Comparative study of Huntington’s disease using biochemical, immunocytochemical and molecular genetic methods on the mouse, minipig and human tissues and cells

Project facts

Project promoter:
Institute of Animal Physiology and Genetics AS CR
Project Number:
CZ09-0016
Target groups
Researchers or scientists
Status:
Completed
Initial project cost:
€936,566
Final project cost:
€841,207
From Norway Grants:
€ 715,026
The project is carried out in:
Středočeský kraj

Description

Incidence of neurodegenerative disease in Western population increases substantially and it represents a serious health problem. There is no available therapy of HD, which can stop the disease progression till now. The project is based on comparative studies of tissues (from brain), somatic cells (skin fibroblasts) and germ cells (spermatozoa) isolated from rodent model, biomedical model (transgenic minipigs for human mutated huntingtin) and HD patients. The study will offer an opportunity to gain better understanding of HD development from the gene mutation to the neurological decline. Biomedical model of HD, transgenic minipigs, will provide a chance to test safety and efficiency of all new approaches how to cure HD. The biological material will be examined by a complex set of biochemical, immunocytochemical and molecular genetic methods in cooperation of four laboratories in Czech and Norway. HD has worldwide prevalence of 3-10 affected individuals per 100,000 persons in Western Europe and North America. For these patients and their families, the study brings hope that better understanding of pathophysiology of HD may lead to a future effective therapy.

Summary of project results

The project was based on the comparative study of Huntington’s disease (HD), a fatal progressive neurodegenerative disorder that is inherited in an autosomal dominant manner. HD is caused by a polymorphic trinucleotide CAG repeat expansion in exon 1 of huntingtin gene (HTT). Up to date there is no curative treatment. Moreover, an exact mechanism, how mutant HTT induces selective neurodegeneration, still remains to be elucidated. We found that the presence of mutant HTT significantly changes the response on double strand breaks (DSBs) in age-dependent manner in primary dermal fibroblast from TgHD minipigs. Furthermore, the dermal fibroblasts from old TgHD minipigs have similar defects in DSBs response as primary dermal fibroblasts from adult patients. We proposed four mitochondrial metabolism parameters which were significantly diminished in TgHD spermatozoa of pre-symptomatic TgHD minipigs as potential biomarkers of HD. Longitudinal study of mitochondrial bioenergetics and ultrastructure in TgHD minipigs showed that muscle wasting started at the age of 48 month, before the disease development. Isolated lymphocytes from peripheral blood of HD patients showed significantly decreased activities and content of selected mitochondrial genes and proteins in comparison with controls. Genes associated with aerobic metabolism, including DNA repair, and the mitochondrial DNA damage were reduced. In contrast, nuclear DNA damage was increased. We also described tissue specific increased formation of toxic N-terminal fragments and proteolytic enzymes in aging minipigs correlated with R6/2 mice model. We characterized the testicular degeneration in TgHD minipigs, the phenotype also described in R6/2 mice and HD patients. Taking all together, we described patients’ lymphocytes as a suitable noninvasive material to monitor progression of HD. We established new methods to detect phenotype development and described the progression of the disease in TgHD animals in order to be able to use this model for testing potential therapies. Thanks to our results from this project we have initiated preclinical testing of gene therapy (human Htt lowering based on RNA interference) to help proceed with clinical trials to postpone or cure HD.

Summary of bilateral results

The four main project objectives have been achieved which helped to attract the pharma. WoS impact factor, primary peer reviewed, primary ERIH peer reviewed publications, numerous lectures, posters, MoU and RCAs with industry, number of experimental data. Partners had a chance to present experimental data at international conferences and penetrate community in an area of neurodegenerative diseases. PhD students had a chance to work in Oslo, in Prague and realize specific measurements in Munich. The Norwegian teams had a chance to experiment with the unique Huntington disease human and minipig samples. PhD student passed the stay in the CZ2 lab specialized on advanced biochemical methods. All parties entered the project with their knowhow and unique methodologies. All equally contributed to the results. The great effort in organization of workshops and conferences in the Czech Republic helped the overall cooperation and I fully value the “human factor” involved in cooperation of all teams. It was a unique example for Czech PhD students and postdocs to see what it is creative research atmosphere. including gender equality actions, ethical issues, and wider awareness? If the common effort of this project will result a causative treatment of HD, the mission of the intensive experimental work is completely filled.