Re-assessment of PrP(Sc) distribution in sporadic and variant CJD

Abstract

Human prion diseases are fatal neurodegenerative disorders associated with an accumulation of PrP(Sc) in the central nervous system (CNS). Of the human prion diseases, sporadic Creutzfeldt-Jakob disease (sCJD), which has no known origin, is the most common form while variant CJD (vCJD) is an acquired human prion disease reported to differ from other human prion diseases in its neurological, neuropathological, and biochemical phenotype. Peripheral tissue involvement in prion disease, as judged by PrP(Sc) accumulation in the tonsil, spleen, and lymph node has been reported in vCJD as well as several animal models of prion diseases. However, this distribution of PrP(Sc) has not been consistently reported for sCJD. We reexamined CNS and non-CNS tissue distribution and levels of PrP(Sc) in both sCJD and vCJD. Using a sensitive immunoassay, termed SOFIA, we also assessed PrP(Sc) levels in human body fluids from sCJD as well as in vCJD-infected humanized transgenic mice (Tg666). Unexpectedly, the levels of PrP(Sc) in non-CNS human tissues (spleens, lymph nodes, tonsils) from both sCJD and vCJD did not differ significantly and, as expected, were several logs lower than in the brain. Using protein misfolding cyclic amplification (PMCA) followed by SOFIA, PrP(Sc) was detected in cerebrospinal fluid (CSF), but not in urine or blood, in sCJD patients. In addition, using PMCA and SOFIA, we demonstrated that blood from vCJD-infected Tg666 mice showing clinical disease contained prion disease-associated seeding activity although the data was not statistically significant likely due to the limited number of samples examined. These studies provide a comparison of PrP(Sc) in sCJD vs. vCJD as well as analysis of body fluids. Further, these studies also provide circumstantial evidence that in human prion diseases, as in the animal prion diseases, a direct comparison and intraspecies correlation cannot be made between the levels of PrP(Sc) and infectivity.

Figure 1. Western blot analysis of PrP^C from normal human tissues.

Ten percent homogenates from normal human brain (Br), spleen (Sp), tonsil (Ts), and lymph node (LN) were prepared and ultracentrifuged through a sucrose cushion as described in Materials and Methods. Pellets were resuspended and either untreated or PK-treated prior to western blot analysis with Mab 3F4.

Figure 2. Partially purified, protease-resistant PrP^Sc titration from sCJD tissues.

Homogenates of sCJD brain, spleen, tonsil, and lymph node were prepared, concentrated by ultracentrifugation and the pellets were resuspended as described in Materials and Methods. Undiluted (und) and two-fold serial dilutions of the PK-treated resuspended pellets were western blotted and immunostained for PrP^Sc using Mab 3F4. A PK-untreated sample is shown for each tissue to demonstrate the completeness of the PK treatment. Asterisk (*) denotes that the amount of brain tissue equivalents loaded in each lane is 1/100 the amount relative to spleen, tonsil, and lymph node.

Figure 3. Partially purified, protease-resistant PrP^Sc titration from vCJD tissues.

Homogenates of vCJD brain, spleen, tonsil, and lymph node were prepared and concentrated by ultracentrifugation as described in Materials and Methods. Undiluted (und) and two-fold serial dilutions of the PK-treated resuspended pellets were western blotted and immunostained for PrP^Sc using Mab 3F4. A PK-untreated sample is shown for each tissue to demonstrate the completeness of the PK treatment. Asterisk (*) denotes that the amount of brain tissue equivalents loaded in each lane is 1/100 the amount relative to spleen, tonsil, and lymph node.

Figure 4. Effects of sCJD and vCJD brain dilution on PrP^Sc detection by SOFIA.

Ten percent homogenates from all the normal human (N), vCJD (V), and sCJD brain tissues were prepared as described in Materials and Methods. Serial 10-fold dilutions were prepared from each brain tissue sample and analyzed in triplicate by SOFIA as described. For each sample, data was calculated as the mean fluorescent signal±standard deviation and expressed as the sample to background ratio (signal intensity).

Figure 5. Detection of PrP^Sc from sCJD by SOFIA following PASA of human CSF.

PASA was carried out using normal human and sCJD CSF as the source of the seeding material. Serial PMCA was carried out individually on all of the normal human and sCJD CSF for a maximum of 200 cycles with samples collected at 0, 40, 80, 160, and 200 cycles for IP and analysis in triplicate by SOFIA. For each sample, data was calculated as the mean fluorescent signal±standard deviation and expressed as the sample to background ratio (signal intensity). Signal intensities from SOFIA were adjusted based on the dilution of samples throughout sPMCA.

Figure 6. PrP^Sc Detection by SOFIA Following PASA (sPMCA₂₀₀) in urine and blood.

PASA was evaluated in human urine and blood from each sCJD case and blood from vCJD-infected Tg666 mice. Following sPMCA₂₀₀, samples were analyzed in triplicate by SOFIA for the presence of PrP^Sc. Signal intensities from SOFIA were adjusted based on the dilution of samples throughout sPMCA₂₀₀, calculated as the mean fluorescent signal±standard deviation and expressed as the signal/background ratio.

Figure 7. Western blot analysis of PrP^Sc from vCJD-infected Tg666 mice.

Ten percent brain homogenates from two vCJD-infected Tg666 mice were prepared in lysis buffer and centrifuged at 13,000×g for 10 min. Twenty µl of supernatant from each of the two mice (1, 2) was either untreated or PK-treated prior to western blotting and immunostaining with Mab 3F4 as described in Materials and Methods.