Protease-sensitive prions with 144-bp insertion mutations

Insertion of 144-base pair (bp) containing six extra octapeptide repeats between residues 51 and 91 of prion protein (PrP) gene is associated with inherited prion diseases. Most cases linked to this insertion examined by Western blotting showed detectable proteinase K-resistant PrPSc (rPrPSc) resembling PrPSc type 1 and type 2 in sporadic Creutzfeldt-Jakob disease (sCJD), or PrP7-8 in Gerstmann-Sträussler-Scheinker disease. However, cases lacking detectable rPrPSc also have been reported. Which PrP conformer is associated with neuropathological changes in the cases without detectable rPrPSc remains to be determined. Here we report that while all six but one subjects with the 144-bp insertion mutations examined display the pathognomonic PrP patches in the cerebellum, one of them exhibits no detectable typical rPrPSc even in PrPSc-enriched preparations. Instead, a large amount of abnormal PrP is captured from this case by gene 5 protein and sodium phosphotungstate, reagents that have been proved to specifically capture abnormal PrP. All captured abnormal PrP from the cerebellum and other brain regions is virtually sensitive to PK-digestion (termed sPrPSc). The presence of the predominant sPrPSc but absence of rPrPSc in this 144-bp insertion-linked inherited CJD case suggests that mutant sPrPSc is the main component of the PrP deposit patches and sPrPSc is sufficient to cause neurotoxicity and prion disease.


INTRODUCTION
including a β-sheet-rich structure, resistance to proteinase K (PK) digestion, insolubility in nondenaturing detergents, and infectivity [2]. It has been well-documented that the co-existence of PrP C and PKresistant PrP Sc (rPrP Sc ) is a prerequisite for the pathogenesis of various prion diseases; however, what type of PrP Sc conformers are directly responsible for the PrP deposition in the brain and the neuropathological changes in the prion diseases remains poorly understood [11].
Phenotypes of inherited prion diseases are mainly determined by specific mutations and a polymorphism at codon 129 (methionine (M) and valine (V)) of PRNP [12]. Most point mutations of PRNP are associated with inherited conditions exhibiting phenotypes similar to the well-characterized sporadic Creutzfeldt-Jakob disease (sCJD) with a fairly rapid course, deposition of a typical rPrP Sc (designated PrP27-30 including PrP Sc type 1 or type 2) in CNS, and widespread of spongiform degeneration. However, a few point mutations and a non-sense mutation are linked to the phenotype of Gerstmann-Sträussler-Scheinker disease (GSS), characterized by a relatively chronic clinical course and the presence of intense amyloid plaques composed of unique rPrP Sc fragments (designated PrP7-8) in the affected brains. A third set of inherited prion diseases are associated with insertions of one to nine octapeptide repeats, except for three that has never been reported [13][14][15][16][17][18]. These mutations are located in a nonapeptide (R1) and four octapeptides (R2 to R4) of the form P(H/Q)GGG(-/G)WGQ between residues 51 and 91 of PrP Wt [19][20]. The phenotypic heterogeneity and allelic origin of PrP Sc linked to insertion of 6 octapeptide repeats of PrP have been extensively characterized [21-25, 5, 26-29]. Although deposition of PrP in the cerebellum of affected brains is strikingly consistent, phenotypes, neuropathological changes, levels of rPrP Sc and transmissibility between individuals are variable, at least the electrophoretic pattern of PrP. PrP species in the insertion cases with undetectable rPrP Sc by the conventional assay [24] have not been further characterized. Addressing these important issues may shed light on the correlation between highly variable neuropathological changes and the chameleon-like conformations of PrP Sc .
Here we examined brain PrP and neuropathological changes in six cases carrying the 144-bp PrP insertion mutation. Neuropathologically, these cases exhibited spongiform degeneration, astrocytosis and multicore plaques with or without neuronal loss although the severity of these changes differed between cases or between areas of the same brain. However, all but one consistently had the deposits of PrP patches orientated perpendicular to the pial surface in the molecular layer of the cerebellum. Surprisingly, one of the cases displaying the cerebellar PrP patches revealed virtually no brain rPrP Sc that was well represented in the other five cases. In contrast, this variant case was associated with a large amount of PrP species that was PKsensitive but was captured by reagents including gene 5 protein (g5p) and sodium phosphotungstate (NaPTA), proven to specifically bind to insoluble and aggregated PrP regardless of its PK resistance [30][31][32][33].

Clinical information of the six subjects examined
Six cases with 144-bp insertion mutation (fCJD Ins ) were collected between 2000 and 2006 at the NPDPSC ( Table 1). All six cases were female with average age at onset of 38.5 ± 9.8 years and highly variable disease durations ranging between 3 and 180 months. Two cases were methionine/methionine (M/M) homozygous at residue 129 of PrP, three valine/valine (V/V) homozygous and one M/V heterozygous. The insertion mutant allele was coupled with the 129M in the M/V heterozygous subject. The octarepeat region in the six fCJD Ins cases examined included two types of sequences ( Table 1).

Detection of rPrP Sc by conventional Western blot analysis
In the samples without PK-treatment, an extra band was observed migrating at ~38-40 kDa in the five cases with fCJD Ins but not in PrP Sc type 1 and type 2 controls from sporadic CJD (sCJD) (Fig. 1A, indicated by the arrow head). This extra high band represents the diglycosylated PrP Ins with six extra octapeptide repeats and the monoglycosylated and non-glycosylated PrP species carrying the insertion were mixed with the three wild-type PrP bands, as indicated by the presence of multiple bands between 28 and 38 kDa compared to the relatively pure three bands in the samples from PrP Sc type 1 and type 2 of sCJD (Fig. 1A). The bands below 29-30 kDa were endogenously N-terminally truncated PrP fragments such as in cases 2 and 3 (Fig. 1A). The amounts of samples loaded were monitored by the detection of β-actin (Fig. 1B). PK-resistant rPrP Sc was detected in the brain homogenates from five out of six cases with fCJD Ins by conventional Western blotting with 3F4, although in case 5 rPrP Sc bands became visible only in the over-exposed film ( Fig. 1C and 1D). The lower PrP band of rPrP Sc had the gel mobility of 21 kDa (identical to that of PrP Sc type 1) for cases 1, 2, and 4, or ~19 kDa (identical to that of PrP Sc type 2) for cases 3 and 5 ( Fig. 1B and 1C; Table 1). Case 6 exhibited no typical rPrP Sc by the conventional Western blot analysis (Table 1), which was further characterized extensively by enrichment using g5p and NaPTA, ultracentrifugation-based sedimentation, and twodimensional gel electrophoresis as described in detail below. www.impactaging.com

Detection of PrP Sc in the case with no typical rPrP Sc
Case 6 was different from other five cases with the 144bp insertion mutation (Fig. 1C) as no rPrP Sc was detected by conventional Western blot in the brain homogenates from frontal cortex and cerebellum ( Fig.  2A). Instead, there were multiple PrP bands prior to PKtreatment although the upper PrP band was also higher than that from non-CJD and sCJD controls ( Fig. 2A).
These observations were in agreement with the genetic finding that fCJD Ins contains a PrP Ins (Table 1). The PrP Ins molecule may form additional set of three glycoforms including diglycosylated, monoglycosylated, and unglycosylated PrP Ins . Hence, there are at least 6 PrP bands that are assumed to be detected by 3F4 antibody in the brain homogenate from fCJD Ins including differently glycosylated PrP Wt and PrP Ins . Nevertheless, it was surprising that no PK-resistant PrP fragments were detectable by the conventional analysis with 3F4 antibody (Fig. 2A). In addition, anti-C antibody also failed to detect PK-resistant PrP fragments (data not shown).
NaPTA is a reagent that has been demonstrated to specifically precipitate both sPrP Sc and rPrP Sc [31,32,34]. In order to increase sensitivity of detection by Western blotting and ELISA, NaPTA has been used to enrich small amounts of PrP Sc in prion-infected peripheral organs where no PK-resistant PrP can be detected by the conventional assays [32,35,36]. To detect sPrP Sc species and determine if there is a small amount of rPrP Sc in this fCJD subject, a relatively large No typical signs of prion disease; severe brain atrophy 4 years after disease onset.
Similar dementia in her father who became demented at age 42 and died at age 52. of β-actin, which was used to monitor the amounts of samples from each case. (C) The samples were treated with PK prior to SDS-PAGE and immunoblotting. The gel mobility of the PK-resistant PrP from the cases 1, 2, and 4 was similar to that of PrP Sc type 1 control migrating at 21 kDa while the case 3 was similar to PrP Sc type 2 migrating at ~19 kDa. No PK-resistant PrP was visible in the case 5. (D) An over exposed smaller blot from the left part of the blot shown in C. The PK-resistant PrP bands from case 5 became detectable, the gel mobility of which was similar to that of case 2 migrating at ~19 kDa. www.impactaging.com amount of brain homogenate was used to incubate with NaPTA. Compared to the normal control, a larger amount of PrP was precipitated by NaPTA (Fig. 2B). Surprisingly, the precipitated PrP was virtually completely digested by PK (Fig. 2B). No typical rPrP Sc was detectable even in the over-exposed film (data not shown).
Using the anti-PrP monoclonal antibody 1E4 that has an epitope N-terminally adjacent to the 3F4 epitope [37,38], our recent study identified a novel PK-resistant PrP Sc characterized by the presence of dominant PKsensitive PrP Sc in an atypical human prion disease termed variably protease-sensitive prionopathy (VPSPr) [34,39]. Upon PK-treatment, virtually no rPrP Sc was detected by conventional Western blotting with 3F4, whereas PrP with a five-step ladder-like gel profile was detected preferentially by 1E4 in VPSPr [34,39]. On the 3F4 blots, similar to non-CJD and VPSPr, no rPrP Sc was detectable in brain homogenates from this fCJD Ins , whereas PK-rPrP Sc was detected in sCJD type 1 and type 2 cases as well as in case 3 of fCJD Ins (Fig. 3A). In contrast, on the 1E4 blots, except for non-CJD, PKresistant PrP Sc was detectable in cases 6 and 3 of fCJD Ins , VPSPr, sCJD type 1 and type 2. Nevertheless, rPrP Sc in case 6 was detectable in the samples treated at PK concentration equal to or less than 10 µg/ml and decreased significantly at PK equal to or greater than 25 µg/ml (Fig. 3B). The amount and gel profile of rPrP Sc in case 3 was very similar to those of rPrP Sc in sCJD type 2. As demonstrated in our previous study [34,39], rPrP Sc from VPSPr exhibited a five-step ladder-like gel profile (Fig. 3B). The rPrP Sc in case 6 detected by 1E4 was more similar to that of sCJD type 1 at low PK concentrations. However, at high PK concentration, no Western blotting of PrP treated with or without PK in case 6. Fr: frontal cortex; Cr: cerebellum. No PrP was observed after PK treatment in the samples from both fCJD Ins (case 6) and non-CJD.
The PK-resistant PrP27-30 was indicated in the sample from sCJD. The migration of PrP from the cerebellum of case 6 was slightly slower than that of PrP from both non-CJD and sCJD controls. (B) Precipitation of abnormal PrP by NaPTA. S1 from non-CJD (500 µl), sCJD (8 µl), and case 6 (three brain regions: 500 µl each) was incubated with NaPTA and then was subjected to SDS-PAGE and immunoblotting with 3F4. Although a small amount of PrP was precipitated from non-CJD brain sample (500 µl of S1), no PK-resistant PrP fragments were detected. NaPTA was able to precipitate PrP from 8 µl of sCJD S1 (62.5-fold less than non-CJD S1) and the precipitated PrP was resistant to PKdigestion. Compared to non-CJD sample, NaPTA precipitated large amounts of PrP from three different brain regions of case 6 including the cerebellum (Cr), occipital cortex (Oc) and brain stem (BS). After PK-treatment of the NaPTA-precipitated PrP from case 6, no PrP bands were observed. , VPSPr with 129VV, sCJD type 1, sCJD type 2, and fCJD Ins+rPrPSc were treated with a variety of concentrations of PK prior to SDS-PAGE and Western blotting with 3F4. PK-resistant PrP was only detected in sCJD type 1, type 2, or fCJD Ins+rPrP but not in other cases. B: The PK-treated PrP from these cases were detected with 1E4. In contrast, PK-resistant PrP was also detected in both fCJD Ins+sPrPSc and VPSPr, in addition to sCJD type 1, type 2, and fCJD Ins+rPrPSc . However, the PK-resistant PrP in fCJD Ins+sPrPSc was only detected when treated with lower amounts of PK less than 25 µg/ml. www.impactaging.com rPrP Sc was detected from case 6 while the profile of typical PrP Sc type 1 appeared in the sCJD type 1 case (Fig. 3B). Therefore, although no typical rPrP Sc was detected in both fCJD Ins of case 6 and VPSPr cases, the gel profile of rPrP Sc detected by 1E4 is clearly different from that detected in VPSPr, classic sCJD, and fCJD Ins of case 3. Importantly, the amounts and PK-sensitivity of rPrP Sc detected by 1E4 in this fCJD Ins (case 6) were also significantly less than those in VPSPr, sCJD and typical fCJD Ins (case 3).

Comparison of PrP oligomeric state between the two types of fCJD cases with the 144-bp insertion mutations
To determine whether the PrP molecule in case 6 has a different oligomeric state compared to other fCJD Ins cases, we further conducted the sedimentation of PrP Sc in the sucrose step gradients. PrP in three cases with readily detectable rPrP Sc was mostly recovered in bottom fractions 9-12, but not in top fractions ( Fig. 4A and 4C), similar to classic sCJD [33]. In contrast, PrP in case 6 was mostly recovered in top fractions 1 and 2, while no significant increases in the amounts of PrP were detected in bottom fractions ( Fig. 4B and 4C).
To investigate whether there are any rPrP Sc species in different fractions in case 6, we treated PrP in the fractions with an extremely low concentration of PK. After treatment with PK at 0.5 µg/ml, no PrP was detected in all fractions from case 6 while PrP was detected in bottom fractions from case 3 ( Fig. 4D and 4E). Our result suggested that PrP Sc from fCJD Ins case 3 was sensitive to PK even at 0.5 µg/ml.

Detection of allelic compositions in PrP C and PrP Sc from fCJD Ins with or without rPrP Sc by sedimentation in detergents
To dissect the differences in the composition of PrP Wt and PrP Ins between the two types of fCJD Ins with rPrP Sc or sPrP Sc (fCJD Ins+rPrPSc or fCJD Ins+sPrPSc ), we analyzed the composition of PrP Wt and PrP Ins . We took advantage of the most effective method by which the soluble PrP C and insoluble PrP Sc can be separated after ultracentrifugation in detergent buffer. After ultracentrifugation, PrP in the soluble and insoluble fractions was deglycosylated with PNGase F prior to By densitometric analysis, we quantified distributions of PrP Wt and PrP Ins in S2 and P2 fractions in the two types fCJD Ins+rPrPSc and fCJD Ins+sPrPSc . In both types of fCJD Ins , the ratio of PrP Wt or PrP Ins to the total PrP was the same: PrP Wt accounted for 63%, whereas PrP Ins accounted for 37% (Fig. 5C). Moreover, there were no differences in ratios of soluble and insoluble PrP Ins to the total PrP Ins between the two types of fCJD Ins (Fig. 5C). In contrast, the ratio of soluble PrP Wt to total PrP Wt was significantly greater in fCJD Ins+sPrPSc than in fCJD Ins+rPrPSc , whereas the ratio of insoluble PrP Wt to total PrP Wt was significantly smaller in fCJD Ins+sPrPSc than in fCJD Ins+rPrP (55% vs. 38% or 45% vs. 62%) (Fig. 5C).
Since fCJD Ins contains only a single PrP Wt allele whereas non-CJD or sCJD contains two PrP Wt alleles, we assumed that the amount of PrP Wt in fCJD Ins should account for approximately half of total PrP Wt detected in non-CJD or sCJD. To test for this possibility, we also quantified the intensity of the single PrP Wt allele from fCJD Ins and of total two PrP Wt alleles from non-CJD and sCJD in both S2 and P2. Surprisingly, although fCJD Ins contains only a single PrP Wt allele, whereas non-CJD or sCJD contains two PrP Wt alleles, the intensity of PrP Wt detected in both S2 and P2 were similar between fCJD Ins and non-CJD or sCJD (p > 0.05) (Fig. 5A, 5B and 5D).

Detection of allelic compositions of PrP C and PrP Sc from the fCJD Ins containing no rPrP Sc by conformation-specific binding reagents
Taking advantage of anti-PrP antibody 6H4 and g5p that specifically recognizes either native PrP C or misfolded PrP [40,41,30,33], we dissected allelic composition of sPrP Sc and ratio of sPrP Sc to total PrP in the detergent-insoluble fraction (P2) rich in PrP Sc from fCJD Ins . After specific capture of either PrP C by 6H4 or PrP Sc by g5p, the samples were treated with or without PNGase F that significantly decreases the heterogeneity of the protein by removal of the glycans (Fig. 6A). www.impactaging.com Moreover, probing the captured proteins with anti-N antibody would simplify the recognition of the fulllength PrP Ins and PrP Wt . Without PNGase F treatment, several bands migrating between 28-39 kDa were detected in both 6H4-captured preparation from S2 preparation rich in soluble normal PrP and g5p-captured preparation from P2 preparation rich in insoluble abnormal PrP (Fig. 6A). These bands represent glycosylated and unglycosylated full-length PrP Wt and PrP Ins . Notably, although g5p captured a large amount of misfolded PrP from P2 as expected, it also captured a small amount of PrP (a thin band) migrating at ~33-34 kDa that represents the unglycosylated full-length PrP Ins .
In the detergent-soluble fraction (S2) treated with PNGase F, two PrP bands were visualized by anti-N antibody in the preparation immunoprecipitated by 6H4: one with weaker intensity migrating at ~33-34 kDa corresponding to unglycosylated full-length PrP Ins and another with stronger intensity migrating at ~28-29 kDa corresponding to unglycosylated full-length PrP Wt , while only one thin PrP band corresponding to unglycosylated full-length PrP Ins was captured by g5p (Fig. 6A). In the P2 fraction rich in detergent-insoluble PrP, there were also two bands visualized by anti-N antibody in the sample precipitated by g5p. The gel mobility of the captured two bands was similar to that of the bands immunoprecipitated by 6H4 from S2 (Fig.  6A). Therefore, not only PrP Ins but also PrP Wt molecule participated in the formation of sPrP Sc in the fCJD with the PrP insertion mutation.
Quantitative analysis by densitometry from three independent experiments revealed that PrP Ins precipitated by 6H4 in S2 and by g5p in S2 and P2 accounted for ~40 % of total PrP while PrP Wt precipitated by the two reagents accounted for ~60% of total PrP (Fig 6B), which was in agreement with those observed by the direct loading of soluble and insoluble PrP in Fig. 5A through C, suggesting that most PrP Ins and PrP Wt molecules in either normal or pathological isoforms were recovered by 6H4 and g5p. A detailed quantitative analysis was conducted in order to dissect the PrP composition of various PrP species (Fig. 6B). We observed that the majority of PrP Ins (~62%) was converted into PrP Sc while the minority (~38%) remained as PrP C (Fig. 6B). In contrast, the majority of PrP Wt (~64%) remained as PrP C while ~36% of it was converted into PrP Sc . Also ~half of PrP Sc was derived from PrP Ins and another half from PrP Wt (52% vs. 48%). However, PrP C was composed of ~72% of PrP Wt and ~28% of PrP Ins . Thus, these data were consistent with the results obtained in Fig. 5 and confirmed that the majority of PrP Wt indeed was not converted into PrP Sc .

Two-dimensional gel electrophoresis of PrP
To have a high resolution profile of PrP from fCJD Ins containing both PrP Wt and PrP Ins , the PrP molecule treated with or without PNGase F was subjected to the two-dimensional (2D) gel electrophoresis that is capable of separating proteins based on both molecular weight and charge.  www.impactaging.com On 2D blots, PrP from non-CJD brain sample mainly was composed of two sets of PrP spots (Fig. 7B). The first set consisted of 13-14 PrP spots migrating between 33 and 42 kDa with pI 5.5-9.7, corresponding to diglycosylated, full-length PrP species (designated PrP 2D spots I). The second set comprised 9-10 PrP spots and was distributed between 29 and 32 kDa, pI 4.5-7.0, corresponding to diglycosylated, truncated PrP species (designated PrP 2D spots IV). This group of PrP species constituted the middle band of PrP on 1D blot.
The 2D profile of PrP from case 6 with fCJD Ins was basically similar to that of PrP from non-CJD (Fig. 7A). However, several differences between the two were still detectable. For instance, pIs of the predominant PrP species from fCJD Ins were different from those of non-CJD sample. Most PrP from 2D spots I of fCJD Ins were mainly localized between pI 6.5 and 7.3 while intense PrP spots from that of non-CJD spread from pI 6.3-9.1. Compared to PrP from non-CJD, slight increases in the intensity of PrP 2D spots II, III, and IV were also observed (Fig. 7A). Moreover, the PrP 2D spots IV were more intense in fCJD Ins than in non-CJD. These differences may result from the intrinsic nature of PrP in fCJD Ins , which is a mixture of PrP Wt and PrP Ins .
Various PrP species from fCJD Ins and non-CJD were also compared on 2D blot after deglycosylation that often profoundly decreases PrP heterogeneity. As expected, by probing with 3F4 antibody, two major sets of PrP spots were observed in the deglycosylated PrP from non-CJD: PrP 2D spots III migrating at 27-29 kDa with pI 7.0-9.6 corresponding to full-length PrP, and PrP 2D spots VI migrating at ~19-22 kDa with pI 6.1-8.1 corresponding to the N-terminally truncated PrP (Fig. 7D). However, in contrast to PrP from non-CJD, PrP from fCJD Ins had an additional set migrating at 31-33 kDa with pI 9.0-9.5 designated PrP 2D spots III' (Fig. 7C), which fitted well with the full-length PrP Ins molecule with 6 extra repeats in terms of their molecular weight. In addition, we also observed that there was a new set of PrP spots designated PrP 2D spots VII present in the two conditions including fCJD Ins and non-CJD, migrating at ~26-27 kDa with pI 5.0-8.1 (Fig. 7C and 7D). This group of PrP spots was N-terminally truncated PrP species missing ~10 residues from the farthest N-terminal portion. While PrP 2D spots III from the non-CJD brain sample were predominant, PrP 2D spots VI from fCJD Ins were predominant. It is also intriguing to note that each of the PrP 2D spots III from the two conditions had different pI of the most intense PrP spot. For example, pI of the most intense PrP spot in fCJD Ins was about 9.6, and ~9.0 in non-CJD. In view of their identical full-length PrP sequence, whether this difference is associated with distinct anchors remains to be further determined.

Histopathology and immunohistochemistry
The five fCJD Ins cases associated with rPrP Sc showed various degree of spongiform degeneration (SD) and variable astrogliosis and neuronal loss in the cerebral cortex and basal ganglia ( Table 2 and Figure 8). Plaques with single or multiple cores were detected only in one case of the four cases with adequate number of slides in the molecular layer of the cerebellum. Typical kuru plaques were observed in the cerebellar granule cell layer and white matter.
PrP immunostaining showed the "synaptic" pattern in the cerebral cortex of all the cases of fCJD Ins associated with rPrP Sc (Table 2). However, the three cases VV-129 also showed the presence of prominent granules, micro plaques or plaque-like formations. Perineuronal staining was seen in the MV-129M and two VV-129V. Real plaques, often multicore, were seen in the 129-MM (case 6) and 129-VV (case 5) but not in the other cases. www.impactaging.com Four of the five cases in which the cerebellum was available showed the distinctive stripe pattern in the molecular layer but the stripes of cases 3 and 4 of Table  2 were shorter or not completely formed. Stripes in the molecular layer could be either alone or associated with the synaptic or plaque-like formations in the granule cell layer and superficial white matter (VV-129, case 5).
In the case lacking the stripe staining pattern, the staining of the molecular layer was synaptic (VV-129, case 1).
In fCJD Ins case 6 lacking rPrP Sc , the histology of the brain areas examined revealed no SD (Fig. 8A). Several multicore plaques were present in the molecular layer of the cerebellum with focal distribution (Fig. 8B).
PrP immunostaining demonstrated weak staining with a focal synaptic pattern and occasional loose fine granular aggregates in the cerebral cortex (Fig. 8C). The molecular layer of the cerebellum had a remarkable combination of stripe-like staining so called PrP patches that are pathognostic for insertion mutation (Fig. 8D, ins. 1) and multicore plaques (Fig. 8D, ins. 2).
In conclusion, the case with sPrP Sc (case 6) differed from those associated with rPrP Sc by the lack of typical SD, and the presence of multicore plaques in limited regions of the cerebellar molecular layer. However had similar PrP stripes in the cerebellar molecular layer as the fCJD cases associated with rPrP Sc .

Histoblotting
We investigated the PK-resistance of the PrP Sc forming the stripes with two histoblot procedures: one based on IHC principles, the other similar to WB. Without PKtreatment, PrP was detected in the blots from cases with or without rPrP Sc by both methods (Fig. 9, A-D). After PK-treatment with 10 or more µg/ml, PrP was detected only in cases with rPrP Sc (Fig. 9B and 9D) but not in case 6 with sPrP Sc (Fig. 9A and 9C). In contrast, after treatment with thermolysin, PrP was detected in all cases with fCJD Ins regardless of the presence or absence of rPrP Sc but not in normal controls (Fig. 9 E through G). Therefore, the histoblot results were consistent with the data from WB and with the conclusion that in case 6 the cerebellar stripes were made of sPrP Sc .

DISCUSSION
Neurodegenerative disorders are all associated with misfolding of various cellular proteins [42][43][44]. Human prion diseases including sporadic, inherited and infectious forms are highly heterogeneous in terms of their broad range of clinical and pathological phenotypes [12]. In addition to the transmissible spongiform encephalopathy (TSE), non-transmissible prion diseases have also been reported [45][46][47], which has brought about a proposal that the spectrum of prion diseases should be beyond the classic definition of TSE [48]. The high heterogeneity of prion diseases may be associated with the chameleon-like conformations of the PrP Sc molecule [11], the only component identified in the infectious prion pathogen to date [2]. Presence of a variety of rPrP Sc including PrP Sc type 1 and type 2 in sCJD and PrP7-8 in GSS detected by Western blotting www.impactaging.com and distinct brain PrP deposits detected by immunohistochemistry might be attributable to the variable PrP Sc conformation [11,12]. Recently, novel PK-resistant PrP species with a distinctive ladder-like gel profile have also been identified in a new prion disease termed VPSPr [34,39]. These newly-identified PK-resistant PrP fragments are preferentially detected by 1E4 but much less immunoreactivity with the widely used 3F4, which is similar to those PK-resistant PrP species detected in the normal brain and uninfected cultured cells [33,37,49,50]. Studies on the correlation between the phenotypic heterogeneity of the diseases and the chameleon-like conformation of PrP Sc molecule are often complicated by the diversity in the etiologies of the diseases. Therefore, an investigation on cases with a single etiology will be critical for understanding the molecular mechanism responsible for this high heterogeneity in disease phenotypes.
Inherited CJD linked to the 144 bp insertion mutation provides us with an excellent opportunity to limit the influence of variable etiologies on phenotype. Since the disease is significantly and tightly linked to this single mutation [21,22], the phenotypes of the disease should be mainly determined by the de novo generatedpathogen itself, mutation-containing PrP Sc

Effect of the levels and types of rPrP Sc and sPrP Sc on deposits of PrP patches and neuropathological changes
So far more than 100 individuals from at least eight families affected by the 144-bp insertion mutation have been identified around the world [21,22,51,16,[24][25][26][27][28][29]. However, the reported examination of the brain PrP with Western blotting has been no more than nine cases in total to date, only six of which exhibited a detectable rPrP Sc [24][25][26]28]. The rPrP Sc species examined with Western blotting were three heterozygotes with 129-M/V polymorphism and 144-bp insertion, in which two were similar to the Gambetti et al. sCJD PrP Sc type 1 in both size and ratio of the three major PrP glycoforms [25] and one similar to PrP7-8 detected in GSS [28]. Although the two fCJD cases had an identical PRNP, one with a 4-year course exhibited only minimal focal spongiform degeneration and another with a 10-year course showed significant astrocytosis, neuronal loss and pronounced spongiform degeneration [25]. The levels of rPrP Sc were five-fold less in the former than in the latter. Nevertheless, the deposits of PrP patches were equally detected in the molecular layer of the cerebellum of the two cases. In another study involving the largest known kindred so far with 86 affected individuals  [24]. In addition, three out of five cases examined revealed no detectable rPrP Sc by conventional Western blotting [24]. Interestingly, the heterozygote with Collinge et al. type 2 and a 19-year course (VI. 23 or their case 10) and the homozygote (129-M/M) with no rPrP Sc and a 9-year course (VII.25 or their case 4) shared the same intense PrP patches in the cerebellum while the former exhibited slightly severer spongiosis and astrocytosis [23,24]. Another case with144-bp insertion examined by Western blotting was reported by Gelpi et al. [26]. This case showed rPrP Sc in a pattern resembling Gambetti et al. PrP Sc type 1 although PrP Sc type 2 might also be present. Neurohistologically, this case exhibited numerous eosinophilic globular structures in the molecular layer and the parahippocampal gyrus in addition to the spongiform changes, slight neuronal loss and gliosis as well as PrP diffuse synaptic staining [26]. The latest case examined by Vital et al was a heterozygote with 129-M/V polymorphism and was of GSS phenotype with PrP7-8 [28].
Our current study confirmed the marked variability in severity of neuropathological changes and levels of rPrP Sc and the predictability in PrP patches staining, similar to previous observations [23][24][25][26]28]. Nevertheless, our cases did not seem to reveal the close correlation either between the amount of rPrP Sc and the length of disease duration or between the amount of rPrP Sc and the severity of neuropathological changes. For instance, although the cases 2 and 3 had the longest and second longest disease durations (15-and 11-year, respectively) among the six cases we examined, the amounts of rPrP Sc in the two cases were not the largest. The current characterization on six fCJD Ins cases including two PrP Sc type 2 and three PrP Sc type 1 and one with no typical rPrP Sc clearly demonstrated that the similar PrP patches are always equally detectable in www.impactaging.com fCJD Ins regardless of the levels and types of rPrP Sc except a case. It is conceivable that rPrP Sc conformer should not be the main component of this type of PrP deposits and the abnormal sPrP Sc conformer containing PrP Ins participates in the formation of the PrP patches instead.
It is known that Western blot analysis is more sensitive than immunohistochemistry in terms of detection of a protein. However, for detection of the abnormal PrP, it has been reported in at least two conditions that immunohistochemistry readily detected the abnormal PrP staining in the samples in which the conventional Western blot analysis showed no detectable rPrP Sc [34,36]. For instance, by immunohistochemistry PrP deposition was readily detected in the olfactory mucosa of sCJD, where the amount of rPrP Sc only accounted for as small as 8% of brain rPrP Sc [36]. In these tissues, no rPrP Sc was detected by conventional Western blotting. It is possible that the majority of PrP Sc was composed of sPrP Sc in the olfactory mucosa. Indeed, this was the case in VPSPr [34,39]. Although there was strong PrP immunostaining with 3F4 in brains from the subjects with VPSPr, no rPrP Sc was detected by the conventional Western blotting probed with the same antibody. The quantitative analysis revealed that the amount of rPrP Sc was very small (as small as 10% of brain rPrP Sc in sCJD) [34]. Therefore, it is most likely that PrP staining in the tissue section detected by immunohistochemistry is the signature of sPrP Sc instead of rPrP Sc alone. The pathognostic PrP patches in the fCJD Ins may comprise sPrP Sc deriving from both PrP Ins and PrP Wt . Indeed, using histoblotting after PK-or thermolysin-treatment, we confirmed that staining of sPrP Sc can be eliminated by PK but not by thermolysin. The latter is the enzyme that has been reported to specifically degrade PrP C but not PrP Sc including sPrP Sc [52,53]. Thus, our results suggest that sPrP Sc may preferentially attack the cerebellum compared to the cerebrum.

Allelic composition of total PrP and PrP Sc in fCJD Ins
By using a PK-sensitivity assay, the rPrP Sc molecule in fCJD with 144-bp insertion has been demonstrated to comprise both PrP Ins and PrP Wt alleles in a case with detectable rPrP Sc [5]. However, whether both PrP Wt and PrP Ins participate in the formation of sPrP Sc or not remains unknown. The ratios of PrP Wt and PrP Ins to the total PrP in fCJD Ins are also unclear. We demonstrated that like rPrP Sc in the typical fCJD Ins+rPrPSc , sPrP Sc in fCJD Ins+sPrPSc also derived from both PrP Wt (~48%) and PrP Ins (~52%). Although PrP Wt and PrP Ins accounted for 60% and 40% of total PrP, respectively, most of PrP Ins (~62%) was converted into sPrP Sc and 64% of PrP Wt remained as PrP C . It is worth noting that less PrP Wt became insoluble in the fCJD Ins+sPrPSc case than in fCJD Ins+rPrPSc (PrP Wt : ~45% vs. ~62%). Whether this is the reason that the insoluble PrP Wt and PrP Ins in the fCJD Ins+sPrPSc did not form the typical rPrP Sc remains to be confirmed. Compared to the previously reported case [5], the current study showed a greater percentage of insoluble PrP Wt (~62% vs. ~57%) and smaller PrP Ins (~70% vs. ~94%) in fCJD Ins+rPrP [5]. The difference between the current study and the previous one may result from that we examined five cases here while only one case was examined in the previous study. Our 2D study further confirmed that PrP Wt is predominant in fCJD Ins+sPrPSc . Whether PrP Ins is less-expressed or it is readily degraded in fCJD Ins remains to be determined. Surprisingly, both full-length PrP Ins and PrP Wt molecules share a similar pI at 9.0-9.5 and the difference between the two seems to be in the molecular weight only but not in the molecular charge. Another interesting finding is that although there are both PrP Wt and PrP Ins in fCJD Ins , the amount of PrP Wt in fCJD Ins is almost similar to that of PrP Wt in non-CJD and sCJD.

Pathophysiology of rPrP Sc and sPrP Sc species
The correlation between rPrP Sc and the neuropathological changes is still controversial. The rPrP Sc species detected in prion-infected brains are surprisingly not neurotoxic and PrP-knockout mice are resistant to prion infection [54,55]. Moreover, subclinical forms of prion diseases have been observed in experimentally or naturally infected animals that harbor high levels of infectivity and PrP Sc but are asymptomatic during a normal life-span [56,57]. Conversely, wild-type mice inoculated with PrP Sc of bovine spongiform encephalopathy showed no detectable rPrP Sc in the brain despite the presence of neurological symptoms and neuronal death [58]. These conditions were observed not only in animals but also in humans. Fatal familial insomnia or GSS with substitution of valine for alanine at residue 117 (A117V) revealed striking clinical manifestations but little or undetectable PK-resistant PrP [59,60]. Therefore, the molecular features of the neurotoxic forms of PrP remain to be determined. Several potentially toxic PrP isoforms have been studied in prion-infected transgenic mice, rodents and humans including transmembrane, cytosolic and PK-sensitive forms of abnormal PrP [61,31,62,30]. Based on the "refolding" or "seeding" models, PrP C may unfold to an intermediate before it refolds under the influence of PrP Sc or the conversion of PrP C into PrP Sc requires a PrP Sc -like form (PrP*) [63,64]. The intermediates have been widely observed in cell-based and cell-free models [65,66,41,67]. These intermediates generated in the www.impactaging.com process of conversion of PrP C to PrP Sc could be the neurotoxic PrP species.
The PK-sensitive sPrP Sc was initially proposed in experimentally infected animals using PTA-based ELISA by Safar et al. [31]. To our knowledge, our previous study was the first to demonstrate that human PrP Sc is composed of both rPrP Sc and sPrP Sc and that the majority of PrP Sc in GSS is sensitive to PK-digestion by using a PrP Sc -specific antibody-based Western blot analysis [30]. Currently the physiochemical features and pathophysiology of sPrP Sc are poorly understood. The possibility cannot be ruled out that sPrP Sc is an intermediate in the formation of the terminal product rPrP Sc and it is responsible for the prion-related neurotoxicity. Remarkably, VPSPr-129VV that we recently identified is rich in sPrP Sc and virally lacks the typical rPrP Sc type 1 and type 2 and PrP7-8 in the cerebral cortex by conventional Western blot analysis [34,39]. In the scrapie-infected hamsters, it has been shown that sPrP Sc forms smaller oligomers while the rPrP Sc forms the larger aggregates [62]. Our current finding that an fCJD Ins with typical clinical and neuropathological characteristics is lack of typical rPrP Sc but full of sPrP Sc favors the hypothesis that the sPrP Sc comprising small oligomers is most likely responsible for the neuropathological changes. Indeed, in Alzheimer's disease, the toxicity was originally thought to be a property of the fibrillar form of Aβ, consistent with the widespread notion at the time that the amyloid fibril itself was pathogenic [68,69]. However, subsequent studies revealed that Aβ fractions containing protofibrillar comprising soluble oligomers, but not fibrillar, material retained their toxicity [70][71][72].
Like in non-CJD, uninfected cultured cells, and VPSPr, we detected rPrP in fCJD Ins+sPrPSc as well when the 1E4 antibody-based Western blotting was used [33,37,34,39,50]. Although the gel profile of rPrP detected in non-CJD with 1E4 is similar to that of fCJD Ins+sPrPSc , the intensity of rPrP is much lower in non-CJD than in fCJD Ins+sPrPSc . The rPrP species in non-CJD was only visible in over-exposed films (data not shown). The gel profile of rPrP in fCJD Ins+sPrPSc is different from that of VPSPr. Interestingly, it is more similar to that of rPrP Sc in sCJD type 1at the lower PK concentrations ranging from 5 to 10 µg/ml with an unglycosylated PrP band migrating at ~19 kDa similar to sCJD type 2 while the typical gel profile of sCJD type 1 with an unglycosylated PrP band migrating at ~21 kDa becomes dominant at higher PK-concentrations greater than 25 µg/ml. It would be interesting to further investigate whether all 1E4-preferentially detected rPrP species originate from a similar precursor.
Brains, sent to the NPDPSC for suspected prion disease diagnosis, were obtained at autopsy and one half was immediately frozen and stores at -80 °C. The remaining tissue was fixed in formalin for 10 days, kept in 98 % formic acid for 1 h and again in formalin until sampling for neuropathological examination and PrP immunohistochemistry [39]. The presence of PrP Sc from frozen tissues of frontal (FC) and occipital (OC) cortices, brain stem (BS), and cerebellum (CE) were determined by western blotting. In addition, paraffin blocks of tissues from FC, OC, BS, and CE were prepared for histology and immunohistochemistry. Molecular genetics. The genomic DNA was extracted from frozen brain tissues. The open reading frame (ORF) of the PRNP was amplified by the polymerase chain reaction (PCR) using 20 ng of genomic DNA and primers PrPO-F (GTCATYATGGCGAACCTTGG, Y=C+T) and PrPO-R (CTCATCCCACKATCAGGAAG, K=T+G) (PCR cycles: 94°C for 3 min; 94°C for 1 min, 57°C for 1 min, 72°C for 1 min, 30 cycles; 72°C for 10 min). The PCR products were separated on a 1.0% www.impactaging.com agarose gel. Both the larger band (~0.9 kb) and the wild type size band (0.77 kb) were recovered separately from the gel using the QIAGEN gel extraction kit, and subjected to automated sequencing with primers PrPO-F, PrPO-R, and HP306R (CATGTTGGTTTTTGGCTTAC TC). For some samples where direct sequencing of PCR products did not give conclusive sequences, the PCR products were cloned then sequenced. The sequences were compared with that of the wild type human PrP using the LALIGN program (http://www2.igh.cnrs.fr/). The sequence of R3g is CCC CAT GGT GGT GGC TGG GGg CAG as defined by Goldfarb et al. [74].
Preparation of brain homogenate, S2, and P2 fractions. The 10% (w/v) brain homogenates were prepared in 9 volumes of lysis buffer (10mM Tris, 150 mM NaCl, 0.5% Nonidet P-40, 0.5% deoxycholate, 5mM EDTA, pH 7.4) with pestle on ice. When required, brain homogenates were centrifuged at 1,000 g for 10 min at 4°C. In order to prepare S2 and P2 fractions, the supernatants (S1) were further centrifuged at 35,000 rpm (100,000 g) for 1 h at 4°C. After the ultracentrifugation, the detergent-soluble fraction was recovered in the supernatants (S2) while the detergentinsoluble fraction (P2) was recovered in the pellets that were resuspended in lysis buffer as described [33].
Specific capture of PrP C and PrP Sc by 6H4 and g5p. The anti-PrP antibody 6H4 or DNA binding protein g5p (100 µg each) were conjugated to 7X10 8 tosyl activated superparamagnetic beads (Dynabeads M-280, Dynal Co.) in 1 ml of phosphate-buffered saline (PBS) at 37 °C for 20 h, respectively. The conjugated beads were incubated with 0.1 % bovine serum albumin (BSA) in 0.2 M Tris-HCl at pH 8.5 to block non-specific binding. The prepared beads were stable for at least 3 months at 4 o C. Brain homogenate (10%, w/v) was prepared in lysis buffer, followed by centrifugation at 3,000 g for 10 min at 4ºC to remove debris. The specific capture of PrP C or PrP Sc by 6H4 or g5p was performed as described [41,33] using brain homogenate and conjugated beads (10 μg mAb or g5p/6X10 7 beads) in 1 ml of binding buffer (3% Tween-20, 3% Nonidet-40 in PBS, pH 7.4). After incubation with constant rotation for 3 h at room temperature, the beads were attracted to the sidewall of the plastic tubes by external magnetic force, allowing easy removal of all unbound materials in the solution. After three washes in wash buffer (2% Tween-20 and 2% Nonidet P-40 in PBS, pH 7.5), the beads were collected and were heated at 95 o C for 5 min in SDS sample buffer (3% sodium dodecyl sulfate (SDS), 2 mM EDTA, 10% glycerol, 50 mM Tris-HCl, pH 6.8). The proteins eluted from the beads were subjected to SDS-PAGE and immunoblotting as described below.
Precipitation of PrP Sc by sodium phosphotungstate. Precipitation of PrP Sc by sodium phosphotungstate (NaPTA) was conducted as described [32,33] with mild modification. Briefly, 10% (w/v) brain homogenates from brain tissues were prepared in Dulbecco's sterile phosphate buffered saline (PBS) lacking Ca2+ and Mg2+. The gross cellular debris was removed by centrifugation at 1,000 rpm (80 g) for 1 min. Supernatant (500 µl) was mixed with an equal volume of 4% (w/v) sarkosyl prepared in PBS pH 7.4 and incubated for 10 min at 37ºC with constant agitation. Samples were adjusted to final concentrations of 50 units/ml Benzonase (Benzon nuclease, purity 1; Merck) and 1 mM MgCl2 and incubated for 30 min at 37ºC with constant agitation. Subsequently, the samples were adjusted with 81.3 µl of a stock solution containing 4% (w/v) NaPTA and 170 mM MgCl2 (prepared in water and titrated to pH 7.4 with sodium hydroxide) to give a final concentration in the sample of 0.3% (w/v) NaPTA. This stock solution was pre-warmed to 37ºC before use. Samples were incubated at 37ºC for 30 min with constant agitation before centrifugation at 14 000 rpm for 30 min. After careful isolation of the supernatant, the pellet was resuspended to 60 µl final volume of 1 X lysis buffer. The samples were incubated with PK at a final concentration of 50 µg/ml at 37ºC for 1 h. Digestion was terminated by the addition of PMSF (3 mM final concentration) and boiling for 10 minutes in an equal volume of electrophoresis sample buffer (3% SDS, 2mM EDTA, 10% glycerol, 2.5% βmercaptoethanol in 62.5 mM Tris, pH 6.8). After cooling for 2 min, the samples were incubated with a five-fold volume of pre-chilled methanol at -20ºC for 2 h and centrifuged at 14,000 rpm for 20 min at 4ºC. The supernatant was discarded and the pellet was resuspended in 30 µl sample buffer. The latter was subjected to SDS-PAGE and immunoblotting.
Velocity sedimentation in sucrose step gradients. Brain homogenates (10%, w/v) in 1X Dulbecco's PBS pH 7.4 were mixed with an equal volume of 2X lysis buffer, then centrifuged for 10 min at 3,000 rpm at 4°C. Supernatants were collected and sarkosyl was added to 1% final concentration. Each sample was loaded atop of 10-60% step sucrose gradients and centrifuged at 200,000 x g in the SW55 rotor for 1 h at 4ºC as described with minor modification [33]. After centrifugation, the content of the centrifuge tubes was sequentially removed from the top to the bottom to collect 12 fractions which were subjected to immunoblotting as described below.

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One-and two-dimensional gel electrophoresis and immunoblotting. Brain homogenates treated with or without PK were resolved either on 15% Tris-HCl Criterion (Bio-Rad) for one-dimensional (1D) PAGE or on pH gradient (IPG) strips for two-dimensional (2D) PAGE. The latter was performed as described by the supplier with minor modifications using the PROTEIN IEF cell (Bio-Rad) [33]. Briefly, for the 1D PAGE, samples boiled in 2X electrophoresis sample buffer were precipitated by 5-fold volume of pre-chilled methanol at -20 °C for 2 h, followed by centrifugation at 14,000 rpm for 20 min at 4°C. Pellets were resuspended in reducing buffer (8 M urea, 2% CHAPS, 5 mM TBP, and 20 mM Tris, pH 8.0) for 1 h at room temperature and then incubated with 20 mM IAA for 1 h. The samples were incubated with 5-fold volume of pre-chilled methanol at -20°C for 2 h and centrifuged at 14,000 rpm for 20 min at 4°C. Pellets were resuspended in 200 μl of rehydration buffer (7M urea, 2 M thiourea, 1% DTT, 1% CHAPS, 1% Triton X-100, 1% Ampholine pH 3-10, and trace amounts of bromophenol blue). Samples dissolved in rehydration buffer were incubated with IPG strips for 14 h at room temperature with shaking. The rehydrated strips were focused for about 40 kVh. For the second dimension, the focused IPG strips were equilibrated for 15 min in equilibration buffer 1 containing 6M urea, 2% SDS, 20% glycerol, 130 mM DTT, and 375 mM Tris pH 8.8, and then in equilibration buffer 2 containing 6M urea, 2%SDS, 20% glycerol, 135 mM iodoacetamide and 375 mM Tris pH 8.8 for another 15 min. The equilibrated strips were loaded onto 8-16% Tris-glycine Criterion gel (Bio-Rad).
The proteins on the gels were transferred to either Immobilon-P (PVDF, Millipore) or Immobilon-FL membranes (PVDF, LI-COR) for 2 h at 70V. For probing the PrP molecule, the membranes were incubated for 2 h at room temperature with 3F4 (1:40,000) or anti-C-terminal antibody (1:4,000) as primary antibody. Incubation with a secondary antibody was performed either with the horseradish peroxidaseconjugated goat anti-mouse antibody (1: 3,000) or IRDye 800CW conjugated goat anti-mouse (LI-COR). The PrP bands or PrP spots were visualized on either Kodak film by the ECL Plus as described by the manufacturer or Odyssey infrared imaging system (LI-COR ® Biosciences, NE, USA).
PrP Immunohistochemistry and histoblotting. Tissue was fixed in formalin for 3 weeks. The following procedures were performed as described [33]. For histological preparations, brain sections were embedded in paraffin and stained with hematoxylin-eosin. For immunohistochemistry sections were deparaffinized, rehydrated, and immersed in 98% formic acid for 1 h at room temperature. Endogenous peroxidase was blocked by immersion in 8% hydrogen peroxide in methanol for 10 min. Sections were completely immersed in 1.5 mM hydrochloric acid and microwaved for 10 min. After rinsing, they were incubated with the mouse monoclonal antibody 3F4 at 1:600, washed and incubated with bridge antibody (goat anti-mouse, Cappel, 1:50) followed by incubation with mouse PAP complex (Sternberger, Meyer Immunocytochemicals, 1:250). Diaminobenzidine tetrahydrochloride was used to visualize the immunoreactivity.