Detection and localization of PrPSc in the skeletal muscle of patients with variant, iatrogenic, and sporadic forms of Creutzfeldt-Jakob disease

Abstract

Variant Creutzfeldt-Jakob disease (vCJD) differs from other human prion diseases in that the pathogenic prion protein PrP(Sc) can be detected to a greater extent at extraneuronal sites throughout the body, principally within lymphoid tissues. However, a recent study using a high-sensitivity Western blotting technique revealed low levels of PrP(Sc) in skeletal muscle from a quarter of Swiss patients with sporadic CJD (sCJD). This posed the question of whether PrP(Sc) in muscle could also be detected in vCJD, sCJD, and iatrogenic (iCJD) patients from other populations. Therefore, we have used the same high-sensitivity Western blotting technique, in combination with paraffin-embedded tissue blotting, to screen for PrP(Sc) in muscle tissue specimens taken at autopsy from 49 CJD patients in the United Kingdom. These techniques identified muscle PrP(Sc) in 8 of 17 vCJD, 7 of 26 sCJD, and 2 of 5 iCJD patients. Paraffin-embedded tissue blotting analysis showed PrP(Sc) in skeletal muscle in localized anatomical structures that had the morphological and immunohistochemical characteristics of nerve fibers. The detection of PrP(Sc) in muscle tissue from all forms of CJD indicates the possible presence of infectivity in these tissues, suggesting important implications for assessing the potential risk of iatrogenic spread via contaminated surgical instruments.

Figure 1

Western blots of vCJD patient muscle samples positive for PrP^res. Samples of muscle homogenate equivalent to 50 mg wet weight of tissue were precipitated with NaPTA, digested with PK, and immunoblotted with anti-PrP antibody 3F4 as the primary reagent, as described in the Materials and Methods section. Top: A typical immunoblot for assessing CJD patient muscle for the presence of PrP^res. Two muscle samples from the vCJD patient V9 are compared with muscle from non-CJD neurological disease control patient, one of which has been spiked with 100 μg of vCJD brain homogenate. The bands that we infer to be PrP^res are indicated with asterisks. These precipitates were run in parallel with vCJD brain standards before (−) and after (+) PK digestion. The molecular weights in kd of the MagicMark XP standards (M) are indicated on the left. Bottom: A composite image taken from six separate immunoblots showing muscle samples from the seven other vCJD patients assigned as positive by Western blotting, aligned with vCJD brain standard (100 μg). The gaps in the upper bar indicate where the separate immunoblots have been aligned.

Figure 2

Western blots of sCJD patient muscle samples positive for PrP^res. Samples of muscle from six patients assigned as positive are compared with muscle from a non-CJD neurological control patient and vCJD control brain homogenate (100 μg) diluted into non-CJD control muscle homogenate. Two of the patients, S7 and S10, were also positive by PET blotting (see Figure 4). As in Figure 1, the bands that we infer to be PrP^res are indicated with asterisks. The gaps in the upper bar indicate where separate blots have been aligned. For comparison, the PrP^res signal obtained from spinal cord from patient S10 after NaPTA precipitation and Western blotting is shown in the final lane.

Figure 3

Western blots of iCJD patient muscle samples positive for PrP^res. Three samples of muscle from an iCJD patient (I4) are compared with 100 μg of vCJD brain homogenate diluted in non-CJD control muscle homogenate, NaPTA precipitated, and digested with PK (vCJD spike). Also shown are samples of vCJD brain homogenate before (−) and after (+) digestion with PK (50 μg and 200 μg, respectively) without NaPTA precipitation. Nonspiked, non-CJD muscle homogenate, digested with PK, is shown in the last lane as a negative control. As noted previously there is a slight upward shift in the mobility of PrP^res after NaPTA precipitation (vCJD spike lane).

Figure 4

PET blot and immunohistochemical analysis of PrP in sections of sCJD skeletal muscle. A: PET blot preparation showing labeling of PrP^res in longitudinal nerve fibers within a muscle sample from sCJD patient S10. B: Paraffin section adjacent to A showing the presence of a nerve fiber bundle in the site of the PrP^res labeling (H&E). C: Paraffin section adjacent to A showing a similar pattern of staining in large diameter axons to the positive labeling for PrP^res after immunocytochemistry for neurofilament protein. D: PET blot preparation showing labeling of PrP^res in an oblique cross-section of large diameter axons within a muscle sample from sCJD patient S7. Original magnifications: ×200 (A–C); ×400 (D).

Figure 5

Clinical durations of sCJD and vCJD patients positive for PrP^res in muscle. The distribution of the clinical durations (in months) of the 17 vCJD patients and the 26 sCJD patients in this study are represented by box and whisker diagrams. Patients with durations of illness greater than [75th percentile + (1.5 × the interquartile range)] were defined as outliers and are indicated by filled circles. The positions of the vCJD and sCJD patients positive for PrP^res in muscle within these distributions are indicated with asterisks.