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Research article2017Peer reviewedOpen access

Basal Autophagy Is Altered in Lagotto Romagnolo Dogs with an ATG4D Mutation

Syrjae, Pernilla; Anwar, Tahira; Jokinen, Tarja; Kyoestilae, Kaisa; Jaederlund, Karin Hultin; Cozzi, Francesca; Rohdin, Cecilia; Hahn, Kerstin; Wohlsein, Peter; Baumgaertner, Wolfgang; Henke, Diana; Oevermann, Anna; Sukura, Antti; Leeb, Tosso; Lohi, Hannes; Eskelinen, Eeva-Liisa

Abstract

A missense variant in the autophagy-related ATG4D-gene has been associated with a progressive degenerative neurological disease in Lagotto Romagnolo (LR) dogs. In addition to neural lesions, affected dogs show an extraneural histopathological phenotype characterized by severe cytoplasmic vacuolization, a finding not previously linked with disturbed autophagy in animals. Here we aimed at testing the hypothesis that autophagy is altered in the affected dogs, at reporting the histopathology of extraneural tissues and at excluding lysosomal storage diseases. Basal and starvation-induced autophagy were monitored by Western blotting and immunofluorescence of microtubule associated protein 1A/B light chain3 (LC3) in fibroblasts from 2 affected dogs. The extraneural findings of 9 euthanized LRs and skin biopsies from 4 living affected LRs were examined by light microscopy, electron microscopy, and immunohistochemistry (IHC), using antibodies against autophagosomal membranes (LC3), autophagic cargo (p62), and lysosomal membranes (LAMP2). Biochemical screening of urine and fibroblasts of 2 affected dogs was performed. Under basal conditions, the affected fibroblasts contained significantly more LC3-II and LC3-positive vesicles than did the controls. Morphologically, several cells, including serous secretory epithelium, endothelial cells, pericytes, plasma cells, and macrophages, contained cytoplasmic vacuoles with an ultrastructure resembling enlarged amphisomes, endosomes, or multivesicular bodies. IHC showed strong membranous LAMP2 positivity only in sweat glands. The results show that basal but not induced autophagy is altered in affected fibroblasts. The ultrastructure of affected cells is compatible with altered autophagic and endo-lysosomal vesicular traffic. The findings in this spontaneous disease provide insight into possible tissue-specific roles of basal autophagy.

Keywords

basal autophagy; Western blot; immunofluorescence; electron microscopy; cytoplasmic vacuolization; pathology; ATG4D; dog

Published in

Veterinary Pathology
2017, Volume: 54, number: 6, pages: 953-963
Publisher: SAGE PUBLICATIONS INC

    UKÄ Subject classification

    Clinical Science

    Publication identifier

    DOI: https://doi.org/10.1177/0300985817712793

    Permanent link to this page (URI)

    https://res.slu.se/id/publ/93042