Life and death in injured chick spinal cord with development: a functional genomics and proteomics approach
Lead Research Organisation:
University College London
Department Name: Institute of Child Health
Abstract
Injury of the adult central nervous system (brain and spinal cord) of mammals and birds, due either to insult or disease, usually leads to irreversible damage and severe disability. In contrast, in young embryos and in certain fish and amphibia the central nervous system is able to regenerate. Several factors are likely to contribute to loss of regenerative ability and their identification may help us to understand why such capability is lost and how it may be recovered. A key requisite for central nervous system repair is either to maintain a high rate of survival in injured neurons or to be able to produce new neurones that, given appropriate environmental cues, will then re-grow their processes and re-establish the appropriate connections. These events do not usually occur in the adult central nervous system of birds and mammals where injury causes extensive death of neurones, and chemical and morphologoical changes which impair axonal regrowth from surviving neurones. In the embryo however, even a rather well developed spinal cord can regenerate, though eventually this capability is lost at late stages of development. At present the molecular mechanisms underlying the differences between regenerating and non regenerating spinal cords are not fully understood. We wish to use modern techniques that allow us to identify genes and proteins that are differentially expressed to define the molecular changes occurring at the transition between regeneration-competent and incompetent stages of development in normal spinal cord and in response to injury using the chick as a model. The great advantage of the chick embryo is that, being easily accessible, it is amenable to manipulations in ovo (a window can be cut in the egg and then closed with cellotape after surgery/ pharmacological treatments) and will allow us to test the role of molecules identified in our screenings. We anticipate that our screenings will identify several molecules that are differently regulated in response to injury at developmental stages permissive and non-permissive for regeneration. We will select some of these molecules for functional studies, initially focusing on those likely to be involved in i) controlling whether a cell is going to survive or die and ii) controlling neurogenesis (formation of new neurones). For example, we will assess whether we can increase neural cell survival and / or neurogenesis by modulating the activity of these molecules with appropriate drugs. Identification of molecules that play a role in these processes, and that can be modulated pharmacologically to increase neurone numbers before treatment with agents which can stimulate targeted nerve growth, is a key step towards attaining significant neural repair.
Technical Summary
Our overall aim is to gain a better understanding of the mechanisms underlying the loss of regenerative capability within the central nervous system that occurs with development in amniotes using the chick embryonic spinal cord as a model. This model has the unique advantage, as compared to rodent embryos, of being easily accessible and amenable to manipulations in ovo during development. We will study changes occurring between a regeneration competent stage when spinal cord development is very advanced (E11), and a regeneration incompetent stage (E15). Although myelin components appear to have a role in the loss of regenerative capability, as they can inhibit axonal regeneration, failure to regenerate the mature spinal cord is likely due to multiple factors. We will focus this study on early events occurring in response to injury (2 and 24 hours), rather than on the issue of axonal regrowth, as we have already evidence for changes in this response with development. We have found that injury at E11 can activate developmental programmes (e.g. up-regulation and phosphorylation of the early neuroblast marker doublecortin) and that injury-induced apoptosis is much more extensive in non regenerating spinal cords than in regenerating ones. Extensive apoptosis correlates with formation of large cavities that impair axon regeneration. The specific aims of this study are 1) to identify molecules/pathways which may underlie regenerative capability in E11 embryonic spinal cord, and define molecular changes occurring at the transition between regeneration-competent and incompetent stages of development in normal and injured spinal cords; 2) to study the function of selected molecules in the regenerative process. To this purpose we will profile gene expression using DNA microarray screening technology in sham operated and injured spinal cords at E11 and E15. As there is already evidence of changes in protein phosphorylation induced in response to injury, which may be of crucial importance and would be missed by examining only gene profiles, we will use a proteomic approach to profile changes in phosphorylation. The analysis of molecules showing the most striking changes in expression will be in two stages. (i) Modulation of expression during development and in response to injury will be confirmed by RT-PCR/in situ hybridization/immunohistochemistry. (ii) Promising molecules will be selected for functional studies both in organotypic spinal cord cultures and in vivo. We will prioritize the analysis of molecules involved in cell cycle control/apoptosis, and neuronal differentiation, and overall focus on those molecules that can be more easily manipulated pharmacologically and are most likely to be of value for therapeutic developments. We believe that the combination of transcription and protein profiling with functional analysis proposed here will allow us to identify developmentally-regulated events leading to loss of regenerative ability, and provide the ground for functional analysis of selected molecules by pharmacological manipulation.
Publications
Ferretti P
(2011)
Is there a relationship between adult neurogenesis and neuron generation following injury across evolution?
in The European journal of neuroscience
Ferretti P
(2014)
Protein Deimination in Human Health and Disease
Ferretti P
(2015)
Neural Stem Cells in Development, Adulthood and Disease
Ferretti P
(2008)
Successful neural regeneration in amniotes: the developing chick spinal cord.
in Cellular and molecular life sciences : CMLS
Gögel S
(2010)
Post-translational regulation of Crmp in developing and regenerating chick spinal cord.
in Developmental neurobiology
Lange S
(2011)
Protein deiminases: new players in the developmentally regulated loss of neural regenerative ability.
in Developmental biology
Tanaka EM
(2009)
Considering the evolution of regeneration in the central nervous system.
in Nature reviews. Neuroscience
U KP
(2014)
Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase-AIF pathway.
in Biochimica et biophysica acta
Whalley K
(2009)
Changes in progenitor populations and ongoing neurogenesis in the regenerating chick spinal cord.
in Developmental biology
Description | The chick model of spinal cord regeneration was used to address the question of why regenerative capability is lost with development and identify possible strategies to reverse it in species that do not regenerate. Focus was on the potential role of neurogenesis in the transition between regeneration-permissive (E11) and non-permissive (E15) stages of development and identification of molecular changes underlying this transition using proteomic and genomic screens that may provide novel targets. The in vivo findings in the chick were validated both in chick and human in vitro models, with the view to reducing animal usage and move swiftly to a meaningful human in vitro model. |
Exploitation Route | The research started within the grant can lead to: development of novel putative therapeutic agent(s) development of diagnostics / enzymatic assays use of the in vitro human neural damage model for studying other pathways Citrullination/PAD role(s) in cell death were investigated in other models, including human neural cells, to validate its importance as therapeutic target. As the PAD inhibitor used in the chick study is not drug-like, a collaboration with a medicinal chemist, Prof. C. Marson, was started in order to develop a new class of PAD inhibitors; several putative neuroprotective agents were generated and currently under study. |
Sectors | Chemicals Education Healthcare Pharmaceuticals and Medical Biotechnology |
Description | New perspective in cell biology and neuroprotection |
Geographic Reach | Local/Municipal/Regional |
Policy Influence Type | Influenced training of practitioners or researchers |
Description | Modelling neural stem cell response to insult and pharmacological agents |
Amount | £67,500 (GBP) |
Organisation | Child Health Research Appeal Trust (CHRAT) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2011 |
End | 09/2014 |
Title | Established in vitro models that validated in vivo findings to dissect novel cell death pathways in vitro |
Description | Established chick and human in vitro models to study neural damage and assess novel putative neuroprotective agents. |
Type Of Material | Model of mechanisms or symptoms - in vitro |
Year Produced | 2014 |
Provided To Others? | Yes |
Impact | The model is currently used by a collaborator to study toxic agents. |
Title | Gene profiling data |
Description | Gene profiling of regenerating and non-regenerating chick spinal cord |
Type Of Material | Database/Collection of data |
Provided To Others? | No |
Impact | No actual impacts realised to date. A paper with data generated is in preparation. |
Description | Analysis of citrullination |
Organisation | University of Alabama at Birmingham |
Country | United States |
Sector | Academic/University |
PI Contribution | Changes in protein citrullination following spinal cord injury |
Start Year | 2009 |
Description | Development of novel PAD inhibitors |
Organisation | University College London |
Department | Department of Chemistry |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Establishment of models to study neuroprotection, enzymatic assays and PAD gene expression manipulation that we use for the characterization of novel PAD inhibitors. |
Collaborator Contribution | synthesis of drug-like putative inhibitors of PAD enzymes |
Impact | Joint paper published (below) and patent filed Discovery of a structurally novel, drug-like and potent inhibitor of peptidylarginine deiminase Patrizia Ferretti,a Kin Pong U, Barbora Vagaska, Rohan Merchant Christopher J. Matthews and Charles M. Marson Med. Chem. Commun., 2013,4, 1109-1113 DOI: 10.1039/C3MD00091E |
Start Year | 2009 |
Description | Inhibition of PAD enzymes |
Organisation | Scripps Research Institute |
Country | United States |
Sector | Charity/Non Profit |
PI Contribution | Effect of PAD inhibition on spinal cord injury; This collaborator provided a PAD inhibitor originally synthesized in his laboratory and a plasmid carrying the human PAD3 gene. |
Start Year | 2009 |
Title | Inhibitors of peptidylarginine deiminases (PADs) and their use in therapy |
Description | The invention relates to compounds and their uses. In particular, though not exclusively, it concerns compounds as inhibitors of peptidylarginine deiminases (PADs) and their use in therapy. |
IP Reference | GB1309180.6 |
Protection | Patent granted |
Year Protection Granted | 2013 |
Licensed | No |
Impact | Novel compounds are under development and being tested for neuroprotective activity. |
Title | Neuroprotection |
Description | Drug-like inhibitors of peptidylarginine diminases (PADs) are being developed through a collaboration with a medicinal chemist, as we had shown that a general non-drug-like PAD inhibitor could reduce neural cell loss in our chick spinal cord injury model and in our in vitro human neural injury model. A number of this compound are potent inhibitors of cell death in the human model. Source of funding has been via UCLB and a charity studentship. |
Type | Therapeutic Intervention - Drug |
Current Stage Of Development | Initial development |
Year Development Stage Completed | 2014 |
Development Status | Actively seeking support |
Impact | Assessment of full potential of PAD inhibitors for neuroprotection will require more extensive studies, including in vivo studies, that require more substantial funding than currently available. |
Title | Use of chick and human cells to model injury responses in vitro |
Description | Set up assays to mimic neural damage and test putative neuroprotective agents |
Type Of Technology | New/Improved Technique/Technology |
Year Produced | 2013 |
Impact | identification of putative neuroprotective compounds |
Description | British Neuroscience Association Spring Symposium |
Form Of Engagement Activity | Scientific meeting (conference/symposium etc.) |
Part Of Official Scheme? | No |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Invited Speaker - British Neuroscience Association Spring Symposium From development to regeneration no actual impacts realised to date |
Year(s) Of Engagement Activity | 2010 |
Description | EMBO Conference Regeneration |
Form Of Engagement Activity | Scientific meeting (conference/symposium etc.) |
Part Of Official Scheme? | No |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Invited Speaker - The Molecular & Cellular Basis of Regeneration & Tissue Repair Loss of regenerative capability with development: PAD3 - a novel mediator of cell death in injured spinal cords and neural stem cells. Asked to co-organize the following EMBO conference in the series. |
Year(s) Of Engagement Activity | 2012 |
Description | Fundraising House of Lords |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Enquiry about our work followed. resulted in visits to my lab from charities / potential donors |
Year(s) Of Engagement Activity | 2011 |
Description | International Chick Meeting 2007 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | extensive discussion after talk on "Life and death in the regenerating chick spinal cord" stimulated interest in my joining my lab |
Year(s) Of Engagement Activity | 2007 |
Description | International Conference Singapore |
Form Of Engagement Activity | Scientific meeting (conference/symposium etc.) |
Part Of Official Scheme? | No |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Changes in spinal cord plasticity with development: cellular and molecular mechanisms affecting regenerative capability. Plenary Lecture I have been contacted by students finishing their PhD and postdocs about the possibility of working in my laboratory. |
Year(s) Of Engagement Activity | 2010 |
Description | Meeting Netherlands |
Form Of Engagement Activity | Scientific meeting (conference/symposium etc.) |
Part Of Official Scheme? | No |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Invited Speaker - 'From the cradle to the grave' Biologica Symposium Regeneration: a balance between life and death Lecture Increased request in joining my laboratory |
Year(s) Of Engagement Activity | 2008 |
Description | Meeting Germany |
Form Of Engagement Activity | Scientific meeting (conference/symposium etc.) |
Part Of Official Scheme? | No |
Type Of Presentation | Keynote/Invited Speaker |
Geographic Reach | international |
Primary Audience | Participants in your research or patient groups |
Results and Impact | Invited Speaker - Biopharmaceutical Summit 2013 Modulation of Human Neural Stem Cell Death/Survival by Post-Translational Modifiers - A New Pathway For Therapeutic Intervention Following Neural Damage Lecture - Abstract no actual impacts realised to date |
Year(s) Of Engagement Activity | 2013 |
Description | Neuroscience Meeting 2010 |
Form Of Engagement Activity | Scientific meeting (conference/symposium etc.) |
Part Of Official Scheme? | No |
Type Of Presentation | Paper Presentation |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Neuroscience Minisymposium: Protein Deimination in Ophthalmic and Neurological Disease Protein deiminases: new players in the developmentally regulated loss of neural regenerative ability Talk - Abstract no actual impacts realised to date |
Year(s) Of Engagement Activity | 2010 |
Description | Press briefing session |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Short presentation and Q&A session at a press gathering at the Science Media Centre at the Royal Society The presentations/discussions at the briefing session were mentioned in national newspapers. no actual impacts realised to date |
Year(s) Of Engagement Activity | 2010 |
Description | Regeneration Conference USA |
Form Of Engagement Activity | Scientific meeting (conference/symposium etc.) |
Part Of Official Scheme? | No |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | Invited speaker - The Regeneration Project II Meeting Mechanisms underlying successful regeneration in the chick spinal cord Lecture networking |
Year(s) Of Engagement Activity | 2009 |