Multiorgan detection and characterization of protease-resistant prion protein in a case of variant CJD examined in the United States

Abstract

Background: Variant Creutzfeldt-Jakob disease (vCJD) is a prion disease thought to be acquired by the consumption of prion-contaminated beef products. To date, over 200 cases have been identified around the world, but mainly in the United Kingdom. Three cases have been identified in the United States; however, these subjects were likely exposed to prion infection elsewhere. Here we report on the first of these subjects.

Methodology/principal findings: Neuropathological and genetic examinations were carried out using standard procedures. We assessed the presence and characteristics of protease-resistant prion protein (PrP(res)) in brain and 23 other organs and tissues using immunoblots performed directly on total homogenate or following sodium phosphotungstate precipitation to increase PrP(res) detectability. The brain showed a lack of typical spongiform degeneration and had large plaques, likely stemming from the extensive neuronal loss caused by the long duration (32 months) of the disease. The PrP(res) found in the brain had the typical characteristics of the PrP(res) present in vCJD. In addition to the brain and other organs known to be prion positive in vCJD, such as the lymphoreticular system, pituitary and adrenal glands, and gastrointestinal tract, PrP(res) was also detected for the first time in the dura mater, liver, pancreas, kidney, ovary, uterus, and skin.

Conclusions/significance: Our results indicate that the number of organs affected in vCJD is greater than previously realized and further underscore the risk of iatrogenic transmission in vCJD.

Figure 1. Histological and immunohistochemical features.

A and B. The cerebral cortex is markedly disrupted with prominent astrogliosis and presence of amyloid plaques (arrows in B); frontal cortex; H.E. C and D: PrP immunostaining shows the presence of many plaques (arrows) and plaque-like aggregates in superficial and deep cortical regions with a punctate background staining; frontal cortex. E: Distinctive spot-like PrP immunostaining in the molecular layer and many plaques especially evident in the Purkinje cell layer of the cerebellum. F: Plaques, often in a row are present in the superficial white matter; cerebellum. C–F: Monoclonal antibody (mAb) 3F4.

Figure 2. Detection and characterization of PrP^res and PK-sensitive PrP in brain.

A: Immunoblot of total homogenates (TH), treated with proteinase K (PK), obtained from the frontal cortex of sCJDMM1, sCJDMM2 (representing PrP^res types 1 and 2, respectively) and the present case showing the over-representation of the upper band (Diglyc.) containing the diglycosylated form, and the co-migration of the lowest band (Unglyc.), containing the unglycosylated form, with the corresponding band of sCJDMM2. B: Immunoblot of TH from the four regions of the cerebral cortex and the cerebellum, treated with PK as indicated. The cerebellar unglycosylated PrP^res isoform generates a thicker and overall slightly faster migrating band than the corresponding PrP^res from the cerebral cortex. C: A high-resolution immunoblot (15%, 15 cm long gel) confirms that the monoglycosylated and unglycosylated PrP^res isoforms from the cerebellum have a faster electrophoretic mobility than the corresponding forms from the cerebral cortex, and shows that the cerebellar unglycosylated isoform resolves into three fragments including a 19 kDa band, corresponding to PrP^res type 2, and two additional bands of slightly lower relative molecular weight (arrowheads); T: Temporal; Cb: Cerebellum. In A–C membranes were probed with the mAb 3F4. D and E: Ratios of the PrP^res glycoforms (D) and of the total PrP and PrP^res (E) obtained from the same brain regions examined in panel B. Each bar represents the mean ± SD of three densitometric determinations on each of two tissue samples.

Figure 3. Detection of PrP^res in non-nervous tissues.

A: PrP^res from dura mater and frontal cortex (1∶24 dilution) from the present case is compared to PrP^res of the frontal cortex from sCJDMM1 (type 1) and sCJDMM2 (type 2). B: Two film exposures of immunoblots from non-nervous tissues compared with that of the frontal cortex (1∶10 dilution). Pituitary gland, adrenal gland and uterus are clearly positive while the bands in the remaining preparations are considered to be non specific. C: PrP^res from skin (double TH loading, equivalent to 4 mg of wet tissue) is barely detectable in TH (Lane 3) compared with frontal cortex TH, which is diluted 1∶4 (lane 1) or 1∶130 (lane 2). Skin PrP^res is better detectable along with the kidney PrP^res after sodium phosphotungstate (NaPTA) precipitation of PrP^res (Lanes 4 and 5), especially after long exposure (Lanes 4L and 5L) (kidney TH loaded in double amount; probed with mAb 3F4). D: PrP^res from mesenteric lymph nodes and other visceral organs recovered following NaPTA precipitation and compared with TH from the frontal cortex (1∶120 dilution) and sCJDMM1 following two film exposures. All organs but ascending colon are positive. Of note, unglycosylated isoform is underrepresented in all NaPTA precipitated samples as compared with that of the TH preparations (see panel A, lanes 3 and 4 and panel B). A–D: Membranes were probed with the mAb 3F4.

Figure 4. Characteristics of PK-sensitive PrP.

Immunoblot analysis of total homogenate from brain, pituitary gland and uterus are shown. The samples, with or without previous PK treatment, were deglycosylated with PNGase F. Membranes were probed with the mAbs 3F4 and 2301 as indicated. A: The brain has relatively large amounts of full-length isoform and PrP^res C2 fragment but the N-terminus truncated PrP^C fragment (C1) is poorly represented. In addition, brain preparation shows a previously unreported PK-sensitive fragment with molecular weight of 25 kDa (arrow), detectable only in deglycosylated samples, of undetermined origin. B: The C1 fragment is relatively better represented in the pituitary gland C: while it is overly abundant in the uterus.