Dominant negative ATP5F1A variants disrupt oxidative phosphorylation causing neurological disorders

Abstract

ATP5F1A encodes the α-subunit of complex V of the respiratory chain, which is responsible for mitochondrial ATP synthesis. We describe 6 probands with heterozygous de novo missense ATP5F1A variants that presented with developmental delay, intellectual disability, and movement disorders. Functional evaluation in C. elegans revealed that all variants tested were damaging to gene function via a dominant negative genetic mechanism. Biochemical and proteomics studies showed a marked reduction in complex V abundance and activity in proband-derived blood cells and fibroblasts. Mitochondrial physiology studies in fibroblasts revealed increased oxygen consumption, yet decreased mitochondrial membrane potential and ATP levels indicative of uncoupled oxidative phosphorylation as a pathophysiologic mechanism. Our findings contrast functionally and clinically with the previously reported ATP5F1A variant, p.Arg207His, suggesting a distinct pathological mechanism. This study therefore expands the phenotypic and genotypic spectrum of ATP5F1A-associated conditions and highlights how functional studies can provide understanding of the genetic, molecular, and cellular mechanisms of ATP5F1A variants of uncertain significance. With 12 heterozygous individuals now reported, ATP5F1A is the most frequent nuclear genome cause of complex V deficiency.

Keywords: ATP synthase; ATP5F1A; C. elegans; Complex V; developmental delay; dystonia; intellectual disability; mitochondriopathy; oxidative phosphorylation; variant.

Figure 1.. A schematic of ATP synthase (Complex V).

Human mitochondrial ATP synthase is comprised of two domains, F₁ and F_o. F₁ consists of α, β, γ, δ, and ε-subunits at a ratio of 3:3:1:1:1, and forms the catalytic activity domain where ATP synthesis occurs. F_o forms the proton pore and consists of a membrane embedded central ring organized by repeating c subunits, and a, e, f, g, j, k, and 8 subunits. A peripheral arm consisting of the b, d, and F6 subunits links to the catalytic head through the ATP5PO (also known as oligomycin sensitivity-conferring protein (OSCP)) subunit, and is embedded in the inner mitochondrial membrane.

Figure 2.. Proband ATP5F1A variants likely disrupt interaction between α, β, and γ subunits.

Proband variants are located at the interfaces of α:β, and α:γ subunits. Locations of the four variants in a single α–β dimer (A), or in the hexameric complex with the γ subunit (B). α-subunit is depicted in dark blue, β-subunit in orange, γ-subunit in yellow, and variant locations are shown in cyan. (C) Close up schematic of wild type and variant interactions in the holocomplex. Wild type residues in cyan, proband variant residues in magenta, other residues in α-, β- and γ-subunits are in dark blue, orange, and yellow, respectively. The P331L, S346F and R182Q substitutions are predicted to destroy H-bonds and/or impact the conformation and interaction between α, β, and γ subunits. The L109S substitution creates two novel H-bonds between α, and β subunits which likely alters the interaction between these subunits.

Figure 3.. P316L, S331F, and R167Q variants are damaging to atp-1 function in C. elegans.

(A) R182, P331, and S346 residues are in areas of high conservation across multiple species, and all three residues are conserved in the C. elegans homolog atp-1. hs- human, mm- mouse, rn- rat, xt- xenopus, dr- zebrafish, dm- drosophila, ce- C. elegans. (B) Schematic of how variant modeling in C. elegans aid in molecular diagnosis of rare disease. (C) Schematic of atp-1 extra copy lines generated that have endogenous atp-1 on chromosome I, and a single copy transgene insertion of wild type atp-1 on chromosome IV. (D) Development of heterozygous variant animals scored at 72 hours after embryo laying. Average of 4 experiments graphed, 60 embryos were counted in each replicate, except for R167Q, where at least 35 embryos were used. More information on the experiment and the crossing scheme used to generate these animals can be found in the Materials and Methods and in Fig. S3. (E) Representative images of animals in D. Scale bar, 100μm. (F) Length of heterozygous variant animals 24 hours post L4 larval stage as measured by WormLab over a two-minute recording. (G) Crawling speed of heterozygous variant animals 24 hours post L4 larval stage from the same recording as F. * p <0.05, ** p<0.01, *** p<0.001, **** p < 0.0001.

Figure 4.. atp-1 P316L, S331F, and R167Q variants induce mitochondrial stress.

(A) Schematic of the hsp-6p::gfp reporter expressing strain. GFP is not expressed under normal conditions. However, upon mitochondrial stress, the hsp-6p::gfp reporter is transcriptionally upregulated in the intestine of the animal. (B-D) Normalized GFP fluorescence expression in 1-day adult animals. (E-F) Representative images of animals quantified in B-D. Scale bar, 100μm. See Fig S7 for crossing scheme to generate these animals. ns – not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Figure 5.. atp-1 P316L variant alters mitochondrial morphology.

Mitochondrial morphology is altered in P316L animals. (A) Representative images of mitochondrial marker bcIs80 (myo-3p::mito-gfp) expressed in the body wall muscles. Scale bar, 5μm. (B) Quantification of average area and minor axis length per animal, calculated using MitoSegNet. 21–23 animals imaged per genotype. ns – not significant, ** p<0.01, *** p<0.001, **** p < 0.0001.

Figure 6.. Proband fibroblasts show reduced complex V abundance and activity.

(A) Schematic of mitochondrial functional studies performed on proband fibroblasts. (B) BN-PAGE analysis of complexes of the electron transport chain of proband 1 (p.Arg182Gln), proband 4 (p.Ser346Phe) and proband 6 (p.Leu109Ser) fibroblasts. (C) Western blots analysis of ATP5F1A, ATP5F1B and ATP5PO of proband and control fibroblasts. cs, citrate synthase, loading control. (D) Relative Complex Abundance (RCA) of OXPHOS complexes from proteomics data of Proband 2 (p.Arg182Gln) LCLs compared to controls (N=4) showing an isolated Complex V defect. Middle bar represents mean complex abundance. Upper and lower bars represent 95% confidence interval. Significance was calculated from a two-sided t-test between the individual protein means. *** = p<0.001, ns = not significant, p>0.05. (E) Protein range for ATP5F1A in LCLs of Proband 2 (p.Arg182Gln) (red dot) and controls (N=4, purple dots) showing a standard deviation of 8.7 from the control median. (F) Topographical heatmap of the log2 fold-change abundances of Proband 2 relative to controls onto the cryo-EM structure of the dimer complex V structure (PDB: 7AJD).

Figure 7.

ATP5F1A p.R182Q proband fibroblasts show reduced ATPase activity and uncoupled oxidative phosphorylation. (A) Representative oxygen consumption rate (OCR) of proband derived fibroblasts compared to age, race, and sex matched control fibroblasts measured by Seahorse in intact cells. (B) Normalized average basal, maximal, and spare OCR from three biological replicates. Individual graphs from each experiment are shown in Fig. S6. (C) Membrane potential of proband and control fibroblasts as measured by TMRE fluorescence with and without FCCP treatment. (D) Total cellular ATP content taken from lysed proband and control fibroblasts using a luciferase reporter. ns – not significant, * p <0.05, ** p<0.01, *** p<0.001, **** p < 0.0001.