Darwin Ernst

Neuron-specific expression of a hamster prion protein minigene in transgenic mice induces susceptibility to hamster scrapie agent

Abstract To study the effect of cell type-restricted hamster PrP expression on susceptibility to the hamster scrapie agent, we generated transgenic mice using a 1 kb hamster cDNA clone containing the 0.76 kb HPrP open reading frame under control of the neuron-specific enolase promoter. In these mice, expression of HPrP was detected only in brain tissue, with highest levels found in neurons of the cerebellu, hippocampus, thalamus, and cerebral cortex. These transgenic mice were susceptible to infection by the 263K strain of hamster scrapie with an average incubation period of 93 days, compared to 72 days in normal hamsters. In contrast, nontransgenic mice were not susceptible to this agent. These results indicate that neuron-specific expression of the 1 kb HPrP minigene including the HPrP open-reading frame is sufficient to mediate susceptibility to hamster scraple, and that HPrP expression in nonneuronal brain cells is not necessary to overcome the TSE species barrier.

Detection of Prion Protein in Formalin-Fixed Brain by Hydrated Autoclaving Immunohistochemistry for the Diagnosis of Scrapie in Sheep

4The diagnosis of scrapie has traditionally relied on histopatholog ic demonstration of characteristi c lesions in brain sections. In recent years, however, increasing attention has been given to the use of immunodiagnos tic methods that focus on detection of a specific protein known as the prion protein (PrP). 7 This protein, which is believed to play an essential role in the pathogenesis of scrapie, is present in normal brain as a protease-sensitive isoform (PrP-sen). In contrast, most of the PrP in scrapie brain is protease resistant (PrP-res), probably because of a posttranslational alteration affecting the conformation of normal PrP. An immunohistoch emical (IHC) method to detect PrP-res in periodate, lysine, paraformaldehyde (PLP)-fixed brains from sheep with scrapie has been described. 5 Researchers working on bovine spongiform encephalopathy have recently reported that PrP-res also can be detected in formalin-fixe d brain if tissue sections are autoclaved prior to immunostaining. 1 This novel method of antigen enhancement, termed hydrated autoclaving, was originally devised to improve the immunoreactivity of certain brain proteins. 10 Hydrated autoclaving has not been used to detect other antigens. However, antigen enhancement is believed to result from heat denaturation of the protein, and the same mechanism has been suggested as the basis for antigen retrieval methods that utilize microwave immunohistochemistry. 6 In the present report, we describe diagnosis of scrapie using hydrated autoclaving IHC on formalin-fixe d sheep brains. For comparative purposes, some brains also were tested for PrP-res by western blot assay or by IHC on PLP-fixed brain. Brain samples from 186 scrapie suspect sheep were submitted to veterinary medical colleges, state veterinary diagnostic laboratories, or the National Veterinary Services Laboratories. Additional brain samples from 10 sheep in a scrapiefree flock were used as controls. Tissue sections were cut from paraffin blocks of formalin-fixe d brain stem (usually obex or midmedulla) and mounted on positively charged

Analysis of linkage between scrapie incubation period and the prion protein gene in mice.

A single gene is known to have a predominant influence on scrapie incubation period in mice. In crosses between strains that give a short incubation period, such as NZW mice, and those which give a long incubation period, such as I/LnJ mice, long incubation period was dominant using a Chandler scrapie agent isolate. Recently a close linkage was found between the incubation period gene and the prion protein (PrP) structural gene in I/LnJ mice crossed to NZW mice. Because this linkage suggested an important role for PrP in the pathogenesis of scrapie we sought to verify the linkage between these genes and extended the analysis to three additional mouse strains. All four of the mouse strains that we evaluated, I/LnJ, P/J, MA/MyJ, and RIIIS/J, had incubation periods longer than those of the NZW mice to which they were crossed. In addition, all four strains shared an XbaI restriction enzyme polymorphism, which suggested that all four strains might also exhibit linkage between the incubation period and the PrP structural gene. Very strong linkage between PrP and incubation period was found in I/LnJ and P/J mice crossed to NZW mice, whereas less obvious linkage was demonstrated for MA/MyJ mice crossed to NZW mice. In MA/MyJ mice genes other than PrP also had an obvious influence on incubation period. In RIIIS/J mice no linkage was shown. Although linkage between PrP and incubation period was very significant in I/LnJ and P/J mice, a few animals were identified in both crosses that represented potential recombinants in which PrP and incubation period did not segregate together. Therefore, although these phenotypes are certainly linked in I/LnJ and P/J mice, it is possible that PrP and incubation period are controlled by separate genes.

Detection of proteinase K-resistant prion protein and infectivity in mouse spleen by 2 weeks after scrapie agent inoculation

The sequential accumulation of the protease-resistant form of the endogenous prion protein (PrP-res) was compared to levels of scrapie infectivity in the spleen and brain of scrapie-infected mice at various times after inoculation. In mouse spleen PrP-res was detected 1 week after inoculation, and increased 65-fold between 1 and 3 weeks post-inoculation and an additional 15-fold during the next 17 weeks. Infectivity in spleen reached a maximum plateau level by 3 weeks. In contrast, in mouse brain PrP-res was not detected until 8 weeks after inoculation and then increased 200-fold during the next 12 weeks. During this same time, infectivity increased approximately 10,000-fold. Therefore, in both spleen and brain of scrapie-infected mice accumulation of PrP-res and infectivity appear to be associated. However, it was not possible to show quantitative correlations between PrP-res detection and infectivity, perhaps owing to the inaccuracy of the infectivity assay.

Proteinase K-Resistant Prion Protein Detection in Animal Tissues and In Vitro

Transmissible spongiform encephalopathies (TSEs) of man and animals are caused by unique transmissible agents that are unusually resistant to chemical inactivation and treatments that destroy or modify nucleic acids (1–4). In preparations enriched for infectivity, a predominant proteinase K-resistant form (PrP-res) of an endogenous protein (PrP-sen) has been identified (5–7). A great deal of controversy exists about the relationship between PrP-res and the infectious scrapie agent. In fact, some investigators believe that PrP-res and the infectious scrapie agent are the same (5, 8). Regardless of the true relationship, virtually all workers in the field recognize that a close association exists between the detection of PrP-res and the presence of spongiform encephalopathy. This association has led in recent years to new diagnostic tests for scrapie based on PrP-res detection by either immunoblotting (9, 10) or immunohistochemistry (11, 12). Both techniques offer distinct advantages over diagnosis based on microscopic evaluation of brain. We summarize here several of our studies that utilized PrP-res detection as a diagnostic test for scrapie. In addition, we report initial results of a study in which PrP-res was sought in brain of cattle presented with clinical symptoms suggestive of bovine spongiform encephalopathy (BSE).