Background: Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. Objective: We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. Methods: In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. Results: Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. Conclusions: The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt.
Background: Huntington disease (HD) is a fatal neurodegenerative disorder involving reduced muscle coordination, mental and behavioral changes, and testicular degeneration. In order to further clarify the decreased fertility and penetration ability of the spermatozoa of transgenic HD minipig boars (TgHD), we applied a set of mitochondrial metabolism (MM) parameter measurements to this promising biological material, which can be collected noninvasively in longitudinal studies. Objective: We aimed to optimize methods for MM measurements in spermatozoa and to establish possible biomarkers of HD in TgHD spermatozoa expressing the N-terminal part of mutated human huntingtin. Methods: Semen samples from 12 TgHD and wild-type animals, aged 12-65 months, were obtained repeatedly during the study. Respiration was measured by polarography, MM was assessed by the detection of oxidation of radiolabeled substrates (mitochondrial energy-generating system; MEGS), and the content of the oxidative phosphorylation system subunits was detected by Western blot. Three possibly interfering factors were statistically analyzed: the effect of HD, generation and aging. Results: We found 5 MM parameters which were significantly diminished in TgHD spermatozoa and propose 3 specific MEGS incubations and complex I-dependent respiration as potential biomarkers of HD in TgHD spermatozoa. Conclusions: Our results suggest a link between the gain of toxic function of mutated huntingtin in TgHD spermatozoa and the observed MM and/or glycolytic impairment. We determined 4 biomarkers useful for HD phenotyping and experimental therapy monitoring studies in TgHD minipigs.
The transcription factors Cat8 and Sip4 were described in Saccharomyces cerevisiae and Kluyveromyces lactis to have very similar DNA binding domains and to be necessary for derepression of a variety of genes under non-fermentative growth conditions via binding to the carbon source responsive elements (CSREs). The methylotrophic yeast Komagataella phaffii (syn Pichia pastoris) has two transcription factors (TFs), which are putative homologs of Cat8 based on sequence similarity, termed Cat8-1 and Cat8-2. It is yet unclear in which cellular processes they are involved and if one of them is actually the homolog of Sip4. To study the roles of the Cat8 homologs in K. phaffii, overexpression or deletion strains were generated for the two TFs. The ability of these mutant strains to grow on different carbon sources was tested, and transcript levels of selected genes from the carbon metabolism were quantified. Our experiments showed that the TFs are required for the growth of K. phaffii on C2 carbon sources, but not on glucose, glycerol or methanol. K. phaffii deleted for Cat8-1 showed impaired growth on acetate, while both Cat8-1 and Cat8-2 are involved in the growth of K. phaffii on ethanol. Correspondingly, both TFs are participating in the activation of ADH2, ALD4 and ACS1, three genes encoding enzymes important for the assimilation of ethanol. Different from S. cerevisiae and K. lactis, Cat8-1 is not regulating the transcription of the putative Sip4-family member Cat8-2 in K. phaffii. Furthermore, Cat8-1 is necessary for the activation of genes from the glyoxylate cycle, whereas Cat8-2 is necessary for the activation of genes from the carnitine shuttle. Neither Cat8-1 nor Cat8-2 are required for the activation of gluconeogenesis genes. Finally, the CAT8-2 gene is repressed by the Mig1-2 transcription factor on glucose and autorepressed by the Cat8-2 protein on all tested carbon sources. Our study identified the involvement of K. phaffii Cat8-1 and Cat8-2 in C2-metabolism, and highlighted similarities and differences to their homologs in other yeast species.
At present, we are probably the only research facility to be breeding transgenic Huntington's disease minipigs (TgHD). These minipigs express N-terminal part of human mutated huntingtin including 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. We take different approaches, some of which are unique for large animal models, to study the phenotype development comparing WT and TgHD siblings. In this chapter, we review these approaches and the phenotype progression in the minipigs. Additionally, we outline perspectives in generation of new models using novel methodology and the potential of pig models in preclinical HD studies.
Testicular degeneration of Huntington's disease involves the reduction of germ cells and wall thickening of seminiferous tubule. Since changes in ejaculates of patients and mammalians models has not yet been tested, the aim of our study was to analyse motility, amount, fertilising capacity and mitochondrial function of spermatozoa from two HD minipig boars from F1 generation and two WT control animals from 12 to 24 month of age. Random analysis of HD boars from F2 generation at the age of 8 to 12 month was also performed. After semen collections of transgenic and control boars, spermatozoa were prepared for in vitro penetration test. The HD F1 boars showed significantly decreasing tendency in motility, total sperm number and fertilising capacity staring at about 13 month of age in comparison with control boars. The slight depression in sperm quality and quantity was observed in two of four tested HD F2 boars. Evaluating the sperm quantity, motility and fertilising capacity, we can conclude the age related depression in all observed parameters in HD F1 transgenic boars. Mitochondrial energy generating system capacity was analysed in homogenates of minipig boar spermatozoa by measuring oxidation rates and oxygen consumption was determined by high resolution polarography. Decreased oxidation rates were found in spermatozoa of the transgenic F1 minipig boars at the age of 18, 21 and 24 months in comparison with controls. Oxidation rate of succinate and decreased oxygen consumption after addition of succinate indicate impaired function of complex II. Our results demonstrate disturbed bioenergetic function of mitochondria in spermatozoa of transgenic boars.FundingSupported by PRVOUK P24/LF1/3 and TAČR Alfa: TA0101146.
BackgroundHuntington’s disease (HD) is characterised by involuntary chorea like movements, poor balance, slurred speech, difficulty swallowing, personality change and difficulty thinking manifesting in the middle age of the patients. Changes in behaviour, motor skills and learning correlate with progression of the disease. Thus it is necessary to monitor these changes in new animal models. In 2009, we have generated transgenic minipigs (TgHD) expressing N-terminal (548 aa) part of human huntingtin (Htt) containing 126 CAG/CAA repeats. AimsTo follow disease development in TgHD minipigs of F0, F1 and F3 generation using motoric and behavioural tests. MethodsIn order to follow behavioural and motoric changes we introduced stability pull back test, balance bean, seesaw test, up and down step test, and tunnel test. We also initiated to use accelerometers to monitor walking differences. ResultsWe observed higher fear or inability to perform certain tests, delayed learning period mainly in situations inducing stress in our oldest TgHD animals (F0, F1 generations). The significant difference between WT and TgHD minipigs was noticed in the ability to step on and to cross the elevated balance beam. Pull back stability test showed a significant difference in TgHD animals. Wobbly movements of back legs were observed in the five years old animals. The weight of all the oldest animals dropped in 10% in the last year. ConclusionsThe minipig model starts to show a motoric impairment phenotype, and thus to be a promising model for preclinical testing of new therapeutic approaches of HD. Acknowledgement“The research leading to these results has received funding from the Norwegian Financial Mechanism 2009–2014 and the Ministry of Education, Youth and Sports Project under Contract no. MSMT-28477/2014” (7F14308/2014), and National Sustainability Programme, project number LO1609 (Czech Ministry of Education, Youth and Sports) and RVO: 67985904
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