Drug repositioning as a therapeutic strategy for neurodegenerations associated with OPA1 mutations

Abstract

OPA1 mutations are the major cause of dominant optic atrophy (DOA) and the syndromic form DOA plus, pathologies for which there is no established cure. We used a 'drug repurposing' approach to identify FDA-approved molecules able to rescue the mitochondrial dysfunctions induced by OPA1 mutations. We screened two different chemical libraries by using two yeast strains carrying the mgm1I322M and the chim3P646L mutations, identifying 26 drugs able to rescue their oxidative growth phenotype. Six of them, able to reduce the mitochondrial DNA instability in yeast, have been then tested in Opa1 deleted mouse embryonic fibroblasts expressing the human OPA1 isoform 1 bearing the R445H and D603H mutations. Some of these molecules were able to ameliorate the energetic functions and/or the mitochondrial network morphology, depending on the type of OPA1 mutation. The final validation has been performed in patients' fibroblasts, allowing to select the most effective molecules. Our current results are instrumental to rapidly translating the findings of this drug repurposing approach into clinical trial for DOA and other neurodegenerations caused by OPA1 mutations.

Figure 1

Yeast drugs screenings. (A) Spot assay of mgm1^I322M mutant strain on YP medium supplemented with 2% ethanol or 2% glycerol at 37°C. 4 × 10⁴, 4 × 10³, 4 × 10² and 4 × 10¹ cells were present in each serial spot. (B) Petite frequency and (C) oxygen consumption rate normalized to the wild type MGM1 strain of the mgm1^I322M mutant strain at 37°C. (D) Spot assay on YP medium supplemented with 2% ethanol and 2% glycerol of the chim3 mutant strains at 28 and 37°C. (E) Petite frequency of the mgm1^I322M mutant strain treated with ORMs (32 μm ORM0, 420 nM ORM2, 16 μm ORM 11, 24 μm ORM12, 92 μm OMR14 and 1 μm ORMA) or with DMSO at 28°C. ^**Denotes P < 0.01; ^***denotes P < 0.001.

Figure 2

Viability and ATP content of MEFs bearing the indicated OPA1 mutations incubated with ORMs. (A–C) Number of viable cells of ISO1, R445H and D603H MEFs, after incubation for 24 h in DMEM-galactose in the absence or presence of ORMs. Data are expressed as percent of the SRB absorbance measured in DMEM-glucose at time = 0, considered as 100% value. Data are means ± SEM of 19 experiments in DMEM-galactose and 3–7 experiments in DMEM-galactose with ORMs treatment (ORMs 0, 2, 12: n = 3; ORM14, A: n = 7). (D–F) Cellular ATP levels of ISO1, R445H and D603H MEFs after incubation for 24 h in DMEM-galactose ± ORMs. Data are expressed as percentage of ATP content measured in DMEM-glucose at time = 0. Values are means ± SEM of 14 experiments in DMEM-galactose and 3–4 experiments in DMEM-galactose + ORMs (ORMs 0, 2, 12, 14 n = 4, ORMA n = 3). ^*Denotes P < 0.05.

Figure 3

Mitochondrial network morphology of MEFs bearing the indicated OPA1 mutations incubated with ORMs. (A, D) Representative images of mitochondrial network of ISO1 and D603H MEFs after labeling with Mitotracker Red. MEFs lines were incubated for 24 h in DMEM (B, C) or in DMEM-galactose (E, F) in absence or presence of the ORMs. Cells were scored in three categories on the basis of mitochondrial network morphologies: cells with filamentous and interconnected network (filamentous), cells with short filamentous mitochondria (intermediate) and cells with fragmented mitochondria (fragmented). 100–120 cells were analyzed for ISO1 (B–E) and D603H (C–F) MEFs in each condition and for each experiment. Data are means ± SEM of 3–4 independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 4

Levels of mitochondrial shaping proteins in MEFs bearing the indicated OPA1 mutations incubated with ORMs. MEFs were incubated for 24 h in DMEM-galactose in the absence or presence of ORMs. (A, D, F, H) Representative blots of OPA1, MFN2, DRP1 and DRP1-P-S616. ACTIN or TUBULIN were used as a loading control. (B, E, G) Densitometric analysis of OPA1, MFN2 AND DRP1 protein levels. Data are normalized to those of untreated cells in DMEM-galactose. Values are means ± SEM (n = 3–6). ^*P < 0.05, one simple t test. (C) Densitometric analysis of OPA1 long-/short-forms ratio. Values are means ± SEM (n = 4–6). (I) Densitometric analysis of DRP1-P-S616/DRP1 ratio. Values are means ± SEM (n = 3).

Figure 5

Mitochondrial protein content of MEFs bearing the indicated OPA1 mutations incubated with ORMs. MEFs were incubated for 24 h in DMEM-galactose in the absence or presence of ORMs. (A) Western blot of representative proteins of outer (TOM20) and inner (TIM23) mitochondrial membrane and of the matrix (citrate synthase and Hsp60). ACTIN or GAPDH were used as a loading control. (B–E) Densitometric analysis of the mitochondrial mass proteins; data are normalized to those of untreated cells in DMEM-galactose. Values are means ± SEM (n = 4–8). ^*P < 0.05, one simple t test. (F) mtDNA copy number. Data are means ± SEM (n = 3).

Figure 6

Levels of OXPHOS proteins of MEFs bearing the indicated OPA1 mutations incubated with ORMs. MEFs were incubated for 24 h in DMEM-galactose in the absence or presence of ORMs. (A) Western blot of representative subunits of complex I (NDUFA9), complex II (SDHA), complex III (UQCRC2), complex IV (COXIV) and complex V (ATP5A). ACTIN was used as a loading control. (B–F) Densitometric analysis of the OXPHOS subunits levels. Values are means ± SEM (n = 3–9) and are normalized to those of untreated cells in DMEM-galactose. ^*P < 0.05, one simple t test.

Figure 7

Levels of autophagy markers. Western blot of LC3 in DMEM and DMEM-galactose (A) and in DMEM-galactose in the absence or presence of ORMs (C) in MEFs. (B, D) Densitometric analysis of LC3II/I ratio. Values are means ± SEM (n = 3–4). ^*P < 0.05, student’s two tail t-test. (E, G) Representative blots of ATG5 and ATG7. (F, H) Densitometric analysis of ATG5 and AT7 protein levels. Values are means ± SEM (n = 3) and are normalized to those of untreated cells in DMEM-galactose. ACTIN or GAPDH were used as a loading control.

Figure 8

Effects of the ORMs in DOA patients’ fibroblasts. (A) Representative images of mitochondrial network of fibroblasts loaded with Mitotracker Red. (B, C) 60–80 cells were analyzed for R445H and D603H fibroblasts in each condition. Data are means ± SEM of three independent experiments. (D, E) Cellular ATP levels of R445H and D603H fibroblasts after 24 h incubation in DMEM-galactose in the absence or presence of ORMs. Data are expressed as percent of ATP content measured in DMEM-glucose at time = 0; values are means ± SEM of three experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 9

(A) Summary of the step-by-step experimental procedures leading to ORMs validation. (B) Hypothetical targets and mechanisms of action of the ORMs.