RESUMO
Huntington's disease (HD) is a neurodegenerative genetic disorder caused by an expansion in the CAG repeat tract of the huntingtin (HTT) gene resulting in behavioural, cognitive, and motor defects. Current knowledge of disease pathogenesis remains incomplete, and no disease course-modifying interventions are in clinical use. We have previously reported the development and characterisation of the OVT73 transgenic sheep model of HD. The 73 polyglutamine repeat is somatically stable and therefore likely captures a prodromal phase of the disease with an absence of motor symptomatology even at 5-years of age and no detectable striatal cell loss. To better understand the disease-initiating events we have undertaken a single nuclei transcriptome study of the striatum of an extensively studied cohort of 5-year-old OVT73 HD sheep and age matched wild-type controls. We have identified transcriptional upregulation of genes encoding N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in medium spiny neurons, the cell type preferentially lost early in HD. Further, we observed an upregulation of astrocytic glutamate uptake transporters and medium spiny neuron GABAA receptors, which may maintain glutamate homeostasis. Taken together, these observations support the glutamate excitotoxicity hypothesis as an early neurodegeneration cascade-initiating process but the threshold of toxicity may be regulated by several protective mechanisms. Addressing this biochemical defect early may prevent neuronal loss and avoid the more complex secondary consequences precipitated by cell death.
Assuntos
Modelos Animais de Doenças , Ácido Glutâmico , Doença de Huntington , Neurônios , Receptores de N-Metil-D-Aspartato , Animais , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Ovinos , Neurônios/metabolismo , Neurônios/patologia , Ácido Glutâmico/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , RNA-Seq , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Morte Celular/genética , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Animais Geneticamente Modificados , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Humanos , Transcriptoma/genética , Receptores de Ácido Caínico/genética , Receptores de Ácido Caínico/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/genética , Neurônios Espinhosos MédiosRESUMO
Melatonin is a pleiotrophic hormone, synthesised primarily by the pineal gland under the control of the suprachiasmatic nuclei (SCN). It not only provides a hormonal signal of darkness but also has neuroprotective properties. Huntington's disease (HD) is a progressive neurodegenerative disorder characterised by abnormal motor, cognitive and psychiatric symptoms. There is growing evidence, particularly from animal models, that circadian rhythms may also be disturbed in HD. We measured two circadian-regulated hormones, melatonin and cortisol, in plasma samples collected around-the-clock from normal and presymptomatic transgenic HD sheep (Ovis aries) at 5 and 7 years of age, to assess SCN-driven rhythms and the effect of genotype, sex and age. Melatonin-related precursors and metabolites (tryptophan, serotonin, kynurenine) were also measured by liquid chromatography (LC)-mass spectrometry (MS). At 5 years of age in both rams and ewes, plasma melatonin levels were significantly elevated in HD sheep. In ewes measured 2 years later, there was still a significant elevation of nocturnal melatonin. Furthermore, the daytime baseline levels of melatonin were significantly higher in HD sheep. Since increased melatonin could have global beneficial effects on brain function, we suggest that the increased melatonin measured in presymptomatic HD sheep is part of an autoprotective response to mutant huntingtin toxicity that may account, at least in part, for the late onset of disease that characterises HD.
Assuntos
Ritmo Circadiano , Doença de Huntington/sangue , Melatonina/sangue , Neuroproteção , Ovinos/sangue , Animais , Modelos Animais de Doenças , Feminino , Humanos , MasculinoRESUMO
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.
Assuntos
Corpo Estriado/metabolismo , Doença de Huntington/metabolismo , Ureia/metabolismo , Adulto , Animais , Animais Geneticamente Modificados , Corpo Estriado/patologia , Modelos Animais de Doenças , Feminino , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/patologia , Masculino , Ovinos , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Insidious changes in behaviour herald the onset of progressive neurodegenerative disorders such as Huntington's disease (HD), sometimes years before overt symptoms are seen. Sleep and circadian disturbances are particularly disruptive symptoms in patients with neurological disorders, but they are difficult to measure in humans. Here we studied circadian behaviour in transgenic HD sheep expressing the full-length human huntingtin protein with an expanded CAG repeat mutation in the juvenile range. Young HD sheep with no other symptoms exhibited circadian behavioural abnormalities that worsened with age. The most obvious change was a disturbed evening behaviour reminiscent of 'sundowning' that is seen in some patients with dementia. There were no structural abnormalities seen with magnetic resonance imaging, even in 5-year-old HD sheep. Interestingly, detection of the circadian abnormalities depended upon their social grouping. Abnormalities emerged in sheep kept in an 'HD-only' flock, whereas the behaviour of HD sheep kept mixed with normal sheep was relatively normal. Sleep-wake abnormalities in HD patients are also likely to be hidden, and may precede overt symptoms by many years. Sleep disruption has deleterious effects, even in normal people. The knock-on effects of sleep-wake disturbance may exacerbate, or even cause symptoms such as irritability and depression that are common in early stage HD patients. HD sheep will be useful models for probing the mechanisms underlying circadian behavioural disorder in HD.
Assuntos
Ritmo Circadiano/fisiologia , Doença de Huntington/fisiopatologia , Meio Social , Animais , OvinosRESUMO
Somatic instability of the huntingtin (HTT) CAG repeat mutation modifies age-at-onset of Huntington's disease (HD). Understanding the mechanism and pathogenic consequences of instability may reveal therapeutic targets. Using small-pool PCR we analyzed CAG instability in the OVT73 sheep model which expresses a full-length human cDNA HTT transgene. Analyses of five- and ten-year old sheep revealed the transgene (CAG)69 repeat was remarkably stable in liver, striatum, and other brain tissues. As OVT73 sheep at ten years old have minimal cell death and behavioral changes, our findings support instability of the HTT expanded-CAG repeat as being required for the progression of HD.
Assuntos
Doença de Huntington , Animais , Ovinos/genética , Humanos , Criança , Pré-Escolar , Doença de Huntington/metabolismo , Corpo Estriado/metabolismo , Neostriado/metabolismo , Mutação , Idade de Início , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , Modelos Animais de DoençasRESUMO
Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene [Huntington's Disease Collaborative Research Group (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's 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.
Assuntos
Animais Geneticamente Modificados/genética , Modelos Animais de Doenças , Doença de Huntington/genética , Ovinos/genética , Animais , Gânglios da Base/metabolismo , Gânglios da Base/patologia , Cromossomos de Mamíferos/genética , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Feminino , Efeito Fundador , Humanos , Proteína Huntingtina , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Linhagem , Receptor CB1 de Canabinoide/metabolismo , Transgenes/genéticaRESUMO
Nutrition during the periconceptional period influences postnatal cardiovascular health. We determined whether in vitro embryo culture and transfer, which are manipulations of the nutritional environment during the periconceptional period, dysregulate postnatal blood pressure and blood pressure regulatory mechanisms. Embryos were either transferred to an intermediate recipient ewe (ET) or cultured in vitro in the absence (IVC) or presence of human serum (IVCHS) and a methyl donor (IVCHS+M) for 6 days. Basal blood pressure was recorded at 19-20 weeks after birth. Mean arterial pressure (MAP) and heart rate (HR) were measured before and after varying doses of phenylephrine (PE). mRNA expression of signaling molecules involved in blood pressure regulation was measured in the renal artery. Basal MAP did not differ between groups. Baroreflex sensitivity, set point, and upper plateau were also maintained in all groups after PE stimulation. Adrenergic receptors alpha-1A (αAR1A), alpha-1B (αAR1B), and angiotensin II receptor type 1 (AT1R) mRNA expression were not different from controls in the renal artery. These results suggest there is no programmed effect of ET or IVC on basal blood pressure or the baroreflex control mechanisms in adolescence, but future studies are required to determine the impact of ET and IVC on these mechanisms later in the life course when developmental programming effects may be unmasked by age.
Assuntos
Pressão Sanguínea/fisiologia , Técnicas de Cultura Embrionária/métodos , Ovinos/fisiologia , Animais , Modelos Animais de Doenças , Técnicas de Cultura Embrionária/estatística & dados numéricos , Ovinos/metabolismoRESUMO
BACKGROUND: The pathological mechanism of cellular dysfunction and death in Huntington's disease (HD) is not well defined. Our transgenic HD sheep model (OVT73) was generated to investigate these mechanisms and for therapeutic testing. One particular cohort of animals has undergone focused investigation resulting in a large interrelated multi-omic dataset, with statistically significant changes observed comparing OVT73 and control 'omic' profiles and reported in literature. OBJECTIVE: Here we make this dataset publicly available for the advancement of HD pathogenic mechanism discovery. METHODS: To enable investigation in a user-friendly format, we integrated seven multi-omic datasets from a cohort of 5-year-old OVT73 (nâ=â6) and control (nâ=â6) sheep into a single database utilising the programming language R. It includes high-throughput transcriptomic, metabolomic and proteomic data from blood, brain, and other tissues. RESULTS: We present the 'multi-omic' HD sheep database as a queryable web-based platform that can be used by the wider HD research community (https://hdsheep.cer.auckland.ac.nz/). The database is supported with a suite of simple automated statistical analysis functions for rapid exploratory analyses. We present examples of its use that validates the integrity relative to results previously reported. The data may also be downloaded for user determined analysis. CONCLUSION: We propose the use of this online database as a hypothesis generator and method to confirm/refute findings made from patient samples and alternate model systems, to expand our understanding of HD pathogenesis. Importantly, additional tissue samples are available for further investigation of this cohort.
Assuntos
Doença de Huntington , Animais , Encéfalo , Humanos , Doença de Huntington/genética , Proteômica , OvinosRESUMO
In mammals, females generally live longer than males. Nevertheless, the mechanisms underpinning sex-dependent longevity are currently unclear. Epigenetic clocks are powerful biological biomarkers capable of precisely estimating chronological age and identifying novel factors influencing the aging rate using only DNA methylation data. In this study, we developed the first epigenetic clock for domesticated sheep (Ovis aries), which can predict chronological age with a median absolute error of 5.1 months. We have discovered that castrated male sheep have a decelerated aging rate compared to intact males, mediated at least in part by the removal of androgens. Furthermore, we identified several androgen-sensitive CpG dinucleotides that become progressively hypomethylated with age in intact males, but remain stable in castrated males and females. Comparable sex-specific methylation differences in MKLN1 also exist in bat skin and a range of mouse tissues that have high androgen receptor expression, indicating that it may drive androgen-dependent hypomethylation in divergent mammalian species. In characterizing these sites, we identify biologically plausible mechanisms explaining how androgens drive male-accelerated aging.
Assuntos
Envelhecimento/genética , Androgênios/deficiência , Metilação de DNA , Epigênese Genética , Feminização/veterinária , Orquiectomia/veterinária , Carneiro Doméstico/fisiologia , Animais , Relógios Biológicos , Feminino , Feminização/metabolismo , Masculino , Carneiro Doméstico/cirurgiaRESUMO
Although Huntington's disease (HD) is a well studied Mendelian genetic disorder, less is known about its associated epigenetic changes. Here, we characterize DNA methylation levels in six different tissues from 3 species: a mouse huntingtin (Htt) gene knock-in model, a transgenic HTT sheep model, and humans. Our epigenome-wide association study (EWAS) of human blood reveals that HD mutation status is significantly (p < 10-7) associated with 33 CpG sites, including the HTT gene (p = 6.5 × 10-26). These Htt/HTT associations were replicated in the Q175 Htt knock-in mouse model (p = 6.0 × 10-8) and in the transgenic sheep model (p = 2.4 × 10-88). We define a measure of HD motor score progression among manifest HD cases based on multiple clinical assessments. EWAS of motor progression in manifest HD cases exhibits significant (p < 10-7) associations with methylation levels at three loci: near PEX14 (p = 9.3 × 10-9), GRIK4 (p = 3.0 × 10-8), and COX4I2 (p = 6.5 × 10-8). We conclude that HD is accompanied by profound changes of DNA methylation levels in three mammalian species.
Assuntos
Metilação de DNA , Epigênese Genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Animais Geneticamente Modificados , Comportamento Animal , Ilhas de CpG/genética , Estudos Transversais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Seguimentos , Técnicas de Introdução de Genes , Loci Gênicos , Estudo de Associação Genômica Ampla , Carga Global da Doença , Humanos , Doença de Huntington/sangue , Doença de Huntington/diagnóstico , Doença de Huntington/epidemiologia , Estudos Longitudinais , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Estudos Prospectivos , Proteínas Recombinantes/genética , Sistema de Registros/estatística & dados numéricos , Índice de Gravidade de Doença , Ovinos , Adulto JovemRESUMO
The substantia nigra is an integral component of the basal ganglia circuitry for limbic and motor functions. Dysfunction and degeneration of the basal ganglia are fundamental aspects of neurodegenerative diseases such as Parkinson's disease and Huntington's disease. With the increasing use of sheep to model neurological diseases, it is crucial to understand the anatomy and neurochemistry of these key basal ganglia nuclei in the normal sheep brain and how they compare to the human brain. Therefore, studies of the gross anatomy, cellular morphology, and neurochemical expression patterns within the sheep substantia nigra were performed. We show that the sheep substantia nigra reflects all important aspects of the anatomy and neurochemistry of the human substantia nigra, with only minor inter-species differences evident. Many neurochemicals that are central to the functioning of the SN, and wider basal ganglia circuitry, are present throughout the sheep SN. In a wider context, the results of this study provide evidence that the sheep substantia nigra accurately reflects the anatomy of the human substantia nigra, which validates the use of sheep models of basal ganglia neurological disorders.
Assuntos
Ovinos , Substância Negra/anatomia & histologia , Substância Negra/metabolismo , AnimaisRESUMO
The pronounced cachexia (unexplained wasting) seen in Huntington's disease (HD) patients suggests that metabolic dysregulation plays a role in HD pathogenesis, although evidence of metabolic abnormalities in HD patients is inconsistent. We performed metabolic profiling of plasma from presymptomatic HD transgenic and control sheep. Metabolites were quantified in sequential plasma samples taken over a 25 h period using a targeted LC/MS metabolomics approach. Significant changes with respect to genotype were observed in 89/130 identified metabolites, including sphingolipids, biogenic amines, amino acids and urea. Citrulline and arginine increased significantly in HD compared to control sheep. Ten other amino acids decreased in presymptomatic HD sheep, including branched chain amino acids (isoleucine, leucine and valine) that have been identified previously as potential biomarkers of HD. Significant increases in urea, arginine, citrulline, asymmetric and symmetric dimethylarginine, alongside decreases in sphingolipids, indicate that both the urea cycle and nitric oxide pathways are dysregulated at early stages in HD. Logistic prediction modelling identified a set of 8 biomarkers that can identify 80% of the presymptomatic HD sheep as transgenic, with 90% confidence. This level of sensitivity, using minimally invasive methods, offers novel opportunities for monitoring disease progression in HD patients.
Assuntos
Biomarcadores/metabolismo , Doença de Huntington/patologia , Metabolômica , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Animais Geneticamente Modificados/genética , Área Sob a Curva , Arginina/análogos & derivados , Arginina/metabolismo , Citrulina/metabolismo , Genótipo , Doença de Huntington/metabolismo , Doença de Huntington/veterinária , Modelos Logísticos , Curva ROC , OvinosRESUMO
This study reports the identification and characterization of markers of Alzheimer's 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-ß (Aß) protein is conserved between sheep and human, and sheep Aß1-42/Aß1-40 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.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Animais Geneticamente Modificados , Modelos Animais de Doenças , Proteínas de Neurofilamentos/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Biomarcadores/líquido cefalorraquidiano , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Emaranhados Neurofibrilares , Placa Amiloide/metabolismo , Placa Amiloide/patologia , OvinosRESUMO
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.
Assuntos
Cerebelo/metabolismo , Hipocampo/metabolismo , Doença de Huntington/metabolismo , Fígado/metabolismo , Metaboloma , Córtex Motor/metabolismo , Aminoácidos/metabolismo , Animais , Animais Geneticamente Modificados , Doenças Assintomáticas , Cerebelo/fisiopatologia , Ritmo Circadiano , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Hipocampo/fisiopatologia , Humanos , Doença de Huntington/fisiopatologia , Fígado/fisiopatologia , Masculino , Córtex Motor/fisiopatologia , Especificidade de Órgãos , Carneiro DomésticoRESUMO
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.
Assuntos
Encéfalo/patologia , Progressão da Doença , Imageamento por Ressonância Magnética , Proteínas de Membrana/genética , Lipofuscinoses Ceroides Neuronais/patologia , Animais , Atrofia , Peso Corporal , Feminino , Masculino , Tamanho do Órgão , Fenótipo , OvinosRESUMO
Recent studies have indicated that there is a decrease in perinatal survival of apparently normal animals produced by somatic-cell nuclear transfer. Here we report that the cortisol and adrenocorticotrophic hormone (ACTH) profiles of cloned lambs in the first 4 weeks of life are significantly different to that of control lambs. The growth of cloned lambs however was not different to controls. These findings demonstrate that endocrine development may be altered in apparently "normal" clones.
Assuntos
Hormônio Adrenocorticotrópico/sangue , Clonagem de Organismos , Hidrocortisona/sangue , Ovinos/sangue , Ovinos/crescimento & desenvolvimento , Envelhecimento , Animais , Animais Recém-Nascidos , Transferência Embrionária , Feminino , Técnicas de Transferência Nuclear , GravidezRESUMO
BACKGROUND: Huntington's 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.
Assuntos
Animais Geneticamente Modificados , Córtex Cerebral/patologia , Modelos Animais de Doenças , Doença de Huntington/patologia , Carneiro Doméstico/genética , Animais , Humanos , Proteína Huntingtina , Corpos de Inclusão Intranuclear/patologia , Proteínas do Tecido Nervoso/genéticaRESUMO
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.
Assuntos
Atividade Motora/fisiologia , Ovinos/fisiologia , Animais , Feminino , Sistemas de Informação Geográfica , Locomoção/fisiologiaRESUMO
We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically interpreted translocations and inversions. We confirm that the recently described phenomenon of 'chromothripsis' (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline, where it can resolve to a relatively balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign copy-number variants (CNVs). We compared these results to experimentally generated DNA breakage-repair by sequencing seven transgenic animals, revealing extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion was the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations.