β-RA reduces DMQ/CoQ ratio and rescues the encephalopathic phenotype in Coq9R239X mice

Abstract

Coenzyme Q (CoQ) deficiency has been associated with primary defects in the CoQ biosynthetic pathway or to secondary events. In some cases, the exogenous CoQ supplementation has limited efficacy. In the Coq9^R239X mouse model with fatal mitochondrial encephalopathy due to CoQ deficiency, we have tested the therapeutic potential of β-resorcylic acid (β-RA), a structural analog of the CoQ precursor 4-hydroxybenzoic acid and the anti-inflammatory salicylic acid. β-RA noticeably rescued the phenotypic, morphological, and histopathological signs of the encephalopathy, leading to a significant increase in the survival. Those effects were due to the decrease of the levels of demethoxyubiquinone-9 (DMQ₉) and the increase of mitochondrial bioenergetics in peripheral tissues. However, neither CoQ biosynthesis nor mitochondrial function changed in the brain after the therapy, suggesting that some endocrine interactions may induce the reduction of the astrogliosis, spongiosis, and the secondary down-regulation of astrocytes-related neuroinflammatory genes. Because the therapeutic outcomes of β-RA administration were superior to those after CoQ₁₀ supplementation, its use in the clinic should be considered in CoQ deficiencies.

Keywords: 2,4‐dihydroxybenzoic acid; Q synthome; astrogliosis; mitochondrial encephalopathy; spongiosis.

Figure EV1. CoQ biosynthetic pathway, including the bypass hypothesis for defects in COQ9 or COQ7

4‐HB = 4‐hydroxybenzoic acid; DMQ = demethoxyubiquinone or 2‐polyprenyl‐6‐methoxy‐3‐methyl‐1,4‐benzoquinone; DMeQ = 2‐polyprenyl‐5‐hydroxy‐6‐methoxy‐3‐methyl‐1,4‐benzoquinone; β‐RA = β‐resorcylic acid. The purple circle indicates the OH group present in the β‐RA and absent in the 4‐HB. The hydroxylation of DMQ is normally catalyzed by COQ7, which needs COQ9 for its stability and activity. This step is compromised in patients with defects in COQ7 or COQ9. The pathway is exampled with the production of CoQ₁₀.

Figure 1. Survival and phenotypic characterization of Coq9 ^R239X mice after β‐RA treatment

1. A

   Survival curve of the Coq9 ^+/+ mice, Coq9 ^R239X mice, and Coq9 ^R239X mice after β‐RA treatment and Coq9 ^R239X after ubiquinol‐10 treatment. The treatments started at 1 month of age [Log‐rank (Mantel‐Cox) test or Gehan–Breslow–Wilcoxon test; Coq9 ^+/+, n = 9; Coq9 ^R239X, Coq9 ^R239X after β‐RA treatment, and Coq9 ^R239X after ubiquinol‐10 treatment, n = 15 for each group].

2. B

   Survival curve of the Coq9 ^R239X mice and Coq9 ^R239X mice after β‐RA treatment started at 3 months of age (Coq9 ^R239X, n = 15; Coq9 ^R239X after β‐RA treatment, n = 10).

3. C, D

   Body weight of males (C) and females (D) Coq9 ^+/+ mice, Coq9 ^R239X mice, and Coq9 ^R239X mice after β‐RA treatment [(C): Coq9 ^+/+, n = from 31 to 4; Coq9 ^R239X, n = from 13 to 13; Coq9 ^R239X after β‐RA treatment, n = from 31 to 7. (D): Coq9 ^+/+, n = from 22 to 3; Coq9 ^R239X, n = from 13 to 12; Coq9 ^R239X after β‐RA treatment, n = from 24 to 3].

4. E, F

   Rotarod test of male (E) and female (F) Coq9 ^+/+ mice, Coq9 ^R239X mice, and Coq9 ^R239X mice after β‐RA treatment [(E): Coq9 ^+/+, n = from 11 to 8; Coq9 ^R239X, n = from 10 to 10; Coq9 ^R239X after β‐RA treatment, n = from 11 to 9. (F): Coq9 ^+/+, n = from 5 to 5; Coq9 ^R239X, n = from 4 to 4; Coq9 ^R239X after β‐RA treatment, n = from 11 to 6].

5. G

   Blood pressure of Coq9 ^+/+ mice, Coq9 ^R239X mice, and Coq9 ^R239X mice after β‐RA treatment. Data from male and female mice are represented together (Coq9 ^+/+, n = 6; Coq9 ^R239X, n = 6; Coq9 ^R239X after β‐RA treatment, n = 7). The whiskers down and up of the boxes indicate the minimum and maximum value, respectively. Horizontal lines inside the boxes mark the median.

6. H

   Comparative image of a Coq9 ^R239X mouse and a Coq9 ^R239X mouse after β‐RA treatment at 4 months of age.

Data information: Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; Coq9 ^R239X or Coq9 ^R239X after β‐RA treatment versus Coq9 ^+/+. ⁺ P < 0.05; ^{+ +} P < 0.01; Coq9 ^R239X versus Coq9 ^R239X after β‐RA treatment (one‐way ANOVA with a Tukey's post hoc test or t‐test; see Appendix Table S2 for exact P‐values).

Figure 2. Pathological features in the brain of Coq9 ^R239X mice after β‐RA treatment

1. A–U

   (A₁–F₁) H&E stain in the diencephalon of Coq9 ^+/+ mice (A₁ and D₁), Coq9 ^R239X mice (B₁ and E₁), and Coq9 ^R239X mice after β‐RA treatment (C₁ and F₁). (G₁–L₁) Anti‐GFAP stain in the diencephalon of Coq9 ^+/+ mice (G₁ and J₁), Coq9 ^R239X mice (H₁ and K₁), and Coq9 ^R239X mice after β‐RA treatment (I₁ and L₁). (M₁–R₁) Anti‐Iba1 stain in the diencephalon of Coq9 ^+/+ mice (M₁ and P₁), Coq9 ^R239X mice (N₁ and Q₁), and Coq9 ^R239X mice after β‐RA treatment (O₁ and R₁). (S₁–U₁) Protein oxidation in the diencephalon of Coq9 ^+/+ mice (S₁), Coq9 ^R239X mice (T₁), and Coq9 ^R239X mice after β‐RA treatment (U₁). (A₂–C₂) Magnetic Resonance Images of the diencephalon of Coq9 ^+/+ mice (A₂), Coq9 ^R239X mice (B₂), and Coq9 ^R239X mice after β‐RA treatment (C₂). (D₂–F₂) Magnetic Resonance Images of the pons of Coq9 ^+/+ mice (D₂), Coq9 ^R239X mice (E₂), and Coq9 ^R239X mice after β‐RA treatment (F₂). (G₂–I₂) Lactate peak in the brain of Coq9 ^+/+ mice (G₂), Coq9 ^R239X mice (H₂), and Coq9 ^R239X mice after β‐RA treatment (I₂). Scale bars: 1 mm (A₁–C₁); 100 μm (D₁–F₁); 200 μm (G₁–I₁); 100 μm (J₁–L₁); 200 μm (M₁–O₁); 100 μm (P₁–R₁); 100 μm (S₁–U₁). The yellow arrows indicated the areas of increased T2 signal, which is characteristic of lesions in specific brain areas.

Figure EV2. COX and SDH staining in gastrocnemius

1. A–C

   COX stains in gastrocnemius of Coq9 ^+/+ mice (A), Coq9 ^R239X mice (B), and Coq9 ^R239X mice after β‐RA treatment (C).

2. D–F

   SDH stain in gastrocnemius of Coq9 ^+/+ mice (D), Coq9 ^R239X mice (E), and Coq9 ^R239X mice after β‐RA treatment (F).

Data information: “I” indicated fiber types I and “II” indicates fiber types II.

Figure 3. Effect of the β‐RA treatment on genes expression profiles and neuroinflammatory‐related features

1. A

   Global differences in the levels of gene expression between experimental groups.

2. B

   Only pathways with more than one gene significantly altered in Coq9 ^R239X compared to Coq9 ^+/+ mice are considered for this figure. The y‐axis represents the percentage of genes corresponding to a pathway in the sample of genes whose expression level is significantly altered in a comparison between mice samples.

3. C

   Heatmap showing the comparative landscape of the expression level per sample of the 27 genes whose expression was altered by the mutation and normalized by the β‐RA treatment. The column numbers correspond to the different samples and the row names are gene codes composed of the ENSEMBL gene ID and the gene symbol (between parentheses). The green means comparatively higher, red means comparatively lower, and black means no difference in expression level (n = 5 for each group).

4. D

   iNOS expression levels in RAW cells after stimulation with LPS (n = 3 for each group).

5. E

   Cytokine levels in the culture supernatant of RAW cells after stimulation with LPS (n = 3 for each group).

6. F

   Cytokine levels in the brainstem of Coq9 ^+/+ mice, Coq9 ^R239X mice, and Coq9 ^R239X mice treated with β‐RA (n = 7 for each group).

7. G, H

   Mortality rate (G) and (H) percentage of malformations in zebrafish embryos treated with MPT and the effect of β‐RA treatment.

8. I

   Survival curve of Ndufs4 knockout mice and Ndufs4 knockout mice treated with β‐RA [Log‐rank (Mantel‐Cox) test; n = 5 for each group; see Appendix Table S3 for exact P‐values].

Data information: Data in panels (D–F) are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; LPS or LPS + β‐RA versus control. ⁺ P < 0.05; LPS or LPS + β‐RA versus control (one‐way ANOVA with a Tukey's post hoc test; see Appendix Table S3 for exact P‐values).

Figure 4. Tissue‐specific differences in mitochondrial function after β‐RA treatment in Coq9 ^R239X mice

1. A–O

   (A, F, K, M) CoQ‐dependent Complex I+III activities in brain (A) and kidneys (F), skeletal muscle (K), and heart (M). (A): Coq9 ^+/+, n = 5; Coq9 ^R239X, n = 6; Coq9 ^R239X after β‐RA treatment, n = 7. (F): Coq9 ^+/+, n = 4; Coq9 ^R239X, n = 6; Coq9 ^R239X after β‐RA treatment, n = 4. (K): Coq9 ^+/+, n = 3; Coq9 ^R239X, n = 6; Coq9 ^R239X after β‐RA treatment, n = 3. (M): Coq9 ^+/+, n = 5; Coq9 ^R239X, n = 6; Coq9 ^R239X after β‐RA treatment, n = 6). (B, G, L, N) CoQ‐dependent Complex II+III activities in brain (B) and kidneys (G), skeletal muscle (L) and heart (N). (B): Coq9 ^+/+, n = 5; Coq9 ^R239X, n = 6; Coq9 ^R239X after β‐RA treatment, n = 7. (G): Coq9 ^+/+, n = 5; Coq9 ^R239X, n = 6; Coq9 ^R239X after β‐RA treatment, n = 6. (L): Coq9 ^+/+, n = 5; Coq9 ^R239X, n = 6; Coq9 ^R239X after β‐RA treatment, n = 7. (N): Coq9 ^+/+, n = 6; Coq9 ^R239X, n = 6; Coq9 ^R239X after β‐RA treatment, n = 6. (C, H, O) Representative Western blot and quantitation of Western blot bands of SQOR in mitochondrial fractions of brain (C) and homogenates extracts from kidneys (H) and heart (O). (C): Coq9 ^+/+, n = 6; Coq9 ^R239X, n = 6; Coq9 ^R239X after β‐RA treatment, n = 6. (H): Coq9 ^+/+, n = 4; Coq9 ^R239X, n = 3; Coq9 ^R239X after β‐RA treatment, n = 3. (O): Coq9 ^+/+, n = 4; Coq9 ^R239X, n = 3; Coq9 ^R239X after β‐RA treatment, n = 4. (D, I) Blue‐native gel electrophoresis (BNGE) followed by C‐III immunoblotting analysis of mitochondrial supercomplexes in brain (D) and kidneys (I). (D): Coq9 ^+/+, n = 9; Coq9 ^R239X, n = 10; Coq9 ^R239X after β‐RA treatment, n = 11. (I): Coq9 ^+/+, n = 5; Coq9 ^R239X, n = 6; Coq9 ^R239X after β‐RA treatment, n = 7. (E, J) Mitochondrial oxygen consumption rate (represented as State 3o, in the presence of ADP and substrates) in brain (E) and kidneys (J). The data represent three technical replicates and the figures are representative of three biological replicates. Coq9 ^+/+ mice, Coq9 ^R239X mice, and Coq9 ^R239X mice after β‐RA treatment were included in all graphs. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; Coq9 ^R239X or Coq9 ^R239X after β‐RA treatment versus Coq9 ^+/+. ⁺ P < 0.05; Coq9 ^R239X versus and Coq9 ^R239X after β‐RA treatment (one‐way ANOVA with a Tukey's post hoc test; see Appendix Table S4 for exact P‐values).

Source data are available online for this figure.

Figure 5. Tissue‐specific differences in the levels of CoQ₉, CoQ_10, and DMQ ₉, and in the DMQ ₉/CoQ₉ ratio after β‐RA treatment in Coq9 ^R239X mice

1. A–T

   Levels of CoQ₉ (A, E, I, M, Q), CoQ₁₀ (B, F, J, N, R), and DMQ₉ (C, G, K, O, S), as well as and DMQ₉/CoQ₉ ratio (D, H, L, P, T) in brain (A–D), kidney (E–H), liver (I–L), skeletal muscle (M–P), and heart (Q–T) of Coq9 ^+/+ mice, Coq9 ^R239X mice, and Coq9 ^R239X mice after β‐RA treatment. Data are expressed as mean ± SD. **P < 0.01; ***P < 0.001; Coq9 ^R239X or Coq9 ^R239X after β‐RA treatment versus Coq9 ^+/+. ⁺ P < 0.05; ⁺⁺ P < 0.01; Coq9 ^R239X versus Coq9 ^R239X after β‐RA treatment (one‐way ANOVA with a Tukey's post hoc test or t‐test; n = 6 for each group; see Appendix Table S5 for exact P‐values). Note that DMQ9 was undetectable in tissues from Coq9 ^+/+ mice as well as in muscle of Coq9 ^R239X mice treated with β‐RA.

Figure 6. Effect of β‐RA treatment in the tissue levels of CoQ biosynthetic proteins

1. A–O

   Representative images of Western blots of the CoQ biosynthetic proteins COQ4, COQ5, COQ6, COQ7, and COQ8A and the quantitation of the protein bands in the brain (A–E), kidneys (F–J), and heart (K–O) of Coq9 ^+/+, Coq9 ^R239X, and Coq9 ^R239X mice treated with β‐RA. Coq9 ^+/+ mice, Coq9 ^R239X mice, and Coq9 ^R239X mice after β‐RA treatment were included in all graphs. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; Coq9 ^R239X or Coq9 ^R239X after β‐RA treatment versus Coq9 ^+/+. ⁺ P < 0.05; Coq9 ^R239X versus and Coq9 ^R239X after β‐RA treatment (one‐way ANOVA with a Tukey's post hoc test; n = 5 for each group; see Appendix Table S6 for exact P‐values).

Source data are available online for this figure.

Figure 7. DMQ ₉/CoQ₉ ratio in other experimental conditions

1. A–F

   (A) DMQ₉/CoQ₉ ratio in tissues from Coq9 ^R239X and Coq9 ^Q95X mice at 1 month of age. (C) DMQ₉/CoQ₉ ratio in tissues from Coq9 ^R239X and Coq9 ^Q95X mice at 3 months of age. (E) DMQ₉/CoQ₉ ratio in tissues from Coq9 ^R239X mice and Coq9 ^R239X mice treated with ubiquinol‐10 during 2 months. (B, D, F) Representative chromatographs showing the different quinones in the kidneys. Data in panels (A, C, E) are expressed as mean ± SD. **P < 0.01; ***P < 0.001; Coq9 ^Q95X versus Coq9 ^R239X (t‐test; n = 5 for each group; see Appendix Table S7 for exact P‐values).