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Testicular Degeneration of Transgenic Porcine Model of Huntington's Disease
Skřivánková, Monika ; Motlík, Jan (advisor) ; Roth, Jan (referee) ; Petr, Jaroslav (referee)
Huntington's disease is an autosomal dominant neurodegenerative disorder caused by an extended (≥36) CAG repeat in the huntingtin gene. Its hallmark is brain athrophy, but huntingtin is widely deposited in all tissues of the body, most notably in the brain and testes. Its pathogenic effect is conditioned by the formation of cytotoxic forms of aggregates and fragments, which occur in both brain and peripheral tissues. Testicular atrophy has been demonstrated in postmortem samples from human patients with Huntington's disease and in transgenic mouse models. We investigated reproductive decline in a large animal model of Huntington's disease. A transgenic (tgHD) minipig model was created by inserting a lentiviral vector into the genome of a pig. Vector contained a truncated form of the N terminal part of huntingtin gene. Boars of this transgenic line showed a reduced ability to produce offspring from 13 months of age. We confirmed apoptosis of seminiferous epithelial cells and Sertoli cells, and a production of morphologically damaged spermatozoa, which were unable to efficiently fertilize the oocyte under in vitro conditions. We found a reduction of mitochondrial metabolism parameters in the sperm of tgHD boars. These changes were not dependent on the age of the boars., It is directly related to the...
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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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Modelovanie ochorenia a štúdium regeneračných procesov v Huntingtonovej chorobe a ALS in vivo
Hruška-Plocháň, Marián
Neurological disorders affect more than 14% of the population worldwide and together with traumatic brain and spinal cord injuries represent major health, public and economic burden of the society. Incidence of inherited and idiopathic neurodegenerative disorders and acute CNS injuries is growing globally while neuroscience society is being challenged by numerous unanswered questions. Therefore, research of the CNS disorders is essential. Since animal models of the CNS diseases and injuries represent the key step in the conversion of the basic research to the clinics, we focused our work on generation of new animal models and on their use in pre-clinical research. We generated and characterized transgenic minipig model of Huntington's disease (HD) which represents the only successful establishment of a transgenic model of HD in minipig which should be valuable for testing of long term safety of HD therapeutics. Next, we crossed the well characterized R6/2 mouse HD model with the gad mouse model which lacks the expression of UCHL1 which led to results that support the theory of "protective" role of mutant huntingtin aggregates and suggest that UCHL1 function(s) may be affected in HD disturbing certain branches of Ubiquitin Proteasome System. Traumatic spinal cord injury and Amyotrophic Lateral...
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The Generation of Transgenic Huntington's Disease Miniature Pig
Baxa, Monika
Huntingtons's disease (HD) is devastating neurodegenerative disorder manifesting by motor disturbances, cognitive decline and personal changes. The huge effort to find a cure for HD has brought several promising therapeutic treatments on the scene. Each of the prospective approaches needs to be investigated for safety, tolerability and efficacy. Mouse and rat models were a lot helpful in examination of pathological mechanisms of HD, but they are not sufficient for completion of pre-clinical testing. Therefore, we aimed to generate transgenic HD minipig to overcome the gap between rodents and humans. Minipig transgenic for the first 548 aminoacids of human mutant huntingtin gene (TgHD) under the control of human HD promotor was manipulated by lentiviral transduction of porcine one-cell stage embryos. Currently, six generations of minipigs expressing single copy of N-truncated human mutant huntingtin protein (mtHtt) with a repetition of 124 glutamines are at disposal. The more the model simulates the disease symptoms the better it is for translational research as the efficacy of the cure can be finer evaluated. Hence, the second aim was to demonstrate HD-like phenotype in our model. Testicular degeneration that preceded the clinical symptoms onset was observed as a consequence of expression of mtHtt....
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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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Trimeric G protein-regulated signaling in neurodegenerative processes
Daňková, Karolína ; Novotný, Jiří (advisor) ; Weissová, Romana (referee)
Members of the large family of G proteins and their coupled receptors are involved in a variety of transduction processes the cell uses to respond to a received signal. Depending on their specific structure and function, they influence a wide range of effector molecules. A large body of research has shown that many neurodegenerative diseases have a negative impact on the signal pathways controlled by G proteins. Due to ageing population, neurodegenerative diseases are currently imposing a risk for growing numbers of people. The sequelae observed in the pathological development of such diseases include especially changes in membrane receptors representation or receptor uncoupling from G protein, which inhibits G subunits activation. The undesirable inhibition or over-stimulation of G proteins results in the increase or decrease in effector activity, which subsequently impacts the production of second messengers and the activity of subsequent members of the signal cascade. As a result, these alterations lead to an increase in intracellular concentration of Ca2+ ions, which then influence receptors responsible for excitotoxicity, and contribute to apoptosis and necrosis of neuronal population. The thesis summarizes the defects of signalling pathways controlled by trimeric G proteins in association...
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The role of proteostatic mechanisms in neurodegenerative diseases
Zezulová, Kristýna ; Vodička, Petr (advisor) ; Marková, Vendula (referee)
Protein homeostasis (proteostasis) plays an important role in maintaining normal cell function and viability. Neurons are particularly vulnerable to proteostasis dysregulation, resulting in damage, dysfunction, and neuronal death, as manifested in many neurodegenerative diseases. One of them is Huntington disease, hereditary neurodegeneration with autosomal dominant inheritance. Expansion of the CAG repeats in the huntingtin gene is translated into an abnormally long glutamine chain in huntingtin protein, leading to disruption of neuronal proteostasis. The primary affected area of the brain is the striatum of the basal ganglia. Disease is progressive, the onset of symptoms usually occurs in adulthood, and after many years leads to the death of the patient. Despite intensive research, disease pathology is still not fully understood, treatment is still only symptomatic and new studies, together with a deeper understanding, also raise many new questions. Through the complexity of the issue, the study of proteostasis in neurodegeneration can bring not only possible implications for therapy, but also could go deeper into the understanding of stress, memory or aging processes.
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The Generation of Transgenic Huntington's Disease Miniature Pig
Baxa, Monika
Huntingtons's disease (HD) is devastating neurodegenerative disorder manifesting by motor disturbances, cognitive decline and personal changes. The huge effort to find a cure for HD has brought several promising therapeutic treatments on the scene. Each of the prospective approaches needs to be investigated for safety, tolerability and efficacy. Mouse and rat models were a lot helpful in examination of pathological mechanisms of HD, but they are not sufficient for completion of pre-clinical testing. Therefore, we aimed to generate transgenic HD minipig to overcome the gap between rodents and humans. Minipig transgenic for the first 548 aminoacids of human mutant huntingtin gene (TgHD) under the control of human HD promotor was manipulated by lentiviral transduction of porcine one-cell stage embryos. Currently, six generations of minipigs expressing single copy of N-truncated human mutant huntingtin protein (mtHtt) with a repetition of 124 glutamines are at disposal. The more the model simulates the disease symptoms the better it is for translational research as the efficacy of the cure can be finer evaluated. Hence, the second aim was to demonstrate HD-like phenotype in our model. Testicular degeneration that preceded the clinical symptoms onset was observed as a consequence of expression of mtHtt....
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The Generation of Transgenic Huntington's Disease Miniature Pig
Baxa, Monika ; Motlík, Jan (advisor) ; Procházka, Jan (referee) ; Petr, Jaroslav (referee)
Huntingtons's disease (HD) is devastating neurodegenerative disorder manifesting by motor disturbances, cognitive decline and personal changes. The huge effort to find a cure for HD has brought several promising therapeutic treatments on the scene. Each of the prospective approaches needs to be investigated for safety, tolerability and efficacy. Mouse and rat models were a lot helpful in examination of pathological mechanisms of HD, but they are not sufficient for completion of pre-clinical testing. Therefore, we aimed to generate transgenic HD minipig to overcome the gap between rodents and humans. Minipig transgenic for the first 548 aminoacids of human mutant huntingtin gene (TgHD) under the control of human HD promotor was manipulated by lentiviral transduction of porcine one-cell stage embryos. Currently, six generations of minipigs expressing single copy of N-truncated human mutant huntingtin protein (mtHtt) with a repetition of 124 glutamines are at disposal. The more the model simulates the disease symptoms the better it is for translational research as the efficacy of the cure can be finer evaluated. Hence, the second aim was to demonstrate HD-like phenotype in our model. Testicular degeneration that preceded the clinical symptoms onset was observed as a consequence of expression of mtHtt....
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