Elesclomol alleviates Menkes pathology and mortality by escorting Cu to cuproenzymes in mice

Abstract

Loss-of-function mutations in the copper (Cu) transporter ATP7A cause Menkes disease. Menkes is an infantile, fatal, hereditary copper-deficiency disorder that is characterized by progressive neurological injury culminating in death, typically by 3 years of age. Severe copper deficiency leads to multiple pathologies, including impaired energy generation caused by cytochrome c oxidase dysfunction in the mitochondria. Here we report that the small molecule elesclomol escorted copper to the mitochondria and increased cytochrome c oxidase levels in the brain. Through this mechanism, elesclomol prevented detrimental neurodegenerative changes and improved the survival of the mottled-brindled mouse-a murine model of severe Menkes disease. Thus, elesclomol holds promise for the treatment of Menkes and associated disorders of hereditary copper deficiency.

Fig. 1.. Effects of ES treatment in cardiac Ctrl KO mice.

(A) Gross appearance of mice at PND 10. Asterisks (***) indicate moribund KO vehicle mouse. (B) Kaplan-Meier survival curve. (C) Growth curves. WT and WT ES mice exhibited identical growth curves (WT ES omitted for clarity; see fig. S3A). Cohorts consisted of WT vehicle (n = 3 mice), WT ES (n = 7), KO vehicle (n = 3), and KO ES (n = 9). (D) Gross appearance of hearts at PND 10. Asterisks (***) indicate hypertrophied KO vehicle mouse heart. (E) Heart-to-body weight ratio. Cohorts consisted of n = 6, 6, 8, and 9 animals, respectively. (F) Heart [Cu] levels. Cohorts consisted of n = 4 per treatment. (G) Heart COX1 levels. (H) Quantification of relative COX1 levels. Data are reported as means ± SD with statistical significance assessed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test or Welch one-way ANOVA with Tukey’s post hoc test. NS, not significant; *P < 0.05; ***P < 0.001. Western blot images in (G) were analyzed with ImageJ software.

Fig. 2.. Effects of ES-Cu²⁺ treatment in mo-br mice.

(A) Mo-br hemizygous males and WT littermate at PND 5 before intervention. (B) Pigmentation changes in mo-br males administered ES-Cu²⁺ compared with vehicle (***) on PND 12. (C) Moribund mo-br vehicle (***) mouse on PND 14. (D and E) Mo-br ES-Cu²⁺ (D) and WT littermate (E) at PND 70. (F) Kaplan-Meier survival curve. All WT mice survived experimental protocol. (G) Growth curves of indicated groups: WT and WT ES-Cu²⁺ mice exhibited near identical growth curves (WT ES-Cu²⁺ omitted for clarity; see fig. S8E). Cohorts consisted of WT vehicle (n = 15), WT ES-Cu²⁺ (n = 13), mo-br vehicle (n = 9), mo-br ES (n = 6), mo-br HIS-Cu²⁺ (n = 6), and mo-br ES-Cu²⁺ (n = 27). Inset shows a close-up of the curves in the region indicated by the dashed-line box, including the curves of mo-br cohorts only. Data are reported as means ± SD with statistical significance assessed by one-way ANOVA test or Welch one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars in (A) to (C), 1 cm.

Fig. 3.. Neuromotor tests of 10-week-old mice.

Mice were assessed by grip strength, rotarod, gait treadmill, and open field at 10 weeks of age. (A) Forelimb grip strength. (B) Rotarod. (C) Gait treadmill. DigiGait-generated ataxia coefficients of shoulder and pelvic girdles were statistically insignificant. (D to F) Open field: movement time (D), rest time (E), and total distance (F). Cohorts consisted of WT (n = 8) and mo-br (n = 13). Data are reported as means ± SD with statistical significance assessed by unpaired t test. ***P < 0.001.

Fig. 4.. Neuropathology of 2- and 10-week-old mice.

(A) Hematoxylin and eosin (H&E)-stained sections of cortex and hippocampus from PND 14 WT and mo-br mice, administered vehicle, ES-Cu²⁺, or HIS-Cu²⁺. Somatomotor cortical neurons in mo-br vehicle and HIS-Cu²⁺ cohorts exhibit marked, diffuse neurodegenerative changes, characterized by numerous pyknotic nuclei with eosinophilic cytoplasm (yellow arrows). In the hippocampus, the pyramidal neuron layer of region CA1 demonstrates degenerative changes including necrotic neurons in vehicle and HIS-Cu²⁺ mo-br mice (yellow arrows). (B) Cerebellar peduncles from PND 70 WT and mo-br ES-Cu²⁺ mice revealed preservation of continuous Purkinje neuron layer. Scale bars in (A), 25 μm. Scale bars in (B), 50 μm.

Fig. 5.. ES-Cu²⁺ rescues biochemical phenotypes in 2- and 10-week-old mo-br mice.

(A) Serum [Cu] at PND 14 (all cohorts, n = 4). (B) Brain [Cu] at PND 14 (all cohorts, n = 4). (C) Brain weights at PND 14 (WT, n = 12; mo-br Vehicle, n = 12; mo-br ES-Cu²⁺, n = 14; mo-br HIS-Cu²+, n = 11). (D and E) Brain COX1 at PND 14 (all cohorts, n = 3). (F and G) Brain COX1 at PND 70 (all cohorts, n = 5). (H) Serum [Cu] at PND 70 (WT, n = 4; mo-br ES-Cu²⁺, n = 6). (I) Brain [Cu] at PND 70 (WT, n = 5; mo-br ES-Cu²⁺, n = 8). (J) Brain weights at PND 70 (WT, n = 8; mo-br ES-Cu²⁺, n = 13). Data are reported as means ± SD with statistical significance assessed by one-way ANOVA or Welch one-way ANOVA with Tukey’s post hoc test. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Western blot images in (D) and (F) were analyzed with ImageJ software.