Dominant optic atrophy (ADOA) is one of the commonest mitochondrial optic neuropathies (MONs) caused by mutation in a nuclear mitochondrial gene, OPA1. There are currently no effective treatments, and patients become blind due to the death of retinal ganglion cells (RGCs), which take information from the eye to the brain via the optic nerves. Mitochondria are structures within the cell that convert food into cellular energy (ATP), with vision being of particularly high demand.
In the recent past extensive research has been carried out on low level laser light therapy, also known as photobiomodulation. It has been hypothesised that laser light works by targeting cytochrome c oxidase in mitochondria and may increase ATP generation.
In this project we will evaluate the effects of photobiomodulation on mitochondrial optic neuropathy, using a range of cell -based models, including a 3D cell culture model. We will be using mouse embryonic stem cell from Opa1+/- mutant mouse model, differentiate them into retinal ganglion cells. Embryonic stem cells at E3.5 will be isolated from embryos derived from mating wild type and Opa1+/- mutant mouse. Following standard protocols, embryos will be cultured in KSOMaag, supplemented with knockout serum replacement until isolation (Vila-Cejudo et al 2017). Differentiation of mESC into retinal ganglion cells will be carried out (Tanaka et al 2016). The cells will be examined for differentiation using differential markers (Brn3a, Brn3b, Brn3c, Math5, Rx). We will also edit OPA1 mutant alleles (frameshift mutations/ knockouts) in a reference cell line e.g. KOLF2, and use that as an isogenic control (e.g. 3 heterozygous mutant clones and 3 WT clones for comparison). Specific mutations will be made using CRISPR/Cas9 using ssDNA with the desired sequence as a homology repair template.
The genotyped differentiated cells will be exposed to photobiomodulation. Assays of mitochondrial ATP synthesis, oxidative phosphorylation/ enzymology (Complex I+ II+III + IV activities and levels of Complex IV protein subunits; bioenergetic studies) and mitochondrial morphological studies will be undertaken.
This is a self funded project.
Must have previous experience in cell and molecular biology and a first class or upper second degree in a biological science, molecular biology, biochemistry or related subject.
Applicants should apply to the Doctor of Philosophy in Vision Sciences.
Please include an up-to-date CV, a personal statement and two references.
We reserve the right to close the opportunity early should a sufficient number of suitable applications be received.
The successful candidate will begin the PhD/MPhil in Cardiff at the start of: April, July, October 2021
1. Davies VJ, Hollins AJ, Piechota MJ, Yip W, Davies JR, White KE, Nichols PP, Boulton ME, Votruba M. Opa1 deficiency in a mouse model of Autosomal Dominant Optic Atrophy impairs mitochondrial morphology, optic nerve structure and visual function. 2007. Hum Molecular Genet 16: 1307
2. Sun S, Erchova I, Sengpiel F, Votruba M. Opa1 deficiency leads to diminished mitochondrial bioenergetics with compensatory increased mitochondrial motility. 2020. Invest Ophthalmol Vis Sci. 61:42. doi: 10.1167/iovs.61.6.42
3. Smith TG, Seto S, Ganne P, Votruba M. A randomized, placebo-controlled trial of the benzoquinone idebenone in a mouse model of OPA1-related dominant optic atrophy reveals a limited therapeutic effect on retinal ganglion cell dendropathy and visual function. 2016. Neuroscience 319:92
4. Williams PA, Morgan JE, Votruba M. Opa1 deficiency in a mouse model of dominant optic atrophy leads to retinal ganglion cell dendropathy. 2010. Brain. 133:2942
5. Williams PA, Piechota M, von Ruhland C, Taylor E, Morgan JE, Votruba M. Opa1 is essential for retinal ganglion cell synaptic architecture and connectivity. 2012. Brain. 135:493
6. Varricchio C, Beirne K, Heard C, Newland B, Rozanowska M, Brancale A, Votruba M. The yin and yang of idebenone: Not too little, not too much – cell death in NQO1 deficient cells and the mouse retina. 2019. Free Radic Biol Med. doi: 10.1016/j.freeradbiomed.2019.11.030. PMID: 31775023
7. Beirne K, Rozanowska M, Votruba M. Red Light Treatment in an Axotomy Model of Neurodegeneration. 2016. Photochem Photobiol. 92:624