Mammary tumor–associated amyloidosis (MTAA) in dogs is characterized by amyloid deposition in the stroma of mammary adenoma or carcinoma; however, the amyloid precursor protein remains unknown. We attempted to identify an amyloid precursor protein and elucidated its etiology by characterizing 5 cases of canine MTAA. Proteomic analyses of amyloid extracts from formalin-fixed paraffin-embedded specimens revealed α-S1-casein (CASA1) as a prime candidate and showed the N-terminal truncation of canine CASA1. Both immunohistochemistry and immunoelectron microscopy showed that amyloid deposits or fibrils in MTAA cases were positive for CASA1. Reverse transcription-polymerase chain reaction and quantitative polymerase chain reaction revealed the complete mRNA sequence encoding CASA1, whose expression was significantly higher in the amyloid-positive group. The recombinant protein of the N-terminal–truncated canine CASA1 and the synthetic peptides derived from canine and human CASA1 formed amyloid-like fibrils in vitro. Structural prediction suggested that the N-terminal region of CASA1 was disordered. Previously, full-length CASA1 was reported to inhibit the amyloidogenesis of other proteins; however, we demonstrated that CASA1 acquires amyloidogenicity via excessive synthesis followed by truncation of its disordered N-terminal region. By identifying a novel in vivo amyloidogenic protein in animals and revealing key mechanistic details of its associated pathology, this study provides valuable insights into the integrated understanding of related proteopathies.

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Amyloid-producing ameloblastoma (APAB) is characterized by abundant amyloid deposits in ameloblastoma, but the amyloid precursor protein is unknown. To explore this, we conducted histopathologic and proteomic analyses on formalin-fixed and paraffin-embedded samples from five cases of APAB (three dogs and two cats). Histologically, the samples exhibited a proliferation of the odontogenic epithelium, with moderate to severe interstitial amyloid deposits. By using Congo red and polarized light, the amyloid deposits were found to show characteristic birefringence. Amyloid deposits were dissected from tissue sections and analyzed by LC/MS/MS, and high levels of ameloblastin were detected in all tissues. Mass spectrometry also revealed that the N-terminal region of ameloblastin is predominantly present in amyloid deposits. Immunohistochemistry was performed using two anti-ameloblastin (N terminal, middle region) antibodies and showed that amyloid deposits were positive for ameloblastin N terminal but negative for ameloblastin middle region. These results suggest that ameloblastin is the amyloid precursor protein of APABs in dogs and cats, and the N-terminal region may be involved in the amyloidogenesis of ameloblastin.

Fibrinogen Aα‐chain amyloidosis is a hereditary systemic amyloidosis characterized by glomerular amyloid depositions, which are derived from the fibrinogen Aα‐chain variant in humans. Despite its unique pathology, the pathogenic mechanisms of this disease are only partially understood. This is in part because comparative pathological studies on fibrinogen Aα‐chain amyloidosis are currently unavailable as there is a lack of reported cases in animals other than humans. In this study, mass spectrometry‐based proteomic analyses of Japanese squirrels (Sciurus lis) that died in five Japanese zoos showed that they developed glomerular‐associated fibrinogen Aα‐chain amyloidosis with an extremely high incidence rate (29/38 cases, 76.3%). The condition was found to be age‐dependent in the Japanese squirrels, with 89% of individuals over 4 years of age affected. Mass spectrometry revealed that the C‐terminal region of the fibrinogen Aα‐chain was involved in amyloidogenesis in Japanese squirrels as well as humans. No gene variations were identified between amyloid‐positive and amyloid‐negative squirrels, which contrasted with the available data for humans. The results indicate that fibrinogen Aα‐chain amyloidosis is a senile amyloidosis in Japanese squirrels. The results have also provided comparative pathological support that the amyloidogenic C‐terminal region of the fibrinogen Aα‐chain is involved in the characteristic glomerular pathology, regardless of the animal species. This study elucidates the potential causes of death in Japanese squirrels and will contribute to future comparative pathological studies of fibrinogen Aα‐chain amyloidosis. © 2023 The Pathological Society of Great Britain and Ireland.