Mitochondrial DNA instability and dysregulated mitochondrial quality control contribute to progressive retinal ganglion cell loss in dominant optic atrophy

Abstract

Purpose: Autosomal dominant optic atrophy (DOA) is a mitochondrial optic neuropathy caused by pathogenic mutations within the OPA1 gene (3q28-q29), which encodes for an inner mitochondrial membrane protein. The neuronal loss observed in DOA represents an attractive monogenic disease model to dissect the overlapping pathological pathways in more complex adult-onset neurodegenerative disorders. However, the fundamental mechanisms by which OPA1 mutations ultimately precipitate retinal ganglion cell (RGC) loss have still not been clarified.

Methods: We made use of a comprehensive OPA1 tissue biobank comprised of muscle biopsy specimens (n=30) and primary fibroblast cell lines (n=40) established from patients with both isolated optic atrophy and the more severe syndromic “DOA plus” phenotypes. By using a series of validated experimental assays, the following cellular parameters were assessed: (i) mitochondrial respiratory chain integrity; (ii) mitochondrial DNA (mtDNA) instability with real-time PCR and ultra-deep next generation sequencing; (iii) the kinetics of mitochondrial genome replication following a period of mtDNA depletion induced with ethidium bromide; and (iv) mitochondrial quality control (mitophagy) using a high-throughput flow cytometry protocol that allows rapid co-localisation of autophagosomes and quantification of LC3-positive puncta.

Results: OPA1 mutations result in a significant mitochondrial biochemical defect in both skeletal muscle and fibroblasts, and a clear correlation was observed with clinical disease severity. An accumulation of secondary mtDNA abnormalities was also observed, implicating marked mitochondrial genome instability secondary to an imbalance between mitochondrial fusion and fission. This impairment in the cell’s mtDNA replication machinery was further exacerbated following a period of mtDNA depletion induced with ethidium bromide treatment. Importantly, there was a dramatic reduction in the cell’s ability to handle exogenous pro-apoptotic stress due to the inappropriate activation of mitophagic pathways in a compensatory effort to eliminate dysfunctional mitochondria.

Conclusions: MtDNA instability and impaired mitochondrial quality control are important synergistic mechanisms that contribute to neuronal loss in DOA, and they represent attractive disease pathways amenable to therapeutic intervention.