Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice

Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1–4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain.

Introduction pathway. Notably, alterations of the number of octapeptide repeats [23,24] have been shown to abrogate the Cu 2+ -dependent endocytosis of PrP, indicating that, although there are nonoctarepeat Cu 2+ -binding sites in PrP C , it is binding of the metal at the octapeptide repeats that is essential for its internalization. Thus, loss of Cu 2+ -mediated endocytosis of PrP C may also contribute to the disease phenotype.
Information on the role of metal ion binding to PrP and its impact on the conversion to the aberrant isoform is skewed towards studies utilizing either peptides corresponding to the octarepeats or bacterially-expressed recombinant PrP (rPrP). Based on the concept that the Nterminal region of metal-free PrP C is natively unstructured but that different stoichiometries of metal binding can impart different structures upon the OR [25], Lau et al. recently created new PrP alleles which either lock the OR in an extended conformation conductive to binding up to 4 copper ions per OR or in a compact conformation that can only bind one copper ion per OR [26] to restrict inter-conversion and study their biological properties individually. In this study, we altered the OR and binding sites 1-5 in a different way, by substituting the histidine residues with glycine in order to prevent loading of metal ions ("metallation"), and monitored the formation of misfolded PrP in vivo in these newly generated transgenic mice.

Generation of transgenic mice
We used a derivative of the half-genomic vector phg-Prnp [5,27] designated phg-Sce1 wherein a 3000 bp KnpI-NarI-fragment, which contains the entire Prnp ORF and part of the 3'-UTR was modified by using by a synthetic double-stranded I-SceI recognition sequence derived from the complementary primer pair 5'-CGCCTAGGGATAACAGGGTAATGTAC-3' and 5'-ATTACCCTGTTATCCCTA-GG-3' (a gift from H. Jacobsen). XhoI-XbaI-fragments from pCi-PrP-TetraH>G and pCi-PrP-H95G, respectively [28], comprising the mutant Prnp ORFs were treated with T4-DNA polymerase to fill in 5'-overhangs at both ends and ligated into the phg-Sce-I vector, which had been cleaved with I-SceI and treated with S1-nuclease to remove 3'-overhanging nucleotides. From these plasmids, called phg-PrP-TetraH>G and phg-PrP-H95G, respectively, a NotI-SalI fragment was injected into the male pronucleus of fertilized oocytes. Blastulae were transferred to the uteri of foster mothers according to standard techniques. This procedure and the initial screening of tail biopsies of the offspring for the integration of the desired transgene were performed at RCC Ltd (Füllinsdorf, Switzerland). To screen founders, genomic DNA was extracted from tail biopsies and used as template for PCR reactions employing primer pair Exon2 (5'-caaccgagctgaagcattctgcct-3') and Mut217 (5'-cctgggactccttctggtactgggtgacgc-3'). Transgenic animals were characterized by the generation of a 700 bp fragment during these reactions. Founders generated on a 129Sv genetic background were identified and crossed back into PrP -/mice (C57BL/ 6J × 129/Sv genetic background; [28]) to yield Prnp 0/0 lines homozygous for the PrP-encoding transgene array. In doing so, one Prnp 0/0 lines homozygous for the PrP(TetraH>G)-encoding transgene array, designated lines 34, and three Prnp 0/0 lines homozygous for the PrP(H95G)encoding transgene array, termed lines 4, 11, and 13, respectively, were identified.

Intracerebral inoculations and scrapie diagnosis
Groups of eight mice each comprising PrP(TetraH>G) +/+ mice (lines 34) as well as PrP (H95G) +/+ mice of three different founders on PrP -/background (lines 4, 11, and 13) and wild-type mice (C57BL/6J × 129Sv genetic background), respectively, were intracerebrally (i. c.) inoculated with mouse-adapted scrapie strain RML (NIH Rocky Mountain Laboratory, Hamilton, MT) using a dose of 30 μl of 10% (w/v) brain homogenate in sterile phosphate buffered saline (PBS). In parallel a 10% (w/v) brain homogenate from uninfected control mice ("mock" homogenate) was used. Inoculation was performed with a Luer lock system (catalog #807.001C; Robert Helwig, Berlin, Germany), and diethylether was used as an anesthetic. At that time the experiments were performed, the use of diethylether to anesthetize mice followed the animal protection regulations at the Ludwig Maximilians University but we note that ether is not any longer advised for use in this role due to potential adverse effects. All animals were kept under standard diurnal pathogen-free conditions and allowed access to food and water ad libitum. Mice were monitored every second day for the onset of clinical symptoms, and TSE was diagnosed according to standard clinical criteria used to identify animals exhibiting signs of scrapie, including ataxia, kyphosis, and hind leg paresis [29,30]. Mice were euthanized at the onset of clinical signs associated with terminal prion disease and extracted brain tissues were subjected to histopathological analyses. The animal experiments were performed in accordance with animal protection standards and were approved by the government of Upper Bavaria (protocol number 209.1-2531-31/02). Every effort was made to reduce the number of animals used and to ensure they were free of pain and discomfort.

Histopathology and immunohistochemistry
Brains from terminally diseased mice were fixed with 4% buffered formalin, subsequently inactivated with 100% formic acid for 1 h, paraffin-embedded and then subjected to routine staining procedures using a Benchmark staining machine with a standard 3',3-diaminobenzidine (DAB) detection system according to the manufacturer's instructions (Ventana Medical Systems, Arizona, USA). For detection of PrP Sc monoclonal CDC1 antibody raised against full-length mouse PrP was used (1: 500 in PBS-Tween). Spongiform changes and gliosis were visualized using Hematoxylin and Eosin staining and anti-GFAP polyclonal antiserum (1:1600, DAKO, Germany), respectively. Histological sections were scored in a blinded fashion for spongiform changes in nine standard grey matter areas and three white matter areas as described earlier [31,32] and lesion profiles were constructed [33,34]. Scoring of pathological change was performed in the same regions using the following score: 0 = "not detectable", 1 ="mild", 2 ="medium", 3 ="high", for levels of gliosis or PrP Sc deposition, respectively.
Cell-free conversion of 35 S-labelled PrP C Adherent rabbit kidney epithelial (RK13) cells [37] were co-transfected with vectors pcDNA3.1/Hygro(+) (Invitrogen) and pCI-neo (Promega) carrying either full-length mouse PrP or PrP(TetraH>G) as described previously [38]. Single cell clones resistant to hygromycin B (500 μg/ml, Roche) were expanded and the level of PrP C expression was monitored by western blot analysis after SDS-PAGE of cell lysates.
Labelling of PrP C with 35 S and purification of PrP C from cell lysates was performed as described previously [38]. Briefly, cell clones were incubated for 1 h at 37˚C with 5% CO 2 in culture medium lacking cysteine and methionine. Tunicamycin was also added in a concentration of 27 μg/ml in order to obtain deglycosylated PrP C . After 1 h starvation, Redivue-Promix (GE Healthcare, Munich, Germany) containing 35 S-methionine and 35 S-cysteine was added at 0.23 mCi/ml. Cells were lysed after 5 h incubation at 37˚C with 5% CO 2 . Lysates were centrifuged for 5 min at 4˚C at 1000 × g, the supernatant precipitated with four volumes of cold methanol and the pellet resuspended into a detergent lipid protein complex (DLPC) buffer (150 mM NaCl, 50 mM Tris/HCl pH 8.0, 2% N-laurylsarcosine, 0.4% L-α-phosphatidylcholine) by sonicating three times for 20 s with 70% output intensity using the ultrasound-generator Sonoplus HD2200-UW2200 with BR30 cup-horn sonicator (Bandelin, Berlin, Germany).
PrP C was purified from the DLPC suspension by immunoprecipitation as described by Caughey et al. [39] using the antibody RA3153 [40]. PrP C was eluted from the protein-A sepharose beads with 0.1 M acetic acid, transferred into 1.5 ml low-binding tubes (Eppendorf, Hamburg, Germany) and stored at 4˚C. The activity of purified PrP C samples was measured using the β-counter TRI-CARB 2900TR (Perkin Elmer, Rodgau, Germany). Purification of PrP Sc was performed as described previously [41]. The final pellet was resuspended in phosphate buffered saline (PBS) with 0.5% zwittergent sulfobetaine 3-14 (100 μl/2 g brain tissue) by sonication. The resulting suspension was transferred into 1.5 ml low-binding tubes (Eppendorf, Hamburg, Germany) and stored at 4˚C. PrP Sc was purified from brains of terminally-diseased tga20 mice infected with RML.
In vitro conversion reactions with purified PrP Sc and 35 S-labeled PrP C were performed in a reaction volume of 30 μl as described previously [38]. Briefly, 15,000 cpm 35 S-PrP C was incubated for 3 days at 37˚C with 4 μl of PrP Sc in conversion buffer (200 mM KCl, 5 mM MgCl 2 , 0.625% N-laurylsarcosine, 50 mM sodium citrate pH 6.0) including 0.7 M guanidine hydrochloride (GndHCl). 90% of the reaction volume was digested with proteinase K (PK) for 1 h at 37˚C (20 μg/ml) and the remaining 10% was left untreated. Further sample preparation was performed as described [39]. After electrophoresis of untreated and PK-digested samples gels were incubated in fixing solution (isopropanol, H 2 O and acetic acid in a ratio (v/v) of 25:65:10, respectively) for 30 min and subsequently incubated in Amplify (Amersham Biosciences, Freiburg, Germany) for additional 30 min. Pre-treated gels were dried and exposed using a Fujifilm Phosphorimager BAS 1800 II and analyzed with AIDA V3.44.035 densitometry software (Raytest, Straubenhardt, Germany). The band intensity of the samples with (I˚+ PK ) and without (I˚-PK ) PK treatment were measured after background subtraction. With respect to samples treated with proteinase K, only bands within the molecular weight range of 18-24 kDa were used for evaluation. The conversion efficiency (CVE) was calculated using the formula CVE [%] = [I˚+ PK˚/˚( I˚-PK Ã 10)] Ã 100.
FCS/SIFT measurements. FCS and SIFT measurements were carried out on an Insight Reader (Evotec-Technologies, Germany) with dual-color excitation at 488 and 633 nm using a 40× of 1.2 NA microscope objective (Olympus, Japan) and a pinhole diameter of 70 μm at FIDA setting exactly as described in Giese et al. [42]. The fluorescence data were analyzed by correlation analysis using the FCSPPEvaluation software version 2.0 (Evotec OAI, Germany). Two color cross-correlation amplitudes G(0) were determined using the same software. Evaluation of SIFT data in two-dimensional intensity distribution histograms was performed as described [42].
Aggregation assay. A stock solution of fluorescently-labeled rPrP was prepared in 10 mM NaP i , pH 7.2, containing 0.2% sodium dodecyl sulfate (SDS, Sigma) to keep the protein in an unaggregated state at the beginning of the measurements. The concentrations of rPrP-A-lexa488 and rPrP-Alexa647 were adjusted to approximately 10 molecules per focal volume and 20 molecules per focal volume, respectively. Experiments were started by diluting this stock solution into NaP i -buffer containing 0-0.2% SDS to obtain final SDS concentrations ranging from 0.0125% to 0.2% and a final concentration of labeled rPrP of approximately 5 nM. In some experiments, metal ions (CuCl 2 , MnCl 2 ) or EDTA were added to the stock solutions of monomeric rPrP in 0.2% SDS. Experiments were performed in 96-well plates with a cover slide bottom (Evotec Munich, Germany). To reduce evaporation, the 96-well plates were sealed with adhesive film. Typically, aggregation was monitored for 8 h in four parallel identical samples for each experimental group.

Statistics
Statistical analysis and graphics were done with SigmaStat 2.03, SigmaPlot 6.0 and Microsoft Excel. Data of two independent groups following a normal distribution with equal variances were analyzed with the unpaired Student´s t-test, p 0.05 was considered statistically significant.

Generation and characterization of PrP transgenic mice
We generated transgenic mouse lines encoding full-length mouse PrP with all four histidine residues within the OR region (PrP(TetraH>G)) or at position 95 (PrP(H95G)) replaced with glycine. To this we used the "half genomic" vector, wherein the protein coding sequence of interest is placed under the control of the mouse Prnp promoter [5]. Constructs were microinjected into fertilized oocytes on a PrP knockout genetic background (Prnp 0/0 , 129/Sv-C57/ Bl6), with offspring of four founder lines expanded for further study. PrP(TetraH>G) (line 34) and PrP(H95G)(line 13) mice were examined in detail and found to express the mutant proteins in the brain at a level comparable to full-length PrP C in wt control mice ( Fig 1A). Moreover, deglycosylation revealed that PrP C encoded by the PrP(TetraH>G) and PrP(H95G) transgenes was represented by a C1 endoproteolytic fragment as well as full-length protein (i.e. was processed in a manner analogous to that of wt PrP C ; Fig 1B). Notably, transgenic mice presented with a wild-type-like distribution of mutant PrP C in the brain, and showed no signs of spontaneous neurodegeneration for an observation period of up to 600 days (exemplarily shown in S1

Susceptibility of mice expressing mutant PrP to scrapie prions
To examine the susceptibility of the novel transgenic mice to scrapie, mice descended from PrP(TetraH>G) founder animals (line 34) as well as corresponding wt controls (129/Sv-C57/ Bl6) were inoculated intracerebrally (i.c.) with mouse-adapted Rocky Mountain Laboratory (RML) prion isolate and monitored for the onset of disease-associated symptoms and for survival. Similarly, mice descended from three different PrP(H95G) founders (lines 4, 11, and 13) were also subjected to prion challenge. We found that TgPrP(TetraH>G) mice were susceptible to scrapie, but succumbed to disease significantly later than wt controls (Table 1, "primary passages"). Clinical signs did not appear until 292 days post infection (dpi; line 34), indicating protracted disease duration, but were otherwise similar to those of RML-infected wt mice, with signs including ataxia, kyphosis and difficulties in righting. In contrast, TgPrP(H95G) mice of all three founder lines presented with clinical signs of scrapie, indistinguishable from that of wt control mice, but died after a significantly shortened mean incubation period ranging from 87 to 116 dpi ( Table 1, "primary passages"). Notably, PrP C expression in both Tg (TetraH>G) and TgPrP(H95G) lines were comparable to wt levels.

Immunohistochemical analyses
Brain sections from terminally ill RML-infected transgenic and wt control mice were examined for pathological changes. Wild-type mice (n = 6) presented with spongiform changes that were prominent in the hippocampus, thalamus, septum, cortex and throughout all white matter areas (Fig 2A, upper two rows). This was accompanied by distinct synaptic PrP Sc deposits. Diseased TgPrP(TetraH>G) mice demonstrated a wild-type-like distribution of spongiform changes with considerably less severe spongiosis (Fig 2A, lower two rows, third column). Concordantly, there was a tendency to milder gliosis in some mice (Fig 2A, lower two rows, fourth column). Immunostaining, however, revealed a PrP Sc deposition pattern in TgPrP(TetraH>G) mice markedly different from the predominantly synaptic pattern seen in wt controls. Instead, plaque-like accumulations of PrP Sc with a dense globular core were observed throughout all sections, particularly noticeable adjacent to the corpus callosum (Fig 2A, lower two rows, first and second columns).
Regarding terminally ill TgPrP(H95G) mice, spongiform changes were significantly less pronounced in all grey and white matter areas analyzed. In all three TgPrP(H95G) transgenic lines, the most extensive spongiform changes were found in the hippocampus and thalamus, while cortex and white matter areas were relatively spared (Fig 2B, rows 2-4, third column). Compared to RML-infected wt mice (Fig 2B, upper row) the thalamus, hippocampus and septum of TgPrP(H95G) mice were similarly affected by gliosis, whereas the medulla and the thalamic cortex tended to higher or lower scoring values, respectively (Fig 2B, rows 2-4, third column). Again, striking observations applied to the staining for PrP Sc deposits, which in TgPrP(H95G) mice appeared as feathery plaque-like structures on a faint synaptic background (Fig 2B, rows 2-4, second column), with accumulations also revealed by the PET-blot technique ( Fig 2B, rows 2-4, first column). The most prominent pathology was recognized in TgPrP(H95G) mouse line 13, followed by lines 4 and 11. Prior studies have indicated that clinical and pathological manfestations of experimental scrapie disease in mice are little affected  by chronological age of the host [43,44], indicating that these different patterns of onset and pathology more likely relate to intrinsic properties of the agent than age-dependent host responses.

Lesion profiling
Using established protocols, we scored the extent of spongiosis and gliosis within 12 brain regions of the infected mice in a blinded fashion. These analyses, to assess allelic effects upon neuropathological endpoints, are shown in Fig 3. With the exception of region 1 (dorsal medulla), the lesion profiles of spongiform change and gliosis in PrP(TetraH>G) mice occurring after i.c. inoculation of RML were similar in shape but with smaller magnitude than in non-Tg mice (Fig 3A and 3C). For the H95G allele, the overall profile of lesions resembled wt mice of the C57/129 genetic background but, again, the intensity of the lesions was attenuated ( Fig 3B); gliosis was similar to wt ( Fig 3D).

Immunodetection of PrP Sc
Western blot analysis of brain tissue derived from terminally ill transgenic mice revealed that all animals harboured protease-resistant PrP Sc in their brains (Fig 4). TgPrP(TetraH>G) line 34 mice propagated PrP Sc with similar ratios of putative di-, mono-and unglycosylated forms to those seen in RML-infected wt mice (Fig 4A). This was also the case for TgPrP(H95G) mice descended from three different founder animals (Fig 4B). In terms of quantity, the amount of PrP Sc was notably diminished versus wt for TgPrP(TetraH>G) line 34.

Properties of prions after sequential passage in TgPrP(TetraH>G) and TgPrP(H95G) mice
We next investigated if sequential passages of a prion isolate in Tg mice resulted in the emergence of novel biological properties. Brain tissues derived from PrP(TetraH>G) and PrP (H95G) mice that were scored as positive for prion infection (by typical clinical signs, abnormal PrP immunohistochemistry, and the presence of PrP Sc on western blot analysis) were used to i.c. inoculate recipient mice of the same genotype. In one set of analyses, PrP(TetraH>G) (line 34) and wt mice were inoculated with brain homogenate extracted from two RMLinfected PrP(TetraH>G) line 34 mice. In TgPrP(TetraH>G) mice challenged with PrP(Tetra-H>G)-passaged prions, the prolonged incubation times observed with the primary transmission of RML prions in corresponding host mice were again apparent, exceeding 350 days ( Table 1, "secondary passages"). In contrast, wt mice challenged with PrP(TetraH>G)-passaged prions presented with survival times that were not significantly different from the corresponding RML-inoculated group (p = 0.08 for inoculum #258 and p = 0.47 for inoculum #260); see Table 1, "secondary passages"), indicating no loss in infectious titre. PrP(H95G) (line 13) and wt animals were infected with brain homogenate derived from two terminally ill line 13 PrP(H95G) mice. In this secondary passage experiment, a non-significant trend towards reduced survival times was observed in PrP(H95G) mice compared to the initial RML-infected group ( Table 1, "secondary passages") however, no significant change in survival time could be observed with wt mice inoculated with PrP(H95G)-passaged prions compared to the corresponding RML-infected control group (Table 1, "secondary passages"). Neuropathological lesions and the pattern of PrP Sc deposition in the brains of secondary passage transgenic mice and wt control animals resembled those noted for the corresponding RML-infected groups (Fig 5). Moreover, western blot analyses of brain tissue derived from transgenic mice expressing mutant PrP as well as wt mice inoculated with PrP(Tetra-H>G)and PrP(H95G)-passaged prions, revealed a banding pattern corresponding to total PrP and PrP Sc , respectively, which resembled the initially RML-infected groups (not shown).

In vitro conversion capability of full-length PrP(TetraH>G)
To assess whether extended incubation times in Tg PrP(Tetra-H>G) mice represented a compromised ability of the mutant PrP C to convert to the PK-resistant conformation ("PrP res "), we performed in vitro reactions in the presence of purified brain-derived PrP Sc using the method of Kocisko et al. [4]. In this approach, the altered PrP C variant as well as wild-type PrP C from mouse were expressed in PrP C -deficient cells in the presence of 35 S-methionine and 35 S-cysteine, purified by immunoprecipitation, and used together with purified PrP Sc from RML infected mouse brain for in vitro conversion reactions. We found that substitution of all four OR histidines reduced the conversion efficiency obtained with wt PrP almost two-fold (Fig 6A; p = 0.07 and 0.03 using two-sided and one-sided T-test analyses, respectively). There was no detectable PrP res in unseeded reactions (Fig 6B), indicating that neither mouse wt PrP nor PrP(TetraH>G) developed intrinsic resistance towards proteinase K digestion under these assay conditions.

Single molecule analysis of glycine-substituted PrP alleles
In a final set of analyses, we assessed the ability of single molecules of recombinant full-length wt PrP and the two mutant proteins to undergo structural conversion and aggregate formation in the presence of putative co-factors [45,46]. To quantify and characterize rPrP aggregates formed, we used fluorescently-labelled PrP and cross-correlation analysis as well as scanning for intensely fluorescent targets in a confocal single molecule detection system, as described previously for recombinant full-length human PrP [42]. The use of single particle fluorescence in combination with a measurement setup optimized for high-throughput measurements in multi-well plates allowed a large number of individual measurements to be performed, a potentially advantageous approach given inherent variability often observed in assays of de novo protein aggregation.

Fig 6. Conformational changes measured by in vitro conversion reactions. (A)
In vitro conversion reactions have been performed with radiolabeled wild-type and PrP C (TetraH>G) purified from RK13 cells and PrP Sc purified from brains of RML-infected Tga20 mice as described [4]. Samples were analyzed by SDS-PAGE-fluorography, and relative conversion efficiencies (CVE) were calculated from band intensities before and after digestion with proteinase K using the formula CVE [%] = [I˚+ PK / (I˚-PK *10)]*100. PrP C with substituted OR histidines (PrP C (TetraH>G)) is only half as efficient in converting to the misfolded, PK-resistant conformer than wt PrP C . Mean values ± standard error (SEM) were determined from 11 independent experiments for each PrP C type. P-values Wild-type rPrP and the two mutant proteins (i.e. TetraH>G-rPrP and H95G-rPrP) were all found in the non-aggregated state in 0.2% SDS at pH 7.2 in the absence of additional copper (S4 Fig, "0.2% SDS"), the results for the wt allele being anticipated from prior studies [45,46]. After SDS was diluted out below 0.05% to induce aggregate formation, an increase in the cross-correlation amplitude was observed, which is a direct measure for the presence of double-labeled particles present in the sample, and is thus indicative of de novo oligomerization. Thus, at SDS-concentrations of 0.02%, wt rPrP reached a significant higher cross correlation amplitude than TetraH>G-rPrP and in particular H95G-rPrP (Fig 7, "0.02% SDS"), indicating that the mutations reduced the oligomerization propensity of wt PrP.
Disturbances in the levels of copper and manganese have been described in prion-infected brain tissue [47][48][49] and changes in brain copper levels have been suggested to influence incubation time in experimental prion disease [11,13]. To assess whether metal ions such as Cu 2+ and Mn 2+ affect the aggregation properties of wt and mutant rPrP, we added different amounts of CuCl 2 , MnCl 2 , and EDTA to aggregation reactions in the presence of the 0.02% concentration of SDS that is permissive to formation of PrP multimers. As shown in Fig 7, in the presence of 0.02% SDS, copper concentrations of 1 μM (in molar excess to recombinant protein) repressed the aggregation of wt-rPrP about 70%, whereas the aggregation of both Tet-raH>G-rPrP and H95G-rPrP was less affected (Fig 7, "1 μM Cu 2+ "). Higher copper concentrations (10 μM and 50 μM) were able to further decrease the aggregation efficiency of wt-rPrP but again showed no significant effect when added to mutant rPrPs (data not shown). These data indicate that sequences within and adjacent to the OR region in unmetallated PrP (i.e., apo-PrP) facilitate aggregation, but this effect is offset by metallation. TetraH>G-rPrP and H95G-rPrP assessed under the same conditions have lower baseline measures for aggregation that are little affected by Cu 2+ addition. To assess whether a reported stimulatory effect of Mn 2+ on aggregation of human rPrP(23-231) [42] is also applicable for full-length mouse rPrP, we added MnCl 2 in the same type of protein aggregation experiments. We found that manganese concentrations of 30 μM were able to further increase the aggregation of full-length wt rPrP bỹ 15%, with a mild proportional increase noted for the TetraH>G-rPrP allele but not the H95G-rPrP allele (Fig 7, "30 μM Mn 2+ "). To assess whether the buffer used for aggregation assays contained traces of divalent metal ions that affect outcome measures, we performed controls using the chelating agent EDTA. In the presence of 100 μM EDTA wt-rPrP aggregated~40% less than with 0.02% SDS alone (Fig 7, columns labelled "100 μM EDTA"). Addition of the chelating agent did not completely abolish the inhibitory effect seen with Cu 2+ , nor the stimulating effect seen with Mn 2+ . Again, no affect was seen on the aggregation of the mutant proteins.
Taken together, our data indicate that H>G replacements in the OR region and at position 95 resulted in a decreased tendency for aggregation of full-length mutant proteins versus the wt point of reference.

Multiple activites of the PrP C OR
Prior work has defined complex relationships between the PrP C OR region and prion disease pathogenesis. Though the OR region comprises the main Cu-binding region of PrP C , accommodating up to 4 Cu (II) ions per polypeptide chain, a direct causative link between copper and prion disease was challenged by the observation that transgenic mice completely lacking the OR region (PrPΔ32-93) were still susceptible to infection with laboratory prion isolates [6]. Elimination of the OR region slowed the rate of disease progression in these transgenic mice, indicating that the octarepeats are not necessary for sustaining prion replication and pathogenesis, but do modulate the extent of neuropathological degeneration and disease presentation. Genetic prion diseases are associated with extra octarepeats (i.e., above the typical wt complement of one histidine-lacking OR followed by four histidine-containing ORs), with these mutant PrP alleles likely originating from replication fork slippage on tandem direct repeats during DNA replication. For these open reading frame expansions, the more octapeptide repeats are present in PrP, the more prone the protein is to misfolding [50,51], which correlates with earlier disease onset and shorter disease duration in affected patients [52]. Also, when supernumerary octarepeat insertions rise above a threshold, they give rise to GSS-like disease with cerebral amyloid deposition instead of the clinical picture of familial CJD [53]. This effect, seen with histidine-containing wt OR units, has been ascribed to a conformational aspect of metal-binding to the OR region, where insertions impede the ability to transition from a configuration denoted "component 3" to a multivalent configuration, "component 1" [54]. Overall, an extensive literature contains complex, and sometimes contrasting, findings [55][56][57][58]; taken together one may surmise that the OR region has pleiotropic effects, i.e. modulating prion replication and pathogenicity, as well as producing a protective effect against oxidative stress [59,60] and modulating copper-dependent multimerization [61]. To address these complex relationships and the in vivo role of site 5 we created two new strains of transgenic mice. The resulting Tg lines revealed a number of unexpected features as well as some parallels to mice with structure-guided re-design of the PrP OR region [26].

Biological properties of PrP(TetraH>G)
We made a line of PrP(TetraH>G) mice that expressed PrP C at a level slightly less than that seen in wt control animals. Here, the histidine to glycine substitutions within the OR region did not influence α-cleavage in the brain to make C1 fragment (Fig 1); others have noted that copper-stimulated PrP internalization requires both the octapeptide repeat domain and the palindromic sequence starting at the α-cleavage site (amino acids 110/111 in mouse PrP) to be intact [18,23,62]. In terms of cell biological effects, PrP(TetraH>G) had a diminished capacity to initiate cell death when exposed to an environment containing PrP Sc and under cell culture conditions without copper-supplementation, PrP(TetraH>G) trafficked to lipid rafts like wt PrP C (S2 and S3 Figs). While we cannot exclude that Cu-dependent endocytosis would be impaired for this allele, this process has been modeled in other studies by supplementation of culture medium with copper concentrations of 100-500 μM, considerably higher than for the other studies described here [23,63].
For the biology of metal speciation, Miura et al. have suggested that a possible role of the wt octarepeat motif is to stabilize the α-helical structure of PrP C in the presence of copper [64]. Also, using hybrid density functional theory (DFT) calculations, Hodak et al. have inferred that at physiologically-relevant low Cu(II) concentrations, a structural alteration of the Cubonded octarepeat region creates flexible turns in the areas separating copper-bound residues, which increases the rigidity of the entire N-terminal domain to make it more resistant to misfolding [12]. On the other hand, and bearing in mind that mature wt PrP C is normally considered to exist a monomer, Yen et al. reported that copper-induced structural conversion of wt PrP C predisposes it for oligomerization that is associated with toxicity [58]. Thus, we arrive at opposite predictions that in the presence of copper, the OR region of the PrP(TetraH>G) allele is now susceptible to misfolding under low Cu(II) occupancy or is prevented from Cu-dependent oligomerization. In practice, and making an assumption that PrP C can become charged with endogenous bioavailable metal [65], we could not detect a spontaneous neurodegenerative disease in the aged PrP(TetraH>G) Tg mice (not shown). In the case of prion-infection initiated with i.c. delivery of PrP Sc -a situation where PrP C does misfold and multimerizereplacement of the four wt histidine residues within the OR region with glycine residues resulted in prolonged survival times of scrapie-infected PrP(TetraH>G) mice (Table 1). These results fit well to the finding that treatment of mice with the copper chelator D-penicillamine (D-PEN) delays onset of prion disease [13]. In addition, the diseased animals show reduced PrP Sc levels and serial passage of infectivity from the brains of mice with end-stage disease resulted in prolonged incubation times in comparison to the parental stock of RML prions, indicative of a drop in infectious titre. Our overall results with PrP(TetraH>G) line 34 mice indicate that the amino acid exchange not only influenced the OR itself but also induced a persistent conformational change in the C-terminal protease-resistant core portion of the protein.
These results can be reconciled with our in vitro data showing that recombinant PrP(Tetra-H>G) was less susceptible to SDS-induced aggregation than wt rPrP, and that cellular PrP (TetraH>G) had a reduced propensity for conformational in vitro conversion to the protease resistant isoform (Figs 6 and 7, S4 Fig). Perhaps most remarkably, we found plaque-like PrP Sc deposits with a dense globular core in infected TgPrP(TetraH>G) mice. A second PrP(Tetra-H>G) Tg line designated HG1-2CL was profiled less extensively but behaved in a similar manner to line 34 in the accumulation of plaque deposits (as well as protease-resistant PrP profile after RML inoculation and in the PMCA conversion assay; not shown). As the dimorphism L108F, T189V that defines the mouse Prnp b allele is typically required to allow certain prion isolates to make amyloid plaques in mice [44,66,67], this phenomenon is most unusual for prion infections where the PrP C precursor has C-terminal sequences identical to the mouse Prnp a allele.

Biological properties of PrP(H95G)
In contrast to the prolonged incubation times observed with scrapie-infected PrP(TetraH>G) mice compared to wt mice, TgPrP(H95G) mice had more rapid disease progression. Cox et al. have suggested from ab initio electronic structure calculations of rPrP(89-230) that detachment of copper bound to H95 might be a pivotal step in the conversion pathway [68]. According to this model, ablation of copper binding to H95 with Ala, Tyr or Gly substitutions should present with shortened incubation times; for the H95G allele here, this was indeed the case (Table 1). In vitro analyses of site 5 mutations using extended X-ray absorption fine structure spectroscopy (EXAFS) along with studies in cultured cells provided some overlapping findings and conclusions [69]. These authors showed enhanced PK-resistance of an epitope-tagged mouse PrP equivalent to H95Y when expressed in chronically infected neuroblastoma cells and also that this allele expressed in uninfected cells generated misfolded PrP that could "seed" uninfected recipient N2a cells. These studies proposed a mechanism underlying these effects wherein a pH drop caused by trafficking into endosomal/lysosomal compartments causes H95Y PrP to "switch" to a pathological conformation with enriched β-sheet content in the segment spanning residues 105-116 and the adjacent palindromic motif. Conversely, WT PrP with the non-OR site 5 region coordinated in the 2His state (i.e. copper coordinated by His95 and His110) is stabilized by an attenuation of contacts between residues 89-126 and the C-terminal structured domain. Furthermore, according to the computational model in Cox et al., copper coordination to H95 can protect PrP from prion conversion as in their copper binding at this position is not compatible with formation of β-strands in a model for PrP Sc involving left-handed helices [68]. However, not exactly in accord with these in silico and in vitro analyses [68,69], the shortened disease progression noted in TgPrP(H95G) mice could neither be ascribed to an increased tendency to undergo structural conversion and aggregate formation triggered by a reduction of SDS concentration from 0.2 to 0.02% in an in vitro system, nor to increased PrP Sc levels in the brains of terminally ill mice (assessed by western and PET blot analyses, respectively). Given that PrP Sc itself is not necessarily the primary cause of neurodegeneration and that aggregation, toxicity and infectivity are distinct processes (summarized in [70]), it appears either that PrP(H95G) aggregates are more toxic than aggregated wt (or for that matter PrP(TetraH>G)) or that copper depletion at position 95 may play a role in conferring PrP Sc neurotoxicity. In accord with the concept of altered toxic behaviour, diffuse PrP deposition observed in TgPrP(H95G) mice differed from the synaptic pattern seen in scrapie infected wt mice and, in contrast to TgPrP(TetraH>G) mice, more closely resembling genetic human prion diseases arising from point mutations in the C-terminal domain [71].

Relationships between PrP N-terminal function and disease pathogenesis
Although studies here and in a related paper [26] were undertaken to explore different predictions from in vitro studies, overlaps were seen in outcomes measured in the brains of Tg mice. The PrP(S3) allele made by Lau et al. has changes (within each OR of the OR region) that will accentuate and lock "component 3" binding geometry [26]; this geometry is associated with nM Cu-binding affinity and a metal: polypeptide stoichiometry of 1:1. A PrP S1 allele made by Lau et al. has a different OR region alteration to disfavor this type of copper binding. Here, the PrP(H95G) substitution C-terminal to the OR region was designed to attenuate "site 5" copper binding (also with a metal:polypeptide stoichiometry of 1:1 and which has a K d estimated between 10 −9 -10 −14 M in its wt form [72,73]). In practice, and accommodating for transgene expression levels, both PrP(S3) and PrP(H95G) alleles led to curtailed incubation periods upon prion inoculation, with a focus upon altered toxicity rather than elevated infectious titre; indeed, serial passage of infectivity and lower spot counts in a scrapie cell assay indicated depressed infectious titres for PrP(S3) [26] while inocula from TgPrP(H95G) mice produced typical incubation times in wt mice (Table 1). For PrP(TetraH>G) and PrP(S1), these alleles were designed to alter the OR region in different ways: in the case of PrP(TetraH>G), to remove OR histidines and prevent any metallation (and hence any metal-dependent structural compaction) of the OR region while in the case of PrP(S1), to hold all histidine-containing OR's in an extended conformation [25,26]. Here, in practice, and again accommodating for transgene expression level, both alleles were associated with reduced PrP Sc levels, reduced titre and prolonged incubation times subsequent to prion inoculation.
One school of thought is that distinct redox activites of PrP are involved in the mechanisms underlying prion diseases by modulating reactions leading to ROS-mediated β-cleavage and initiating beta-sheet formation. However, the finding that cleavage in the vicinity of the β site can be greatly enhanced by a process independent of metal binding to the OR region [26,74] gives pause to the view that redox processes are necessarily front and centre in PrP biology. Instead, conformational changes may lie to the fore. While the OR region and site 5 are natively unstructured in apo-PrP, we conclude that cis interactions between the OR region or the region corresponding to site 5 and the globular C-terminal domain can modulate pathogenic outcomes. This conclusion is based in part upon in vitro studies where the OR region in zinc-loaded PrP interacts with negatively charged residues on helices 2 and 3 [75] and where Cu-loaded OR region (in component 3 geometry and with site 5 inactivated by histidines to tyrosine replacements) interacts with helix 3 [76]. Using a different approach, Thakur et al. inferred that in the presence of copper the PrP OR region interacts with helix 2 and the site 5 region interacts in cis with helix 1 and the preceeding loop [77]. For the folding of PrP expressed in the brain, the PrP Sc from TgPrP(S3) mice with an altered OR had a change in accessibility of the 12B2 epitope at residues 89-93 closely adjacent to H95 [26] and cis effects upon the globular domain could be inferred from altered infectivity titres, proteinase K-resistance and responses in conformational stability assays. We infer that similar long-range remodeling effects likely drive the outcome measures in Tg.PrP(TetraH>G) and Tg.PrP (H95G) mice. Moreover, since the vicinity of site 5 is also a major binding site for PrP C /oligomeric Aβ interactions [78,79] further work to understand these interactions may be useful above and beyond the immediate context of prion infections. Prior to PMCA, healthy brain homogenates were adjusted to 60 ng PrP C /μl with 10% (wt/vol) PrP 0/0 brain homogenate, spiked 1:50 with 10% (wt/vol) RML homogenate and subjected to 10 rounds of PMCA. In vitro generated wt PrPres (C57/129Sv; lanes 1-4), PrPres(TetraH>G) lines 34 (lanes 5-8), as well as PrPΔ32-93 (C4; lanes 9-12) was detected using monoclonal antibody 4H11. In order to rule out degradation of PrP C before PMCA, the corresponding undigested starting material