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A Transgenic Minipig Model of Huntington's Disease Shows Early Signs of Behavioral and Molecular Pathologies

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A Transgenic Minipig Model of Huntington's Disease Shows Early Signs of Behavioral and Molecular Pathologies

Georgina Askeland et al. Dis Model Mech.

Abstract

Huntington's disease (HD) is a monogenic, progressive, neurodegenerative disorder with currently no available treatment. The Libechov transgenic minipig model for HD (TgHD) displays neuroanatomical similarities to humans and exhibits slow disease progression, and is therefore more powerful than available mouse models for the development of therapy. The phenotypic characterization of this model is still ongoing, and it is essential to validate biomarkers to monitor disease progression and intervention. In this study, the behavioral phenotype (cognitive, motor and behavior) of the TgHD model was assessed, along with biomarkers for mitochondrial capacity, oxidative stress, DNA integrity and DNA repair at different ages (24, 36 and 48 months), and compared with age-matched controls. The TgHD minipigs showed progressive accumulation of the mutant huntingtin (mHTT) fragment in brain tissue and exhibited locomotor functional decline at 48 months. Interestingly, this neuropathology progressed without any significant age-dependent changes in any of the other biomarkers assessed. Rather, we observed genotype-specific effects on mitochondrial DNA (mtDNA) damage, mtDNA copy number, 8-oxoguanine DNA glycosylase activity and global level of the epigenetic marker 5-methylcytosine that we believe is indicative of a metabolic alteration that manifests in progressive neuropathology. Peripheral blood mononuclear cells (PBMCs) were relatively spared in the TgHD minipig, probably due to the lack of detectable mHTT. Our data demonstrate that neuropathology in the TgHD model has an age of onset of 48 months, and that oxidative damage and electron transport chain impairment represent later states of the disease that are not optimal for assessing interventions.This article has an associated First Person interview with the first author of the paper.

Keywords: DNA damage; DNA repair; HD large animal model; Huntington's disease; Mitochondrial function.

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
The TgHD minipig model shows behavioral abnormalities consistent with early neurodegeneration. (A) Behavioral testing with tests specially designed for minipigs revealed a deficit in performing the tunnel test (P=0.04) and indications of a deficit in walking (nonsignificant). (B) Representative images (clockwise from top) of the balance beam, hurdle, seesaw and tunnel tests. Mann–Whitney test and two-way ANOVA, *P<0.05. Box plots and whiskers indicate minimum to maximum values, with hinges representing the 25th and 75th percentiles and the median indicated by the centerline. Sample size [female (F)+male (M)] distribution: balance beam, WT 4F+2M, TgHD 4F+2M; hurdle, WT 3F+2M, TgHD 4F+1M; seesaw, WT 3F+2M, TgHD 3F+2M; tunnel, WT 3F+2M, TgHD 4F+1M; walking, WT 4F+2M, TgHD 4F+2M.
Fig. 2.
Fig. 2.
Tissue-specific changes in genome integrity in the TgHD minipig model. (A) mtDNA damage analysis revealed lower damage levels in the basal ganglia in TgHD than in WT (P=0.01), but no changes in PBMCs or the frontal cortex. (B) nDNA damage analysis demonstrated that nDNA integrity is apparently unaffected in the TgHD model. (C) Quantification of mtDNA copy number shows a significant decrease in the frontal cortex (P=0.04) and, in contrast, an increase in the basal ganglia (P=0.01) in the TgHD minipigs, relative to WT. No differences were seen in PBMCs. (D) mtDNA mutation frequency analysis indicates higher levels in the basal ganglia in TgHD (nonsignificant) than in WT, but normal levels in PBMCs and the frontal cortex. (E) The methylation mark 5-me(dC) was increased in the frontal cortex in TgHD compared with WT (P=0.047). No differences in 5-me(dC) levels were seen in PBMCs or the basal ganglia. Student's t-test, *P<0.05. Box plots and whiskers indicate minimum to maximum values, with hinges representing the 25th and 75th percentiles and the median indicated by the centerline. Sample sizes: (A) basal ganglia, WT n=4, TgHD n=5; PBMCs, WT n=17, TgHD n=14; frontal cortex, WT n=10, TgHD n=7; (B) PBMCs, WT n=17, TgHD n=14; frontal cortex, WT n=10, TgHD n=7; basal ganglia, WT n=4, TgHD n=5; (C) frontal cortex, WT n=10, TgHD n=5; basal ganglia, WT n=4, TgHD n=5; PBMCs, WT n=17, TgHD n=13; (D) basal ganglia, WT n=2, TgHD n=2; PBMCs, WT n=7, TgHD n=4; frontal cortex, WT n=10, TgHD n=6; (E) frontal cortex, WT n=10, TgHD n=7; PBMCs, WT n=10, TgHD n=6; basal ganglia, WT n=2, TgHD n=5.
Fig. 3.
Fig. 3.
Tissue-specific alterations in oxidative DNA damage and repair in TgHD minipigs. (A) Mass spectrometry analysis of the oxidative damage marker 8-oxoG [as nucleoside 8-oxo(dG)] showed increased levels of PBMCs in TgHD relative to WT (P=0.03), whereas the frontal cortex and basal ganglia displayed similar levels in the two genotypes. (B) The level of malondialdehyde (MDA) was determined in the specified brain subregions. No significant differences were found between WT and TgHD in any of the groups. Arbitrary units (A.U.) represent levels of MDA (μg), showing the extent of lipid peroxidation. (C) Representative image of the DNA glycosylase activity assay, showing incision of 8-oxoG containing 32P-endlabeled oligonucleotide. Extracts were collected from individual animals. (D) DNA glycosylase activity toward 8-oxoG in nuclear protein extracts from PBMCs and different brain subregions. The analysis of substrate cleavage by DNA glycosylase enzyme OGG1 revealed reduced activity in the frontal cortex in TgHD (P=0.03), and indicates a defect in DNA repair of oxidative DNA damage. No genotype differences were seen in other brain regions. Data are presented as relative to the average of the WT activities. Student's t-test, *P<0.05. Box plots and whiskers indicate minimum to maximum values, with hinges representing the 25th and 75th percentiles and the median indicated by the centerline. Samples sizes: (A) PBMCs, WT n=10, TgHD n=6; frontal cortex, WT n=10, TgHD n=7; basal ganglia, WT n=2, TgHD n=5; (B) frontal cortex, WT n=9, TgHD n=9; putamen, WT n=4, TgHD n=4; caudate nucleus, WT n=2, TgHD n=3; hippocampus, WT n=7, TgHD n=7; (D) PBMCs, WT n=7, TgHD n=8; frontal cortex, WT n=9, TgHD n=10; putamen, WT n=4, TgHD n=4; caudate nucleus, WT n=2, TgHD n=3; hippocampus, WT n=7, TgHD n=8.
Fig. 4.
Fig. 4.
Mitochondrial status in TgHD minipig brain subregions and PBMCs. (A,B) Mitochondrial ETC complexes I-IV (A), pyruvate dehydrogenase (PDH) and citrate synthase (CS) (B) all showed normal activity in TgHD compared with WT in the frontal cortex. (C,D) In the basal ganglia, mitochondrial ETC complexes I-IV (C), PDH and CS (D) all showed normal activity in TgHD compared with WT. (E) In addition, mitochondrial ETC complexes I, II and IV and CS showed normal activity in TgHD compared with WT in PBMCs. Arbitrary units (A.U.) represent enzymatic activity in nmol/min/mg. Student's t-test was used to assess significance. Box plots and whiskers indicate minimum to maximum values, with hinges representing the 25th and 75th percentiles and the median indicated by the centerline. Sample sizes: (A,B) frontal cortex: complex I, WT n=19, TgHD n=20; complex II, III and IV, all WT n=19, TgHD n=21; PDH, WT n=16, TgHD n=17; CS, WT n=19, TgHD n=21; (C,D) basal ganglia: complex I-IV, all WT n=10, TgHD n=9; PDH, WT n=9, TgHD n=7; CS, WT n=10, TgHD n=9; (E) PBMCs: complex I, II and IV and CS, all WT n=18, TgHD n=19.
Fig. 5.
Fig. 5.
TgHD minipig PBMCs do not express mHTT. (A-D) Western blot analyses of individual minipigs and subsequent quantification revealed that mHTT and endogenous HTT were expressed in a tissue- and age-specific manner in the frontal cortex (A,B) (mHTT: TgHD 24 vs 48 months, P=0.04; PolyQ: TgHD 24 vs 48 months, P=0.02; TgHD 24 vs 36 months, P=0.02; HTT: TgHD 24 vs 48 months, P=0.03; TgHD 36 vs 48 months, P=0.02; ANOVA WT vs TgHD, P=0.004) and putamen (C,D) (PolyQ: TgHD 24 vs 48 months, P=0.04; TgHD 36 vs 48 months, P=0.004; HTT: WT 36 vs 48 months, P=0.04; TgHD 36 vs 48 months, P=0.009; ANOVA WT vs TgHD, P=0.001) in TgHD minipigs, and confirmed the absence of mHTT and PolyQ in WT animals. (E) mHTT was not expressed in minipig TgHD PBMCs. Representative western blot of mHTT in PBMCs from HD patients (HD) and controls (Ctr), and PBMCs from WT and TgHD minipigs, as indicated. Three distinct antibodies were used to identify mHTT and PolyQ fragments and endogenous HTT protein (see Materials and Methods). Mann–Whitney test and ANOVA, *P<0.05, **P<0.01, ***P<0.001. Sample sizes: (A,B) mHTT and PolyQ: TgHD 24 months, n=4; TgHD 36 months, n=5; TgHD 48 months, n=6; HTT: WT 24 months, n=3; WT 36 months, n=4; WT 48 months, n=6; TgHD 24 months, n=4; TgHD 36 months, n=5; TgHD 48 months, n=5; (C,D) mHTT and PolyQ: TgHD 24 months, n=3; TgHD 36 months, n=6; TgHD 48 months, n=5; HTT: WT 24 months, n=2; 36 months, n=3; 48 months, n=5; TgHD 24 months, n=3; 36 months: n=6, 48 months, n=5.

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