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

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.

Pathologic Changes and Autophagy: New Insights for the Pathogenesis of Animal Diseases

“…In the current issue, Syrjä et al 9 investigated alterations in basal autophagy in LR dogs with progressive cerebello-vestibular dysfunction using an in vitro fibroblast-culture system from affected LR dogs. The authors’ research group has also identified a missense change in the cysteine proteinase gene ATG4D genotype (c.1288G>A; p.Ala430Thr) in dogs of this breed.…”

“…1) and is also associated with the conversion between LC3-I and LC3-II. The authors 3,9 indicate that abnormalities of the autophagy system associated with ATG4D gene mutation cause neuronal vacuolar changes and spheroid-like lesions. In addition, the current article in Veterinary Pathology 9 focused on the vacuolar changes in visceral organs other than the brain in LR dogs.…”

“…In the current issue, dysfunction of autophagy is described in an article on canine neurodegenerative disorder of Lagotto Romagnolo (LR) dogs. 9…”

Multifaceted Roles of Autophagy in Health and Disease

“…5 In veterinary medicine, changes in autophagy have recently been associated with degenerative myelopathy in Pembroke Welsh Corgis 6 and progressive neurologic disease of Lagotto Romagnolo dogs. 8 Specifically, Ogawa et al 6 have demonstrated that Pembroke Welsh Corgis with degenerative myelopathy have increased accumulation of the autophagosome-associated protein LC3 in neurites and the autophagosome adaptor protein p62 in the neuropil of affected spinal cords. Accumulations of these proteins were associated with reduced expression of Beclin1 and Atg16 L, both of which play roles in autophagosome isolation membrane formation, collectively suggesting that affected animals may have disruptions in autophagy pathways.…”

“…6 Mutations in the autophagy-related gene ATG4D have been identified in Lagotto Romagnolo dogs with progressive neurodegenerative disease and have been associated with altered basal autophagy. 8 In cancer, autophagy plays complex and context-specific roles. During tumor initiation, autophagy can prevent neoplastic transformation by removing damaged proteins, DNA, or organelles and by reducing oxidative damage.…”