Neurobiology of Aging 35 (2014) 1177e1188

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Association of prion protein genotype and scrapie prion protein type with cellular prion protein charge isoform profiles in cerebrospinal fluid of humans with sporadic or familial prion diseases Matthias Schmitz a, *, Katharina Lüllmann a, Saima Zafar a, Elisabeth Ebert a, Marie Wohlhage a, Panteleimon Oikonomou a, Markus Schlomm a, Eva Mitrova b, Michael Beekes c, Inga Zerr a a

Department of Neurology, Clinical Dementia Center and DZNE Georg-August University, Göttingen, Germany Slovak Medical University, Bratislava, Slovakia c Robert Koch-Institute, FG 14 e AG 5: Work Group Unconventional Pathogens and Their Inactivation, Division of Applied Infection Control and Nosocomial Hygiene, Berlin, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 July 2013 Received in revised form 31 October 2013 Accepted 10 November 2013 Available online 16 November 2013

The present study investigates whether posttranslational modifications of cellular prion protein (PrPC) in the cerebrospinal fluid (CSF) of humans with prion diseases are associated with methionine (M) and/or valine (V) polymorphism at codon 129 of the prion protein gene (PRNP), scrapie prion protein (PrPSc) type in sporadic Creutzfeldt-Jakob disease (sCJD), or PRNP mutations in familial Creutzfeldt-Jakob disease (fCJD/E200K), and fatal familial insomnia (FFI). We performed comparative 2-dimensional immunoblotting of PrPC charge isoforms in CSF samples from cohorts of diseased and control donors. Mean levels of total PrPC were significantly lower in the CSF from fCJD patients than from those with sCJD or FFI. Of the 12 most abundant PrPC isoforms in the examined CSF, one (IF12) was relatively decreased in (1) sCJD with VV (vs. MM or MV) at PRNP codon 129; (2) in sCJD with PrPSc type 2 (vs. PrPSc type 1); and (3) in FFI versus sCJD or fCJD. Furthermore, truncated PrPC species were detected in sCJD and control samples without discernible differences. Finally, serine 43 of PrPC in the CSF and brain tissue from CJD patients showed more pronounced phosphorylation than in control donors. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Biomarker Creutzfeldt-Jakob disease PrPC charge isoforms Truncated PrPC forms

1. Introduction Creutzfeldt-Jakob disease (CJD), Gerstmann-StrausslerScheinker syndrome, fatal familial insomnia (FFI), and kuru (Gambetti et al., 2003a, 2003b) are human transmissible spongiform encephalopathies (TSE), or prion diseases. CJD occurs in sporadic, familial, and iatrogenic forms. Familial CJD (fCJD) is linked to pathogenic mutations in the prion protein (PrP) gene (PRNP). The most common fCJD mutation is located at codon 200 and results in a substitution of lysine for glutamate (called E200K mutation) (Hsiao et al., 1991). Two allelic forms of PRNP encode either methionine (M) or valine (V) at codon 129. With respect to this polymorphism at PRNP codon 129, about 40%

* Corresponding author at: Department of Neurology, Georg-August-University, National Reference Center for TSE, Robert-Koch-Str.40, 37075 Gottingen, Germany. Tel.: þ49 551 39 10454; fax: þ49 551 39 7020. E-mail address: [email protected] (M. Schmitz). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2013.11.010

of the Caucasian population is homozygous for methionine, about 50% methionine and/or valine heterozygous, and about 10% homozygous for valine (these genotypes are referred to in the following text as MM, MV, and VV genotypes). Six molecular subtypes of sporadic CJD (sCJD) have been defined based on a combination of these genotypes, with 2 forms of pathologic PrP (known as PrPSc type 1 and PrPSc type 2) that are found in sCJD patients (Cali et al., 2006; Parchi et al., 1999). The physiological function(s) of PrPC are not yet completely understood. PrPC is expressed in caveolin-containing microdomains (Peters et al., 2003), which are involved in signal transduction processes (Althaus et al., 2008; Schmitz et al., 2010a, 2011). The protein may also have a protective function under cellular stress conditions (Brown et al., 1997; Martins and Brentani, 2002; Weise et al., 2004, 2006) and play a role in Alzheimer’s Disease (Schmitz et al., 2014). A variety of PrPC isoforms is generated by posttranslational modifications that include differential glycosylation and phosphorylation. Human PrPC contains 2 N-glycosylation sites at

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residue 181 and 197 (Lawson et al., 2005; Rogers et al., 1990). Diglycosylated (36 kDa), monoglycosylated (33 kDa), and unglycosylated cerebral PrPC isoforms (27 kDa) (Beringue et al., 2003; Zou et al., 2003) can be distinguished with western blotting. In a previous study we showed that human cerebrospinal fluid (CSF) contains several different forms of truncated PrPC in addition to full-length PrPC (Schmitz et al., 2010b). Such truncated PrPC fragments may be generated in a late compartment of the secretory pathway (Walmsley et al., 2009) and after reaching the cell surface (Watt et al., 2005). It has been reported that PrPC is mainly endoproteolysed at amino-acids 110/111 and 90/91, resulting in a 18 kDa carboxyl-terminal C1-, a 9 kDa amino-terminal N1-, a 21 kDa C2-, and a 7 kDa N2 fragment (Chen et al., 1995; Sunyach et al., 2007). The physiological function of these N-terminally truncated PrPC forms is unclear. One possibility might be a role in signal transduction (Chen et al., 2003; Mouillet-Richard et al., 2000). Additionally, it has been suggested that the C1 and C2 fragments of PrPC exert protection against oxidative stress (Sunyach et al., 2007; Watt and Hooper, 2005; Watt et al., 2005). The composition of differentially glycosylated or truncated PrPC forms in CSF from patients with human prion diseases has been examined in only a few studies so far (Castagna et al., 2002; Schmitz et al., 2010b). Because CSF may provide a suitable analyte for both the detection of TSE biomarkers (Gawinecka et al., 2012; Zerr et al., 1998) and a better understanding of neurodegenerative brain diseases (Tapiola et al., 2009; Zerr et al., 1996), this scarcity of studies appears somewhat surprising. We therefore performed a comprehensive analysis of PrPC and its isoforms in CSF from patients with different human prion diseases and PRNP genotypes: We determined the concentrations of total PrPC in CSF from patients with sCJD (MM, MV, and VV genotypes), fCJD (E200K), or FFI, and in donor controls. PrPC charge isoform patterns were then comparatively characterized by 2dimensional immunoblotting using different PrP antibodies and CSF samples from cohorts of sCJD, fCJD, or FFI patients and donors without prion disease. Finally, we examined the occurrence of truncated PrPC isoforms and the phosphorylation of PrPC at serine 43 in CSF from sCJD patients and control donors. 2. Methods 2.1. Patients and CSF sample collection The cohort of patients in this study consisted of 46 sCJD, 16 fCJD (E200K), and 12 FFI (D178N) cases, and 30 control donors without prion disease. All sCJD (26 female, 20 male; aged 23e85 years; mean age 65  1.5 years at notification; 16 MM, 15 MV, 15 VV), E200K (9 MM and 7 MV, 10 female, 6 male; aged 52e73 years; mean age 57  1.4 years), and FFI patients (9 males, 3 female; aged 55e85 years; 9 MM, 3 MV) were classified as definite cases by neuropathological examinations or as probable CJD cases according to diagnostic consensus criteria (WHO, 1998; Zerr et al., 2000, 2009). CSF from E200K cases was provided by the Department of Prion Diseases, Slovak Medical University, Bratislava, Slovakia. Control donors (16 female, 14 male; aged 33e87 years; mean age 67  2.7 years at notification) are patients with an either clinically or pathologically defined alternative diagnosis. The PRNP codon 129 genotypes were matched. After collection, CSF was aliquoted in 1 mL portions in Eppendorf cups (1.5 mL) and stored at 80  C. Blood-stained CSF samples were excluded from the study and we avoided thawing and refreezing the same samples more than 8 times. The analysis of the codon 129 genotype of PRNP was performed after isolation of genomic DNA from blood samples according to standard methods (Windl et al., 1999).

The study was conducted according to the revised Declaration of Helsinki and Good Clinical Practice guidelines. Informed consent was given by all study participants or their legal next of kin. 2.2. Brain samples Brain tissue was obtained from patients with sCJD and from control subjects without prion disease. All sCJD (3 female, 5 male; aged 42e77 years; mean age 65  2.5 years; 4 MM1 and 4 VV). Control donors (2 female, 6 male; aged 46e59 years; mean age 54  2.7 years) are patients with an either clinically or pathologically defined alternative diagnosis. 2.3. Enzymatic deglycosylation of PrPC CSF samples were deglycosylated using a glycoprotein deglycosylation kit (Calbiochem and Merck, Darmstadt, Hesse, Germany). The procedure was carried out following the manufacturer’s instructions. After addition of reaction and denaturation buffer, CSF samples were heated for 5 minutes at 95  C. After cooling to room temperature N-glycosidase F was added. Thereafter, the samples were incubated for 3 hours at 37  C. 2.4. Determination of the PrP concentration We used an enzyme linked immunosorbent assay (ELISA), which is based on the Luminex xMAP technology, to determine the PrP concentration. All chemicals and equipment were obtained from (Bio-Rad, Munich, Germany). Sodium azide was removed by using Micro Bio-Spin 6 Chromatography Columns. The PrP-specific antibody 8G8 (SPI-Bio, Montigny Le Bretonneux Paris, France), used as a detection antibody, was coupled using an Amine Coupling Kit on COOH-Beads 044 following the manufacturer’s instruction. Bead-coupling rate was determined by using a biotinylated antibody (1:1000, Goat Anti Mouse IgG, Label: Biotin, Thermo Scientific and Pierce, Bonn, Germany) that varies between 15,000 and 20,000 fluorescence intensities (FI). The PrP-specific antibody SAF32 (SPI-Bio), used as a quantitative antibody, was biotinylated by using the ChromaLink Biotin Labeling Kit (Solulink, San Diego, USA). CSF samples (50 mL, diluted 1:100) were incubated overnight with the bead-coupled detection antibody (diluted 1:50). After washing in Bioplex ProII Wash Station, the samples were incubated with 50 mL of biotinylated antibody (SAF32, diluted 1:200). PrPC concentration was measured by using the Bioplex 200 system. In order to confirm our results, a statistically robust number of samples were analyzed by a commercial BetaPrion BSE-ELISA Test Kit (AJ Roboscreen, Leipzig, Saxony, Germany). The kit is based on ELISA techniques and is eligible for a rapid BSE test for the qualitative determination of resistant prion protein in the brain of cattle and sheep. We omitted the proteinase (PK) digestion step. Our aim was to measure the concentration of total PrP (which is almost identical to PrPC in our assay because the amount of misfolded prion protein in CSF is extremely low) in the CSF as reported before (Meyne et al., 2009). The CSF samples were applied undiluted and measured according to the manufacturer’s instruction. 2.5. Two-dimensional gel electrophoresis For 2-dimensional analysis of CSF, samples were precipitated with methanol and acetone at the ratio of 9:1 overnight. The supernatant was removed after centrifugation for 30 minutes at 14,000 rpm (Eppendorf Rotor F45-30-11, Hamburg, Germany). Afterward, cell pellets were dried for 5 minutes and resolved in 150-mL rehydration buffer (7 M urea, 2 M thiourea, 15 mM

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dithiothreitol [DTT], 4% CHAPS, 0.2% Bio-Lyte) for first-dimension isoelectric focusing (IEF). IEF was performed on a 7-cm immobilized pH gradient strip (pH 3e10, non-linear); for SAF70 (pH 3e10 linear) (Bio-Rad) by applying approximately 40e50 mg of total proteins per strip (w15 ng PrPC per strip). The IEF was initiated at 200 V for 1 hour, followed by ramping at 500 V for 1 hour, 1000 V for 1.5 hour, and final focusing at 5000 V for a total of 50,000-volt hours. Subsequently, proteins were reduced on the immobilized pH gradient strip in a denaturation buffer containing 6 M urea, 2% sodium dodecyl sulphate (SDS), 30% glycerol, 2% DTT for 20 minutes and alkylated in the same buffer supplemented with bromophenol blue, and 2.5% iodoacetamide instead of DTT for further 20 minutes. Equilibrated strips were placed on top of vertical 12% polyacrylamide gels and electrophoresis was started. 2.6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting For western blotting we used the following PrP antibodies: SAF32, SAF70, 12F10, and Ser 43 P-PrP (SPI-Bio, Montigny Le Bretonneux Paris, France) diluted 1:300-1:500; 3B5 (Walter Bodemer, Primatenzentrum, Goettingen, Lower Saxony, Germany) and 3F4 (RKI, Berlin, Germany) diluted 1:1000. Samples were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (12%e15% wt/vol polyacrylamide) and transferred to polyvinylidene difluoride hydrobond-P membranes (Amersham, Freiburg, Baden Württemberg, Germany) using a semi-dry transblot cell for 70 minutes at 12 volts (Bio-Rad). Protein-labeled polyvinylidene difluoridemembranes were blocked with 5% dried milk in phosphate buffered saline and 0.1% Tween-20 (PBST) for 1 hour at room temperature. Subsequently, membranes were probed with the antibodies of interest overnight at 4  C. Membranes were rinsed in phosphate-buffered saline with 0.05% Tween 20 and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, Leipzig, Saxony, Germany) (diluted 1:10,000) for 1 hour. Protein bands were visualized after immersion of the membranes in enhanced chemiluminescence detection system solution using Chemicon system (Bio-Rad). 2.7. Signal quantification and statistical analysis Quantification of the signal intensities of all PrPC isoforms was performed using the software Total Lab Quant. Total PrPC signals of all PrPC forms were defined as 100%. Each PrPC spot was calculated as percentage of the total PrPC amount. Using the statistics program GraphPad Prism (version 6.0), the PrPC isoform ratios of different patient groups were tested for significant differences. Because our values were not normally distributed we used the Wilcoxon-Mann-Whitney Test (Mann-Whitney-U Test) for statistical data analysis. p-values were calculated from means of independently performed replicates, and resulting p-values below 0.05 were considered to indicate significant differences between compared sample groups. The standard deviation of the mean was calculated for individual samples to assess the variations between individuals of the same group and depicted as error bars. 3. Results 3.1. Decreased mean levels of total PrPC in CSF from fCJD (E200K) patients Limited PK digestion of CSF from a control donor and sCJD patient using the PrP antibody 3F4 showed similar PK sensitivity

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of PrP. The limit of PK tolerance was at approximately 3 mg PK/mL. We were not able to detect PK-resistant PrPSc in the CSF, not from sCJD (Fig. 1A), fCJD or from FFI patients (data not shown). Thus we concluded that the western blot signals in our subsequent analyses represented PrPC. To characterize the glycosylation status of PrPC, we examined a non-infectious brain sample and 2 CSF samples from sCJD (MM) and control patients by western blotting. In brain tissue we detected PrPC isoforms that represented 3 different states of glycosylation. The band at approximately 36 kDa is considered to be diglycosylated, that at 33 kDa as being monoglycosylated, and that at 27 kDa as unglycosylated PrPC. In CSF we identified 2 PrPC bands at approximately 36 kDa and 27 kDa, considered to represent diglycosylated and unglycosylated PrPC, respectively. This is substantiated by enzymatic deglycosylation of the CSF samples, which produced a shift of the upper molecular band from 36 kDa to 27 kDa, corresponding from the molecular weight to unglycosylated recombinant PrPC (Fig. 1B). To determine whether the concentration of total PrPC in CSF varies with the PRNP genotype, PrPSc type or between sporadic and familial human prion diseases, CSF samples were analyzed using a PrP assay based on the Luminex xMAP technology, with 8G8 as a detection and SAF32 as a quantitative antibody. This revealed a significantly lower mean concentration of PrPC in CSF from fCJD patients than in sCJD and FFI patients as well as control donors (Fig. 1C1). Control donors showed the highest concentrations of total PrPC in the CSF (Fig. 1C1). For sCJD, mean concentrations of total PrPC in CSF were not found to vary significantly with the PRNP codon 129 genotype or the PrPSc type (Fig. 1C2 and C3). To exclude possible artifacts because of sample storage, we investigated the stability of PrPC under different storage conditions (Supplementary Fig. 4). We found that PrPC concentrations were stable for at least 4 days (no loss of more than 20% total PrPC) at room temperature and 4  C, as well as for up to 8 freezing and thawing cycles (Supplementary Fig. 4AeC).

3.2. Two-dimensional western blot mapping of PrPC charge isoforms in CSF by different PrP antibodies Because 2-dimensional western blot mapping of PrPC depends on the primary antibodies used for immunolabeling (Zanusso et al., 2002), we analyzed a CSF sample from the same sCJD patient using 5 different PrP antibodies recognizing different PrP epitopes: Antibodies 3B5 and SAF32 were raised against the octarepeat region and bind to epitopes located between amino acids (AAs) 51e89 and 59e89, respectively; 3F4 recognizes an epitope within the central part of PrP (AAs; 108e111), whereas 12F10 and Ab SAF70 are directed against the core-region of PrP (AAs; 142e160). The antibodies 3B5, SAF32, and 3F3 showed a similar PrPC charge isoform profile, consisting of 12 prominently stained PrPC isoforms. The isoelectric point (pI) of all isoforms spans a pH ranges from 4.0 to 9. The molecular weights ranged between 27 and 36 kDa, most probably representing full-length PrPC forms which can be either glycosylated (33e36 kDa) or unglycosylated (27 kDa) (Fig. 2). In addition to full-length PrPC, antibodies 12F10 and SAF70 also recognized several different N-terminally truncated PrPC species. Each of these antibodies was able to distinguish between more than 20 different forms of PrPC. One fraction of truncated PrPC forms showed a molecular weight between 21 kDa and 18 kDa within a pI range from pH 4 to 6. Additionally, spots of truncated PrPC were detectable at a molecular weight of 12e15 kDa within a narrow acidic pI range between pH 5 and 7 (Fig. 2). A significant impact of gender and age (between 60 and 80 years) of

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Fig. 1. Analysis of proteinase K (PK)-sensitivity, glycosylation, and concentration of cellular prion protein (PrPC) in human cerebrospinal fluid (CSF). (A) A limited digestion of CSF from sporadic Creutzfeldt-Jakob disease (sCJD) and control patients using different concentrations of PK revealed similar PK sensitivity of prion protein (PrP) in these samples (western blot results with anti-PrP 3F4). No PK-resistant PrP could be detected by this method in CSF. The limit of tolerance to PK was at about 2.5e3.0 mg PK/mL. (B) Glycosylation status of PrPC in brain and CSF was analyzed by western blotting. The PrP antibody SAF32 showed 3 PrPC isoforms (di-, mono-, and unglycosylated PrPC) in the brain tissue and 2 PrPC isoforms in the CSF. Enzymatic deglycosylation with N-glycosidase F (PNGase F) produced unglycosylated PrPC with the same molecular weight (approximately 27 kDa) as recombinant PrPC. All experiments were performed at least 3 times. (C) Concentration of PrPC in CSF was determined by a quantitative PrP assay based on the Luminex X-MAP technology. Familial CJD (E200K) patients exhibited a significantly decreased PrPC concentration as compared with sCJD and fatal familial insomnia (FFI) patients as well as compared with control donors (1). No significant differences could be observed when the CSF from sCJD patients with different PRNP codon 129 genotypes (MM, MV and VV) (2) or different types of PrPSc were compared (3). Error bars represent standard deviations of the mean (SEM). The number of stars indicates the significance level: 1 star (*) for p < 0.05, 2 (**) for p < 0.01, and 3 (***) for p < 0.001. Statistics were performed using the Wilcoxon-Mann-Whitney Test. Abbreviations: CSF, cerebrospinal fluid; FFI, fatal familial insomnia; PK, proteinase K; PrP, prion protein; PrPC, cellular prion protein; sCJD, sporadic Creutzfeldt-Jakob disease; SEM, standard deviations of the mean.

sCJD patients on the PrPC isoform pattern could not be observed (Supplementary Fig. 1). 3.3. Impact of codon 129 polymorphism and PrPSc type on PrPC charge isoform profile Using 2-dimensional immunoblot technique and antibody SAF32, we investigated PrPC charge isoform profiles in CSF from sCJD patients with different PRNP codon 129 genotypes (Fig. 3A). The intensities of the 12 most abundantly expressed PrPC charge isoforms were densitometrically quantified and calculated as percentage of the total PrPC (that comprised all isoforms) (Fig. 3A,B). In all groups isoform 10 (IF 10, marked with “10” in Fig. 3A) showed a characteristic pattern, which we used as an electrophoretic reference for the sequential numbering of charge isoforms on the blots from the left to the right. PrPC isoforms 1 to 12 showed partially different staining intensities between each other. When we analyzed each of the 12 PrPC charge isoforms individually for their respective abundance in samples from MM, MV, and VV patients, none of the isoforms 1e11 showed significant variations between the 3 groups. However, the staining intensity of PrPC isoform 12 (IF12) was significantly lower (on background level) in samples from VV patients as compared with samples from MM and MV patients (Fig. 3B). These observations could be confirmed with an alternative PrP antibody 3F4 (Supplementary Fig. 2). In CSF derived from controls, all PRNP codon 129 genotypes (MM, MV, and VV) showed identical respective staining intensities of all PrPC charge isoforms (including IF12) (Supplementary Fig. 3).

Subsequently, we compared PrPC charge isoform profiles in CSF from sCJD patients exhibiting PrPSc types 1 and 2 independently from the PRNP codon 129 genotype (Fig. 4A). This analysis revealed that the respective staining intensities of IFs 1e11 were similar for both groups of patients, while the signal intensity of IF12 was significantly lower in sCJD patients with PrPSc type 2 as compared with those with PrPSc type 1 (Fig. 4B). 3.4. Association of sporadic and familial prion diseases with PrPC charge isoform profiles in CSF PrPC charge isoform profiles were comparatively examined in CSF samples from patients with sCJD, fCJD (E200K mutation) or FFI, as well as from controls without prion disease (Fig. 5A). No differences in the staining intensities of IFs 1e11 could be observed between the different groups. Interestingly, IF12 produced consistently weaker signals in CSF samples from FFI patients than those derived from sCJD, fCJD (E200K) patients or controls (Fig. 5B). 3.5. Detection and charge isoform characterization of truncated PrPC in CSF from sCJD and control patients To investigate the specific occurrence of truncated PrPC charge isoforms in sCJD, we subjected CSF samples derived from sCJD (MM) patients and control (MM) donors to 2-dimensional immunoblot analysis. By the use of the antibody 12F10, we detected and quantified 12 full-length and 10 truncated PrPC species (Fig. 6A). To obtain a quantitative PrPC charge isoform profile, we calculated

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Fig. 2. Two-dimensional immunoblot mapping of cellular prion protein (PrPC) charge isoforms in cerebrospinal fluid (CSF) by using antibodies against different PrPC epitopes. PrPC charge isoforms in CSF from a sporadic Creutzfeldt-Jakob disease (sCJD) patient were analyzed by 2-D western blotting. Antibodies recognizing the octarepeat region of PrPC such as 3B5 and SAF32, and antibody 3F4 which recognizes an epitope within the central part of PrPC, showed similar profiles of different charge isoforms of PrPC with an isoelectric point between pH 4 and 9. Core region-specific antibodies 12F10 and SAF70 revealed distinct PrPC charge isoform profiles and truncated PrPC species. All experiments were performed at least 3 times. Full-length and truncated isoforms were separately labeled from 1e12 and 1e10, respectively, and isoform (IF)-labels marked with a star (*) indicates the absence of the respective isoform in the tested sample. Abbreviations: CSF, cerebrospinal fluid; IF, isoform; PrPC, cellular prion protein; sCJD, sporadic Creutzfeldt-Jakob disease.

their relative abundance as a percentage of the total PrPC (Fig. 6A,B). When we compared the expression of 10 truncated PrPC charge isoforms in the CSF of sCJD patients to control donors, we found no significant differences in PrPC cleavage process (Fig. 6B). All truncated PrPC species in the CSF of sCJD patients were PK sensitive (data not shown).

3.6. Increased phosphorylation of PrPC at serine 43 in CSF from sCJD patients For the further characterization of PrPC isoforms in the CSF we used a PrP antibody raised against serine 43 phosphorylated PrP (Ser43 P-PrP). Immunoblot analysis of CSF and brain samples from CJD patients and controls revealed the presence of Ser43 P-PrP in both groups (Fig. 7AeC). As revealed by densitometric quantification of the band intensities using the software “Scion Image”, the amount of Ser43 P-PrP was significantly higher in the CSF from sCJD and fCJD (E200K) patients when compared with control donors without prion disease (Fig. 7A1e2). An increase of Ser43 P-PrP could be also observed in brain tissue derived from sCJD patients (Fig. 7B). Two-dimensional immunoblot analysis of CSF for Ser43 P-PrP showed the presence of at least 5 charge isoforms in sCJD and control samples with a molecular weight of approximately 26 kDa and a pI range between pH 6 and 8 (Fig. 7C).

4.1. Decreased PrPC levels in CSF of fCJD patients Because there is only limited knowledge about the physiological regulation of PrPC in humans, we measured the total PrPC level in the CSF of sCJD patients with different PRNP genotypes and PrPSc types, of fCJD (E200K) patients and of FFI patients. PRNP codon 129 MM homozygosity is a risk factor for human prion disease (Alperovitch et al., 1999) and influences the susceptibility to and progression of sCJD. However, we observed no significant impact of PRNP codon 129 polymorphisms or the PrPSc type on the mean concentration of PrPC in CSF of sCJD patients. When we compared mean PrPC levels in CSF from sCJD, fCJD (E200K), and FFI patients, we observed a significantly decreased mean concentration of PrPC in CSF from fCJD (E200K) patients versus all other groups of donors. A decrease of PrPC levels in sCJD patients as compared with control donors was previously observed (Meyne et al., 2009) and could be confirmed by us. One explanation for the decrease of mean PrPC levels in fCJD (E200K) patients might be that a proportion of PrP is associated with cerebral PrPSc aggregates. However, PrP expression levels may also depend on other factors, such as gender (Meyne et al., 2009), or unknown metabolic variations. In the light of the detected decrease of the mean concentration of PrPC in CSF from fCJD patients we further examined the PrPC charge isoform profiles in our CSF samples. We hypothesized a possible association with the PRNP codon 129 genotype, PRNP mutations or specific forms of prion disease.

4. Discussion 4.2. Detection of PrPC charge isoforms in human CSF In inherited forms of human TSE, specific mutations of the prion protein gene (e.g., E200K) cause the disease by interfering with the normal metabolism of PrPC. Increased generation of truncated PrPC fragments (Capellari et al., 2000) and changed glycoforms ratios of PrPSc in the brains (Hill et al., 2006) of patients with pathogenic PRNP mutations seem to substantiate this notion. However, the exact mechanisms that molecularly translate pathogenic PRNP mutations into prion disease are unclear. In the present study we examined CSF samples obtained from patients of different prion diseases to elucidate whether the metabolism of PrPC varies with PRNP codon 129 polymorphisms in sCJD or pathogenic PRNP mutations in fCJD and FFI.

The presence of PrPC in human CSF had already been demonstrated by Tagliavini et al. (Tagliavini et al., 1992). However, for a detailed characterization of PrPC patterns putative variations between the antibody recognition patterns of PrPC (Zanusso et al., 1998) have to be taken into consideration. In our two-dimensional immunoblot analysis we therefore used different PrP antibodies raised against different epitopes of PrPC. Our study revealed the presence of a number of full-length and truncated PrPC species in human CSF. These PrPC isoforms are generated by physiological or pathophysiological posttranslational processing of PrPC and are not artificial postmortem

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Fig. 3. Association of PRNP codon 129 polymorphism with cellular prion protein (PrPC) charge isoform profiles in cerebrospinal fluid (CSF) of sporadic Creutzfeldt-Jakob disease (sCJD) patients. (A) PrPC charge isoforms were detected in the CSF of sCJD with different PRNP codon 129 genotypes (MM, MV and VV) by 2-D western blotting using the antibody SAF32. The 12 most abundant charge isoforms (IF1eIF12) were quantified by Total Lab Quant and calculated as a percentage of the total PrP. Isoform IF1-12 were marked for orientation. (B) The level of one specific charge isoform, IF12, in CSF was found to be significantly lower in sCJD patients with VV at PRNP codon 129 than in sCJD patients with MM or MV at this PRNP codon. Repeated gel runs with samples from 8 different patients (n ¼ 8; 8xPrPSc type 1 and 8xPrPSc type 2, 8x with no confirmed PrPSc type) per group confirmed the consistency of the shown profiles. Error bars represent standard deviations of the mean (SEM). The number of stars indicates the significance level: 1 star (*) for p < 0.05, 2 (**) for p < 0.01, and 3 (***) for p < 0.001. Statistics were performed using the Wilcoxon-Mann-Whitney Test. Abbreviations: CSF, cerebrospinal fluid; IF, isoform; PrPC, cellular prion protein; sCJD, sporadic Creutzfeldt-Jakob disease; SEM, standard deviations of the mean.

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Fig. 4. Association of PrPSc type with cellular prion protein (PrPC) charge isoform profiles in cerebrospinal fluid (CSF) of sporadic Creutzfeldt-Jakob disease (sCJD) patients. (A) PrPC charge isoforms were detected in the CSF of sCJD patients with PrPSc types 1 or 2 by 2-dimensional western blotting using the antibody SAF32. The 12 most abundant PrPC charge isoforms were quantified by Total Lab Quant and calculated as a percentage of the total PrP. Isoforms (IF) were labeled from 1e12, and IF-labels marked with a star (*) indicate the absence of the respective isoform in the tested sample. (B) The level of one specific charge isoform, IF12, in CSF was found to be significantly lower in sCJD patients with PrPSc type 2 as compared with sCJD patients with PrPSc type 1. Repeated gel runs with samples from 8 different patients (n ¼ 8) per group (type 1: 3xMM1, 3xMV1, and 2xVV1; type 2: 4xMV2 and 4xVV2) confirmed the consistency of the shown profiles. Error bars represent standard deviations of the mean (SEM). The number of stars indicates the significance level: 1 star (*) for p < 0.05, 2 (**) for p < 0.01, and 3 (***) for p < 0.001. Statistics were performed using the Wilcoxon-Mann-Whitney Test. Abbreviations: CSF, cerebrospinal fluid; IF, isoform; PrPC, cellular prion protein; sCJD, sporadic Creutzfeldt-Jakob disease; SEM, standard deviations of the mean.

products induced by autolysis. Their pI range was between 4 and 9, which reflects the presence of neutral and sialated complex-type glycans linked to PrPC (Pan et al., 2002; Rudd et al., 1999). Enzymatic deglycosylation of these PrPC species resulted in a marked decrease in different charge isoforms (Zanusso et al., 2002). We detected more than 12 different full-length PrPC charge isoforms using antibodies recognizing the octarepeat or central region of PrPC. The individual charge isoforms could be distinguished by the variances in their pI. The higher preference to glycosylated PrPC of these kinds of antibodies has previously been described (Kuczius et al., 2007; Schmitz et al., 2010b). In contrast, antibodies directed against the core region of PrPC, such as 12F10

and SAF70, additionally detected N-terminally truncated PrPC forms in human CSF. These PrPC fragments exhibited a pI range from pH 4 to 6, suggesting the loss of highly charged amino acids in truncated PrPC forms. Some fragments corresponded to the molecular weight of the C2 fragment (21 kDa) and the C1 fragment (18 kDa). Both had been described as being generated by the alpha and beta cleavage of PrPC at residue 90/91 and 111/112 in the brain, respectively (Chen et al., 1995; Cissé et al., 2005; Jiménez-Huete et al., 1998; Mangé et al., 2004). Smaller truncated PrPC fragments with a molecular weight of approximately 12e13 kDa, whose presence has already been described in human CSF (Schmitz et al., 2010b), could be detected as different charge

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isoforms exhibiting a pI in the pH range of approximately 5e7. The biochemical properties of the PrPC forms that we detected in human CSF are consistent with previous studies (Castagna et al., 2002; Zanusso et al., 2002). However, the PrPC charge isoform profiles in brain and CSF differ from each other (Castagna et al., 2002). Most PrPC in CSF is likely to originate from brain tissue, but not all of the PrPC isoforms from brain tissue are detectable in CSF (Castagna et al., 2002; Zanusso et al., 2002).

4.3. Association of PRNP genotype, PrPSc type and prion disease type with PrPC charge isoform profiles The PRNP codon 129 polymorphism can influence the clinical and pathologic phenotype of sCJD (Collins et al., 2006; Parchi et al., 1999) and has an impact on the conformation of PrPC as well as on the PK resistance of PrPSc in brain tissue (Pham et al., 2008; UroCoste et al., 2008). Pathogenic point mutations in the PRNP gene,

Fig. 5. Association of sporadic and familial prion diseases with cellular prion protein (PrPC) charge isoform profiles in cerebrospinal fluid (CSF). (A) PrPC charge isoforms were detected in CSF from sporadic Creutzfeldt-Jakob disease (sCJD), familial Creutzfeldt-Jakob disease (fCJD) (E200K) and fatal familial insomnia (FFI) patients as well as from control donors using 2-dimensional western blotting with antibody SAF32. The twelve most abundant PrPC charge isoforms were quantified by Total Lab Quant and calculated as a percentage of the total PrP. Isoforms (IF) were labeled from 1 to 12, and IF-labels marked with a star (*) indicate the absence of the respective isoform in the tested sample. (B) The level of one specific charge isoform, IF12, in CSF was found to be significantly lower in FFI patients as compared with sCJD and fCJD patients or control donors. Repeated gel runs with samples from at least 6 different patients per group (sCJD: 6x MM and 2xMV; fCJD: 4xMM and 2 MV; FFI 6xMM; control: 4xMM, 3xMV, and 1xVV) confirmed the consistency of the shown profiles. Error bars represent standard deviations of the mean (SEM). The number of stars usually indicates the significance level: 1 star (*) for p < 0.05, 2 (**) for p < 0.01 and 3 (***) for p < 0.001. Statistics were performed by using the Wilcoxon-Mann-Whitney Test. Abbreviations: CSF, cerebrospinal fluid; IF, isoform; PrPC, cellular prion protein; fCJD, familial Creutzfeldt-Jakob disease; FFI, fatal familial insomnia; sCJD, sporadic Creutzfeldt-Jakob disease; SEM, standard deviations of the mean.

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such as E200K in fCJD or D178N in FFI, are thought to change the biochemical aggregation properties of PrPC, which influences the development of disease. However, it is still unclear in which way the polymorphism at PRNP codon 129 or pathogenic PRNP mutations exactly modify the metabolism of PrPC. Therefore, we examined the PrPC metabolism in more detail using 2-dimensional PrPC charge isoform mapping of CSF samples from diseased and control patients. Our data provide evidence for the first time that subtle changes in the metabolism of PrPC in sCJD patients are associated with the PRNP codon 129 genotype. Valine-homozygosity at this gene position was associated with a decreased level of 1 specific PrPC charge isoform (IF12). The presence of the same charge isoform was also reduced in sCJD patients with PrPSc type 2 and in FFI patients. Because most of the sCJD (VV) patients (Parchi et al., 1999) and FFI patients (MM and MV) typically express PrPSc type 2 (Montagna et al., 2003), we suggest that IF12 is associated with PrPSc type 2, indicating subtle change in the physiological metabolism of PrPC in prion disease patients exhibiting PrPSc type 2. In control donors, a differential association of IF12 levels with the PRNP genotype could not be observed, which further supports this hypothesis. However,

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whether the reduced levels of IF12 were dependent on the PrPSc type, the PRNP codon 129 genotype, or both of these factors could not be definitely resolved in our study. The observed differences in the PrPC patterns were obtained by a sensitive analysis of PrPC charge isoforms. This may be the reason why immunoblot studies on PrPC isoforms in human CSF and peripheral blood mononuclear cells (Schmitz et al., 2010b; Segarra et al., 2009) previously failed to detect these subtle differences in PrPC metabolism. 4.4. Increased phosphorylation of PrPC at serine 43 in CSF from sCJD patients Protein phosphorylation is thought to be essentially involved in the pathogenesis of neurodegenerative protein aggregation disorders such as Alzheimer’s and Parkinson’s disease. In our study we investigated the phosphorylation of PrP at serine 43 with two-dimensional western blotting and found 5 different P-PrP charge isoforms in human CSF. The amount of Ser43 P-PrP was significantly increased in CSF of sCJD as well as in fCJD (E200K) patients compared with the control group. In addition, we

Fig. 6. Analysis of truncated cellular prion protein (PrPC) charge isoforms in cerebrospinal fluid (CSF) from sporadic Creutzfeldt-Jakob disease (sCJD) patients and control donors. (A) Full-length and truncated PrPC charge isoforms were detected in CSF from sCJD patients and control donors using 2-dimensional western blotting with the antibody 12F10. PrPC charge isoforms were quantified by Total Lab Quant and calculated as a percentage of the total PrP. Full-length and truncated isoforms were separately labeled from 1 to 12 and 1 to 10, respectively, and IF-labels marked with a star (*) indicate the absence of the respective isoform in the tested sample. (B) Samples from sCJD patients and control donors (n ¼ 4 each, all with MM at PRNP codon 129) showed indistinguishable profiles of truncated PrP charge isoforms. Error bars represent standard deviations of the mean (SEM). The number of stars indicates the significance level: 1 star (*) for p < 0.05, 2 (**) for p < 0.01, and 3 (***) for p < 0.001. Statistics were performed using the Wilcoxon-Mann-Whitney Test. Abbreviations: CSF, cerebrospinal fluid; PrPC, cellular prion protein; sCJD, sporadic Creutzfeldt-Jakob disease; SEM, standard deviations of the mean.

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Fig. 7. Detection of cellular prion protein (PrPC) phosphorylation at serine 43. The level of PrPC phosphorylated at serine 43 was investigated in sporadic Creutzfeldt-Jakob disease (sCJD) and familial Creutzfeldt-Jakob disease (fCJD) (E200K) in comparison to control samples by 1-dimensional western blotting in CSF (A1-2), in brain tissue of sCJD patients versus controls (B) and in CSF of a sCJD patient by 2-dimensional western blotting (C). (A) The mean level of PrPC phosphorylated at serine 43 was significantly higher in CSF from sCJD (1) and fCJD (E200K) (2) patients than from control donors (n ¼ 8 per group). (B) Immunoblot analysis of brain tissue revealed a significant increase of Ser43 P-PrP in sCJD brain samples in comparison to control donors (n ¼ 8 per group). (C) 2-dimensional immunoblot analysis showed the presence of at least 5 Ser43 P-PrP charge isoforms in sCJD and control samples, which were all more intensely stained in the sCJD sample. The number of stars indicates the significance level: 1 star (*) for p < 0.05, 2 (**) for p < 0.01, and 3 (***) for p < 0.001. Statistics were performed by using the Wilcoxon-Mann-Whitney Test. Abbreviations: CSF, cerebrospinal fluid; PrPC, cellular prion protein; fCJD, familial Creutzfeldt-Jakob disease; sCJD, sporadic Creutzfeldt-Jakob disease.

found the expression of Ser43 P-PrP enhanced in brain tissue of sCJD patients, which is in line with the CJD-dependent increase of Ser43 in the CSF.

This finding has 2 potential implications: first, Ser43 P-PrP in CSF may possibly be used in the future as a biomarker for substantiating the diagnosis of suspected sCJD in a proportion of cases that

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exceeds a specific cut-off. Second, PrP phosphorylation may be involved in the pathogenesis of sCJD. In a previous study the phosphorylation of PrP at serine 43 had been examined in a cell model (Giannopoulos et al., 2009). This study showed that phosphorylation of PrP at serine 43, provoked by neuronal cyclin-dependent kinase 5, may induce structural changes of PrPC resulting in an increased structural conversion of PrP (Giannopoulos et al., 2009). In this reaction, the negative charges of the phosphate group at serine 43 may have acted similarly to other anionic molecules such as RNA that have been previously shown to induce PrP conversion in vitro (Deleault et al., 2007). The occurrence of PrP phosphorylation at serine 43 phosphorylation has also been shown in vivo, for example in the brains of scrapie-infected mice (PrP in the brains of uninfected control mice did not show such phosphorylation; Giannopoulos et al., 2009). This raises the question of whether phosphorylation of PrP at serine 43 might directly trigger the conversion of PrPC into PrPSc. 5. Conclusion In conclusion, our study revealed an association between PrPC charge isoform profiles in human CSF and PRNP codon 129 genotype, PrPSc type and the type of human prion disease. Whether this association results from a modulation of the metabolism of PrPC by one or more of these factors remains to be established. Moreover, Ser43 P-PrP might provide a new molecular marker for the refinement of sCJD diagnostics. Disclosure statement The authors have no actual or potential conflicts of interest. Acknowledgements This work was supported by a grant from the European Commission: protecting the food chain from prions: shaping European priorities through basic and applied research (PRIORITY, N 222887) Project number: FP7-KBBE-2007-2A. The study was performed within the recently established Clinical Dementia Center at the University Medical Center Göttingen and was partly supported by grants from the JPND program (DEMTEST [Biomarker based diagnosis of rapid progressive dementias-optimization of diagnostic protocols, 01ED1201 A]) and from the Federal Ministry of Education and Research grant within the German Network for Degenerative Dementia, KNDD-2, 2011-2013, determinants for disease progression in AD. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2013.11.010. References Alperovitch, A., Zerr, I., Pocchiari, M., Mitrova, E., de Pedro Cuesta, J., Hegyi, I., Collins, S., Kretzschmar, H., van Duijn, C., Will, R.G., 1999. Codon 129 prion protein genotype and sporadic Creutzfeldt-Jakob disease. Lancet 353, 1673e1674. Althaus, H.H., Klöppner, S., Klopfleisch, S., Schmitz, M., 2008. Oligodendroglial cells and neurotrophins: a polyphonic cantata in major and minor. J. Mol. Neurosci. 35, 65e79. Beringue, V., Mallinson, G., Kaisar, M., Tayebi, M., Sattar, Z., Jackson, G., Anstee, D., Collinge, J., Hawke, S., 2003. Regional heterogeneity of cellular prion protein isoforms in the mouse brain. Brain 126, 2065e2073. Brown, D.R., Schmidt, B., Kretzschmar, H.A., 1997. Effects of oxidative stress on prion protein expression in PC12 cells. Int. J. Dev. Neurosci. 15, 961e972.

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