Recent discoveries in the field of regeneration research inspire great optimism for future therapies aimed at repairing the injured or diseased human brain. Of particular clinical significance is the approach to promote neurogenesis from endogenous parenchymal cells independent of the use of embryonic or induced pluripotent stem cells. Perhaps the most attractive strategy appears to be direct in vivo cell reprogramming to induce a neuronal fate. However, the initial enthusiasm in the field has been tempered, as quantity and quality of replenished neuronal populations have remained low. An exciting new study by Magdalena Goetz and co-workers (1) published in Cell Stem Cell sheds new light on the problem of cell death at the time of fate conversion and emphasizes the importance of coordinated metabolic reprogramming to increase neurogenesis successfully. In this editorial we summarize these critical new findings and consider them in the context of the adult brain’s potential for plasticity and self-regeneration.
Creating an ectopic neurogenic niche to drive neurogenesis de novo endogenously
The discovery that adult neurogenesis persists throughout life has stirred great optimism as a potential therapeutic mechanism that can be applied to the entire brain. External stimuli (e.g., physical exercise, exposure to an enriched environment) (2,3); and intrinsic signaling pathways [e.g., neurotransmitters such as serotonin, growth and neurotrophic factors such as the brain-derived neurotrophic factor (BDNF) reviewed in (4)]; modulate neurogenesis in concert with cell-autonomous survival and differentiation factors (5). Although frequently expressed simultaneously within the same cell, transcription factors differ widely in their effects, i.e., on cell proliferation, differentiation, cell death and cell survival, with most of them showing a transient expression pattern [reviewed in (6)]. An intensely studied pro-neurogenic stimulus is physical exercise which has been shown to robustly increase the number of proliferating progenitor cells in the dentate gyrus of the hippocampus (7,8), thus increasing the reservoir of cells that can be quickly recruited into a neuronal fate (9). Interestingly, voluntary running also increases the number of proliferating Neuron-glia2 (NG2) glia precursor cells in the amygdala (10). Enlarging the glia pool may be especially important in demyelinating diseases, while therapeutically enforced neurogenesis is needed in neurodegenerative disorders and in stroke. Cell engineering to drive neurogenesis de novo from stem and non-stem cells of the (diseased) brain is desirable, as neurons may be lost in neurological and neurosurgical conditions such as traumatic brain injury, stroke as well as the classical neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease.
The capacity of the adult brain to self-regenerate upon injury is limited. Replacement of neurons only takes place in a few spatially restricted neurogenic regions, where neural stem cells retain fate plasticity [the subventricular zone and the subgranular zone of the dentate gyrus in rodents, the adult human hippocampus and striatum; reviewed in (11)]. Outside these neurogenic regions, proliferating NG2 glia precursors are the endogenous source of cells with multipotent potential (12,13), but they remain glial-restricted in situ. Hence, most of the brain consists of non-neurogenic regions apparently incapable of repair. The knowledge gained from neurogenic niches can be used to drive cell fate of (non-neurogenic) resident progenitor cells. Previous in vivo work in rodents has provided insights into initiation of adult neurogenesis in models of targeted apoptotic cell death (14,15), as well as after acute brain injury (stab lesion) (16,17). Although the goal of generating new neurons from endogenous precursor cells was achieved, these approaches required specific forms of tissue damage. However, non-selective injury, as in the case of traumatic brain injury, never, or in the case of brain ischemia, barely (18) leads to endogenous neuronal cell birth.
Direct in vivo reprogramming into the desired phenotype
‘Direct reprogramming’ has become a popular approach to induce a neuronal fate. Direct reprogramming of somatic cells into pluripotent stem cells in vitro (19) has opened up entirely new possibilities in regeneration research. Successful direct in vivo reprogramming of desired tissue-specific cells was achieved in the pancreas (20) and myocardium (21). Recent research has refined technologies to induce neurogenesis using gene therapy. In the adult brain, gene delivery and lineage tracing approaches were used to create a neurogenic niche and to drive cell fate in vivo. Using one of the several factors studied [e.g., BDNF, Sox2, Pax6, Olig2, achaete-scute complex homolog-like 1 (Ascl1), Bcl-2], cell genesis or enlargement of the progenitor cell pool was accomplished in neurogenic niches and following stroke. It has been shown that selectively modifying transcriptional cues is sufficient to induce a cell fate switch in the dentate gyrus neurogenic niche and striatum: e.g., conversion of neural stem/progenitor cells into oligodendrocytes by Ascl1, also known as Mash1 (22); enlargement of the pool of glia cells that may support a cell-replacement strategy aimed at remyelination after injury (23); reprogramming of striatal astrocytes into neuroblasts by viral-mediated expression of Sox2 (24); and retroviral delivery of the lineage-instruction factors Olig2 and Pax6 altering the neurogenesis-gliogenesis ratio (25). Although retroviral overexpression of the pro-neuronal factor Pax6 pushes precursor cells in the dentate gyrus into a neuronal phenotype, survival of these engineered cells is significantly reduced (25). In models of mild cerebral ischemia, induced cell death was delayed by altering cell cycle regulatory mechanisms (26), and gene delivery of fate determinants induced the birth of immature neurons in the non-neurogenic injured striatum (18). Newly generated cells after brain injury or stroke were identified by co-expression of the thymidine analog bromodeoxyuridine (BrdU) and doublecortin. Known as a marker of immature neurons in neurogenic regions, doublecortin has distinctive characteristics dependent on the niche environment (27).
Altogether, only few (typically immature) cells are generated upon viral gene delivery of fate determinants. Furthermore, bona fide neurons commonly identified by co-expression of BrdU and the neuronal nuclear marker NeuN may possibly represent an artifact (28). Indeed, aberrant DNA synthesis may lead to incorporation of BrdU and, thus, may indicate the induction of cell death rather than the birth of a new neuron (26,28). Another challenge besides ectopic induction of neurogenesis is to generate neurons that become functionally integrated. So far, these efforts have met with limited success, although resident glia cells have been shown to differentiate into spiking neuroblasts in models of stroke (18,29). Cleary, outside the canonical neurogenic brain region, newly generated immature neurons lack crucial signals within the non-neurogenic and injured region. The niche environment therefore needs to be primed as well for neuronal fate conversion and subsequent neuronal differentiation, which, in turn, requires coordinated and cell type-specific transcription of the genes involved alongside the release of neurotrophic factors to foster a pro-neurogenic milieu.
Taken together, the existing literature suggest that driving neurogenesis de novo from stem/progenitor cells in vivo has its downsides as it often leads to cell death, disruption of homeostasis within the niche, and not to the desired recovery of the region. In their study, Magdalena Goetz and co-workers define cell death as the ‘main limiting factor of neuronal reprogramming’ and study coordinated metabolic reprogramming at the time of cell fate transition. At this ‘metabolic checkpoint’, key neuronal genes are rapidly regulated leading to either cell death or survival. Gascón et al. made use of the highly advanced method of continuous single-cell imaging to track fate conversion of (I) Ascl1-transfected astrocytes and (II) after brain injury in vivo. Thereby, the focus of their study was on metabolic features common to all cell types that may improve cellular reprogramming (1).
Redox homeostasis in neuronal reprogramming
Reactive oxygen species (ROS) production, lipid peroxidation, or cell death-regulating proteins such as Bcl-2 are emerging as potent contributors to cell death or survival decisions. Redox homeostasis, the interplay of oxidative stress and antioxidant signaling (30), is a biological feature common to all cell types. It marks a metabolic interface for intrinsic vs. environmental signals and can command cell death programs. At neuronal fate conversion, reprogramming from glycolytic to oxidative metabolism formidably challenges redox homeostasis. High oxidation levels lead to apoptosis or ferroptosis (an oxidative stress-dependent/caspase-independent form of cell death characterized by accumulation of lipid peroxidation products), thereby limiting the number of cells adopting a neuronal phenotype. This finding may not come as a big surprise to researchers at the bench, as we already know from daily cell culture work that neuronal progenitors almost literally hate oxidation. In the study by Gascón and co-workers, treatment with liproxstatin-1, a ferroptosis inhibitor, was shown to not only prevent cell death but also to improve reprogramming efficiency. This was accompanied by suppression of ROS production and lipid peroxidation, leading to increased survival of new neurons. Another efficient molecule to reduce lipid peroxidation is forskolin. When added to Ascl1-transduced astrocytes, a cell type-independent increase in fate transition was observed which Nrf2 anti-oxidative pathways supported (1).
Yet another regulator of the metabolic switch is the anti-apoptotic protein Bcl-2 which improves metabolic conversion at the time of direct neuronal reprogramming. In their report, Magdalena Goetz and coworkers demonstrate that Bcl-2 co-expression in Ascl1-transfected astroglia cells facilitates neuronal conversion in tandem with increased cell survival via the non-canonical pathway (Bcl-2 inhibits apoptosis by sequestering Bax/Bak when phosphorylated at serine 69/70) (1). Thereby, Bcl-2 improves maturation uncoupled from cell proliferation leading to successful neuronal transition: up to 80% of Ascl1-transduced astrocytes turned into neurons upon Bcl-2 (1). Furthermore, both Bcl-2 and forskolin together enhanced conversion efficiency in the presence of neuronal fate determinants (e.g., Neurog2) (1).
So far, the replenishment of neuronal populations in the injured brain has fallen short both in quantity and in quality. The oxidative stress response along with lipid peroxidation plays a key role in directed reprogramming as shown in vitro (1). In situ, injury profoundly affects the niche environment inducing inflammation and thereby shifting redox homeostasis. Previous in vivo work by the same group had already provided invaluable insights into initiation of adult neurogenesis in the injured brain (16,17). Here, the authors followed up on their previous experiments and tested whether Bcl-2 and forskolin are capable of enhancing neuronal transition after acute stab wound injury. Excitingly, retroviral gene delivery and singe-cell imaging revealed that direct reprogramming of resident cortical progenitor cells with Bcl-2 (in the presence of the fate determinant Neurog2) increased neuronal conversion and accelerated neuronal maturation at 10 days post-injection. Importantly, two-thirds of transduced cells in vivo were shown to express mature neuronal marker NeuN (1).
Intriguingly, co-transfection with another anti-oxidant and ‘top transcriptional regulator’, calcitriol receptor (a.k.a., vitamin D receptor), further improved neuron transition and complexity. Calcitriol-dependent anti-oxidant signaling protected against lipid peroxidation by a mechanism that was independent of mitochondria (1). This exciting discovery raises questions on whether vitamin D itself may also increase cell survival or whether it acts indirectly, for example, via inhibition of the immune system.
In conclusion, the new findings by Gascón and co-workers will have a profound impact on regeneration research by taking the field a huge step forward toward increasing the pool of newly generated neurons. Firm control of metabolic reprogramming in concert with refined timing of genetic factors involved in neuronal fate choice offers great promise for the repair of the injured brain.
Funding: This work was supported by the Deutsche Forschungsgemeinschaft (GE2576/3-1 to K Gertz; KR2956/5-1 to G Kronenberg), the Bundesministerium für Bildung und Forschung (Center for Stroke Research Berlin to G Kronenberg and K Gertz), the European Union’s Seventh Framework Programme (FP7/HEALTH.2013.2.4.2-1) under grant agreement No. 602354 (counterstroke consortium to K Gertz), and Rahel Hirsch Fellowship Charité Berlin to F Klempin.
Conflicts of Interest: The authors have no conflicts of interest to declare.
- Gascón S, Murenu E, Masserdotti G, et al. Identification and Successful Negotiation of a Metabolic Checkpoint in Direct Neuronal Reprogramming. Cell Stem Cell 2016;18:396-409. [Crossref] [PubMed]
- Kempermann G, Gage FH. Experience-dependent regulation of adult hippocampal neurogenesis: effects of long-term stimulation and stimulus withdrawal. Hippocampus 1999;9:321-32. [Crossref] [PubMed]
- van Praag H, Kempermann G, Gage FH. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci 1999;2:266-70. [Crossref] [PubMed]
- Alenina N, Klempin F. The role of serotonin in adult hippocampal neurogenesis. Behav Brain Res 2015;277:49-57. [Crossref] [PubMed]
- Ramírez-Rodríguez G, Babu H, Klempin F, et al. The α crystallin domain of small heat shock protein b8 (Hspb8) acts as survival and differentiation factor in adult hippocampal neurogenesis. J Neurosci 2013;33:5785-96. [Crossref] [PubMed]
- Gonçalves JT, Schafer ST, Gage FH. Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior. Cell 2016;167:897-914. [Crossref] [PubMed]
- Klempin F, Beis D, Mosienko V, et al. Serotonin is required for exercise-induced adult hippocampal neurogenesis. J Neurosci 2013;33:8270-5. [Crossref] [PubMed]
- Kronenberg G, Reuter K, Steiner B, et al. Subpopulations of proliferating cells of the adult hippocampus respond differently to physiologic neurogenic stimuli. J Comp Neurol 2003;467:455-63. [Crossref] [PubMed]
- Fabel K, Wolf SA, Ehninger D, et al. Additive effects of physical exercise and environmental enrichment on adult hippocampal neurogenesis in mice. Front Neurosci 2009;3:50. [PubMed]
- Ehninger D, Wang LP, Klempin F, et al. Enriched environment and physical activity reduce microglia and influence the fate of NG2 cells in the amygdala of adult mice. Cell Tissue Res 2011;345:69-86. [Crossref] [PubMed]
- Bond AM, Ming GL, Song H. Adult Mammalian Neural Stem Cells and Neurogenesis: Five Decades Later. Cell Stem Cell 2015;17:385-95. [Crossref] [PubMed]
- Belachew S, Chittajallu R, Aguirre AA, et al. Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons. J Cell Biol 2003;161:169-86. [Crossref] [PubMed]
- Sellers DL, Horner PJ. Instructive niches: environmental instructions that confound NG2 proteoglycan expression and the fate-restriction of CNS progenitors. J Anat 2005;207:727-34. [Crossref] [PubMed]
- Magavi SS, Leavitt BR, Macklis JD. Induction of neurogenesis in the neocortex of adult mice. Nature 2000;405:951-5. [Crossref] [PubMed]
- Snyder EY, Yoon C, Flax JD, et al. Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex. Proc Natl Acad Sci USA 1997;94:11663-8. [Crossref] [PubMed]
- Buffo A, Vosko MR, Ertürk D, et al. Expression pattern of the transcription factor Olig2 in response to brain injuries: implications for neuronal repair. Proc Natl Acad Sci USA 2005;102:18183-8. [Crossref] [PubMed]
- Heinrich C, Bergami M, Gascon S, et al. Sox2-mediated conversion of NG2 glia into induced neurons in the injured adult cerebral cortex. Stem Cell Reports 2014;3:1000-14. [Crossref] [PubMed]
- Kronenberg G, Gertz K, Cheung G, et al. Modulation of fate determinants Olig2 and Pax6 in resident glia evokes spiking neuroblasts in a model of mild brain ischemia. Stroke 2010;41:2944-9. [Crossref] [PubMed]
- Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663-76. [Crossref] [PubMed]
- Zhou Q, Brown J, Kanarek A, et al. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature 2008;455:627-32. [Crossref] [PubMed]
- Song K, Nam YJ, Luo X, et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature 2012;485:599-604. [Crossref] [PubMed]
- Jessberger S, Toni N, Clemenson GD, et al. Directed differentiation of hippocampal stem/progenitor cells in the adult brain. Nat Neurosci 2008;11:888-93. [Crossref] [PubMed]
- Braun SM, Pilz GA, Machado RA, et al. Programming Hippocampal Neural Stem/Progenitor Cells into Oligodendrocytes Enhances Remyelination in the Adult Brain after Injury. Cell Rep 2015;11:1679-85. [Crossref] [PubMed]
- Niu W, Zang T, Zou Y, et al. In vivo reprogramming of astrocytes to neuroblasts in the adult brain. Nat Cell Biol 2013;15:1164-75. [Crossref] [PubMed]
- Klempin F, Marr RA, Peterson DA. Modification of pax6 and olig2 expression in adult hippocampal neurogenesis selectively induces stem cell fate and alters both neuronal and glial populations. Stem Cells (Dayton, Ohio) 2012;30:500-9. [Crossref] [PubMed]
- Katchanov J, Harms C, Gertz K, et al. Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death. J Neurosci 2001;21:5045-53. [PubMed]
- Klempin F, Kronenberg G, Cheung G, et al. Properties of doublecortin-(DCX)-expressing cells in the piriform cortex compared to the neurogenic dentate gyrus of adult mice. PloS One 2011;6:e25760. [Crossref] [PubMed]
- Burns KA, Ayoub AE, Breunig JJ, et al. Nestin-CreER mice reveal DNA synthesis by nonapoptotic neurons following cerebral ischemia hypoxia. Cerebral Cortex 2007;17:2585-92. [Crossref] [PubMed]
- Magnusson JP, Goritz C, Tatarishvili J, et al. A latent neurogenic program in astrocytes regulated by Notch signaling in the mouse. Science 2014;346:237-41. [Crossref] [PubMed]
- Ursini F, Maiorino M, Forman HJ. Redox homeostasis: The Golden Mean of healthy living. Redox Biology 2016;8:205-15. [Crossref] [PubMed]
Cite this article as: Klempin F, Gertz K, Kronenberg G. Redox homeostasis: unlocking the bottleneck in glia-to-neuron conversion. Stem Cell Investig 2017;4:7.