Sunday, February 5, 2012

Cellular prion protein participates in amyloid-β transcytosis across the blood–brain barrier

Brief Communication

Journal of Cerebral Blood Flow & Metabolism , (1 February 2012) | doi:10.1038/jcbfm.2012.7

Cellular prion protein participates in amyloid-β transcytosis across the blood–brain barrier

Thorsten Pflanzner, Benjamin Petsch, Bettina André-Dohmen, Andreas Müller-Schiffmann, Sabrina Tschickardt, Sascha Weggen, Lothar Stitz, Carsten Korth and Claus U Pietrzik

The blood–brain barrier (BBB) facilitates amyloid-β (Aβ) exchange between the blood and the brain. Here, we found that the cellular prion protein (PrPc), a putative receptor implicated in mediating Aβ neurotoxicity in Alzheimer's disease (AD), participates in Aβ transcytosis across the BBB. Using an in vitro BBB model, [125I]-Aβ1−40 transcytosis was reduced by genetic knockout of PrPc or after addition of a competing PrPc-specific antibody. Furthermore, we provide evidence that PrPc is expressed in endothelial cells and, that monomeric Aβ1−40 binds to PrPc. These observations provide new mechanistic insights into the role of PrPc in AD.


Supplementary methods

Generation of monoclonal antibody 51.2

Monoclonal antibody 51.2 was generated by immunization of Prnp0/0 mice with SDS-purified sodium phosphotungstic acid (NaPTA)-precipitated Rocky mountain laboratory (RML) strain scrapie brain material (Bueler et al 1992). 250 µl of a 10 % brain homogenate of terminally RML-infected CD1 mice, were precipitated with NaPTA as described elsewhere (Wadsworth et al 2001). The resulting pellet was mixed with SDS-PAGE loading puffer (Roti-load 1, Carl Roth, Karlsruhe, Germany) heated at 95 °C for 10 minutes and separated on a SDS-gel. Gel slices corresponding to molecular weight from 16 to 30 kDa were cut from the gel and homogenized using mortar and liquid nitrogen. Mice were immunized using an equivalent to 250 µl of 10 % homogenate subcutaneously per mouse. Mice were immunized 4-times s.c. and i.p.boostered four days prior to fusion of spleen cells of immunized mice to X63.Ag8.8163 cells as described before by Kohler and Milstein (Kohler and Milstein 1975). Hybridoma supernatants were screened by ELISA using recombinant prion protein expressed in E. coli.
Flow cytometric analysis
Binding of monoclonal antibody 51.2 to prion protein on cell surface was determined using neuroblastoma cell lines Bos2 or ScN2a (Bosque and Prusiner 2000) as described before (Petsch et al 2011). The prion protein specific monoclonal antibody W226 was used as positive control (Petsch et al 2011).
Primary mouse brain capillary endothelial cell (pMBCEC) transwell transport model
Meninges-free forebrains of 6-10 week old C57Bl6 wild-type (WT) and cellular prion protein (PrPc) knock out (KO; Prnp0/0) mice were pooled, mechanically dissociated and, subsequently enzymatically digested (Bueler et al 1992; Pflanzner et al 2011). Endothelial capillaries were separated on a 33% continuous Percoll (GE Healthcare, Munich, Germany) gradient, collected and plated in 24 well transwell dishes (membrane pore size 0.4 µm and growth surface area 31.2 mm2; Greiner Bio-One, Frickenhausen, Germany) coated with collagen IV/fibronectin (Sigma, Schnelldorf, Germany). Hence, the lower abluminal compartment represents the brain side and the upper luminal compartment the blood side. Cultures were maintained in DMEM supplemented with 20% plasma-derived bovine serum (PDS; First Link, Birmingham, UK), 100 U/ml penicillin and 100 µg/ml streptomycin, 2 mM L-glutamine (all from Gibco, Darmstadt, Germany) and 1 ng/ml basic fibroblast growth factor (R&D Sytems, Wiesbaden, Germany) at 37 °C and 5% CO2. After reaching confluence on day 5, the culture medium was removed and serum-free DMEM/Ham’s F12 (Gibco) medium, containing 1 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 550 nM hydrocortisone (HC; Sigma) was added to induce high transendothelial electrical resistance for 24 hours.
Co-immunoprecipitation, sodium dodecylsulfate-polyacrylamide gel and Western blot analysis
Expression of PrPc and low density lipoprotein receptor-related protein 1 (LRP1) was analyzed in freshly isolated pMBCECs, LRP1-deficient mouse embryonic fibroblasts (MEFs) and brain homogenates of PrPc WT and KO mice in VRL buffer (50 mM HEPES pH 7.5, 100 mM KAc, 5 mM MgCl2, 250 mM sucrose and 1% Triton X-100). Cells were lyzed in NP-40 buffer containing proteinase inhibitors (complete; Roche Applied Science, Mannheim, Germany) and protein concentration was determined by the BCA method (Pierce, Bonn, Germany). For pMBCECs, 98 µg protein representing all capillaries derived from 10 mice, were analyzed.
LRP1 was immunoprecipitated from pMBCEC and LRP1-deficient chinese hamster ovary cell (13-5-1) lysates with rabbit polyclonal anti-LRP1 (1704) antibody at a final dilution of 1:100 in the presence of Protein A Agarose beads (Invitrogen, Darmstadt, Germany) (Pietrzik et al 2002). Prior to immunoprecipitation, 13-5-1 cells were transiently transfected with human PrPc in pcDNA3.1 for 48 hours with FuGeneHD (Roche) transfection reagent according to manufacturer instructions.
Proteins were separated on a 12% tris-glycine gel under denaturing and reducing conditions and transferred to a PVDF membrane (Millipore, Schwalbach, Germany) for 2 hours at 70 V on ice. Membrane was blocked in Tris-buffered saline containing 0.1% Tween 20 (v/v) and 5% milk (w/v). Mouse monoclonal anti-PrPc (1 µg/ml; W226) and anti-α-tubulin (clone DM1A 1:5000; Sigma), rabbit polyclonal anti-LRP1 (1:10000; 1704) and anti-actin (1:1000; Sigma) were used as primary antibodies (Petsch et al 2011). Secondary goat-anti-mouse and goat-anti-rabbitt IgGs linked to horseradish peroxidase (dilution 1:10000; The Jackson Laboratory, Sulzfeld, Germany) were used to visualize protein bands by ECL (Millipore) reagent.

Supplementary results
Generation and binding characteristics of monoclonal antibody 51.2
The monoclonal antibody 51.2 was generated by immunization of Prnp0/0mice using NaPTA-precipitated pathological prion protein that was further separated by SDS-PAGE as described under supplementary methods (Bueler et al 1992). Monoclonal antibody 51.2 reacted with recombinant prion protein in ELISA (data not shown) and detected prion protein in western blotting using brain homogenate of scrapie infected (RML) or control mice (Supplementary figure 1a). Both cellular and pathological prion protein were recognized by the monoclonal antibody 51.2, displaying the characteristic banding pattern in western blotting, staining the three glycoforms (non-, mono- and di-glycosylated prion protein) and C2-fragment of the prion protein (Supplementary figure 1a) (Chen et al 1995). The characteristic “down-shift” of the three glycoforms of the pathological prion protein PrPsc after proteinase K digestion strongly supports the specificity of the monoclonal antibody 51.2 for the prion protein.
The epitope of monoclonal antibody 51.2 was determined using a membrane-bound peptide library as described before (Petsch et al 2011), and identified to be a linear epitope located between amino acids (aa) 93-101 comprising the amino acid sequence GGTHNQWNK of the prion protein (numbering of aa referring to the sequence of hamster prion protein). Interestingly, the 51.2 epitope overlaps with the epitope of the monoclonal antibody 6D11 ( aa 93-110), an antibody that has previously been described to block the interaction between Aβ42 oligomers and PrPc (Lauren et al 2009).
The binding of monoclonal antibody 51.2 to prion protein on the surface of cells was demonstrated using Bos2 and ScN2a cells (Supplementary figure 1b (Bos 2) and 1c (ScN2a cells)). Unstained cells or cells stained with secondary antibody alone did not result in positive signals. In contrast thereto, cells stained with monoclonal antibody 51.2 or prion protein specific control antibody W226 both resulted in staining of distinct populations of cells (Petsch et al 2011), both in Bos2 or ScN2a cells, demonstrating the capacity of monoclonal antibody 51.2 to bind to the prion protein on the surface of both infected and non-infected prion protein expressing cells.

Supplementary references

Titles and legends to supplementary figures
Supplementary Figure 1 The monoclonal antibody (Ab) 51.2 recognizes the prion protein in western blotting and on the surface of neuroblastoma cells.(a) Brain homogenate of scrapie infected (RML: +) and control mice (RML: -) were separated by SDS-PAGE and subsequently used for western blotting. Samples of brain homogenates were digested with proteinase K (PK: +) to eliminate cellular prion protein PrPc. Banding pattern in undigested scrapie-infected brain homogenate corresponds to di-, mono-, non-glycosylated or C2-fragment of prion protein (Chen et al 1995). (b and c) Binding of monoclonal antibody 51.2 to prion protein on the surface of neuroblastoma cell line Bos2 (b) or ScN2a (c) by flow cytometric analysis. Surface staining of PrPc by mAb 51.2 is demonstrated by shift of negative population in negative controls (unstained and secondary antibody (sec control)) from lower left quadrant to upper left quadrant using mAb 51.2 or W226.
Supplementary figure 1

Wednesday, January 18, 2012

Government seeking $1T campaign against Alzheimer's


No comments: