Phenotypic study Huntington's disease TgHD minipigs: Appearance and progress of reproductive and biochemical changes

Huntington's disease (HD) is one of the incurable and fatal diseases. HD belongs to the monogenic neurodegenerative diseases. According to the number of the CAG repetitions in the gene coding huntingtin, the onset of the disease is in childhood (5%), in the middle age, which is the most common (90%) and in the older age (5%). Beginning of the disease is manifested by changes in behavior; including problems with coordination and movement. Later, there is a psychological change. The disease leads to death. Until now, there is no effective curative treatment. In 2009, we created a model of the transgenic minipigs (TgHD) carrying the N - terminal part of the human mutant huntingtin (mtHtt) at our Institute in Liběchov. The number of offsprings and the resemblance in physiology and morphology between the pig (Sus scrofa) and humans (Homo sapiens) give us opportunities not only to study changes not only in central nerve organs, but also in peripheral tissues. The reproductive problems of TgHD boars were observed as the first phenotypic changes. Therefore, my work focuses at first on a study of the reproduction parameters of TgHD boars as well as ultrastructural, immunocytochemical and biochemical changes in testes and spermatozoa. In PhD thesis, I described in details the reproductive defects in TgHD..

Fenotypová studie Huntingtonové choroby TgHD miiniprasat: nástup a průběh reprodukčních a biochemických změn

Huntingtonova choroba (HCH) patrí k nevyliečiteľným ochoreniam s fatálnym koncom. Zaraďujeme ju k monogénnym neurodegeneratívnym ochoreniam. Podľa počtu opakovaní CAG repetícii v géne kódujúcom huntingtín, nástup ochorenia môže byť v detskom veku (5%), strednom veku - najčastejšie (90%) a vyššom veku (5%). Začiatok choroby sa prejaví zmenami v správaní, ku ktorým sa pridružia problémy s koordináciou a pohybom. Neskôr dochádza k psychickým zmenám. Ochorenie končí smrťou. V dnešnej dobe zatiaľ nie je vyvinutá účinná liečba. Doktori momentálne len príznaky farmakologicky utlmujú, ale nelieči sa príčina choroby. Na našom ústave v Liběchove sme v roku 2009 vytvorili model transgénneho miniprasaťa (TgHD) nesúceho N - terminálnu časť ľudského mutovaného huntingtínu (mtHtt). Dostatok potomkov a veľká príbuznosť fyziológie a morfológie medzi prasaťom (Sus scrofa) a človekom (Homo sapiens) nám dáva dostatok možnosti na študovanie zmien v centrálnych nervových orgánoch, ale i v periférnych tkanivách. Ako prvé zmeny fenotypu sme pozorovali reprodukčné problémy TgHD kancov. Preto sa moja práca v prvom rade zamerala na komplexné preštudovanie reprodukčných parametrov TgHD kancov, rovnako ako na ultraštrukturálne, imunocytochemické a biochemické zmeny v tkanivách semenníkov a v spermiách. V tejto práci som...Huntington's disease (HD) is one of the incurable and fatal diseases. HD belongs to the monogenic neurodegenerative diseases. According to the number of the CAG repetitions in the gene coding huntingtin, the onset of the disease is in childhood (5%), in the middle age, which is the most common (90%) and in the older age (5%). Beginning of the disease is manifested by changes in behavior; including problems with coordination and movement. Later, there is a psychological change. The disease leads to death. Until now, there is no effective curative treatment. In 2009, we created a model of the transgenic minipigs (TgHD) carrying the N - terminal part of the human mutant huntingtin (mtHtt) at our Institute in Liběchov. The number of offsprings and the resemblance in physiology and morphology between the pig (Sus scrofa) and humans (Homo sapiens) give us opportunities not only to study changes not only in central nerve organs, but also in peripheral tissues. The reproductive problems of TgHD boars were observed as the first phenotypic changes. Therefore, my work focuses at first on a study of the reproduction parameters of TgHD boars as well as ultrastructural, immunocytochemical and biochemical changes in testes and spermatozoa. In PhD thesis, I described in details the reproductive defects in TgHD...Department of Cell BiologyKatedra buněčné biologieFaculty of SciencePřírodovědecká fakult

Evaluation of efficacy and bio-distribution of AAV5-miHTT in HD minipig brain

Healthy minipigs (n=29) were pre-screened for serum AAV5 neutralizing antibodies (NABs) in serum by uniQure. Conclusions: Intrastriatal/intrathalamic bilateral application of AAV5-CAG-miHtt(3e13/1e13gctotal) or PBS/Sucrose (54μl/216μl) similarly to AA5-CAG-GFP didn´t cause any neurological deficit in transgenic as well as wild type animals. The body weight increased in all animals 84 days after AAV5-CAG-miHtt/PBS-Sucroseintrastriatal/intrathalamic delivery with two exceptions TGanimal L616 (AAV5-CAG-miHtt1e 13g ctotal) and WTanimal L485 (AAV5-CAG-miHtt3e13gc total). The TG boar N176 from group VII (PBS/Sucrose) died during narcosis 56 days after IP/IT application), shortly after CSF and blood collection.The boar breathed heavily and intermittently, then the heart stopped beat and resuscitation failed. We immediately flushed them by 20 L of PBS with heparin and collect all tissues. All animals had normal food consumption. Inflammatory proces in AAV5-CAG-miHtt (1e13 gc total) seems to be similar to PBS/Sucrose injected animals accompanied with no detectable histological pathologies

Deterioration of mitochondrial bioenergetics and ultrastructure impairment in skeletal muscle of a transgenic minipig model in the early stages of Huntington's disease

Skeletal muscle wasting and atrophy is one of the more severe clinical impairments resulting from the progression of Huntington's disease (HD). Mitochondrial dysfunction may play a significant role in the etiology of HD, but the specific condition of mitochondria in muscle has not been widely studied during the development of HD. To determine the role of mitochondria in skeletal muscle during the early stages of HD, we analyzed quadriceps femoris muscle from 24-, 36-, 48- and 66-month-old transgenic minipigs that expressed the N-terminal portion of mutated human huntingtin protein (TgHD) and age-matched wild-type (WT) siblings. We found altered ultrastructure of TgHD muscle tissue and mitochondria. There was also significant reduction of activity of citrate synthase and respiratory chain complexes (RCCs) I, II and IV, decreased quantity of oligomycin-sensitivity conferring protein (OSCP) and the E2 subunit of pyruvate dehydrogenase (PDHE2), and differential expression of optic atrophy 1 protein (OPA1) and dynamin-related protein 1 (DRP1) in the skeletal muscle of TgHD minipigs. Statistical analysis identified several parameters that were dependent only on HD status and could therefore be used as potential biomarkers of disease progression. In particular, the reduction of biomarker RCCII subunit SDH30 quantity suggests that similar pathogenic mechanisms underlie disease progression in TgHD minipigs and HD patients. The perturbed biochemical phenotype was detectable in TgHD minipigs prior to the development of ultrastructural changes and locomotor impairment, which become evident at the age of 48 months. Mitochondrial disturbances may contribute to energetic depression in skeletal muscle in HD, which is in concordance with the mobility problems observed in this model