At present, we are probably the only research facility to be breeding transgenic Huntington's disease minipigs (TgHD). These minipigs express N-terminal part of human mutated huntingtin including 124Q under the control of human huntingtin promoter. The founder animal, born in 2009, gave birth to four subsequent generations with an equal contribution of wild-type (WT) and transgenic (TgHD) piglets in all litters. We take different approaches, some of which are unique for large animal models, to study the phenotype development comparing WT and TgHD siblings. In this chapter, we review these approaches and the phenotype progression in the minipigs. Additionally, we outline perspectives in generation of new models using novel methodology and the potential of pig models in preclinical HD studies.
BackgroundHuntington’s disease (HD) is progressive neurodegenerative disorder caused by the mutation in the huntingtin gene giving rise to mutated form of huntingtin protein (mHTT). Recent findings suggest that mHTT may also affect DNA damage response. However, it is not clear whether mHTT compromises detection of double strand DNA breaks (DSBs) or repair mechanism itself.AimsThe main aim of this study is to characterise DSBs response in primary fibroblasts isolated from transgenic minipig HD model.MethodsIn order to study DNA damage we monitored kinetics of γH2AX and 53BP1 by immunofluorescence in primary dermal fibroblasts isolated from wild-type and HD transgenic minipigs of age 8, 24 and 48 months after treatment with radiomimetic drug neocarzinostatin (NCS). The quantitative analysis of confocal images was done by FindFoci algorithm (available as an ImageJ plugin).ResultsAnalysis showed decreased number of γH2AX foci in 24 and 48 months HD fibroblasts after NCS treatment compared to wild-type fibroblasts, suggesting that the ability to recognise new DSBs in HD fibroblasts is compromised. Moreover, we observed that NCS treatment in 8 and 48 months fibroblasts caused significantly higher number of 53BP1 foci shortly after DSBs induction suggesting that the DSBs repair in HD fibroblasts is impaired.ConclusionTaken together, we found that HD fibroblasts exhibit compromised ability to recognise new DSBs and that significant change in dynamics of loading repair factor 53BP1 is present. These data confirmed that transgenic minipig HD model exhibits similar defects in DSBs response as human patients and mouse models.AcknowledgementThis study was supported by CHDI Foundation (A-5378) and by National Sustainability Programme, project number LO1609 (Czech Ministry of Education, Youth and Sports).The research leading to these results has received funding from the Norwegian Financial Mechanism 2009–2014 and the Ministry of Education, Youth and Sports under Project Contract no. MSMT-28477/2014 (project ID 7F14308).
BackgroundHuntington's disease (HD) is a dominant inherited disorder caused by mutation in huntingtin (htt) protein that causes massive neural cell death leading to neurodegeneration. Mutated huntingtin (mt htt) induces a cascade of events that stimulates DNA breaks and the activation of DNA damage response pathway. This genotoxic stress is followed by activation of the MDC1 and H2AX proteins, in the early stages of HD pathogenesis. Expression of htt occurs in cells of neuronal and non-neuronal origin.AimsThe aim of our research is to study genetoxic stress of mt htt on fibroblasts and mesenchymal stem cells (MSCs) of our miniature pig model for HD.MethodsCurrently, we have three generations of transgenic (tg) minipigs for N-terminal part of the human mt htt (548 aa, 124 Q). Each litter presents wild type (wt) and tg siblings at an approximate ratio 1:1 with the same genetic background. DNA damage was examined on fibroblasts and MSCs isolated from tg and wt minipigs using confocal microscopy, immunocytochemistry, Western blot analysis and the dynamic of tg and wt cell proliferation was tested by life cell imaging.ResultsThe significant increase of MDC1 and γH2AX proteins, indicating DNA damage, was proved by immunocytochemistry and also Western blot analysis. Proliferation tests could not clearly establish the genotoxic stress of tg fibroblasts and MSCs.ConclusionsWe will continue in our studies to observe changes in genotoxic stress during the lifespan of our minipigs. HD tg miniature pig represents a perspective large animal model for the research of molecular mechanisms of mt htt.
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