Abstract Beta amyloid (Aβ) deposits are seen in aged individuals in many of the mammalian species that possess the same Aβ amino acid sequence as humans. Conversely, neurofibrillary tangles (NFT), the other hallmark lesion of Alzheimer’s disease (AD), are extremely rare in these animals. We detected Aβ deposits in the brains of Tsushima leopard cats (Prionailurus bengalensis euptilurus) that live exclusively on Tsushima Island, Japan. Aβ42 was deposited in a granular pattern in the neuropil of the pyramidal cell layer, but did not form argyrophilic senile plaques. These Aβ deposits were not immunolabeled with antibodies to the N-terminal of human Aβ. Sequence analysis of the amyloid precursor protein revealed an amino acid substitution at the 7th residue of the Aβ peptide. In a comparison with other mammalian animals that do develop argyrophilic senile plaques, we concluded that the alternative Aβ amino acid sequence displayed by leopard cats is likely to be related to its distinctive deposition pattern. Interestingly, most of the animals with these Aβ deposits also developed NFTs. The distributions of hyperphosphorylated tau-positive cells and the two major isoforms of aggregated tau proteins were quite similar to those seen in Alzheimer’s disease. In addition, the unphosphorylated form of GSK-3β colocalized with hyperphosphorylated tau within the affected neurons. In conclusion, this animal species develops AD-type NFTs without argyrophilic senile plaques.

Citation: Chambers JK, Uchida K, Harada T, Tsuboi M, Sato M, Kubo M, et al. (2012) Neurofibrillary Tangles and the Deposition of a Beta Amyloid Peptide with a Novel N-Terminal Epitope in the Brains of Wild Tsushima Leopard Cats. PLoS ONE 7(10): e46452. https://doi.org/10.1371/journal.pone.0046452 Editor: Chad A. Dickey, University of South Florida Alzheimer’s Institute, United States of America Received: July 18, 2012; Accepted: August 30, 2012; Published: October 3, 2012 Copyright: © Chambers et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by Grants-in-Aid for Scientific Research, Japan Society for the Promotion of Science (research project number: 24780283) [http://www.jsps.go.jp/english/e-grants/index.html]. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Neurofibrillary tangles (NFT), one of the diagnostic lesions of Alzheimer’s disease (AD), are rarely found in non-human animal brains. Although the etiology of AD is yet to be elucidated, the “amyloid hypothesis” is widely accepted to explain its pathogenesis [1]. According to this hypothesis, the age-dependent accumulation of beta amyloid (Aβ) peptides in the brain induces a subsequent cascade that culminates in NFT formation. Argyrophilic aggregates of Aβ peptide are called senile plaques, which are another diagnostic lesion of AD. The AD-related alterations that occur in the brains of animals such as monkeys and dogs have been well studied [2], [3], [4], [5], [6]. However, although these animals frequently form senile plaques with aging, they rarely develop NFT [7], [8], [9]. Even in the few reported animal cases of NFT, no pathological examinations were performed to exclude other diseases that could have caused the NFT to develop [10], [11]. Therefore, it has been a major interest whether AD is a human-specific disease [12], [13]. One of the authors (JKC) has previously reported the occurrence of NFT in the brains of captive cheetahs (Acinonyx jubatus) [11]. Subsequently, we have detected NFT and Aβ deposits in the brains of wild Tsushima leopard cats (Prionailurus bengalensis euptilurus). According to the phylogenetic tree of living cat species (Felidae), these two species belong to two closely related lineages that diverged approximately 6.7 million years ago [14]. The NFT of the leopard cats were consistent with the pathological characteristics of human AD and were also accompanied by diffuse granular Aβ42 deposits. Interestingly, unlike other animals such as monkeys and dogs [15], aged cheetahs and leopard cats do not develop argyrophilic senile plaques despite the fact that they develop diffuse Aβ deposits in their brains. In the present study, analysis of the leopard cat APP gene detected a base substitution, which altered the N-terminal amino acid sequence of the Aβ protein. Interestingly, many higher mammals that develop argyrophilic plaques, including dogs and monkeys, possess the same Aβ amino acid sequence as humans [16], [17], [18]. The present study provides biological insights into the pathogenesis of AD. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Immunohistochemical scoring of Aβ42 and AT8. https://doi.org/10.1371/journal.pone.0046452.t001 PPT PowerPoint slide

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larger image TIFF original image Download: Table 2. Primary antibodies used in the present study. https://doi.org/10.1371/journal.pone.0046452.t002

Supporting Information Figure S1. Double immunofluorescence staining of tau/hyperphosphorylated tau (AT100) (A), tau/3R-tau (B), tau/4R-tau (C), and Aβ/Aβ42. (A, B, C) Hyperphosphorylated tau, 3R-tau and 4R tau colocalized with pan tau antibody-positive aggregates. Bar = 20 µm. (D) Granular staining of Aβ42 colocalized with pan Aβ antibody. Bar = 100 µm. https://doi.org/10.1371/journal.pone.0046452.s001 (TIF)

Acknowledgments This study was performed with the approval of the Japanese Ministry of the Environment and was supported by Kyushu Regional Environment Office. The authors thank Ms. Hanae Yamamoto of Tsushima Wildlife Conservation Center and Ms. Maki Okamura of Iriomote Wildlife Conservation Center for their generous cooperation.

Author Contributions Conceived and designed the experiments: JKC KU HN. Performed the experiments: JKC TH MT. Analyzed the data: JKC KU HN. Contributed reagents/materials/analysis tools: MS MK HK NM HT. Wrote the paper: JKC.