- Open Access
Myositis facilitates preclinical accumulation of pathological prion protein in muscle
Acta Neuropathologica Communications volume 1, Article number: 78 (2013)
In human and animal prion diseases, pathological prion protein, PrPSc, as well as prion infectivity is mainly found in the central nervous system, but also in lymphoid organs and muscle. Pathophysiology of prion colonization of lymphoid organs has been studied intensively, yet how myositis influences prion accumulation in muscle is unknown.
We have investigated the influence of myositis on PrPSc accumulation and prion infectivity in two distinct mouse models of experimental autoimmune myositis. Furthermore, we have addressed the relevance of PrPC expression in the lymphoreticular system in myositis by generating bone marrow chimeras.
Here we show that myositis positively influences muscular PrPSc accumulation at preclinical time points and that PrPC-expression in the lymphoid system is critical for this. In muscle, PrPSc and prion infectivity are uncoupled with detectable PrPSc but no prion infectivity at preclinical time points. Muscle has an intrinsically high ability to clear PrPSc once myositis has ceased, possibly involving autophagy.
Our findings provide new insights into the pathophysiology of prion colonization in muscle pointing out that myositis leads to enhanced prion colonization of muscle in subclinical prion disease.
Prion diseases are characterized by the accumulation of misfolded prion protein (PrPSc), a posttranslationally modified form of the host-encoded prion protein (PrPC) . Accumulation of PrPSc correlates with neurodegeneration, and PrPSc represents an essential part of the infectious agent causing prion disease [2–4]. Prion diseases occur in animals as well as in humans and are transmissible within and more rarely between mammalian species. Prion diseases in animals include scrapie in sheep and goat , chronic wasting disease in elk and deer , and bovine spongiform encephalopathy in cattle . Among the human prion diseases, three distinct etiologies are defined: they either arise sporadically as in sporadic Creutzfeldt-Jakob disease (sCJD), as autosomal dominantly inherited diseases as in genetic Creutzfeldt-Jakob disease (gCJD) or as acquired conditions in iatrogenic or variant Creutzfeldt-Jakob disease (iCJD, vCJD) [8, 9].
In prion diseases, accumulation of PrPSc and prion infectivity is not confined to the nervous system. PrPSc and prion infectivity are inevitably detectable in the lymphoreticular system (LRS) or in the skeletal muscle of terminally diseased individuals or animals [10–14]. In the subclinical phase of disease, the situation differs. Here PrPSc and prion infectivity are readily detectable in the LRS in the majority of instances, whereas prion accumulation in muscle occurs to a lesser extent both quantitatively and qualitatively [15–18].
Our knowledge on the pathophysiology of PrPSc accumulation between LRS and muscle differs as well. For prion colonization of the LRS, the molecular events have been worked out in great detail. Here, follicular dendritic cells (FDCs) residing in germinal centers of lymphoid follicles accumulate PrPSc before prions find their way to the central nervous system via peripheral nerves [19–21].
PrPC expression in muscle has been studied and it could be shown that myocytes as well as muscle macrophages express PrPC  and that the expression in muscle is highly regulated and fibre-type specific . Overexpression of PrPC in muscle leads to a myopathy and a wide range of myopathies are characterized by increased PrPC-levels [23–27]. However, in muscle much less is known regarding molecular determinants of PrPSc accumulation. PrPSc accumulation is muscle-type specific with hind limbs showing higher PrPSc content than fore limb muscles . Thus, the molecular mechanisms underlying accumulation of PrPSc and prion infectivity in skeletal muscle are poorly understood. Case studies from patients with myositis and prion disease suggest that inflammation may promote PrPSc accumulation . In fact, recent data indicate that ectopic follicular inflammation is able to support prion accumulation even in non-lymphoid tissue . On the other hand, peripheral nerves or muscle spindles or myocytes, have been shown to accumulate PrPSc even in the absence of inflammation [13, 15, 31].
Here, we show that PrPSc accumulation in skeletal muscle of mice was enhanced upon induction of experimental autoimmune myositis (EAM) in early subclinical prion disease. Our data suggest that accumulation of PrPSc correlates with elevated levels of PrPC at the peak of myositis originating from infiltrating lymphocytes. Once myositis ceased, PrPSc was rapidly cleared from muscle most likely by autophagy which is upregulated in muscle compared to spleen and brain. Accumulation of PrPSc in inflamed muscle required presence of PrPC on the LRS. Interestingly, titers of infectious prions as measured by bioassay were unchanged between the myositis and the control cohort pointing to an uncoupling of PrPSc loads and titers of infectious prions in our experimental model.
Five to six weeks old SJL/J and C57Bl/6 mice were purchased from Charles River Laboratory (Sulzfeld, Germany). Prion protein-deficient mice (Prnp0/0)  were bred in house. Mice were sacrificed in groups of 4 animals at day 35, day 60, and day 90 after inoculation with Rocky Mountain Laboratory (RML) prions or when clinical signs of terminal prion disease (tail rigidity, weight loss, ataxia, and roughened fur) occurred. All animal procedures were performed in accordance with the institutional guidelines from the animal facility of the University Medical Center Hamburg-Eppendorf.
Generation of bone marrow chimeras
C57Bl/6 mice were irradiated with a dosage of 8 Gy with constant 1 Gy/min using a BIOBEAM 2000 Cs-137 chloride gamma irradiator (Eckert & Ziegler, Berlin, Germany) one day prior to bone marrow reconstitution. Bone marrow was harvested in serum-free media (D-MEM) at 4°C from femur and tibia of the hind-legs of Prnp0/0 and C57Bl/6 mice using a syringe with a 23 G needle. The cell suspension was transferred to a 100 μm strainer and the flow-through was centrifuged at 300 × g for 10 min at 4°C. Cell pellets were solubilised in 1 ml of erythrocytes-lysis-buffer containing 0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA (pH = 7.2 to 7.4) and were then incubated for 5 min on ice. Afterwards cells were washed with PBS and transferred to a 40 μm strainer. The flow-through was directly used for reconstitution. Each mouse was injected intravenously with 300 μl of the cell suspension containing about 107 bone marrow cells in total and reconstitution efficiency was assessed by FACS analysis (see below).
Induction of an experimental autoimmune myositis (EAM)
In order to induce an EAM, SJL/J mice were treated as described before . Briefly, purified myosin from rabbit skeletal muscle (6.6 mg/ml; Sigma-Aldrich, Munich, Germany) was emulsified with an equal amount of Complete Freund's adjuvant (CFA) (Difco Laboratories, Detroit, USA) with 3.3 mg/ml Mycobacterium butyricum (Difco Laboratories, Detroit, USA). Mice were anesthetized using a CO2/O2 mixture and were then immunized subcutaneously with 100 μl of the emulsion into four locations (total, 400 μl) on the back on days 0, 7, and 14. On day 21 and 54 mice were either sacrificed for histological and FACS analysis or they were inoculated at day 21 with RML prions. In order to introduce an EAM in C57Bl/6 mice, in addition animals were injected intraperitoneally with 0.5 μg Pertussis toxin in PBS at all time points of immunizations.
Inoculations, determination of incubation time and prion titers
At d ay 21 mice were anesthetized using an intraperitoneal injection of ketamine (12 mg/ml) mixed with xylazine (1.6 mg/ml) with a dosage of 100 μl solution per 10 g bodyweight. Afterwards mice were inoculated intraperitoneally with 100 μl PBS containing 6 logLD50 units of RML scrapie strain. In order to determine the incubation time to onset of terminal prion disease, mice were kept until the day of onset of terminal clinical signs of prion disease (see above). Incubation time in days starting from the day of scrapie administration until the day of death was determined and plotted against the survival probability in % in Kaplan-Meier survival curves using SPSS statistic software.
Bioassays to determine titers of prion infectivity were performed on 1% homogenates of either spleen or muscle tissue. Spleens from one single animal per group and muscle tissues from either 1 animal per group (muscle control samples but day 90 and all chimeras), 2 animals per group (SJL/J mice day 35, day 60 and terminal diseased) or 3 animals per group (SJLJ/ mice day 90) were homogenized in 0.32 M sucrose using a FastPrep FP120 homogenizer (Qbiogene, Cedex, France), diluted in 5% BSA in PBS and centrifuged for 5 min at 500 × g. 30 μl of each supernatant were inoculated intracerebrally into groups of 4 highly prion susceptible tga20 mice . The relationship y = 11.45-0.088*x (y, log LD50 per milliliter of homogenate; x, incubation time in days to terminal disease) was used to calculate prion titers [35, 36].
Muscle and brain tissues were either frozen in liquid nitrogen-cooled 2-methyl-butan (Sigma Aldrich, Munich, Germany) and cut into 8 μm sections with a cryostat (CM1950, Leica, Wetzlar, Germany; see Figures 1a and 2), or were fixed using 3.5% of formaldehyde solution buffered according to Lillie for 15 hrs (see Figures 3c, 4c and 5b). If appropriate, tissues were prion-inactivated using 98% formic acid for 1 hr followed by 3.5% of formaldehyde solution for at least 12 hrs. Tissues were processed to paraffin blocks using an ASP300S dehydration machine (Leica, Wetzlar, Germany) and an EG1160 tissue embedding system (Leica, Wetzlar, Germany). Paraffin blocks were cut into 4 μm sections, which were stained with hematoxylin and eosin following standard laboratory procedures. Sections from frozen tissue were also stained with Elastica-von-Giesson standard staining solutions. For immunohistochemical staining the Ventana Benchmark XT machine (Ventana, Tuscon, Arizona, USA) was used. Briefly, deparaffinised sections were boiled for 30 to 60 min in 10 mM citrate buffer, pH 6.0, for antigen retrieval. Primary antibodies were diluted in 5% goat serum (Dianova Immundiagnostic, Hamburg, Germany), 45% Tris buffered saline pH 7.6 (TBS) and 0.1% Triton X-100 in antibody diluent solution (Zytomed, Berlin, Germany). Sections were then incubated with primary antibody for 1 hr (see also Table 1). Anti-rabbit or anti-goat histofine Simple Stain MAX PO Universal immunoperoxidase polymer (Nichirei Biosciences, Wedel, Germany) were used as secondary antibodies. Detection of secondary antibodies and counter staining was performed with an ultraview universal DAB detection kit from Ventana (Ventana, Tuscon, Arizona, USA) according to the standard settings of the machine. The staining of FDCs was performed according manufactures’ instructions. All sections were cover-slipped using TissueTek® glove mounting media (Sakura Finetek, Staufen, Germany), and dried in an incubator at 60°C. Pictures were taken using a light microscope (Axioskop 40, Zeiss, Jena, Germany or Olympus BH-2, Hamburg, Germany) equipped with a digital camera (AxioCam ICc3 Zeiss, Jena Germany).
Sodium phosphotungstic acid (NaPTA) precipitation
NaPTA precipitation was performed according to a previously, slightly modified method [14, 37]. Briefly, 100 mg of frozen tissue was thawed and put into 900 μl of dissociation buffer containing 25 mM HEPES (pH 7.2), 0.3 M sucrose and 53.6 μg Liberase Blendzyme 2 (Roche, Penzberg, Germany). Samples were incubated for 30 min at 37°C with a ribolyzing step performed every 10 min, until completely homogenized. To each 500 μl of 10% (w/v) tissue homogenates an equal volume of 4% (w/v) sarkosyl-PBS was added, vortexed, and incubated for 10 min at 37°C with constant agitation. 50 U/ml of benzonase (Novagen, Darmstadt, Germany) and 1 mM MgCl2 were added, and incubated at 37°C for 30 min with vigorous agitation. Afterwards, 81.3 μl of a pre-warmed (to 37°C) 4% (w/v) NaPTA/170 mM MgCl2 solution (pH 7.4) was added, vortexed, and incubated with vigorous agitation for 30 min at 37°C. Samples were then centrifuged at 16,000 × g for 30 min, after which supernatants were carefully removed and the remaining pellets resuspended in 22.5 μl of 0.1% sarkosyl-PBS. For detection of PrPSc only, samples were digested with proteinase K (Invitrogen, Karlsruhe, Germany) for 60 min at 37°C with a final concentration of 20 μg/ml. Since PrPC is proteinase K sensitive it is digested completely after this treatment, whereas PrPSc is proteinase K resistant and therefore is still detectable. Digestion was stopped by adding CVL-sample buffer (1% (w/v) SDS; 25 mM Tris/HCl, pH 7.4; 2,5% (v/v) β-mercaptoethanol; 1.5% (w/v) sucrose; 0.02% (w/v) brome-phenol-blue) and boiling for 10 min prior to Western blot analysis. As positive control RML 5.0 standard inoculum brain homogenate (RML) was spiked into uninfected tissue and digested as described.
Frozen tissues were thawed, homogenized and ~50 μg of total protein were separated by SDS-PAGE on a 12% SDS-PAGE gel, transferred to PVDF membranes (Biorad, Munich, Germany) at 400 mA for 1 hr in a wet-blot chamber (Biorad, Munich, Germany), and blocked for 1 hr at room temperature in 5% (w/v) milk powder in PBS containing 0.1% of Tween 20. Membranes were incubated overnight at 4°C using anti-PrP antibody POM1 or for two days using the anti-LC3 antibody, both in blocking buffer (see also Table 1). After incubation for 1 hour at room temperature with an HRP-conjugated anti-mouse secondary antibody (Promega GmbH, Mannheim, Germany, 1:5000 in blocking buffer), proteins were detected using ECL femto reagent (Pierce, Rockford USA). Samples were recorded and chemiluminescence signals were quantified using a chemiluminescence reader (Biorad, Munich, Germany). Detection of beta-actin (Sigma-Aldrich, Seelze, Germany) served as an internal loading control.
FACS analysis of blood samples
Two drops of blood, drawn by submandibular puncture were transferred into 5 ml of FACS buffer containing 2% (v/v) FCS, 0.01 M EDTA (pH 8) and 0.1% (w/v) NaN3 in PBS. After centrifugation of the samples for 10 min at 1000 rpm supernatants were discharged and 100 μl of primary antibody (biotinylated 6H4, Prionics, Planegg-Martinsried, Germany) diluted 1:100 in FACS buffer was added to the pellets and incubated at 4°C for 30 min. Afterwards samples were washed and incubated with fluorochrome-labelled streptavidin diluted 1:100 in FACS buffer at 4°C for 30 min. After another washing step samples were counterstained with an antibody against T-cells (PE-anti-CD3 (2 μl per sample)) by incubating at 4°C for 30 min. Following rigorous washing steps, samples were resuspended in 0.5 ml of FACS buffer and were analyzed using a LSRII FACS machine (BD Bioscience, Heidelberg, Germany).
FACS analysis of muscle samples
Quadriceps femoris muscles of immunized mice were dissected, minced and put into 5 ml of digestion buffer before incubation at 37°C for 30 min. Then, cooled (4°C) samples were washed and passed through a 100 μm cell strainer as well as through a 30 μm pre-separation filter (Miltenyi Biotec, Bergisch Gladbach, Germany). The flow-through was resuspended in 2.5 ml of a 30% Percoll solution and underlayed with 2 ml of a 78% Percoll solution before centrifugation for 30 min at 2500 rpm. Cells lying at the interface of the gradient were removed and washed several times with FACS buffer. In order to block Fc receptors before FACS analysis, pellets were incubated for 10 min in 50 μl of FACS buffer containing 1 μl of Fc-block (eBioscience, Frankfurt, Germany). After washing cells were stained for 1 hr using 10 μl of each of the following antibodies: Gr1-FITC, NU1.1-PE, B220-PECP-Cy5.5, CD11c-PECy7, CD11b-APC, CD45-APCCy7, and CD3-PaBkuc. After washing cells were resuspended in 400 μl of FACS buffer and were analyzed using a LSRII FACS machine (BD Bioscience, Heidelberg, Germany).
In all experiments, mean +/− SEM is reported. Statistical comparisons among groups were determined using Student’s t test.
Induction of experimental autoimmune myositis (EAM) in SJL/J mice
EAM was induced by subcutaneous injection of myosin and CFA at days 0, 7 and 14. On day 21 and 54, quadriceps femoris muscles of hind-legs were analyzed by FACS or histology (Figure 6). As shown in Table 2, FACS revealed T-cell dominated myositis (50.3 and 21.3% of all infiltrating cells at day 21 and 54 were CD3-positive; 17.3 and 1.4% of all infiltrating cells at day 21 and 54 were B220-positive) in EAM mice when compared to controls (5.6% of all infiltrating cells were CD3-positive; 1.2% of all infiltrating cells were B220-positive). Neutrophil granulocytes and other lymphocytes were not changed between EAM and controls. Up-regulation of T- and B-cells in EAM could be confirmed histologically (Figure 1a), where we found prominent lymphocytic infiltrates as early as day 21. Immunohistochemical staining with antibodies against CD3 for T-cells, B220 for B-cells and milk fat globule protein-epidermal growth factor-8 (MFG-E8) for FDCs demonstrated high numbers of T- and B-cells as well occasional MFG-E8-positive cells in EAM when compared to control tissue. Less pronounced infiltrates of T- and B-cells, but no MFG-E8-positive cells could be detected at day 54 after immunization. At both time points, morphological signs of chronic myositis such as necrotic muscle fibers and fibrosis were found in EAM but not in controls (Figure 1a). Interestingly, myositis did not lead to a reduction in muscle strength as demonstrated by hanging wire test (Additional file 1: Figure S1). For further analysis, prion inoculations were performed at peak of inflammation (21 days after the first immunization). For reasons of clarity, day of RML/sham (CD1 brain homogenate) inoculation was set to day 0, see Figure 6.
EAM leads to enhanced expression of PrPC in muscle
To investigate if EAM leads to an up-regulation of PrPC in muscle, we analyzed muscle tissue for its PrPC content by Western blotting and compared data to PrPC-levels in spleen and brain. As shown in Figure 1, we could detect a slight up-regulation of PrPC at day 60 after sham immunization in muscle (Figure 1b,c; p = 0.1). In all other muscle samples we could not find any changes in PrPC expression. As expected, brain showed high levels of PrPC irrespective of EAM (Figure 1b,c). PrPC-levels in spleen showed a considerable variation with a peak of PrPC expression at day 60 after sham inoculation (Figure 1b,c, p = 0.04).
EAM does not affect incubation time until onset of terminal prion disease
Having established a reliable EAM-model, we analyzed if EAM changed incubation time until onset of terminal prion disease. For this, we induced EAM and inoculated mice at day 21 after immunization with RML prions or sham. At identical time points, non-immunized SJL/J mice were treated equally as control. There was no difference in incubation times until onset of terminal prion disease between myosin-treated and non-treated mice (Figure 3a). EAM mice came down with disease after 182 +/− 8 days and non-immunized mice after 177 +/− 3 days.
After a proteinase K digestion of tissue homogenates we could detect presence of variable amounts of PrPSc by Western blot analysis in muscle, brain and spleen with no obvious differences in PrPSc loads and PrPSc glycotypes between EAM and controls (Figure 3b, Additional file 2: Figure S2).
In terminally sick mice, where the peak of active myositis has passed, morphological changes such as necrotic muscle fibers or fibrosis, typical for post-myositic muscles, were observed in EAM mice only (Figure 3c). In brain, spongiform changes and astrogliosis, typical for terminal prion disease were observed in both cohorts at comparable extent (Figure 3c).
EAM leads to transient PrPSc accumulation in muscle in subclinical prion disease
In order to analyze if EAM leads to changed kinetics of PrPSc accumulation in muscle, we investigated PrPSc loads by NaPTA-precipitation and Western blot analysis after proteinase K digestion to detect PrPSc at 35, 60 and 90 days post prion challenge (Figure 4a). At 35 as well as at 60 days after prion inoculation, we detected prominent PrPSc accumulation only in EAM mice, whereas we could not detect significant levels of PrPSc in control mice. Interestingly, at 90 days after prion inoculation, none of the muscle samples showed any PrPSc accumulation (Figure 4a), whereas at terminal disease, PrPSc accumulation was equally strong in EAM and control mice (Figure 3b). In brain and spleen PrPSc accumulation was not influenced by EAM with high PrPSc contents in spleen at all examined time points and rising PrPSc contents in brain detectable from day 90 onwards (Figure 4a).
Since autophagy has been shown to clear PrPSc effectively, we investigated if increased autophagy may help to clear PrPSc in muscle. For this, we determined LC3 levels in C57/Bl6 mice at day 0 in order to monitor if autophagy is per se activated in muscle tissue versus brain and spleen. As shown in Figure 4b, there was a high up regulation of LC3 II, a marker for autophagy, in muscle when compared to brain and spleen.
Histologically, signs of myositis could be observed in muscle of EAM mice, but not in control animals at days 35 and 60. At day 90 only EAM mice showed necrotic muscle fibers and fibrosis, both of which are routinely observed as late signs of myositis (Figure 4c). In brain no obvious pathological changes could be detected at preclinical stages (Figure 4c).
Dissociation of PrPSc loads and prion infectivity titers in muscle
PrPSc is thought to constitute an essential component of prion infectivity. Nevertheless, presence of prion infectivity does not strictly correlate with PK-resistant PrPSc load and protease-sensitive PrPSc species harbor significant amounts of prion infectivity . Thus we assessed prion titers of muscle and spleen by bioassay (Figure 4c). Titers of infectious prions in muscle were under the detection limit of the assay at early time points demonstrating partial dissociation of PrPSc loads and prion titer. At day 90, prion titers were around LD50 for both cohorts with one EAM mouse (at day 129) and two control mice (at days 123 and 160) showing signs of prion disease. However, at terminal disease stages high infectivity titers (4.1 log LD50/ml of tissue homogenate for EAM, 4.3 log LD50/ml of tissue homogenate for control) could be measured in muscle. As expected, in spleen high infectivity titers (3.8, 4.6, 4.8 and 5.2 log LD50/ml tissue homogenate for EAM, and 4.5, 4.8, 4.7 and 4.9 log LD50/ml tissue homogenate for controls) at days 30, 60, 90, and at terminal disease could be detected irrespective of the presence of EAM.
PrPSc accumulation in muscle at early time points requires a PrPC expressing lymphoreticular system
Next, we set out to examine which cell type in muscle tissue is responsible for accumulation of PrPSc. Since we could show that elevated PrPC levels in muscle occur during peak of myositis (Figure 1c), cells of the hematopoietic compartment were likely candidates. In order to further investigate the role of PrPC in the LRS we generated bone marrow chimeras with a PrPC-expressing and PrPC-deficient LRS. Since Prnp0/0 mice are kept on a C57Bl/6 background, and the majority of EAM models use a SJL/J background , we first had to establish a protocol for the induction of EAM in C57Bl/6 mice, thus allowing transplantation of syngenic bone marrow.
EAM in C57Bl/6 mice was confirmed by FACS analysis of muscle at 21 days post induction showing increased presence of T-cells (9.1% CD3-positive cells for EAM; 4.4% CD3-positive cells for controls, see Table 3). Upon histological analysis, we could observe T-cell dominant perimysial infiltrates in EAM in C57Bl/6 mice (Figure 2). Interestingly, we could not observe MFG-E8-positive FDCs and only a few B-cells.
To study PrPSc accumulation, we lethally irradiated C57Bl/6 mice and reconstituted their bone marrow with bone marrow of either Prnp0/0 or C57Bl/6 mice as a control. The reconstitution efficiency was assessed by FACS analysis of blood samples taken three weeks after lethal irradiation (Additional file 3: Figure S3). Afterwards, we induced EAM in 50% of the mice of both groups (Prnp0/0 → C57Bl/6; C57Bl/6 → C57Bl/6) and challenged them with either RML prions or mock at the peak of myositis. Accumulation of PrPSc and infectious prions was investigated by serially sacrificing mice at defined time points (day 35 and 90) and detection of muscular PrPSc loads by NaPTA-precipitation and Western blots. Only in mice with a PrPC-expressing hematopoietic compartment and EAM, we were able to observe significant accumulation of PrPSc in muscle at day 35 (Figure 5a). At day 90, no significant accumulation of PrPSc could be detected in either group. In spleen, PrPSc could be detected at day 35 and day 90 irrespective of the PrPC status of the hematopoietic compartment (Figure 5a).
In brain, as expected at this preclinical state of the disease, no spongiform changes or astrogliosis was seen (Figure 5b). In muscle, signs of myositis could be observed in both chimeric cohorts of mice after EAM but not in control mice at day 35 and 90 (Figure 5b). Since myositis is very mild in C57/Bl6 mouse lines compared to SJL/J mice, immune cells infiltrate singular muscle fibres (first, third and fourth row of pictures) or only the interstitial space (third row of pictures). Most infiltrating cells are T-cells as shown with the immunohistochemical staining of marker CD3. PrPC loads are not detectable in muscle tissue by standard PrPC staining, since expression levels are under the detection limit of this method.
To study the correlation between PrPSc loads and titers of infectious prions, we assayed prion titers of muscle and spleen by bioassay. As shown in Figure 5c, no infectivity was detectable at day 35 whereas at day 90 individual mice came down with prion disease with an attack rate below 50%, indicating that prion titers in these tissues were at the detection limit of the assay. As expected, in spleen high infectivity titers could be detected at all given time points irrespective of PrPC expression in the LRS and myositis.
Although, the central nervous system is the principal site of prion accumulation and replication, and the only site where prion-related tissue damage is seen, PrPSc and prion infectivity can be found in peripheral compartments such as spleen and muscle. Research focusing on mechanisms of prion accumulation in the periphery has yielded important insights into the temporal kinetics and prerequisites of prion accumulation . Presence of PrPSc and prion infectivity in muscle has been highlighted by a number of reports describing its presence in a wide range of instances such as sporadic and variant CJD, BSE, CWD and Scrapie [12, 13, 15, 29, 40, 41]. PrPSc in muscle can be found preclinically in prion-infected rodents and primates [31, 42]. Both, nerve fibres and myocytes have been shown to harbour PrPSc within muscle at terminal disease stages [13, 15, 42], yet in one patient with prion disease and myositis, PrPSc accumulation in muscle was surprisingly high . In summary, the pathophysiological events underlying accumulation of PrPSc and prion infectivity in muscle specifically also in myocytes is poorly understood and it is likely that events predisposing to prion colonization of muscle at terminal disease differ from those at early subclinical time points. Detailed knowledge of the mechanisms controlling prion colonization of muscle may help to explain the role of muscle in neuroinvasion of prions from the periphery to the brain [43, 44]. Here, we studied the temporal kinetics of PrPSc accumulation and prion titers in two murine EAM models and generated bone marrow chimeras to investigate the role of PrPC expression in the lymphoreticular system in myositis.
As expected, in our EAM models, the peak of myositis was reached 21 days following immunization . At this time point, we could observe significantly elevated levels of PrPC in muscle by Western blot yet not by immunohistochemical methods. Increased expression of PrPC has been linked to a number of diseases of the muscle such as inclusion body myositis, dermato-, and polymyositis [25, 26]. Our data imply that this increase is transient coinciding with inflammation. Although, presence of myositis did not influence disease kinetics with regard to incubation times until terminal prion disease, we could observe that presence of PrPSc at early subclinical disease is augmented in EAM. Differences were most drastic at days 35 and 60 following prion challenge. Here, we could only detect very low amounts of PrPSc by NaPTA-enhanced Western blotting in controls, whereas mice with EAM showed high (at day 35) and moderate (day 60) PrPSc loads in muscle. Surprisingly, at a time point where the peak of myositis has passed (day 90), PrPSc was completely cleared from muscle. Although, we did not investigate the mechanisms of PrPSc clearance in detail, autophagy may contribute to this as cell culture data  and our in vivo data show higher rate of basal autophagy in muscle.
Preclinical PrPSc accumulation in muscle has been demonstrated in a number of instances, yet we are not aware of any study showing clearance of PrPSc from this compartment after prion colonization has been established [31, 42, 46].
We could observe an uncoupling of PrPSc and prion infectivity in muscle at day 35 and 60, when significant levels of PrPSc in EAM muscle occur in the absence of prion infectivity. Similar findings have recently been reported in the brain  and spleen . Thus, our data expand the range of tissues where PrPSc and prion infectivity are not congruent and support the concept that non-PrPSc species considerably contribute to prion infectivity [47, 48].
The fact that EAM does not influence incubation time until onset of terminal prion disease is not surprising. Even if absence of PrPSc accumulation or prion infectivity in lymphoid tissue dramatically slows down development of prion disease, augmentation of prion replication even in ectopic sites does not lead to shortening of incubation time until onset of terminal prion disease [49, 50]. At terminal prion disease, PrPSc and prion infectivity could be observed irrespective of the presence of EAM. This is in line with a wealth of papers detecting PrPSc in muscle [12, 13, 29]. The fact that PrPSc is cleared from muscle and reappears at terminal disease fits to a concept where preclinical PrPSc results from lymphatic spread whereas PrPSc in diseased animals is due to centrifugal spread from the central nervous system [17, 43].
It has been suggested that glycosylation patterns of PrPSc differ between muscle and CNS . We could not observe such differences with muscular and brain-derived PrPSc showing similar glycosylation patterns.
In the LRS, accumulation of PrPSc and prion infectivity has been associated with FDC networks [20, 51]. Since myositis leads to diffuse and not follicular lymphoid infiltrates, we did not find FDC networks, but rather single MFG-E8-positive cells in our EAM muscles. It is questionable if these represent bona fide FDCs or merely MFG-E8-positive macrophages. In either case, accumulation of PrPSc in muscle of EAM mice at early time points occurs in the absence of FDC-networks.
Our experiments with bone marrow chimeras generating mice with a PrPC-deficient LRS indicate that PrPC expression on cells of the LRS is decisive for early, EAM-dependent accumulation of PrPSc in muscle. This sets early inflammation-modulated PrPSc accumulation in muscle apart from early PrPSc accumulation in spleen where PrPSc accumulates irrespective of the PrPC status of the LRS . In the spleen this property has been attributed to FDCs, which are of non-hematopoietic origin and radio-resistant . Thus, it is plausible that in muscle, lack of FDC-networks creates a situation, where a PrPC-expressing LRS is needed for PrPSc accumulation.
These data imply that myocytes are unlikely candidates for accumulation of PrPSc in EAM at early time points. A hypothesis that would accommodate current experimental data, would conceive PrPSc accumulation in muscle as a heterogeneous, disease stage-dependent event. In early disease stages, PrPSc accumulation is driven by cells of hematopoietic origin, whereas at late disease stages, non-hematopoietic cells such as nerve fibres or myocytes represent the main sites for PrPSc accumulation [13, 15, 17, 18, 31, 52]. There have been major concerns regarding biosaftey aspects of muscle biopsy in subclinically prion-diseased patients [12, 29]. Although our data indicate that enhanced PrPSc-loads in muscle may not be reflected by elevated prion titers it is not possible to interpolate our data from mouse experiments to the human situation and more research efforts are needed to assess biosaftey aspects of muscle biopsy in demented patients.
Our data show that: (i) myositis positively influences PrPSc accumulation in homogenized muscle tissue at preclinical time points and that a PrPC-expressing LRS is a prerequisite for this, (ii) PrPSc and prion infectivity are uncoupled in muscle with detectable PrPSc in the absence of prion infectivity at preclinical time points, (iii) muscle, unlike the LRS, has an intrinsically high ability to clear PrPSc once myositis has ceased, possibly involving autophagy.
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This research was supported by grants from the DFG GL 589/2-1 to MG.
The authors declare that they have no competing interests.
MN: carried out experiments; analysed data; drafted the manuscript. SK: carried out experiments; analysed data; drafted the manuscript. KSch: carried out experiments. KSt: carried out experiments; analysed data. MG: analysed data; drafted the manuscript. All authors read and approved the final manuscript.
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Neumann, M., Krasemann, S., Schröck, K. et al. Myositis facilitates preclinical accumulation of pathological prion protein in muscle. acta neuropathol commun 1, 78 (2013). https://doi.org/10.1186/2051-5960-1-78
- Bovine Spongiform Encephalopathy
- Inclusion Body Myositis
- FACS Buffer