Contrasting pathophysiological mechanisms of OPA1 mutations in autosomal dominant optic atrophy

Abstract

Autosomal dominant optic atrophy (ADOA) caused by mutations in the nuclear-encoded OPA1 gene result in the preferential loss of retinal ganglion cells (RGCs) and progressive optic nerve degeneration. The severity of ADOA can be highly variable. This study compared the pathophysiological consequences of the c.1034 G > A OPA1 missense mutation and the c.1305+2delGT OPA1 deletion. There was a significant correlation between the severity of visual loss and the extent of macular RGC loss as determined by optical coherence tomography imaging. In cells transfected with the c.1034 G > A mutant, the percentage of fragmented mitochondria was greater than 60% with cytochrome c (cyt c) overflow, and significantly elevated levels of reactive oxygen species (ROS) and apoptosis. In contrast, the c.1305+2delGT mutant caused mitochondrial fragmentation in ~ 20% of HeLa cells, resulting in less cyt c overflow and apoptosis. The extent of mitochondrial network fragmentation and apoptosis increased with decreasing WT OPA1 mRNA expression levels. The c.1034 G > A OPA1 missense mutation is likely to induce a dominant-negative effect compared with haploinsufficiency with the c.1305+2delGT OPA1 deletion. These contrasting pathophysiological mechanisms could influence disease severity in ADOA through their differential consequences on mitochondrial structure and function. The small drug molecule Paromomycin was able to rescue the mitochondrial fragmentation induced by the c.1034 G > A mutation, providing proof-of-concept for further therapeutic validation in ADOA.

Fig. 1. Clinical Features of ADOA Families.

A, C, D Pedigree of the 3 families with ADOA indicating the segregation of the identified OPA1 variants. Black symbols indicate patients with optic neuropathy; gray symbols indicate patients with subclinical disease. Arrows indicate family index cases. B Ophthalmologic features in family 1: an unaffected family member (F1-2) and a severely affected member (F1-6). E Ophthalmologic features in family 2: an unaffected family member (F2-4), four mildly affected family members (F2-6,7,5,3), and a severely affected family member (F2-1). F–K Correlation analysis between visual acuity and various parameters, including relative mRNA levels of normal and mutant OPA1, and optical coherence tomography measurements. The results showed a significant correlation. For each family member, the posterior fundus, RNFL thickness map and macular cube are shown. OD =“Oculus Dexter” or right eye; OS = “Oculus Sinister” or left eye.

Fig. 2. Genetic and Structure Analysis of OPA1 Mutations.

A Sequencing electropherograms showing the c.1034 G > A OPA1 missense mutation in family 1. B, C Location of the c.1034 G > A OPA1 missense mutation and c.1305+2delGT OPA1 deletion and its impact on transcript processing. D–F The OPA1 protein structure was predicted using Alphafold2 derived for wild-type (WT) (D), c.1034 G > A (E), and c.1305+2delGT (F) transcripts. The amino acid residues that were predicted to be skipped as a result of the c.1305+2delGT OPA1 deletion have been highlighted in yellow on the WT protein. G Protein secondary structure elements were monitored throughout the simulation with alpha-helices in orange and beta-strands in blue. H, I Stability and flexibility of the OPA1 proteins over the course of 100 ns. H Root mean square deviation (RMSD). I Root mean square fluctuation (RMSF).

Fig. 3. Mitochondrial Morphology and Function analysis of OPA1 Mutations.

A, B Mitochondrial morphology in HeLa and RGC5 cells overexpressing OPA1 mutants. Mitochondria were marked by red fluorescence. The cell nucleus was stained by DAPI. C–F Quantitative analysis of mitochondrial morphology classified as filamentous (white), intermediate (gray), and fragmented (black). The HeLa and RGC5 cells transfected with the c.1034 G > A mutant displayed a fragmented mitochondrial network. G, H Colocalization between cytochrome (cyt) c (green fluorescence) and mitochondrial in HeLa and RGC5 cells overexpressing OPA1 mutants. The transfected cells have been indicated with stars. I–L Quantitative analysis of colocalization coefficients of cyt c and mitochondria. The cells transfected with the c.1034 G > A mutant displayed a decreased coefficient. Asterisks indicate statistical significance (*adjusted p < 0.05, **adjusted p < 0.01, ***adjusted p < 0.001).

Fig. 4. Apoptosis, ROS level and colocalization analysis of OPA1 Mutations.

A TUNEL assay of apoptosis in HeLa and RGC5 cells overexpressing OPA1 mutants. B, C Quantitative analysis of the level of apoptosis. D–F Western blot of the cleaved-caspase3 expression level in HeLa and RGC5 cells overexpressing OPA1 mutants. G–I Quantitative analysis of the level of apoptosis. Overexpression of c.1034 G > A mutant resulted in a significant increase in apoptosis. J, K Cellular reactive oxygen species (ROS) level in HeLa and RGC5 cells overexpressing OPA1 mutants. L, M Quantitative analysis of the cellular ROS level. Both HeLa and RGC5 cells overexpressing OPA1 c.1034 G > A had relatively higher cellular ROS levels. N, O Colocalization between OPA1 mutants and fragmented mitochondrial in HeLa and RGC5 cells. The OPA1 mutant was tagged by flag, shown in green fluorescence. P, Q Quantitative analysis of colocalization coefficients. OPA1 c.1034 G > A was significantly colocalized with fragmented mitochondria.

Fig. 5. OPA1 Expression Level related analysis.

A Western blot of the OPA1 isoforms and expression level in HeLa and RGC5 cells overexpressing different OPA1 mutants. The two OPA1 isoforms were tagged by flag. B, C Quantitative analysis of the expression level of OPA1 mutant proteins. The expression level of OPA1 mutant proteins decreased significantly. D–G OPA1 mRNA expression levels in blood samples from ADOA family members. Unaffected family members are show in white, mildly affected family members in light purple, and the proband in dark blue. H Mitochondrial morphology in HeLa cells transfected with different concentrations of OPA1 siRNA. I OPA1 mRNA expression level detection by qPCR. J, K Quantification of OPA1 with Western blot. L, M Quantitative analysis of mitochondrial morphology. Loss of OPA1 induced fragmentation of the mitochondrial network. N, O Western blot of the c-cas3 expression level in HeLa cells transfected with different concentrations of OPA1 siRNA. Decreased OPA1 led to mitochondrial fragmentation with a significant increase in apoptosis.

Fig. 6. High-Throughput Screening for c.1034 G > A Mutant.

A High-throughput screening of about 1,500 FDA-approved small drug molecules was performed using the GLIDE module of Schrödinger Suites centered on the OPA1 missense mutation site. B Venn diagram showed the number of small molecules bound to WT and the OPA1 c.1034 G > A protein. C, D Molecular docking poses for paromomycin and OPA1 c.1034 G > A protein. E Mitochondrial morphology in HeLa cells overexpressing the c.1034 G > A mutant when treated with paromomycin. F–H Quantitative analysis of mitochondrial morphology. Paromomycin (500ug/ml) was able to resist the dominant-negative mechanism of the c.1034 G > A mutant by competitive binding.