De Biase et al. BMC Veterinary Research (2017) 13:102 DOI 10.1186/s12917-017-1028-1 RESEARCH ARTICLE Open Access Amyloid precursor protein, lipofuscin accumulation and expression of autophagy markers in aged bovine brain D. De Biase1, A. Costagliola1*, T.B. Pagano1, G. Piegari1, S. Wojcik2, J. Dziewiątkowski2, E. Grieco3, G. Mattace Raso4, V. Russo1, S. Papparella1 and O. Paciello1 Abstract Background: Autophagy is a highly regulated process involving the bulk degradation of cytoplasmic macromolecules and organelles in mammalian cells via the lysosomal system. Dysregulation of autophagy is implicated in the pathogenesis of many neurodegenerative diseases and integrity of the autophagosomal - lysosomal network appears to be critical in the progression of aging. Our aim was to survey the expression of autophagy markers and Amyloid precursor protein (APP) in aged bovine brains. For our study, we collected samples from the brain of old (aged 11–20 years) and young (aged 1–5 years) Podolic dairy cows. Formalin-fixed and paraffin embedded sections were stained with routine and special staining techniques. Primary antibodies for APP and autophagy markers such as Beclin-1 and LC3 were used to perform immunofluorescence and Western blot analysis. Results: Histologically, the most consistent morphological finding was the age-related accumulation of intraneuronal lipofuscin. Furthermore, in aged bovine brains, immunofluorescence detected a strongly positive immunoreaction to APP and LC3. Beclin-1 immunoreaction was weak or absent. In young controls, the immunoreaction for Beclin-1 and LC3 was mild while the immunoreaction for APP was absent. Western blot analysis confirmed an increased APP expression and LC3-II/LC3-I ratio and a decreased expression of Beclin-1 in aged cows. Conclusions: These data suggest that, in aged bovine, autophagy is significantly impaired if compared to young animals and they confirm that intraneuronal APP deposition increases with age. Keywords: Autophagy, Brain, Neuropathology, Ageing, Bovine Background targeting, lysosome docking and autophagosome-lysosome Autophagy is a self-degradative, highly regulated process fusion, vesicle breakdown and recycling. During Nucle- that involves the non-specific degradation of cytoplasmic ation, the activity of specific autophagy effectors including macromolecules and organelles via the lysosomal system. Beclin-1 (Atg6 orthologue) and LC3 (microtubule-associ- There are three different autophagic pathways based on ated protein 1 light chain 3) generates an active phago- the mechanisms for delivery of cargo to lysosomes: macro- phore (or isolation membrane) [2]. The nascent membrane autophagy, microautophagy and chaperone-mediated wraps around a portion of cytoplasm (including the soluble autophagy (CMA) [1]. Macroautophagy (herein referred to proteins, aggregates, or organelle) to eventually form a as autophagy) is the major lysosomal pathway for the turn- double membrane-bounded structure called “autophago- over of cytoplasmic components. Specifically, this process some”. The nascent autophagosome subsequently fuse with consists of the following steps: induction or initiation and lysosomes to form an autophagolysosome in which the cargo selection, vesicle nucleation and expansion, lysosome cytoplasmic cargo is degraded by lysosome hydrolases; degradation products are recycled for the synthesis of new molecules [2]. Emerging evidence indicates that autophagy * Correspondence: [email protected] protects cells by removing long-lived proteins, aggregated 1Department of Veterinary Medicine and Animal Production, University of Naples Federico II via Delpino, 1, 80137 Naples, Italy protein complexes, and excess or damaged organelles [3]. Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. De Biase et al. BMC Veterinary Research (2017) 13:102 Page 2 of 9 Defects in autophagy, therefore, are associated to vari- p < 0.001) and this relation was strong (d Somer’s statis- ous pathological conditions within organisms, includ- tics =0.6611; p < 0.001). ing tumorigenesis, defects in developmental programs and the build-up of toxic, protein aggregates involved in neurodegeneration [3] such as Amyloid precursor Immunofluorescence protein (APP). It has been recently suggested that the In all samples, autophagy induction was evaluated by progressive age-related decline of autophagic and Beclin-1 immunofluorescence and showed a progressive lysosomal activity may also be responsible for the age-related decrease of intraneuronal Beclin-1 expres- continuous intraneuronal accumulation of lipofuscin, sion. In young animals (Group B), the percentage of or “age pigment” [4]. For this study, we aimed to in- Beclin-1 immuno-positive neurons and astrocytes as well vestigate the expression of autophagic markers such as Beclin-1 staining intensity was of mild degree and as Beclin 1 and LC3 and the accumulation of patho- widespread. Beclin-1 staining intensity as well as per- logic proteins such as APP and lipofuscin in aged bo- centage of Beclin-1 immuno-positive cells were signifi- vine brains. cantly more noticeable among the young animals (p < 0.01and p < 0.001, respectively). On the contrary, in Results 76% (n = 14) of samples from aged cows (group A) no Histopathology Beclin-1 positivity was detected (Fig. 2a, b). In remaining In aged bovine, microscopic evaluation revealed a mod- 24% of cases percentage of Beclin-1 immuno-positive erate to severe gliosis and an increase of the number of cells as well as Beclin-1 staining intensity was of mild glial cells around neurons (satellitosis) in the gray mat- degree. A Beclin-1 positivity was mainly observed in ter. Moreover, we observed scattered, strongly eosino- neurons in the hippocampus. There was a statistically philic and shrunken neurons with nuclear pyknosis of a significant negative association between age and increase centrally placed nucleus. This findings were not ob- of percentage of Beclin-1 immunoreactive cells served in young animals. The striking and constant find- (G = −0.9521; p < 0.001) and this relation was strong (d ing in all aged brains (group A) was the accumulation of Somer’s statistics = −0.6653; p < 0.001). There was also a small perinuclear and granular yellow-brown, Periodic statistically significant negative association between age acid – Schiff (PAS) stain positive (Fig. 1a) and autofluor- and increase of staining intensity of Beclin-1 immunore- escent deposits (Fig. 1b) consisting of lipofuscin. The activity (G = −0.8523; p < 0.001) and this relation was amount of lipofuscin was moderate to severe, with a strong (d Somer’s statistics = −0.6276; p < 0.001). wide distribution in both cerebral cortex and hippocam- Moreover, strongly LC3 immuno-positive neurons and pus regions. Lipofuscin accumulation was found within astrocytes were observed in aged animals (Fig. 3a), indicat- the perikarya and sometimes the proximal dendritic tree ing possibly an excessive accumulation of autophago- of neurons and within the cytoplasm of astrocytes. No somes. In younger animals, LC3 staining intensity was evidence of lipofuscin accumulation was observed in the mostly absent (Fig 3b), even though a low percentage of neurons of young animals (Fig. 1c).Lipofuscin accumula- LC3 widespread immune-positive neurons was rarely tion was significantly more noticeable among the older observed. These parameters were significantly more animals (group A) (p < 0.001). There was a statistically noticeable among the aged animals (p < 0.01and significant positive association between age and increase p < 0.001, respectively). There was a statistically sig- in severity of lipofuscin accumulation (G = 0.7980; nificant positive association between age and increase Fig. 1 a Lipofuscin storage, hippocampus, dentate gyrus. Pyramidal neurons showing an abundant granular, PAS positive, intracytoplasmatic storage material. Periodic acid-Schiff (PAS) stain, 40X. b Intraneuronal storage material exhibits green-yellow autofluorescence by fluorescence microscopy. Fluorescence microscope; FITC filter (excitation, 455–500 nm; emission, 500–570 nm), 40X. c Neurons of young controls does not exhibit PAS positive and intracytoplasmatic storage material. Periodic acid-Schiff (PAS) stain, 40X De Biase et al. BMC Veterinary Research (2017) 13:102 Page 3 of 9 Fig. 2 Beclin 1 expression, hippocampus, dentate gyrus. a Immunostaining inyoung and b aged brain. TRITC filter (excitation, 543 nm; emission 560 nm). DAPI counterstain, 40X. c Protein expression in young and aged bovine brains. Densitometric values shows that BECN1 is expressed in higher amount in young controls when compared to aged brain samples (P < 0,001 vs controls). Bars refers to mean values. Actin protein levels confirm the amount of protein loading in each lane of these parameters (G = 1; p < 0.001) and this relation 1(represented as 60 kDa band) and two isoforms of LC3 was moderate