Comprehensive behavioral testing in the R6/2 mouse model of Huntington's disease shows no benefit from CoQ10 or minocycline

Previous studies of the effects of coenzyme Q10 and minocycline on mouse models of Huntington’s disease have produced conflicting results regarding their efficacy in behavioral tests. Using our recently published best practices for husbandry and testing for mouse models of Huntington’s disease, we report that neither coenzyme Q10 nor minocycline had significant beneficial effects on measures of motor function, general health (open field, rotarod, grip strength, rearing-climbing, body weight and survival) in the R6/2 mouse model. The higher doses of minocycline, on the contrary, reduced survival. We were thus unable to confirm the previously reported benefits for these two drugs, and we discuss potential reasons for these discrepancies, such as the effects of husbandry and nutrition

Effect of the rd1 mutation on motor performance in R6/2 and wild type mice

Homozygosis for the rd1 mutation in the Pbe6b gene results in the loss of the rod beta-subunit of the cyclic GMP phosphodiesterase and, eventually, of all rod and cone photoreceptors. The R6/2 mouse line is a widely used model of Huntington’s disease (HD). The original line was made available on a mixed background obtained by crossing, via ovarian transplant, female R6/2 (on a B6CBA mixed background) with male B6CBAF1/J mice. As the CBA/J strain used in the US is homozygous for the rd1 mutation and the breeding scheme does not ensure heterozygosis for the mutation, a significant percentage of the offspring on this mixed background is expected to be homozygous for the rd1 mutation. We investigate here the effect of rd1 homozygosis on motor function and examined the effects of the mutation on the R6/2 phenotype. Homozygosis for the rd1 mutation resulted in increased activity in the open field test and reduced rotarod test performance. In addition, rd1 mutation absence or heterozygosis reduced the differences between the R6/2 and the WT mice. Our recommendation for the neurodegeneration field, and for all mouse studies in general, is to carefully control homozygosis for retinal degeneration mutation, even when using tests of motor function

Mean plasma and brain levels of minocycline.

<p><b>Experiment 3.</b> Both plasma and brain minocycline levels were higher in WT mice compared to R6/2 mice (Plasma: F(1,19) = 1.4, p<0.01; brain: genotype main effect: F(1,10) = 19.6, p<0.002). <b>Experiment 4.</b> Brain and plasma concentrations of minocycline in R6/2 mice were significantly higher than in WT treated mice (Brain: treatment×genotype main effect: F(1,16) = 7.67, p<0.02, simple main effects: p<0.0001; plasma: genotype main effect: F(1,17) = 27.89, p<0.03) in the two 0.375% dosing groups.</p

Effect of daily i.p. administration of 5mg/kg of minocycline in R6/2 mice.

<p>WT and R6/2 mice received either 5mg/kg of minocycline or its vehicle by intraperitoneal injection. Minocycline had no effects on survival (A) on either R6/2 or WT mice, but transiently increased the body weight (B) of mutant male mice at 8, 10, 11 and 12 weeks of age (treatment×gender×age interaction: F(22,312) = 4.2, p<0.0001; simple main effects: ps<0.05). In the WT group, minocycline did not produce significant effects in body weight. In the rotarod test (C), although there was a triple interaction of treatment, age and day for R6/2 mice, simple main effects did not reveal a consistent pattern (overall effects: treatment×age×day interaction: F(10,143) = 3.2, p<0.001). There were no effects for the WT mice. Minocycline transiently increased locomotion (D) in R6/2 male mice (treatment×gender×age: F(6,87) = 4.5; p<0.0005; simple main effects: ps<0.05 at 4 weeks of age) but decreased locomotion in the male WT mice (treatment×gender×week interaction, F(6,102) = 2.6, p<0.03, simple main effects and post hocs, ps<0.05 at 4 weeks of age). Rearing frequency (E) was transiently decreased by minocycline in female R6/2 mice at 6 weeks, but increased in male R6/2 mice at 6 and 8 weeks of age (treatment×gender interaction: F(1,33) = 11.2, p<0.002; treatment×age interaction: F(3,87) = 6.4, p<0.001; treatment×gender×age interaction: F(6,87) = 5.4, p<0.0001; simple main effects: ps<0.05). Minocycline decreased rearing in WT mice at 8 and 12 weeks of age especially in the male group (treatment×gender interaction, F(1,37) = 9.9, p<0.0005; treatment×age interaction: F(3,102) = 8.9, p<0.0001, simple main effects, ps<0.05). Minocycline did not affect grip strength (F) or rearing (G) in either R6/2 or WT mice although the latter showed a tendency to rear less (p<.07).</p

Effect of the administration of 0.6% of CoQ10 in food in R6/2 mice.

<p>WT mice and R6/2 mice were fed with a diet with HydroQsorb (HQD) alone or with 0.6% CoQ10 in HQD. There was no treatment effect on survival (A). CoQ10 significantly decreased the body weight (B) of R6/2 males between 6 and 10 weeks of age (treatment×age×gender interaction: F(22,330) = 4.1, p<0.0001; simple main effects: ps<0.05). There were no effects of treatment on the latency to fall from the rotarod (C). In mutant females, CoQ10 diminished locomotor activity in the open field (D). Treated R6/2 females were significantly hypoactive at 4 weeks (0–5 min), 6 weeks (last 10 min), at 12 weeks of age (20 to 25 min; treatment×gender interaction: F(1,35) = 6.7, p<0.02; simple main effects: p<0.007; treatment×age×time interaction: F(30,460) = 3.4, p<0.0001; simple main effects: ps<0.05). R6/2 treated mice reared significantly less in the open field (E) at 6 weeks of age compared with untreated mutant mice (treatment×age interaction: F(3,92) = 3.2, p<0.03; simple main effects: p<0.035). There were no treatment effects on either grip strength (F) or in the rearing climbing test (G). CoQ10 had no effects in WT mice (A–F).</p

Effect of the administration of 0.2% of CoQ10 in food in R6/2 mice.

<p>WT mice received unsupplemented diet (UD) and R6/2 mice received either UD or 0.2% CoQ10. There were no significant treatment effects on survival (A), body weight (B) or rotarod (C: latency to fall). Deleterious effects of 0.2% CoQ10 were observed in the total distance traveled in the open field (D) at 4 and 12 weeks of age, mainly in the first 5 min of the session (treatment×age interaction: F(3,213) = 7.5, p<0.0001; treatment×session time interaction: F(5,375) = 2.5, p<0.04; simple main effects: p<0.05). There were no effects on the rearing frequency in the open field (E); CoQ10 impaired grip strength (F) of female mice at 12 weeks of age (treatment×gender interaction (F(1,76) = 11.3, p<0.002, simple main effects: p<0.001) and decreased the number of mice that climbed in the rearing climbing test (G, p<0.04).</p