Community Research and Development Information Service - CORDIS

FP7

KON-TIKIGENET Report Summary

Project ID: 622082
Funded under: FP7-PEOPLE
Country: United Kingdom

Final Report Summary - KON-TIKIGENET (Kon-Tiki gene network for CNS regeneration)

PUBLISHABLE SUMMARY

Neuron Glia antigen 2 (NG2)-positive glia are repair cells that proliferate upon central nervous system (CNS) damage, promoting functional recovery. However, repair is limited due to the failure of the newly produced glial cells fail to differentiate. It is a key goal to discover how to regulate NG2 to enable glial proliferation and differentiation conducive to repair. Drosophila has an NG2 homologue called kon-tiki (kon), of unknown CNS function. We show that kon promotes repair, and identify the underlying mechanism. Crush injury up-regulates kon expression downstream of Notch. Kon in turn induces glial proliferation and initiates glial differentiation by activating glial genes and prospero (pros). Two negative feedback loops with Notch and Pros, allow Kon to drive the homeostatic regulation required for repair. By modulating Kon levels in glia, we could prevent or promote CNS repair. Thus, the functional links between Kon, Notch and Pros are essential for, and can drive, repair. Analogous mechanisms could promote CNS repair in mammals.

MAIN ACHIEVEMENTS AND OBJECTIVES

Detailed results on each section will be described following the order in the proposed work plan. We have obtained convincing evidence that Kon-tiki (Kon) functions in glia in CNS development and in injury repair. We have established a novel crush injury paradigm in the living larva, and demonstrated that -like stabbing injury - it elicits a glial regenerative response. All experiment where performed focused in the larval Ventral Nerve Cord (VNC, equivalent to the spinal cord) as a model to study the Glial Regenerative Response (GRR) to the injury in the CNS.

RESEARCH OBJECTIVE 1 (RO1): To characterise the cells expressing kon-tiki (kon), test whether kon is linked to Pros and/or Notch, and investigate its functions in the normal CNS (Central Nervous System).

We have found that kon is expressed in the CNS, and it is required in glial cells. The levels of kon expression change throughout the development being higher at the embryonic and the pupal stages. In embryonic development Kon is expressed in the Lateral Glioblast (LGB) in each segment of the developing VNC. The LGB is the precursor cell of the Neuropile associated Glial (NG), involved in the GRR. During the pupal stages kon expression progressively increases in the NG cells.
Through analyzing loss of kon function knock-down using targeted RNAi, and gain of function in over-expression we could describe Kon functions in the normal CNS.

We have found that Kon is required for glial cell division. Knocking-down kon decreased the number of glial cells in the VNC, while over-expressing kon increased glial cell number. To test if this effect in glial number was due to an alteration on glial proliferation we carried out a BrdU pulse experiment. BrdU incorporates into DNA in the S-phase of the cell cycle, and when applied in a pulse reveals cells undergoing cell cycle progression at that time point. When we over-expressed kon in third instar larvae we observed an increase on BrdU incorporation in NG cells. These data demonstrate that Kon induces NG cell division.
We also found that Kon positively regulates glial marker expression and cell shape of the Prospero (Pros) positive NG. Pros-negative NG were not affected by changes in kon expression. To address this question we visualised the effect of altering kon levels on the expression of NG markers. When we knocked-down kon there was a decrease in the number of Ebony+ cells (which are the Pros+ NG cells). Also when we over-expressed kon there was an increase in the number of Ebony+ cells, additionally there was also an increase in other NG glial markers such as Glutamine Sintetase 2 (GS2) and Nazgul (Naz). Furthermore the over-expression of kon produced a change in the morphology of these cells that generate thicker projections into the neuropile. When we manipulate kon levels in the Pros-negative NG cells there was no effect on the glial markers.

However, Kon was not sufficient to induce glial proliferation, as it could not induce cell division in Pros-negative glia. This further suggested that the function of Kon in glia is linked to Notch and Pros.

We were able to describe how Kon is functionally linked to the GRR gene network involving Pros and Notch. To do so, we first studied the relationship of Kon with Pros and Notch in the normal CNS, where they regulate differentiation and proliferation respectively, and used genetic epistasis analysis to work out their relationships. We found that Kon activates pros expression, and pros represses kon. Kon knock-down showed a decrease in the number of Pros+ cells while in pros mutants the levels of kon were reduced. Pros is required for glial differentiation, thus by activating pros as well as glial differentiation markers, Kon initiates glial differentiation in daughter cells. Kon promotes and Pros inhibits cell proliferation, thus by activating its own inhibitor, Kon exerts negative feedback, restoring cell number homeostasis.

We have also seen that Notch signalling activates kon expression. Expression levels of kon rise when the activated form of Notch, Notch intracellular domain (NotchICD) is over-expressed. Kon also inhibits Notch, since downregulation of kon enhances the over-expression of Notch phenotype and over-expression of kon reduces the activation on Notch in the NG cells. Therefore our results indicate that Notch activates kon and Kon promotes cell proliferation.
Following cell division, Kon activates glial markers inducing the onset of glial cell differentiation, and activates pros, which maintains glial differentiation. Structural homeostasis comes about with negative feedback, as Kon represses Notch, and Pros represses kon, restoring quiescence in daughter cells and cell number homeostasis.

These findings are reported in the research article: Losada-Perez, Harrison and Hidalgo (in press) “Molecular mechanism of central nervous system repair by the Drosophila NG2 homologue kon-tiki”. Journal of Cell Biology, accepted 25 July 2016. Impact Factor (2015): 9.834.

RESEARCH OBJECTIVE 2 (RO2): To investigate whether Kon influences the glial and axonal regenerative responses to CNS injury. The outcome will be a profile of Kon functions upon a

To test whether Kon is involved in the glial regenerative response to injury (GRR), we developed a novel crushing injury method in the living larval CNS. The CNS was visualised with GFP using the G9 exon-trap reporter, which reveals all CNS axons. Crushing injury was applied to the larval VNC under UV light, with fine forceps, with a swift closing of the forceps tips on the VNC, and larvae were kept alive for up to two days post-injury.
The same cellular events observed in stabbing injury were observed in crushing injury: glial cells proliferated, they changed their morphology expanding their cytoplasm and generating more lamelipodia, cellular debris was engulfed by glia and the wound progressesed in a similar fashion with an initial expansion followed by shrinkage.

To test whether Kon is required for the glial response and whether Kon can influence axonal repair after injury we first analyzed kon expression upon injury. Injury induced a dramatic rise in kon expression levels. Moreover we could visualize strong expression of Kon in the NG cells after performing the crushing injury. We have also shown that this increase depends on Notch, since the levels of kon expression don’t rise up so much in a Notch mutant background. Therefore Kon functions downstream of Notch.

Next we analyzed injury size progression when manipulating kon levels in the NG cells, in loss of function or gain of function conditions for kon. While in control animals the wound followed a stereotypic progression, it first expanded and then shrunk, in larvae where kon was knocked-down the wound expanded progressively and did not shrink. Moreover in larvae where kon was over-expressed the wound did not expand and most dramatically, resulted in virtually complete repair by 48h. This results demonstrate that Kon is required in NG for, and can promote, CNS repair.

These findings are reported in the research article: Losada-Perez, Harrison and Hidalgo (in press) “Molecular mechanism of central nervous system repair by the Drosophila NG2 homologue kon-tiki”. Journal of Cell Biology, accepted 25 July 2016. Impact Factor (2015): 9.834.

RESEARCH OBJECTIVE 3 (RO3): To test if candidate genes expressed in neurons interact with glial kon. The outcome will be the identification of Kon partners that regulate CNS regeneration or repair.

To identify neuronal proteins that might interact with Kon, we have first investigated the previously reported candidate Ptp99A.Using Ptp99A antibodies we observed that Ptp99A is distributed in neurons throughout the axonal neuropile. We have also shown that the over expression of kon in neurons produces an increase of Ptp99A levels. We have generated molecular tools to investigate the relationship between Kon and Ptp99A. These data are preliminary, and not ready for publication yet. They form part of collaborative work with Dr Neale Harrison, also in the host lab at the University of Birmingham. We anticipate that together our findings will result in another future publication within the next year.

POTENTIAL SOCIO-ECONOMIC IMPACT

Kon-tiki is the Drosophila homologue of NG2. The NG2+ cells currently present the most promising approach to CNS injury and damage. The human CNS does not regenerate upon injury. As a consequence, brain damage and spinal cord (stroke, injury, multiple sclerosis and neurodegenerative diseases, e.g. Alzheimer’s, Parkinson’s) result in permanent disability. These conditions are devastating personal tragedies, and constitute the greatest burden of disease. The cost of brain disease in Europe doubles that of cancer and of neurodegenerative diseases alone is expected to double every twenty years as the population ages. A promising approach to CNS repair is to understand how NG2+ cells divide and differentiate and how this process is regulated. Knowing so it will be possible to manipulate these cells in the hope that they divide and differentiate appropriately enabling functional repair in spinal cord injury and multiple sclerosis. Drosophila is the most powerful organism to investigate the genetic mechanisms underlying a biological processes. Studying the Drosophila homologue of NG2, Kon-tiki, we can predict possible functions of NG2 in mammals. With the results obtained with this project we are one step closer to fully understand how NG2 cells divide and differentiate to enable the CNS repair.

The findings from our research will be of interest to the Medical Research community, to find ways to direct NG2 cells and stem cells to promote CNS regeneration and repair. It will be of interest to the drug discovery community, to identify drugs that might regulate the function of NG2 in relationship with ProsX1 and Notch1 to enhance the glial regenerative response to injury and other forms of demyelination.

Finally, a beneficiary of the socio-economic impact was the research fellow, Dr Maria Losada-Perez, who returned back to Spain to take up a position at the Universidad Autonoma de Madrid.

This project resulted in one major publication: Losada-Perez, Harrison and Hidalgo (in press) “Molecular mechanism of central nervous system repair by the Drosophila NG2 homologue kon-tiki”. Journal of Cell Biology, accepted 25 July 2016. Impact Factor (2015): 9.834.

A summary of this project will be published in the Hidalgo Lab web-site at:

http://www.biosciences-labs.bham.ac.uk/hidalgo/Alicia_Hidalgo_Lab_Home.html

http://www.biosciences-labs.bham.ac.uk/hidalgo/Projects.html

update of this website was pending acceptance of the research article in a peer review journal. The article was accepted yesterday 25 July, so we will update the website soon.

Related information

Contact

Xavier Rodde, (Head of Research Information and European Funding Support)
Tel.: +441214158202
Fax: +441214146056
E-mail
Record Number: 189516 / Last updated on: 2016-10-06