Award details

Life and death in injured chick spinal cord with development: a functional genomics and proteomics approach

Reference	BB/D013755/1
Principal Investigator / Supervisor	Professor Patrizia Ferretti
Co-Investigators / Co-Supervisors	Professor Nicholas Greene
Institution	University College London
Department	Institute of Child Health
Funding type	Research
Value (£)	352,304
Status	Completed
Type	Research Grant
Start date	22/05/2006
End date	21/02/2010
Duration	45 months

Abstract

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.

Summary

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.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Neuroscience and Behaviour, Regenerative Biology, Stem Cells
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search