Alzheimer’s disease (AD) is a progressive neurodegenerative disease that presents with debilitating memory and cognitive impairments, and accounts for between 60–80% of all dementias worldwide . Whilst the prevalence and the consequent economic and social impacts of AD are predicted to increase each year as the global population ages, the exact aetiology of AD is still unknown, making the development of effective treatments extremely challenging . Accumulations of β-amyloid (Aβ) exhibited as extracellular plaques and cerebral amyloid angiopathy (CAA), and accumulations of hyperphosphorylated tau present as paired helical filaments (PHFs) and neurofibrillary tangles (NFTs) are accompanied by significant neuronal loss and constitute the primary histopathological hallmarks of the disease [11, 19].
Rodents do not naturally exhibit either Aβ or tau pathology as they age . This presents a particular challenge when using rodents as a species for preclinical studies. To overcome this obstacle, it has been necessary to create transgenic mouse models overexpressing Aβ . Such models have been used to elucidate the significance of mutations in the amyloid precursor (APP), and presenilin proteins (PSEN1 and PSEN2) that cause familial AD (FAD) . Mutations in these proteins affect APP metabolism, leading to an increased production of the amyloidogenic form of Aβ (Aβ-42), and impaired Aβ clearance . These events form the basis of the ‘amyloid cascade hypothesis’ to explain the pathogenesis of AD, in which it is proposed that accumulation of Aβ-42 is the primary pathological event that drives all other associated pathologies (including tau pathology, inflammation, vascular damage, and neuronal loss) . This hypothesis, however, does not explain the mechanisms by which soluble and/or insoluble forms of intracellular and/or extracellular aggregates of Aβ and tau differentially affect one another [47, 68]. Indeed, while transgenic mouse models using APP and PSEN mutations present with significant Aβ pathology between three and six months of age they often fail to exhibit significant tau pathology or neuron loss . Consequently, tau pathologies within these rodent models are achieved only by introducing tau mutations that cause tau pathology in other dementias, namely those associated with frontotemporal dementia with parkinsonism-17 (FTDP-17) . This, combined with the relatively short lifespan of rodents means that the capacity of transgenic mouse models to fully reflect the aetiological mechanisms and temporal progression of familial AD is limited . Furthermore, given the lack of naturally occurring age-related neuropathology, the ability of mouse models to recapitulate mechanisms associated with ‘sporadic’ AD (that is not associated with mutations in any of the FAD genes and accounts for more than 95% of AD cases), is also extremely restricted .
Increasing recognition of the limitations associated with rodent models of AD has led to the investigation of species that have a longer lifespan, a more physically and functionally differentiated brain, and a propensity to naturally develop both Aβ and tau pathology with age [22, 73]. Non-human primates such as the chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta) and common marmoset (Callithrix jacchus) are of particular interest because they naturally develop some AD-like pathology . For example, diffuse and dense-cored Aβ plaques, and CAA have been detected in aged chimpanzees [24, 26, 28, 71], rhesus macaques [72, 88] and marmosets [33, 54], with quantities of Aβ in aged individuals comparable to levels seen in AD patients. NFTs have also been described within the entorhinal cortex of aged chimpanzees and rhesus macaques , while in marmosets, abnormally phosphorylated tau has been identified as early as adolescence . Other species that exhibit AD-like pathology include the domestic dog (Canis familiaris) [16, 17, 73, 79] and cat (Felis catus) . Both dogs and cats develop cognitive decline alongside diffuse Aβ plaques in old age, however, tau pathology rarely accompanies Aβ deposition and dense-cored Aβ plaques and NFTs are not consistently detected. Whilst these species have the potential to mitigate the limitations of current rodent AD models, they are limited in terms of their ethical use and therefore their numbers available to facilitate robust experimental design.
We chose to focus on sheep as a potential animal model of human AD for a number of reasons. Sheep have a moderate lifespan (fifteen-twenty years), an extensively annotated reference genome , are numerous within a well-established agricultural production infrastructure, and can be kept in a naturalised environment. Sheep also have a highly gyrencephalic neocortex with differentiated cortical and subcortical structures . This species has also been used as a model for studying the neurodevelopmental and cognitive effects of hormone manipulation [42, 69], Huntington’s disease [43, 58, 59, 78], and Batten disease [6, 23, 75]. Sheep between eight and fourteen years of age have also been shown to spontaneously develop both diffuse Aβ plaques  and NFTs [7, 60, 61]. These findings were confirmed in a pilot study (within this study) and suggest that sheep could provide a more robust model of AD than species that do not naturally develop this pathology. While these studies point towards sheep as a potential AD model, few molecular studies of AD-relevant proteins have been conducted in sheep in order to fully assess the translatability of the model. For example, whereas humans express six central nervous system (CNS) tau isoforms formed from the alternative splicing of three exons, the tau isoform expression of sheep has been predicted but not confirmed experimentally [44, 62]. Furthermore, whilst in humans over seventy different possible phosphorylation sites of the tau protein have been identified and associated with different time points of AD progression , similar information (that may be critical in identifying early tau dysfunction during AD aetiology) is extremely limited in the ovine model [7, 60, 61]. The aims of this study, therefore, were to characterise Aβ and tau histopathology, including tau phosphorylation, in sheep of a range of ages, and to investigate the nature of tau isoform expression in sheep. The results of the aforementioned pilot study are also described.