Pathfinder: Identification of axonal- and synaptic-protection mutants using real-time confocal microendoscopy

Lead Research Organisation: University of Edinburgh
Department Name: Unknown

Abstract

Publication of the findings will be via the normal channels of peer-review in journals and scientific conferences. After publication, the results will be disseminated in public lectures and public access web pages. Details of phenotype will be published on line in the Harwell mutagenesis database (htttp://mgu.har.mrc.ac.uk/mutabase) as soon as the founder lines are identified.

Technical Summary

The aim is to identify novel mutations in mice that protect synapses and axons from degeneration. New technology will be used to effect high-throughput, high-resolution analysis of neuromuscular junctions in mice mutagenised by ENU injection, exploiting a sensitised background: the WldS mutant. Heterozygotes in this line show robust protection of axons from degeneration following nerve injury. Hence they constitute an ideal background for screening novel mutations that either a) additionally protect synapses; or b) that counteract the axon protection produced by the WldS background; or c) that produce other covert or overt neuromuscular synaptic phenotypes. Two technological developments underpin the likely success of this project. The first is that the sensitized background mouse strain will also be transgenic for thy1.2:YFP. Thus, our WldS/YFP mice contain motor neurones that are both protected from degeneration and endogenously fluorescent . The second is the Cell viZio confocal microendoscope, a new instrument that facilitates optical access to living tissue, via a fibre-optic probe (probe diameter 1500 micrometres, resolution 5 micrometres). We have already used endogenous expression of fluorescent proteins to study axonal and synaptic degeneration in mutant thy1.2:YFP-WldS mice. ENU-mutagenised C57Bl6 mice will be crossbred with thy1.2:YFP-WldS mice. Systematic confocal-microendoscopic screening of 25-50 F1 progeny mouse lines per week will have a high chance of success in identifying dominant mutations that protect synapses from axotomy-induced degeneration. Significant phenotypes will be inheritance-tested through further genetic breeding. Subsequent analysis will be refined using histological, cellular and molecular techniques. The research will contribute to understanding and treatment of neurodegenerative disease.

Publications

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Babetto E (2010) Targeting NMNAT1 to axons and synapses transforms its neuroprotective potency in vivo. in The Journal of neuroscience : the official journal of the Society for Neuroscience

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Beirowski B (2009) Non-nuclear Wld(S) determines its neuroprotective efficacy for axons and synapses in vivo. in The Journal of neuroscience : the official journal of the Society for Neuroscience

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Blanco G (2012) Confocal Microendoscopy of Neuromuscular Synapses in Living Mice. in Current protocols in mouse biology

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Brown R (2014) Endomicroscopy and electromyography of neuromuscular junctions in situ. in Annals of clinical and translational neurology

 
Description MNDA Project Grant
Amount £120,000 (GBP)
Organisation Motor Neurone Disease Association (MND) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2010 
End 08/2013
 
Title CEMOP_S5 mutant 
Description ENU mutant with Mendelian inheritance of a characteristic that protects synapses from degeneration when co-expressed with the natural WldS mutation. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Funding required to capitalise on this 
 
Title Ex-vivo assay of neuromuscular synaptic degeneration 
Description We found that culture of isolated nerve-muscle preparations dissected from mice, in physiological saline maintained at 32ºC differentiated between rapid Wallerian degeneration,which occurs in normal mice following nerve injury, and slow Wallerian degeneration which occurs following axotomy of muscles from WldS mutant mice. This generated a novel paradigm for assessing chemical and environmental factors, including use or disuse of synapses, on the vulnerability of synapses to degeneration. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2014 
Provided To Others? Yes  
Impact Too early to say. 
 
Description Charities AGM 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Supporters
Results and Impact 50 members of the Scottish Motor Neurone Disease Association attended the AGM. I gave an accessible research talk about the research leading to the formation of the Euan MacDonald Centre for MND Research, that I spearheaded, and included images and movies of the research done on the MRC project. There was discussion afterwards.

N/A
Year(s) Of Engagement Activity 2008