Prion protein with an insertional mutation accumulates on axonal and dendritic plasmalemma and is associated with distinctive ultrastructural changes.

Prion diseases are fatal neurological diseases characterized by central nervous system deposition of abnormal forms of a membrane glycoprotein designated PrP (prion protein). Tg(PG14) transgenic mice express PrP that harbor a nine-octapeptide insertional mutation homologous to one described in a familial prion disease of humans. Tg(PG14) mice spontaneously develop a fatal neurological illness accompanied by massive apoptosis of cerebellar granule neurons and accumulation of an aggregated and weakly protease-resistant form of PrP that is not infectious. Previous light microscopic analyses of these mice left open questions regarding the subcellular distribution of the mutant protein and the nature of the neuropathological lesions produced. To address these questions, we undertook an immunogold electron microscopic study of Tg(PG14) mice. We found that mutant PrP is localized primarily on the plasma membrane of dendrites and unmyelinated axons in the hippocampus and cerebellum, with little labeling of either neuronal cell bodies or intracellular organelles. PrP deposits were shown to be associated with degenerative changes in dendritic structure. We also describe for the first time marked pathology in myelinated axons, and alterations in the axon/oligodendrocyte interface. Taken together, our results suggest cellular mechanisms by which mutant PrPs produce pathology. In addition, they highlight distinctions between familial and infectious prion disorders at the ultrastructural level that correlate with differences in cellular trafficking of the disease-associated PrP forms.

Strain-associated variations in abnormal PrP trafficking of sheep scrapie.

Prion diseases are associated with the accumulation of an abnormal form of the host-coded prion protein (PrP). It is postulated that different tertiary or quaternary structures of infectious PrP provide the information necessary to code for strain properties. We show here that different light microscopic types of abnormal PrP (PrP(d)) accumulation found in each of 10 sheep scrapie cases correspond ultrastructurally with abnormal endocytosis, increased endo-lysosomes, microfolding of plasma membranes, extracellular PrP(d) release and intercellular PrP(d) transfer of neurons and/or glia. The same accumulation patterns of PrP(d) and associated subcellular lesions were present in each of two scrapie strains present, but they were present in different proportions. The observations suggest that different trafficking pathways of PrP(d) are influenced by strain and cell type and that a single prion strain causes several PrP(d)-protein interactions at the cell membrane. These results imply that strains may contain or result in production of multiple isoforms of PrP(d).

Sub-cellular pathology of scrapie: coated pits are increased in PrP codon 136 alanine homozygous scrapie-affected sheep.

Sub-cellular studies of transmissible spongiform encephalopathies (TSEs) have been carried out on several animal species and human beings. However, studies of optimal perfusion-fixed tissues have largely been confined to examination of rodents. Using a recently developed technique, heads of scrapie-affected sheep and controls were perfusion fixed with mixed aldehydes. The obexes were immunohistochemically labelled with PrP antibodies, and the dorsal motor nucleus of the vagal nerve was examined by electron microscopy. Irregular neuritic profiles with highly invaginated membranes, associated with coated pits were found in all scrapie-affected sheep, but not in controls. Interestingly, they were consistently more frequent in the homozygous A(136) sheep. This is the first report describing sub-cellular differences in pathology associated with different PrP genotypes. Rarely, amorphous material, or sparse fibrillar structures, were present in the extracellular space. The changes were often associated with irregular plasmalemma and frequent coated pits. Vacuolation typical of TSEs, dystrophic neurites and variable gliosis were present. Herniation of membranes and organelles from apparently healthy processes into adjacent vacuoles and dendrites was also observed. We suggest that the increase in coated pits and plasmalemma invagination is related to an attempted internalisation of aggregated disease-specific PrP, or protofilaments, from the extracellular space.

Relationships between ultrastructural scrapie pathology and patterns of abnormal prion protein accumulation.

On immunohistochemical examination several morphological types of disease-specific prion protein (PrP(d)) accumulation are recognised in the brain of sheep suffering from scrapie. The present study examined the relationship between the type of PrP(d) deposits seen by light microscopy and ultrastructural changes in the olivary nuclei and the dorsal motor nucleus of the vagus (DMNV) in naturally infected sheep with clinical scrapie. The nature and magnitude of sub-cellular morphological changes found in the olivary nuclei differed from the patterns of degeneration previously described in the DMNV. In the olivary nuclei, lamellar bodies in the neuronal perikaryon were found to correlate with marked intraneuronal PrP(d) accumulation. Bizarre, coated, spiral invaginations of the plasmalemma were only found in A(136) homozygous sheep in this nucleus, where few coated pits were usually observed. Neuropil vacuolation in the olivary nuclei was mild and correlated with sparse extracellular PrP(d) deposition. In the DMNV, the magnitude of extracellular immunolabelling in the neuropil was prominent. These extracellular PrP(d )aggregates coincided with intense neuropil vacuolation, increased numbers of coated pits, and with the presence of pre-amyloid changes and infrequent short fibrils in the extracellular space. Scrapie-infected neurons in the two neuroanatomic sites examined, therefore, appear to process and respond to the presence of PrP(d) differently. We hypothesise that vacuolation, coated pits and spiral invaginations of the plasmalemma may be responses to extracellular PrP(d) molecules, and that lamellar bodies are changes associated with the high levels of intraneuronal PrP(d).

Scrapie-specific neuronal lesions are independent of neuronal PrP expression.

In the transmissible spongiform encephalopathies (TSE), accumulation of the abnormal disease-specific prion protein is associated with neurodegeneration. Previous data suggested that abnormal prion protein (PrP) could induce neuronal pathology only when neurons expressed the normal form of PrP, but conflicting evidence also has been reported. Understanding whether neuronal PrP expression is required for TSE neuropathological damage in vivo is essential for determining the mechanism of TSE pathogenesis. Therefore, these experiments were designed to study scrapie pathogenesis in vivo in the absence of neuronal PrP expression. Hamster scrapie (strain 263K) was used to infect transgenic mice expressing hamster PrP in the brain only in astrocytes. These mice previously were shown to develop clinical scrapie, but it was unclear whether the brain pathology was caused by damage to astrocytes, neurons, or other cell types. In this electron microscopic study, neurons demonstrated TSE-specific pathology despite lacking PrP expression. Abnormal PrP was identified around astrocytes, primarily in the extracellular spaces of the neuropil, but astrocytes showed only reactive changes and no damage. Therefore, in this model the pathogenesis of the disease appeared to involve neuronal damage associated with extracellular astrocytic accumulation of abnormal PrP acting upon nearby PrP-negative neurons or triggering the release of non-PrP neurotoxic factors from astrocytes.