RESUMO
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.
Assuntos
Comportamento Animal/fisiologia , Cognição/fisiologia , Doença de Huntington/fisiopatologia , Atividade Motora , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Doença de Huntington/complicações , Estudos Longitudinais , Condicionamento Físico Animal , Estresse Psicológico/complicações , Suínos , Porco Miniatura , LínguaRESUMO
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 70-100â 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 60-70â 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 48â 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 60-70â 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.
Assuntos
Doença de Huntington/patologia , Degeneração Neural/patologia , Envelhecimento/patologia , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Índice de Massa Corporal , Núcleo Caudado/patologia , Núcleo Caudado/ultraestrutura , Modelos Animais de Doenças , Feminino , Genótipo , Humanos , Proteína Huntingtina/metabolismo , Masculino , Córtex Motor/patologia , Córtex Motor/ultraestrutura , Bainha de Mielina/metabolismo , Agregados Proteicos , Suínos , Porco Miniatura , Redução de Peso , Substância Branca/patologia , Substância Branca/ultraestruturaRESUMO
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 48â 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 48â 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 48â 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.
Assuntos
Comportamento Animal , Doença de Huntington/patologia , 8-Hidroxi-2'-Desoxiguanosina , Animais , Animais Geneticamente Modificados , Dano ao DNA , Reparo do DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Genoma , Humanos , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Mitocôndrias/metabolismo , Degeneração Neural/patologia , Especificidade de Órgãos , Suínos , Porco MiniaturaRESUMO
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.
Assuntos
Encéfalo/metabolismo , Proteína Huntingtina/genética , Doença de Huntington/genética , Proteínas do Tecido Nervoso/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Proteínas de Membrana/metabolismo , Proteínas Nucleares/genética , Fenótipo , Suínos , Porco MiniaturaRESUMO
BACKGROUND: Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. OBJECTIVE: We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. METHODS: In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. RESULTS: Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. CONCLUSIONS: The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt.
Assuntos
Proteína Huntingtina/metabolismo , Mutação , Espermatozoides/metabolismo , Espermatozoides/patologia , Testículo/metabolismo , Testículo/patologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Animais Geneticamente Modificados , Proliferação de Células/fisiologia , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Proteína Huntingtina/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Lentivirus/genética , Masculino , Contagem de Espermatozoides , Suínos , Porco MiniaturaRESUMO
BACKGROUND: Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. OBJECTIVE: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of human HTT promoter. METHODS: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. RESULTS: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. CONCLUSIONS: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.