Prion diseases, also known as transmissible spongiform encephalopathies (TSE), are a group of rare, rapidly progressive, and fatal neurologic diseases. Precisely prions are proteinaceous infectious particles that lack nucleic acids. This field has been intense areas of research owing to the capacity of the molecule to self proliferate without a nucleic acid. Very little is known about this molecular enigma though a small amount of information is available. Dr. Stanley Prusiner was awarded the noble prize for his work on prions.
Photo: Stanley B. Prusiner
It is now widely agreed that the prions are structural abnormal isoform (PrPsc) of host proteins designated as PrP through a post translational mechanism. PrP is a protein found in maximum concentration in the central nervous system of many different vertebrates and highly conserved. The protein is found predominantly in synapses at cholesterol rich microdomains or caveolae and also in immune system. A complete analysis of prion structure with Fourier transform infrared (FTIR) spectroscopic methods reveals that the beta pleating of the protein molecule is abnormally high. (well that explains a lot!!!!)
Under experimental mixing conditions the PrPsc is used as a seed to convert the PrPc to form protease resistant forms. However the concentration of seed used for the experiment is high which makes the assay of new infective forms very difficult. For experimental protocols swainsonine which is an inhibitor of complex glycosylation is used to block prion formation, though all strains are not equally susceptible. The general idea here is the biochemical amplification of protease-resistant PrPSc-like protein (PrPres) using a protein-misfolding cyclic amplification method.
So our basic understanding is that the prions are misfolded proteins that have an unusual high tendency to resist degradation and its subsequent accumulation. In addition they cause conversion of other normal proteins to abnormal phenotypes. Now thats really a rogue molecule.
CJD is a neurodegenerative disorder, which is the most common of prion diseases. It was first described by German neurologist Hans Gerhard Creutzfeldt. The prion molecule involved is human PrPsc from PrPc encoded by a gene PRNP. The human PRNP gene is located on the short (p) arm of chromosome 20 between the end (terminus) of the arm and position 12, from base pair 4,615,068 to base pair 4,630,233.PRNP has also recently been designated CD230. CJD can be subdivided into three different subtypes – genetic, acquired and sporadic. The PrPsc molecule is resistant to normal cellular degradation process. This accumulates to form an amyloid aggregate. PrPsc also forms tiny fibers called scrapie-associated fibrils (SAFs).
Laboratory Diagnosis of Prion diseases
Prions are simply proteins and the diagnosis implies finding the unique pathotypes in the specimens. Almost universally the prions are found in the nervous tissue (probably because most of them arise from PRNP exclusively active in nervous tissue.) Though a few studies have reported isolation and detection of prion particles from other sites they are less diagnostically sensitive, (except in vCJD tonsilar biopsy is highly diagnostic). The Prions are identified for its presence by resistance to proteinase K. An extracted material is subjected to enzymatic digestion to proteinase K and the remaining tissue is extracted for proteins. They are then subjected to a western blot. A further identification is possible by Proteomic analysis and Protein sequencing.
Culture of the prions is a Hercules tool for research. This can be done by seeding prion protein (from the sample) to a susceptible tissue possibly into a monolayer maintained by the tissue culture methods. This however requires time and the process may be very slow. The sensitivity and specificity of the method is quite low. The newer innovative technique is by use of PMCA (Protein Mis-fold cyclic amplification). PMCA is an amplification technique (conceptually like PCR without use of nucleotides) to multiply misfolded prions. The technique initially incubates a small amount of abnormal prion with an excess of normal protein, so that some conversion takes place. The growing chain of misfolded protein is then blasted with ultrasound, breaking it down into smaller chains and so rapidly increasing the amount of abnormal protein available to cause conversions. By repeating the cycle, the mass of normal protein is rapidly changed into mis-folded prion.
1. Stanley B prusiner. Prions, PNAS, Vol. 95, pp. 13363–13383, November 1998. Nobel Lecture.
2. Alison etal. 14-3-3 in the cerebrospinal fluid of patients with variant and sporadic Creutzfeldt–Jakob disease measured using capture assay able to detect low levels of 14-3-3 protein. Neuroscience Letters 324 (2002) 57–60.