Mitochondrial protein sorting as a therapeutic target for ATP synthase disorders

Abstract

Mitochondrial diseases are systemic, prevalent and often fatal; yet treatments remain scarce. Identifying molecular intervention points that can be therapeutically targeted remains a major challenge, which we confronted via a screening assay we developed. Using yeast models of mitochondrial ATP synthase disorders, we screened a drug repurposing library, and applied genomic and biochemical techniques to identify pathways of interest. Here we demonstrate that modulating the sorting of nuclear-encoded proteins into mitochondria, mediated by the TIM23 complex, proves therapeutic in both yeast and patient-derived cells exhibiting ATP synthase deficiency. Targeting TIM23-dependent protein sorting improves an array of phenotypes associated with ATP synthase disorders, including biogenesis and activity of the oxidative phosphorylation machinery. Our study establishes mitochondrial protein sorting as an intervention point for ATP synthase disorders, and because of the central role of this pathway in mitochondrial biogenesis, it holds broad value for the treatment of mitochondrial diseases.

Figure 1. Identification of NaPT in a drug screen and a potential new therapeutic target revealed by a genomic screen.

(a) NaPT rescues respiratory growth of the fmc1Δ mutant, a yeast model for ATP synthase disorders, in a dose-dependent manner. fmc1Δ cells were spread onto solid rich glycerol medium, indicated amounts of NaPT were spotted onto filters, and plates were incubated at 36 °C for 6 days. High concentrations of NaPT are toxic, as indicated by the regions with no growth immediately surrounding the filters. The halos of growth at dose-dependent distances from the filters correspond to the lower therapeutic concentrations. DMSO (−), compound vehicle. (b) NaPT treatment improves survival of atp6-T8993G (JCP239) cybrids derived from ATP synthase disorder patients in glucose-deprived medium in a dose-dependent manner. Data from three replicates per condition are indicated by points; height of the bar represents the mean. Statistical significance: *P<0.05, (Wilcoxon’s test) relative to the untreated sample (0). DHLA, positive control, drug currently in clinical trials for mitochondrial encephalopathies. (c) The chemical–genomic profile of NaPT identifies mitochondrial protein sorting as a potential target. On treatment of the yeast genome-wide deletion collection with 8.35 μM NaPT, TIM17 and TIM23, essential genes involved in mitochondrial protein import, displayed pronounced deletion sensitivity/haploinsufficiency (calculated relative to the same strain collection treated with DMSO as a negative control; see Methods). Increasing data density is depicted as lighter shades of blue. (d) TIM17-deletion sensitivity is specific to NaPT. Frequency distribution of the TIM17^+/− mutant z-scores (see Methods) from 726 published chemical–genomic profiles across 332 compounds compared with the score obtained for NaPT.

Figure 2. NaPT selectively and differentially modulates mitochondrial protein sorting via the presequence translocase TIM23.

(a) Import of in vitro-synthesized, radiolabelled cytochrome b₂Δ-DHFR, a model protein targeted to the mitochondrial matrix, into isolated yeast mitochondria (p=precursor; i=intermediate) in the presence or absence of 100 μM NaPT. The lower panel displays quantifications of the bands detected by autoradiography in three independent experiments (imported, protease-resistant i-form). The signal obtained in the presence DMSO after the longest incubation time was set to 100%. Error bars represent the s.e.m. Δψ, mitochondrial inner membrane potential. (b) Import of in vitro synthesized, radiolabelled cytochrome b₂-DHFR, targeted to the inner membrane, into isolated mitochondria (m=mature). The lower panel displays quantifications of the bands (imported, protease-resistant m-form) as in a. (c) Importing saturating amounts of recombinantly expressed and purified cytochrome b₂Δ-DHFR causes a pronounced, concentration-dependent inhibition by NaPT. Import reactions were analysed by immunoblotting using an antibody against DHFR.

Figure 3. Genetic modulation of mitochondrial protein sorting rescues yeast and human ATP synthase disorder models.

(a,b) Tim21 overexpression induces NaPT-like modulation of TIM23-mediated mitochondrial protein import. Assays as in Fig. 2a,b were performed with mitochondria isolated from fmc1Δ cells carrying Tim21 overexpression (Tim21↑) versus empty plasmids. Data are representative of three independent experiments. (c) Tim21 overexpression suppresses the respiratory growth defect of fmc1Δ yeast. Strains were grown in liquid rich glycerol media (YPG) at 36 °C until stationary phase; their maximum growth rate (in generations per day, see Methods) is displayed in a boxplot (WT (wild type), n=3; fmc1Δ, n=5). (d) Overexpression of TIMM21, the human ortholog of yeast TIM21, improves cybrid survival. NARP atp6-T8993G cybrids were transduced with lentiviral particles carrying TIMM21 and GFP or RFP alone as a negative control. After 6 days, surviving cells were counted using flow cytometry. Individual replicates are displayed as points; height of the bar represents the mean; * indicates statistical significance (P<0.05, Wilcoxon’s test).

Figure 4. Modulation of mitochondrial protein sorting considerably restores the bioenergetic capacity of a yeast model of ATP synthase disorders.

(a,b) Tim21 overexpression accelerates the incorporation of subunits Qcr8 and Su h into cytochrome bc₁-containing respiratory chain supercomplexes and ATP synthase, respectively, in fmc1Δ mitochondria. Radiolabelled, in vitro-synthesized proteins were imported into isolated mitochondria and their assembly into their respective complexes (indicated with arrows) was followed using BN-PAGE and autoradiography. Δψ, mitochondrial inner membrane potential; III₂/IV₂, III₂/IV₁, III₂: respiratory chain supercomplexes formed by complex III (cytochrome bc₁) and IV (cytochrome c oxidase, COX); V₁ and V₂, monomeric and dimeric F₁F_o-ATP synthase (complex V), respectively. (c) Genetic modulation of mitochondrial protein sorting improves the mitochondrial inner membrane potential (Δψ) of fmc1Δ yeast. The Δψ of indicated mitochondria was monitored using a DiSC₃(5) fluorescence quenching assay (see Methods). The difference in fluorescence before and after addition of valinomycin indicates the magnitude of Δψ of the analysed mitochondria. (d) Genetic modulation of mitochondrial protein sorting improves the bioenergetic capacity of fmc1Δ yeast. The following parameters were measured in isolated mitochondria: maximal/uncoupled respiratory rate (upper left), maximal/uncoupled cytochrome c oxidase (COX) activity with ascorbate/TMPD as a substrate (upper right), oligomycin-sensitive ATP hydrolysis using a colorimetric ATPase assay (lower left), and oligomycin-sensitive ATP synthesis using a luciferase assay (lower right). Bar height corresponds to median value of individual measurements displayed as points (both technical and biological replicates). Significance according to a t-test is represented as **P<0.01 or *P<0.05.