The N-terminal sequence of mutant htt with 148 polyglutamine repeats (N63-148Q-myc) was inducibly expressed in PC12 cells by withdrawal of doxycycline in culture medium in Tet-off system. Cells were differentiated by adding NGF (50 ng/ml) in culture medium. Cell toxicity was assessed by LDH release from dead cells. *p<0.05 compared to the values of Dox(+) group; **p<0.05 compared to the values of Dox(−) group. Three individual experiments with triplicates in each experiment. Standard Student’s t-test was used for statistical analysis.

Tiagabine was administered to mice at doses of 2 and 5 mg/kg/d. Survival was monitored daily. (a) Tiagabine at 5 mg/kg (red line) significantly extended survival of N171-82Q mice. p=0.028 comparison between tiagabine 5mg/kg group and vehicle group by log-rank test. (b) tiagabine did not affect body weight in N171-82Q mice. Motor behavioral performance was evaluated by an accelerating rotarod apparatus in mice at 16 weeks of age (c) and 18 weeks of age (d). n=10–15. Values are the mean and SE. *p<0.05 compared to the value of WT-vehicle group; ^#p<0.05, compared to the value of HD-vehicle group by standard student t-tests.

Tiagabine was administered to HD mice beginning at 8 weeks of age, mice were perfused at 18 weeks old, brain sections were processed for Nissl staining (a) and neurosilver staining for detecting degenerative neurons (b). (a) Representative photographs of brain sections of Nissl staining from indicated group. Scale bar=120 µm. note that N171-82Q mouse brain exhibited enlarged ventricles and shrunk striatum. Tiagabine treatment reversed the brain atrophy in HD mice. (b–c) Quantification data of brain atrophy. Tiagabine treatment significantly attenuated the brain atrophy in N171-82Q mice. n=4 mice. *p< 0.05, compared to the values of nontransgenic-vehicle group ( Nontg-veh); **p< 0.05, compared to the values of N171-82Q mice treated with vehicle (HD-veh) by standard student t-tests. (d) Representative silver staining results, the dark staining neurons represent cells undergoing neurodegeneration. Note more dark stained neurons were found in vehicle-treated brain compared to tiagabine-treated brain in both cortex and striatum regions. Scale bars = 20 µm. (e–f) Quantification of silver positive neurons in piriform cortex and striatum. There were fewer silver-positive neurons in both piriform cortex and striatum in tiagabine-treated samples. n=4 mice. *p< 0.05, compared to the values of vehicle group by standard student t-tests.

HD mice were treated with vehicle (saline) or tiagabine (5 mg/kg) from 8 weeks of age. Mice were perfused for EM48 immunostaining to detect the mutant huntingtin aggregates at 18 weeks of age. (a) Representative photographs of brain sections stained with EM48 antibody from indicated group. Scale bars=20 µm. (b–c) There was no difference in the density of cells with htt aggregates both in piriform cortex and hippocampus between N171-82Q mice treated with vehicle and tiagabine. n=4 mice.

(a) 6-week-old R6/2 mice were injected with either vehicle (saline) or tiagabine (5 mg/kg) daily, and survival was monitored daily. Tiagabine at 5 mg/kg (red line) significantly increased survival. P<0.05 comparison between R6/2 –vehicle ( black line) and R6/2 tiagabine-treated group ( red line) by log rank test. (b) tiagabine did not affect body weight in R6/2 mice. (c) Motor behavioral performance was evaluated by an accelerating rotarod apparatus with mice at indicated ages, n=8–15. Values are the mean and SE. *p<0.05 compared to the value of WT-vehicle group; ^#p<0.05, compared to the value of HD-vehicle group by standard student t-tests.

(a) Representative photographs of brain sections of Nissl staining from indicated group. Scale bar=120 µm. Note that R6/2 mouse brain showed enlarged ventricles and smaller striatum. Tiagabine treatment reversed the brain atrophy in R6/2 mice. (b–c) Quantification of brain atrophy. Tiagabine treatment significantly attenuated the brain atrophy in R6/2 mice. n=4 mice. *p< 0.05, compared to the values of nontransgenic-vehicle group (Nontg-veh); **p< 0.05, compared to the values of R6/2 mice treated with vehicle (R6/2-veh) by standard student t-tests. (d) Representative photographs of brain sections stained with EM48 antibody from indicated group. Scale bars=20 µm. (e–f) There was no difference in the density of cells with htt aggregates both in frontal cortex and striatum between R6/2 mice treated with vehicle and tiagabine. n= 4 mice.