Glial kon/NG2 gene network for central nervous system repair

Maria Losada-Perez, Neale Harrison Alicia Hidalgo

1 School of Biosciences, University of Birmingham, Birmingham, England, UK

2 Universidad Autónoma de Madrid, Madrid, Spain

Glial kon/NG2 gene network for central nervous system repair

Maria Losada-Perez1,2, Neale Harrison1, Alicia Hidalgo1,＊

1 School of Biosciences, University of Birmingham, Birmingham, England, UK

2 Universidad Autónoma de Madrid, Madrid, Spain

How to cite this article:Losada-Perez M, Harrison N, Hidalgo A (2017) Glial kon/NG2 gene network for central nervous system repair. Neural Regen Res 12(1):31-34.

Open access statement:is is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

e glial regenerative response to central nervous system (CNS) injury, although limited, can be harnessed to promote regeneration and repair. Injury provokes the proliferation of ensheathing glial cells, which can di ff erentiate to remyelinate axons, and partially restore function.is response is evolutionarily conserved, strongly implying an underlying genetic mechanism. In mammals, it is elicited by NG2 glia, but most oen newly generated cells fail to di ff erentiate.us an important goal had been to fi nd out how to promote glial di ff erentiation following the proliferative response. A gene network involving Notch and prospero (pros) controls the balance between glial proliferation and di ff erentiation in fl ies and mice, and promotes CNS repair at least in fruit‐ fl ies. A key missing link had been how to relate the function of NG2 to this gene network. Recent fi ndings by Losada‐Perez et al., published in JCB, demonstrated that the Drosophila NG2 homologue kon‐tiki (kon) is functionally linked to Notch and pros in glia. By engaging in two feedback loops with Notch and Pros, in response to injury, Kon can regulate both glial cell number and glial shape homeostasis, essential for repair. Drosophila o ff ers powerful genetics to unravel the control of stem and progenitor cells for regeneration and repair.

NG2; kon-tiki; glia; Drosophila; injury; regeneration; repair; CNS

Accepted: 2017-01-13

Introduction

Regenerative responses to central nervous system (CNS) injury, although limited, reveal underlying nat‐ural mechanisms that could be harnessed to promote regeneration and repair. Conversely, these same mech‐anisms may promote CNS structural robustness and homeostasis during normal growth and adult life, and their impairment could underlie the disregulation that accompanies ageing, neurodegenerative diseases and brain tumours. Demyelinating diseases, like multiple sclerosis, and traumatic brain and spinal cord injury, provoke a spontaneous, natural regenerative response in ensheathing glial cell progenitors (i.e., in mammals, oligodendrocyte progenitor cells, OPCs) (Franklin and Ffrench‐Constant, 2008). In response to damage, en‐sheathing glia proliferate and re‐enwrap axons, leading to recovery of behavior.is response is very limited in humans, partly due to the fact that newly produced cells can fail to differentiate (Franklin and Ffrench‐Con‐stant, 2008).us a key goal has long been to fi nd out how to promote differentiation of oligodendrocytes (OLs) following the regenerative proliferation of their progenitors. Importantly, the regenerative response of ensheathing glia is evolutionarily conserved, and occurs in cockroach, fruit‐flies, fish and rodents too (Smith et al., 1987; Dubois‐Dalcq et al., 2008; Franklin and Ffrench‐Constant, 2008; Kato et al., 2011), implying there is an underlying genetic mechanism.e discov‐ery of gene networks underlying regenerative responses to injury is key to understand how cells achieve and maintain normal body integrity. Importantly, they will enable the manipulation of stem cells, progenitor cells and neural circuits for therapeutic aims.

The fruit‐flyDrosophilahas recurrently proven to be an extremely powerful model organism to discover evolutionarily conserved gene networks with relevance for humans. The Hidalgo lab demonstrated that the glial regenerative response to CNS injury inDrosophiladepends on a gene network involving the genesNotchandprospero (pros), whereby Notch promotes glial pro‐liferation and Pros glial differentiation (Griffiths and Hidalgo, 2004; Gri ffiths et al., 2007; Kato et al., 2011).is was an important fi nding: in mammals Notch was known to maintain OPCs proliferative, but upon injury, Notch prevents OL di ff erentiation (Wang et al., 1998),and a challenge was to discover genes related to Notch that could promote glial differentiation. We demon‐strated that similarly toprosinDrosophila, its homo‐logueprox1in the mouse is also required for OL di ff er‐entiation (Kato et al., 2015). A key missing link was the yet unknown relationship of Notch1 and Prox1 to NG2.

Figure 1 Glialkon/NG2gene network for central nervous system (CNS) repair.

In mammals, the glial regenerative response to CNS injury is carried out by NG2+OPCs, also known as NG2‐glia (Zuo and Nishiyama, 2013). The NG2 protein is required for the glial regenerative response: like Notch1, NG2 levels are also up‐regulated upon injury, and mice lacking NG2 have reduced OPC proliferation during nor‐mal development, and in response to injury (Kucharova and Stallcup, 2010; Kucharova et al., 2011). Moreover, the size of demyelinating lesions decreases over time in wild type animals as the CNS tends to repair naturally, but in NG2 knock‐out mice lesions fail to shrink, due to reduced OPC proliferation and resulting depletion in OLs (Kucha‐rova et al., 2011).us,NG2is a crucial factor involved in the regulation of the regenerative response of OPCs. However, finding out what genes related to NG2 might enable the di ff erentiation of ensheathing glial cells follow‐ing their injury‐induced proliferation, was still a crucial missing link.

We approached this question by investigating whether anNG2homologue might operate inDrosophilaglia, in response to injury. NG2 is an evolutionarily conserved, extracellular protein, with two N‐terminal Laminin Neurexin Sex‐hormone Globulin (LNS) motifs, a sin‐gle transmembrane domain and a small intracellular PDZ domain (Trotter et al., 2010). Cleavage can release four protein products, including a large secreted ecto‐domain, and an intracellular domain, which functions as a transcription factor (Trotter et al., 2010).Drosophilahas anNG2homologue, calledkon-tiki (kon)orperdido(Estrada et al., 2007; Schnorrer et al., 2007). Both the extracellular domain and intracellular PDZ motif of NG2 and Kon are highly conserved (Estrada et al., 2007; Schnorrer et al., 2007; Trotter et al., 2010). Pre‐vious work on Kon had centered on its role in muscle (Stegmuller et al., 2003; Estrada et al., 2007; Schnorrer et al., 2007), but whether it had functions in the CNS was unknown.

To investigate a potential link between the Notch‐Pros glial gene network and Kon, we first developed a new crush injury paradigm in theDrosophilaventral nerve cord (VNC, equivalent to the vertebrate spinal cord) (Losada‐Perez et al., 2016). We had carried out stabbing injury before (Kato et al., 2011; Kato and Hidalgo, 2013), but we reasoned crush injury might mimic more closely natural accidental injury. Most importantly, we found that both methods induced an equivalent regenerative re‐sponse in glial cells. Firstly, neuropile associated glial cells (which normally enwrap CNS axons) proliferate upon both types of injury. Secondly, these glial cells are also in‐volved in phagocytosis and clearance of cell debris resulting from the injury. This function is normally accomplishedby microglia/macrophages in mammals, which can also express NG2. And third, in both injury types, we could increase or prevent repair by manipulating gene expres‐sion in neuropile associated glial cells.is indicated that the regenerative response to injury is robustly induced inDrosophila, opening the opportunity to develop further types of CNS injury, which could be even more amenable to genetic analysis.

A Gene Network for kon/NG2 to Promote CNS Repair

Using this novel crush injury method in the VNC of the fruit‐ fl y larval CNS, we asked whetherKonwas involved in the glial regenerative response (Losada‐Perez et al., 2016) (Figure 1). We found that in the normal CNS,konis hardly expressed, but injury induces its up‐regulation in neuropile glia.is is analogous to the injury‐induced up‐regulation of NG2 levels in OPCs in vertebrates (Kucharova et al., 2011). Injury also provokes the activa‐tion of Notch1 signalling, and of the pro‐inflammatory tumor necrosis factor (TNF)/ TNF receptor (TNFR)/ nuclear factor‐κB (NF‐κB) pathway in mammals to trig‐ger OPC proliferation (Figure 1B). We had previously shown that, similarly, inDrosophila, the TNF homo‐logue Egr through its TNFR receptor Wengen induced the translocation of Dorsal/NF‐κB in response to injury, and together with Notch, promoted glial proliferation (Kato et al., 2011). Blocking NF‐κB translocation (by over‐expressing cactus, a known inhibitor of NF‐κB), Kon levels did not rise aer injury, meaning that NF‐κB is linked to the up‐regulation of Kon (Losada‐Perez et al., 2016) (Figure 1B). Kon activation also depends on Notch, since levels ofkonalso failed to rise aer injury inNotchmutants (Figure 1B). Furthermore, Kon is suf‐ fi cient to promote glial proliferation in Pros+neuropile glia cells, indicating that ultimately the increase in Kon levels upon injury is the trigger for glial cell division. Importantly, Kon cannot induce proliferation of neu‐ropile glia that do not express both Notch and Pros. Notch and Pros together maintain glial progenitors qui‐escent in G1, with proliferative potential (Gri ffiths and Hidalgo, 2004; Kato et al., 2011). Neuropile glia lacking Notch and Pros fully exit the cell cycle, and cannot di‐vide, even in the presence of Kon. Together, this further meant that Kon function is linked to those of Pros and Notch (Losada‐Perez et al., 2016). Notch and Kon act together as cell cycle activators breaking the Notch‐Pros balance and pushing the Pros+glia to divide (Figure 1B).

Usingin vivofunctional genetic analysis, we uncovered the relationship between Kon, Notch and Pros (Losa‐da‐Perez et al., 2016)(Figure 1B, C). Kon functions in two feedback loops. In the fi rst one, Notch activates the expression ofkon, while Kon feeds back and represses Notch signaling or expression.is Notch—Kon feedback loop enables and limits glial proliferation (Figure 1B). In a second feedback loop, Kon activatespros, andprosrepresseskonexpression. This feedback loop constrains the lifetime ofkonexpression, and restores cell number homeostasis (Figure 1C).is constraint is important as aer injury cell division enables repair, but uncontrolled cell division would lead to tumours. In fact, NG2 is a key glioma marker in humans. By activatingprosexpres‐sion, Kon enables the onset of glial differentiation. Kon also regulates the expression of the glial differentiation markersebonyandGS2, involved in the recycling of neu‐rotransmitters. Interfering with Kon function also alters the shape Pros+glia (Losada‐Perez et al., 2016).us, Kon is required for glial activation and the onset of glial di ff er‐entiation, which depends on Pros (Gri ffi ths and Hidalgo, 2004; Kato et al., 2011; Losada‐Perez et al., 2016). The Kon—Pros feedback loop enables the transition from glial proliferation to di ff erentiation and restores cell shape ho‐meostasis (Losada‐Perez et al., 2016). Most importantly, both feedback loops are homeostatic: they enable change, but within constraints, restoring structural integrity whilst avoiding tumours. Finally, Kon promotes repair, since manipulating Kon levels alters injury size progres‐sion. Crush injury induces a typical progression of wound expansion followed by shrinkage (Kato et al., 2011; Losa‐da‐Perez et al., 2016). Uponkonknock‐down, the wound fails to shrink, and whenkonis over‐expressed, there is a signi fi cant reduction in wound size (Losada‐Perez et al., 2016).

Our work has revealed a key functional link between Notch, Kon and Pros for CNS repair, which could be evo‐lutionarily conserved (Losada‐Perez et al., 2016) (Figure 1). Importantly, our findings showed evolutionary con‐servation in the functions of NG2 and Kon in the glial re‐generative response to CNS injury: both are up‐regulated upon injury, both are required for glial proliferation and for the regenerative reduction in lesion size ‐ in mammals and fruit‐flies (Kucharova et al., 2011; Losada‐Perez et al., 2016). In mammals, Prox1 is also co‐distributed to‐gether with Notch1 in NG2+OPCs, Prox1 levels increase as OPCs di ff erentiate into OLs, and Prox1 is required for OL differentiation (Cahoy et al., 2008; Kato et al., 2015). We have shown that Kon drives the onset of glial di ff eren‐tiation by activating ‐ as well as other glial markers ‐prosexpression. We had also previously shown that Pros in fl ies, and Prox1 in the mouse, are required for neuropile glia and oligodendrocyte di ff erentiation, respectively (Gri ffi ths and Hidalgo, 2004; Kato et al., 2011, 2015). Thus our findingsstrongly suggest that Prox1 may be the key target of NG2 to manipulate in glioma, progenitors and stem cells to modulate and exploit the pro‐regenerative potential of NG2‐glia.

To conclude,Drosophilagenetics offers a powerful means to investigatein vivofundamental biology, regen‐eration and repair, and discover gene networks as they functionin vivo. Our discovery of the gene network un‐derlying the regulation of glial proliferationvs. glial di ff er‐entiation in response to injury may not only be relevant in the context of the glial regenerative response. It could also provide important insights for the understanding of glioma, and of how to manipulate glial progenitors and stem cells for CNS regeneration and repair.

Author contributions:MLP, NH, and AH all contributed to writing this manuscript.

Con fl icts of interest:None declared.

Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Chris‐topherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA,ompson WJ, Barres BA (2008) A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 28:264‐278.

Dubois‐Dalcq M, Williams A, Stadelmann C, Stanko ff B, Zalc B, Lubetzki C (2008) From fi sh to man: understanding endoge‐nous remyelination in central nervous system demyelinating diseases. Brain 131:1686‐1700.

Estrada B, Gisselbrecht SS, Michelson AM (2007)e transmem‐brane protein Perdido interacts with Grip and integrins to mediate myotube projection and attachment in the Drosophila embryo. Development 134:4469‐4478.

Franklin RJ, Ffrench‐Constant C (2008) Remyelination in the CNS: from biology to therapy. Nat Rev Neurosci 9:839‐855.

Griffiths RL, Hidalgo A (2004) Prospero maintains the mitotic potential of glial precursors enabling them to respond to neu‐rons. EMBO J 23:2440‐2450.

Kato K, Hidalgo A (2013) An injury paradigm to investigate cen‐tral nerovus system repair in Drosophila. JoVE 73:e50306.

Kato K, Forero MG, Fenton JC, Hidalgo A (2011) The glial regenerative response to central nervous system injury is en‐abled by pros‐notch and pros‐NFkappaB feedback. PLoS Biol 9:e1001133.

Kato K, Konno D, Berry M, Matsuzaki F, Logan A, Hidalgo A (2015) Prox1 inhibits proliferation and is required for di ff eren‐tiation of the oligodendrocyte cell lineage in the mouse. PLoS One 10:e0145334.

Kucharova K, Stallcup WB (2010) The NG2 proteoglycan pro‐motes oligodendrocyte progenitor proliferation and develop‐mental myelination. Neuroscience 166:185‐194.

Kucharova K, Chang Y, Boor A, Yong VW, Stallcup WB (2011) Reduced in fl ammation accompanies diminished myelin dam‐ age and repair in the NG2 null mouse spinal cord. J Neuroin‐fl ammation 8:158.

Losada‐Perez M, Harrison N, Hidalgo A (2016) Molecular mech‐anism of central nervous system repair by the Drosophila NG2 homologue kon‐tiki. J Cell Biol 214:587‐601.

Schnorrer F, Kalchhauser I, Dickson BJ (2007) The transmem‐brane protein Kon‐tiki couples to Dgrip to mediate myotube targeting in Drosophila. Dev Cell 12:751‐766.

Smith PJ, Howes EA, Treherne JE (1987) Mechanisms of glial regeneration in an insect central nervous system. J Exp Biol 132:59‐78.

Stegmuller J, Werner H, Nave KA, Trotter J (2003)e proteogly‐can NG2 is complexed with alpha‐amino‐3‐hydroxy‐5‐meth‐yl‐4‐isoxazolepropionic acid (AMPA) receptors by the PDZ glutamate receptor interaction protein (GRIP) in glial progeni‐tor cells. Implications for glial‐neuronal signaling. J Biol Chem 278:3590‐3598.

Trotter J, Karram K, Nishiyama A (2010) NG2 cells: properties, progeny and origin. Brain Res Rev 63:72‐82.

Wang S, Sdrulla AD, diSibio G, Bush G, Nofziger D, Hicks C, Weinmaster G, Barres BA (1998) Notch receptor activation in‐hibits oligodendrocyte di ff erentiation. Neuron 21:63‐75.

Zuo H, Nishiyama A (2013) Polydendrocytes in development and myelin repair. Neurosci Bull 29:165‐176.

Alicia Hidalgo, Ph.D., a.hidalgo@bham.ac.uk.

10.4103/1673-5374.198969

＊< class="emphasis_italic">Correspondence to: Alicia Hidalgo, Ph.D., a.hidalgo@bham.ac.uk.

orcid: 0000-0001-8041-5764 (Alicia Hidalgo)