Effects of the Pimelic Diphenylamide Histone Deacetylase Inhibitor HDACi 4b on the R6/2 and N171-82Q Mouse Models of Huntington's Disease

Abstract

This report represents a detailed description of experiments designed to replicate and extend the findings of a published study on the effects of treating the R6/2 Huntington's disease (HD) mouse model with ~300 CAG repeats using the pimelic diphenylamide histone deacetylase (HDAC) inhibitor, HDACi 4b (Thomas et al., 2008). In addition to testing the R6/2 mice, similar experiments examined the effects of the drug on a second transgenic HD mouse model, the N171-82Q mice. As in the original study, the drug was delivered in the drinking water. In the present study we tested larger groups of mice than in the original study. The results indicated that we were unable to replicate the significant behavioral effects of oral HDACi 4b treatment in the R6/2 mice. There were however, non-significant trends for the treated R6/2 mice to be less affected on some of the measures and there were instances of phenotype progression being delayed in these treated mice. In contrast, we did replicate the protection from striatal atrophy in the R6/2 mice. We also did not observe any beneficial effects of HDACi 4b treatment in the N171-82Q mice. Although the behavioral procedures were replicated and an automated activity assessment was added, there were several unexpected complications in terms of solubility of the drug, CAG repeat length differences and gender differences in progression of the phenotype that could have affected outcomes. Clearly more studies will have to be performed using other methods of delivery as well as assessing effects in more slowly progressing HD models to better evaluate the effects of this HDAC inhibitor.

Cumulative survival

A. There were no significant differences in survival between R6/2-vehicle and R6/2-HDACi 4b mice with less than 300 CAG repeats. B. Although more N171-vehicle mice died compared to N171-HDACi 4bH mice, the difference in survival was not statistically significant.

Water consumption

A. Water consumption over the duration of drug administration. There was a gradual increase in water consumption for R6/2 mice over time. B. Overall, R6/2-HDACi 4b mice consumed significantly more water than WT mice (p<0.01). C. Initially, both N171-vehicle and N171-HDACi 4bH groups consumed more water than WTs. However, this was not seen during the later stages of drug administration. D. On average, N171-vehicle mice consumed significantly more water than WT-vehicle and WT-HDACi 4bH mice. For A and C, significant differences between mutant and WT mice are indicated by * (p<0.05). For B and D, significant differences in average water consumption are indicated by * (p<0.05) and ** (p<0.01).

Change in body weight of male and female R6/2 mice

A, B. Average body weight of male and female R6/2 mice measured weekly from baseline to the end of drug administration. Male R6/2 mice consistently weighed less than their WT littermates while body weight differences in female R6/2 mice were only seen in the late stages of drug administration. C, D. Percent change in body weight of male and female R6/2 mice. Progressive weight loss was observed only in male R6/2 mice. In this figure, significant differences between R6/2 and WT mice are indicated by * (p<0.05).

R6/2 activity for the entire open field session

A. Total movement distance. B. Total velocity of movement. C. Total number of stereotypic movements. D. Total number of vertical plane entries. In this and other figures, data were divided into an early epoch (14-21 weeks) and a late epoch (23-27 weeks). Both groups of R6/2 mice had decreased movement distance, slower velocity, more stereotypic movements, and fewer vertical plane entries than WTs in the late epoch (right). In this and subsequent figures for R6/2 mice, gray lines highlight differences between R6/2 and WT mice in each time epoch while colored lines show differences between the early and late epochs of each group. Significant differences are indicated by * (p<0.05), ** (p<0.01), and *** (p<0.001).

Gender differences in activity of the entire session

A. Total movement distance. B. Total velocity of movement. C. Total number of stereotypic movements. Significant differences in total activity were only see in male R6/2 mice (left two panels) while there were no differences in activity of female R/62 mice (right two panels). Both groups of R6/2 males had decreased movement distance, slower velocity, and more stereotypies than WT males in the late epoch.

R6/2 activity during the first 3 minutes of the open field session

A. Movement distance. B. Velocity of movement. C. Number of stereotypic movements. D. Number of vertical plane entries. In the early epoch (left), R6/2-vehicle mice moved less and were slower than WT mice. In the late epoch (right), both R6/2-vehicle and HDACi 4b mice had decreased movement distance, slower velocity, more stereotypic movements, and less vertical plane entries than WT mice.

Gender differences during the first 3 min of open field

A. Movement distance. B. Velocity of movement. C. Number of stereotypic movements. D. Number of vertical plane entries. During the early epoch (first and third group of bars), there was a significant decrease in movement distance and velocity that was only observed in female R6/2-vehicle mice. During the late epoch (second and fourth group of bars), both male and female R6/2-vehicle mice, as well as male R6/2-HDACi 4b mice, showed significant differences in all areas of activity measured. There were no significant differences in stereotypies or vertical plane entries of female R6/2-HDACi 4b mice in the late epoch.

Rotarod performance and clasping score of R6/2 mice

A. Average latency to fall from an accelerating rotarod. There were no significant differences in rotarod performance between groups during the early epoch. In the late epoch, R6/2-HDACi 4b mice fell off the rotarod sooner than WT mice. B. Average clasping scores. R6/2-vehicle mice had a higher clasping score than WTs during the early epoch that persisted in the late epoch.

R6/2 kyphosis and activity from the videotaped open field

A. Average kyphosis scores. Kyphosis was apparent in R6/2-vehicle and HDACi 4b mice during both the early and late epochs. B. General locomotor activity scores. R6/2-HDACi 4b mice displayed impaired locomotor activity during the early and late epochs. C. Gender differences in general locomotor activity. Significant differences in locomotor activity were seen in male R6/2 mice during both epochs. There were no differences in activity between groups of female mice.

Changes in striatal and ventricular volumes of R6/2 mice

A. Representative images of the striatum from WT, R6/2-vehicle, and R6/2-HDACi 4b mice. Scale bar is 1 mm. B. Average striatal volumes. R6/2-vehicle mice had smaller striatal volumes than WT and R6/2-HDACi 4b mice. C. Average ventricle volumes. R6/2-vehicle mice also had larger ventricles than WTs.

Changes in body weight of N171-82Q mice

A, B. Average body weight. C, D. Percent change in body weight over time. Both male and female WT mice gained weight while there was little to no weight change in N171 mice over the duration of drug treatment. Significant differences in body weight between N171 and WT mice are indicated by * (p<0.05).

N171-82Q activity for the entire open field session

A. Total movement distance. B. Total velocity of movement. C. Total stereotypic movements. D. Total vertical plane entries. There were no differences in distance moved, velocity, or stereotypies between N171 and/or WT mice. N171-HDACi 4bH mice had fewer vertical plane entries than WT-vehicle and WT-HDACi 4bH mice during both the early and late epochs. In this and subsequent figures for N171 mice, dark gray lines highlight differences between N171 and WT mice in each time epoch while colored lines show differences between the early and late epochs of each group. Significant differences are indicated by * (p<0.05), ** (p<0.01), and *** (p<0.001).

N171-82Q activity for the first 3 minutes of the open field session

A. Movement distance. B. Velocity of movement. C. Number of stereotypic movements. D. Number of vertical plane entries. Overall, N171-HDACi 4bH mice moved less, displayed lower velocity of movement, and fewer vertical plane entries than WTs during the early and late epochs. Similar differences are also seen in N171-vehicle mice in the late epoch.

Rotarod, clasping, kyphosis, and general activity of N171-82Q mice

A. Latency to fall on an accelerating rotarod. N171-HDACi 4bH mice fell off the rotarod significantly faster than WT-vehicle and WT-HDACi 4bH mice during the late epoch. B. Average clasping score. Both groups of N171 mice had higher clasping scores than WT mice throughout the duration of treatment. C. Average kyphosis score. Significant kyphosis of N171 mice was observed only in the late epoch. D. General locomotor activity. In the early and late epochs, N171 mice displayed decreased locomotor activity compared to WTs. WT-HDACi 4bH mice also showed higher activity scores than WT-vehicle mice in the late epoch. There were no significant differences on any activity measure between N171-vehicle and N171-HDACi 4bH mice.