Molecular Biology of the Spongiform Encephalopathies

The Diseases

There are seven Spongiform Encephalopathies described in man and animals. In man: kuru, Creutzfeldt-Jakob disease (CJ), Gerstrnann-Straussler syndrome (GSS); in sheep and goats: Scrapie; in cows: Bovine Spongiform Encephalopathy (BSE); in mink: Transmissible Mink Encephalopathy; and in mule deer and elk: chronic wasting disease. These diseases are characterised by similar pathological features including vacuolation, gliosis and amyloid plaque formation. Ml are transmissible by intracerebral inoculation into experimental animals (chiefly syrian hamsters). The disease process is characterised by a long latent period and absence of an immune response.

The nature of the agent

The unusual nature of the agent was realised several decades ago. Activity is filterable suggesting a viral component but is resistant to treatments that inactivate nucleic acids. Radiation inactivation experiments suggested that if the target is nucleic add it could only be of the order of 30 base pairs in size. Infectivity purified with a protein component of amyloid rods found in scrapie infected material. This was composed of a 27-30K molecular weight protein termed PrPsc

The Prion Hypothesis

Due to the unusual properties of the agent much speculation as to the nature of the pathogen has arisen. The agent was termed an unconventional virus or virion to distinguish it from normal viruses. Other hypotheses proposed that the agent was a replicating membrane fragment because of its apparent association with membrane material. It was also proposed that the agent was a protein termed prion (small proteinaceous infectious particle). This proposal provoked much controversy but subsequently experimental evidence has accumulated supporting this hypothesis. This evidence has arisen from molecular genetic and transgenic studies.

Molecular genetic studies

Purification of infective material had revealed that the major protein component was PrPsc Partial sequencing enabled enough amino acid sequence to be determined so that cDNA dones coding for PrPsc could be isolated. cDNA clones for PrPsc failed to hybridise to purified scrapie prions. However, the done hybridised to normal cellular DNA and RNA. This protein was found to be the product of a normal cellular gene that was transcribed in normal cells to give a 35K molecular weight protein called PrPc. Proteolytic treatment during purification of the scrapie agent was responsible for the smaller molecular weight of PrPsc The PrPc gene Is transcribed normally in scrapie infected brains.

The proteins PrPsc and PrPC differ in several physical characteristics:

a) Sensitivity to proteases, PrPsc is resistant to proteolytic attack in contrast to PrPc.

b) Cellular location, PrPsc is intracellular and not released from the cell surface by treatment with enzymes that hydrolyse glycosyl~phosphatidyl-inositol anchors unlike PrPc.

Genetics of Susceptibility

Studies of infectivity of inbred strains of mice identified alleles rendering increased susceptibility to infection. These alleles (Pm-i) were closely linked to the structural gene for PrPc. Studies in man of inherited forms of CJ and GSS identified mutations in the PrPc gene that were associated with the disease. Three different mutations have been identified in affected individuals so far. This is strong evidence for the intrinsic infectivity of mutant forms of PrPc. Experiments with transgenic mice in which the human mutation was introduced into the endogenous mouse gene supported this hypothesis. These mice exhibited a spontaneous neurodegenerative disease with pathological changes similar to GSS, unlike their normal littermates.

Possible Mechanisms of Pathogenesis

While there is strong evidence for the prion hypothesis, the mechanism of infectivity remain elusive. Experiments using mice transgenic for the syrian hamster PrPc gene led to a hypothesis of prion replication. These mice were much more susceptible to infection with hamster prions (75 days to exhibition of symptoms) compared to nontransgenics (>500 days). The exact incubation time was dependent on the amount of mRNA synthesised for hamster PrPc. Incubation of transgenic mice with mouse prions resulted in a pattern of infection similar to nontransgenic mice (incubation time of 140480 days). Characterisation of material from hamster and mouse prion infected transgenic animals showed that the specificity of the resulting prions was dependent on the initial inoculating species - ie hamster prion inoculated transgenic mice only formed hamster prions and mouse prion inoculated animals only mouse prions. The mechanism of replication of the prion was therefore postulated to involve a species specific interaction between the inoculating PrPsc and the homologous PrPc