Therapeutic effects of cystamine in a murine model of Huntington's disease

Abstract

The precise cause of neuronal death in Huntington's disease (HD) is unknown. Proteolytic products of the huntingtin protein can contribute to toxic cellular aggregates that may be formed in part by tissue transglutaminase (Tgase). Tgase activity is increased in HD brain. Treatment in R6/2 transgenic HD mice, using the transglutaminase inhibitor cystamine, significantly extended survival, improved body weight and motor performance, and delayed the neuropathological sequela. Tgase activity and N(Sigma)-(gamma-L-glutamyl)-L-lysine (GGEL) levels were significantly altered in HD mice. Free GGEL, a specific biochemical marker of Tgase activity, was markedly elevated in the neocortex and caudate nucleus in HD patients. Both Tgase and GGEL immunoreactivities colocalized to huntingtin aggregates. Cystamine treatment normalized transglutaminase and GGEL levels in R6/2 mice. These findings are consistent with the hypothesis that transglutaminase activity may play a role in the pathogenesis of HD, and they identify cystamine as a potential therapeutic strategy for treating HD patients.

Fig. 1.

Survival in cystamine-treated R6/2 mice. Kaplan–Meier probability of survival analysis for cystamine treatment using intraperitoneal injection of 112 and 225 mg/kg in R6/2 mice and untreated R6/2 mice showing cumulative survival (A). Survival analysis of oral treatment using 225 mg/kg (B). Both intraperitoneal and oral cystamine treatment significantly extended survival in R6/2 transgenic mice (p < 0.001).

Fig. 2.

Motor performance and body weight analysis in cystamine-treated R6/2 mice. Effects of intraperitoneal cystamine treatment (112 mg/kg) on rotarod performance (A) significantly improved motor performance in R6/2 HD transgenic mice throughout the temporal sequence of the experiment (4–16 weeks). Effects of intraperitoneal (112 and 225 mg/kg) (B) and oral (225 mg/kg) (C) cystamine treatment on body weight in R6/2 HD transgenic mice. Greater body weight improvement was observed in both the intraperitoneal and oral paradigms.

Fig. 3.

Gross brain and histopathological neuroprotection with cystamine treatment. Photomicrographs of coronal sections through the rostral neostriatum at the level of the anterior commissure in a wild-type littermate mouse (A), cystamine-treated (C), and untreated (E) R6/2 HD transgenic mice at 90 d. Note the generalized gross atrophy of the brain in the untreated R6/2 mouse along with enlargement of the lateral ventricles (E). In contrast, the cystamine-treated R6/2 mouse at 90 d (C) shows significantly less atrophy and ventricular enlargement than the unsupplemented mouse. Corresponding Nissl-stained tissue sections from the dorsomedial aspect of the neostriatum (B, D, F) with A, C, and E, respectively. Note the reduced neuronal size in the unsupplemented R6/2 mouse, with delayed neuronal atrophy in the cystamine-treated R6/2 mouse, in comparison with the control (A). Scale bars: (in A) A, C, E, 2 mm; (inB) B, D, F, 50 μm.

Fig. 4.

Huntingtin immunoreactivity in cystamine-treated R6/2 mice. htt-immunostained tissue sections from the neostriatum and layer six of the neocortex in untreated (A, C, respectively) and cystamine-treated (B, D, respectively) R6/2 HD transgenic mice at 90 d. Although there is diffuse immunoreactivity within nuclei in the cystamine-treated mice, the number and size of htt aggregates is significantly greater in the untreated R6/2 mice, in comparison with the cystamine-treated R6/2 mice. Diffuse nuclear immunostaining is present in the cystamine-treated mice. Scale bar, 100 μm.

Fig. 5.

Transglutaminase activities in cystamine-treated R6/2 mice. Tgase activity is significantly increased in unsupplemented R6/2 mice in comparison with wild-type littermate control mice (A). Cystamine treatment reduces Tgase activity to control levels (A). Tgase immunoreactivity in a wild-type littermate mouse (B) and an R6/2 mouse (C) at 90 d. There is light immunostaining in the wild-type control with increased immunoreactivity in the R6/2 mouse. Intense aggregate-like figures (arrows) are present in neurons and the neuropil of R6/2 mice (C). Combined immunofluorescence within the same tissue section of an R6/2 mouse for Tgase (red) (D) and huntingtin (green) (E) immunoreactivities show that there is partial colocalization between htt-positive aggregates and Tgase-positive aggregate figures (yellow) within the merged figures (F). Scale bars: (in B)B, C, 100 μm; (inF) D, E,F, 20 μm.

Fig. 6.

Brain GGEL levels in Huntington's disease patients and R6/2 mice. Free GGEL levels in both the neocortex and caudate nucleus in severe grades of HD were markedly elevated in HD patients as compared with non-neurologic control patients (A). Free GGEL levels in unsupplemented R6/2 mice, however, were significantly reduced in comparison with WT littermate control mice, with improved GGEL levels in cystamine-treated R6/2 mice. htt aggregate formation is markedly greater in R6/2 mice than in HD patients. GGEL is colocalized with and sequestered in insoluble htt aggregates. This may result in artificially lowered free GGEL levels observed in R6/2 mice.

Fig. 7.

Protein-bound GGEL immunoreactivity in R6/2 mice and HD patients. GGEL immunocytochemical findings in R6/2 mice (A–C) show a marked increase in GGEL immunointensity in brain sections from R6/2 mice at 90 d (B), in comparison with wild-type littermate control mouse (A). GGEL immunoreactivity was found in neurons and the vasculature in R6/2 mice, with intensely immunostained aggregate-like structures in both neurons and the neuropil (arrows). In contrast, cystamine-treated R6/2 mice show reduced GGEL immunoreactivity and fewer aggregates (C), consistent with reduced htt aggregates in treated R6/2 mice seen in Figure 4. The neocortex (lamina 6) from a grade 3 HD patient shows a similar increase in GGEL immunoreactivity (E), in comparison with an age-matched control (D). GGEL-positive aggregates are present in the HD neocortex (arrows). Scale bar, 50 μm.

Fig. 8.

Combined GGEL and mutant htt immunofluorescence in R6/2 mice and HD patients. Combined immunofluorescence for htt (green) (A) and GGEL (red) (B) immunoreactivities within the same tissue specimens from the neostriatum of a 90-d-old R6/2 mouse show colocalization of htt aggregates and GGEL immunostaining in the merged figure (yellow) (C). Scale bar, 100 μm.