eISSN: 1509-572x
ISSN: 1641-4640
Folia Neuropathologica
Current issue Archive Manuscripts accepted About the journal Abstracting and indexing Subscription Contact Instructions for authors
SCImago Journal & Country Rank
vol. 56
Original paper

Dystrophic neurites accumulating autophagic vacuoles show early stages of neuritic destruction

Paweł P. Liberski, Agata Gajos, Beata Sikorska

Folia Neuropathol 2018; 56 (3): 175-178
Online publish date: 2018/09/28
Article file
- Dystrophic.pdf  [0.19 MB]
Get citation
JabRef, Mendeley
Papers, Reference Manager, RefWorks, Zotero


Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease or syndrome (GSS) are prototypic human prion diseases [1]; for which several experimental models in hamsters, mice or bank voles are currently available. The best known, not transgenic, models are mice infected with the K. Fu (Fujisaki) strain of GSS and the Echigo-1 strain of CJD [12,13,24,27]. The ultrastructural pathology of rodent models of human prion diseases are characterized by a spongiform change, dystrophic neurites containing abundant autophagic vacuoles and lysosomal dense bodies and the presence of tubulovesicular structures (TVS), disease-specific particles of unknown significance [10,15]. We [12-14,24,25,27] and then others [5] pioneered the research on autophagy in prion diseases; however, the exact role of autophagy has never been clearly elucidated [2,7].
In diseases of protein misfolding, including prion diseases, autophagy seems to play a protective role by removal of toxic protein aggregates [19,22,29] and the name “a guardian against neurodegeneration” was coined for this function [3]. However, the role of autophagy in neurodegeneration was also considered.
Recently, a role for autophagy in Drosophila flies transfected with a construct encoding A-42, a major amyloidogenic peptide in Alzheimer disease (AD) was published [18] and demonstrated the presence of dystrophic neurites, not unlike those seen in AD [6,16,30], filled with autophagic vacuoles and lysosomal electron-dense bodies, and showing areas of the cytoplasmic clearance. The latter finding suggests the leakage of lysosomal enzymes form autolysosomes that initiate degeneration and, probably, the loss of neurites.
The latter publication prompted us to re-examine our database of some 20,000 electron micrographs from Echigo-1 CJD-infected, 263K-strain and 22C-H strain of scrapie-infected hamsters to investigate evidence of cytoplasmic clearance as found in the Drosophila model.

Material and methods

Creutzfeldt-Jakob disease strain, animals, incubation period of illness

Outbred 6-week-old golden Syrian hamsters (Medical University of Lodz, Department of Oncology, Lodz, Poland) were inoculated intracerebrally with 0.05 ml of a 10% (w/v) centrifugation-clarified hamster brain suspension containing the Echigo-1 strain of the CJD agent [23]. Control animals were sham inoculated intracerebrally with the same volume of saline. We used five hamsters for each experiment; the control group consisted of 2 hamsters. In the first passage in our laboratory (7th serial passage after initial isolation), the incubation period was approximately six months. The experiment was repeated twice with the similar results.

Scrapie strains

Hamsters were inoculated with the 263 K or 22C-H strains of scrapie [9,17]. These strains are widely used experimental tools primarily because of the relatively short incubation periods which, for mice, ranged from 16 to 18 weeks and for hamsters from 9 to 10 weeks for the 263K strain and 24-26 weeks for the 22C-H strain. Appropriate control animals were sham inoculated with saline.

Electron microscopy

Hamsters in the terminal stage of CJD and control sham-inoculated hamsters at the same interval after inoculation were anaesthetized with ketamine. They were perfused by an intracardiac injection with saline followed by 150 ml of 1.25% glutaraldehyde and 1% paraformaldehyde prepared in cacodylate buffer (pH 7.4) and then by 50 ml of 5% glutaraldehyde and 4% paraformaldehyde.
Perfused animal carcasses were held at 4°C for at least 2 hours, after which brains were removed and several 1-mm3 samples were dissected under a binocular microscope from parietal cortex, corpus callosum, CA2 region of the hippocampus, thalamus, cerebellum and the brain stem. Those samples were postfixed in 1% osmium tetroxide for 1-2 hours, dehydrated through a series of graded ethanols and propylene oxide, and then embedded in Epon resin (Serva). Semi-thin sections were stained with toluidine blue, blocks trimmed, and ultrathin sections stained with lead citrate and uranyl acetate. Specimens were examined using a JEM 100 C transmission electron microscope.


The ultrastructural picture of the Echigo-1, 263-K and 22C-H infected hamsters were described elsewhere [13,14,24]. Briefly, spongiform change, astrocytosis, TVS and dystrophic neurites were readily found.
We reevaluated the largest database in the world of photographed dystrophic neurites for the presence of cytoplasmic clearance as shown in transgenic fruit flies transfected with A-42 [18]. In several neurites, we found electron-lucent areas not bound by any membranes or only partially bound; thus, they were not autophagic vacuoles as the latter are membrane-bound and contain cargo (Figs. 1-4). Those changes were not observed in every examined neurite and no correlation with any other changes was noticed. In some neurites, which could be traced over several sections, the electron-lucent areas were evident to change size, i.e. to expand.


We report here that dystrophic neurites in prion diseases in hamsters showed accumulation of lysosomes and autolysosomes and autophagic vacuoles. This phenomenon, albeit neglected in studies of prion diseases, is reminiscent of another protein misfolding disease, AD. This was shown in a classical paper by Lampert [6] and later elucidated in several reports including ours [8,26]. Recently numerous transgenic models of AD have been developed and enabled more detailed studies of the development of autophagic vacuoles. For instance, in APP single transgenic and APP co-expressing knocked-in mutant PS1 mice [28], severe neuroaxonal dystrophy was reported. Numerous dystrophic neurites were seen in the vicinity of plaques and in the regions distant from plaques and were stained by anti-phosphorylated APP and anti-phosphorylated 200 kDA neurofilament antibodies. This is analogous to our study on expression of phosphorylated neurofilaments in CJD [11,26].
Massive accumulations of lysosomes and autophagosomes were observed within dystrophic neuritis around plaques in transgenic APP/PS1 mouse models of AD [4]. Axonal lysosomes appeared early in the developing of the disease in a transgenic AD model and it is not the end-stage of pathology. Those authors suggested that dystrophic neuritis accumulating autophagic vacuoles resulted from merging of endosome and autophagosome pathways and it seems that they travel along the axons toward the somata of neurons [20,21]. Interestingly, in variant CJD we also observed abundant dystrophic neurites filled with lysosomes and autophagic vacuoles similar to those seen in neuritic plaques in human brains with AD. The mechanism of dystrophic neurite clearance is unknown but such a process may lead to their removal and it may contribute to deficits associated with neuroaxonal dystrophy.


The authors declare no conflict of interest.


1. Aguzzi A, Calella AM. Prions: protein aggregation and infectious diseases. Physiol Rev 2009; 89: 1105-1152.
2. Cherra SJ 3rd, Dagda RK, Chu CT. Review: autophagy and neurodegeneration: survival at a cost? Neuropathol Appl Neurobiol 2010; 36: 125-132.
3. García-Arencibia M, Hochfeld WE, Toh PP, Rubinsztein DC. Autophagy, a guardian against neurodegeneration. Semin Cell Dev Biol 2010; 21: 691-698.
4. Gowrishankar S, Yuan P, Wu Y, Schrag M, Paradise S, Grutzendler J, De Camilli P, Ferguson SM. Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer’s disease amyloid plaques. Proc Natl Acad Sci USA 2015; 112: E3699-708.
5. Heiseke A, Aguib Y, Schatzl HM. Autophagy, prion infection and their mutual interactions. Curr Issues Mol Biol 2010; 12: 87-97.
6. Lampert P. Fine structural changes of neurites in Alzheimer’s disease. Acta Neuropathol 1971; 5 Suppl 5: 49-53.
7. Lansbury PT, Lashuel HA. A century-old debate on protein aggregation and neurodegeneration enters the clinic. Nature 2006; 443: 774-779.
8. Liberski PP. Transmissible cerebral amyloidoses as a model for Alzheimer’s disease. An ultrastructural perspective. Mol Neurobiol 1994; 8: 67-77.
9. Liberski PP, Asher DM, Yanagihara R, Gibbs CJ Jr, Gajdusek DC. Serial ultrastructural studies of scrapie in hamsters. J Comp Pathol 1989; 101: 429-442.
10. Liberski PP, Brown P. Disease-specific particles without prion protein in prion diseases – phenomenon or epiphenomenon? Neuropathol Appl Neurobiol 2007; 33: 395-397.
11. Liberski PP, Budka H, Yanagihara R, Gajdusek DC. Neuroaxonal dystrophy in experimental Creutzfeldt-Jakob disease: electron microscopical and immunohistochemical demonstration of neurofilament accumulations within affected neurites. J Comp Pathol 1995; 112: 243-255.
12. Liberski PP, Hainfellner JA, Sikorska B, Budka H. Prion protein (PrP) deposits in the tectum of experimental Gerstmann-Sträussler-Scheinker disease following intraocular inoculation. Folia Neuropathol 2012; 50: 85-88.
13. Liberski PP, Hainfellner JA, Sikorska B, Mori S, Budka H. Echigo-1: a panencephalopathic strain of creutzfeldt-jakob disease. I. neuropathological and immunohistochemical studies. Folia Neuropathol 2004; 42 Suppl B: 161-166.
14. Liberski PP, Sikorska B, Gibson P, Brown P. Autophagy contributes to widespread neuronal degeneration in hamsters infected with the Echigo-1 strain of Creutzfeldt-Jakob disease and mice infected with the Fujisaki strain of Gerstmann-Sträussler-Scheinker (GSS) syndrome. Ultrastruct Pathol 2011; 35: 31-36.
15. Liberski PP, Sikorska B, Hauw JJ, Kopp N, Streichenberger N, Giraud P, Budka H, Boellaard JW, Brown P. Tubulovesicular structures are a consistent (and unexplained) finding in the brains of humans with prion diseases. Virus Res 2008; 132: 226-228.
16. Liberski PP, Yanagihara R, Gibbs CJ Jr, Gajdusek DC. Neuroaxonal dystrophy: an ultrastructural link between subacute spongiform virus encephalopathies and Alzheimer’s disease. Prog Clin Biol Res 1989; 317: 549-557.
17. Liberski PP, Yanagihara R, Gibbs CJ Jr, Gajdusek DC. Scrapie as a model for neuroaxonal dystrophy: ultrastructural studies. Exp Neurol 1989; 106: 133-141.
18. Ling D, Song HJ, Garza D, Neufeld TP, Salvaterra PM. Abeta42-induced neurodegeneration via an age-dependent autophagic-lysosomal injury in Drosophila. PLoS One 2009; 4: e4201.
19. Llorens F, Thüne K, Sikorska B, Schmitz M, Tahir W, Fernández-Borges N, Cramm M, Gotzmann N, Carmona M, Streichenberger N, Michel U, Zafar S, Schuetz AL, Rajput A, Andréoletti O, Bonn S, Fischer A, Liberski PP, Torres JM, Ferrer I, Zerr I. Altered Ca2+ homeostasis induces Calpain-Cathepsin axis activation in sporadic Creutzfeldt-Jakob disease. Acta Neuropathol Commun 2017; 5: 35.
20. Maday S. Mechanisms of neuronal homeostasis: Autophagy in the axon. Brain Res 2016; 1649 (Pt B): 143-150.
21. Maday S, Wallace KE, Holzbaur EL. Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons. J Cell Biol 2012; 196: 407-417.
22. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature 2008; 451: 1069-1075.
23. Mori S, Hamada C, Kumanishi T, Fukuhara N, Ichihashi Y, Ikuta F, Miyatake T, Tsubaki T. A Creutzfeldt-Jakob disease agent (Echigo-1 strain) recovered from brain tissue showing the ‘panencephalopathic type’ disease. Neurology 1989; 39: 1337-1142.
24. Sikorska B, Hainfellner JA, Mori S, Bratosiewicz J, Liberski PP, Budka H. Echigo-1: a panencephalopathic strain of Creutzfeldt-Jakob disease. II. Ultrastructural studies in hamsters. Folia Neuropathol 2004; 42 Suppl B: 167-175.
25. Sikorska B, Liberski PP, Giraud P, Kopp N, Brown P. Autophagy is a part of ultrastructural synaptic pathology in Creutzfeldt-Jakob disease: a brain biopsy study. Int J Biochem Cell Biol 2004; 36: 2563-2573.
26. Sikorska B, Liberski PP, Sobów T, Budka H, Ironside JW. Ultrastructural study of florid plaques in variant Creutzfeldt-Jakob disease: a comparison with amyloid plaques in kuru, sporadic Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker disease. Neuropathol Appl Neurobiol 2009; 35: 46-59.
27. Sikorska B, Waliś A, Bratosiewicz-Wasik J, Brown P, Liberski PP. Fate of myelinated fibres in the optic nerves in experimental Creutzfeldt-Jakob disease in rodents: an ultrastructural study. Folia Neuropathol 2004; 42: 101-105.
28. Stilund M, Gjelstrup MC, Petersen T, Møller HJ, Rasmussen PV, Christensen T. Biomarkers of inflammation and axonal degeneration/damage in patients with newly diagnosed multiple sclerosis: contributions of the soluble CD163 CSF/serum ratio to a biomarker panel. PLoS One 2015; 10: e0119681.
29. Williams A, Jahreiss L, Sarkar S, Saiki S, Menzies FM, Ravikumar B, Rubinsztein DC. Aggregate-prone proteins are cleared from the cytosol by autophagy: therapeutic implications. Curr Top Dev Biol 2006; 76: 89-101.
30. Xu Y, Tian C, Wang SB, Xie WL, Guo Y, Zhang J, Shi Q, Chen C, Dong XP. Activation of the macroautophagic system in scrapie-infected experimental animals and human genetic prion diseases. Autophagy 2012; 8: 1604-1620.
Copyright: © 2018 Mossakowski Medical Research Centre Polish Academy of Sciences and the Polish Association of Neuropathologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
Quick links
© 2018 Termedia Sp. z o.o. All rights reserved.
Developed by Bentus.
PayU - płatności internetowe