Background Huntington'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.
Aims The 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.
Methods Currently, 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.
Results The 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.
Conclusions We 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.
- Genotoxic stress