Astroglia from the postnatal cerebral cortex can be reprogrammed in vitro to generate neurons following forced expression of neurogenic transcription factors, thus opening new avenues towards a potential use of endogenous astroglia for brain repair. However, in previous attempts astroglia-derived neurons failed to establish functional synapses, a severe limitation towards functional neurogenesis. It remained therefore also unknown whether neurons derived from reprogrammed astroglia could be directed towards distinct neuronal subtype identities by selective expression of distinct neurogenic fate determinants. Here we show that strong and persistent expression of neurogenic fate determinants driven by silencing-resistant retroviral vectors instructs astroglia from the postnatal cortex in vitro to mature into fully functional, synapse-forming neurons. Importantly, the neurotransmitter fate choice of astroglia-derived neurons can be controlled by selective expression of distinct neurogenic transcription factors: forced expression of the dorsal telencephalic fate determinant neurogenin-2 (Neurog2) directs cortical astroglia to generate synapse-forming glutamatergic neurons; in contrast, the ventral telencephalic fate determinant Dlx2 induces a GABAergic identity, although the overall efficiency of Dlx2-mediated neuronal reprogramming is much lower compared to Neurog2, suggesting that cortical astroglia possess a higher competence to respond to the dorsal telencephalic fate determinant. Interestingly, however, reprogramming of astroglia towards the generation of GABAergic neurons was greatly facilitated when the astroglial cells were first expanded as neurosphere cells prior to transduction with Dlx2. Importantly, this approach of expansion under neurosphere conditions and subsequent reprogramming with distinct neurogenic transcription factors can also be extended to reactive astroglia isolated from the adult injured cerebral cortex, allowing for the selective generation of glutamatergic or GABAergic neurons. These data provide evidence that cortical astroglia can undergo a conversion across cell lineages by forced expression of a single neurogenic transcription factor, stably generating fully differentiated neurons. Moreover, neuronal reprogramming of astroglia is not restricted to postnatal stages but can also be achieved from terminally differentiated astroglia of the adult cerebral cortex following injury-induced reactivation.

The brain consists of two major cell types: neurons, which transmit information, and glial cells, which support and protect neurons. Interestingly, evidence suggests that some glial cells, including astroglia, can be directly converted into neurons by specific proteins, a transformation that may aid in the functional repair of damaged brain tissue. However, in order for the repaired brain areas to function properly, it is important that astroglia be directed into appropriate neuronal subclasses. In this study, we show that non-neurogenic astroglia from the cerebral cortex can be reprogrammed in vitro using just a single transcription factor to yield fully functional excitatory or inhibitory neurons. We achieved this result through forced expression of the same transcription factors that instruct the genesis of these distinct neuronal subtypes during embryonic forebrain development. Moreover we demonstrate that reactive astroglia isolated from the adult cortex after local injury can be reprogrammed into synapse-forming excitatory or inhibitory neurons following a similar strategy. Our findings provide evidence that endogenous glial cells may prove a promising strategy for replacing neurons that have degenerated due to trauma or disease.

Funding: This work was supported by grants from the Deutsche Forschungsgemeinschaft [ http://www.dfg.de/ ] to BB and MG (BE 4182/1-3 and GO 640/9-1) and to TS (SCHR 1142/1-1), the Bavarian State Ministry of Sciences, Research, and the Arts (ForNeuroCell)[ http://www.bayfor.org/de/geschaeftsbereiche/forschungsverbuende/welt-des-lebens/forneurocell.html ] to BB and MG, and the Exzellenzcluster 114 Munich Center for Integrated Protein Science Munich (CIPSM) [ http://www.cipsm.de/en/index.html ], the SFB 596 ( http://www.sfb596.med.uni-muenchen.de/index.html ), the SFB 870 ( http://www.sfb870.mcn.uni-muenchen.de/index.html ), and European Transcriptome, Regulome & Cellular Commitment Consortium (EUTRACC) [ http://www.eutracc.eu/ ] to MG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2010 Heinrich et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Towards this end, we first aimed at a more potent neuronal reprogramming by inducing higher and more persistent expression of neurogenic fate determinants in astroglial cells. This allowed us not only to obtain fully functional neurons that also establish synapses from astroglial cells in vitro but also to demonstrate that distinct neurogenic transcription factors, such as on the one hand Neurog2 and on the other Dlx2 alone or in combination with Mash1, can indeed instruct the selective generation of different neuronal subtypes, such as glutamatergic and GABAergic neurons, respectively. Moreover, we found that the reprogramming efficiency of postnatal cortical astroglia towards GABAergic neurons by Dlx2 could be enhanced by first expanding the astroglial cells under neurosphere conditions prior to forced expression of Dlx2. Given that following brain injury reactive astroglia from the adult cerebral cortex de-differentiate, resume proliferation, and can give rise to self-renewing neurospheres in vitro [16] , we finally show that neuronal reprogramming and subtype specification are not restricted to postnatal stages but can also be achieved from adult cortical astroglia responding to injury.

However, several major obstacles remained to be overcome to fully exploit the potential of reprogrammed astroglia as an endogenous cellular source for neuronal repair. Firstly, reprogramming of astroglia towards neurons remained incomplete as the astroglia-derived neurons failed to establish a functional presynaptic output [7] , an obvious hurdle towards functional repair that requires participation in a neural network. Secondly, given the lack of functional presynaptic output, we could not determine the neuronal subtype generated by the reprogrammed astroglial cells [7] . This raises the conceptual concern of whether neurons derived from astroglial cells in a given brain region may be restricted towards the generation of a respective neuronal subtype. During development of the forebrain in rodents, stem/progenitor cells in the dorsal telencephalon generate exclusively excitatory glutamatergic neurons, directed by Pax6 and Neurog1/2 [8] – [10] , while stem/progenitor cells in the ventral telencephalon give rise primarily to inhibitory GABAergic neurons, governed by the fate determinants mammalian achaete-schute homolog 1 (Mash1) [11] , [12] and Dlx1/2 [13] . Region-specific fate restriction also seems to apply for adult neural stem cells that are intrinsically specified towards the generation of distinct neuronal subtypes [14] . This implies that despite their multipotent nature in regard to generating different glial cell types and neurons, the subtype identity of the neurons generated from these stem cells is predetermined (see also [15] ) and is not altered following transplantation [14] . This raises the important question of whether neuronal reprogramming of astroglia derived from the cerebral cortex, a region derived from the dorsal telencephalon, may be restricted towards the generation of glutamatergic neurons, or whether this region-specific bias could be overcome by forced expression of the appropriate neurogenic fate determinants. Such an ability to generate both glutamatergic and GABAergic neurons from astroglia may be a crucial step towards restoring a damaged or imbalanced neuronal network.

While exerting diverse functions within the brain parenchyma [1] , astroglia are remarkable in that they also function as neural stem or progenitor cells in specific regions of the postnatal and adult brain [2] , such as the ventricular subependymal zone [3] and the subgranular zone of the hippocampus [4] , [5] . This raises the possibility that even astroglia from non-neurogenic regions such as the cerebral cortex may be reprogrammed towards neurogenesis when provided with the appropriate transcriptional cues. Indeed, we could previously show that astroglia from the early postnatal cerebral cortex can be reprogrammed in vitro towards the generation of neurons capable of action potential (AP) firing by a single transcription factor, such as Pax6 or its target, the pro-neural transcription factor neurogenin-2 (Neurog2) [6] , [7] . These findings may open interesting avenues towards the potential activation of endogenous astroglia for neuronal repair of injured brain tissue.

Results

Genetic Fate Mapping Demonstrates Reprogramming of Postnatal Cortical Astroglia into Glutamatergic Neurons In order to ascertain the astroglial nature of the cells that gave rise to functional glutamatergic synapses following reprogramming by Neurog2, we took advantage of a transgenic mouse line in which GFP expression can be induced in astroglia and is maintained in their progeny. Heterozygous mice in which the expression of a tamoxifen-inducible Cre recombinase is driven by the astroglia specific L-glutamate/L-aspartate transporter promoter (GLAST::CreERT2) [23] were crossed to a reporter mouse line (Z/EG) [24] to generate double heterozygous mutants (GLAST::CreERT2/Z/EG) that were used in the present study. Cre-mediated recombination of the reporter locus was induced via tamoxifen administration from postnatal day 2 (P2) until sacrifice (P5–P7). Astroglia cultures were prepared as described above and, 1 wk later, cells were passaged onto glass coverslips. The vast majority of GFP reporter-positive cells were immunoreactive for GFAP (98.7%±0.7%) at 1 d after plating, with few cells being positive for the oligodendroglial markers NG2/O4 (1.2%±0.7%) and none (0.1%±0.1%) for the neuronal marker βIII tubulin (Figure 3A; n = 3 independent experiments, n = 1,560 GFP-positive cells counted). These data indicate that, under our culture conditions, most reporter-positive cells at the time of transduction possess an astroglial identity. These cells largely remain within their astroglial lineage (86.9%±12.7% of GFP-positive cells expressing GFAP) when analysed at later stages (Figure 3A'; n = 4 independent experiments, n = 1,363 GFP-positive cells counted; 9–21 d following plating). We noted, however, a slight increase in the number of NG2/O4-positive cells (13.0%±12.8%), likely due to the expansion of few reporter-positive clones of oligodendrocyte precursors. Also at later stages reporter-positive cells did not give rise to βIII tubulin-positive neurons (0.1%±0.1%; Figure 3A'). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Fate-mapped astroglia from the postnatal cortex can be reprogrammed to generate neurons following forced expression of Neurog2. (A–A') The histograms show the percentage of GFP reporter-positive cells from GLAST::CreERT2/Z/EG mice immunoreactive for the astroglial marker GFAP, oligodendroglial markers NG2/O4, and the neuronal marker βIII tubulin 1 d after plating (A) and 9–21 d after plating (A'), respectively. (B–B”) Cortical astroglia transduced with control retrovirus remain in the glial lineage. (B) The micrograph depicts GFP-positive, fate-mapped astroglia derived from postnatally induced GLAST::CreERT2/Z/EG mice. (B'–B”) Micrographs of the same field of view as shown in (B) showing that fate-mapped astroglia transduced with a control retrovirus encoding DsRed only (pCAG-IRES-DsRed) exhibit a glial morphology and express GFAP (B”). (C) Representative example of a GFP-positive neuron, i.e. derived from a fate-mapped astroglia, prepared from the postnatal cortex of tamoxifen-induced GLAST::CreERT2/Z/EG mice. (C') DsRed expression in the same cell indicating forced expression of Neurog2. (C”) The same astroglia-derived neuron as shown in (C) and (C') is immunoreactive for the mature neuronal marker MAP2 (27 DPI). (D–D') Direct visualisation of Neurog2-induced reprogramming of fate-mapped astroglia by time-lapse video microscopy. (D) Sequence of bright field images, overlaid with GFP reporter fluorescence, depicting the same field of view over the time course of 5 d as indicated in the corresponding panels below in (D'). The arrow points to a reporter-positive cell that divided once giving rise to two daughter cells that subsequently underwent reprogramming into neurons. The arrowhead points to another fate-mapped reprogrammed cell that entered the field of view at a later time point. (D') Corresponding sequence of DsRed fluorescence images taken at the same time points as the bright field images shown in (D). Note the expression of DsRed (encoded by Neurog2-IRES-DsRed) in the fate-mapped cell (arrow). Note that besides fate-mapped cells, several other Neurog2-transduced cells also became reprogrammed into neurons. Scale bars: B–B”: 57 µm; C–C”: 26 µm. https://doi.org/10.1371/journal.pbio.1000373.g003 To determine the identity of fate-mapped astroglial cells following retroviral transduction, we performed immunostaining for GFP (identifying cells of astroglial origin), DsRed (identifying transduced cells), and either βIII tubulin, MAP2, or GFAP (identifying neuronal and astroglial cells, respectively). Notably, the stochastic infection of the subset of genetically recombined cells results in a limited number of double-targeted cells. When cultures of adherent astroglia were transduced with the control retrovirus encoding DsRed only, fate-mapped astroglial cells co-expressing GFP and DsRed remained in the glial lineage, as revealed by their astroglial morphology and GFAP expression 1 mo after transduction (Figure 3B–3B”). In sharp contrast, when cultures of tamoxifen-induced astroglia were transduced with the new retrovirus encoding Neurog2 and DsRed, most GFP/DsRed-double-positive fate-mapped astroglia were reprogrammed into neurons expressing the neuronal markers βIII tubulin and MAP2 (67.3%±12.7% among GFP/DsRed-double positive cells at 8.0±1.0 DPI, n = 3 independent experiments, n = 217 double-positive cells counted; Figure 3C–3C”). Single cell tracking of GFP-reporter positive cells following Neurog2-transduction allowed the direct visualisation of the glia-to-neuron conversion of fate-mapped cells over the time course of 5 d (Figure 3D and 3D'; Video S1 and Video S2). Perforated patch clamp recordings of these fate-mapped astroglia-derived cells reprogrammed by Neurog2 revealed their functional neuronal identity as these cells fired APs following step-current injection in current clamp (n = 8; Figure 4A–4C). In the next set of experiments, we assessed whether neurons derived from fate-mapped astroglia could give rise to functional glutamatergic autapses (Figure 4D–4I). Step-depolarisation of GFP/DsRed-double-positive neurons at 0.05 Hz evoked a sequence of both autaptic and polysynaptic components (2 out of 8 cells recorded) consistent with the excitatory nature of the recorded neurons (average age of the cells: 18.1±2.2 DPI; Figure 4D–4I, insets), while at higher stimulation frequency (1 Hz) the autaptic component with a short decay time typical of glutamatergic synaptic transmission [25] could be observed in isolation (Figure 4F and 4I). Consistent with their glutamatergic nature, fate-mapped astroglia reprogrammed by forced expression of Neurog2 also exhibited a dense labelling of vGluT1-positive puncta (Figure 4J and 4K). These data clearly demonstrate that Neurog2 instructs fate-mapped astroglia from the postnatal cerebral cortex to acquire a glutamatergic identity. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Fate-mapped cortical astroglia reprogrammed by forced expression of Neurog2 establish functional synaptic connections. (A) Fluorescence micrograph depicting a GFP-positive neuron derived from a fate-mapped astroglia, prepared from postnatal cortex of tamoxifen-induced GLAST::CreERT2/Z/EG mice, 14 d after transduction with Neurog2-DsRed. The inset shows control untransduced GFP-positive astroglia. (B) DsRed expression in the same cell indicating forced expression of Neurog2. (C) Step-current injection into the cell shown in (A) and (B) results in repetitive firing of action potentials. (D–E) Fluorescence micrographs depicting another GFP-positive neuron, derived from a fate-mapped astroglia, following Neurog2-induced reprogramming 19 d after transduction. (F) Step-depolarisation at 1 Hz of the neuron shown in (D) and (E) evokes an autaptic response exhibiting a short decay time (90%–10%) typical of glutamatergic synaptic transmission (5.2 ms). The inset shows a single response evoked at 0.05 Hz revealing both an autaptic and a polysynaptic response (asterisk) due to recruitment of other neurons in the cultured network, indicating the excitatory nature of the fate-mapped reprogrammed neuron. (G–H) Another example of a GFP-positive neuron derived from a fate-mapped astroglia following Neurog2-induced reprogramming 27 DPI. The inset in (H) shows an enlargement of the boxed area revealing a DsRed-positive axon (arrowheads) originating from another DsRed-positive neuron and meandering along the dendrites and the soma of the recorded cell, after the patch pipette had been withdrawn and the cell died. (I) Step-depolarisation of the neuron shown in (G) and (H) evokes a sequence of both autaptic and polysynaptic responses. The black traces show individual autaptic responses observed in isolation when evoked at 1 Hz to eliminate polysynaptic components (the average trace is shown in red). The autapse exhibited a short decay time (90%–10%) typical of glutamatergic synaptic transmission (7.7 ms). The inset shows two individual responses evoked at 0.05 Hz (black and red traces) revealing both autaptic and polysynaptic responses (asterisk) due to recruitment of other neurons in the cultured network, indicating the excitatory nature of the fate-mapped reprogrammed neuron. (J–K) Expression of the vesicular glutamate transporter 1 (vGluT1) by fate-mapped postnatal astroglia reprogrammed by Neurog2. (J) Micrograph depicting a GFP and DsRed double-positive neuron derived from a fate-mapped astroglia, 27 d after transduction with a retrovirus encoding Neurog2 and DsRed. Note the DsRed-negative fate-mapped astrocyte. (K) Immunocytochemistry for vGluT1 reveals that the fate-mapped astroglia-derived neuron reprogrammed by Neurog2 exhibits a dense labelling of vGluT1-positive puncta outlining its cell body and dendrites. The insets show higher magnification views of the soma and dendrites illustrating the punctuate staining for vGluT1. Scale bars: J-K: 30 µm. https://doi.org/10.1371/journal.pbio.1000373.g004