Mitochondrial shaping proteins in models of optic neuropathy

Lead Research Organisation: Cardiff University
Department Name: Research and Commercial Division

Abstract

Inherited optic neuropathies are amongst the most common causes of childhood blindness in the developed world. Not only are inherited optic neuropathies common but they are also currently completely untreatable and incurable. The purpose of this work is to cast light on strategies for preventing optic neuropathy based on understanding the expression, regulation and modulation of mitochondrial fusion/ fission pathways and mitochondrial shaping genes. There are an increasing number of genes known to be involved in controlling the shape, and hence the function of, mitochondria in the cell. Some have all ready been identified as being involved in causing inherited optic neuropathy. In the short term, new genes need to be discovered to permit diagnostic screening, clinical and molecular diagnosis and management, assessment of genotype/ phenotype correlation and prognostic counselling, and hence improve the global management of these conditions for today?s patients. Concrete outcomes of this research will be to broaden the genetic spectrum of inherited optic neuropathy and cast light on the expression and function of such genes in neural tissue. The research will also focus on developing real animal models of disease by reducing or removing the expression of selected of these genes in the retina and studying the effect this has on mitochondrial shaping and retinal and optic nerve histology and morphology. This will enable an understanding of what role these genes have specifically in causing blindness by their putative effects on retinal ganglion cells. Only by broadening our understanding of this group of physiological vital genes will we be able to understand why abnormalities in this delicate mechanism may lead specifically to blindness.

Technical Summary

Mitochondrial shaping proteins are fundamental to the control of normal cellular physiolgy, especially in neural tissue, and those identified to date play a significant role in the pathophysiology of human inherited optic neuropathy. However, there is evidence from both genetic studies and basic cell biology for as yet unidentified proteins acting on the mitochondrial inner/ outer membrane in health and disease. The relative expression levels of mitochiondrial pro fusion/ fission genes may dictate mitochondrial morphology in cellular differentiation and during development and may thus play a role in the pathophysiology of disease. This will be explored by addressing the following specific research aims and questions:
Aim 1: identify evidence for new fusion/ fission genes and explore if any are involved in inherited optic neuropathy.
Aim 2: explore the spatio-temporal expression profile for mitochondrial shaping genes and proteins in the normal developing and adult mammalian eye.
Aim 3: generate murine models utilising in vivo knockdown in the eye.
Aim 4: and comprehensively characterise these mutant effects and interactions.

New mitochondrial shaping genes will be identified by homology from other species, or from research on mitochondrial membrane dynamics, and explored as candidate genes for human disease. Candidate gene screening for mutations in a population of patients DNA with inherited or sporadic optic atrophy, in whom mutations in the previously identified genes, OPA1 and OPA3 have been excluded, will be carried out. A novel gene mapped to a novel locus by the Applicant ( OPA7 ) will be identified and screened in the same samples. In order to explore the spatio-temporal expression profile for these genes and proteins in the normal developing and adult mammalian eye the plan is to use RT-PCR, in situ hybridization, immunohistochemistry and western blotting for candidates, including the novel gene discovered by the Applicant. Tissue localisation in adult mouse and human retinal sections, optic nerve and brain will be studied by immunohistochemistry and the overall expression of the gene, and its isoforms, in different tissues will be assessed by RT-PCR using tissue-specific cDNA. Lastly, in vivo modelling in the eye will be achieved utilising knowdown with siRNA in order to study mitochondrial shaping proteins and their pathophysiology. Once this has been achieved there will be a comprehensive characterisation of the mutant line(s) in order to permit the in vivo phenotypic assessment of the pathophysiology of this protein and hence gain invaluable insights into normal function.

Publications

10 25 50
 
Guideline Title Testing for inherited optic neuropathy
Description OPA1 gene test
Geographic Reach National 
Policy Influence Type Citation in clinical guidelines
Impact Improved service delivery of genetic testing for inherited optic neurpathy
 
Description UK EGG
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Fight for Sight PhD Studentship
Amount £95,000 (GBP)
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 09/2016
 
Description National Eye Research Centre Project Grant
Amount £50,000 (GBP)
Organisation National Eye Research Centre 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 09/2014
 
Description PhD Student
Amount £25,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2009 
End 09/2012
 
Description PhD Student
Amount £25,000 (GBP)
Organisation Cardiff University 
Sector Academic/University
Country United Kingdom
Start 10/2009 
End 09/2012
 
Description Small Grants Award
Amount £15,000 (GBP)
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2012 
End 10/2014
 
Title New -80 
Description New equipment 
Type Of Material Improvements to research infrastructure 
Year Produced 2009 
Provided To Others? Yes  
Impact Not yet 
 
Title Opa1 W616CinsLR 
Description Mouse model with new Opa1 mutation leading to Opa1 mislocalization and embryonic lethality when homozygous. heterozygotes bred on to C57Bl/6J line and ocular phenotype analysed. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Seconf Opa1 mutant line. 
 
Title Opa1 mutant mouse 
Description Novel mutant B6;C3-Opa1 Q285 STOP 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2007 
Provided To Others? Yes  
Impact ongoing research 
 
Title Opa3 mutant mouse 
Description B6;C3-Opa3 L122P 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2008 
Provided To Others? Yes  
Impact ongoing reearch 
 
Description JP Oxford 
Organisation Oxford University Hospitals NHS Foundation Trust
Country United Kingdom 
Sector Academic/University 
PI Contribution The collaboration is a basic science programe on mitophagy in Opa1 mutant mice.
Collaborator Contribution The provision of live mice (Opa1+/-) and tissues and other reagents. Planning and writing joint papers and grants.
Impact A number of grant applications are pending. One paper is under review.
Start Year 2011
 
Description NCL PYWM 
Organisation Newcastle University
Department Mitochondrial Research Group
Country United Kingdom 
Sector Academic/University 
PI Contribution Providing Opa1 mutant embryos and tissues as well as DNA, RNA and technical help. Also providing patient data.
Collaborator Contribution Screening samples from tissues and embryos from Opa1 mutant mice for mitochondrial DNA deletions and also screening patients and phenotypes
Impact Two publications submitted. • Yu-Wai-Man P, Griffiths PG, Gorman GS, Lourenco CM, Wright AF, Auer-Grumbach M, Toscano A, Musumeci O, Valentino ML, Caporali L, Lamperti C, Tallaksen CM, Duffey P, Miller J, Whittaker RG, Baker MR, Jackson MJ, Clarke MP, Dhillon B, Czermin B, Stewart JD, Hudson G, Reynier P, Bonneau D, Marques W Jr, Lenaers G, McFarland R, Taylor RW, Turnbull DM, Votruba M, Zeviani M, Carelli V, Bindoff LA, Horvath R, Amati-Bonneau P, Chinnery PF. Multi-system neurological disease is common in patients with OPA1 mutations. Brain 2010; 133: 771-786 • Yu-Wai-Man P, Davies VJ, Piechota MJ, Cree LM, Votruba M, Chinnery PF. Secondary mtDNA defects do not cause optic nerve dysfunction in a mouse model of dominant optic atrophy. Invest Ophthalmol Vis Sci. 2009; 50: 4561-4566
Start Year 2008
 
Description University of Cologne 
Organisation University of Cologne
Country Germany 
Sector Academic/University 
PI Contribution Sharing of DNA and resources exome sequencing
Collaborator Contribution Exome squencing
Impact none yet
Start Year 2013
 
Description School visit Cardiff 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 35 pupils attended an activity centered around genetics.

Positive feedback.
Year(s) Of Engagement Activity 2010
 
Description UK EGG 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Health professionals
Results and Impact Approx. 90 health and allied professional attended meeting on eye genetics organised and delivered by MV.

Excellent feedback,networking and plans to hold seconf meeting.
Year(s) Of Engagement Activity 2008