Biochemical characterization of PrP^CGPIThy-1 mice.

(A) Schematic representation of the PrPCGPIThy-1 fusion protein. PrPC comprises an N-terminal and a C-terminal (GPI-anchor) signal sequence, both of them not being present in the mature protein. Positions of an octameric repeat region (OR), a hydrophobic domain (HD), two N-glycans (N), a disulfide bridge, the 3F4 tag and epitope (present in mouse lines L27 and L16) and of the cleavage site for the ADAM10-mediated shedding (scissors) are indicated. The substitution of the GPI-SS of PrPC for the one of Thy-1 is shown in the dotted box. The substitution of the GPI-SS of PrPC for the one of Thy-1 is indicated. (B) Relative amount of PrPC mRNA from WTPrPC (n = 5) and PrPCGPIThy-1 (n = 5) measured by RT-qPCR. WTPrPC mRNA is set to one. Error bars are SEM. (C) Representative western blot showing PrPCGPIThy-1 protein expression in brain compared to WTPrPC. Bar chart shows the mean of PrP relative intensity related to actin intensity (used as a loading control). WTPrPC is set to 100%. Error bars are SEM. (D) Triton X-114 phase partitioning assay. Both, WTPrPC and PrPCGPIThy-1 were mainly found in the insoluble phase (ip), indicating that both carry a GPI-anchor as described [28] (dp: detergent phase; sp: soluble phase). Diagram shows the relative signal intensity of PrP from 3 independent experiments. WTPrPC ip is set to 100%. Error bars are SEM. (E) Representative western blot showing total homogenates (TH) from PrP KO (Prnp0/0, used as a negative control), WTPrPC and PrPCGPIThy-1 brains used for PNGase treatment (to eliminate N-glycans) followed by PrP immunoprecipitation (IP), shown in the adjacent western blot. PNGase+IP treated samples were then used to isolate the GPI-anchors, showed in (F). (F) Dot blot analysis of the GPI-anchors from deglycosylated, immunoprecipitated PrP and PK digested samples from mouse brain. Phosphatidylinositol (PI), mannose (man) and sialic acid (sial. acid) were detected as described in Methods. Note that the amounts of PI and mannose are similar between PrPCGPIThy-1 and WTPrPC whereas sialic acid is almost absent in PrPCGPIThy-1. Sialic acid background signal in PrPCGPIThy-1 could be explained by deglycosylation not being 100% achieved as shown in blot in (E).</p

Graphical summary of research aim and key findings.

(A) To study the role of the GPI-anchor signal sequence (GPI-SS) in determining localization and biology of a protein, the GPI-SS of PrPC (dark green) was exchanged for the one of Thy-1 (red). Both proteins, WTPrPC and the PrPCGPIThy-1 mutant, are expressed at the cell surface and comprise a GPI-anchor. A sialic acid modification (green asterisk) typical for the GPI-anchor of PrPC is lacking in the GPI-anchor of PrPCGPIThy-1 (note its red GPI-anchor referring to the GPI-anchor of Thy-1 [in B]). This suggests that an altered GPI-SS in the mRNA results in a different GPI-anchor of the mature protein. (B) PrPC (green) and Thy-1 (blue) are both known residents of lipid rafts/DRMs. While PrPC is located in their periphery and able to leave and re-enter these membrane subdomains, Thy-1 has been shown to occupy more central regions therein. Despite several biochemical methods applied in our study, we were unable to demonstrate a relative re-distribution of PrPCGPIThy-1 towards Thy-1 compared to WTPrPC. (C) Nevertheless, the overall cellular sorting is altered with PrPCGPIThy-1 being relatively more transported towards the apical compartment compared to WTPrPC with its predominant basolateral sorting in a polarized epithelial cell model such as MDCK cells. This also holds true in our transgenic mice and translates to an increased axonal sorting of PrPCGPIThy-1 in primary neurons compared to a mainly somatodendritic presence of PrPC in wild-type neurons. (D) Altered GPI-anchor composition and sorting of PrPCGPIThy-1 results in different biological consequences (in comparison to WTPrPC): (i) Endogenous proteolytic shedding by the metalloprotease ADAM10 (orange) at the cell surface is reduced. (ii) Although PrPCGPIThy-1, in principle, is able to transduce PrPSc-associated toxic signaling (e.g. via cleaved caspase-3), signaling via the MAP kinase ERK1/2 is reduced upon prion infection. Though not investigated here, p38 signaling may be reduced at early time points, contributing to delay to terminal disease (showed as p38?). Key hallmarks of prion-associated neuropathology are also altered in the transgenic mice including (iii) decreased PrPSc production and deposition, (iv) reduced vacuolization (spongiosis) of the brain parenchyma, and (v) reduced induction of astrocytes and microglia (reactive gliosis). These changes are accompanied with prolonged survival of the PrPCGPIThy-1 mice and support a relevant impact of the GPI-SS on a GPI-AP`s biology.</p

PrP^CGPIThy-1 can induce prion-associated apoptosis in cells.

(A) SH-SY5Y cells expressing the indicated PrP constructs (PrP-CD4 is a transmembrane version of PrP and served as a negative control) were co-cultured with ScN2a or N2a cells for 16 h. For quantification of apoptotic cell death, SH-SY5Y cells were fixed, permeabilized and stained for active caspase-3. Quantifications are based on triplicates of at least three independent experiments. (B) Comparable expression of PrP versions was confirmed by western blot using the anti-PrP antibody 3F4.</p

PrP^CGPIThy-1 shows altered sorting.

(A) Confocal microscopy showing expression of WTPrPC and PrPCGPIThy-1 (green) in fully polarized MDCK epithelial cells. ZO1 (red) is expressed at the tight junction and delimitates the apical (a)/basolateral (b) side. Note that PrPCGPIThy-1 relocalizes to the apical side compared to WTPrPC, which is predominantly basolaterally located (scale bar is 5 μm). (B) Confocal microscopy of primary neuronal cultures stained with an antibody against PrP (POM1; green) under non-permeabilizing conditions. Both WTPrPC and PrPCGPIThy-1 are expressed at the plasma membrane (scale bars are 10 μm). (C) Representative confocal microscopy pictures of primary neuronal cultures stained with antibodies against PrP (POM1; green) and tau (red). (i) The squares indicate selected areas of the dendrites (where tau is absent) showing decreased PrPCGPIThy-1 amounts whereas staining is present in WTPrPC. (ii) Same staining as in (i) but focusing on tau-positive axons (the squares indicate magnifications where the relative increase in PrP staining at the axons in PrPCGPIThy-1 neurons can be observed). (iii) Bar diagram of a semi-automated quantification showing that the amount of PrPCGPIThy-1 present in tau-positive axons is significantly increased compared to WTPrPC (***p = 0.0001).</p

PrP^CGPIThy-1 L150 mice show a delay to terminal disease and altered neuropathology upon prion infection.

(A) Representative blot of PrPGPIThy-1 L150 (generated for infection experiments) and WTPrPC mouse brain homogenates showing no relevant differences in the amount of PrPCGPIThy-1 and WTPrPC in the brain. Chart underneath shows mean relative intensity of PrP referred to actin where WTPrPC samples are set to 100% as a reference (error bars are SEM; n = 5 for each genotype). (B) Kaplan-Meier survival curve. Mice were inoculated intracerebrally (i.c.) with RML prions and sacrificed at terminal disease. Note the significant delay (****pCGPIThy-1 L150 (n = 10) and WTPrPC mice (n = 8) incubation time. (C) Neuropathological characterization of terminally diseased mice. Note that PrPCGPIThy-1 L150 mouse brains show a general decrease in spongiosis as observed with HE staining. Gliosis is also decreased in the transgenic mice as manifested by reduced GFAP (astrocytes) and Iba1 staining (microglia) in all the studied regions. (D) Lesion profile after semiquantitative assessment (n = 3 or n = 4 for each genotype) of spongiosis and gliosis (FC: frontal cortex; H: hippocampus; S: striatum; P: pons; Cb: cerebellum). Note the general decrease in lesion severity for the PrPCGPIThy-1 L150 mice (red line), compared to WTPrPC profile (green line).</p

The amount of PK-resistant PrP is decreased, and the glycopattern is changed in PrP^CGPIThy-1 L150 mouse brains after RML prion infection.

Representative blots of total brain homogenates from terminally sick WTPrPC and PrPCGPIThy-1 L150 mice infected with RML prions, without (A) and after (B) PK digestion at 37°C and detected with POM1 antibody. Note that there is a significant overall decrease in the total amounts of PrPCGPIThy-1 in the infected brains (n = 4) which is more conspicuous after digestion with PK (B). (C) Bar chart representing the mean relative intensity of PK-digested samples related to the undigested amounts. Note the significant decrease in the amount of resistant PrPSc for PrPCGPIThy-1 compared to WTPrPC (**p = 0.0021). (D) Quantification of the glycoform banding pattern of PrPSc. The intensity of each band in the PK-digested samples was measured and referred to the total amount of PrP as a percentage. The glycopattern is significantly changed in PrPCGPIThy-1 L150 after RML infection, where the monoglycosylated isoform is less PK-resistant compared to WTPrPC (*p = 0.019).</p

Shedding of PrP is decreased in PrP^CGPIThy-1 L150 mice but increases after RML infection.

(A) Representative western blot showing shed PrP (detected with our new antibody described in [36]) and total PrP levels (detected with POM1). The blots for shed PrP and total homogenates were run in parallel (n = 4 for each genotype). The diglycosylated band is preferentially shed in both WTPrPC and PrPCGPIThy-1 L150 brains, but in the latter, shedding is reduced to 27% (***p = 0.0005) as shown in the bar chart (B). To quantify, each blot (shed and total PrP) was first referred to actin prior to the relative quantification of shed PrP referred to total PrP. WTPrPC is set to 100% and error bars are SEM. (C) Representative western blot of shed PrP and total PrP from RML infected brain homogenates of terminally sick mice (n = 4 for WTPrPC and n = 5 for PrPCGPIThy-1 L150). Note that in the infected brains all the isoforms are shed, in both WTPrPC and PrPCGPIThy-1 L150. (D) Bar chart showing the quantification of shed PrP referred to actin. WTPrPC is set to 100%. Note that shedding in PrPCGPIThy-1 L150 is still decreased compared to WTPrPC (***p = 0.0004). (E) Bar chart showing that when shed PrP is referred to the total PrP in RML infected mice, there are no significant differences in the relative amount of shedding, implying a relatively increased shedding in infected PrPCGPIThy-1 L150 brains. Blots were run in parallel and first referred to its actin. WTPrPC is set to 100%. (F) Quantification of the shed PrP glycopattern. Each band intensity was referred to the total amount of shed PrP. In infected brains, the monoglycosylated isoform is significantly less shed in PrPCGPIThy-1 L150 mice (*p = 0.0158).</p

Phosphorylated ERK (ERK-P) is decreased in terminally prion-diseased PrP^CGPIThy-1 L150 mice.

(A) Representative western blots of total brain homogenates from prion diseased mice infected with RML prions, incubated with antibodies against total ERK and ERK-P (upper part) as well as total p38 and p38-P (lower part). (B) Quantifications of the relative intensity of ERK-P signal related to total ERK signal and p38-P signal (upper part) related to the total p38 signal (lower part). (WTPrPC, n = 4; PrPCGPIThy-1, n = 5). For the quantification, each signal was first related to the corresponding actin signal. Note that there is a significant decrease in ERK-P signal (*p = 0.031) in PrPCGPIThy-1 RML infected mice compared to WTPrPC mice, whereas no changes are observed for p38-P.</p

Additional file 1: of Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

(.jpg) The 3F4-tag in PrPC does not alter the ADAM10-mediated shedding. Western blot analysis of forebrain homogenates comparing PrPC shedding between mice expressing endogenous wild-type PrPC (PrPCWT) and knock-in mice expressing 3F4-tagged PrPC instead (PrP3F4KI). Quantification was done by referring the sPrP signal to the respective fl-PrP signal (POM2 Ab) of the re-probed blot and is shown on the right (n = 3). As in other parts of this study, forebrain homogenates of Prnp0/0, ADAM10 cKO and tga20 mice served as specificity controls. The position of air bubbles on the membrane (indicated by arrows) further supports the slight molecular weight shift between sPrP and fl-PrP described in Fig. 1b. To prove genotypes of PrPCWT and PrP3F4KI mice, in a parallel blot shown below, PrPC was first detected with an antibody directed against the 3F4 epitope and re-probed with POM2. (JPEG 225 kb

Additional file 4: of Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

(.jpg) Differences in the glycopattern between total and cell surface PrPC in N2a cells. (A) Western blot analysis and (B) densitometric quantification of glycoform proportions of total (cell lysates) versus cell surface fl-PrP (biotinylated samples) using POM2 antibody for detection. Absence of actin and almost exclusive expression of mature ADAM10 (with almost no premature ADAM10) in the biotinylated samples confirm technical soundness of the assay (the upwards shift of ADAM10 in gel likely results from the assay protocol). Quantification reveals that the fraction of diglycosylated PrP at the cell surface is increased compared to total PrP in cell lysates (diglycosylated: 68.0 ± 0.7% (surface PrP) vs. 55.1 ± 2.4% (total PrP); monoglycosylated: 23.5 ± 0.8% (surface PrP) vs. 29.3 ± 0.6% (total PrP); unglycosylated: 8.5 ± 0.2% (surface PrP) vs. 15.5 ± 1.9% (total PrP); n = 3; ±SD). (JPEG 610 kb