What disease mechanisms contribute to multisystem tissue involvement in dominant optic atrophy due to OPA1 mutations?

Autosomal dominant optic atrophy (DOA) affects the optic nerve, causing insidious visual loss from early childhood. It is the most common inherited optic nerve disorder and the majority of patients carry mutations in OPA1, a nuclear gene critical for normal mitochondrial function. Mitochondria are essential components of all human cells, containing multiple copies of their own DNA (MtDNA), and forming long branching networks which fuel the cell‘s energy requirements.

About 20% of patients with OPA1 mutations will develop a more severe form of the disease (DOA+), associated with a worse visual prognosis and additional neurological complications. The risk of developing DOA+ is three times higher with missense mutations located within the GTPase gene region. Using a range of human tissue samples, I will investigate whether this specific mutational subgroup results in greater fragmentation of the mitochondrial network and more marked mtDNA damage, thus contributing to accelerated cell death. I will also explore the possibility of rescuing the fragmentation defect by manipulating the level of expression of OPA1 and other mitochondrial proteins involved in network fusion and fission. Visual loss in DOA is progressive and the neurological features develop in later life, providing a clear window of opportunity for therapeutic intervention.

Autosomal dominant optic atrophy (DOA) is the most common inherited optic nerve disorder and the majority of patients harbour pathogenic OPA1 mutations. Although DOA is characterised by the preferential loss of retinal ganglion cells (RGC), about 20% of OPA1 carriers will develop a more severe form of the disease (DOA+), associated with a worse visual prognosis and additional debilitating neurological complications. The risk of developing DOA+ is three times higher with missense OPA1 mutations affecting the catatytic GTPase domain. I will explore the pathogenenetic mechanisms underlying this key observation by comparing tissue samples; skeletal muscle biopsies, fibroblasts, and myoblasts, collected from DOA+ patients with missense GTPase mutations (n=10), and pure DOA patients carrying frameshift deletions (n=5) and splice site mutations (n=5).

(i) Do missense OPA1 GTPase mutations result in increased mitochondrial network fragmentation?
OPA1 is a mitochondrial inner membrane protein with important pro-fusional properties. In conjunction with the mitochondrial outer membrane proteins MFN1 and MFN2, OPA1 counterbalances the influence of hFIS1 and DRP1, two pro-fission proteins found in the mitochondrial outer membrane and cytosol, respectively. Our preliminary data suggest that missense GTPase mutations lead to greater mitochondrial fragmentation compared with other OPA1 mutational subgroups. I will confirm these initial findings, investigating at the same time the pathological consequences of the observed fragmentation defect on cellular function and viability.

(ii) Do missense OPA1 GTPase mutations lead to greater mitochondrial DNA (mtDNA) instability?
Our pilot data, based on next-generation ultra-deep-sequencing, suggest that OPA1 GTPase mutations result in greater mtDNA instability. I will therefore compare the pattern and mutational burden of somatic mtDNA abnormalities in a larger group of DOA+ and pure DOA patients, relating them to two classical disorders of mtDNA maintenance (POLG1 and PEO1), and to normal ageing. I will use the generated biological data to model possible OPA1 disease mechanisms leading to accelerated mtDNA mutagenesis and the development of DOA+ features.

(iii) Are these deleterious consequences secondary to a dominant-negative effect?
HEK293 cells will be transfected with mutant cDNA extracted from OPA1 fibroblasts harbouring missense GTPase mutations. If these mutations exert a dominant-negative effect, mitochondrial fragmentation will be observed despite normal levels of the endogenous, wild-type OPA1 protein. Finally, I will explore the possibility of rescuing the fragmentation phenotype by manipulating the expression of the major pro-fusion and pro-fission mitochondrial proteins. These experiments could pave the way for gene therapy aimed initially at halting RGC loss and progressive visual decline.