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. Author manuscript; available in PMC: 2022 Jul 29.
Published in final edited form as: Dev Neurobiol. 2022 May 23;82(5):367–374. doi: 10.1002/dneu.22880

In vivo glia-to-neuron conversion: pitfalls and solutions

Lei-Lei Wang 1, Chun-Li Zhang 1
PMCID: PMC9337910  NIHMSID: NIHMS1824369  PMID: 35535734

Abstract

Neuron loss and disruption of neural circuits are associated with many neurological conditions. A key question is how to rebuild neural circuits for functional improvements. In vivo glia-to-neuron (GtN) conversion emerges as a potential solution for regeneration-based therapeutics. This approach takes advantage of the regenerative ability of resident glial cells to produce new neurons through cell fate reprogramming. Significant progress has been made over the years in this emerging field. However, inappropriate analysis often leads to misleading conclusions that create confusion and hype. In this perspective, we point out the most salient pitfalls associated with some recent studies and provide solutions to prevent them in the future. The goal is to foster healthy development of this promising field and lay a solid cellular foundation for future regeneration-based medicine.

Keywords: BrdU, glia-to-neuron (GtN) conversion, in vivo reprogramming, lineage tracing, NEUROD1, PTBP1

1 |. INTRODUCTION

Traumatic injury or neurodegeneration frequently leads to permanent loss of neurons and irreversible long-term impairments. Since most regions of the adult mammalian central nervous system (CNS) lack neurogenic ability, a key question is how to repair the disrupted neural circuits for functional improvements. In the past decade, a paradigm-shifting regenerative strategy has emerged. This is to reprogram resident glial cells into neurons in vivo (Barker et al., 2018; G. Chen et al., 2015; Gascon et al., 2017; Götz & Bocchi, 2021; Tai et al., 2020; Wang & Zhang, 2018). These glial cells include not only the lineage-related astrocytes, Müller glia, NG2 glia (also known as oligodendrocyte precursor cells, OPCs) but also the lineage-distant microglia (Y. C. Chen et al., 2020; Gascón et al., 2016; Ge et al., 2020; Hoang et al., 2020; Jorstad et al., 2017; Lentini et al., 2021; M. H. Liu et al., 2020; Y. Liu et al., 2015; Matsuda et al., 2019; Mattugini et al., 2019; Niu et al., 2013; Pereira et al., 2017; Qian et al., 2020; Rivetti di Val Cervo et al., 2017; Z. Su et al., 2014; Tai et al., 2021; Torper et al., 2015; Wang et al., 2016; Wu et al., 2020; Zhou et al., 2020). Such glia-to-neuron (GtN) conversions are accomplished through controlling the expression of a single or a combination of fate-determining factors.

2 |. PERFECT “NEW” NEURONS FROM RESIDENT GLIA?

While a DCX-positive immature neuronal stage was observed in some of the in vivo glia reprogramming studies (Grande et al., 2013; Guo et al., 2014; Heinrich et al., 2014; Islam et al., 2015; Lentini et al., 2021; Maimon et al., 2021; Matsuda et al., 2019; Niu et al., 2015, 2013; Z. Su et al., 2014; Tai et al., 2021; Wang et al., 2016; Zhang et al., 2022), such an intermediate stage was not reported in many of the other studies especially those using adeno-associated viruses (AAVs). For example, AAV-mediated ectopic expression of NEUROD1 or knockdown of PTBP1 was reported to induce highly efficient astrocyte-to-neuron conversion, with rapid appearance of viral reporter-labeled mature neurons exhibiting perfect regional identity, electrophysiology, axonal projections, and connectivity (Y. C. Chen et al., 2020; Qian et al., 2020; Wu et al., 2020; Zhou et al., 2020). Astonishingly, studies with NEUROD1 or PTBP1 also reported greatly improved behavioral functions in many animal disease models including ischemic brain injury (Y. C. Chen et al., 2020; Tang et al., 2021), retinal injury (Zhou et al., 2020), Hungtington’s disease (Wu et al., 2020), temporal lobe epilepsy (Zheng et al., 2022), and Parkinson disease (Qian et al., 2020; Zhou et al., 2020). Based on these remarkable discoveries, a cureall, regeneration-based, therapeutic strategy is on the horizon for neural injuries, neurological diseases or symptoms. Such phenomenal discoveries immediately attracted huge financial investments for clinical translation.

Although behavioral improvements are all attributed to the perfect “new” neurons through GtN conversion, a completely overlooked key question is how manipulation of a generic factor, such as NEUROD1 or PTBP1, can theoretically generate diverse neuronal subtypes with precise regional identity and connectivity. Such a phenomenon, if true, will completely rewrite the whole history of developmental neuroscience, as we learned through decades of basic research that neuronal subtypes are specified through precise controls of genetic and epigenetic regulators and that axonal projections are guided by both attractive and repulsive cues. Even if glial cells might exhibit regional identity that could facilitate the generation of region-dependent neurons, how could reactive glial cells still maintain such identity and be converted to cortical layer-specific neurons after stroke injury that leads to loss of a big chunk of brain tissue (Y. C. Chen et al., 2020)? Are we at the dawn of a new era of neuroscience?

3 |. PERFECT “NEW” NEURONS ARE PRE-EXISTING NEURONS

Setting aside the theoretical puzzlements, what is the key evidence for the conclusion that “new” neurons are regenerated through GtN conversion in the case of NEUROD1 or PTBP1? Invariably, those perfect “new” neurons are identified solely based on the virus-expressed fluorescence reporter such as GFP or mCherry (Y. C. Chen et al., 2020; Qian et al., 2020; Wu et al., 2020; Zhou et al., 2020). These viral reporters are under the direct control of hGFAP promoter or driven by the strong ubiquitous CAG promoter but in a Cre-dependent fashion. The Cre recombinase, on the other hand, is regulated by the hGFAP promoter in the virus or mGfap promoter in a transgenic mouse line. Although the viral reporters are mainly restricted to astrocytes in the control virus group such as mCherry or RFP alone, they are highly efficiently detected in mature neurons with perfect regional identity in the NEUROD1 or PTBP1 group (Y. C. Chen et al., 2020; Qian et al., 2020; Wu et al., 2020; Zhou et al., 2020). As such, the simplest and straight-forward conclusion is that perfect “new” neurons are regenerated through GtN conversion by controlling the expression of NEUROD1 or PTBP1.

Is that simple? Is there any direct evidence showing that astrocytes are indeed the cell origin for those perfect “new” neurons? Has any of those studies excluded the possibility that the perfect “new” neurons are actually pre-existing neurons? The answers to these questions, unfortunately, are a resounding no. By prelabeling astrocytes with a genetically encoded reporter in genetic lineage tracing mouse lines and using a different reporter for the later injected AAVs (Wang et al., 2020; Wang et al., 2021), our recent study unambiguously demonstrated that resident astrocytes are not the cell origin for those viral reporter-labeled “new” neurons that were observed in the studies of NEUROD1 (Y. C. Chen et al., 2020; Tang et al., 2021; Wu et al., 2020) or PTBP1 (Qian et al., 2020; Zhou et al., 2020). This conclusion is further supported by the results of several independent studies on either NEUROD1 (Leib et al., 2022) or PTBP1 (W. Chen et al., 2021; Hoang et al., 2021; Leib et al., 2022). Conversely, by prelabeling endogenous neurons through a retrograde approach and using a different reporter for subsequently injected AAVs, our results rather clearly show that those presumed glia-converted perfect “new” neurons are endogenous pre-existing neurons (Wang et al., 2020, 2021). Such a result explains why those viral reporter-labeled “new” neurons exhibit perfect regional identity and have precise axonal projections and connections.

Without glia-converted new neurons as the cellular basis, how could the behaviors of those impaired animals be improved by controlling the expression of NEUROD1 or PTBP1 (Y. C. Chen et al., 2020; Qian et al., 2020; Tang et al., 2021; Wu et al., 2020; Zheng et al., 2022; Zhou et al., 2020)? This question awaits future independent studies for confirmations and mechanisms.

4 |. UNRELIABLE DETECTION SYSTEM LEADS TO MISLEADING INTERPRETATIONS

How virus-expressed reporters could be highly expressed in endogenous neurons under certain conditions such as in the NEUROD1 group but not in the control GFP or mCherry group? This phenomenon seems perplexing but has been long known in the neuroscience field (M. Su et al., 2004). Cell type-specificity of the hGFAP promoter can be significantly influenced by the downstream expressed gene sequences. For example, in transgenic animal studies, while GFP expression is restricted to astrocytes and the expression of lacZ is moderately detected in neurons, cathepsin A (a lysosome enzyme) showed particularly strong and widespread neuronal expression under the hGFAP promoter (M. Su et al., 2004). Consistent with these results, we also showed that NEUROD1, as well as several other examined transcription factors such as ASCL1, PAX6, and MYC, can lead to neuronal expression of the AAV-expressed reporter (Wang et al., 2020, 2021). The underlying molecular mechanism is not clear; nonetheless, NEUROD1’s neurogenic activity is not required (Wang et al., 2021). The viral reporter, on the other hand, seems to be cis-regulated by the expressed NEUROD1 sequences at least in the early time points (Wang et al., 2021). Such cis-regulation, however, could become pseudo trans-regulation, since AAVs can time-dependently concatemorize in head-to-head, head-to-tail, and tail-to-tail fashions (Yang et al., 1999). Interestingly, intermolecular recombination between AAVs can also occur and add additional complexity of gene regulations. Concatemorization and intermolecular recombination among AAVs may explain why the viral reporter could be more efficiently expressed in endogenous neurons after co-injections of AAVs expressing two or more genes such as NEUROD1 and DLX2 in the striatum (Wu et al., 2020).

The Cre-dependent FLEX (flip-excision), DiO (doublefloxed inverse orientation), or LSL (loxP-STOP-loxP) system, which is broadly employed in GtN conversions, is similarly subject to leakage in endogenous neurons (Fischer et al., 2019). Such basal neuronal leakage of the above systems may be further exacerbated by the downstream gene sequences, especially when combined with the AAV-expressed hGFAP-Cre that potentially form a feedforward loop to enhance expression of the viral reporter in endogenous neurons (Y. C. Chen et al., 2020; Wu et al., 2020).

Like in transgenic animals (M. Su et al., 2004), gene-dependent neuronal leakage of the viral reporter may also show preferences for certain brain regions and neuronal subtypes, suggesting that cellular context plays a similarly important role in determining cell type-specificity of the virusdriven reporters. After all, gene expression is governed by multiple factors for transcription, translation, and stability of RNAs and proteins. All these regulations are determined by the cellular context. Of note, neuronal leakage of the viral reporter is not only an issue with AAVs but also happens with the lentivirus system (Rao et al., 2021), although it is not clear how such leakage occurs. Therefore, conclusions cannot be clearly drawn from results that are solely based on the virus-expressed reporters.

5 |. IDENTIFICATION OF REACTIVE GLIA-CONVERTED NEW NEURONS

How to identify the newly generated neurons through GtN conversion? A simple and broadly applicable gold standard is BrdU- or EdU-based labeling (Figure 1a), which has been widely used for decades in stem cell biology and neurogenesis (Kuhn & Cooper-Kuhn, 2007; Taupin, 2007). As a nucleoside analog of thymidine, BrdU or EdU is incorporated into DNA of dividing cells during S-phase of the cell cycle. These cells can then be identified through an antibody- or nonantibody-based method. Since endogenous neurons are postmitotic, they will not be traced by such a method. This method, when combined with neuron-specific markers such as NeuN, MAP2, or SYN1, can uniquely identify new neurons that are converted from reactive glial cells undergoing proliferation or with a history of proliferation or when the GtN conversion passes through a proliferative progenitor stage.

FIGURE 1.

FIGURE 1

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Stringent methods for studying glia-to-neuron (GtN) conversion in vivo. (a) BrdU/EdU-based labeling of proliferative cells, which may be injury-induced reactive glia or reprogramming-induced neuroblasts. New neurons are then identified by staining of BrdU/EdU and a marker for mature neurons such as NeuN or MAP2. (b) Genetic lineage tracing of the reprogramming-induced new neurons. Resident glia are genetically labeled in mice harboring a glia-specific Cre and a Cre-dependent reporter that is under a constitutively active promoter. In most cases, the tamoxifen-inducible CreER™ or CreERT2 is preferred since the reporter can be selectively turned on in the adult stage. Reprogramming-induced new neurons from the reporter-positive glia can then be identified by immunohistochemistry. (c) Time-lapse in vivo imaging of GtN conversion. Resident glial cells are specifically labeled with a Cre-dependent reporter, as described in (b). After virus injection to initiate the reprogramming process, those virus-infected glial cells are then under time-lapse in vivo imaging for days or even weeks. Postimaging immunohistochemistry is then conducted to verify the neuronal identity of those converted glia. (d) Single cell RNA-sequencing (scRNA-seq) analysis of GtN conversion. As described in (b), resident glial cells are specifically traced with a reporter. At a predefined time point post-reprogramming, single reporter-positive cells are isolated through fluorescence-activated cell sorting (FACS) and processed for scRNA-seq. Pseudotime analysis will reveal a sequential order of cell state progression reflected by a trajectory of transcriptional states during GtN conversion. It may include resident glia, virus-infected but not yet converted glia, glia-converted immature and mature neurons

Through ectopic SOX2 expression, our group has reported that a large number of BrdU-labeled new neurons can be reprogrammed from resident glia in both the striatum and the spinal cord of adult mice (Niu et al., 2013; Z. Su et al., 2014; Tai et al., 2021; Wang et al., 2016; Zhang et al., 2022). The underlying mechanism is that glial cells are reprogrammed by SOX2 into proliferative progenitors and immature neurons. Consistent with results in the neurogenesis field, our ability to detect BrdU-labeled new neurons dismisses those concerns that BrdU might be toxic to glia-converted neurons.

What if the GtN conversion is direct without passing through a proliferative progenitor stage? Since a key promise of in vivo reprogramming is to target those reactive glial cells after traumatic brain injury, stroke, or neurodegeneration (Y. C. Chen et al., 2020; Qian et al., 2020; Tang et al., 2021; Wu et al., 2020; Zheng et al., 2022; Zhou et al., 2020), these proliferative glial cells can be prelabeled with BrdU/EdU and then subject to direct GtN conversions. Although some damaged DNAs or mitochondria might also be labeled (Taupin, 2007), this could easily be identified by examining the subcellular localization and labeling intensity and patterns. Therefore, BrdU/EdU-based labeling can serve as a broad and unambiguous approach to differentiate reactive glia-converted new neurons from those pre-existing mature neurons in the adult CNS.

Through specific targeting of proliferating cells, retroviruses can similarly be used to identify new neurons that are converted from reactive glial cells (Gascón et al., 2016; Heinrich et al., 2014; Lentini et al., 2021). This is normally accomplished by using the retrovirus-expressed reprogramming factors and a fluorescence reporter under a constitutively active promoter. Although such an approach is generally specific, it is known that retrovirus-transduced microglia can fuse with neighboring endogenous neurons, thereby leading to their labeling by the viral reporter (Ackman et al., 2006). These cell fusion-labeled endogenous neurons could be excluded by careful analysis of the morphology, contacting microglia, and nucleus staining.

Additional caveat with BrdU/EdU- or retrovirus-based approach is that neural stem cell-derived neurons can also be traced, especially when these cells can migrate to the lesion sites after injury or neurodegeneration (Benner et al., 2013). Attention should be paid to exclude the possibility of these stem cell-derived neurons being inadvertently considered as the glia-converted. One way is to inject the reprogramming virus into regions away from those neurogenic regions (Niu et al., 2013). Alternatively, neural stem cells and their derived neurons can be genetically traced and then excluded as the cell origin for GtN conversions (Niu et al., 2018; Niu et al., 2013; Tai et al., 2021; Zhang et al., 2022). The tamoxifen-inducible Nes-CreERT2 (Lagace et al., 2007) and Ascl1-CreERT2 (Kim et al., 2011) mouse lines are excellent for this purpose. Nonetheless, if the goal is to expand neurogenesis no matter what the cell source is, neural stem cells could be an excellent target since they are more plastic than any other somatic glial cells.

6 |. TRACING THE GLIAL CELL ORIGIN

How to identify or confirm the exact cell source for the presumed glia-converted neurons? As described above, the virus-expressed reporters cannot be used as lineage tracers due to leakiness of the short viral promoter, broad tropism of the viruses, and cis-regulation by the downstream genes. Fortunately, decades of studies on molecular and developmental biology have developed unique tools to genetically tag and follow the fates of resident glial cells. The glial cells in the CNS broadly refer to astrocytes, Müller glia, NG2 glia, oligodendrocytes, or microglia. Each of these cell types can be genetically and permanently traced with a reporter base on the Cre-loxP system. For example, the inducible Aldh1l1-CreERT2 (Srinivasan et al., 2016) and Pdgfra-CreER™ (Kang et al., 2010) can be employed to trace astrocytes and NG2 glia, respectively, when combined with a Cre-dependent genetic reporter under a ubiquitous and constitutively active promoter. Such a genetic reporter will continuously be expressed even after GtN conversions and thus mark the glial cell origin and their derivatives (Figure 1b). With genetic lineage tracings, our group and others have clearly showed that astrocytes or NG2 glia can be in vivo reprogrammed to produce new neurons in the adult mouse brain or spinal cord (Heinrich et al., 2014; Niu et al., 2013; Z. Su et al., 2014; Tai et al., 2021; Wang et al., 2016; Zhang et al., 2022).

An advantage of using lineage tracing mouse lines is that they can be thoroughly examined for cell type-specificity of the genetic reporters. Such examinations are essential since variable leaky expression in neurons can occur in certain Cre-containing mouse lines, especially when combined with a high efficiency reporter line such as the R26R-tdTomato (Madisen et al., 2010). Therefore, regions of interests should be broadly examined, and those animals with neuronal leaky expression of the reporter should be excluded from further analysis, as shown in our recent publication for using the Aldh1l1-CreERT2;R26R-tdTomato mouse line (Wang et al., 2021). Another example is the mGfap-Cre line (Gregorian et al., 2009), which exhibits leaky neuronal expression in multiple brain regions including the cortex and the midbrain (Wang et al., 2021); therefore, these regions should not have been used for GtN conversion in this mouse line (Qian et al., 2020).

Recently, it was argued that the Cre-loxP lineage tracing system cannot be used for examining GtN conversions due to a higher barrier (Zheng et al., 2022); however, such an argument is not supported by any available scientific data. The Cre-loxP system has been well applied to the neuroscience field for nearly three decades (Tsien, 2016), and it was also successfully employed to trace the glial cell origin for GtN conversions in the mouse brain, spinal cord, or eye (Heinrich et al., 2014; Hoang et al., 2020; Niu et al., 2013; Z. Su et al., 2014; Tai et al., 2021; Wang et al., 2016; Zhang et al., 2022). The simple reason for not being genetically traced for the viral reporter-positive neurons in those NEUROD1 or PTBP1 studies is that they are pre-existing neurons, not converted from resident glial cells (W. Chen et al., 2021; Hoang et al., 2021; Leib et al., 2022; Wang et al., 2020, 2021).

Another way to utilize genetic lineage tracing is to conduct in vivo time-lapse imaging, as beautifully demonstrated in studying adult hippocampal neurogenesis (Pilz et al., 2018). The traced cells can be chronically followed by two-photon confocal imaging, and their final identity is then confirmed by post hoc immunohistochemical analysis (Figure 1c). In combination with genetic lineage tracing, single cell RNA sequencing (scRNA-seq) is also a great tool to examine cell lineage progression from traced glial cells to converted neurons (Figure 1d) (Hoang et al., 2020; Todd et al., 2021; Zhang et al., 2022). It may also reveal the underlying gene regulatory networks and identify key factors for GtN conversions.

7 |. CONCLUDING REMARKS

In vivo GtN conversion has huge potentials for regenerative medicine. However, we must not be dumbfounded by certain artifacts brought by inappropriate analysis. A clear understanding of the cellular basis for GtN conversion is a prerequisite for its future clinical applications. We propose that stringent lineage tracing methods should be broadly applied to such studies (Calzolari & Berninger, 2021; Leaman et al., 2022; Svendsen & Sofroniew, 2022; Wang & Zhang, 2022). Genetic lineage tracing in combination with in vivo time-lapse imaging or scRNA-seq is superior, though they may be technically challenging and costly prohibitive. On the other hand, traditional genetic lineage tracing in well-characterized mouse lines and prelabeling of reactive glial cells with BrdU or EdU can be readily conducted in most research labs. Once the GtN conversion process is well characterized in genetically modified animal models, it can then be applied to preclinical studies with high confidence in large animals with which the genetic tools might not be available. The future for in vivo GtN conversion is bright, but we must be vigilant and diligent.

ACKNOWLEDGMENTS

We thank members of the Zhang laboratory for discussions. Chun-Li Zhang is a W. W. Caruth, Jr. Scholar in Biomedical Research. The work in Zhang laboratory was supported by the Decherd Foundation, the Texas Alzheimer’s Research and Care Consortium (TARCC2020), and NIH Grants (NS099073, NS092616, NS111776, and NS117065).

Funding information

Decherd Foundation; Texas Alzheimer’s Research and Care Consortium (TARCC2020); NIH Grants, Grant/Award Numbers: NS099073, NS092616, NS111776, NS117065, NS127375

DATA AVAILABILITY STATEMENT

No data are included in this study.

Footnotes

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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