-marked RFP+ cells were in the ventral tegmental area; the DA neuron marker ALDH1A1 was detected in both substantia nigra and ventral tegmental area (i; scale bar, 25 μm). j, Quantification based on 3 mice with at least 100 cells counted. Data are mean ± s.e.m. Results further support the generation of different subtypes of DA neurons. k, Minimal leaky Cre expression in endogenous DA neurons in midbrain. As Gfap-cre is known to show a degree of leaky expression in neurons, raising a concern that AAV-shPTB might infect some endogenous DA neurons, mice treated with AAV-empty (which expresses RFP but not shPTB) were examined carefully. Scale bar, 30 μm. Compared with AAV-shPTB treated mice, few RFP+ cells stained positively for either NeuN or TH in the midbrain of mice transduced with AAV-empty, as quantified on the right, based on 3 mice with at least 100 cells counted in each. Data are mean ± s.e.m. Results show little, if any, leaky Cre expression in endogenous DA neurons and in midbrain regions of mice at the age (two months old) used in our studies.
a, b, Schematic depiction of patch recording of converted neurons in midbrain (a). According to this scheme, the fluorescent dye Neurobiotin 488 (green) loaded in the electrode was used to mark cell bodies in substantia nigra for patch clamp recording on brain slices. b, After recording, the patched cells were confirmed to be RFP+TH+ to demonstrate the recording being performed on newly converted neurons (scale bar, 20 μm). Experiments were independently repeated 4 times with similar results. c–e, Detection of spontaneous action potential (c) and relatively wider action potential generated by newly converted neurons in comparison with endogenous GABAergic neurons (d). e, Notably, hyperpolarization-activated currents of HCN channels (Ih currents) were recorded at 12 weeks after, but not 6 weeks after, AAV-shPTB-induced neuronal conversion; these currents could be specifically blocked with CsCl. The numbers of cells that showed the recorded activity versus the total number of cells examined are indicated. Note that the bottom trace is also shown in Fig. 2h. f, g, Extracellular recording showing more converted neurons firing spontaneous action potentials at 12 weeks after transduction with AAV-shPTB than at 6 weeks after transduction. The numbers of cells that showed the recorded activity versus the total number of cells examined are indicated. g, The frequency of spontaneous spikes that increased upon further maturation was further quantified. Data were based on a total of 31 cells from 4 mice. Results show progressive maturation of newly converted DA neurons in the brain. Statistical significance was determined by two-sided Student’s t-test. h, Cortical neurons generated in AAV-shPTB-transduced cortex, in contrast to a large population of RFP+TH+ cells in midbrain. As a control, AAV-shPTB was injected in cortex. After 12 weeks, RFP+ cells were co-stained with the cortical neuron marker CTIP2 (top) and CUX1 (bottom). Scale bars, 40 μm (main); 15 μm (magnified inset). Note that RFP+ CUX1+ cells are rare in comparison to RFP+CTIP2+ cells, indicative of different conversion efficiency in different layers of cortex. Experiments were independently repeated twice with similar results.
Extended Data Fig. 7 Characterization of cortical astrocyte-derived neurons compared with midbrain astrocyte-derived neurons.
a–c, A small fraction of cortical astrocyte-derived neurons express DA neuron markers. a, RT–qPCR showed the induction of DA neuron-specific genes Slc6a3 and Foxa2 in isolated cortical astrocytes treated with lentiviral shPTB. These DA-like neurons were further characterized by immunostaining for additional DA neuron markers DAT and VMAT2 (b; scale bar, 20 μm) and quantified among TUJ1+ cells based on 3 biological repeats with at least 100 cells counted in each (c). Two-sided Student’s t-test; mean ± s.e.m. P-values are indicated. Results indicate that although cortex does not contain DA neurons and RFP+TH+ DA-like neurons were never detected in AAV-shPTB-transduced cortex in the brain, isolated cortical astrocytes were able to give rise to a fraction of DA-like neurons in vitro. This implies that astrocytes may become more plastic in culture than within specific brain environments. d, Additional immunochemical evidence for the expression of DA neuron-specific markers (LMX1A, PITX3 and DDC) in a subpopulation of TUJ1+ cells derived from cortical astrocytes. Scale bar, 20 μm. Experiments were independently repeated 3 times with similar results. e–g, TH staining of TUJ1+ neurons derived from midbrain astrocytes and comparison with neurons derived from cortical astrocytes. e, Lentiviral shPTB, but not control shRNA, converted midbrain astrocytes into TH+ DA neurons in culture. Scale bar, 25 μm. f, Conversion efficiencies of cortical and midbrain astrocytes, showing similar high percentage of TUJ1+ neurons (left), but a significantly higher percentage of DA neurons converted from midbrain astrocytes compared with cortical astrocytes (right). Data are based on 3 biological repeats with at least 200 cells counted in each. Statistical significance was determined by two-sided Student’s t-test; mean ± s.e.m. P-values are indicated. g, Western blotting analysis of a pan-neuronal marker (TUJ1) and two specific markers for DA neurons (TH and VMAT2) in shPTB-reprogrammed astrocytes from cortex and midbrain, showing much higher levels of the DA neuron markers in neurons generated from midbrain astrocytes compared to cortical astrocytes. Experiments were independently repeated twice with similar results. Together, these data strongly suggest intrinsic cellular differences that are responsible for the generation of different neuron subtypes from astrocytes in different brain regions.
Extended Data Fig. 8 Cell-autonomous mechanisms for the regional specificity in neuronal conversion.
a, TH+ neurons generated from cortical astrocytes with normal and conditioned media from cultured midbrain astrocytes. Scale bar, 100 μm. b, Quantification of cells in a. Three biological repeats with at least 100 cells counted in each. Statistical significance was determined by ANOVA; mean ± s.e.m. c–f, RT–qPCR analysis of DA neuron-specific transcription factors in cortical and midbrain astrocytes before and after lentiviral shPTB-induced neuronal conversion. c, The indicated transcription factors were quantified by real-time PCR and normalized against β-actin mRNA. d, To ensure that the isolated astrocytes were free of contaminated neurons, RT–qPCR was also performed with the 3 indicated pan-neuron markers with isolated neurons as control. e, In response to PTB knockdown, astrocyte-specific genes S100b and Gfap were repressed, whereas pan-neuronal transcription factors Myt1l and Ascl1 were activated in astrocytes derived from both cortex and midbrain. Dashed lines indicate levels before shPTB treatment, which was set to 1 for comparison with levels after shPTB treatment. f, Under the same conditions, the 4 DA-neuron-specific transcription factors were more robustly induced in response to PTB depletion in midbrain astrocytes compared to cortical astrocytes. Statistical significance was determined by ANOVA with post hoc Tukey test (d) or two-sided Student’s t-test (c, e, f), based on 3 biological repeats; mean ± s.e.m. P-values are indicated. Results suggest higher basal levels and more robust induction of DA neuron-specific transcription factors in midbrain astrocytes compared to cortical astrocytes, providing evidence for the differences in cell-intrinsic gene expression programs in giving rise to distinct subtypes of neurons. g–i, Schematic of amperometric recording of monoamine release, showing the placement of a carbon fibre electrode on a midbrain astrocyte-derived neuron (g). Scale bar, 30 μm. h, Spike-like events were captured by holding the electrode at +750 mV after K+ (25 mM) stimulation. i, A high-resolution view of dopamine release events in h. Results demonstrate a key functional property of midbrain astrocyte-derived DA neurons. Experiments were independently repeated twice with similar results.
a, Schematic of coronal sections for analysing fibre density in the nigrostriatal pathway. b–d, Sphere-determined density of RFP+ fibres that were progressively increased along the nigrostriatal bundle (NSB). Shown are low-magnification views (b; scale bar, 150 μm) and enlarged views (c; scale bar, 35 μm). IC, internal capsule. d, Quantification of RFP+ (left) or RFP+TH+ fibres (right), based on 3 independent biological sections. Statistical significance was determined by ANOVA with post hoc Tukey test; mean ± s.e.m. P-values are indicated. Results show time-dependent increase in fibre density, a portion of which also exhibits colocalization of the DA neuron marker TH. e, Low-magnification view of striatum innervated by RFP+ projections. Scale bar, 300 μm. Smaller panels show magnified views of RFP+ projections in different regions. Scale bar, 15 μm. Note the bright RFP signals in septal nuclei. f, Three selected regions were further amplified to highlight a fraction of RFP+ fibres with (arrowheads) or without (arrows) co-staining with the DA neuron marker TH. Scale bar, 5 μm. Results emphasize that converted DA neurons targeted broader regions in striatum than endogenous DA neurons, which might cause side effects—a potential caveat of neuronal reprogramming experiments that requires investigation in future studies. g, h, Retrograde tracing of TH+ neurons from striatum to substantia nigra. Depicted is the AAV-shPTB injection site at day 0 and the retrobead injection site at day 30 (g). Retrograde tracing was monitored 24 h after injection of retrobeads. After treatment with AAV-shPTB for 30 days, TH+ cells, but not TH+RFP+ cells, in substantia nigra were labelled with retrograde beads (h). Arrowheads, RFP+ cells; arrows, cell bodies of endogenous TH+ DA neurons labelled with retrobeads. Scale bar, 20 μm. These data provide a critical control for AAV-shPTB-converted DA neurons that could be traced from striatum to substantia nigra, as described in the main text. All experiments shown in this figure were independently repeated 3 times with similar results.
Extended Data Fig. 10 shPTB-converted neurons replenish lost dopaminergic neurons in substantia nigra.
a, Schematic of the experimental schedule for 6-OHDA-induced lesion followed by reprogramming with AAV-PTB and then TH staining. b, c, Low-magnification views of unlesioned substantia nigra stained for TH (b) and substantia nigra lesioned with 6-OHDA and transduced with AAV-shPTB (c). Scale bars, 80 μm. These data were used to provide the quantitative information shown in Fig. 4f, g. d, Enlarged view of RFP+ cells that co-expressed TH in substantia nigra. Two RFP+TH+ cell bodies are highlighted by orthogonal views of z-stacked images, attached on the right and bottom of the main image in each panel. Scale bar, 10 μm. Results show the generation of TH+ DA neurons in a highly region-specific manner in substantia nigra, as a large population of RFP+ cells were not labelled by TH staining in the same image. All experiments shown in this figure were independently repeated 3 times with similar results.
a, b, Schematic of the coronal section of striatum and images of uninjured control and lesioned striatum treated on the right side of the brain with either AAV-empty or AAV-shPTB (a). Scale bar, 500 μm. b, Magnified images showed extensive colocalization of TH with RFP-labelled fibres. Scale bar, 10 μm. Results show a significant degree of restoration of TH+ fibres in striatum. Experiments were independently repeated 3 times with similar results. c, d, Quantitative analysis of TH+ fibres in striatum under different treatment conditions. TH staining of striatum under different treatment conditions, as indicated (c). Scale bar, 10 μm. d, Quantification of total TH+ or TH+RFP– fibre density in striatum under different treatment conditions based on 3 biological repeats. Statistical significance was determined by ANOVA with post hoc Tukey test; mean ± s.e.m. P-values are indicated. Results show that most TH+ fibres seem to derive from AAV-shPTB-converted dopaminergic neurons; however, the data do not rule out the possibility that the axons of some endogenous neurons also responded to the environment created by newly converted neurons.
Extended Data Fig. 12 Reconstruction of the nigrostriatal pathway by converted dopaminergic neurons.
a, Schematic of the experimental schedule for 6-OHDA-induced lesion and reconstruction of the nigrostriatal pathway. b–f, Images of RFP+ projections extending from substantia nigra to striatum. The schematic diagram shows the dorso-ventral level of the horizontal section. Scale bar, 100 μm. Magnified views show indicated brain regions (c–f). Scale bar, 25 μm. g, Amplified views of RFP-positive fibres that co-stained with TH in CPu and globus pallidus. Scale bar, 20 μm. These data were used to provide the quantitative information shown in Fig. 4h, i. Experiments were independently repeated twice with similar results.
Extended Data Fig. 13 Measurement of striatal dopamine by HPLC and controls with AAV-shGFP and AAV-hM4Di.
a, b, Dopamine levels in brain detected by HPLC with two different doses of spiked dopamine (a). b, Standard curve generated from the spiked dopamine. This set of experiments was performed only once. c, Controls for behavioural tests, showing that expressing an anti-GFP control shRNA alone did not rescue chemical-induced behavioural deficits based on apomorphine-induced rotation (left) and cylinder test (right). d, Controls for behavioural tests, showing that the expression of hM4Di in non-reprogrammed astrocytes did not trigger detectable behaviour change in non-lesioned mice in the presence of CNO. Statistical significance was determined by ANOVA (c, d); mean ± s.e.m. Six mice were analysed in each group. P-values are indicated.
Extended Data Fig. 14 Electrophysiological analysis of PTB ASO-induced neurons in vitro and in brain.
a–c, Converted neurons showed large currents from voltage-dependent sodium and potassium channels (a), repetitive action potentials (b) and spontaneous postsynaptic currents (c). The numbers of cells that showed the recorded activity versus the total number of cells examined are indicated on the top right in each panel. d, Schematic of transgenic mice used to trace astrocytes in vivo. e, Three weeks after tamoxifen treatment, none of the tdTomato-labelled cells in the midbrain of Gfap-cre ER:Rosa-tdTomato mice stained positive for NeuN (left), and all were GFAP+ (right). Scale bar, 50 μm. f–i, Converted neurons in brain slices showed large currents from voltage-dependent sodium and potassium channels (f), repetitive action potentials (g), spontaneous action potentials (h) and spontaneous postsynaptic currents (i). The numbers of cells that showed the recorded activity versus the total number of cells examined are indicated. The results show that functional neurons are induced by PTB ASO both in culture and in mouse brain. All experiments shown in this figure were independently repeated twice with similar results.
Supplementary Figure 1: Uncropped scans of source data for immunoblots. Shown are original Western blots with specific cropped bands used in main and Extended data figures in this study.
Supplementary Table 1: List of significantly up- and down-regulated genes in response to PTB depletion in cortical versus midbrain astrocytes. RNA-seq analysis in cortical and midbrain astrocytes before and after PTB depletion. n=2 biological repeats under each experimental conditions. Significantly up- and down-regulated genes were identified based on FDR<0.05. Data were sorted based on fold-changes from high to low.
Supplementary Table 2: Report on independently verified experiments and repeats All raw data and experimental repeated as tabulated were independently verified by one of the authors Roy Maimon who did not involve in generating any of the data or writing of any of the reversion during the review process.
Supplementary Table 3: List of antibodies. Listed are all antibodies used in this study, including lot numbers from individual vendors and specific dilutions used. Supplementary Table 4: List of primers. Listed are all primers for RT-qPCR analyses reported in this study. Supplementary Table 5: Statistical report on all figure panels. Reported are specific p-values, confidential intervals, and degree of freedom for individual experiments reported in this study.
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Qian, H., Kang, X., Hu, J. et al. Reversing a model of Parkinson’s disease with in situ converted nigral neurons. Nature 582, 550–556 (2020). https://doi.org/10.1038/s41586-020-2388-4
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