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

Autophagy is one of the major protein degradation systems widely preserved in organisms from yeasts to mammals. ⁶ Recent autophagy research concerning molecular mechanisms and physiologic functions ¹¹ gives new insights for the understanding of the pathogenesis of various diseases including degenerative, inflammatory, and neoplastic conditions. Several research articles ^{1,4,5,7 –9} have been published in Veterinary Pathology and indicated roles of the autophagy system in various diseases and/or pathologic conditions of animals. In the current issue, dysfunction of autophagy is described in an article on canine neurodegenerative disorder of Lagotto Romagnolo (LR) dogs. ⁹

Autophagy is broadly classified into inducible autophagy and basal autophagy. Inducible autophagy occurs in response to several forms of cellular damage including starvation conditions. In contrast, basal autophagy is important for the constitutive turnover of cytosolic components via the degradation of denatured proteins. Dysfunction of basal autophagy is especially implicated as the key event in various neurodegenerative diseases, ^2,11 such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. In addition, dysregulation of the autophagy system also results in cell death known as type II programmed cell death.

The basic molecular pathway of the major autophagy system has been recently elucidated in both yeast ⁶ and mammalian ^2,11 cells. Figure 1 is a schematic overview of the autophagy pathway in mammalian cells. Briefly, protein degradation by the autophagy system begins with the formation of a complex of Unc-51-like kinases ¹ (ULK1) identified as a mammalian homolog to yeast autophagy-related gene (ATG)-1 and followed by the formation of Beclin1-Vps34 phosphatidylinositol 3-kinase complex. The Atg12-Atg5-Atg16L complex forms a double membrane known as the isolation membrane. Atg 4, Atg 3, and Atg 7 are associated with the cleavage of microtubule-associated protein light chain 3 (LC3), identified as a mammalian homolog to yeast ATG8, and with the conversion of LC3 from LC3-I to LC3-II. Then, the isolation membrane is formed around denatured proteins or damaged organelles together with adaptor proteins, such as p62/A170/SQSTM1 (p62) and neighbor of BRCA gene 1 (NBR1). The isolation membrane is elongated and finally forms an autophagosome. The denatured molecules in the autolysosomes formed by the fusion of autophagosomes and lysosomes are degraded by lysosomal enzymes (Fig. 1). Thus, Beclin1, LC3, p62, NBR1, and ubiquitin are commonly used as useful molecules to evaluate the function or alteration of the autophagy system.

Figure 1.

A schematic overview of the autophagy system in mammalian cells, including the major molecules associated with autophagy from induction to autolysosome formation.

Autophagy and Neurodegenerative Disorders

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. ³ The gene product ATG4D cleaves LC3 from proLC3 (Fig. 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. It is interesting that the level of inducible autophagy response of cultured fibroblasts from affected LR dogs was almost equal to that of normal controls. However, under basal conditions, LC3-II levels were higher in fibroblasts from affected dogs when compared with the control cells. ⁹ Together with extensive morphologic examinations, the authors concluded that the alteration of basal autophagy might also be associated with vacuolar changes in the visceral organs in affected LR dogs, although the clinical significance of these changes in visceral organs remains unknown.

Previously, similar brain lesions have been reported in ATG4B knockout mice. ⁷ The gene product Atg4b (autophagin-1) has the strongest proteolytic activity for human LC3 among the 4 mammalian ATG4 gene products (Atg4a, b, c, and d). The ATG4B-knockout mice exhibited mild neurologic signs, and pathologic examinations revealed numerous spheroid-like bodies that were immunopositive for ubiquitin in the cerebellar nucleus. Although vacuolar changes were not observed in either the nervous system or the visceral organs of ATG4B-knockout mice, these observations may further support the hypothesis that alterations of the ATG4 gene products can lead to similar neurodegeneration in LR dogs.

Cytoplasmic vacuolization is also one of the hallmark changes in several lysosomal storage diseases, in which a disease-specific substance accumulates because of a defect of the degradative enzyme. It has been well elucidated that the dysregulated autophagy system is secondarily involved in lysosomal storage diseases. ¹¹ Thus, Syrjä et al. ⁹ performed careful exclusion of major lysosomal storage diseases in dogs. Based on the results of several screenings, the authors concluded that the affected LR dogs did not suffer from lysosomal storage disease. In addition, spheroid formation is frequently found in other types of neurodegenerative conditions, such as neuroaxonal dystrophy (NAD), or in aging brains. In Papillon dogs with NAD, for which a missense mutation PLA2G6 c.1579G>A was recently identified, ¹⁰ marked accumulations of autophagy-associated molecules including LC3, P62, ATG5, ATG16L, and ubiquitin are found predominately within the spheroids (Figs. 2, 3). The role of autophagy is not fully elucidated in Papillon dogs with NAD; however, alteration of the autophagy system may ordinarily occur as a secondary event following excess storage of plasmalemmal phospholipids caused by dysfunction of PLA2G6 c gene products. Thus, to make conclusions about the pathologic role of the autophagy system in this type of lesion, further careful morphologic and physiologic investigations will be needed, similar to the studies in LR dogs. ^3,9

Figures 2 and 3. Neuroaxonal dystrophy, brain, Papillon dog. Figure 2. Numerous spheroids in the cerebellar nucleus of the affected dog. Figure 3. Those spheroids are intensely immunolabeled for microtubule-associated protein light chain 3.

Autophagy and Neoplastic Diseases

Chen and Yin ¹ discussed topics concerning the coordination of autophagy and proteasomes in resolving endoplasmic reticulum stress in cancer cells in an earlier review article in Veterinary Pathology and concluded that autophagy seems to be more beneficial for cancer cells to survive through stressful conditions. Since inducible autophagy may occur in some cancer cells undergoing cellular damage (eg, ischemia, hypoxia, chemotherapy, and radiation therapy ¹ ), the autophagy system can facilitate cancer cells to survive and develop antidrug resistance. Thus, synergy between antiautophagy and anticancer drugs might have some benefit as new chemotherapy, especially for drug-resistant cancers. Massimini et al. ⁴ indicated the possible strategy of anticancer chemotherapy based on the findings of in vitro studies using canine osteosarcoma cell lines. Previously, the role of autophagy in canine lingual granular cell tumors (Fig. 4) has been investigated, showing increased autophagy (Fig. 5a, b, c) in tumor cells with characteristic cytoplasmic granules in both the original canine tumors and xenotransplanted tumors in nude mice. ⁸ Electron microscopic examination revealed an autophagosome-like membranous structure containing several organelles in the cytoplasm. These findings may indicate that up-regulated autophagy can be associated with some specific morphologic lesions such as granular or vacuolar changes and the survival of tumor cells.

Figure 4. Lingual granular cell tumor, dog. Unique morphology of this tumor characterized by numerous cytoplasmic granules. Figure 5. Xenotransplanted tumors in nude mice resulting from injection of cells cultured from a canine lingual granular cell tumor. Cytoplasmic granules of the tumor cells are intensely immunopositive for light chain 3 (a), p62/A170/SQSTM1 (b), and neighbor of BRCA gene 1 (c) in the foci developing granular cell appearance. The immunolabeling pattern may indicate increased autophagy in tumor cells with characteristic cytoplasmic granules. The original data have been published by Suzuki et al. ⁸

Conclusion

Dysregulation of the autophagy system may occur primarily or secondarily in any type of morphologic changes induced by intracellular substance deposition, such as cytoplasmic vacuolization, granular or oncocytic changes, and aggregation of misfolding proteins (eg, highly phosphorylated tau in Alzheimer’s disease and alpha-synuclein in Parkinson’s disease). ^2,11 For a pathologist, the current findings of autophagy may give a new theoretical concept to understand the pathogenesis of these morphologic changes. We need to understand autophagy because it is so often involved in the diseases involving accumulation of intracellular substances. Since the information concerning the roles of autophagy in diseases is still limited in veterinary fields, further multifaceted examinations will be required to evaluate the significance of the autophagy system in animal diseases.