Defective mitochondrial fusion, altered respiratory function, and distorted cristae structure in skin fibroblasts with heterozygous OPA1 mutations

Abstract

Deleterious consequences of heterozygous OPA1 mutations responsible for autosomal dominant optic atrophy remain a matter of debate. Primary skin fibroblasts derived from patients have shown diverse mitochondrial alterations that were however difficult to resolve in a unifying scheme. To address the potential use of these cells as disease model, we undertook parallel and quantitative analyses of the diverse reported alterations in four fibroblast lines harboring different OPA1 mutations, nonsense or missense, in the guanosine triphosphatase or the C-terminal coiled-coil domains. We tackled several factors potentially underlying discordant reports and showed that fibroblasts with heterozygous OPA1 mutations present with several mitochondrial alterations. These included defective mitochondrial fusion during pharmacological challenge with the protonophore carbonyl cyanide m-chlorophenyl hydrazone, significant mitochondrial elongation with decreased OPA1 and DRP1 proteins, and abnormal mitochondrial fragmentation during glycolysis shortage or exogenous oxidative stress. Respiratory complex IV activity and subunits steady-state were decreased without alteration of the mitochondrial deoxyribonucleic acid size, amount or transcription. Physical link between OPA1 protein and oxidative phosphorylation was shown by reciprocal immunoprecipitation. Altered cristae structure coexisted with normal response to pro-apoptotic stimuli and expression of Bax or Bcl2 proteins. Skin fibroblasts with heterozygous OPA1 mutations thus share significant mitochondrial remodeling, and may therefore be useful for analyzing disease pathophysiology. Identifying whether the observed alterations are also present in ganglion retinal cells, and which of them underlies their degeneration process remains however an essential goal for therapeutic strategy.

Syndromic parkinsonism and dementia associated with OPA1 missense mutations

“…We and others have previously shown this feature in fibroblasts carrying OPA1 mutations leading to haploinsufficiency linked to nonsyndromic DOA 31, 44. Western blot analysis revealed the somewhat surprising observation that, as also reported for DOA‐associated nonsense mutations, both these OPA1 missense mutations led to decreased OPA1 protein amount, suggesting haploinsufficiency.…”

Impaired complex IV activity in response to loss of LRPPRC function can be compensated by mitochondrial hyperfusion

“…Alternatively, complex IV may directly affect Opa1, which localizes to the inner mitochondrial membrane. It has recently been shown that mammalian Opa1 can interact with some of the subunits of the ETC, including subunits of complex IV (21). Therefore, it is feasible that complex IV activates mitochondrial fusion by physically interacting with Opa1.…”

“…For example, inactivation in HeLa cells of Drp1, which is required for mitochondrial fission, causes a decrease in mitochondrial membrane potential, respiration, and cellular ATP (20). Similarly, primary skin cells derived from patients with Optic Dominant Atrophy, who carry mutations in the mitochondrial fusion protein Opa1, exhibit reduced levels of complex IV subunits, resulting in decreased complex IV activity (21). Furthermore, the inactivation of the mitochondrial fusion protein Mfn2 in muscle cells grown in culture causes a reduction in the levels of subunits of several ETC complexes, as well as a decrease in mitochondrial membrane potential and respiration (22).…”

The connection between inner membrane topology and mitochondrial function

“…Caspase 8 cleaved BID (a precursor to fully truncated BID) disrupts Opa1 oligomers and widens the junction openings [24]. Opa1 defects in humans are associated with a serious neuropathy, autosomal dominant optic atrophy, and skin fibroblasts from these patients have distorted crista structure [25]. Functional mutants of Opa1 in Caenorhabditis elegans have a slow metabolism and their mitochondria contain numerous detached cristae in the matrix [26].…”