Changes in the localization of brain prion proteins during scrapie infection

Abstract

Prion proteins (PrP) were localized in the brains of normal and scrapie-infected hamsters by immunohistochemistry and Western blotting. PrP monoclonal antibodies and monospecific anti-PrP peptide sera, which react with both the cellular (PrPC) and scrapie (PrPSc) isoforms of the prion protein, were used to locate PrP in tissue sections. In normal hamsters, PrPC was located primarily in nerve cell bodies throughout the CNS; whereas, in the terminal stages of scrapie, PrP immunoreactivity was shifted to the neuropil and was absent from most nerve cell bodies. Prion proteins were not uniformly dispersed throughout the gray matter of scrapie-infected hamster brains; rather, they were concentrated in those regions that exhibited spongiform degeneration and reactive astrogliosis. Since earlier studies showed that the level of PrPC remains constant during scrapie infection as measured in whole brain homogenates and no antibodies are presently available that can distinguish PrPC from PrPSc, we analyzed individual brain regions by Western blotting. Analysis of proteinase K-digested homogenates of dissected brain regions showed that most of the regional changes in PrP immunoreactivity that are seen during scrapie infection are due to the accumulation of PrPSc. These observations indicate that the tissue pathology of scrapie can be directly correlated with the accumulation of PrPSc in the neuropil, and they suggest that the synthesis and distribution of the prion protein has a central role in the pathogenesis of this disorder.

Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

“…The mammalian Prnp gene has been described as a ubiquitous gene, with little known regarding gene regulation (26,31,32). The cellular specificity of the Prnp gene is based mainly on the data from PrP immunohistochemistry studies (14,16,33) and in situ hybridization (13,20,21,34). However, some data are contradictory and may be explained by the techniques used or by the low level of PrP expression in nonneural cells.…”

“…Total RNA was extracted and purified with the RNA NOW kit (Biogenetex, Seabrook, TX) as recommended by the supplier. For cDNA synthesis, 2 g of total RNA was heat-denatured for 3 min at 70°C and reverse-transcribed with 200 units of Superscript II RNaseH Ϫ Reverse Transcriptase (Life Technologies, Paisley, Scotland) in 20 l of 1ϫ incubation buffer (provided by the supplier) supplemented with 10 mM DTT, 1 mM dNTP, 1 unit͞ l RNAsin (Promega), and 0.5 g of (dT) [12][13][14][15][16][17][18] primers (Amersham Pharmacia). Two microliters of each cDNA reaction was subjected to PCR amplification with 25 pmol each of the sense and antisense primers in 50 l 1ϫ PCR buffer, 0.2 mM dNTP, and 1 unit recombinant Taq DNA polymerase (Life Technologies).…”

“…PrP c has been shown to be present in various regions of the hamster brain, including cortex, hippocampus, striatum, olfactory bulb, hypothalamus, midbrain, cerebellum, and brainstem (14). However, some regional differences in the abundance and localization have been found, according to different authors (16,17). PrP c has been detected by Northern blot and͞or Western blot in a large variety of peripheral tissues, including heart, lung, pancreas, testes, and kidney in the rodent (18,19) and also skeletal muscle and uterus in sheep (15).…”

Mice deficient for prion protein exhibit normal neuronal excitability and synaptic transmission in the hippocampus.

“…Further experiments will be needed to clarify this issue. Thus, the function of PrPc in the normal hippocampus, where this protein is expressed at higher levels than many other regions of the central nervous system (30), remains enigmatic. Our results suggest that, whatever the role of PrPC might be, its absence has little or no impact on the excitability of CAI pyramidal cells as well as synaptic transmission and supports the hypothesis that it is the accumulation of PrPsc and not the loss of PrPC that is responsible for the pathology in prion diseases (9,15).…”

Molecular characterization of proteolytic cleavage sites of the Pseudomonas syringae effector AvrRpt2

“…PrP C expression in brain was defined by standard immunohistochemistry (208) and by histoblotting in the brains of uninfected controls (209). Immunostaining of PrP C in the SHa brain was most intense in the stratum radiatum and stratum oriens of the CA1 region of the hippocampus and was virtually absent from the granule cell layer of the dentate gyrus and the pyramidal cell layer throughout Ammon's horn.…”