Skye R. Rudiger

An ovine transgenic Huntington's disease model

Huntingtons disease (HD) is an inherited autosomal dominant neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene [Huntingtons Disease Collaborative Research Group (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntingtons disease chromosomes. The Huntingtons Disease Collaborative Research Group. Cell, 72, 971-983]. Despite identification of the gene in 1993, the underlying life-long disease process and effective treatments to prevent or delay it remain elusive. In an effort to fast-track treatment strategies for HD into clinical trials, we have developed a new large-animal HD transgenic ovine model. Sheep, Ovis aries L., were selected because the developmental pattern of the ovine basal ganglia and cortex (the regions primarily affected in HD) is similar to the analogous regions of the human brain. Microinjection of a full-length human HTT cDNA containing 73 polyglutamine repeats under the control of the human promotor resulted in six transgenic founders varying in copy number of the transgene. Analysis of offspring (at 1 and 7 months of age) from one of the founders showed robust expression of the full-length human HTT protein in both CNS and non-CNS tissue. Further, preliminary immunohistochemical analysis demonstrated the organization of the caudate nucleus and putamen and revealed decreased expression of medium size spiny neuron marker DARPP-32 at 7 months of age. It is anticipated that this novel transgenic animal will represent a practical model for drug/clinical trials and surgical interventions especially aimed at delaying or preventing HD initiation. New sequence accession number for ovine HTT mRNA: FJ457100.

Effect of Nutrition of Oocyte Donor on the Outcomes of Somatic Cell Nuclear Transfer in the Sheep

Abstract The purpose of this study was to determine if the nutrition of the oocyte donor ewe influenced the success of somatic cell cloning. Merino ewes were fed at either a high- or a low-nutrition level for 3–5 mo before superovulation treatments. Freshly ovulated oocytes were enucleated and fused with serum-starved adult granulosa cells, and resulting reconstructed embryos were cultured for 6 days in modified synthetic oviduct fluid. Embryo cleavage and development to blastocysts were recorded, and good-quality embryos were transferred to synchronized recipient ewes either fresh or, on a few occasions, after vitrification. Pregnancies were monitored by ultrasonography from Day 40 of pregnancy, and offspring were delivered by either cesarean section or vaginal delivery. No differences occurred in the numbers of follicles aspirated, of oocytes recovered, or of oocytes utilizable for cloning between the high and low groups. Neither were there treatment differences in development to the blastocyst stage. However, transfer of embryos from the high group led to significantly more pregnancies and implanted fetuses. Also, more of the established pregnancies from the high group were carried to term, although this difference was not statistically significant. Lamb mortality was high, with half the live-born perishing soon after birth and more succumbing to various infections within days or weeks of birth, but no clear association between the offspring fate and the treatment group could be established. These results suggest that more research into the effect of nutrition on oocyte quality and its subsequent effect on cloning is warranted.

Further Molecular Characterisation of the OVT73 Transgenic Sheep Model of Huntington's Disease Identifies Cortical Aggregates

BACKGROUND Huntingtons disease is a neurodegenerative disorder, typically with clinical manifestations in adult years, caused by an expanded polyglutamine-coding repeat in HTT. There are no treatments that delay or prevent the onset or progression of this devastating disease. OBJECTIVE AND METHODS In order to study its pre-symptomatic molecular progression and provide a large mammalian model for determining natural history of the disease and for therapeutic testing, we generated and previously reported on lines of transgenic sheep carrying a full length human HTT cDNA transgene, with expression driven by a minimal HTT promoter. We report here further characterization of our preferred line, OVT73. RESULTS This line reliably expresses the expanded human huntingtin protein at modest, but readily detectable levels throughout the brain, including the striatum and cortex. Transmission of the 73 unit glutamine coding repeat was relatively stable over three generations. At the first time-point of a longitudinal study, animals sacrificed at 6 months (7 transgenic, 7 control) showed reduced striatum GABAA α1 receptor, and globus pallidus leu-enkephalin immunoreactivity. Two of three 18 month old animals sacrificed revealed cortical neuropil aggregates. Furthermore, neuronal intranuclear inclusions were identified in the piriform cortex of a single 36 month old animal in addition to cortical neuropil aggregates. CONCLUSIONS Taken together, these data indicate that the OVT73 transgenic sheep line will progressively reveal early HD pathology and allow therapeutic testing over a period of time relevant to human patients.

Metabolic disruption identified in the Huntington’s disease transgenic sheep model

Huntington’s disease (HD) is a dominantly inherited, progressive neurodegenerative disorder caused by a CAG repeat expansion within exon 1 of HTT, encoding huntingtin. There are no therapies that can delay the progression of this devastating disease. One feature of HD that may play a critical role in its pathogenesis is metabolic disruption. Consequently, we undertook a comparative study of metabolites in our transgenic sheep model of HD (OVT73). This model does not display overt symptoms of HD but has circadian rhythm alterations and molecular changes characteristic of the early phase disease. Quantitative metabolite profiles were generated from the motor cortex, hippocampus, cerebellum and liver tissue of 5 year old transgenic sheep and matched controls by gas chromatography-mass spectrometry. Differentially abundant metabolites were evident in the cerebellum and liver. There was striking tissue-specificity, with predominantly amino acids affected in the transgenic cerebellum and fatty acids in the transgenic liver, which together may indicate a hyper-metabolic state. Furthermore, there were more strong pair-wise correlations of metabolite abundance in transgenic than in wild-type cerebellum and liver, suggesting altered metabolic constraints. Together these differences indicate a metabolic disruption in the sheep model of HD and could provide insight into the presymptomatic human disease.

Data-loggers carried on a harness do not adversely affect sheep locomotion.

The use of externally fitted motion sensors to animal subjects has the potential for allowing researchers to investigate subtle changes in animal movement that may occur with the onset of specific diseases. However, it is crucial to consider whether or not the use of such technology has an effect on the variables measured. Here, we examine the effect of a body harness data logging device on the locomotive patterns of female Merino sheep, Ovis aries. We extracted locomotion variables typical of motion sensor data (stride frequency, stride length, gait type, speed, and limb velocity) from high-definition video collected under controlled conditions. We found no significant difference between the variables measured in the harnessed and unharnessed conditions. Overall, our experiment demonstrates that data-loggers carried on a harness do not adversely affect sheep locomotion, and extended periods of habituation post-instalment of devices should ensure consistency and accuracy of data in future experiments.

Rapid and Progressive Regional Brain Atrophy in CLN6 Batten Disease Affected Sheep Measured with Longitudinal Magnetic Resonance Imaging.

Variant late-infantile Batten disease is a neuronal ceroid lipofuscinosis caused by mutations in CLN6. It is a recessive genetic lysosomal storage disease characterised by progressive neurodegeneration. It starts insidiously and leads to blindness, epilepsy and dementia in affected children. Sheep that are homozygous for a natural mutation in CLN6 have an ovine form of Batten disease Here, we used in vivo magnetic resonance imaging to track brain changes in 4 unaffected carriers and 6 affected Batten disease sheep. We scanned each sheep 4 times, between 17 and 22 months of age. Cortical atrophy in all sheep was pronounced at the baseline scan in all affected Batten disease sheep. Significant atrophy was also present in other brain regions (caudate, putamen and amygdala). Atrophy continued measurably in all of these regions during the study. Longitudinal MRI in sheep was sensitive enough to measure significant volume changes over the relatively short study period, even in the cortex, where nearly 40% of volume was already lost at the start of the study. Thus longitudinal MRI could be used to study the dynamics of progression of neurodegenerative changes in sheep models of Batten disease, as well as to assess therapeutic efficacy.

Alzheimer's disease markers in the aged sheep (Ovis aries)

This study reports the identification and characterization of markers of Alzheimers disease (AD) in aged sheep (Ovis aries) as a preliminary step toward making a genetically modified large animal model of AD. Importantly, the sequences of key proteins involved in AD pathogenesis are highly conserved between sheep and human. The processing of the amyloid-β (Aβ) protein is conserved between sheep and human, and sheep Aβ1-42/Aβ1-40 ratios in cerebrospinal fluid (CSF) are also very similar to human. In addition, total tau and neurofilament light levels in CSF are comparable with those found in human. The presence of neurofibrillary tangles in aged sheep brain has previously been established; here, we report for the first time that plaques, the other pathologic hallmark of AD, are also present in the aged sheep brain. In summary, the biological machinery to generate the key neuropathologic features of AD is conserved between the human and sheep, making the sheep a good candidate for future genetic manipulation to accelerate the condition for use in pathophysiological discovery and therapeutic testing.

Brain urea increase is an early Huntington’s disease pathogenic event observed in a prodromal transgenic sheep model and HD cases

Significance We present evidence for the presymptomatic dysregulation of urea metabolism in Huntington’s disease (HD). We identified increased levels of a urea transporter transcript and other osmotic regulators in the striatum of our prodromal sheep model of HD and a concomitant increase in striatal and cerebellar urea. Elevated urea was also detected in brain tissue from postmortem HD cases, including cases with low-level cell loss, implying that increased brain urea in HD is not just a product of end-stage cachexia. Disruption of urea metabolism is known to cause neurologic impairment and could initiate neurodegeneration and the symptoms of HD. Our findings suggest that lowering brain levels of urea and/or ammonia would be a worthwhile therapeutic target in HD. The neurodegenerative disorder Huntington’s disease (HD) is typically characterized by extensive loss of striatal neurons and the midlife onset of debilitating and progressive chorea, dementia, and psychological disturbance. HD is caused by a CAG repeat expansion in the Huntingtin (HTT) gene, translating to an elongated glutamine tract in the huntingtin protein. The pathogenic mechanism resulting in cell dysfunction and death beyond the causative mutation is not well defined. To further delineate the early molecular events in HD, we performed RNA-sequencing (RNA-seq) on striatal tissue from a cohort of 5-y-old OVT73-line sheep expressing a human CAG-expansion HTT cDNA transgene. Our HD OVT73 sheep are a prodromal model and exhibit minimal pathology and no detectable neuronal loss. We identified significantly increased levels of the urea transporter SLC14A1 in the OVT73 striatum, along with other important osmotic regulators. Further investigation revealed elevated levels of the metabolite urea in the OVT73 striatum and cerebellum, consistent with our recently published observation of increased urea in postmortem human brain from HD cases. Extending that finding, we demonstrate that postmortem human brain urea levels are elevated in a larger cohort of HD cases, including those with low-level neuropathology (Vonsattel grade 0/1). This elevation indicates increased protein catabolism, possibly as an alternate energy source given the generalized metabolic defect in HD. Increased urea and ammonia levels due to dysregulation of the urea cycle are known to cause neurologic impairment. Taken together, our findings indicate that aberrant urea metabolism could be the primary biochemical disruption initiating neuropathogenesis in HD.