Mitochondrial shaping proteins in models of optic neuropathy

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.

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.