A Mouse Model of Familial ALS Has Increased CNS Levels of Endogenous Ubiquinol_9/10 and Does Not Benefit from Exogenous Administration of Ubiquinol₁₀

<div><p>Oxidative stress and mitochondrial impairment are the main pathogenic mechanisms of Amyotrophic Lateral Sclerosis (ALS), a severe neurodegenerative disease still lacking of effective therapy. Recently, the coenzyme-Q (CoQ) complex, a key component of mitochondrial function and redox-state modulator, has raised interest for ALS treatment. However, while the oxidized form ubiquinone₁₀ was ineffective in ALS patients and modestly effective in mouse models of ALS, no evidence was reported on the effect of the reduced form ubiquinol₁₀, which has better bioavailability and antioxidant properties. In this study we compared the effects of ubiquinone₁₀ and a new stabilized formulation of ubiquinol₁₀ on the disease course of SOD1^G93A transgenic mice, an experimental model of fALS. Chronic treatments (800 mg/kg/day orally) started from the onset of disease until death, to mimic the clinical trials that only include patients with definite ALS symptoms. Although the plasma levels of CoQ₁₀ were significantly increased by both treatments (from <0.20 to 3.0–3.4 µg/mL), no effect was found on the disease progression and survival of SOD1^G93A mice. The levels of CoQ₁₀ in the brain and spinal cord of ubiquinone₁₀- or ubiquinol₁₀-treated mice were only slightly higher (≤10%) than the endogenous levels in vehicle-treated mice, indicating poor CNS availability after oral dosing and possibly explaining the lack of pharmacological effects. To further examine this issue, we measured the oxidized and reduced forms of CoQ_9/10 in the plasma, brain and spinal cord of symptomatic SOD1^G93A mice, in comparison with age-matched SOD1^WT. Levels of ubiquinol_9/10, but not ubiquinone_9/10, were significantly higher in the CNS, but not in plasma, of SOD1^G93A mice, suggesting that CoQ redox system might participate in the mechanisms trying to counteract the pathology progression. Therefore, the very low increases of CoQ₁₀ induced by oral treatments in CNS might be not sufficient to provide significant neuroprotection in SOD1^G93A mice.</p></div

Unchanged plasma levels of ubiquinol₉ in symptomatic SOD1^G93A mice.

<p>Levels of CoQ₉ (A), ubiquinol₉ (B) and ubiquinone₉ (C) in plasma of male 16 week-old 129Sv SOD1^WT and SOD1^G93A mice are shown as percentage of the levels measured in age-matched 129Sv non-transgenic mice (table S1). Each value is mean±SEM of 4–7 mice.</p

Increased levels of ubiquinol_9/10 in the brain of symptomatic SOD1^G93A mice.

<p>Endogenous brain levels of CoQ₉ (A–C) and CoQ₁₀ (D–F) in 16 week-old male 129Sv SOD1^WT and SOD1^G93A mice are shown as percentage of the levels measured in age-matched 129Sv non-transgenic mice (absolute values in table S1). Panels A, D show the total CoQs levels, whereas panels B, E and panels C, F show the reduced and oxidized forms, respectively. Each value is the mean±SEM of 4–7 mice. * P<0.05, ** P<0.01 compared to SOD1^WT mice (Student’s T-test).</p

Increased levels of ubiquinol_9/10 in the lumbar spinal cord of symptomatic SOD1^G93A mice.

<p>Endogenous levels of CoQ₉ (A–C) and CoQ₁₀ (D–F) in the lumbar spinal cord of male 16 week-old 129Sv SOD1^WT and SOD1^G93A mice are shown as percentage of the levels measured in age-matched 129Sv non-transgenic mice (absolute values in table S1). Panels A, D show the total CoQs levels, whereas panels B, E and panels C, F show the reduced and oxidized forms, respectively. Each value is mean±SEM of 4–7 mice. * P<0.05, ** P<0.01 compared to SOD1^WT mice; ° P<0.05 compared to 129Sv NTg mice (Student’s T-test).</p

Ubiquinol₁₀ chronic treatment has no effect on disease course in SOD1^G93A mice.

<p>Effect of oral treatment with 800 mg/kg/day ubiquinone₁₀, ubiquinol₁₀ or vehicle, on motor dysfunction and disease progression of 129Sv SOD1^G93A mice (n = 15 mice per group). Treatment started at the age of 91 days (arrows) until the sacrifice (at the end-stage of the disease, when mice were unable to right themselves within 10 seconds after being placed on both sides). Treatment with ubiquinone₁₀ or ubiquinol₁₀ had no significant effects on body weight (A), on the latency of rotarod (B) and PaGE test (C) (Two-way ANOVA), or on disease onset (D) and survival length (E). Each point represents the mean; for sake of clarity standard deviations are not indicated but they were always less than 15% of the value. Table reports the mean and standard deviations of symptoms onset and life-span for each group.</p

Levels of CoQ_9/10 in the plasma of SOD1^G93A mice treated chronically with ubiquinone₁₀ or ubiquinol₁₀.

<p>Female 129Sv SOD1^G93A mice were treated orally with 800 mg/kg of ubiquinol₁₀ or ubiquinone₁₀, or vehicle (sunflower seed oil), once a day, starting from age of 91 days until the last stage of the disease (age of 125 days, on average) when they were sacrificed, two hours after the last treatment. Values are means±SEM of (n) mice.</p

G93A-SOD1 C57BL/6 mice treated with EPA at the pre-symptomatic stage of the disease have an increase in vacuolization in the spinal cord.

<p>(A–B) Wild type littermates on the control diet have very sparse vacuoles within the spinal cord. (C–D) G93A-SOD1 mice on the control diet have vacuoles predominantly in the ventral horn. (E–F) Wild type littermates on the EPA diet have a slight increase in vacuoles within the spinal cord. (G–H) G93A-SOD1 mice on the EPA diet have a marked increase in vacuoles in the dorsal and ventral horn. (I–J) Quantitative analysis of vacuolisation in the spinal cord. Scale bar = 50 µm.</p

G93A-SOD1 C57BL/6 mice with dietary EPA at the symptomatic stage of the disease do not have a significantly different development of the disease compared to animals on the control diet.

<p>(A) Consumption of food is slightly increased when diet is enriched with EPA. (B) The body weight of mice was not affected as the disease evolves (C) Survival and (D) disease duration were not affected by dietary EPA. (E) The Rotarod and (F) The grip strength were not affected by dietary EPA.</p

Astrocyte activation in G93A-SOD1 C57BL/6 mice treated with EPA at the pre-symptomatic stage of the disease is reduced by treatment.

<p>Some astrocytes can be observed in the wild-type littermate spinal cord (A–C). In the spinal cord of G93A-SOD1 mice, there is a significant increase in the number of astrocytes (D–F). The treatment with EPA did not significantly alter the number of astrocytes in wild type mice (G–I), but significantly reduced astrocytic activation in G93A-SOD1 mice (J–L) in comparison to the corresponding control group. Quantitative analysis of astrocytic cell number (M) and staining intensity (N) in the ventral horn of the mice spinal cord: GFAP (astrocytes, red), Nissl stain (neurons, green) and DAPI (nuclei, blue). Scale bar = 100 µm.</p

G93A-SOD1 C57BL/6 mice treated with dietary EPA at the pre-symptomatic stage of ALS have an accelerated development of the disease compared to G93A-SOD1 mice on the control diet.

<p>(A) Consumption of food is increased in animals on the EPA enriched diet. (B) The body weight of mice with EPA diet decreases as the disease evolves. (C) Survival and (D) disease duration are both reduced with dietary EPA. (E) The Rotarod and (F) Hindlimb extension reflex are both affected by dietary EPA. (G) The rate at which the disease starts is not affected by dietary EPA. (H) The grip strength was not affected by dietary EPA.</p