A genetic approach to the study of the neuroprotective role of cysteine string protein during normal ageing

A reduction in neuronal function and loss of neurons occurs during normal ageing and is accelerated in neurodegenerative diseases. Much effort has been put into understanding how mutated genes can lead to acceleration of neurodegeneration in disease but much less work has been done on understanding the basic mechanisms for the protection of neurons during normal ageing processes. Increasing evidence has begun to identify specific proteins that are involved in the protection of neurons from damage that can occur due to their high level of ongoing activity. A key aspect of communication between neurons is due to the release of small neurotransmitters at the junctions (synapses) between neurons. Proteins within the synapses are crucial for the release of neurotransmitters; these are used multiple times and have to be recycled for use very rapidly. The functions of proteins are often protected by so called chaperone proteins. One such chaperone is cysteine string protein (CSP) that is found in synapses and interacts with the proteins responsible for neurotransmitter release. A key physiological role for CSP in neuroprotection emerged in study of mice in which its gene had been disrupted. These mice were born normal but showed progressive abnormalities and died after a few weeks. Evidence for neurodegeneration was seen in these mice and also in flies when the equivalent gene was disrupted. We have recently studied the CSP present in the nematode worm Caenorhabditis elegans. C. elegans has been widely used as a model organism due to its relative simplicity, the ease of genetic manipulation and the availability of simple functional assays. Importantly, many of the basic mechanisms underlying biological processes such as ageing are conserved in organisms from worms to man and involve the equivalent proteins in all species. Our recent work has shown that worms lacking functional CSP show age-dependent defects in movement, reduced life-span and a progressive loss of neurons during ageing. This suggests that CSP is involved in an evolutionarily conserved basic mechanism required to prevent the degeneration and death of neurons. We will use the power of worm genetic approaches to dissect the pathways by which CSP acts and to identify new regulators of the neurodegeneration that occurs in its absence. These studies will give insight into pathways of physiological importance in neuroprotection during normal ageing.

We will use genetic approaches to study the basic mechanisms underlying the neuroprotective function of cysteine string protein (CSP) during normal ageing. This synaptic chaperone protein has an essential physiological role in preventing presynaptic neurodegeneration. This function was known to be conserved from flies to man and our recent work has shown it is also conserved in Caenorhabditis elegans based on study of mutants alleles of the CSP orthologue dnj-14. Elucidation of the basic mechanisms and regulators of the CSP-dependent pathway in worms could illuminate general aspects of normal neuroprotective pathways. C. elegans is an ideal model for such an investigation due to its simple organisation and the ease of genetic manipulation. In addition, genetic and regulatory factors that influence ageing are conserved from worm to mammals. Mechanistic insight into how CSP exerts its neuroprotective role and why CSP absence results in neurodegeneration is limited although recent work has implicated interactions with the synaptic protein SNAP-25. This proposal aims to use the genetic power of C. elegans to test SNAP-25 (RIC-4 in the worm) as a potential target for CSP action. Biochemical analyses will be used to assess RIC-4 levels and ubiquitination in dnj-14 mutant strains and an epistasis analysis will be carried out by crossing the dnj-14 mutant with ric-4 mutants and other control worm lines. We will aim to identify regulators of the neurodegeneration pathway triggered by knock-out of the CSP orthologue DNJ-14. This will be based on mutagenesis of dnj-14 mutant strains that express GFP in all neurons. Following mutagenesis the whole genomes of selected mutants and the parent strain will be sequenced. Following bioinformatic analysis candidate mutations that act as suppressors of the dnj-14 mutant phenotype (loss of fluorescent neurons and reduced life-span) will be characterised using genetic approaches to confirm the functional significance of the mutated gene.

This project will require the appointment of a post-doctoral research assistant to perform the bulk of the experimental work. This individual will receive extensive and high quality training in a variety of biochemical, molecular biological and genetic techniques relevant to many types of projects using a key model organism. In addition to laboratory training the post-doctoral researcher will also be expected to extend their transferrable skills base through involvement in the communication of scientific data generated from the project both in the form of written journal articles and audio-visual presentations at scientific meetings. Participation in University training modules in a range of transferable skills will also be required. The development of a highly trained and skilled scientist through these channels will benefit the individual but also contribute more broadly through skill transfer as the person moves on to other posts in the academic or commercial sectors.

The subject matter of our research is to improve our understanding the molecular details underlying protection of neurons from neurodegeneration. It is possible that proteins and regulatory pathway that we identify could be important not only for a basic understanding of processes during normal ageing but could become therapeutic targets in the treatment of human disease. This proposal is not directed at examining potential therapeutic applications of the work but the PI will extend the long-term impact of our work by contacting pharmaceuticals companies interested in developing small-drug based therapeutic near the end of the project. Application of our findings in this way could in the long term, have the potential to positively impact upon both wealth creation and the public health of the UK.

The University of Liverpool runs several open days each year which are attended by large numbers of the general public. We participate in these events which showcase our research and provide hands on experience of scientific research for the lay-person. These activities will further impact on the post-doctoral researcher as we will expect participation in such events so as to develop skills necessary for presenting complex scientific information to a lay-audience. Related to these activities, we participate in the Nuffield Bursary Scheme which provides A-level students the opportunity to complete a 4-week research project in our laboratory. We will offer one such project in each year of the grant. An obvious consequence of these activities in the short term is the engagement of young people with science at an early stage of their academic careers such that they eventually continue along this path in more advanced studies. In addition, undergraduate students in the first and second years of their science degrees have also been given the opportunity to undertake short research projects during their summer break. We will aim to take on a minimum of one such summer student in each year of the proposal.

We are actively engaged though the University Press office in publicising the outcomes of our research and for example a press release on a recent paper using the worm model published in Mol Biol Cell was the subject of a University press release and gained widespread attention.