Full motor recovery despite striatal neuron loss and formation of irreversible amyloid-like inclusions in a conditional mouse model of Huntington's disease

Abstract

The primary mechanism responsible for Huntington's disease remains unknown. Postulated early pathogenic events include the following: impaired protein folding, altered protein degradation, mitochondrial dysfunction, and transcriptional dysregulation. Although related therapies can delay disease progression in mouse models, they target downstream and probably indirect effects of mutant-huntingtin expression. Accordingly, in case they prove beneficial in humans, they might only palliate some aspects of disease. Our previous studies in the Tet/HD94 conditional model and the recently reported efficacy of RNA interference against mutant huntingtin in another mouse model support silencing mutant-huntingtin expression as a valid therapeutic approach that has the advantage of targeting toxicity at its root. Here, we address whether gene silencing can still be beneficial in the late stages of disease with detectable striatal neuron loss. Stereological analysis was applied to determine an age at which Tet/HD94 mice show a decrease in the number of striatal neurons. Then, progression of neuropathology and motor phenotype were analyzed in mice that were allowed to continue expressing mutant huntingtin and in mice that no longer expressed it. Neuronal loss did not revert in gene-off mice, but the additional loss that takes place in gene-on mice was prevented. The total number of huntingtin-containing inclusions dramatically reverted, but a small fraction of inclusions positive for the amyloid dye thioflavin-S remained. Interestingly, despite a 20% decrease in striatal neurons and the presence of amyloid-like irreversible inclusions, gene-off mice fully recover from their motor deficit, thus ruling out amyloid-like huntingtin inclusions as the main toxic species and suggesting that gene-silencing therapies might work in late stages of disease.

Figure 1.

Motor dysfunction and neuropathology (including striatal neuron loss) in 17-month-old Tet/HD94 mice. A, B, Rota-Rod analysis of 17-month-old control (open squares; n = 20) and Tet/HD94 (filled circles; n=21) mice. Values in A represent mean ± SEM time spent on Rota-Rod, per daily trial and genotype (see Material and Methods) (^*p < 0.01; ^**p < 0.005). Values in B represent mean ± SEM of the latencies to fall in the eight trials per genotype (^*p < 0.01). C, Immunohistochemistry with anti-N-terminal mutant-htt (CAG53b) antibody revealed the presence of aggregates in the forebrains of Tet/HD94 mice. Scale bar, 20 μm. D, Immunoelectron microscopy analysis of a CAG53b-stained inclusion from a Tet/HD94 mouse. The inset in D corresponds to a 2.0× magnification of the indicated area, showing that inclusions are formed by randomly oriented filamentous structures. Scale bar, 1 μm. E, F, Striatal atrophy can be observed in coronal sections like those stained with anti-Neu-N antibody from control (E) and Tet/HD94 (F) mice. Scale bar: (in E) E, F, 500 μm. G, Stereological measurement of striatal size for control (open bar; n = 4) and Tet/HD94 (filled bar; n = 4) mice. H, Stereological analysis of number (N.) of striatal neurons in control (open bar; n = 4) and Tet/HD94 (filled bar; n = 4) as determined by Neu-N staining. ^*p < 0.01. Error bars indicate SE.

Figure 2.

Silencing mutant-htt expression in Tet/HD94 mice with decreased striatal neurons results in full motor recovery. A-E, Rota-Rod performance of control or Tet/HD94 mice either receiving doxycycline to shut down transgene expression (DOX) or kept on water, from the age of 17 months to the age of 22 months. A-D, Values represent mean ± SEM time spent on Rota-Rod at the ages of 17 (A), 19 (B), 21 (C), and 22 (D) months. E, Mean ± SEM of the latencies to fall in the eight trials per genotype, age, and treatment condition. Motor performance of 22-month-old Tet/HD94 mice that have been on DOX for 5 months improve significantly with respect to their performance at the age of 17 months (^*p < 0.01). F, Western blot analysis of striatal (St) and cortical (Cx) extracts from 22-month-old Tet/HD94 mice (+/- DOX treatment) and from control littermates probed with antibodies against N-terminal htt (CAG53b). Membranes were probed with anti-tubulin antibody to correct for any possible deviation on protein loading. M, Month; α-Tub, anti-tubulin; WT, wild type.

Figure 3.

Silencing mutant-htt expression in Tet/HD94 mice halts additional loss of striatal neurons and boosts DARPP-32 expression in the remaining ones. A, B, Stereological measurement of striatal volume on DARPP-32 immunostained sections (A) or number of striatal neurons on Neu-N-immunostained sections (B). Results are presented as the evolution of percentage of Tet/HD94 values with respect to their control littermates from the age of 17 months (open bar) to the age of 22 months either with continued expression (gene-on; gray bar) or doxycycline-treated (gene-off; black bar). Significant differences between groups were determined by ANOVA: ^***p < 0.001; ^*p < 0.02. Striatal volume values for control mice were as follows: 12.1 ± 0.56 mm³ for 17-month-old, 9.8 ± 0.8 mm³ for untreated 22-month-old, and 10.1 ± 0.6 mm³ for doxycycline-treated 22-month-old mice. Numbers of striatal neurons in control mice were as follows: 1.49 × 10⁶ ± 0.04 neurons in 17-month-old ones, 0.99 × 10⁶ ± 0.01 neurons in untreated 22-month-old ones, and 1.11 × 10⁶ ± 0.02 in doxycycline-treated 22-month-old ones. C, D, Striatal sections from 22-month-old gene-on (C) or gene-off (D) Tet/HD94 mice were immunostained with an antibody against DARPP-32 (brown labeling) and counterstained with Nissl dye (blue labeling). A dramatic increase in the number and intensity of positive DARPP-32 striatal neurons was observed in gene-off compared with gene-on mice. Insets in C and D show a 5× magnification. N., Number; M, month; DOX, doxycycline.

Figure 4.

Silencing mutant-htt expression in Tet/HD94 mice in advanced stages of disease results in dramatic but incomplete reversal of htt- and ubiquitin-immunopositive inclusions. Immunocytochemistry with anti-N-terminal huntingtin antibody (MAB5374) or anti-polyubiquitin (FK2) revealed the presence of inclusions in the striatum (A-D) and cortex (F-I) of gene-on and gene-off Tet/HD94 22-month-old mice. The number of inclusions was significantly reduced on gene-off with respect to gene-on mice. E, Histogram showing quantification of N-terminal htt- or polyubiquitin-positive inclusions in striatum of gene-on (gray bar) or gene-off (black bar) 22-month-old Tet/HD94 mice. J, Histogram showing quantification of N-terminal htt- or polyubiquitin-positive aggregates in cortex of gene-on (gray bar) or gene-off (black bar) 22-month-old Tet/HD94 mice. Statistical significance was determined by ANOVA: ^***p < 0.001. Error bars indicate SE.

Figure 5.

Thioflavin-S amyloid staining and highly ordered core material in the irreversible subset of inclusions. Thioflavin-S staining in the striatum (A, C, E) and cortex (B, D, F) of 22-month-old control or Tet/HD94 mice. Single transgenic BiTetO mice that carry the transgene encoding mutant htt but that do not express it because they lack the tTA transactivator were used as control (A, B). Tet/HD94 mice (C-F) show thioflavin-S-stained inclusions regardless of whether they continued expressing mutant htt (gene-on; C, D) or not (gene-off; E, F). Scale bars: (in E, F) A-F, 50 μm. G, Histogram showing the evolution in number of striatal and cortical thioflavin-S-positive inclusions in Tet/HD94 mice from the age of 17 months (open bar) to the age of 22 months either expressing (gene-on; gray bar) or not expressing mutant htt (gene-off; black bar). Error bars indicate SE. St, Striatal; Cx, cortical. H, Immunoelectron microscopy analysis of N-terminal mutant-htt (CAG53b antibody)-stained sections showing an example of inclusion with two different ultrastructural textures, one consisting of randomly oriented filaments (white filled arrow) and the other consisting of a compact core of highly ordered material (white open arrow). Scale bar, 1 μm.