PHOSPHORYLATION STATUS OF SPINAL CORD AXONS
NADEEM, M., SPITZBARTH, I., HAIST, V., ROHN, K., TAUSCHER, K., ROHN, K., BOSSER, A., LANGEVELD, J., GROSCHUP, M.H., BAUMGÄRTNER, W., FAST, C., GERHAUSER, I.
Abstract
Transmissible spongiform encephalopathies (TSEs) represent a group of progressive diseases that affect the nervous system of humans and various animal species. Recently reported bovine spongiform encephalopathy (BSE) infections in goats in the United Kingdom and France have brought small ruminant species into the focus of prion disease research.
Immunohistochemistry was performed to detect axonal cytoskeletal and transport disturbances during the course of BSE in the spinal cord and autonomous ganglia of experimentally infected goats. Interestingly, the present study demonstrated abnormal expression of non-phosophorylated neurofilament (nNF) in axons of the white matter of the spinal cord, which was restricted to goats with clinical BSE. The results indicate axonal damage and disturbances in axonal transport during BSE in goats. However, whether abnormal nNF accumulations are related to disturbed axonal transport mechanisms in TSEs, the spread of TSE agents, and neuronal degeneration, has to be evaluated in future studies.
Moreover, the study reports for the first time that there is immunohistochemical evidence for PrPSc deposition in spinal cord white matter glial cells of BSE positive goats, indicating the involvement of glial cells in the spread of the agent.
Submitted for publication
AUTHORS CONTRIBUTIONS
NADEEM, M., SPITZBARTH, I., HAIST, V., ROHN, K., TAUSCHER, K., ROHN, K., BOSSER, A., LANGEVELD, J., GROSCHUP, M.H., BAUMGÄRTNER, W., FAST, C., GERHAUSER, I.
BSE INFECTION OF GOATS ALTERS NEUROFILAMENT PHOSPHORYLATION STATUS OF SPINAL CORD AXONS.
Submitted for publication.
M. Nadeem performed immunohistochemistry and electron microscopy, analyzed the data, and drafted the manuscript.
I. Spitzbarth was involved in the coordination of the immunohistochemical studies, performed statistical analysis, and drafted the manuscript.
V. Haist was involved in the coordination of the ultrastructural studies and revised the manuscript.
K. Rohn was involved in performing electron microscopy and generation of ultrastructural photographs.
K. Tauscher performed the animal experiments and obtained tissue samples.
K. Rohn was involved in the statistical analysis of the data.
A. Bosser, J. Langeveld, and M. H. Groschup were involved in the study design, the coordination of the animal experiments, revision of the manuscript, and in obtaining funding.
W. Baumgärtner was involved in the coordination of the immunohistochemical and ultrastructural studies, edited the manuscript, and obtained parts of the funding.
C. Fast was involved in the study design, coordination of the animal experiments, the conduction of immunohistochemistry, and edited the manuscript.
I. Gerhauser was involved in the coordination of the immunohistochemical studies, performed statistical analyses, designed figures, and edited the manuscript.
SHORT COMMUNICATION
BSE infection of goats alters the neurofilament phosphorylation status of spinal cord axons
Muhammad Nadeem1,2,*, Ingo Spitzbarth1,2,*, Verena Haist1, Kerstin Rohn1, Kerstin Tauscher3, Karl Rohn4, Alex Bossers5, Jan Langeveld5, Martin H. Groschup3, Wolfgang Baumgärtner1,2,#,Christine Fast3,+, Ingo Gerhauser1,+
1 Department of Pathology, University of Veterinary Medicine, Hannover, Germany
2 Center for Systems Neuroscience, University of Veterinary Medicine, Hannover, Germany
3 Friedrich Loeffler Institute, Institute of Novel and Emerging Infectious Diseases, Greifswald-Insel Riems, Germany
4 Department of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine, Hannover, Germany
5 Central Veterinary Institute, Wageningen UR, Lelystad, The Netherlands
* both authors contributed equally to this study and should be considered co-first authors (first authors in alphabetical order)
+ both authors contributed equally to this study and should be considered co-last authors (last authors in alphabetical order)
# Corresponding author:
Prof. Dr. Wolfgang Baumgärtner, Ph.D.
University of Veterinary Medicine Hannover, Department of Pathology Bünteweg 17, D-30559 Hannover, Germany
Tel.: +49-(0)-511-953-8620; Fax: +49-(0)-511-953-8675 E-mail: Wolfgang.Baumgaertner@tiho-hannover.de
TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSEs) including bovine spongiform encephalopathy (BSE) are devastating neurodegenerative disorders caused by conversion of the normal cellular prion protein (PrPC) into an abnormal isoform (PrPSc; Prusiner, 1982; Chesebro, 2003). Following the discovery of two goat BSE cases in the UK and France (Eloit and others 2005; Jeffery and others 2006) small ruminants were considered to pose an BSE infection risk/source for cattle and humans in particular. In goats experimental TSE susceptibility strongly depends on polymorphisms in the prion protein gene (PRNP) (Aguilar-Calvo and others 2014; Aguilar-Calvo and others 2015). In particular, goats with R/Q211 polymorphism (IQQ/IRQ) and Q/K222 polymorphism (IRK/IRQ) show delayed or even absent clinical signs compared with wild-type goats (IRQ/IRQ) (Aguilar-Calvo and others 2015). After oral infection of ruminants, the agent spreads via the autonomous nervous system, ultimately resulting in manifest disease in the brain (Van Keulen and others 2002; Hoffmann and others 2007; Kaatz and others 2012). However, the cellular and molecular mechanisms that facilitate the spread of prions within the nervous system and the involvement of the spinal cord in the pathogenesis of TSEs has been subjected to little research so far, even though PrPSc has been detected in the spinal cord (Flechsig and others 2000; Fukuda and others 2012; Kaatz and others 2012).
The aim of the present study was to elucidate potential changes in the antigen expression of various immunohistochemical markers in regions of the sympathetic infection route, which have been demonstrated to represent key localizations for the spread of the BSE agent (Kaatz and others 2012), including the celiac and mesenteric ganglion complex (CMGC) and the spinal cord. Moreover, we sought to characterize ultrastructural changes in neurons of the superior cervical ganglion (SCG).
Samples were collected during an oral BSE challenge (1g BSE-positive homogenized caprine brain) and serial kill study of Alpine-Saanen-mixed breed goats approved by the local authorities of the Federal State of Mecklenburg-Western Pomerania, Germany (LALLF 7221.3-2.5-001/05). The goats carried different PRNP genotypes with differing susceptibility.
Parts of the same population have been described in a previously published study (Aguilar-Calvo and others, 2015; table 1). The animals were killed 6-45 months post infection (mpi;
table 1). Formalin-fixed tissue samples were incubated for 1 h in 98% formic acid prior to paraffin embedding (Kaatz and others 2012). Three-micrometer sections were stained with haematoxylin and eosin (HE).
Table 1: Summary of the animals used in the present study, their clinical BSE status and results of immunohistochemistry for abnormal prion protein in superior cervical ganglia, celiac and mesenteric ganglion complex, and spinal cord of controls, BSE-infected goats, and their offspring (goat kids)
Genotype Animal
m = male, f = female, IHC = immunohistochemistry, SCG = superior cervical ganglion, CMGC = celiac and mesenteric ganglion complex, n.d. = not determined
* - = no clinical signs; + = mild subclinical signs, ++, +++ = moderate to severe clinical signs;
** - = negative in immunohistochemistry for PrPSc; + = positive in immunohistochemistry for PrPSc;
*** = age of the animals (not infected)
PrPSc was visualized using a monoclonal primary antibody as described (clone 6C2; Central Veterinary Institute, Wageningen UR, Lelystad, Netherlands; Kaatz and others 2012).
Immunohistochemistry was additionally performed on CMGC and spinal cord sections using markers for microtubules, axonal motor proteins, axonal damage, intermediate filaments, peripheral and central myelin proteins, and antigen presenting cells as described (Seehusen and Baumgärtner, 2010; Bock and others 2013; Spitzbarth and others 2011; table 2). SCG samples were processed for ultrastructural analyses as described (Baumgärtner and others 1987; Ulrich and others 2008).
Table 2: Summary of the antibodies used to characterize neurons and satellite glial cells of the celiac and mesenteric ganglion complex in controls and BSE infected goats using immunohistochemistry
Detected
antigen Clonality Primary antibody
(Supplier) Pretreatment Blocking
Mouse mc Sigma T 6793 Citric buffer/MW/20' Horse
serum 1:5 1:1500 beta-APP Mouse mc Chemicon M348 Lot
LV1622798 Citric buffer/MW/20' Horse
serum 1:5 1:1250 CNPase Mouse mc Millipore MAB 326 Lot
LV 1567855 Citric buffer/MW/20' Horse
serum 1:5 1:500 GFAP Rabbit pc DAKO Z 0334 Lot
23120676 - Horse
serum 1:5 1:6000
Iba-1 Rabbit pc Wako 019-19741 Citric buffer/MW/20' Horse
serum 1:5 1:500
Kinesin 5 A Rabbit pc Sigma K 0889 Citric buffer/MW/20' Horse
serum 1:5 1:400
MAP-2 Mouse mc Chemicon, MAB 3418 Citric buffer/MW/20' Horse n-NF Mouse mc Sternberger Monoclonals
Inc. SMI 311 Lot 9 Citric buffer/MW/20' Horse
serum 1:5 1:1000 p75NTR Mouse mc Hybridoma, Clone
HB-8737, ATCC, USA - Horse
serum 1:5 1:5 Periaxin Rabbit pc Sigma HPA001868 Citric buffer/MW/20' Horse
serum 1:5 1:2500 p-NF Mouse mc Sternberger Monoclonals
Inc. SMI 312 Lot 9
Triton-X 0,25% in PBS 15'
Horse
serum 1:5 1:8000 Synaptophysin Mouse mc DAKO SY38 Citric buffer/MW/20' Horse
serum 1:5 1:50 Tau-1 Mouse mc Chemicon MAB 3420 Citric buffer/MW/20' Horse
serum 1:5 1:2000 Vimentin Mouse mc DAKO M 0725 Lot
00051105 - Horse
serum 1:5 1:75 βIII TubulinIII Mouse mc Sigma T 8660 Citric buffer/MW/20' Horse
serum 1:5 1:8000 20' = 20 minutes; beta APP = beta amyloid precursor protein; CNPase = 2',cyclic-nucleotide 3'-phosphodiesterase; GAP 43 = growth-associated protein 43; GFAP = glial fibrillar associated protein; Iba-1 = ionized calcium-binding adapter molecule 1; mc = monoclonal; MAP-2 = microtubules associated protein 2;
MHC II = major histocompatibility complex class II; MW = microwave; n-NF = non-phosphorylated neurofilament; p75NTR = low affinity neurotrophin receptor p75; pc = polyclonal; p-NF = phosphorylated neurofilament
Clinical signs typical for BSE were detected in three IRQ/IRQ goats (animal numbers: 9, 11, 12) and three IQQ/IRQ goats (animal numbers: 21, 22, 24), which developed hypersensitivity, weight loss, lethargy, and alopecia at advanced time points beginning at 24 mpi (Aguilar-Calvo and others 2015; table 1). In the spinal cord, BSE-typical vacuolation of the neuropil and PrPSc-deposition were evident in all clinical animals and one subclinical goat (case number: 10). Besides the grey matter, PrPSc-positivity was observed in glial cells but not in axons of the spinal cord white matter in all 7 positive goats (table 3). Despite the lack of histological lesions PrPSc was detected in the SCG of three animals (case numbers: 10, 12, and 21). Unfortunately, SCG tissue was not available from some animals including the 4 remaining PrPSc-positive goats (table 1). In the CMGC, 5 animals displayed PrPSc
immunopositivity (case numbers: 9, 10, 11, 22, and 24) and three of them also neuronal vacuolation (case numbers: 9, 11, and 22). No CGMC tissue was available in goats 12 and 21 (table 1). Strikingly, expression of non-phosphorylated neurofilament (n-NF) was not detected in any spinal cord axon of the PrPSc-negative animals, but was significantly up-regulated in both swollen and normal appearing axons of the spinal cord white matter in 6 PrPSc-positive animals (figure 1). In contrast, expression of all other investigated antigens (table 2) was not influenced by the BSE status. Similarly, synapses were normal in SCG neurons in both control and PrPSc-positive goats at the ultrastructural level. Mitochondrial swelling, cristolysis, and occasionally dilatation of rough endoplasmic reticulum was noted in animals of each genotype, PrPSc-status, and age, thus most probably indicating post-mortem artifacts.
PrPSc deposition white matter (IHC)
ST = spinal tract, AT = ascending tract, DT = descending tract,* = spongiform changes + - +++ mild to severe immunopositivity
The present study demonstrates for the first time that BSE infection of goats is associated with glial PrPSc-deposition in the spinal cord white matter along with unsuspected alterations in the neurofilament phosphorylation status of axons. While absent in normal axons, abnormal neurofilament expression has been shown in various neurologic diseases including Creutzfeldt-Jakob disease (Gajdusek, 1985; Liberski and Budka, 1999; Tsunoda and Fujinami, 2002; Seehusen and Baumgärtner, 2010; Bock and others 2013). Axonal expression of n-NF in the present study is indicative of disturbances in axonal transport processes. Underlining this hypothesis, some immunopositive axons had an increased diameter (figure 1).
Figure 1: Immunohistochemical demonstration of non-phosphorylated neurofilament (n-NF) in axons of a control animal and a goat with experimental infection with bovine spongiform encephalopathy (BSE).
A: Spinal cord from a control animal with absent expression of n-NF in axons of the white matter of the spinal cord. B: Spinal cord of a BSE infected goat revealing increased axonal expression of n-NF in both axons with a normal appearing diameter (arrows) and swollen axons (spheroids, arrowhead) at > 24 months post infection, indicating axonal damage with disrupted axonal transport processes. Immunohistochemistry with the avidin-biotin-peroxidase-complex method. Chromogen: 3,3'-diaminobenzidine. Bars = 20 μm.
Synaptic disorganization and loss has been indicated as a constant feature of prion disease (Clinton and others 1993; Jeffrey and others 2000). Nevertheless, ultrastructural lesions were lacking in the SCG of PrPSc-positive goats in the present study. Consequently, BSE-induced synaptic pathology in goats might be restricted to the brain itself, which has to be confirmed in future investigations. The lack of PrPSc-deposition in IRK/IRQ animals substantiates their low susceptibility to clinical BSE (Aguilar-Calvo and others 2015). In contrast to earlier reports in cattle (Kaatz and others, 2012), PrPSc-deposition was not detected in peripheral ganglia prior to involvement of the spinal cord (table 1). However, ganglia were not available from all animals excluding definitive conclusions on the time course of the infection. In summary, our findings suggest that axonal damage and disturbed axonal transport in the spinal cord white matter might play a so far underestimated role in the pathogenesis of TSEs.
Acknowledgements
This study was in part supported by the Niedersachsen-Research Network on Neuroinfectiology (N-RENNT) of the Ministry of Science and Culture of Lower Saxony and in part by European Union projects (FOOD-CT-2006-36353, GoatBSE, and 219235 ERA-NET EMIDA, GOAT-TSE-FREE). Muhammad Nadeem is thankful to Higher Education Commission (HEC), Pakistan and Deutscher Akademischer Austauschdienst (DAAD), Germany for providing a scholarship. The authors acknowledge the work of Dr. Susanne Niedermeyer, Gesine Kreplin, Bettina Buck, Petra Grünig, Claudia Herrmann, Christiane Namneck, Kerstin Schöne, Caroline Schütz, and Brigitte Behrens.
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