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Porcine models for Huntington disease
Růna Vochozková, Petra ; Motlík, Jan (advisor) ; Bohačiaková, Dáša (referee) ; Fulková, Helena (referee)
The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...
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Mitophagy in Huntington's Disease
Šonský, Ivan ; Hansíková, Hana (advisor) ; Macůrková, Marie (referee)
Mitochondrial dysfunctions contribute to the progression of many neurodegenerative diseases, including Huntington's disease (HD). In HD, mutation in the huntingtin gene (HTT) results in the expansion of CAG repeats, causing the growth of the polyglutamine tract. This growth is responsible for the gain of toxicity function of the protein. The turnover of dysfunctional and damaged mitochondria is mediated via mitophagy - a selective form of autophagy. Additionally, mitophagy impairments have recently been described to play a key role not only in neurodegenerative diseases. The protrusion of mitophagy results in the clustering of defective mitochondria, organelles which are responsible for fulfilling the energetic demands of neural cells. The most distinctive impact of the impairment is on the striatal medium spiny neurons and results in the development of motor and cognitive dysfunctions. This thesis describes how HD affects mitophagy and reveals the biggest obstacle of mitophagy - disruption of mitochondria targeting into emerging autophagosomes caused by the abnormal interaction of mHTT and p62. Induction of mitophagy at this stage could be crucial for the future therapeutic research of HD. Generally, initiation of mitophagy could become a relevant therapeutic target for many other...
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Gradual Molecular Changes in Primary Porcine Cells Expressing Mutated Huntingtin
Šmatlíková, Petra ; Motlík, Jan (advisor) ; Trejbalová, Kateřina (referee) ; Reiniš, Milan (referee)
Huntington's disease (HD) is inherited fatal disorder caused by CAG triplet expansions in the huntingtin gene resulting in the expression of mutated huntingtin protein (mtHtt). The main symptoms of HD are neurodegeneration, osteoporosis, testicular degeneration, loss of muscle tissue and heart muscle malfunction, weight loss, metabolic changes, and sleeping disturbances. Since huntingtin protein (Htt) has a role in several biological processes, many molecular mechanisms, like oxidative stress, mitochondrial dysfunction, DNA-damage, and others, are affected by mtHtt. However, its exact pathogenic mechanisms in HD are still not well understood. Transgenic minipig model of HD (TgHD) serves an opportunity to isolate unlimited number of primary cells and unlike primary cells obtained from HD patients, often in the late stages of the disease, the TgHD minipig model allows to monitor molecular changes occurring gradually with age and progression of the disease. Thus, TgHD minipig model and primary cells isolated from it play an important role in investigating and understanding the underlying mechanistic cause of HD. We focused on molecular and cellular changes in primary cells isolated from TgHD minipigs and their wild type (WT) controls at different ages (24, 36, and 48 months). In mesenchymal stem cells...
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EEG correlates of neurodegenerative disorders
Schlezingerová, Nicol ; Telenský, Petr (advisor) ; Kelemen, Eduard (referee)
Due to the aging of the population, there is an increasing incidence of neurodegenerative diseases. In clinical practice there is a need to for a cheap and noninvasive method for screening and early diagnosis of neurodegenerative disorders. To this end, markers of disease progression and prognosis must be determined. EEG correlates provide information that can be used in the diagnosis and prognosis of neurodegenerative disorders. Individual diseases have their specific EEG abnormalities that are closely related to different stages of the disease. Individual illnesses - Alzheimer's disease, Parkinson's disease, Huntington's disease have their specific changes in the basic rhythms of the brain that correlate with motor and cognitive changes. This work focuses on the quantitative (qEEG) correlates of the above-mentioned diseases. Key words: brain, neural activity, EEG, quantitative EEG analysis, biomarker, connectivity, neurodegeneration, Alzheimer's disease, Parkinson's disease, Huntington's disease.
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Problematic Areas in the Everyday Life of Patients with Huntington's Disease. Subtitle: A Suggestion of Compensatory Strategies in Coping with Cognitive Impairment
Sýkorová, Jitka ; Zemánková, Zuzana (advisor) ; Sládková, Petra (referee)
This diploma thesis explores problematic areas of patients with Huntington's disease in their performance during activities of daily living (ADLs) from the perspective of patients and their caregivers. The aim of the research was also to assess a possible correlation between cognitive impairment and the patient's performance in ADL. Twenty-five patients with their caregivers met the selection criteria for the research. There were used standardized assessment methods available in Czech: the Montreal Cognitive Assessment (MoCA), the Canadian Occupational Performance Measure (COPM) and the questionnaire for caregivers called Bristol Activities of Daily Living Scale (BADLS-CZ). The statistical analyses consisted of methods of the nonparametric statistics, qualitative analysis was processed by data categorizing. Caregivers reported more problematic areas in ADLs which was significantly confirmed in the statistical hypothesis testing (p <0,05). A significant correlation was seen between the results of the questionnaire and the results of the MoCA assessment (rSp = -0,620; p <0,05). For various reasons, patients with Huntington's disease did not mention as many problematic areas in performing ADL as their caregivers. Therefore, it is appropriate in clinical practice to supplement the assessment of the patient's...
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Characterization of immune cells and monitoring changes of inflammatory proteins in minipig model of Huntington's disease
Butalová, Nikola ; Motlík, Jan (advisor) ; Janda, Jozef (referee)
The Huntington disease (HD) is a hereditary neuro-degenerative disorder caused by a mutation of the huntigtin gene that codes a protein of the same name. The mutated form of the huntigtin gene plays its part in many pathological interactions and influences a number of cellular mechanisms, including the immune system that could serve as a modifier of the neuropathology of the disease. The cells of the monocyte-macrophage system express cytokines whose production changes in relation to the activation of the cell. The presence of the mutated huntingtin protein in these cells renders them hyper-responsive to immunity incentives leading to changes in the production of cytokines. These differences are discernible a few years prior to the appearance of the symptoms. Therefore, the changes in the levels of certain cytokines could serve as appropriate biomarkers for monitoring of the onset of the disease and its progression. The HD pathogenesis includes an inflammation of the central neutral system. Inflammatory changes in peripheral tissues could reflect inflammatory processes in the central neural system. A miniature TgHD pig could represent an appropriate model organism for studying of the impact of the mHtt on the immune system. This model enables to observe a slow progression of the disease. Changes in...
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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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Investigation of proteolytic enzymes expression in different tissues at the transgenic animal model of Huntington disease by means of biochemical and immunohistochemical methods
Kocurová, Gabriela ; Dršata, Jaroslav (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Gabriela Kocurová Supervisor: Prof. MUDr. Jaroslav Dršata, CSc. Title of diploma thesis: Investigation of proteolytic enzymes expression in different tissues at the transgenic animal model of Huntington's disease by means of biochemical and immunohistochemical methods Background: Huntington's disease (HD) is a neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the huntingtin (Htt) protein. Because it is known that mutant Htt and especially its small proteolytic fragments are toxic to neurons (particularly those in the striatum and cortex), it has been suggested that proteolysis of mutant huntingtin (mHtt) might play an important role in HD pathogenesis. Therefore, the aim of the present study was to examine the expression of endogenous and mtHtt and possible participation of the proteolytic enzymes from the group of caspases, matrix metalloproteinases (MMPs), kallikreins (KLKs) and calpains in HD pathology of brain tissue. Methods: In this study we used WT and TgHD minipigs for N-terminal part of the human mtHtt (548aaHTT-145Q, both F2 generation, age 36 months; F3 generation, age 48 months in additional experiment), R6/2 mice were used as...
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Protein analysis of selected mitochondrial proteins in the muscle tissue of porcine model of Huntington's disease
Dosoudilová, Žaneta ; Klempíř, Jiří (advisor) ; Baxa, Monika (referee)
Huntington's disease (HD) is an autosomal dominant hereditary neurodegenerative disease characterized by motor, cognitive and behavioral disorders. HD is caused by expansion of CAG triplet (cytosine-adenosine-guanine) located in a gene on the short arm of the fourth chromosome. This expansion encodes an aberrant polyglutamine chain in the protein huntingtin. Physiological and mutated huntingtin (in case of HD) are expressed in almost all tissues and influences many cellular functions. The prevalence of HD in population is about 1 per 10.000. The disease is currently incurable and its mechanisms are not sufficiently understood. Besides affecting the central nervous system HD also affects peripheral tissues, including skeletal muscles. HD disrupts mitochondrial function and damages oxidative phosphorylation system, which has the task of producing energy in the form of ATP in cells. Research of transgenic minipig model for HD could help elucidate the mechanisms of disease's pathogenesis and potential therapeutic strategy. In this diploma thesis, immunodetection with help of specific antibodies to detect changes in amount of 14 selected mitochondrial proteins in skeletal muscle tissue of three age groups of transgenic HD minipigs - 24, 36 and 48 months old was used. Gradual progression in reduced...
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