Induction of functional dopamine neurons from human astrocytes in vitro and mouse astrocytes in a Parkinson's disease model

Journal name:
Nature Biotechnology
Volume:
35,
Pages:
444–452
Year published:
DOI:
doi:10.1038/nbt.3835
Received
Accepted
Published online
Corrected online

Abstract

Cell replacement therapies for neurodegenerative disease have focused on transplantation of the cell types affected by the pathological process. Here we describe an alternative strategy for Parkinson's disease in which dopamine neurons are generated by direct conversion of astrocytes. Using three transcription factors, NEUROD1, ASCL1 and LMX1A, and the microRNA miR218, collectively designated NeAL218, we reprogram human astrocytes in vitro, and mouse astrocytes in vivo, into induced dopamine neurons (iDANs). Reprogramming efficiency in vitro is improved by small molecules that promote chromatin remodeling and activate the TGFβ, Shh and Wnt signaling pathways. The reprogramming efficiency of human astrocytes reaches up to 16%, resulting in iDANs with appropriate midbrain markers and excitability. In a mouse model of Parkinson's disease, NeAL218 alone reprograms adult striatal astrocytes into iDANs that are excitable and correct some aspects of motor behavior in vivo, including gait impairments. With further optimization, this approach may enable clinical therapies for Parkinson's disease by delivery of genes rather than cells.

At a glance

Figures

  1. Human astrocytes can be reprogrammed with NeAL218.
    Figure 1: Human astrocytes can be reprogrammed with NeAL218.

    (a,b) iDAN reprogramming was done with a basic protocol (BP) (a) or a midbrain protocol (MP) (b). (c) Quantification of the percentage of human immortalized astrocytes (hIA) that became TH+ after infection with ALN after 10 d of doxycycline (dox) induction in BP or MP media. BP n = 4; MP n = 5. Two-tailed Student's t-test with unequal sample variance P = 0.016. (d) hIAs treated with ALN-MP, but not ALN-BP, showed presence of immature TH+;TUBB3+ neurons at day 10. (e) Quantification of the percentage of iDANs obtained with different combinations of transcription factors in MP medium at day 10. Untreated, miR218, A218, L, L218, N, N218, Ne, Ne218, AL218, AN218, ALN, NeAL and NeALN218, n = 3; A, AL, AN, ALN218, NeAL218 and NeALN, n = 4. One-way ANOVA followed by Dunnett's post-hoc multiple comparison test against the NeAL218 condition: A, P = 0.0001. NeAL218 vs. AN, P = 0.0001; NeAL218 vs. ALN, P = 0.0059; NeAL218 vs. A218, P = 0.0002. NeAL218 vs. AN218, P = 0.0001; NeAL218 vs. ALN218, P = 0.0347. (f) Quantification of the percentage of mature MAP2+ iDANs obtained with AL-, AL218- or NeAL218-MP (for e and f multiple comparison test against the NeAl218 condition). AL, AL218 and NeAL218, n = 4. One-way ANOVA followed by Dunnett's post-hoc multiple comparison test against the NeAL218 condition: AL, P = 0.0002. NeAL218 vs. AL218, P = 0.0001. (g) TH+ cells in NeAL218-MP cultures were also TUBB3+, MAP2+, DDC+ and SLC6A3+. Replicates are counts in independent experiments performed in duplicate. Data are expressed as box plots showing the median as well as the 25th and 75th percentiles. Scale bars, 50 μm. *P < 0.05, **P < 0.001 and ***P < 0.0001.

  2. Small molecules increase maturation of human-astrocyte-derived iDANs.
    Figure 2: Small molecules increase maturation of human-astrocyte-derived iDANs.

    (a) Remodelling TGF⇐ Midbrain Protocol (RTMP). (b) Confocal images of TH+ iDANs, which were TH+ iDANs were also TUBB3+, MAP2+, SYN1+, DDC+, SLC6A3+, KCNJ6+ and ALDH1A1+ after 13–17 d. Scale bars: upper row, 50 μm; lower row, 10 μm. (c) Comparison of the transcriptional profiles of hIAs and iDANsNeAL218-RTMP at day 10. Selected genes are shown over the threshold (0.05) and above twofold change of expression (dotted lines). Green circles, the reprogramming factors; red circles, selected neuronal and astrocytic genes. (d) Hierarchical clustering of transcriptome expression of hIAs, iDANsNeAL218-RTMP at day 10, and the dopaminergic signature of the human embryonic VM. Scale: Euclidian distance. TPM, transcripts per million. (e) Gene expression analysis by real-time qPCR data of hIAs untreated or treated with NeAL218-MP or NeAL218-RTMP at day 10. P values for Student's t-test between MP and RTMP conditions: *P < 0.05, **P < 0.01. Two-tailed Student's t-test with unequal variance, MP vs. RTMP: EN1 P = 0.0039; NR4A2 P = 0.0032; FOXA2 P = 0.0095; PITX3 P = 0.01; ALDH1A1 P = 0.01; CALB1 P = 0.00001. (f) Quantification of the percentage of iDANsNeAL218-RTMP obtained from human immortalized (hIA) or primary (hPA) astrocytes. hIA and hPA, n = 3. (g) Ca2+ responses in hPA-derived iDANsNeAL218-RTMP: upon depolarization (55 mM KCl, left panel) and 15 pA current injection in the cell with the thickest trace, action potentials (AP) were generated (right panel). (h) Spontaneous electrical activity accompanied by generation of action potentials in hPA-derived iDANsNeAL218-RTMP at days 13–17. (i) Voltage-clamp recordings in hPA cells; left, representative current profiles of cells treated with NeAL218-RTMP; middle and right panels, quantification of amplitudes in NeAL218-RTMP-treated cells (n = 21) and control-RTMP cells (n = 5) for VG outward currents (at command voltage +50 mV, middle) and voltage-gated (VG) inward currents (in peak, right). *P < 0.05 and **P < 0.001. (j) Current-clamp recordings of NeAL218-RTMP-treated hPA cells reveal different firing properties upon current injections: silent cells (no AP), cell generating single AP on depolarization (left panel) and cells generating multiple AP (right panel), also demonstrating the recognizable voltage rectification (sag) in response to the neuron hyperpolarization (gray trace, 2 out of 7 neurons generating multiple AP). Pie chart shows the mean percentage of cells with AP (n = 21). (k) Confocal images showing that hPA-derived iDANsNeAL218-RTMP are immunoreactive to TH and MAP2, RBFOX3, SYN1, ALDH1A1, SLC6A3, DDC, KCNJ6 and PBX1 after 14 d. Scale bars: 10 μm, except for upper left image, 50 μm.

  3. NeAL218 reprograms astrocytes into iDANs in vivo.
    Figure 3: NeAL218 reprograms astrocytes into iDANs in vivo.

    (a) Outline of the in vivo experiment. (b) Diagram depicting the reprogramming process in vivo. (c) Striata of GFAP-tTA adult mice infected with GFP 13 weeks after viral injection. White arrowhead, GFAP+;GFP+;RBFOX3 cell; insets show one cell at higher magnification. (d) iDANs in GFAP-tTA mice infected with NeAL218 show different degrees of reprogramming. Insets show examples of GFAP+;TH+;DAPI+ and GFAP;TH+;DAPI+ cells. Solid arrowhead, TH+;RBFOX3+ cell; empty arrowhead, TH+;DCX+ cell. (e) TH+ iDANs in GFAP-tTA mice infected with NeAL218 were negative for striatal neuron markers. Scale bars, 25 μm; for SLC6A3 in d, 10 μm. Wk, week.

  4. Reprogramming of adult striatal astrocytes into functional iDANs by NeAL218 in vivo.
    Figure 4: Reprogramming of adult striatal astrocytes into functional iDANs by NeAL218 in vivo.

    (a,b) Micrographs of brain cryosections from SLC6A3Tomato+ reporter mice intrastriatally injected with NeAL218 5 weeks after infection. (a) Presence of SLC6A3Tomato+;TH+ iDAN cell bodies and processes in the lesioned and injected striata. (b) SLC6A3Tomato+;TH+ iDANs in mice infected with NeAL218 showed positive staining for RBFOX3, NR4A2 and PBX1. (c) Time-lapse microphotographs showed KCl-induced release of FFN206, a fluorescent dopamine derivative, from SLC6A3Tomato+;TH+ iDANs in 250 μm-thick acute brain slices of GFAP-tTa;SLC6A3Cre/+;Rosa26RTomato or from GFAP-tTa;SLC6A3Cre/+ adult mice 5 weeks after NeAL218 injection. (d) Examples of patch-recorded SLC6A3Tomato+-reprogrammed iDANs patched 5 weeks after viral infection, in 250 μm-thick acute brain slices. (e) Voltage-gated currents at incrementing polarization steps lasting 100 ms. Inset: hyperpolarization-activated currents in voltage-clamp recordings upon hyperpolarization steps to −90 and −100 mV. (f) Current-clamp trace showing the ability of iDANs to generate repetitive action potentials (black) and their voltage sag rectification upon somatic current injection (gray). Scale bars in a: 10 μm; bd: 5 μm.

  5. iDANsNeAL218 induce behavioral recovery in a mouse model of Parkinson's disease.
    Figure 5: iDANsNeAL218 induce behavioral recovery in a mouse model of Parkinson's disease.

    (a) Net rotations (expressed as rotations/min) induced by amphetamine or apomorphine 13 weeks after NeAL218 or GFP injection (GFP n = 6, NeAL218 n = 7). Tukey whiskers plot with the median, 10th, 25th, 70th and 90th percentiles. Mann–Whitney rank sum test for apomorphine P = 0.035 and for amphetamine P = 0.917 (two tailed). (b) Spontaneous ipsilateral rotations (expressed as rotations/15 min) 5 weeks after NeAL218 or GFP injection (naive n = 13, lesion n = 13, GFP n = 6, NeAL218 n = 7). One-way ANOVA, F(3,35) = 6.03; P = 0.002 followed by Holm–Sidak's comparison; lesion vs. naive P = 0.003; GFP vs. naive P = 0.0493 and NeAL218 vs. naive P = 0.843. (cg) Gait analysis showing: (c) total number of gait cycles (naive n = 24, lesion n = 22, GFP n = 11, NeAL218 low dose n = 7, NeAL218 high dose n = 4). Tukey whiskers plot. Kruskal–Wallis test P < 0.0001 followed by Dunn's comparison; lesion vs. naive P < 0.0001; GFP vs. naive P = 0.0012; NeAL218 low vs. naive P = 0.3622 and NeAL218 high vs. naive P > 0.999. (d) Right, examples of consecutive step cycle for all limbs during walk (naive n =10, 6-OHDA-lesion n = 8, lesion-GFP n = 4 and lesion-NeAL218 mice n = 4). Gait was examined using ventral plane videography system. The horizontal bars represent the time during which a paw makes contact with the floor (direction of travel left to right). Asterisks mark hesitations in the gait cycle. (d) Left, diagrams depicting the movement order of the limbs during gait cycles. Black arrows indicate correct ordering of paw-treadmill-contacts. Red arrows highlight step cycle deviations. (e) Percentage of correct paw ordering cycles; box and whiskers graph showing min to max and all individual data. One-way ANOVA, F(3,22) = 5.42; P = 0.006 followed by Dunnett's comparison; lesion vs. naive P = 0.009; GFP vs. naive P = 0.0435; NeAL218 vs. naive P = 0.991. (f) Sagittal gait symmetry (zero: bilaterally symmetric gait). Bar graphs, mean ± s.e.m. Data were analyzed with one sample t-test with theoretical mean equal to 0 (two-tailed). Naive P = 0.383; lesion P = 0.0218; GFP P = 0.0231 and NeAL218 P = 0.178; and (g) coordination index (phase value) representing rear limbs synchronicity in naive n =10, 6-OHDA-lesion n = 8, lesion-GFP n = 4 and lesion-NeAL218 mice n = 4. Tukey whiskers plot, no outlier found. Kruskal–Wallis test P < 0.0001 followed by Dunn's comparison; lesion vs. naive P < 0.0001; GFP vs. naive P = 0.0193; NeAL218 vs. naive P > 0.999. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

  6. Characterization of hIA cultures and midbrain markers after iDAN reprogramming.
    Supplementary Fig. 1: Characterization of hIA cultures and midbrain markers after iDAN reprogramming.

    a. Uninfected hIAs do not spontaneously become TH+ in MP media at day 17. b. hIAs infected with GFP show a high infection efficiency in MP media at day 17 c. Gene expression analysis by real-time q-PCR revealed an increase in multiple mDA markers in hIA cultures treated with AL-MP, AL218-MP or NeAL218-MP at day 10. Data are shown as fold increase over control cells. * p<0.05 and ** p<0.01, n≥3, n.d.: not detectable

  7. Properties of hIA-derived iDANsNeAL218-RTMP cells.
    Supplementary Fig. 2: Properties of hIA-derived iDANsNeAL218-RTMP cells.

    a. Percentage of hIAs that become TH+ after infection with NeAL218 in RTMP conditions at day 4(n=3) and day 10(n=3). b. hIAs infected with NeAL218 in RTMP conditions at day 4 (scale bar 50μm) c. Illustration of the experiment for doxycycline withdrawal. d. hIAs infected with NeAL218 and cultured in RTMP for 13 days, but with only 7 or 10 days of doxycycline supplementation (scale bars 50μm). e. Bright field image of a representative hIA infected with NeAL218 and treated with RTMP media, probed by patch-clamp electrophysiology with its current profile (f) depicted in voltage-clamp mode. g. Current clamp recordings show the presence of hIA-derived iDANsNeAL218-RTMP capable of generating single action potential upon current injection (5, 20 and 30 pA). h. Vertical box plots, reflecting amplitudes of outward and inward currents in control (n=16) and NeAL218-infected hIAs cells with RTMP protocol (n=12). Whitney Rank Sum Test for outward currents p = 0,043; for inward currents p < 0,001. i. Pie chart depicting the fraction of hIA-derived iDANsNeAL218-RTMP generating single action potentials among the 12 cells probed * p<0.05 and ** p<0.01.

  8. Properties of hPA-derived control and iDANsNeAL218-RTMP cells
    Supplementary Fig. 3: Properties of hPA-derived control and iDANsNeAL218-RTMP cells

    a. Efficient expression of GFP by hPAs in RTMP media at day 17. b. Uninfected hPAs do not become TH+ in RTMP media at day 17 (scale bar 50μm). c. Representative voltage-clamp (top) and current-clamp (bottom) recordings from control-RTMP hPAs cells. Control cells did not generate any action potential (AP). d. Comparison of gene expression by real-time q-PCR of hPAs treated with control-RTMP or NeAL218-RTMP at day 10. Data are mean ± s.d. FOXA2 was compared to untreated cells at day 0, as Foxa2 mRNA was undetectable in control-RTMP cells at day 10.

  9. Characterization of the 6-OHDA lesion model, transduction of the GFAP-tTA mice and reporter activity of the GFAP-tTa;SLC6A3cre/+;Rosa26RTomato mice.
    Supplementary Fig. 4: Characterization of the 6-OHDA lesion model, transduction of the GFAP-tTA mice and reporter activity of the GFAP-tTa;SLC6A3cre/+;Rosa26RTomato mice.

    a. Ventral midbrain cryosection of GFAP-tTA adult mice infected in the striatum with NeAL218 for 13 weeks (scale bar 50μm). Comparison of the right and left substantia nigra showing the effect of the 6-OHDA injection in the ipsilateral medial forebrain bundle. b. Striatal cryosections of GFAP-tTA adult mice 2 weeks after unilateral 6-OHDA (scale bar 100μm) showing loss of TH and increase in GFAP in the ipsilateral striatum. c. Striatal cryosections of GFAP-tTA adult mice infected with GFP for 2 weeks (scale bar 25μm). d. Striatal cryosections of GFAP-tTA adult mice infected with GFP for 13 weeks (scale bar 10μm). e. Striatal cryosections of GFAP- tTa;SLC6A3cre/+;Rosa26RTomato adult mice infected with GFP for 5 weeks (scale bar 10μm). f. Cryosection of adult GFAP-tTa;SLC6A3cre/+;Rosa26RTomato mice showing the functionality of the reporter mice in the intact contralateral side of 6-OHDA lesioned mice infected with GFP or NeAL218 for 5 weeks (scale bar 10μm).

  10. Characterization of in vivo iDAN reprogramming by NeAL218.
    Supplementary Fig. 5: Characterization of in vivo iDAN reprogramming by NeAL218.

    a. Diagram depicting am alternative strategy used to visualize in vivo reprogrammed mature iDANs: GFAP-tTA; SLC6A3Cre/+ mice and flex-tdTomato virus. b. Time-lapse microphotographs showing release of a fluorescent dopamine derivative, FFN 206, upon KCl-induced depolarization in acute brain slices of GFAP-tTA adult mice, 5 weeks after NeAL218 or GFP injection (scale bar 25μm). c-e. Electrophysiological recordings at 5 weeks showing NeAL218-induced SLC6A3Tomato+ cells unable to generate action potentials in voltage clamp mode (c: voltage clamp profile, n=19 out of 28) or able to generate single action potentials upon somatic current injection (n=4 out of 28 in d and e, showing voltage-clamp and current clamp profiles, respectively). f. Control GFP+ cells recorded in voltage-clamp configuration (polarization for 100 ms from -100 to + 50 mV, n=9; 2 examples are presented) showed outward voltage-gated currents but no inward voltage gated (sodium/calcium) currents. g. Net rotations after systemic amphetamine administration to control and 6-OHDA lesioned GFAP-tTA mice ± intrastriatatal GFP or NeAL218 for 5, 9 or 13 weeks. GFP n = 6; NeAL218, n = 7. Two Way RM ANOVA with time effect F(4,44) = 34,75; p < 0,0001 and subject matching F(11, 44) = 7,908; p < 0,0001 followed by Fisher’s comparison (for GFP: naïve vs lesion p = 0,0003; W5 vs lesion p = 0,0125; W9 vs lesion p = 0,0039 and W13 vs lesion p = 0,0122 and for NeAL218: naïve vs lesion p < 0,0001; W5 vs lesion p = 0,0813; W9 vs lesion p = 0,0011 and W13 vs lesion p = 0,0649).

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Change history

Corrected online 19 April 2017
In the version of this article initially published, in the paragraph before the Discussion, the sentence that read in part “…the very high levels of DA that are required in the synapses to induce circling behavior…” should have read “…the very high levels of DA that are required in the synapses to reduce circling behavior…” The error has been corrected for the print, PDF and HTML versions of this article.

References

  1. DeMaagd, G. & Philip, A. Parkinson's disease and its management. Part 2: Introduction to the pharmacotherapy of parkinson's disease, with a focus on the use of dopaminergic agents. P&T 40, 591600 (2015).
  2. Brundin, P. et al. Intracerebral grafting of dopamine neurons. Experimental basis for clinical trials in patients with Parkinson's disease. Ann. NY Acad. Sci. 495, 473496 (1987).
  3. Lindvall, O. et al. Fetal dopamine-rich mesencephalic grafts in Parkinson's disease. Lancet 2, 14831484 (1988).
  4. Arenas, E. Towards stem cell replacement therapies for Parkinson's disease. Biochem. Biophys. Res. Commun. 396, 152156 (2010).
  5. Arenas, E., Denham, M. & Villaescusa, J.C. How to make a midbrain dopaminergic neuron. Development 142, 19181936 (2015).
  6. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663676 (2006).
  7. Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 10351041 (2010).
  8. Ladewig, J., Koch, P. & Brustle, O. Leveling Waddington: the emergence of direct reprogramming and the loss of cell fate hierarchies. Nat. Rev. Mol. Cell Biol. 14, 225236 (2013).
  9. Chen, G. et al. In vivo reprogramming for brain and spinal cord repair(1,2,3). eNeuro. 2, e010615.2015 (2015).
  10. Caiazzo, M. et al. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476, 224227 (2011).
  11. Kim, J. et al. Functional integration of dopaminergic neurons directly converted from mouse fibroblasts. Cell Stem Cell 9, 413419 (2011).
  12. Liu, X. et al. Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells. Cell Res. 22, 321332 (2012).
  13. Pfisterer, U. et al. Direct conversion of human fibroblasts to dopaminergic neurons. Proc. Natl. Acad. Sci. USA 108, 1034310348 (2011).
  14. Addis, R.C. et al. Efficient conversion of astrocytes to functional midbrain dopaminergic neurons using a single polycistronic vector. PLoS One 6, e28719 (2011).
  15. Heins, N. et al. Glial cells generate neurons: the role of the transcription factor Pax6. Nat. Neurosci. 5, 308315 (2002).
  16. Heinrich, C. et al. Directing astroglia from the cerebral cortex into subtype specific functional neurons. PLoS Biol. 8, e1000373 (2010).
  17. Heinrich, C. et al. Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex. Nat. Protoc. 6, 214228 (2011).
  18. Torper, O. et al. Generation of induced neurons via direct conversion in vivo. Proc. Natl. Acad. Sci. USA 110, 70387043 (2013).
  19. Su, Z., Niu, W., Liu, M.L., Zou, Y. & Zhang, C.L. In vivo conversion of astrocytes to neurons in the injured adult spinal cord. Nat. Commun. 5, 3338 (2014).
  20. 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, 188202 (2014).
  21. Heinrich, C. et al. Sox2-mediated conversion of NG2 glia into induced neurons in the injured adult cerebral cortex. Stem Cell Reports 3, 10001014 (2014).
  22. Torper, O. et al. In vivo reprogramming of striatal NG2 glia into functional neurons that integrate into local host circuitry. Cell Rep. 12, 474481 (2015).
  23. Gascón, S. et al. Identification and successful negotiation of a metabolic checkpoint in direct neuronal reprogramming. Cell Stem Cell 18, 396409 (2016).
  24. Brambrink, T. et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151159 (2008).
  25. Esteban, M.A. et al. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell 6, 7179 (2010).
  26. Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275280 (2009).
  27. Wernig, M. et al. Tau EGFP embryonic stem cells: an efficient tool for neuronal lineage selection and transplantation. J. Neurosci. Res. 69, 918924 (2002).
  28. Kriks, S. et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480, 547551 (2011).
  29. Pang, Z.P. et al. Induction of human neuronal cells by defined transcription factors. Nature 476, 220223 (2011).
  30. Huang, T., Liu, Y., Huang, M., Zhao, X. & Cheng, L. Wnt1-cre-mediated conditional loss of Dicer results in malformation of the midbrain and cerebellum and failure of neural crest and dopaminergic differentiation in mice. J. Mol. Cell Biol. 2, 152163 (2010).
  31. Huangfu, D. et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat. Biotechnol. 26, 795797 (2008).
  32. Pennarossa, G. et al. Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells. Proc. Natl. Acad. Sci. USA 110, 89488953 (2013).
  33. Chung, T.L. et al. Vitamin C promotes widespread yet specific DNA demethylation of the epigenome in human embryonic stem cells. Stem Cells 28, 18481855 (2010).
  34. Liu, X. et al. Sequential introduction of reprogramming factors reveals a time-sensitive requirement for individual factors and a sequential EMT-MET mechanism for optimal reprogramming. Nat. Cell Biol. 15, 829838 (2013).
  35. La Manno, G. et al. Molecular diversity of midbrain development in mouse, human, and stem cells. Cell 167, 566580.e19 (2016).
  36. Koyano-Nakagawa, N., Kim, J., Anderson, D. & Kintner, C. Hes6 acts in a positive feedback loop with the neurogenins to promote neuronal differentiation. Development 127, 42034216 (2000).
  37. Jhas, S. et al. Hes6 inhibits astrocyte differentiation and promotes neurogenesis through different mechanisms. J. Neurosci. 26, 1106111071 (2006).
  38. Dillon-Carter, O., Conejero, C., Tornatore, C., Poltorak, M. & Freed, W.J. N18-RE-105 cells: differentiation and activation of p53 in response to glutamate and adriamycin is blocked by SV40 large T antigen tsA58. Cell Tissue Res. 291, 191205 (1998).
  39. Lundblad, M., Picconi, B., Lindgren, H. & Cenci, M.A. A model of L-DOPA-induced dyskinesia in 6-hydroxydopamine lesioned mice: relation to motor and cellular parameters of nigrostriatal function. Neurobiol. Dis. 16, 110123 (2004).
  40. Darmopil, S., Muñetón-Gómez, V.C., de Ceballos, M.L., Bernson, M. & Moratalla, R. Tyrosine hydroxylase cells appearing in the mouse striatum after dopamine denervation are likely to be projection neurones regulated by L-DOPA. Eur. J. Neurosci. 27, 580592 (2008).
  41. Masuda, M. et al. Postnatal development of tyrosine hydroxylase mRNA-expressing neurons in mouse neostriatum. Eur. J. Neurosci. 34, 13551367 (2011).
  42. Hu, G. et al. New fluorescent substrate enables quantitative and high-throughput examination of vesicular monoamine transporter 2 (VMAT2). ACS Chem. Biol. 8, 19471954 (2013).
  43. Freyberg, Z. et al. Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain. Nat. Commun. 7, 10652 (2016).
  44. Brooks, S.P. & Dunnett, S.B. Tests to assess motor phenotype in mice: a user's guide. Nat. Rev. Neurosci. 10, 519529 (2009).
  45. Bagga, V., Dunnett, S.B. & Fricker, R.A. The 6-OHDA mouse model of Parkinson's disease - Terminal striatal lesions provide a superior measure of neuronal loss and replacement than median forebrain bundle lesions. Behav. Brain Res. 288, 107117 (2015).
  46. Fasano, A., Aquino, C.C., Krauss, J.K., Honey, C.R. & Bloem, B.R. Axial disability and deep brain stimulation in patients with Parkinson disease. Nat. Rev. Neurol. 11, 98110 (2015).
  47. Kurz, M.J. et al. A chronic mouse model of Parkinson's disease has a reduced gait pattern certainty. Neurosci. Lett. 429, 3942 (2007).
  48. Bonito-Oliva, A., Masini, D. & Fisone, G. A mouse model of non-motor symptoms in Parkinson's disease: focus on pharmacological interventions targeting affective dysfunctions. Front. Behav. Neurosci. 8, 290 (2014).
  49. Amende, I. et al. Gait dynamics in mouse models of Parkinson's disease and Huntington's disease. J. Neuroeng. Rehabil. 2, 20 (2005).
  50. Wang, H., Li, X., Gao, S., Sun, X. & Fang, H. Transdifferentiation via transcription factors or microRNAs: current status and perspective. Differentiation 90, 6976 (2015).
  51. Dickson, D.W. Parkinson's disease and parkinsonism: neuropathology. Cold Spring Harb. Perspect. Med. 2, a009258 (2012).
  52. Zeisel, A. et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 11381142 (2015).
  53. 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).
  54. Wang, J., Lin, W., Popko, B. & Campbell, I.L. Inducible production of interferon-gamma in the developing brain causes cerebellar dysplasia with activation of the Sonic hedgehog pathway. Mol. Cell. Neurosci. 27, 489496 (2004).
  55. Ekstrand, M.I. et al. Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons. Proc. Natl. Acad. Sci. USA 104, 13251330 (2007).

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Author information

Affiliations

  1. Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

    • Pia Rivetti di Val Cervo,
    • Elisa Martín-Montañez,
    • Enrique M Toledo,
    • Gioele La Manno,
    • Sara Padrell Sánchez,
    • Sten Linnarsson &
    • Ernest Arenas
  2. Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria.

    • Roman A Romanov,
    • Christian Pifl &
    • Tibor Harkany
  3. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.

    • Roman A Romanov,
    • Giada Spigolon,
    • Débora Masini,
    • Michael Feyder,
    • Tibor Harkany &
    • Gilberto Fisone
  4. Department of Pharmacology, Faculty of Medicine, Biomedical Research Institute of Malaga (IBIMA), Malaga University, Malaga, Spain.

    • Elisa Martín-Montañez
  5. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California, USA.

    • Yi-Han Ng &
    • Marius Wernig

Contributions

E.A. and P.R.d.V.C. conceived the experiments and wrote the manuscript; P.R.d.V.C., R.A.R., G.S., D.M., E.M.-M., E.M.T., G.L.M., M.F., C.P., Y.-H.N. and S.P.S. performed the experiments; S. L., M.W., T.H., G.F. and E.A. provided expertise and funding.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Characterization of hIA cultures and midbrain markers after iDAN reprogramming. (418 KB)

    a. Uninfected hIAs do not spontaneously become TH+ in MP media at day 17. b. hIAs infected with GFP show a high infection efficiency in MP media at day 17 c. Gene expression analysis by real-time q-PCR revealed an increase in multiple mDA markers in hIA cultures treated with AL-MP, AL218-MP or NeAL218-MP at day 10. Data are shown as fold increase over control cells. * p<0.05 and ** p<0.01, n≥3, n.d.: not detectable

  2. Supplementary Figure 2: Properties of hIA-derived iDANsNeAL218-RTMP cells. (317 KB)

    a. Percentage of hIAs that become TH+ after infection with NeAL218 in RTMP conditions at day 4(n=3) and day 10(n=3). b. hIAs infected with NeAL218 in RTMP conditions at day 4 (scale bar 50μm) c. Illustration of the experiment for doxycycline withdrawal. d. hIAs infected with NeAL218 and cultured in RTMP for 13 days, but with only 7 or 10 days of doxycycline supplementation (scale bars 50μm). e. Bright field image of a representative hIA infected with NeAL218 and treated with RTMP media, probed by patch-clamp electrophysiology with its current profile (f) depicted in voltage-clamp mode. g. Current clamp recordings show the presence of hIA-derived iDANsNeAL218-RTMP capable of generating single action potential upon current injection (5, 20 and 30 pA). h. Vertical box plots, reflecting amplitudes of outward and inward currents in control (n=16) and NeAL218-infected hIAs cells with RTMP protocol (n=12). Whitney Rank Sum Test for outward currents p = 0,043; for inward currents p < 0,001. i. Pie chart depicting the fraction of hIA-derived iDANsNeAL218-RTMP generating single action potentials among the 12 cells probed * p<0.05 and ** p<0.01.

  3. Supplementary Figure 3: Properties of hPA-derived control and iDANsNeAL218-RTMP cells (328 KB)

    a. Efficient expression of GFP by hPAs in RTMP media at day 17. b. Uninfected hPAs do not become TH+ in RTMP media at day 17 (scale bar 50μm). c. Representative voltage-clamp (top) and current-clamp (bottom) recordings from control-RTMP hPAs cells. Control cells did not generate any action potential (AP). d. Comparison of gene expression by real-time q-PCR of hPAs treated with control-RTMP or NeAL218-RTMP at day 10. Data are mean ± s.d. FOXA2 was compared to untreated cells at day 0, as Foxa2 mRNA was undetectable in control-RTMP cells at day 10.

  4. Supplementary Figure 4: Characterization of the 6-OHDA lesion model, transduction of the GFAP-tTA mice and reporter activity of the GFAP-tTa;SLC6A3cre/+;Rosa26RTomato mice. (989 KB)

    a. Ventral midbrain cryosection of GFAP-tTA adult mice infected in the striatum with NeAL218 for 13 weeks (scale bar 50μm). Comparison of the right and left substantia nigra showing the effect of the 6-OHDA injection in the ipsilateral medial forebrain bundle. b. Striatal cryosections of GFAP-tTA adult mice 2 weeks after unilateral 6-OHDA (scale bar 100μm) showing loss of TH and increase in GFAP in the ipsilateral striatum. c. Striatal cryosections of GFAP-tTA adult mice infected with GFP for 2 weeks (scale bar 25μm). d. Striatal cryosections of GFAP-tTA adult mice infected with GFP for 13 weeks (scale bar 10μm). e. Striatal cryosections of GFAP- tTa;SLC6A3cre/+;Rosa26RTomato adult mice infected with GFP for 5 weeks (scale bar 10μm). f. Cryosection of adult GFAP-tTa;SLC6A3cre/+;Rosa26RTomato mice showing the functionality of the reporter mice in the intact contralateral side of 6-OHDA lesioned mice infected with GFP or NeAL218 for 5 weeks (scale bar 10μm).

  5. Supplementary Figure 5: Characterization of in vivo iDAN reprogramming by NeAL218. (375 KB)

    a. Diagram depicting am alternative strategy used to visualize in vivo reprogrammed mature iDANs: GFAP-tTA; SLC6A3Cre/+ mice and flex-tdTomato virus. b. Time-lapse microphotographs showing release of a fluorescent dopamine derivative, FFN 206, upon KCl-induced depolarization in acute brain slices of GFAP-tTA adult mice, 5 weeks after NeAL218 or GFP injection (scale bar 25μm). c-e. Electrophysiological recordings at 5 weeks showing NeAL218-induced SLC6A3Tomato+ cells unable to generate action potentials in voltage clamp mode (c: voltage clamp profile, n=19 out of 28) or able to generate single action potentials upon somatic current injection (n=4 out of 28 in d and e, showing voltage-clamp and current clamp profiles, respectively). f. Control GFP+ cells recorded in voltage-clamp configuration (polarization for 100 ms from -100 to + 50 mV, n=9; 2 examples are presented) showed outward voltage-gated currents but no inward voltage gated (sodium/calcium) currents. g. Net rotations after systemic amphetamine administration to control and 6-OHDA lesioned GFAP-tTA mice ± intrastriatatal GFP or NeAL218 for 5, 9 or 13 weeks. GFP n = 6; NeAL218, n = 7. Two Way RM ANOVA with time effect F(4,44) = 34,75; p < 0,0001 and subject matching F(11, 44) = 7,908; p < 0,0001 followed by Fisher’s comparison (for GFP: naïve vs lesion p = 0,0003; W5 vs lesion p = 0,0125; W9 vs lesion p = 0,0039 and W13 vs lesion p = 0,0122 and for NeAL218: naïve vs lesion p < 0,0001; W5 vs lesion p = 0,0813; W9 vs lesion p = 0,0011 and W13 vs lesion p = 0,0649).

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  1. Supplementary Text and Figures (1,228 KB)

    Supplementary Figures 1–5

Additional data