BACKGROUND: Huntington disease (HD) is an incurable neurodegenerative disease caused by the expansion of a polyglutamine sequence in a gene encoding the huntingtin (Htt) protein, which is expressed in almost all cells of the body. In addition to small animal models, new therapeutic approaches (including gene therapy) require large animal models as their large brains are a more realistic model for translational research. OBJECTIVE: In this study, we describe phenotype development in transgenic minipigs (TgHD) expressing the N-terminal part of mutated human Htt at the age of 24 months. METHODS: TgHD and wild-type littermates were compared. Western blot analysis and subcellular fractionation of different tissues was used to determine the fragmentation of Htt. Immunohistochemistry and optical analysis of coronal sections measuring aggregates, Htt expression, neuroinflammation, and myelination was applied. Furthermore, the expression of Golgi protein acyl-CoA binding domain containing 3 (ACBD3) was analyzed. RESULTS: We found age-correlated Htt fragmentation in the brain. Among various tissues studied, the testes displayed the highest fragmentation, with Htt fragments detectable even in cell nuclei. Also, Golgi protein ACBD3 was upregulated in testes, which is in agreement with previously reported testicular degeneration in TgHD minipigs. Nevertheless, the TgHD-specific mutated Htt fragments were also present in the cytoplasm of striatum and cortex cells. Moreover, microglial cells were activated and myelination was slightly decreased, suggesting the development of a premanifest stage of neurodegeneration in TgHD minipigs. CONCLUSIONS: The gradual development of a neurodegenerative phenotype, ac-companied with testicular degeneration, is observed in 24- month-old TgHD minipigs.

• Klíčová slova
• ACBD3, Aggregates, Fragments, Huntington disease, Mutant huntingtin, Pig model,
• MeSH
• fenotyp MeSH
• geneticky modifikovaná zvířata MeSH
• Huntingtonova nemoc genetika   MeSH
• jaderné proteiny genetika   MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• miniaturní prasata MeSH
• modely nemocí na zvířatech MeSH
• mozek metabolismus   MeSH
• prasata MeSH
• protein huntingtin genetika   MeSH
• proteiny nervové tkáně genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jaderné proteiny MeSH
• membránové proteiny MeSH
• protein huntingtin MeSH
• proteiny nervové tkáně MeSH

BACKGROUND: Large animal models, such as the transgenic (tg) Huntington disease (HD) minipig, have been proposed to improve translational reliability and assessment of safety, efficacy and tolerability in preclinical studies. Minipigs are characterised by high genetic homology and comparable brain structures to humans. In addition, behavioural assessments successfully applied in humans could be explored in minipigs to establish similar endpoints in preclinical and clinical studies. Recently, analysis of voice and speech production was established to characterise HD patients. OBJECTIVE: The aim of this study was to investigate whether vocalisation could also serve as a viable marker for phenotyping minipigs transgenic for Huntington's disease (tgHD) and whether tgHD minipigs reveal changes in this domain compared to wildtype (wt) minipigs. METHODS: While conducting behavioural testing, incidence of vocalisation was assessed for a cohort of 14 tgHD and 18 wt minipigs. Statistical analyses were performed using Fisher's Exact Test for group comparisons and McNemar's Test for intra-visit differences between tgHD and wt minipigs. RESULTS: Vocalisation can easily be documented during phenotyping assessments of minipigs. Differences in vocalisation incidences across behavioural conditions were detected between tgHD and wt minipigs. Influence of the genotype on vocalisation was detectable during a period of 1.5 years. CONCLUSION: Vocalisation may be a viable marker for phenotyping minipigs transgenic for the Huntington gene. Documentation of vocalisation provides a non-invasive opportunity to capture potential disease signs and explore phenotypic development including the age of disease manifestation.

• Klíčová slova
• Large animal model, minipig, phenotyping, preclinical, transgenic, vocalisation,
• MeSH
• časové faktory MeSH
• diskriminace (psychologie) MeSH
• fenotyp * MeSH
• geneticky modifikovaná zvířata * MeSH
• Huntingtonova nemoc * patofyziologie   psychologie   MeSH
• jazyk patofyziologie   MeSH
• lidé MeSH
• longitudinální studie MeSH
• miniaturní prasata * MeSH
• modely nemocí na zvířatech * MeSH
• motorické dovednosti MeSH
• prasata MeSH
• protein huntingtin genetika   metabolismus   MeSH
• reverzní učení MeSH
• vnímání barev MeSH
• vokalizace zvířat * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HTT protein, human MeSH Prohlížeč
• protein huntingtin MeSH

BACKGROUND: While several novel therapeutic approaches for HD are in development, resources to conduct clinical trials are limited. Large animal models have been proposed to improve assessment of safety, tolerability and especially to increase translational reliability of efficacy signals obtained in preclinical studies. They may thus help to select candidates for translation to human studies. We here introduce a battery of novel tests designed to assess the motor, cognitive and behavioral phenotype of a transgenic (tg) HD minipig model. NEW METHODS: A group of tgHD and wildtype (wt) Libechov minipigs (n=36) was available for assessment with (1) a gait test using the GAITRite(®) automated acquisition system, (2) a hurdle-test, (3) a tongue coordination test, (4) a color discrimination test, (5) a startbox back and forth test and (6) a dominance test. Performance of all tests and definition of measures obtained is presented. RESULTS: Minipigs were able to learn performance of all tests. All tests were safe, well tolerated and feasible. Exploratory between group comparisons showed no differences between groups of tgHD and wt minipigs assessed, but low variability within and between groups. COMPARISON WITH EXISTING METHOD(S): So far there are no established or validated assessments to test minipigs in the domains described. CONCLUSIONS: The data shows that the tests presented are safe, well tolerated and all measures defined can be assessed. Prospective longitudinal application of these tests is warranted to determine their test-retest reliability, sensitivity and validity in assessing motor, cognitive and behavioral features of tg and wt minipigs.

• Klíčová slova
• Animal models, Behavioral, Cognitive, Minipig, Motor, Phenotyping, Preclinical research,
• MeSH
• chování zvířat * MeSH
• chůze (způsob) fyziologie   MeSH
• design vybavení MeSH
• diskriminace (psychologie) fyziologie   MeSH
• fenotyp MeSH
• geneticky modifikovaná zvířata * MeSH
• Huntingtonova nemoc genetika   MeSH
• jazyk patofyziologie   MeSH
• lidé MeSH
• miniaturní prasata genetika   fyziologie   psychologie   MeSH
• modely nemocí na zvířatech * MeSH
• neuropsychologické testy MeSH
• pohybová aktivita fyziologie   MeSH
• prasata MeSH
• protein huntingtin genetika   metabolismus   MeSH
• studie proveditelnosti MeSH
• učení fyziologie   MeSH
• vidění barevné fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• HTT protein, human MeSH Prohlížeč
• protein huntingtin MeSH

BACKGROUND: Large animal models of Huntington's disease (HD) may increase the reliability of translating preclinical findings to humans. Long live expectancy offers opportunities particularly for disease modifying approaches, but also challenges. The transgenic (tg) HD minipig model assessed in this study exhibits a high genetic homology with humans, similar body weight, and comparable brain structures. To test long-term safety, tolerability, and efficacy of novel therapeutic approaches in this model reliable assessments applicable longitudinally for several years are warranted for all phenotypical domains relevant in HD. OBJECTIVE: To investigate whether the tests proposed assessing motor, cognitive and behavioral domains can be applied repetitively over a 3-year period in minipigs with acceptable variability or learning effects and whether tgHD minipigs reveal changes in these domains compared to wildtype (wt) minipigs suggesting the development of an HD phenotype. METHODS: A cohort of 14 tgHD and 18 wt minipigs was followed for three years. Tests applied every six months included a tongue coordination and hurdle test for the motor domain, a color discrimination test for cognition, and a dominance test for assessing behavior. Statistical analyses were performed using repeated ANOVA for longitudinal group comparisons and Wilcoxon-tests for intra-visit differences between tgHD and wt minipigs. RESULTS: All tests applied demonstrated feasibility, acceptable variance and good consistency during the three-year period. No significant differences between tgHD and wt minipigs were detected suggesting lack of a phenotype before the age of four years. CONCLUSIONS: The assessment battery presented offers measures in all domains relevant for HD and can be applied in long-term phenotyping studies with tgHD minipigs. The observation of this cohort should be continued to explore the timeline of phenotype development and provide information for future interventional studies.

• MeSH
• chování zvířat fyziologie   MeSH
• geneticky modifikovaná zvířata MeSH
• Huntingtonova nemoc patofyziologie   MeSH
• jazyk fyziologie   MeSH
• lidé MeSH
• miniaturní prasata fyziologie   MeSH
• prasata fyziologie   MeSH
• protein huntingtin genetika   fyziologie   MeSH
• učení fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• pozorovací studie MeSH
• Názvy látek
• protein huntingtin MeSH

Recently developed therapeutic approaches for the treatment of Huntington's disease (HD) require preclinical testing in large animal models. The minipig is a suitable experimental animal because of its large gyrencephalic brain, body weight of 70-100 kg, long lifespan, and anatomical, physiological and metabolic resemblance to humans. The Libechov transgenic minipig model for HD (TgHD) has proven useful for proof of concept of developing new therapies. However, to evaluate the efficacy of different therapies on disease progression, a broader phenotypic characterization of the TgHD minipig is needed. In this study, we analyzed the brain tissues of TgHD minipigs at the age of 48 and 60-70 months, and compared them to wild-type animals. We were able to demonstrate not only an accumulation of different forms of mutant huntingtin (mHTT) in TgHD brain, but also pathological changes associated with cellular damage caused by mHTT. At 48 months, we detected pathological changes that included the demyelination of brain white matter, loss of function of striatal neurons in the putamen and activation of microglia. At 60-70 months, we found a clear marker of neurodegeneration: significant cell loss detected in the caudate nucleus, putamen and cortex. This was accompanied by clusters of structures accumulating in the neurites of some neurons, a sign of their degeneration that is also seen in Alzheimer's disease, and a significant activation of astrocytes. In summary, our data demonstrate age-dependent neuropathology with later onset of neurodegeneration in TgHD minipigs.

• Klíčová slova
• Brain, Huntingtin, Large animal model, Neuropathology, TgHD,
• MeSH
• bílá hmota patologie   ultrastruktura   MeSH
• biologické markery metabolismus   MeSH
• degenerace nervu patologie   MeSH
• geneticky modifikovaná zvířata MeSH
• genotyp MeSH
• hmotnostní úbytek MeSH
• Huntingtonova nemoc patologie   MeSH
• index tělesné hmotnosti MeSH
• lidé MeSH
• miniaturní prasata MeSH
• modely nemocí na zvířatech MeSH
• motorické korové centrum patologie   ultrastruktura   MeSH
• myelinová pochva metabolismus   MeSH
• nucleus caudatus patologie   ultrastruktura   MeSH
• prasata MeSH
• protein huntingtin metabolismus   MeSH
• proteinové agregáty MeSH
• stárnutí patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• Htt protein, mouse MeSH Prohlížeč
• protein huntingtin MeSH
• proteinové agregáty MeSH

Skeletal muscle wasting and atrophy is one of the more severe clinical impairments resulting from the progression of Huntington's disease (HD). Mitochondrial dysfunction may play a significant role in the etiology of HD, but the specific condition of mitochondria in muscle has not been widely studied during the development of HD. To determine the role of mitochondria in skeletal muscle during the early stages of HD, we analyzed quadriceps femoris muscle from 24-, 36-, 48- and 66-month-old transgenic minipigs that expressed the N-terminal portion of mutated human huntingtin protein (TgHD) and age-matched wild-type (WT) siblings. We found altered ultrastructure of TgHD muscle tissue and mitochondria. There was also significant reduction of activity of citrate synthase and respiratory chain complexes (RCCs) I, II and IV, decreased quantity of oligomycin-sensitivity conferring protein (OSCP) and the E2 subunit of pyruvate dehydrogenase (PDHE2), and differential expression of optic atrophy 1 protein (OPA1) and dynamin-related protein 1 (DRP1) in the skeletal muscle of TgHD minipigs. Statistical analysis identified several parameters that were dependent only on HD status and could therefore be used as potential biomarkers of disease progression. In particular, the reduction of biomarker RCCII subunit SDH30 quantity suggests that similar pathogenic mechanisms underlie disease progression in TgHD minipigs and HD patients. The perturbed biochemical phenotype was detectable in TgHD minipigs prior to the development of ultrastructural changes and locomotor impairment, which become evident at the age of 48 months. Mitochondrial disturbances may contribute to energetic depression in skeletal muscle in HD, which is in concordance with the mobility problems observed in this model.This article has an associated First Person interview with the first author of the paper.

• Klíčová slova
• Biomarkers, Disease development, HD large animal model, Huntington's disease, Mitochondrial function, Skeletal muscle, Ultrastructure,
• MeSH
• DNA metabolismus   MeSH
• energetický metabolismus * MeSH
• geneticky modifikovaná zvířata MeSH
• Huntingtonova nemoc metabolismus   patologie   MeSH
• kosterní svaly metabolismus   ultrastruktura   MeSH
• lidé MeSH
• miniaturní prasata MeSH
• mitochondriální proteiny metabolismus   MeSH
• modely nemocí na zvířatech * MeSH
• mutace MeSH
• oxidativní fosforylace MeSH
• prasata MeSH
• progrese nemoci MeSH
• protein huntingtin genetika   MeSH
• svalové mitochondrie metabolismus   ultrastruktura   MeSH
• tělesná hmotnost MeSH
• transport elektronů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• HTT protein, human MeSH Prohlížeč
• mitochondriální proteiny MeSH
• protein huntingtin MeSH

Huntington's disease (HD) is an inherited devastating neurodegenerative disease with no known cure to date. Several therapeutic treatments for HD are in development, but their safety, tolerability and efficacy need to be tested before translation to bedside. The monogenetic nature of this disorder has enabled the generation of transgenic animal models carrying a mutant huntingtin (mHTT) gene causing HD. A large animal model reflecting disease progression in humans would be beneficial for testing the potential therapeutic approaches. Progression of the motor, cognitive and behavioral phenotype was monitored in transgenic Huntington's disease minipigs (TgHD) expressing the N-terminal part of human mHTT. New tests were established to investigate physical activity by telemetry, and to explore the stress-induced behavioral and cognitive changes in minipigs. The longitudinal study revealed significant differences between 6- to 8-year-old TgHD animals and their wild-type (WT) controls in a majority of the tests. The telemetric study showed increased physical activity of 4.6- to 6.5-year-old TgHD boars compared to their WT counterparts during the lunch period as well as in the afternoon. Our phenotypic study indicates progression in adult TgHD minipigs and therefore this model could be suitable for longstanding preclinical studies of HD.This article has an associated First Person interview with the first author of the paper.

• Klíčová slova
• Cognitive and behavioral studies, Huntington's disease, Large animal model, Motor, Phenotyping,
• MeSH
• chování zvířat fyziologie   MeSH
• geneticky modifikovaná zvířata MeSH
• Huntingtonova nemoc komplikace   patofyziologie   MeSH
• jazyk MeSH
• kognice fyziologie   MeSH
• kondiční příprava zvířat MeSH
• longitudinální studie MeSH
• miniaturní prasata MeSH
• modely nemocí na zvířatech MeSH
• pohybová aktivita * MeSH
• prasata MeSH
• psychický stres komplikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• Klíčová slova
• DNA damage, DNA repair, HD large animal model, Huntington's disease, Mitochondrial function,
• MeSH
• 8-hydroxy-2'-deoxyguanosin MeSH
• chování zvířat * MeSH
• degenerace nervu patologie   MeSH
• deoxyguanosin analogy a deriváty   metabolismus   MeSH
• energetický metabolismus MeSH
• geneticky modifikovaná zvířata MeSH
• genom MeSH
• Huntingtonova nemoc metabolismus   patologie   MeSH
• lidé MeSH
• miniaturní prasata MeSH
• mitochondrie metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• oprava DNA MeSH
• orgánová specificita MeSH
• poškození DNA MeSH
• prasata MeSH
• protein huntingtin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 8-hydroxy-2'-deoxyguanosin MeSH
• deoxyguanosin MeSH
• protein huntingtin MeSH

BACKGROUND: As novel treatment approaches for Huntington's disease (HD) evolve, the use of transgenic (tg) large animal models has been considered for preclinical safety and efficacy assessments. It is hoped that large animal models may provide higher reliability in translating preclinical findings to humans, e.g., by using similar endpoints and biomarkers. NEW METHOD: We here investigated the feasibility to conduct MRI assessments in a recently developed tgHD model in the Libechov minipig. The model is characterized by high genetic homology to humans and a similar body mass and compartments. The minipig brain provides anatomical features that are attractive for imaging studies and could be used as endpoints for disease modifying preclinical studies similar to human HD. RESULTS: We demonstrate that complex MRI protocols can be successfully acquired with tgHD and wild type (wt) Libechov minipigs. We show that acquisition of anatomical images applicable for volumetric assessments is feasible and outline the development of a segmented MRI brain atlas. Similarly diffusion-weighted imaging (DWI) including fiber tractography is presented. We also demonstrate the feasibility to conduct in vivo metabolic assessments using MR spectroscopy. COMPARISON WITH EXISTING METHODS: In human HD, these MRI methods are already validated and used as reliable biomarker of disease progression even before the onset of a clinical motor phenotype. CONCLUSIONS: The results show that the minipig brain is well suited for MRI assessments in preclinical studies. We conclude that further characterization of phenotypical differences between tg and wt animals in sufficiently powered cross-sectional and longitudinal studies is warranted.

• Klíčová slova
• Animal models, Brain atlas, MRI, Minipig, Preclinical research,
• MeSH
• geneticky modifikovaná zvířata MeSH
• Huntingtonova nemoc diagnostické zobrazování   metabolismus   MeSH
• magnetická rezonanční tomografie * přístrojové vybavení   metody   MeSH
• miniaturní prasata * MeSH
• modely nemocí na zvířatech * MeSH
• mozek diagnostické zobrazování   metabolismus   MeSH
• neurozobrazování přístrojové vybavení   metody   MeSH
• prasata MeSH
• protonová magnetická rezonanční spektroskopie * přístrojové vybavení   metody   MeSH
• velikost orgánu MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH