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The impact of mutant huntingtin on oxidative stress in primary fibroblasts isolated from a new Huntington's disease knock in porcine model
Sekáč, Dávid ; Ellederová, Zdeňka (advisor) ; Hanzlíková, Hana (referee)
Huntington's chorea is a dominantly inherited disease caused by trinucleotide (Cytosine-Adenine -Guanine) expansion in a gene coding huntingtin protein. Carriers of these mutation show symptoms associated with motor impairment, a cognitive and psychiatric disturbance, which is called Huntington's disease (HD). The major sign of HD is striatal atrophy in the middle age of life. Since it is known that huntingtin protein participates in a lot of cellular processes, such as transcriptional regulation and metabolism, these processes change by its mutation. One of the features observed in HD pathogenesis is the presence of oxidative stress. The aim of the work was to monitor the molecular changes preceding the HD manifestation in the knock-in minipig model. As a material for monitoring molecular changes leading to this condition, primary fibroblasts were used. Whereas, the oxidative stress arises from an imbalance between oxidants and antioxidants, level of reactive species and lipid peroxidation together with expression of antioxidant response associated genes was measured. At the same time, expression of metabolic and DNA repair related genes was monitored. Although the differences in oxidative stress level or the expression of antioxidative response genes were not detected, the changes in the...
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Nanoparticle-Mediated Delivery System for Gene Therapy
Dvořáková, Nikola ; Ellederová, Zdeňka (advisor) ; Šálek, Petr (referee)
Gene editing with the CRISPR/Cas9 system is one of the options that sets a new trend in the development of gene therapy. The most commonly used delivery of DNA into the cells are via viruses. Nevertheless, they are often unable to take CRISPR/Cas9 system, which can be bigger than several kb. Nanoparticles (NPs), as non-viral transporters, seem to be a good alternative delivery system. For this work magnetic Fe3O4 NPs (MNPs) were selected, because of their excellent properties such as multifunctionality, biocompatibility, easy degradation and simple synthesis. The aim of this work was to synthesise MNPs and a complex of MNPs coated with PEI/CRISPR-Cas9 plasmid and to characterize them by physicochemical methods. The created complex MNPs/PEI/CRISPR-Cas9 was defined by exact parameters that are suitable for possible cell uptake. The hypothesis of stabilization of the MNPs/CRISPR-Cas9 plasmid complex by polyethylenimine (PEI), which can also protect plasmid DNA against restriction endonucleases, was verified. Next a stable modified cell line HEK293-TLR3, designed to evaluate the efficacy of double strand break (DSB) repair by nonhomologous end joining (NHEJ) or homologous recombination (HR) was, transfected with the synthesised MNPs/PEI/CRISPR-Cas9 complex. The results indicate a 25% transfection...
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Monitoring of the development of the Huntington's disease in transgenic minipigs with N-terminal part of human mutated huntingtin: biochemical and motoric changes of F0, F1 and F2 generation
Kučerová, Šárka ; Ellederová, Zdeňka (advisor) ; Klempíř, Jiří (referee)
Huntington's disease (HD) belongs to neurodegenerative disorders. It is a monogenic disease caused by trinucleotic CAG expansion in exon 1 of gene coding protein huntingtin. Even though the cause of HD is known since 1993, the pathophysiology and cure for HD reminds to be found. The animal models are being used for better understanding of HD. The most common animal models for HD are rodents, especially mice but it was also important to create large animal models, which will be more like human. Therefore, TgHD minipig was created in Academic of Science in Liběchov in 2009. This model was created by microinjection of lentiviral vector carrying N-terminal part of human HTT with 124 repetitive CAG in exon 1. This model is viable and in every generation, is part of the offspring transgenic. In this thesis, I specialized to biochemical and behavioral changes of this model. I compared transgenic and wild type siblings. I found that biochemical changes are manifested mostly by increased level of mtHtt fragments in testes and brain. In behavioral part of this thesis I established new methods for testing behavioral changes in this model. The introduced methods showed some changes between wild type and transgenic animals at the tested ages but these changes were not significant due to the low number of...
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The impact of mutant huntingtin on oxidative stress in primary fibroblasts isolated from a new Huntington's disease knock in porcine model
Sekáč, Dávid ; Ellederová, Zdeňka (advisor) ; Hanzlíková, Hana (referee)
Huntington's chorea is a dominantly inherited disease caused by trinucleotide (Cytosine-Adenine -Guanine) expansion in a gene coding huntingtin protein. Carriers of these mutation show symptoms associated with motor impairment, a cognitive and psychiatric disturbance, which is called Huntington's disease (HD). The major sign of HD is striatal atrophy in the middle age of life. Since it is known that huntingtin protein participates in a lot of cellular processes, such as transcriptional regulation and metabolism, these processes change by its mutation. One of the features observed in HD pathogenesis is the presence of oxidative stress. The aim of the work was to monitor the molecular changes preceding the HD manifestation in the knock-in minipig model. As a material for monitoring molecular changes leading to this condition, primary fibroblasts were used. Whereas, the oxidative stress arises from an imbalance between oxidants and antioxidants, level of reactive species and lipid peroxidation together with expression of antioxidant response associated genes was measured. At the same time, expression of metabolic and DNA repair related genes was monitored. Although the differences in oxidative stress level or the expression of antioxidative response genes were not detected, the changes in the...
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Nanoparticle-Mediated Delivery System for Gene Therapy
Dvořáková, Nikola ; Ellederová, Zdeňka (advisor) ; Šálek, Petr (referee)
Gene editing with the CRISPR/Cas9 system is one of the options that sets a new trend in the development of gene therapy. The most commonly used delivery of DNA into the cells are via viruses. Nevertheless, they are often unable to take CRISPR/Cas9 system, which can be bigger than several kb. Nanoparticles (NPs), as non-viral transporters, seem to be a good alternative delivery system. For this work magnetic Fe3O4 NPs (MNPs) were selected, because of their excellent properties such as multifunctionality, biocompatibility, easy degradation and simple synthesis. The aim of this work was to synthesise MNPs and a complex of MNPs coated with PEI/CRISPR-Cas9 plasmid and to characterize them by physicochemical methods. The created complex MNPs/PEI/CRISPR-Cas9 was defined by exact parameters that are suitable for possible cell uptake. The hypothesis of stabilization of the MNPs/CRISPR-Cas9 plasmid complex by polyethylenimine (PEI), which can also protect plasmid DNA against restriction endonucleases, was verified. Next a stable modified cell line HEK293-TLR3, designed to evaluate the efficacy of double strand break (DSB) repair by nonhomologous end joining (NHEJ) or homologous recombination (HR) was, transfected with the synthesised MNPs/PEI/CRISPR-Cas9 complex. The results indicate a 25% transfection...
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Phenotypic Analyses of the HD Transgenic Minipig Model (A11609)
Ellederová, Zdeňka
The transgenic Huntington's disease minipigs (TgHD) express N‐terminal part of human mutated huntingtin (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. The model is being used for preclinical huntingtin lowering studies. Here we take different non-invasive and invasive approaches, some of which are unique for large animal models, to study the phenotype development comparing WT and TgHD siblings. We show gradual progression of the disease in these TgHD animals. Moreover, some biomarkers were identified. These markers could serve for monitoring of organism response to HD treatment to assess efficacy and safety in preclinical studies prior to human clinical trials.
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Following the phenotype development of TgHD minipigs by invasive and noninvasive approach
Ellederová, Zdeňka ; Baxa, Monika ; Vidinská, Daniela ; Bohuslavová, Božena ; Vochozková, Petra ; Šmatlíková, Petra ; Klíma, Jiří ; Valeková, Ivona ; Ardan, Taras ; Juhás, Štefan ; Juhásová, Jana ; Konvalinková, R. ; Klempíř, J. ; Pokorný, M. ; Krupička, R. ; Kauler, J. ; Hansíková, H. ; Motlík, Jan
Recent promising treatments for Huntington’s disease (HD) may require pre-clinical testing in large animals. In 2009, we generated HD transgenic (TgHD) minipigs with one copy encoding the N-terminal part (548 aa) of human huntingtin (HTT) with 124 CAG/CAA repeats integrated into chromosome 1 q24-q25. The successful germ line transmission occurred through four successive generations.
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Oxidative stress in primary porcine fibroblasts expressing mutated huntingtin
Šmatlíková, Petra ; Askeland, G. ; Vaškovičová, Michaela ; Klíma, Jiří ; Motlík, Jan ; Eide, L. ; Ellederová, Zdeňka
Molecular events, such as protein aggregation, mitochondrial dysfunction, and transcriptional dysregulation have been linked to Huntington’s disease (HD) pathogenesis. Oxidative stress has been considered as one of the key players in disease progression. Though, it is still not clear whether oxidative stress causes HD, or if it is a consequence of other primary events.
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Mitochondrial phenotype in minipig model transgenic for N-terminal part of human mutated huntingtin
Hansíková, H. ; Rodinová, M. ; Křížová, J. ; Dosoudilová, Z. ; Štufková, H. ; Bohuslavová, Božena ; Klíma, Jiří ; Juhás, Štefan ; Ellederová, Zdeňka ; Motlík, Jan ; Zeman, J.
Huntington’s disease (HD) is neurodegenerative disorder caused by an abnormal expansion of CAG repeat encoding a polyglutamine tract of huntingtin (htt). It has been postulated that mitochondria dysfunction may play significant role in the pathophysiology of the HD. But it is still not known yet in detail how mitochondria are able to cover energy needs of the cells during the progression of the HD.
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