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1 Paul-Ehrlich-Institut, Langen, Germany
2 Department of Virology and Immunology, German Primate Centre, Göttingen, Germany
3 Commissariat à l'Energie Atomique, Fontenay-aux-Roses, France
Correspondence
Edgar Holznagel
holed{at}pei.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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- and -
-positive CSF samples were found around the time of onset of mild neurological signs, but not earlier. In contrast, 14-3-3
and -
isoforms were not detectable. 14-3-3 levels increased with time and were positively correlated with the degree of neurological symptoms. Post-mortem examination of brain samples revealed a positive correlation between PrPres and 14-3-3 levels. Interestingly, florid plaques characteristic of human vCJD could not be detected in diseased monkeys. It was concluded that analysis of 14-3-3 proteins in CSF is a reliable tool to characterize the time course of brain degeneration in simian vCJD. However, there are differences in the clinical course between orally and intracerebrally infected animals that may influence the detection of other biomarkers.
These authors contributed equally to this work. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Cattle are affected by bovine spongiform encephalopathy (BSE) and small ruminants by scrapie. Human prion diseases include sporadic Creutzfeldt–Jakob disease (sCJD) and its variant form, vCJD (Ironside, 1998). In humans, it is likely that vCJD was caused by the consumption of BSE-contaminated food (Ironside, 1998). The causative agent of TSEs is regarded as a conformational variant of the ubiquitous host prion protein (PrPC) (Weissmann, 2005), generally termed PrPSc, as the oldest known TSE is scrapie. PrPSc is defined as an aggregated form of PrPC that is largely resistant to proteinase K (PK) digestion (Weissmann, 2005). Human prion diseases can be transmitted experimentally to non-human primates (NHPs) (Gajdusek et al., 1966; Gajdusek & Gibbs, 1967, 1971, 1975; Gibbs et al., 1968, 1980; Cathala et al., 1975; Masters et al., 1976; Baker et al., 1993; Bons et al., 1999; Williams et al., 2007). BSE transmitted to cynomolgus monkeys (Macaca fascicularis) causes a simian form of vCJD after an incubation period of 3–5 years (Lasmézas et al., 1996, 2001, 2005; Herzog et al., 2004).
A number of isoforms, named , , , 
, 
, 
and , comprise the family of mammalian 14-3-3 proteins. 14-3-3 proteins are crucial for various physiological cellular processes, such as signalling, cell growth, division, adhesion, differentiation, apoptosis and regulation of ion channels. These intracytoplasmic molecules are expressed across a broad range of neuronal and non-neuronal cell types. However, the highest tissue concentration is found in the central nervous system (CNS), where it is present in the cytoplasmic compartment (Berg et al., 2003).
Extensive destruction of the brain leads to leakage of 14-3-3 proteins into the cerebrospinal fluid (CSF), which can be detected by ELISA (Kenney et al., 2000; Green et al., 2002) or Western blotting (Hsich et al., 1996; Takahashi et al., 1999). Elevated 14-3-3 levels are found in the CSF of patients with dementia (Burkhard et al., 2001) or suffering from prion diseases (Hsich et al., 1996; Will et al., 1996; Collins et al., 2000; Zerr et al., 2000; Giraud et al., 2002; Geschwind et al., 2003). In this regard, detection of 14-3-3 proteins in CSF is not disease-specific, but rather reflects the process of damaged neurons spilling their proteins into CSF (Pocchiari et al., 2004; Cuadrado-Corrales et al., 2006).
CSF 14-3-3 immunoassays have been used as ante-mortem tests for both sCJD and vCJD (Hsich et al., 1996; Will et al., 2000; Zerr et al., 2000; Cuadrado-Corrales et al., 2006; Peoc'h et al., 2006; Satoh et al., 2006). However, detection of 14-3-3 proteins in the CSF from vCJD patients has a lower specificity and sensitivity compared with sCJD (Van Everbroeck et al., 2005). Epidemiological studies have shown that 14-3-3-negative CSF test results are significantly correlated with relatively prolonged survival in sCJD (Pocchiari et al., 2004). The total amount of 14-3-3 proteins in the CSF during neurodegeneration is in the range of ng ml–1 (Kenney et al., 2000; Aksamit et al., 2001). In sCJD, the lowest 14-3-3 levels are found at the onset and during the end stage of the disease (Green et al., 2001; Van Everbroeck et al., 2003, 2005; Sanchez-Valle et al., 2004; Shiga et al., 2006). In CJD patients, , , and isotypes can be detected in CSF samples (Wiltfang et al., 1999). 14-3-3 protein isoforms have been found in the endoplasmic reticulum (Shikano et al., 2006) and in the Golgi apparatus (Pietromonaco et al., 1996). Subcellularly, the , , , and isotypes have been found in synaptic vesicle membranes, and there are hints that some isotypes (, and ) might be located at the synaptic junction and bind to the synaptic membrane (Martin et al., 1994; Jones et al., 1995; Baxter et al., 2002). Interestingly, the cellular prion protein (PrPC) is also localized at the synaptic plasma membrane (Moya et al., 2000). 14-3-3 proteins also have a role in the conformational stabilization of other proteins and could therefore be involved in the axonal transport of prion proteins and their delivery through secretory pathways to the cell surface (Wiltfang et al., 1999; Satoh et al., 2005).
Here, we present data from a longitudinal study of BSE-infected cynomolgus monkeys. Our main goals were to determine the isoform pattern, to examine 14-3-3 kinetics and to correlate the clinical course with the occurrence of CSF 14-3-3 proteins in this NHP animal model. For the latter, we used two clinical parameters: neurological signs and body weight.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The 20 animals were housed in a microbiological containment level 3 facility of the Paul-Ehrlich-Institut's primate centre and were maintained in social groups of up to six or seven monkeys comprising stable dominants and stable subordinates. Behavioural disorders could thus be detected easily and early under these housing conditions by changed social behaviour.
The animals tested negative for common primate pathogens. The prion protein gene (PRNP) was sequenced and showed no polymorphism, and all animals were homozygous for methionine at codon 129.
The study was approved by the Hessian Animal Protection Committee before experiments started (local authority permit no. V54–19c 20/15–F107/63). All animal experiments were supervised by local authorities (Regierungspräsidum Darmstadt) and were performed in accordance with section 8 of the German Animal Protection Law in compliance with EC Directive 86/609.
All animals were observed daily for any abnormalities in movement or general behaviour by an animal caretaker and weekly by a veterinarian. EDTA-anticoagulated whole blood samples for complete haematology and CSF samples were collected at regular intervals from anaesthetized monkeys (2.5 mg xylazine/HCl kg–1 and 6 mg ketamine/HCl kg–1 administered intramuscularly). Clinical check-ups included measurements of body weight and temperature.
BSE inoculum.
The inoculum was a pool of homogenized bovine brain stems from 11 naturally infected, histopathologically and immunohistochemically confirmed cases of BSE. The BSE stock tested positive for PK-resistant prion protein (PrPres) by Western immunoblotting. The material was generously provided by the Veterinary Laboratories Agency, Weybridge, UK, and processed at the Institute for Reference Material and Measurements, Geel, Belgium. Briefly, a 90 % (w/v) brain homogenate was prepared in sucrose solution (90 % brain, 10 % sucrose solution) for oral infection, and a 10 % (w/v) brain homogenate was prepared for intracerebral infection.
Experimental BSE infection.
Six monkeys, each 3 years of age, were injected intracerebrally (right frontal lobus) with 0.25 ml 10 % BSE brain homogenate mixture corresponding to 5 mg BSE brain. All animals recovered well from the inoculation procedure. For the dietary exposure study, seven trained monkeys, each 4 years of age, were fed with muesli balls containing 5 g BSE brain homogenate. The monkeys were closely observed during food uptake (within 10 min) to confirm that each consumed the total amount of brain homogenate. Seven animals remained non-inoculated, serving as age- and sex-matched controls.
CSF specimens.
CSF was obtained from the cerebellomedullary cistern (cisterna magna) by suboccipital puncture (Spinocan disposable needle, 0.70x40 mm/22 Gx1.5''; B. Braun Melsungen AG) at regular intervals during the asymptomatic phase (two samples per year). Samples were collected monthly as soon as body weights dropped to significantly lower levels, or when changes in social behaviour were observed. Freshly obtained CSF samples were kept at 4 °C immediately after puncture, carefully tested for cell contamination, centrifuged and aliquotted (50 µl) within 2 h of collection. One aliquot was tested on the day of collection to exclude effects caused by a freeze–thaw cycle and the remaining aliquots were stored at –80 °C within 2 h of collection. Aliquots from each time point were tested five to ten times by Western immunoblotting using a panel of different antisera and monoclonal antibodies against different 14-3-3 protein isoforms to determine the overall reproducibility. Two positive controls (simian brain homogenate and recombinant human 14-3-3 protein) and one negative control (incubation of secondary antibody without primary antibody) were included.
Post-mortem examination.
As a rule, diseased monkeys were sacrificed 
28 days after the onset of gait ataxia. Two orally dosed monkeys showing no neurological symptoms were sacrificed at 365 days and one orally dosed monkey at 1460 days after infection. Two uninfected control animals were sacrificed at the ages of 3 and 5 years, respectively, serving as age-/sex-matched controls. At necropsy, the brain was taken and the left hemisphere was fixed in phosphate-buffered paraformaldehyde solution or in Carnoy's fixative, as described previously (Giaccone et al., 2000). Paraffin-embedded tissues were cut into 5 µm sections and stained with haematoxylin and eosin (H&E). Lesion profiles were determined in H&E-stained serial sections by routine histopathology. The right hemisphere was cut into ten coronary sections (6–8 mm), which were rapidly frozen on dry ice and stored at –80 °C. Tissue samples from defined areas (cortex cerebri, nucleus caudatus, thalamus, hypothalamus, pons, medulla oblongata and deep cerebellar nuclei) were taken from coronary sections of the right hemisphere and tested for the presence of PrPres and 14-3-3 proteins by Western blotting, as described below.
Reagents.
Monoclonal antibodies (mAbs) and polyclonal antisera used for the detection of 14-3-3 proteins are shown in Table 1. A recombinant human 14-3-3 protein containing an N-terminal glutathione S-transferase tag with a total molecular mass of 60 kDa (Biomol) and homogenized simian brain were used as positive controls and to determine the detection limit of our immunoassay.
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Tissue homogenization.
Defined brain areas as indicated above were taken from frozen coronary brain sections, cut into pieces and homogenized with commercially available kits according to the manufacturers' guidelines (beads from Lysing Matrix D, MP Biomedicals; homogenizing buffer from BetaPrion BSE Purification kit, Roboscreen; FastPrep homogenizer from MP Biomedicals). Homogenized tissue was cleared by two centrifugation steps. Supernatant was recovered after the last centrifugation step and protein concentrations were determined (BCA protein assay kit; Pierce). Homogenates were adjusted to a final protein concentration of 10 mg ml–1, aliquotted (50 µl) and stored at –80 °C.
PrPres extraction from homogenized brain and Western blotting.
Lyophilized PK (recombinant PK; Roche Diagnostics) was diluted in a commercially available PK buffer (BetaPrion BSE purification kit; Roboscreen). The sample was added to the PK/buffer solution to obtain a final concentration of 20 µg PK ml–1 and incubated at 37 °C for 20 min. The sample was then processed using a commercially available purification kit (BetaPrion BSE purification kit) according to the manufacturer's guidelines, loaded onto a 12 % Tris/glycine polyacrylamide gel (NuPAGE Novex 12 %; Invitrogen) and run at 200 V for 50 min [XCell SureLock Minicell (Bio-Rad) with NuPAGE MOPS SDS running buffer (Invitrogen)]. Gels were electroblotted onto a nitrocellulose membrane (semi-dry blot apparatus at 25 V for 30 min, XCell SureLock MiniCell), which was subsequently blocked with horse serum (10 %, v/v, in Tris-buffered saline containing 0.1 %, v/v, Tween 20; Gibco) and co-incubated with a 1 : 10 000 dilution of mAb 6H4 in blocking buffer together with a 1 : 100 000 dilution of anti-actin antiserum for 60 min at room temperature. Blots were incubated with a 1 : 10 000 dilution of goat anti-mouse IgG–HRP (Dianova) in blocking buffer for 45 min at room temperature, developed in chemiluminescent substrate (Supersignal, West Pico, Pierce; or ECL kit, Amersham) and visualized using ECL Hyperfilm (Amersham).
Detection and quantification of 14-3-3 proteins by Western blotting.
Ten microlitres of CSF was treated with an equal volume of 2x Laemmli buffer (Bio-Rad) at 100 °C for 5 min, loaded onto a 12 % Tris/glycine polyacrylamide gel and blotted as described above with the following modifications. Gels were electroblotted in a wet-blot apparatus (Invitrogen) and blots were developed in ECL Plus chemiluminescent substrate (Amersham) and visualized with ECL Hyperfilm (Amersham). Western blots were evaluated by visual inspection and by densitometric analyses using UN-SCAN-IT gel software (version 6.1; Silk Scientific). Values were expressed as either the percentage of a loading control (2 ng recombinant 14-3-3) or as a 14-3-3 : actin ratio.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Three other primates dosed orally with BSE were sacrificed during the incubation period on day 365 (n=2, cases #11 and #12) and day 1460 (n=1, case #13) post-infection (p.i.) to examine the PrPres tissue distribution in asymptomatic primates (see below).
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). Time courses are shown in Fig. 1(a) (cases 1, 2, 3 and 8). In five cases (1, 2, 5, 6 and 8), this altered growth preceded the onset of neurological signs (143–246 days earlier, mean 196 days). In two cases, body weight loss was seen in combination with a voracious appetite. A ruffled coat was observed in 10/10 cases long before the onset of neurological symptoms.
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summarizes the clinical data of 10 BSE-inoculated monkeys (cases #1 to #10) that developed vCJD. Another three orally dosed monkeys (cases #11 to #13) were sacrificed during the clinically asymptomatic incubation period (not shown).
The symptomatic phase of simian vCJD could thus be characterized by five different stages based on the predominant clinical symptoms: stage 1 was characterized by a disturbance of body growth, followed by stage 2 dominated by behavioural disorders, combined with a lack of coordination in stage 3. All clinical symptoms worsened in stage 4, and truncal ataxia was the main symptom during this stage. However, appetite remained good in 10/10 diseased monkeys until this stage. One monkey (case #1) rapidly progressed to stage 5 (dementia). The median duration from stage 2 until the time point of necropsy was 99 days and 121 days in intracerebrally and orally dosed animals, respectively (Table 2).
In each of the ten diseased monkeys, spongiform encephalopathy and PrPres deposits were found by histological examination of the brain (data not shown) and Western blotting of brain homogenate samples, respectively (Fig. 1b). However, florid plaques were not seen.
Evaluation of an immunoassay to detect simian 14-3-3 proteins
To establish a simian 14-3-3 protein immunoassay, we electrophoresed simian brain homogenates and blotted samples onto a nitrocellulose membrane, as described in Methods. A number of commercially available mAbs and polyclonal antisera directed against human 14-3-3 proteins (Table 1) were then tested for cross-reactivity. As shown in Fig. 2(a), antisera against the (C-16, lane 2), (FL-246, lane 3), (T-16, lane 4) and (E-12, not shown) isoforms cross-reacted with simian 14-3-3 proteins. Two mAbs recognizing all human isoforms (mAb H-8, lane 8; mAb C23-1, lane 9) also cross-reacted with simian 14-3-3 proteins. Antibody C23-1 could not recognize the simian isoform. However, there was a marked variability in titres among different batches of the above-mentioned polyclonal antisera (data not shown).
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0.2 ng recombinant 14-3-3 protein was detected in a volume of 10 µl (data not shown).
Detection of 14-3-3 proteins in CSF samples from diseased animals
mAb H-8 and polyclonal antisera C-16, C-20, E-12 and T-16 were used as standard antibodies to detect 14-3-3 protein isoforms in CSF samples. First, we analysed CSF samples obtained from BSE-infected animals showing mild neurological signs (Fig. 2b) and from uninfected age- and sex-matched controls. We readily detected 30 kDa bands in animals showing ataxia using C-16 (Figs 2b and 3a), C-20 and H-8 (Fig. 3a). The intensity of the 30 kDa band differed among samples collected at different stages of the clinical phase (Fig. 2b, lanes 3 and 4; Fig. 3). However, the and isoforms could not be detected in any examined CSF samples (data not shown). Faint 30 kDa bands were sometimes visible in CSF samples collected from uninfected controls in specimens free from blood contamination using antiserum C-16 (Fig. 2b, lane 5) or C-20.
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, columns 3 and 5), whereas in others the onset of clinical symptoms could be confirmed by 14-3-3 testing procedures within days (cases #2, #5 and #8). However, in 2/10 cases (#1 and #9), a positive CSF result was obtained distinctly earlier than clinical symptoms. Unfortunately, there was a diagnostic 14-3-3 gap (Table 2, columns 4 and 5) that ranged from 63 days in case #5 (the last time point of a 14-3-3 negative CSF was at 1978 days p.i. and the first time point of a 14-3-3-positive test was 2041 days p.i.) to 372 days in case #2. This was often due to CSF samples that were collected in between being contaminated with blood and unable to be used for 14-3-3 analyses. Nevertheless, two different isoforms could be detected by Western blotting in CSF samples collected over time in diseased primates: and isoforms (Figs 2b and 3a). We found a progressive increase in these two isoforms during the symptomatic phase, as detected by an increase in the optical density of Western blot bands (Fig. 3b, c). This increase was seen in 10/10 monkeys (Fig. 4).
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14-3-3 expression patterns in brain samples
A number of different brain localizations (medulla oblongata, pons, deep cerebellar nuclei, hypothalamus, thalamus, nucleus caudatus and cortex cerebri) were examined for the presence of PrPres and 14-3-3 protein isoforms. PrPres patterns were semi-quantitatively grouped into three classes: negative (–), weakly positive (+) and strongly positive (++). A positive correlation was found between the amount of PrPres and the 14-3-3 (Fig. 5) and - isoforms (not shown) that was not seen for and (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and has been established as an animal model to study the pathogenesis of vCJD (Herzog et al., 2004, 2005; Lasmézas et al., 2005). However, there are only a few studies reporting the detection of 14-3-3 proteins in TSE-infected primates (Hsich et al., 1996). Furthermore, the timing of appearance of 14-3-3 proteins in CSF in simian vCJD was unknown previous to this study. Therefore, we initiated a 14-3-3 protein time-course study in BSE-infected cynomolgus monkeys. As early clinical symptoms are sometimes difficult to detect in an NHP model, detection of 14-3-3 proteins in CSF may be more suitable for diagnosis of the disease during the early stages. Finally, detection of 14-3-3 proteins in CSF was evaluated as a humane end point. There was variation in the incubation periods of intracerebrally dosed primates. In intracerebrally infected animals, this was mostly due to one rapid progressor, whereas the 25th to 75th percentile range was lower: 1338–2123 days p.i. Exogenous factors such as stress may have been responsible for the early onset of clinical signs in this single case. Moreover, the variation in incubation time was much lower in orally dosed primates compared with intracerebrally dosed animals. This was mostly due to the different doses that were given to the monkeys. Orally dosed primates in this study received a high dose, which contributed to a low variability in incubation time, whereas the 5 mg intracerebral dose did not represent a high dose, thereby leading to a prolonged incubation period compared with animals infected with 50 mg each (data not shown).
The symptomatic phase of simian vCJD was characterized by five different stages based on the predominant clinical symptoms. However, stage 1 (disturbed body growth, no neurological signs, CSF-negative for 14-3-3 proteins) was seen in 5/6 intracerebrally inoculated monkeys, but only in 1/4 orally dosed monkeys, indicating that the clinical course may be influenced by the inoculation route. The efficacy of intracerebral versus oral inoculation will be described elsewhere, involving a larger number of infected cynomolgus monkeys.
The underlying mechanisms for the disturbed body growth are unknown. Body weight loss has been described in clinically symptomatic vCJD patients (Zeidler et al., 1997) and in intracerebrally infected primates (Williams et al., 2007), but to our knowledge there has been no description so far of its onset occurring several months before the onset of early neurological signs. Body weight in cynomolgus monkeys is influenced by social ranking when animals are kept together in groups: dominants always have higher body weight than subordinates, explaining the wide range in body weights of the described simian vCJD cases. For example, in one infected subordinate, body weight dropped from 5 to 3 kg, whereas body weight decreased from 8 to 6 kg in an infected dominant (Table 2). We can exclude that the observed changes were caused by alterations in the social ranking of our animal groups. It is tempting to speculate that this phenomenon was caused by disturbed hypothalamic regulation, as food intake is regulated by hypothalamic nuclei (Cota et al., 2006; Morton et al., 2006). We therefore examined the hypothalamus from a monkey that was sacrificed during this stage (case #13), but found no evidence of PrPres. Moreover, CSF samples were negative for 14-3-3 proteins throughout stage 1 in 6/6 simian vCJD cases with altered body weight, indicating that PrPres-induced brain degeneration did not cause this phenomenon. Alternatively, very low amounts of PrPres might cause neuronal defects, leading to disturbed hypothalamic regulation. However, the fact that food intake was not disturbed and appetite remained good in all monkeys during stage 1 also did not support this explanation. Thus, the underlying mechanism remains to be elucidated.
Post-mortem examination of the brain revealed large vacuoles in neurons from different areas of the brain. However, florid plaques typical of human vCJD (Ironside, 1998), which were described in cynomolgus monkeys intracerebrally infected with BSE (Lasmézas et al., 1996), were not found in this study either in intracerebrally or in orally dosed cynomolgus monkeys. Interestingly, Williams et al. (2007) were also unable to detect amyloid plaques in squirrel monkeys that were intracerebrally infected with vCJD brain homogenate when they were adults. They hypothesized that the absence of florid plaques in their animal model was due to the host species, an explanation that cannot account for the absence in our primate model.
Another characteristic was non-glycosylated double bands in brain homogenates digested with PK by Western blotting (Fig. 1b, case #6). These double bands were found in the thalamus of all cases when 4–12 % gels were used (data not shown). We are currently examining different brain areas for the presence of this double band using modified laboratory protocols to explain this phenomenon.
14-3-3 proteins form homo- and heterodimers: the isoform monomer has a molecular mass of 32 kDa (Takahashi et al., 1999) and all other isoform monomers have a molecular mass of 28–30 kDa (Martin et al., 1993). At the beginning of the present study, we used a Western blot assay to detect simian 14-3-3 protein isoforms. For this purpose, a number of commercially available antibodies and antisera were tested for cross-reactivity using simian brain homogenate as antigen. However, there were a number of samples without any red blood cell contaminations from both infected and uninfected monkeys in which sequential testing gave rise to contradictory results: in one instance negative and in the other weakly positive, especially when antiserum C-20 was used. This faint 30 kDa band was regarded as a false-positive, as it also occurred in CSF samples from healthy control animals. Finally, there was great variability in titres among different batches of the above-mentioned commercially available polyclonal antiserum. The use of standardized positive controls is therefore highly recommended to exclude false-negative test results (Aksamit, 2003). False-positive results were observed in samples contaminated with red blood cells. These samples were excluded from further analyses.
All CSF samples collected during stage 1 were negative for all examined 14-3-3 protein isoforms, but CSF samples were positive for and isoforms in animals showing neurological signs (stages 2–5). 14-3-3 and - isoforms could not be detected in any of the ten simian vCJD cases, most probably because concentrations were below the detection limit of our immunoassay or due to their complete absence in the NHP animal model. Concentrations of 14-3-3 proteins progressively increased over time in BSE-infected monkeys. This is not in line with what is known from 14-3-3 kinetics in sCJD patients (Green et al., 2001; Van Everbroeck et al., 2003, 2005; Shiga et al., 2006) and may suggest a difference between sCJD and vCJD.
Neurons constitutively express the , , and isoforms, but do not constantly express the or isoforms (Satoh et al., 2004). 14-3-3 proteins are also expressed by astrocytes, microglia and oligodendrocytes (Satoh et al., 2004). A changed 14-3-3 expression pattern has been described under pathological conditions. The isoform is upregulated in reactive human astrocytes (Satoh et al., 2004) and is detectable in CSF samples from sCJD patients (Takahashi et al., 1999). In murine scrapie, 14-3-3 isoform expression is decreased in some neuroanatomical areas and increased in others (Baxter et al., 2002). Indeed, in this study, we found higher 14-3-3 and - levels (data not shown) in brain areas with large amounts of PrPres deposits. It is tempting to speculate that the large amounts of the isoform was due to reactive astrocytes, but this assumption can only be proved by immunohistochemical methods, which we have recently initiated. However, no differences could be detected when we examined the levels of the 14-3-3 and - isoforms.
We conclude that the detection of 14-3-3 proteins in CSF is a reliable and excellent tool to characterize the course of brain degeneration and to standardize a humane end point in simian vCJD. 14-3-3 levels progressively increased in simian vCJD. However, there were differences in the clinical course between orally and intracerebrally infected animals that may influence the detection of other biomarkers. In addition, the brain pathology of simian vCJD was characterized by the absence of florid plaques.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Aksamit, A. J., Preissner, C. M. & Homburger, H. A. (2001). Quantitation of 14-3-3 and neuron-specific enolase proteins in CSF in Creutzfeldt–Jakob disease. Neurology 57, 728–730.
Baker, H. F., Ridley, R. M. & Wells, G. A. H. (1993). Experimental transmission of BSE and scrapie to the common marmoset. Vet Rec 132, 403–406.[Abstract]
Baxter, H. C., Liu, W. G., Forster, J. L., Aitken, A. & Fraser, J. R. (2002). Immunolocalisation of 14-3-3 isoforms in normal and scrapie-infected murine brain. Neuroscience 109, 5–14.[CrossRef][Medline]
Berg, D., Holzmann, C. & Riess, O. (2003). 14-3-3 proteins in the nervous system. Nat Rev Neurosci 4, 752–762.[CrossRef][Medline]
Bons, N., Mestre-Frances, N., Belli, P., Cathala, F., Gajdusek, D. C. & Brown, P. (1999). Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents. Proc Natl Acad Sci U S A 96, 4046–4051.
Burkhard, P. R., Sanchez, J. C., Landis, T. & Hochstrasser, D. F. (2001). CSF detection of the 14-3-3 protein in unselected patients with dementia. Neurology 56, 1528–1533.
Cathala, F., Court, L., Hauw, J. J., Escourelle, R., Rohmer, F. & Castaigne, P. (1975). Clinical studies in primates inoculated with kuru and Creutzfeldt–Jakob agents. Adv Neurol 10, 319–339.[Medline]
Collins, S., Boyd, A., Fletcher, A., Gonzales, M., McLean, C. A., Byron, K. & Masters, C. L. (2000). Creutzfeldt–Jakob disease: diagnostic utility of 14-3-3 protein immunodetection in cerebrospinal fluid. J Clin Neurosci 7, 203–208.[CrossRef][Medline]
Cota, D., Proulx, K., Blake Smith, K. A., Kozma, S. C., Thomas, G., Woods, S. C. & Seeley, R. J. (2006). Hypothalamic mTOR signaling regulates food intake. Science 312, 927–930.
Cuadrado-Corrales, N., Jimenez-Huete, A., Albo, C., Hortiguela, R., Vega, L., Cerrato, L., Sierra-Moros, M., Rabano, A., de Pedro Cuesta, J. & Calero, M. (2006). Impact of the clinical context on the 14-3-3 test for the diagnosis of sporadic CJD. BMC Neurol 6, 25[CrossRef][Medline]
Gajdusek, D. C. & Gibbs, C. L., Jr (1967). Transmission and passage of experimental "Kuru" to chimpanzees. Science 155, 212–214.[Medline]
Gajdusek, D. C. & Gibbs, C. L., Jr (1971). Transmission of two subacute spongiform encephalopathies of man (kuru and Creutzfeldt–Jakob disease) to new world monkeys. Nature 230, 588–591.[CrossRef][Medline]
Gajdusek, D. C. & Gibbs, C. J., Jr (1975). Familial and sporadic chronic neurological degenerative disorders transmitted from man to primates. Adv Neurol 10, 291–317.[Medline]
Gajdusek, D. C., Gibbs, C. J., Jr & Alpers, M. (1966). Experimental transmission of a Kuru-like syndrome to chimpanzees. Nature 209, 794–795.[CrossRef][Medline]
Geschwind, M. D., Martindale, J., Miller, D., DeArmond, S. J., Uyehara-Lock, J., Gaskin, D., Kramer, J. H., Barbaro, N. M. & Miller, B. L. (2003). Challenging the clinical utility of the 14-3-3 protein for the diagnosis of sporadic Creutzfeldt–Jakob disease. Arch Neurol 60, 813–817.
Giaccone, G., Canciani, B., Puoti, G., Rossi, G., Goffredo, D., Lussich, S., Fociani, P., Tagliavini, F. & Bugiani, O. (2000). Creutzfeldt–Jakob disease: Carnoy's fixative improves the immunohistochemistry of the proteinase K-resistant prion protein. Brain Pathol 10, 31–37.[Medline]
Gibbs, C. J., Jr, Gajdusek, D. C., Asher, D. M., Alpers, M. P., Beck, E., Daniel, P. M. & Matthews, W. B. (1968). Creutzfeldt–Jakob disease (spongiform encephalopathy): transmission to the chimpanzee. Science 161, 388–389.
Gibbs, C. J., Jr, Amyx, H. L., Bacote, A., Masters, C. L. & Gajdusek, D. C. (1980). Oral transmission of kuru, Creutzfeldt–Jakob disease, and scrapie to nonhuman primates. J Infect Dis 142, 205–208.[Medline]
Giraud, P., Biacabe, A. G., Chazot, G., Later, R., Joyeux, O., Moene, Y. & Perret-Liaudet, A. (2002). Increased detection of 14-3-3 protein in cerebrospinal fluid in sporadic Creutzfeldt–Jakob disease during the disease course. Eur Neurol 48, 218–221.[CrossRef][Medline]
Green, A. J., Thompson, E. J., Stewart, G. E., Zeidler, M., McKenzie, J. M., MacLeod, M. A., Ironside, J. W., Will, R. G. & Knight, R. S. (2001). Use of 14-3-3 and other brain-specific proteins in CSF in the diagnosis of variant Creutzfeldt–Jakob disease. J Neurol Neurosurg Psychiatry 70, 744–748.
Green, A. J. E., Ramljak, S., Muller, W. E. G., Knight, R. S. G. & Schroder, H. C. (2002). 14-3-3 in the cerebrospinal fluid of patients with variant and sporadic Creutzfeldt–Jakob disease measured using capture assay able to detect low levels of 14-3-3 protein. Neurosci Lett 324, 57–60.[CrossRef][Medline]
Herzog, C., Sal
s, N., Etchegaray, N., Charbonnier, A., Freire, S., Dormont, D., Deslys, J.-P. & Lasmézas, C. I. (2004). Tissue distribution of bovine spongiform encephalopathy agent in primates after intravenous or oral infection. Lancet 363, 422–428.[CrossRef][Medline]
Herzog, C., Riviere, J., Lescoutra-Etchegaray, N., Charbonnier, A., Leblanc, V., Sales, N., Deslys, J.-P. & Lasmézas, C. I. (2005). PrPTSE distribution in a primate model of variant, sporadic, and iatrogenic Creutzfeldt–Jakob disease. J Virol 79, 14339–14345.
Hsich, G., Kenney, K., Gibbs, C. J., Lee, K. H. & Harrington, M. G. (1996). The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform encephalopathies. N Engl J Med 335, 924–930.
Ironside, J. W. (1998). Prion diseases in man. J Pathol 186, 227–234.[CrossRef][Medline]
Jones, D. H., Martin, H., Madrazo, J., Robinson, K. A., Nielsen, P., Roseboom, P. H., Patel, Y., Howell, S. A. & Aitken, A. (1995). Expression and structural analysis of 14-3-3 proteins. J Mol Biol 245, 375–384.[CrossRef][Medline]
Kenney, K., Brechtel, C., Takahashi, H., Kurohara, K., Anderson, P. & Gibbs, C. J., Jr (2000). An enzyme-linked immunosorbent assay to quantify 14-3-3 proteins in the cerebrospinal fluid of suspected Creutzfeldt–Jakob disease patients. Ann Neurol 48, 395–398.[CrossRef][Medline]
Lasmézas, C. I. (2003). The transmissible spongiform encephalopathies. Rev Sci Tech 22, 23–36.[Medline]
Lasmézas, C. I., Deslys, J.-P., Demaimay, R., Adjou, K. T., Lamoury, F., Dormont, D., Robain, O., Ironside, J. & Hauw, J. J. (1996). BSE transmission to macaques. Nature 381, 743–744.[CrossRef][Medline]
Lasmézas, C. I., Fournier, J.-G., Nouvel, V., Boe, H., Marce, D., Lamoury, F., Kopp, N., Hauw, J.-J., Ironside, J. & other authors (2001). Adaptation of the bovine spongiform encephalopathy agent to primates and comparison with Creutzfeldt–Jakob disease: implications for human health. Proc Natl Acad Sci U S A 98, 4142–4147.
Lasmézas, C. I., Comoy, E., Hawkins, S., Herzog, C., Mouthon, F., Konold, T., Auvré, F., Correia, E., Lescoutra-Etchegaray, N. & other authors (2005). Risk of oral infection with bovine spongiform encephalopathy agent in primates. Lancet 365, 781–783.[Medline]
Martin, H., Patel, Y., Jones, D., Howell, S., Robinson, K. & Aitken, A. (1993). Antibodies against the major brain isoforms of 14-3-3 protein. An antibody specific for the N-acetylated amino-terminus of a protein. FEBS Lett 331, 296–303.[CrossRef][Medline]
Martin, H., Rostas, J., Patel, Y. & Aitken, A. (1994). Subcellular localisation of 14-3-3 isoforms in rat brain using specific antibodies. J Neurochem 63, 2259–2265.[Medline]
Masters, C. L., Kakulas, B. A., Alpers, M. P., Gajdusek, D. C. & Gibbs, C. J., Jr (1976). Preclinical lesions and their progression in the experimental spongiform encephalopathies (kuru and Creutzfeldt–Jakob disease) in primates. J Neuropathol Exp Neurol 35, 593–605.[Medline]
Morton, G. J., Cummings, D. E., Baskin, D. G., Barsh, G. S. & Schwartz, M. W. (2006). Central nervous system control of food intake and body weight. Nature 443, 289–295.[CrossRef][Medline]
Moya, K. L., Sales, N., Hassig, R., Creminon, C., Grassi, J. & Di Giamberardino, L. (2000). Immunolocalization of the cellular prion protein in normal brain. Microsc Res Tech 50, 58–65.[CrossRef][Medline]
Peoc'h, K., Delasnerie-Laupretre, N., Beaudry, P. & Laplanche, J.-L. (2006). Diagnostic value of CSF 14-3-3 detection in sporadic CJD diagnosis according to the age of the patient. Eur J Neurol 13, 427–428.[CrossRef][Medline]
Pietromonaco, S. F., Seluja, G. A., Aitken, A. & Elias, L. (1996). Association of 14-3-3 proteins with centrosomes. Blood Cells Mol Dis 22, 225–237.[CrossRef][Medline]
Pocchiari, M., Puopolo, M., Croes, E. A., Budka, H., Gelpi, E., Collins, S., Lewis, V., Sutcliffe, T., Guilivi, A. & other authors (2004). Predictors of survival in sporadic Creutzfeldt–Jakob disease and other human transmissible spongiform encephalopathies. Brain 127, 2348–2359.
Sanchez-Valle, R., Graus, F. & Saiz, A. (2004). Discrepancies in the clinical utility of the 14-3-3 protein for the diagnosis of sporadic Creutzfeldt–Jakob disease. Arch Neurol 61, 604
Satoh, J., Yamamura, T. & Arima, K. (2004). The 14-3-3 protein epsilon isoform expressed in reactive astrocytes in demyelinating lesions of multiple sclerosis binds to vimentin and glial fibrillary acidic protein in cultured human astrocytes. Am J Pathol 165, 577–592.
Satoh, J., Onue, H., Arima, K. & Yamamura, T. (2005). The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein. J Neuropathol Exp Neurol 64, 858–868.[Medline]
Satoh, K., Shirabe, S., Eguchi, H., Tsujino, A., Eguchi, K., Satoh, A., Tsuhihata, M., Niwa, M., Katamine, S. & other authors (2006). 14-3-3 protein, total tau and phosphorylated tau in cerebrospinal fluid of patients with Creutzfeldt–Jakob disease and neurodegenerative disease in Japan. Cell Mol Neurobiol 26, 45–52.[CrossRef][Medline]
Schätzl, H. M., Da Costa, M. M., Taylor, L., Cohen, F. E. & Prusiner, S. B. (1995). Prion protein gene variation among primates. J Mol Biol 245, 362–374.[CrossRef][Medline]
Shiga, Y., Wakabayashi, H., Miyazawa, K., Kido, H. & Itoyama, Y. (2006). 14-3-3 protein levels and isoform patterns in the cerebrospinal fluid of Creutzfeldt–Jakob disease patients in the progressive and terminal stages. J Clin Neurosci 13, 661–665.[CrossRef][Medline]
Shikano, S., Coblitz, B., Wu, M. & Li, M. (2006). 14-3-3 proteins: regulation of endoplasmic reticulum localization and surface expression of membrane proteins. Trends Cell Biol 16, 370–375.[CrossRef][Medline]
Takahashi, H., Iwata, T., Kitagawa, Y., Takahashi, R. H., Sato, Y., Wakabayashi, H., Takashima, M., Kido, H., Nagashima, K. & other authors (1999). Increased levels of and isoforms of 14-3-3 proteins in cerebrospinal fluid in patients with Creutzfeldt–Jakob disease. Clin Diagn Lab Immunol 6, 983–985.[Medline]
Van Everbroeck, B., Quoilin, S., Boons, J., Martin, J. J. & Cras, P. (2003). A prospective study of CSF markers in 250 patients with possible Creutzfeldt–Jakob disease. J Neurol Neurosurg Psychiatr 74, 1210–1214.
Van Everbroeck, B., Boons, J. & Cras, P. (2005). Cerebrospinal fluid biomarkers in Creutzfeldt–Jakob disease. Clin Neurol Neurosurg 107, 355–360.[CrossRef][Medline]
Weissmann, C. (2005). Birth of a prion protein: spontaneous generation revisited. Cell 122, 165–168.[CrossRef][Medline]
Will, R. G., Zeidler, M., Brown, P., Harrington, M., Lee, K. H. & Kenney, K. L. (1996). Cerebrospinal fluid test for new-variant Creutzfeldt–Jakob disease. Lancet 348, 955[Medline]
Will, R. G., Zeidler, M., Stewart, G. E., Macleod, M. A., Ironside, J. W., Cousens, S. N., Mackenzie, J., Estibeiro, K., Green, A. J. & Knight, R. S. (2000). Diagnosis of new variant Creutzfeldt–Jakob disease. Ann Neurol 47, 575–582.[CrossRef][Medline]
Williams, L., Brown, P., Ironside, J., Gibson, S., Will, R., Ritchie, D., Kreil, T. R. & Abee, C. (2007). Clinical, neuropathological and immunohistochemical features of sporadic and variant forms of Creutzfeldt–Jakob disease in the squirrel monkey (Saimiri sciureus). J Gen Virol 88, 688–695.
Wiltfang, J., Otto, M., Baxter, H. C., Bodemer, M., Steinacker, P., Bahn, E., Zerr, I., Kornhuber, J., Kretzschmar, H. A. & other authors (1999). Isoform pattern of 14-3-3 proteins in the cerebrospinal fluid of patients with Creutzfeldt–Jakob disease. J Neurochem 73, 2485–2490.[CrossRef][Medline]
Zeidler, M., Stewart, G. E., Barraclough, C. R., Bateman, D. E., Bates, D., Burn, D. J., Colchester, A. C., Durward, W., Fletcher, N. A. & other authors (1997). New variant Creutzfeldt–Jakob disease: neurological features and diagnostic tests. Lancet 350, 903–907.[CrossRef][Medline]
Zerr, I., Schulz-Schaeffer, W. J., Giese, A., Bodemer, M., Schroter, A., Henkel, K., Tschampa, H. J., Windl, O., Pfahlberg, A. & other authors (2000). Current clinical diagnosis in Creutzfeldt–Jakob disease: identification of uncommon variants. Ann Neurol 48, 323–329.[CrossRef][Medline]
Received 1 May 2007;
accepted 9 August 2007.
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