Neuropharmacology 58 (2010) 894e902
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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
Review
Making neurons from mature glia: A far-fetched dream?
Benedikt Berninger a, b, *,1
a
b
Institute for Stem Cell Research, National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany
Department of Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, Schillerstrasse 46, D-80336 Munich, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 August 2009
Received in revised form
5 November 2009
Accepted 12 November 2009
The fact that cells with glial characteristics such as forebrain radial glia during development and
astroglial stem cells in the adult neurogenic zones serve as neuronal precursors provokes the question
why glia in most other areas of the adult central nervous system are apparently incapable of generating
new neurons. Besides being of pivotal biological interest answers to this question may also open new
avenues for cell-based therapies of neurodegenerative diseases that involve a permanent loss of neurons
which are not replaced naturally. For if one could indeed instruct glia to generate neurons, such
a strategy would carry the enormous advantage of making use of a large pool of endogenous, and hence
autologous cells, thereby circumventing many of the problems associated with therapeutic strategies
based on transplantation. Accordingly, the recent years have seen increasing effort in assessing the
plasticity of astroglia and other types of resident non-neuronal cells as a potential source for new
neurons in the injured brain or eye. For instance, following injury astroglia in the cerebral cortex and
Müller glia in the retina can de-differentiate and acquire stem or precursor cell like properties. Moreover,
it has been shown that astroglia can be reprogrammed in vitro by forced expression of neurogenic
transcription factors to transgress their lineage restriction and stably acquire a neuronal identity. In this
review I will discuss the status quo of these early attempts, the limitations currently encountered and the
future challenges before the full potential of this approach can be weighed.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Stem cells
Neurogenesis
Proneural genes
Brain repair
Reactive gliosis
Müller glia
There are two experimental strategies for developing cell-based
therapies for neurodegenerative diseases: One aims at replacing
degenerated neurons (and glia) via transplantation of cells that
have been expanded in vitro and subsequently specified into the
desired cell type. The most widely used expandable source for this
experimental approach are embryonic stem (ES) cells as protocols
for directing these cells into distinct neuronal populations such as
midbrain dopaminergic neurons, spinal motor neurons and cortical
pyramidal neurons are becoming gradually more available (for
review see Berninger et al., 2006). These protocols should in principle also work for induced pluripotent stem cells (iPSCs) (Dimos
et al., 2008; Ebert et al., 2009; Wernig et al., 2008), i.e. ES-like cells
that can be derived from somatic cells through cellular reprogramming by defined factors (Takahashi and Yamanaka, 2006). The
differentiation capacity of another potential source for transplantable cells, namely neural stem cells isolated from the adult
subependymal zone (SEZ) is far less well understood. In fact these
* Department of Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, Schillerstrasse 46, D-80336 Munich, Germany. Tel.: þ49
89 2180 75 208; fax: þ49 89 2180 75 216.
E-mail address: benedikt.berninger@lrz.uni-muenchen.de
1
Tel.: þ49 89 3187 3751; fax: þ 49 89 3187 3761.
0028-3908/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2009.11.004
“stem cells”, far from being at liberty in their differentiation
potential, seem to be restricted with regard to neuronal subtype
specification (Merkle et al., 2007). However, despite such fate
restrictions, forced expression of transcriptional fate determinants
can redirect these cells towards different identities in vitro (Berninger et al., 2007b) and in vivo (Brill et al., 2008; Colak et al., 2008;
Hack et al., 2005; Jessberger et al., 2008).
The other principle approach pursues the possibility to recruit
endogenous cells for brain repair. It can be split again into two
distinct strategies, one aiming at the recruitment of endogenously
adult neural stem cells that reside in neurogenic zones such as
the adult SEZ lining the lateral ventricle (Saghatelyan et al., 2004).
Moreover, cells with stem cell-like potential may be present to
varying degree throughout the brain, such as the cortical white
matter (Nunes et al., 2003), the spinal cord (Johansson et al.,
1999) and the periventricular zone (Nakatomi et al., 2002). While
these cells do not generate neurons under physiological conditions, it may be possible to recruit them for regenerative neurogenesis by providing certain stimuli such as growth factor
treatment (Nakatomi et al., 2002; Ohori et al., 2006). The second
approach, which we shall discuss in this review in more detail,
aims at recruiting cells for brain repair that are far more common
than proper stem cells, namely glia, and in particular astroglia.
B. Berninger / Neuropharmacology 58 (2010) 894e902
The reasons for selecting this cell type are manifold: (i) they occur
virtually ubiquitously throughout the nervous system, thus in
contrast to recruitment of stem cells from defined brain regions
such as the SEZ, conscription of glia resident to the parenchyma
would not necessitate complicated, albeit feasible strategies
aiming at re-routing migration (Saghatelyan et al., 2004); (ii)
parenchymal astroglia are direct progeny of and share many
features with radial glia (Campbell and Gotz, 2002), and hence of
cells with neurogenic potential; (iii) parenchymal astroglia are
also related to astroglial stem cells in the adult neurogenic zones
with whom they do not share only a common radial glial origin
(Merkle et al., 2004), but also many ultrastructural and molecular
features (Doetsch et al., 1999; Merkle et al., 2004; Seri et al.,
2001); (iv) and while being engaged in more “earthly” tasks such
as regulating various metabolic functions within the nervous
tissue (Wang and Bordey, 2008) compared to astroglial stem cells
responsible for life-long neurogenesis, they can be re-activated
during injury when they re-acquire certain features of their more
lofty “sisters” of the adult neurogenic zones (Buffo et al., 2008;
Silver and Steindler, 2009).
What strategies can be envisaged for recruiting parenchymal
astroglia for brain repair? In first place, a strategy may aim at taking
advantage of the intrinsic potential of these cells revealed during
reactive astrogliosis (Buffo et al., 2008; Silver and Steindler, 2009).
Indeed, as we will see below following injury normally quiescent
astroglia and Müller glia can resume proliferation, de-differentiate
(Buffo et al., 2008; Karl et al., 2008) and can give rise to neurosphere-forming cells when isolated in vitro (Buffo et al., 2008). A
second approach aims at reprogramming parenchymal astroglia via
direct intervention with the nuclear machinery through forced
expression of transcription factors. Here again in theory two
possibilities may be considered, namely reprogramming astroglia
like other somatic cells towards a pluripotent status, from whence
they could be differentiated into the desired neuronal cell type.
However, such strategy faces the high risk for teratoma formation
from inappropriately differentiated ES-like cells when placed in
vivo. Alternatively, reprogramming may aim at a direct astroglia-toneuron lineage transgression or transdifferentiation, as discussed
below in more detail.
1. Glia as neural stem cells/neuronal precursors
It is a fairly recent notion that most neurons of the telencephalon are derived either directly or indirectly from radial glia
(Anthony et al., 2004; Campbell and Gotz, 2002; Kriegstein and
Alvarez-Buylla, 2009; Malatesta et al., 2003, 2000; Noctor et al.,
2001). The particular features of radial glia, such as their possession
of a long radial process anchoring these cells at the pial surface as
well as a shorter apical process contacting the ventricular surface
are believed to play a pivotal role in integrating signals regulating
cell cycle, symmetric versus asymmetric cell division and cell fate
decision (Gotz and Huttner, 2005). Besides these rather particular
features, radial glia display many features of astroglia. For instance,
in the developing cerebral cortex radial glia express GLAST,
a astrocyte specific glutamate/aspartate transporter (Mori et al.,
2006) and have an active glial fibrillary acidic protein (GFAP)
promoter (Malatesta et al., 2003). While generating neuronal
(basal) progenitors and neurons through asymmetric and
symmetric cell divisions during the periods of neurogenesis (Noctor et al., 2001, 2004), at the end of neurogenesis the remaining
radial glia appear to de-attach their apical (ventricular) process and
migrate towards the cortical plate (Noctor et al., 2004) where they
transform into astroglia (Noctor et al., 2008). With this transformation into astrocytes, the cerebral cortex loses its radial glia
pool and alongside the potential to generate neurons (Kriegstein
895
and Alvarez-Buylla, 2009). At the early stage some of the astroglial
cells still proliferate generating astrocytes which eventually
become postmitotic (for review see (Kriegstein and Alvarez-Buylla,
2009)) and hence do no longer divide in the adult cortex (Buffo
et al., 2008). However, the fact of their direct radial glial descent
could suggest that astroglia may latently still retain some of the
prerequisites for neurogenesis.
In restricted zones of the adult CNS, radial glia does not just give
rise to parenchymal astrocytes, but transform into astroglial stem
cells (Merkle et al., 2004). One of the most surprising features of the
radial glia-derived astroglial stem cells in the adult SEZ is the fact
that they maintain a direct contact to the ventricular surface where
they protrude a single cilium into the ventricular fluid while at the
same time extending a long process establishing contact with blood
vessels (Mirzadeh et al., 2008; Shen et al., 2008; Tavazoie et al.,
2008). These features are so peculiar as to suggest that much of the
secrets regarding the stem cell nature of the SEZ astroglia are
indeed safeguarded by these structural elements, by placing these
cells in intimate contact with a specific regulatory environment
called the stem cell niche. Consistent with a central role of the
contact between SEZ astroglia and the ventricular fluid for stem cell
function is the fact that inferring with ciliogenesis also diminishes
proliferation in the adult SEZ (Kriegstein and Alvarez-Buylla, 2009).
Cilia are known to be enriched in signalling receptors such as those
for the morphogen sonic hedgehog (Shh) and conditional deletion
of cilia and Shh signalling drastically reduces adult neurogenesis
(Han et al., 2008).
2. Postnatal astroglia: an intermediate stage between
radial glia and mature quiescent astroglia
While astroglial stem cells apparently retain features that
characterize radial glia during embryonic development, one may
wonder what happens to their more profane progeny during the
process of radial glia-to-astrocyte transformation. Interestingly,
this transformation appears to be a gradual process during which
the neurogenic potential of the astrocytic progeny becomes
progressively diminished, through mechanisms involving epigenetic silencing of neurogenic fate determinants (Hirabayashi et al.,
2009). Of note, when astroglial cells from the early postnatal
cerebral cortex are grown in vitro in the absence of serum factors,
but in the presence of epidermal growth factor (EGF) and fibroblast
growth factor 2 (FGF2), these cells can still give rise to selfrenewing and multipotent neurospheres (Laywell et al., 2000), an
ability often employed by in vitro assays for assessing stem cell
properties. It will be interesting to know whether the low degree of
spontaneous neurogenesis observed in these astroglia-derived
neurospheres (Laywell et al., 2000) is accompanied by the erasure
of epigenetic marks that secure the silencing of neurogenic fate
determinants (Hirabayashi et al., 2009). Notably, the potential of
astroglia to give rise to neurospheres declines to zero during the
second postnatal week (Laywell et al., 2000) (Berninger, unpublished observation). This data indicate that the assignment of early
postnatal astroglia to their glial fate may not yet be irrevocable
during the first postnatal week, but becomes progressively locked
into their chosen glial fate during the second postnatal week.
It has been recently suggested that early postnatal astroglia in
the cerebral cortex may retain even some capacity for neurogenesis
in vivo (Ganat et al., 2006). A study performed in the Vaccarino lab
showed that when Cre recombinase activity driven by the hGFAP
promoter was induced early postnatally (P5), most fate-mapped
cells in the cerebral cortex comprised astroglia and some oligodendroglia (Ganat et al., 2006). Yet, a very small percentage (<1%)
of the fate-mapped cells had given rise to neurons one month after
recombination. However, this data cannot rule out the alternative
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B. Berninger / Neuropharmacology 58 (2010) 894e902
interpretation that some of the fate-mapped cells had in fact not
been generated locally within the cortex, but had arrived to the
cortex from neurogenic zones such as the postnatal SEZ. Indeed the
observation that many of the postnatally generated neurons were
apparently GABAergic (Ganat et al., 2006), a cell type normally not
generated within the dorsal telencephalon, may suggest a origin
foreign to the cerebral cortex itself.
Interestingly, the same group also reported a truly remarkable
neurogenesis following chronic postnatal hypoxia (Fagel et al.,
2009, 2006). Previous work in the adult cerebral cortex had shown
that locally induced apoptosis can indeed provoke a low degree of
neurogenesis (Magavi et al., 2000), but at early postnatal stages
death of cortical neurons appears to trigger a nearly complete
recovery (Fagel et al., 2006), a phenomenon requiring signalling
through glial FGF receptors 1 (Fagel et al., 2009). Again it remains to
be shown whether this remarkable postnatal plasticity is due to
cortical neurogenesis from local astroglia or rather recruitment of
new neurons from the postnatal SEZ, especially as proliferation in
the SEZ is dramatically increased following chronic hypoxia (Fagel
et al., 2009, 2006). Moreover, there is direct evidence for migration
of newly generated neurons from the SEZ to the cerebral cortex
during postnatal stages (Fagel et al., 2006; Inta et al., 2008). Interestingly, many of the newly generated neurons express the T-box
transcription factor Tbr1, a transcription factor characterizing the
glutamatergic lineage in the telencephalon (Hevner et al., 2006),
which may argue at first sight for local regeneration. However,
recent evidence points to the possibility that Tbr1 positive cells can
also be recruited from the SEZ towards the cerebral cortex following
injury as the dorsal SEZ can serve as a life-long source for glutamatergic neurons (Brill et al., 2009). Taken together, at the current
stage, evidence for neurogenesis from early postnatal astroglia
either in the intact or injured cerebral cortex remains inconclusive.
To distinguish between the two possibilities of maintenance or reacquisition of neurogenic potential by early postnatal cortical
astroglia or the recruitment of astroglial stem cell derived progenitors from neurogenic brain regions such as the SEZ conclusively,
new fate-mapping tools need to be developed that would allow for
unambiguously distinguishing between different astroglial populations in the adult cerebral cortex and neurogenic zones.
However, in order to do so, one needs first to identify genes, which
are selectively expressed in one population, but not the other.
3. Lineage reprogramming of early postnatal astroglia
Based on the observation that Pax6 is expressed in radial glia
and crucial for their proper morphology (Gotz et al., 1998), Magdalena Götz and her laboratory showed that Pax6 is also required
for the genesis of glutamatergic neurons from cortical radial glia
(Heins et al., 2002). This led to the hypothesis that lack of expression of neurogenic transcription factors in astroglia may be one of
the primary causes for the cessation of neurogenesis. Interestingly,
the neurogenic competence of cortical precursor cells appears to be
restricted by the polycomb group (PcG) complex causing the
repression of neurogenins' genes through histone acetylation and
methylation, thereby promoting the transition from a neurogenic
to a astrocytic fate (Hirabayashi et al., 2009). Accordingly, knockout
of key components of the PcG complex such as Ring1B prolong the
neurogenic and delay the onset of the astrogenic phase of corticogenesis (Hirabayashi et al., 2009) Thus, would re-expression of
neurogenic transcription factors re-endow intrinsically astrogenic
precursors with a neurogenic potential? Indeed, retrovirus-mediated re-expression of Pax6 in cultured astroglia isolated from the
cerebral cortex between postnatal days 5e7 resulted in the rapid
down-regulation of astroglial markers such as glial fibrillary acidic
protein (GFAP) and the up-regulation of the early neuronal marker
TuJ1 (Heins et al., 2002). This study however left open whether
these obvious signs of neurogenesis represent a true transdifferentiation or rather reflects a stress response of the transduced
cells. If a full neuronal program was induced by Pax6 one would
expect these cells to acquire also functional properties of neurons,
such as action potential firing. Indeed a subsequent study showed
that not only forced expression of Pax6, but also of other neurogenic transcription factors expressed in the developing forebrain,
such as the proneural proteins Neurogenin2 (Neurog2) and mouse
achaeteescute homologue1 (Mash1), endow astroglial cells with
the hallmark of repetitive action potential firing (Berninger et al.,
2007a). Of note, Neurog2, but neither Pax6 nor Mash1 induced the
expression of Tbr1 indicating that Neurog2 does not only induce
a generic neuronal fate in early postnatal astroglia, but also appears
to specify these cells towards the glutamatergic lineage (Berninger
et al., 2007a).
A particularly important piece of evidence for lineage reprogramming of early postnatal astroglia was provided by single cell
tracking of cells from hGFAP-GFP mice that were subsequently
transduced with Neurog2 (Berninger et al., 2007a). These experiments revealed that the metamorphosis from astroglia to neuron
takes about four to six days and is accompanied by changes in
morphology and in migratory behaviour (Fig. 1) similar to what has
been observed in cortical precursors (LoTurco and Bai, 2006). Such
direct effect of Neurog2 on the morphological and migratory
features of astroglia undergoing neuronal metamorphosis is
consistent with the recent finding that one of Neurog2's direct
targets is the constitutively active small GTP-binding protein Rnd2
thereby regulating the morphology and migratory behaviour of
early cortical progenitors (Heng et al., 2008).
Yet, for stable and complete lineage reprogramming of astroglia
crucial would be evidence that astroglia-derived neurons are
capable of synapse-formation. Our lab could now show that
consistent with the up-regulation of Tbr1, forced expression of
Neurog2 directs astroglia to give rise to neurons forming fully
functional glutamatergic synapses (Heinrich et al., unpublished).
Importantly, fate mapping, using a mouse line expressing a tamoxifen-inducible Cre recombinase driven by the astroglia specific
GLAST promoter, corroborated the genesis of functional neurons
from postnatal astroglia (Heinrich et al, unpublished). Moreover,
forced expression of the mouse distal less homologue Dlx2, a transcription factor crucially involved in the genesis of GABAergic
neurons during embryonic development and adult neurogenesis
(Brill et al., 2008; Petryniak et al., 2007), showed that the same
cortical astroglia can be directed towards the genesis of functional
GABAergic neurons (Heinrich et al., unpublished). These data show
that, albeit lineage restricted under physiological conditions, early
postnatal astroglia can be stably reprogrammed towards distinct
neuronal identities. An important next step will be to assess the
feasibility of neurogenic reprogramming of astroglial cells at early
postnatal stages in vivo. Guided differentiation of inhibitory neurons
from astroglia may be an interesting alternative to transplantation as
a preclinical approach to treat epilepsies (Baraban et al., 2009;
Richardson et al., 2008), especially of the types caused by early
cortical malformations which in humans are often drug-resistant
and can only be treated surgically (Gupta et al., 2004).
4. Neurogenesis from mature astroglia in vivo?
It is necessary to halt here for a moment and to consider in what
respects mature prototypic parenchymal astrocytes differ from
astroglial stem cells such as those residing in the SEZ, as this may
have an important bearing on the question why normal astroglia in
the intact brain lack stem cell properties and do not spontaneously
generate neurons. In fact, parenchymal and stem cell astroglia have
B. Berninger / Neuropharmacology 58 (2010) 894e902
897
Fig. 1. Single cell tracking reveals the metamorphosis from astroglia to neuron. The upper micrographs show a bright field and the corresponding fluorescence image of an astroglia
derived from a P7 cerebral cortex at time point 0 (0:00.00; days:hours:minutes). By 1.5 days following transfection with an expression plasmid encoding Neurog2-IRES-DsRed,
expression of DsRed becomes visible and along with it signs of morphological change (lower micrographs). By 4 days the transfected cell has developed into a neuron (right
micrograph).
been shown to differ substantially in morphology, chemical
phenotype and their physiological characteristics. For instance,
dividing GFAP-positive cells in the adult SEZ were found to exhibit
bipolar and unipolar morphologies unlike non-neurogenic multipolar astroglia (Garcia et al., 2004; Mirzadeh et al., 2008). On
a molecular level, SEZ cells with neurosphere-forming capacity
express on their surface the carbohydrate LewisX/Cd15 (Capela and
Temple, 2002; Imura et al., 2006). Of note, LeX expression was
found in GFAP-positive cells from the adult SEZ, but not the cerebral
cortex (Imura et al., 2006). Finally, electrophysiological analysis
suggests that GFAP-expressing cells in the SEZ display a unique
phenotype between radial glia and parenchymal astrocytes, yet
they can also perform typical astrocytic functions such as potassium and glutamate buffering (Liu et al., 2006b). However, given
the fact that both stem cell and non-stem cell astroglia can be found
in the adult SEZ, it is not certain whether the physiological characteristics described in the latter study are pertinent to either
populations or only one of them.
The very nature of the stem cell residing within the SEZ, i.e.
whether truly astrocytic or not, has been hotly debated for quite
some time (for review see Chojnacki et al., 2009) and the precise
nature of the stem cell is still not fully understood. One of the major
impediments to resolving this issue is lack of a methodology to
prospectively isolate at high purity the stem cell population(s)
within the SEZ, which would allow performing a transcriptome
analysis and thereby a direct comparison to parenchymal astrocytes (Cahoy et al., 2008). Obviously additional differences apart
from the above mentioned (Garcia et al., 2004; Imura et al., 2003,
2006; Liu et al., 2006b) are expected to be found, as even “normal”
astrocytes exhibit substantial degree of heterogeneity according to
their tissue location, i.e. gray or white matter, and there is evidence
that this heterogeneity is transcriptionally specified during development not unlike neuronal subtypes, involving even the same sets
of transcription factors (Hochstim et al., 2008). Besides such type of
astroglial heterogeneity, stem cell and parenchymal astrocytes
differ in the very fact of the proliferative capacity of the former,
while the latter are largely postmitotic in the intact brain (Buffo
et al., 2008). Thus, there must be obviously differences in the
expression of cell cycle related genes between these populations.
Other differences that may be expected on a first glance relate to
specific functions of astrocytes within the parenchyma. For
instance, parenchymal astrocytes participate in the so-called
tripartite synapse, i.e. play a fundamental role in regulating and
modulating synaptic transmission by their ability to sense and
respond to neurotransmitter as well as their capacity to secrete socalled gliotransmitters (Haydon and Carmignoto, 2006; Wang and
Bordey, 2008). However, although no exact replica of a tripartite
synapse can be found in the adult SEZ, astroglial stem cells do
express the glutamate transporter GLAST (Mori et al., 2006; Ninkovic et al., 2007) and it has been proposed that glutamate is
secreted by astroglial stem cells that in turn acts on migrating
neuroblasts (Platel et al., 2008). Conversely, neuroblasts secrete
GABA that can be sensed by the astroglial stem cells modulating
their rate of proliferation (Liu et al., 2005). These data suggest that
astroglial stem cells may not fundamentally differ from parenchymal astroglia with respect to their ability to interact with
neuronal cells via neuro- and gliotransmitters. Another potential
difference may be expected to be found regarding the parenchymal
astrocytes' ability to regulate local blood flow (Gordon et al., 2007)
through calcium elevation in their end feet that engage in contact
with capillaries (Mulligan and MacVicar, 2004). However, again,
astroglial stem cells also engage with blood vessels via long basal
processes at sites devoid of classical astrocytic end feet (Mirzadeh
et al., 2008; Shen et al., 2008; Tavazoie et al., 2008). While this
contact is primarily thought of as exerting an important regulation
of stem cell behaviour, we do not know whether astroglial stem
cells can also wield some influence on these blood vessels. In
summary, astroglial stem cells at least resemble classical parenchymal astrocytes in their engagement with neuronal cells and the
vasculature, but the precise nature of these engagements is likely to
reflect their respective functions, i.e. the regulation of stem cell and
precursor proliferation on one hand and metabolic control and
synaptic modulation on the other requiring specific structural and
molecular adaptations.
So far no data are available on the effect of forced expression of
neurogenic fate determinants in quiescent mature astroglia.
However, preliminary data from our lab have shown that reexpression of Neurog2 in glia from the adult cerebral cortex in
culture is not sufficient to direct these cells towards neurogenesis
(unpublished observation), indicating that at later developmental
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B. Berninger / Neuropharmacology 58 (2010) 894e902
stages forced expression of single transcription factors can no
longer lift a glial fate restriction. This strongly suggests that at that
stage additional epigenetic modification may have occurred
besides silencing of neurogenic fate determinants by the PcG
complex (Hirabayashi et al., 2009) and thus by-passing the latter by
forced expression of the fate determinants themselves is no longer
sufficient to induce the downstream neurogenic program.
One major barrier to reprogramming of mature astroglia by
neurogenic fate determinants may be the fact that these cells do not
proliferate, but are essentially postmitotic (Buffo et al., 2008). Yet,
neurogenic fate determinants normally act in proliferative cells,
meaning that an activated cell cycle may provide a more favourable
context for their action. Following a CNS lesion astrocytes become
reactive and depending on the severity of the injury eventually can
re-enter the cell cycle and proliferate (Buffo et al., 2008; Gadea
et al., 2008, for review see Sofroniew, 2009). Interestingly, reactive
astrocytes up-regulate the expression of the epidermal growth
factor receptor (EGFR) (Codeluppi et al., 2009; Liu et al., 2006a),
therein resembling radial glia (Sun et al., 2005) as well so called
activated astrocytes in the adult SEZ (Doetsch et al., 2002; Pastrana
et al., 2009), i.e. glia with proliferative capacity. Might reactive
astroglia therefore represent a more amenable target for reprogramming by neurogenic fate determinants? In a earlier study, Buffo
and colleagues showed that stab wound or ischemic injury within
the cerebral cortex is followed by a massive up-regulation of the
basic helixeloopehelix transcription factor Olig2 (Buffo et al.,
2005), some of which co-localises with the astroglial marker S100b.
Indeed some of the Olig2-positive cells proliferate (Buffo et al.,
2005) and give rise to new astrocytes (Buffo et al., 2008; Chen et al.,
2008; Tatsumi et al., 2008). There is evidence that maintained
expression of Olig2 contributes to keep the newly generated cells
within the astroglial lineage. When Olig2-mediated transcriptional
repression was converted into transcriptional activation by retroviral expression of a Olig2VP16 fusion protein in proliferating cells,
some of the transduced cells up-regulated doublecortin (DCX) by
7e14 days post infection, an effect which was accompanied by upregulation of Pax6 and could also be mimicked by forced expression
of Pax6 itself (Buffo et al., 2005). However, the neurogenic effect
was only transient, as the number of DCX positive cells had drastically declined after one month, either due to death or because
lineage transgression was only partial and had been reverted again.
While this study revealed an interesting response to forced
expression of fate determinants in proliferating cells within the
injured cerebral cortex, it remained unclear whether the responsive
population indeed comprises astroglia. To address this, the Götz
laboratory then went on to examine the astroglial response to
injury by genetic fate mapping using the above mentioned GLAST::
CreERT2 mice (Buffo et al., 2008). While very few astroglia proliferated in the intact brain, following stab wound injury reporterpositive cells incorporated the thymidine analogue BrdU indicating
that quiescent astroglia do indeed resume proliferation. Importantly, in vivo, reporter-positive cells stayed within the astroglial
lineage as the vast majority of fate-mapped cells expressed astroglial markers. This data would indicate that despite of proliferative
response reactive astroglia are not capable of generating neurons. It
came thus as a surprise that when isolated in vitro and cultured
under serum free conditions in the presence of EGF and FGF2, cells
from the injury site gave rise to self-renewing and multipotent
neurospheres generating astroglia, oligodendroglia and, most
strikingly, also to few neurons (Buffo et al., 2008). Notably, neurosphere formation could also be elicited from reporter-positive cells
indicating that it is indeed reactive astroglia that can initiate neurosphere formation. Of note, neurospheres can also be isolated from
the post-stroke cerebral cortex (Nakagomi et al., 2009a,b). By local
lentiviral infection of the cortical tissue subject to subsequent
stroke induction, the authors of the latter study could show that it
is indeed cells local to the damaged cortex that give rise to neurospheres rather than neural stem cells recruited from the SEZ
(Nakagomi et al., 2009b). Thus, these data suggest that following
injury quiescent astroglia resume proliferation whereby they
de-differentiate and when isolated in vitro assume some of the
hallmark properties of adult astroglial stem cells. It will be interesting to investigate whether these cells can indeed undergo neurogenesis in a more favourable environment such as the adult SEZ
as it may be the absence of specific niche factors which keeps these
cells within the astroglial lineage. What factors account for the
metamorphosis of a quiescent astrocyte into an astroglial cell with
stem cell properties? Two recent studies shed some light on this
intriguing astroglial response. Jiao and Chen (2008) showed that
Shh can induce the formation of neurospheres from dissociated
tissue of the adult cerebral cortex. Importantly, using transgenic
mice expressing GFP driven by the GFAP promoter, Jiao and Chen
(2008) were able to show that Shh induces neurosphere formation
from astroglial cells. While this effect may be a pharmacological,
Amankulor et al. (2009) demonstrated that injury induces Shh
expression in GFAP-positive reactive astrocytes due to pro-inflammatory stimuli provided by macrophages. Notably, the increase in
Shh expression resulted in the local activation of the Shh downstream mediator Gli, a response which was blocked by the Shh
antagonist cyclopamine. Finally, cyclopamine also diminished the
injury-induced proliferation and up-regulation of Olig2. These
studies thus suggest that it may be the injury-induced activation of
the Shh pathway which endows previously quiescent astrocytes
with stem cell like properties, an effect consistent with the fundamental role of Shh in stem and progenitor cell maintenance in the
adult stem cell niches (Ahn and Joyner, 2005; Han et al., 2008;
Machold et al., 2003; Palma et al., 2005). Two new questions arise
then: firstly, whether the acquisition of stem cell properties is
accompanied by or even requires the elaboration of cilia-like
structures in reactive astroglia given the pivotal role of cilia for Shh
signal transduction in stem cells; and secondly, whether the absence
of neurogenesis following injury is partly due to overstimulation of
the Shh pathway resulting in a maintained expression of Olig2 and
hence keeping the proliferating cells within the glial lineage. Alternatively, in vivo de-differentiation of astrocytes during reactive
gliosis may remain incomplete with the consequence that epigenetically silenced genes required for neurogenesis do simply not
become accessible and fail to be re-activated.
5. Lineage reprogramming of retinal pigment epithelium
Other examples of transgression from a non-neuronal towards
the neuronal lineage have been described in the eye. Beneath the
photoreceptors is a layer of non-neural pigmented cells, the so called
retinal pigmented epithelium (RPE). Indeed the first evidence for
physiologically occurring transdifferentiation stems from studies in
urudele amphibians, which showed that upon removal of the neural
retina cells residing in the RPE can de-differentiate and regenerate
the entire neural retinal tissue through a process that recapitulates
development (for review see Lamba et al., 2008a). Studies in the
developing chick embryo have shown that the RPE at early stages of
differentiation can still transdifferentiate following surgical removal
of the retina and exposure to acidic or basic fibroblast growth factor
(FGF2) (Guillemot and Cepko, 1992; Park and Hollenberg, 1989;
Pittack et al., 1991; Sakaguchi et al., 1997). Notably, growth factor
treatment stimulates the expression of Pax6 in RPE and forced
expression of Pax6 is sufficient to drive transdifferentiation in the
absence of neural retina removal and FGF treatment (Azuma et al.,
2005). To a more limited degree, transdifferentiation of RPE tissue
could also be induced by forced expression of Sox2, an effect that
B. Berninger / Neuropharmacology 58 (2010) 894e902
may be due to up-regulation of FGF2 (Ma et al., 2009). Moreover,
forced expression of Neurog2 or atonal homologue 5 can induce the
generation of photoreceptor and retinal ganglion cell-like neurons
in embryonic day 6 chick RPE cultures (Yan et al., 2001). However, it
remains to be shown under which conditions RPE-to-neural retina
transdifferentiation could also be induced in more mature RPE tissue
in vivo, and especially in mammals. Interestingly, similar to reactive
astroglia, quiescent RPE cells resume proliferation after injury in pigs
(Kiilgaard et al., 2007) and neurosphere-like aggregates with limited
neurogenic potential can be isolated from the adult RPE tissue
(Engelhardt et al., 2005). Thus, lineage reprogramming of adult RPE
tissue may eventually become an interesting strategy for a regenerative response following retinal degeneration.
6. Müller glia as a source for new retinal neurons
An even more promising source for regenerating retinal neurons
is the so called Müller glia which span the retinal epithelium and
perform functions similar to astroglia in other parts of the CNS
(Lamba et al., 2008a). Interestingly despite their supportive role for
neuronal function, on a molecular level Müller glial cells exhibit
many similarities with retinal progenitor cells (for review see Jadhav et al., 2009). These finding have led to the notion that Müller
glia may represent a form of late stage retinal progenitor cell, which
acquire some specialized glial functions such as neurotransmitter
recycling, regulation of ion homeostasis and gliaeneuronal
communication, but do not irreversibly leave the progenitor state
(Jadhav et al., 2009). While Müller glia may resemble in some
aspects radial glia, they differ from these by the fact that rather than
being a neuronal precursor Müller glia is the last cell type to be
generated from retinal progenitors (Turner and Cepko, 1987).
Following injury however, Müller glia have been shown to
generate new neurons in non-mammalian vertebrates such as
teleost fish and birds (Fischer and Reh, 2001; Yurco and Cameron,
2005). While in the fish retina repair is nearly complete, regeneration is much less successful in birds. One reason for the incomplete
repair seems to be the persistence of Notch signalling (Hayes et al.,
2007). In the chick, acute injury causes Müller glia cells to resume
proliferation and to undergo de-differentiation (Fischer and
Reh, 2001), which is accompanied by the induction of Notch1 and
Hes5 expression (Hayes et al., 2007). Both proliferation and dedifferentiation were found to be decreased when Notch signalling
was blocked in the early regeneration process, suggesting that
induction of Notch signalling is a critical step in the de-differentiation program. However, when Notch signalling is blocked at later
stages, i.e. after de-differentiation of Müller glia, the number of
newly generated neurons was increased suggesting that Notch signalling exerts a dual role in the regenerative response.
What is the regenerative capacity of Müller glia in the
mammalian retina? There is evidence in young adult rats for
regeneration of some photoreceptor and bipolar cells following
neurotoxic injury (Ooto et al., 2004). Low numbers of Müller glia
were found to proliferate following injury suggesting that also in
the mammalian retina this cell type retains some regenerative
capacity. Both extrinsic and intrinsic factors were found to modulate the regenerative response: while retinoic acid treatment
enhanced the birth of new bipolar cells, forced expression of the
transcription factors NeuroD or Math3 induced the appearance of
newly generated amacrine cells in explants of the injured retinae.
Notably, while Pax6 alone did not promote neurogenesis, the
number of newly generated amacrine cells markedly increased
following co-expression of Pax6 and either NeuroD or Math3 after
two weeks of explant culture. Similarly, co-expression of the
homeobox transcription factor Crx with NeuroD favoured the
genesis of rhodopsin expressing photoreceptor cells.
899
In the adult mouse retina only few Müller glia cells enter the cell
cycle following a lesion paradigm that results in the selective death
of retinal ganglion and amacrine cells. However their proliferation
across the entire retina can be drastically enhanced by injection of
EGF or FGF1 (Karl et al., 2008). Interestingly, a high number of the
proliferating Müller glia were found to up-regulate Pax6 along with
other progenitor specific genes, suggesting de-differentiation of
quiescent Müller glia into retinal progenitor-like cell (Karl et al.,
2008). This response is in stark contrast to the otherwise very
similar response of reactive astroglia in the cerebral cortex, where
no up-regulation of Pax6 was observed (Buffo et al., 2005). Finally,
while the number of BrdU-positive cells declines during the first
week after their production, suggestive of cell death, a small
percentage (<5 %) of the de-differentiated Müller glia survived and
gave rise to new neurons acquiring characteristics of amacrine cells
in vivo. The limited survival rate of the newly generated amacrine
cells may be related to the observation that survival of adult
generated neurons is highly dependent on functional integration
(Petreanu and Alvarez-Buylla, 2002; Tashiro et al., 2006). Given that
Karl and colleagues observed only regeneration of amacrine but not
ganglion cells, the rather high death rate may be due to the fact that
the lack of regenerated ganglion cells severely compromised retinal
function thereby reducing the chance of the newly generated
amacrine cells for functional integration.
On a molecular level, the re-entry into the cell cycle of Müller
glial cells is accompanied by a change in the SWI/SNF chromatin
remodelling complex (Lamba et al., 2008b). This complex uses
energy from ATP hydrolysis to disrupt histone-DNA interactions
resulting in a remodelling of the chromatin structure thereby
regulating gene accessibility (Martens and Winston, 2003). The
SWI/SNF complex is composed of a catalytic ATPase subunit (either
Brg1 or Brm) and other subunits called BAFs (Brg/Brm-associated
factors). The precise composition of the core complex varies
according to the state of cellular differentiation (Lessard et al.,
2007; Yoo and Crabtree, 2009). For instance, the subunit BAF60c is
expressed in progenitors in the developing retina, but becomes
down-regulated upon differentiation and is virtually absent in the
adult retina (Lamba et al., 2008b). Importantly, when Müller glia in
the adult retina re-enter the cell cycle, BAF60c becomes reexpressed. Such change in subunit composition may alter the target
specificity of the SWI/SNF complex, since BAF60c is known to
physically interact with the Notch intracellular domain and Rbp-J,
thereby stabilizing their interaction and potentiating Notch signalling (Takeuchi et al., 2007). Such changes in target specificity of
the SWI/SNF complex may not only be crucial for the proliferative
response of Müller glia, but may also endow the Müller glial cells
with the ability to transgress their glial lineage and acquire
a neuronal phenotype.
7. Challenges ahead
From the above discussion emerges the following picture: at
early postnatal stages, astroglial cells in the cerebral cortex gradually lose the neurogenic potential they inherited from their radial
glial ancestors, most likely through mechanisms involving epigenetic modifications of genes required for running a neurogenic
program. However, initially they retain some degree of plasticity
which enables them to correctly interpret neurogenic cues as
shown by the experiments involving forced expression of neurogenic transcription factors (Berninger et al., 2007a; Heins et al.,
2002; Heinrich et al., unpublished). On reaching adulthood, this
residual capacity is lost. Yet, following injury stimuli, such as the
activation of the Shh pathway, can induce a complex process in the
now mature glia that eventually triggers the re-entry of these cells
into the cell cycle and the de-differentiation into a progenitor-like
900
B. Berninger / Neuropharmacology 58 (2010) 894e902
state (Buffo et al., 2008). The completeness of this de-differentiation as well as environmental factors acting upon the de-differentiated cells then determine whether the reactive glia remains in the
glial lineage or can spontaneously transgress it and generate
neuronal progeny. Within the cerebral cortex, the latter process
appears to occur at best rarely (Buffo et al., 2008; Magavi et al.,
2000), while in the retina limited neuronal regeneration may occur
(Karl et al., 2008). What are the limiting factors to a more efficient
regenerative neurogenic response? First of all, the environment
may provide anti-neurogenic stimuli that force newly generated
cells along the glial lineage despite their a priori ability to undergo
neurogenesis. However, at the same time, de-differentiation may
be only partial with the consequence that genes required for the
induction of neurogenesis are still epigenetically silenced. Such
restriction may be overcome by the forced expression of neurogenic master regulators provided that only these are subject to
epigenetic silencing or that their re-expression can cause the
erasure of epigenetic modifications of their downstream targets. In
early postnatal astroglia this seems to be the case indeed as single
neurogenic transcription factors can elicit the full neurogenic
response (Berninger et al., 2007a; Heinrich et al., unpublished).
However, in adult glia the capacity to execute the full neurogenic
program downstream of these factors appears to be rather limited
(Buffo et al., 2005). Here some approach analogous to the reprogramming of adult somatic cells may have to be considered (Jaenisch
and Young, 2008): pluripotency is superimposed onto a faterestricted somatic cell by re-establishing the entire regulatory
circuitry required for maintaining the pluripotent state through
a limited set of transcription factors. Is there an equivalent regulatory circuitry for a neural stem cell state? Several genes have been
identified that may contribute to the transcriptional network (Liu
et al., 2008; Molofsky et al., 2005; Shi et al., 2004; Suh et al., 2007),
but future studies will have to show whether there is indeed
a limited set of transcription factors that can superimpose a neural
stem cell like status onto a glial cell or any kind of somatic cell for
that matter. Ideally such a set of genes should reprogram endogenous astroglia even in the absence of any lesion and thus not rely on
the partial de-differentiation induced by the inflicted injury. That
such an approach is not condemned to failure a priori is suggested
by akin studies in the pancreas in vivo where the simultaneous coexpression of three different transcription factors was sufficient to
reprogram exocrine a cells into endocrine insulin-secreting b cells
(Zhou et al., 2008). Conceptually, the successful combination of
factors included one factor exerting normally its effects in early
pancreatic progenitors, a second factor involved in b cell specification and finally a third one required for b cell differentiation.
However, for a guided functional reconstitution of a damaged
neuronal circuitry it will be of crucial importance to regenerate not
only distinct neuron types at the same time, but also at a balanced
measure. Thus, to employ the approach of endogenous reprogramming we will have to work out precise strategies not only to
induce a generic neurogenic response but to instruct the diverse
neuron types that constitute a given circuit. In case of the cerebral
cortex, our growing knowledge of the transcription factors involved
in the specification of the diverse neuronal populations (Molyneaux et al., 2007) will hopefully bring us into the position to test
one day whether local astroglia can be selectively driven towards
adopting the identity of the entire spectrum of neurons and
whether these then assemble into a functioning network. This is no
doubt far-fetched, but may be more than just a dream.
Acknowledgement
The author would like to thank Drs. Magdalena Götz, Christophe Heinrich and Aditi Deshpande for discussion and comments
on the manuscript. Furthermore the author is indebted to the two
anonymous reviewers for their constructive criticism on the
manuscript. Work by the author is supported by the DFG, the
BMBF and the Bavarian State Ministry of Sciences, Research and
the Arts (ForNeuroCell).
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