Biol212 Biochemistry of Disease Neurological Disorders: Prions

Biol212 Biochemistry of Disease Neurological Disorders: Prions

Prions Transmissible spongiform encephalopathies (TSEs) are diseases of the central nervous system caused by unconventional infectious agents, prions. Vacuolation of neurons (leading to holes in the brain ) and the accumulation of abnormal, proteaseresistant prion protein in the CNS are characteristics of the TSEs. All attempts to identify a virus or at least a nucleic acid associated with the TSEs have proved unsuccessful. Brain biopsy from a patient showing intra-cytoplasmic vacuoles the morphological change characteristic of all prion diseases.

The TSEs CJD (Creutzfeldt-Jakob disease) characterised by rapidly progressive dementia, patients are usually between 50 and 75 years old. Sporadic CJD accounts for ~ 85% of all cases, has a worldwide distribution with an incidence of ~ 1 new case per million people each year, with no identifiable cause. Atrogenic CJD occurs accidentally as the result of medical procedures (e.g. use of contaminated neurosurgical instruments or therapy with cadaveric pituitary-derived hormones). Familial CJD accounts for 10-20% of all cases and is always associated with mutations in the gene for PrP (prion protein). GSS (Gerstmann-Sträussler-Scheinker syndrome) very rare inherited autosomal dominant disease, associated with mutations in the gene for PrP, characterised by terminal dementia and a long clinical duration (2-10 yr). FFI (Fatal familial insomnia) inherited disease characterised by sleep disturbances. The phenotype depends on an asp178asn mutation in PrP coupled with a met at codon 129.

Scrapie worldwide neurological disease of sheep and goats, first recognised in 1730. The name comes from the observation that affected animals rubbed against fences to stay upright. CWD (Chronic wasting disease) a fatal brain-wasting disease described in deer in the USA since 1980.

BSE (Bovine spongiform encephalopathy - mad cow disease ) first described in 1986 in the UK. vcjd (variant Creutzfeldt- Jakob disease) newly discovered (1996) human TSE related to eating BSEcontaminated food. Mainly affects young adults and characterised by a progressive neuropsychiatric disorder. Prion protein can be detected in the tonsils. Given the lack of information regarding incubation time and level of exposure to contaminated material, it is impossible to make well-founded predictions about the future numbers of vcjd cases.

PrP The key event in TSEs is the conformational change in a host protein, cellular PrP (PrP c ) into a pathological conformer (PrP Sc - after original identification in experimentally scrapie-infected rodents). PrP c is the product of a single host gene and is present in the brain of uninfected animals and constitutively expressed in many cell types. Most, if not all, cases of the familial TSEs are associated with mutations in the gene encoding PrP. cellular PrP Mice devoid of this gene are resistant to scrapie infection i.e. host PrP c is required for disease propagation.

The mechanism through which PrP c undergoes conformational change to PrP Sc remains unknown. The prion hypothesis postulates that in sporadic and genetic TSEs, the initial formation of PrP Sc occurs spontaneously, and, in the genetic forms is facilitated by the substitution of an amino acid. Newly folded PrP Sc then induces the conformational change in other molecules of PrP c via an autocatalytic process. In atrogeneic CJD, kuru, and vcjd (the acquired forms of human TSE s) the formation of PrP Sc is induced by exogenous human or bovine PrP Sc.

The Prion Hypothesis

PrP c is derived from a precursor protein of 252 amino acids. Signal sequences at the amino- (yellow leader sequence, removed during processing) and carboxyl-termini (yellow, cleaved so that a glycosylphosphatidylinositol (GPI) anchor can be added to residue 231). The protein also contains 5 amino-terminal octapeptide repeats, two glycosylation sites and a disulphide bridge.

The GPI anchor attaches the mature protein to the outer surface of the cell membrane. The a-helical content of PrP c is 40% with little or no b-sheet. In contrast, PrP Sc contains 50% b sheet and only 20% a helix. The existence of two stable conformations for a protein with the same amino acid sequence is at odds with the theory that the primary structure of a protein determines a single three-dimensional structure.

Prions are like Transformers

PrP Sc aggregates into amyloid fibrils and accumulates in neural and, often, lymphoreticular cells. Proteolytic treatment of PrP Sc removes 60-70 amino acids from the amino-terminal region of the protein, leaving a protease-resistant core. In contrast, proteolytic treatment of PrP c results in complete digestion. Proteolytic resistance might be a key factor in permitting survival of PrP Sc during digestion and allowing its absorption by the gastrointestinal tract. Immunological and biochemical methods show that PrP Sc and scrapie infectivity co-purify and the concentration of the protein is proportional to the infectivity titre. Moreover, infectivity is neutralized by agents that destroy protein structure, and more importantly, by anti-prp antibodies. The amyloid fibre is a coil of several fibrils perpendicular to a cylindrical axis.

Propagation of PrP Sc PrP Sc propagates at the expense of host PrP c by transferring its toxic conformation to the normal host protein. Two different mechanisms have been suggested to account for this process:

Template-assisted conversion PrP Sc (red) is thermodynamically more stable than PrP c but there is a kinetic barrier to reach this stability. PrP c (green) exists in equilibrium with a transient conformational intermediate PrP* (blue), which, after interaction with a chaperone (protein X) is able to heterodimerize with PrP Sc. Spontaneously this heterodimer is converted into a PrP Sc homodimer. The homodimer can then dissociate to form two templates thereby ensuring the exponential growth of PrP Sc concentratrion.

Nucleation-polymerisation This model proposes the coexistence of PrP c (green) and PrP Sc (red) in equilibrium. PrP Sc is unstable and is only stabilized by aggregation with other PrP Sc molecules. Aggregation and precipitation, diminishes the concentration of PrP Sc in solution thereby displacing the equilibrium in favour of conversion of PrP c to PrP Sc. This explains why the pathological conformation is dominant.

b-sheet blocker/breaker peptides can stimulate the reversion of PrP Sc to a state similar to PrP c - this may represent a new therapeutic approach.

The role of the normal protein is unclear; it is highly concentrated at synapses. PrP c deficient mice show a clear phenotype indicating that they are more sensitive to various kinds of stress. The amino-terminal region, containing the octomeric repeat region, appears to bind up to 5 Cu 2+. Copper can catalyse the interconversion of various reactive oxygen species thus sequestering copper can have protective benefits for cells that are sensitive to oxidative damage. PrP c containing at least 2 Cu 2+ has an activity similar to superoxide dismutase i.e. PrP c may detoxify superoxide radicals preventing oxidative stress.

A role for PrP c in neuronal zinc homeostasis? The brain has the highest zinc content of all organs with an overall concentration of approx. 150mM. There are millimolar concentrations of zinc in the presynaptic vesicles of specific nerve terminals. PrP c is highly expressed in neurons and is concentrated on both pre- and post-synaptic membranes. The concentration of zinc in the extracellular spaces of the brain is higher than that of copper so it is possible the zinc rather than copper binds to the octapeptide repeats in PrP c. i.e. PrP c may be involved in the binding and/or transport of zinc in the brain.

Models for the involvement of PrP c in zinc homeostasis (c caveolin and f Fyn - a tyrosine kinase)

A role for zinc in the pathology of prion diseases? It appears that in prion diseases zinc may be displaced from its normal locations. If PrP c has a role in binding neuronal zinc, then conversion to PrP sc may cause the loss of this function with the consequent redistribution of zinc.

Summary Transmissible spongiform encephalopathies are diseases of the central nervous system caused by unconventional infectious agents, prions. The key event is the conformational change in a host protein, cellular PrP (PrP c ) into a pathological conformer (PrP Sc ). In sporadic and genetic TSEs, the formation of PrP Sc occurs spontaneously; in the genetic forms, it is facilitated by the substitution of an amino acid. Newly folded PrP Sc then induces the conformational change in other molecules of PrP c via an autocatalytic process. Protease-resistant PrP Sc aggregates into amyloid fibrils and accumulates in neural cells. Vacuolation of neurons then occurs leading to holes in the brain.