A fully humanized transgenic mouse model of Huntington disease

Abstract

Silencing the mutant huntingtin gene (muHTT) is a direct and simple therapeutic strategy for the treatment of Huntington disease (HD) in principle. However, targeting the HD mutation presents challenges because it is an expansion of a common genetic element (a CAG tract) that is found throughout the genome. Moreover, the HTT protein is important for neuronal health throughout life, and silencing strategies that also reduce the wild-type HTT allele may not be well tolerated during the long-term treatment of HD. Several HTT silencing strategies are in development that target genetic sites in HTT that are outside of the CAG expansion, including HD mutation-linked single-nucleotide polymorphisms and the HTT promoter. Preclinical testing of these genetic therapies has required the development of a new mouse model of HD that carries these human-specific genetic targets. To generate a fully humanized mouse model of HD, we have cross-bred BACHD and YAC18 on the Hdh(-/-) background. The resulting line, Hu97/18, is the first murine model of HD that fully genetically recapitulates human HD having two human HTT genes, no mouse Hdh genes and heterozygosity of the HD mutation. We find that Hu97/18 mice display many of the behavioral changes associated with HD including motor, psychiatric and cognitive deficits, as well as canonical neuropathological abnormalities. This mouse line will be useful for gaining additional insights into the disease mechanisms of HD as well as for testing genetic therapies targeting human HTT.

Figure 1.

HTT levels in Hu97/18 mice. HTT RNA (A and B) and protein (C–F) were evaluated in cortical lysates from 2-month-old wt, Hu18/18, Hu97/18 and BACHD mice (n = 4 per group). (A) Quantitative reverse transcription polymerase chain reaction (qRT–PCR) detection of human wtHTT RNA, which is similar in Hu18/18 and Hu97/18 mice, but absent from BACHD mice. (B) qRT–PCR detection of human muHTT RNA, which is absent from Hu18/18 mice, and higher in BACHD than Hu97/18 mice. (C) FRET detection of relative levels of the total HTT protein, which is less than wt in Hu18/18 mice, similar to wt in Hu97/18 mice, and higher than wt in BACHD mice. (D) FRET detection of relative levels of muHTT protein, which is absent in wt and Hu18/18 mice and higher in BACHD mice than Hu97/18 mice. (E) Representative allelic separation quantitative immunoblot of wt and muHTT protein (MAB2166) showing that there are similar levels of human wt and muHTT protein in Hu97/18 mice. (F) Quantitation of three animals per group and two replicates per animal. **P < 0.01 and ***P < 0.001.

Figure 2.

Body weight is increased in both Hu18/18 and Hu97/18 mice. Body weight was assessed at 2 month intervals in (A) male and (B) female Hu97/18 and Hu18/18 mice and compared with historical FVB body weight data (unpublished). While Hu97/18 mice are slightly heavier than Hu18/18 mice, both humanized genotypes are significantly heavier than wt mice. Different from FVB: *P < 0.05 and ***P < 0.001; different from Hu18/18: #P < 0.05.

Figure 3.

Hu97/18 mice display a motor learning deficit. (A) Two-month-old mice were trained on a fixed speed rotarod over 3 consecutive days and tested in an accelerating program on the fourth day. The number of falls during training and latency to the first fall during training and testing were scored. On day 1 of training, Hu97/18 mice fell from the rotarod sooner and more frequently. However, during testing on the fourth day, there was no genotypic difference indicating that the difference in training performance is the result of a motor learning deficit. (B) Mice were tested on an accelerating rotarod at 2 month intervals from 2 to 12 months of age. Performance declines with age for both genotypes (age P < 0.001). For males, there is a genotypic difference in performance with Hu18/18 mice scoring slightly better (genotype P < 0.01), but for females, there is no effect of genotype. (C) Regression analysis of rotarod performance and body weight. Linear relationship P < 0.0001 both genotypes, r² = 0.55 for Hu18/18 and 0.58 for Hu97/18, indicating that rotarod performance is partially dependent on body weight. *P < 0.05.

Figure 4.

Hu97/18 mice display a climbing deficit. Climbing was assessed at 2 months of age by placing mice in a wire mesh container and recording activity for 5 min. (A) Hu97/18 mice display a trend toward increased latency to begin climbing and (B) decreased number of climbing events, and (C) a significant decrease in total time spent climbing. *P < 0.05.

Figure 5.

Hu97/18 mice display similar activity but increased stereotypy and decreased jumping during spontaneous activity. Spontaneous activity was assessed by infrared beam breaks at 2-month intervals from 2 to 12 months of age, during a 30 min exploration. Hu97/18 and Hu18/18 mice display similar (A) total distance traveled and (B) time spent during ambulation. Compared with Hu18/18 mice, Hu97/18 mice display (C) increased stereotypy and (D) decreased jumping. *P < 0.05 and **P < 0.01.

Figure 6.

Hu97/18 mice display increased anxiety during open-field exploration. Mice aged 3, 6 or 9 months were allowed to explore an open field under bright lighting for 10 min. Exploration was recorded by a ceiling-mounted video camera and animals were tracked using Ethovision XT 7 software. Both genotypes had similar (A) total distance traveled and (B) mean velocity during the exploration period. Compared with Hu18/18 mice, Hu97/18 mice (B) less frequently entered into the center portion of the field and (D)spent less total time in the center of the field, though these differences only reached significance by post hoc at 3 months of age. *P < 0.05.

Figure 7.

Hu97/18 mice display increased anxiety during elevated plus maze exploration. Mice aged 3, 6 or 9 months were allowed to freely explore an elevated plus maze under normal lighting during 5 min. Exploration was recorded by a ceiling-mounted video camera and animals were tracked using Ethovision XT 7 software. Both genotypes had similar (A) total distance traveled and (B) mean velocity during the exploration period. Compared with Hu18/18 mice, Hu97/18 mice (C) spent less time in the open arms and (D) less frequently dipped their heads off the edges of the open arms, though these differences only reached significance by post hoc at 6 and 9 months of age. *P < 0.05 and **P < 0.01.

Figure 8.

Hu97/18 mice display depressive-like behavior. Twelve-month-old mice were placed in cylinders filled with room temperature water and recorded for 6 min with a video camera. During the final 5 min, compared with Hu18/18 mice, Hu97/18 mice (A) spent more time immobile and (B) less time swimming. *P < 0.05.

Figure 9.

Hu97/18 mice display progressive cognitive deficits. Mice aged 3, 6 or 9 months were evaluated for spatial learning and object recognition using the novel object location and novel object preference learning assays. (A) Diagram of the two tests. In the object location test, the mice are presented with two unknown objects in trial 1, and two known objects (the same objects), one in a novel location, in trial 2. In the object preference test, mice are presented with two known objects (the same from object location testing) in trial 1, and one known and one novel object in trial 2. (B) At 3 months of age, both genotypes display normal spatial learning and object recognition, i.e. they prefer to investigate the target object, either the one in the novel location or the novel object, in trial 2. (C) At 6 months of age, Hu18/18 mice display normal learning. Hu97/18 mice display impaired spatial learning (they do not prefer the target object in trial 2), but normal object recognition. (D) At 9 months of age, Hu18/18 mice display normal learning, while Hu97/18 mice have deficits in both tasks indicating impaired spatial learning and object recognition. Mo, month; *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 10.

Hu97/18 mice display neuropathological abnormalities consistent with HD. The brains of 12-month-old mice were evaluated for physical changes seen in HD. (A) Hu97/18 trend toward reduced forebrain weight (P = 0.07), (B) but have no change in cerebellum weight. Stereological analysis shows that compared with Hu18/18 mice, Hu97/18 mice have significantly decreased (C) striatal and (D) cortical volume and (E) a trend toward reduced corpus callosum volume. (F) There is also a trend toward reduced DARPP-32 staining intensity in the Hu97/18 brain. **P < 0.01.