Neuroprotective effects of creatine in a transgenic mouse model of Huntington's disease

Abstract

Huntington's disease (HD) is a progressive neurodegenerative illness for which there is no effective therapy. We examined whether creatine, which may exert neuroprotective effects by increasing phosphocreatine levels or by stabilizing the mitochondrial permeability transition, has beneficial effects in a transgenic mouse model of HD (line 6/2). Dietary creatine supplementation significantly improved survival, slowed the development of brain atrophy, and delayed atrophy of striatal neurons and the formation of huntingtin-positive aggregates in R6/2 mice. Body weight and motor performance on the rotarod test were significantly improved in creatine-supplemented R6/2 mice, whereas the onset of diabetes was markedly delayed. Nuclear magnetic resonance spectroscopy showed that creatine supplementation significantly increased brain creatine concentrations and delayed decreases in N-acetylaspartate concentrations. These results support a role of metabolic dysfunction in a transgenic mouse model of HD and suggest a novel therapeutic strategy to slow the pathological process.

Fig. 1.

Effects of 1, 2, and 3% creatine on rotarod performance. There was significantly improved performance in R6/2 HD transgenic mice with 2% creatine supplementation throughout the temporal sequence of the experiment (4–13 weeks) (B), from 5–10 weeks in 1% creatine-treated mice (A), with significance only occurring at 6 weeks in the 3% creatine-treated R6/2 mice (C).

Fig. 2.

Effects of 1, 2, and 3% creatine on body weight in R6/2 HD transgenic mice. Whereas significantly greater body weight was observed throughout the measured temporal sequence in 1% (except 6 weeks) and 2% creatine-supplemented R6/2 mice, significance was present only from 10 to 13 weeks in the 3% treated mice.

Fig. 3.

Photomicrographs of coronal sections through the rostral neostriatum at the level of the anterior commissure in R6/2 HD transgenic mice at 42 (A), 63 (B), and 90 (C) d. Note the generalized gross atrophy of the brain over time along with enlargement of the lateral ventricles. In contrast, a 2% creatine-supplemented R6/2 mouse at 90 d (D) shows significantly less atrophy and ventricular enlargement than the unsupplemented mouse (C). Scale bar, 2 mm.

Fig. 4.

Neuronal areas of 2% creatine-supplemented and unsupplemented R6/2 mice in comparison to littermate transgene-negative mice at 28, 42, 63, and 90 d; *p < 0.001.

Fig. 5.

Photomicrographs of Nissl-stained tissue sections from the dorsomedial aspect of the neostriatum (A, C, E, G) and 2% creatine-supplemented (B, D, F, H) R6/2 HD transgene mice at 4 (A, B), 6 (C, D), 9 (E, F), and 13 (G, H) weeks. Note the progressive loss in neuronal size in the unsupplemented R6/2 group, with delayed neuronal atrophy in the 2% creatine-supplemented R6/2 mice. Scale bar, 100 μm.

Fig. 6.

Correlation between creatine and NAA levels in HD transgenic R6/2 mice. Correlation between NAA and CR in unsupplemented mice was not significant.

Fig. 7.

Graphs of the temporal sequence in the number of huntingtin-positive aggregates in the neostriatum (A) and motor neocortex (B) at 4, 6, 9, and 13 weeks. There was a significant delay in the formation of aggregates within the striatum in 2% creatine-supplemented R6/2 mice, in comparison to unsupplemented R6/2 mice. Although a similar trend was observed in the neocortex, significance was not obtained.

Fig. 8.

Photomicrographs of huntingtin-immunostained tissue sections from dorsomedial aspect of the neostriatum at the level of the anterior commissure in 2% creatine-supplemented (A, C, E, G) and unsupplemented (B, D, F, H) R6/2 HD transgene mice at 4 (A, B), 6 (C, D), 9 (E, F), and 13 (G, H) weeks. There is a progressive increase in number and size of huntingtin aggregates over time in the unsupplemented R6/2 mice in comparison to the delay in aggregate formation in the 2% creatine-supplemented R6/2 mice. Scale bar, 50 μm.

Fig. 9.

Photomicrographs of huntingtin-immunostained tissue sections from layer 6 of the motor cortex at the level of the anterior commissure in unsupplemented (A, C, E) and 2% creatine-supplemented (B, D, F) R6/2 HD transgene mice at 4 (A, B), 9 (C, D), and 13 (E, F) weeks. There is a progressive increase in number and size of huntingtin aggregates over time in the unsupplemented R6/2 mice in comparison to the delay in aggregate formation in the 2% creatine-supplemented R6/2 mice. Scale bar, 100 μm.

Fig. 10.

Photomicrographs of islets of Langerhan in the pancreas of 90-d-old unsupplemented (A) and 2% creatine-supplemented (B) R6/2 HD transgenic mice immunostained with EM48 antibody. There is a marked reduction in the huntingtin aggregates within the treated mouse. Scale bar, 50 μm.

Fig. 11.

Effects of 2% creatine supplementation on glucose tolerance in 8.5-week-old R6/2 mice. Creatine administration significantly attenuated abnormal glucose tolerance. *p < 0.05; **p < 0.01.