Regular articleAlzheimer's disease markers in the aged sheep (Ovis aries)
Introduction
Dementia is the most common neurological condition among older adults, with the majority of cases being attributable to Alzheimer's disease (AD). AD is a devastating neurodegenerative disease that causes progressive memory loss, cognitive decline, and finally dementia, leading to premature death and causing considerable stress to families. The prevalence of AD is expected to triple by 2050 due to an aging population (Alzheimers, N.Z, 2012, Alzheimers Ass, 2016) and, therefore, effective treatments for the disease are desperately needed. Mutations in 3 genes; amyloid precursor protein (APP), and presenilin 1 and 2 (PSEN1 and PSEN2) (Levy et al., 1990, Levy-Lahad et al., 1995, Sherrington et al., 1995) are known to cause relatively rare (<1%) familial AD. Each of these mutations results in the enhanced production of, or imbalances favoring the amyloidogenic 42-amino acid-long amyloid-β peptide (Aβ42) form of the APP protein. The risk of developing sporadic or late onset AD has been associated with variations in several genes including apolipoprotein E (APOE) (Harold et al., 2009, Saunders et al., 1993, Schellenberg and Montine, 2012). These genes are also functionally linked to Aβ peptide homeostasis, supporting the “amyloid cascade hypothesis” as an initiating mechanism for AD pathogenesis (Hardy and Higgins, 1992).
Due to the difficulty of making a diagnosis of AD in the earlier phases of the disease, patients recruited for clinical trials have typically been in the mild to moderate dementia stages of the disease (Blennow, 2010). However, it is generally agreed that the most effective treatment window would be early and ideally presymptomatic (Mangialasche et al., 2010). Cerebrospinal fluid (CSF) biomarkers are increasingly being used in the diagnosis of AD and also in the mild cognitive impairment phase of AD (Blennow et al., 2010). These biomarkers are also central in the recent research criteria for AD (Dubois et al., 2014) and preclinical AD (Dubois et al., 2016). Late-onset neurodegenerative diseases such as AD are difficult to model accurately in rodents because of their short lifespans. The commonly used rodent models of AD have been engineered to exhibit rapid and unnatural disease progression (Sabbagh et al., 2013), limiting their applications for early-stage disease research. Indeed, while several compounds have been beneficial in mouse models of AD, translation to humans has been very disappointing (Blennow et al., 2006, Dragunow, 2008, McGonigle, 2014). Successfully translated compounds have been those providing symptomatic relief rather than halting disease progression (McGonigle, 2014).
To enable safer, more effective clinical trials, and to discover the early pathogenic mechanisms of AD, we believe there is a need for a large animal model of AD with a complex brain structure (including a more developed cortex with gyri and sulci) and longevity, which will accurately capture the disease as it progresses, including its presymptomatic phase. Dogs and nonhuman primates have been used as models of aging and show relevant AD pathology, as recently reviewed (Youssef et al., 2016); however, these models are expensive and fraught with ethical issues. A transgenic AD mini pig has been produced by random integration of mutant human APP into the mini pig genome, driven by the PDGFβ promoter to give high levels of expression (Kragh et al., 2009, Sondergaard et al., 2012). Mini pigs are housed individually or in small groups, making long-term preclinical trials relatively expensive.
We see value in modeling AD in sheep (Ovis aries) due to the similarity of its brain structure and size relative to human. Sheep can live for at least 10 years, making them ideal for the study of later-onset diseases such as AD. Importantly, studies have shown AD-associated neurofibrillary accumulation (tau pathology) in normal aged sheep (Braak et al., 1994, Nelson and Saper, 1995); a feature which is absent in wild-type rodents and has made AD modeling challenging in rats and mice (Hardy and Selkoe, 2002). While the rate of naturally occurring dementia in sheep is unknown (as most farmed sheep are culled before reaching old age), sheep with cognitive deficits are studied due to natural mutations in genes causing Battens Disease in humans (Cook et al., 2002, Jolly et al., 1980, Weber and Pearce, 2013). Sheep are readily trainable for use in tests of cognitive function (Morton and Avanzo, 2011) and sheep suffering from a progressive neurological disease can be quantified longitudinally using modern methods, such as EEG (Perentos et al., 2015) and MRI (Sawiak et al., 2015). Sheep have face recognition systems for remembering specific individuals long term comparable with human (Kendrick et al., 2001). Furthermore, sheep can be kept in large numbers in a social environment on a farm, which is ethically more acceptable and cheaper than caged large laboratory animals. Genetically modified flocks can also be expanded relatively quickly from a few founder animals due to the JIVET reproductive technology that has been developed specifically in sheep (Kelly et al., 2005). A transgenic sheep model of the neurodegenerative disorder, Huntington's Disease, has been successfully established by our laboratory in this manner (Jacobsen et al., 2010) and is proving to be a valuable tool in HD research (Handley et al., 2016, Morton et al., 2014, Reid et al., 2013). The sheep genome has now been published and annotated (Jiang et al., 2014), and thus the genome of the sheep can now be precisely manipulated for human disease research.
In this report, we present data on the suitability of sheep as a model for AD. We compare the human and sheep peptide sequences for relevant AD proteins and peptides, and compare the types and levels of common AD biomarkers in CSF that will be relevant for tracking disease progression. We also looked for evidence of plaques and tangles, the hallmarks of human AD, in the aged sheep brain.
Section snippets
Human and sheep protein sequence alignments
Key human AD-associated reference protein sequences (as at July 26, 2016) were used in BLAST analysis (utilizing the blastp algorithm) against all O. aries protein sequences. Where proteins have multiple isoforms, the longest recorded isoform for human was used. For the APP protein, cleavage sites were compared, as well as the amino acid sequence of the Aβ1–42 fragment. The sheep protein with the highest homology against each human sequence is presented in Table 1.
Collection of tissue and CSF samples
Samples were obtained from
Homology of key AD proteins between human and sheep
The amino acid sequences of the key AD-related proteins show high homology between human and sheep, several close to 100% (Table 1). As seen in humans, sheep have multiple isoforms of APP. There are 6 predicted sheep isoforms for APP, ranging from 677–770 amino acid residues. There are 23 amino acid differences between human and sheep within the full-length 770 APP protein compared; however, none of these are close to the C-terminal cleavage sites. The sheep Aβ1–42 mRNA region has 6 nucleotide
Discussion
The aim of this study was to assess the suitability of sheep for future genetic manipulation to produce a large animal model of AD. We examined the similarities between key human and sheep proteins known to be involved in AD and measured the CSF levels of proteins and peptides that are known to be associated with the disease. In addition, we report evidence of plaques and tangles, the neuropathologic hallmarks of the disease, in the aged sheep brain.
Aged sheep naturally develop the
Disclosure statements
The authors have no conflicts of interest to declare.
Acknowledgements
The authors thank the Freemasons of New Zealand for their support and funding for this project. HZ is supported by the Knut and Alice Walleberg Foundation, the Swedish Research Council, and the European Research Council. KB is supported by the Swedish Research Council, the Swedish Alzheimer Foundation, the Swedish Brain Foundation, and the Torsten Söderberg Foundation.
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Authors contributed equally.