A Prion is a normal protein that is found on the membranes of cells. The normal Prion protein (PrPC) consist mainly alpha helix rich 30-35kDa glycoprotein with 209 amino acid sequence and one disulfide bond (1). The disease causing form of normal prion protein named after scrapie (PrPSc) occurs when properly folded normal prion protein change its conformation. Although the exact tertiary structure of PrPSc still remains elusive but the previous studies have shown that the secondary structure of infectious protein contains high proportion of beta sheet in place of normal alpha helix.
The increased content of beta sheet in PrPSc leads to the formation of aggregates resistant to proteases that assemble into amyloid fibers and accumulate to form plaques in brain cells (2). Prions are extremely small unique pathogens and have no nucleic acid. The PrpSc protein cannot self replicate but causes other normal prion proteins to change the conformation making them infectious. The process by which the infectious prion recruits the normal protein is still unclear. All known prion disease commonly known as transmissible spongiform encephalopathies to date is untreatable and fatal.
Some of the common prion disease found in humans includes Creutzfeldt-Jakob disease (CJD), Variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial insomnia, and Kuru. Besides human prions disease is also prevalent in animals causing Bovine Spongiform Encephalopathy (BSC) also known as mad cow disease, Chronic Wasting Disease, Scrapie, Transmissible mink encephalophathy, Feline spongiform encephalophathy. The new human prion disease vCJD was first reported in United Kingdom in 1996 and was believed to come from feeding the meat of cattle infected with mad cow disease.
Prion disease can be transmitted by ingestion of BSC contaminated food products especially beef and may also be transmitted by using contaminated surgical instruments. It can also be transmitted by blood transfusion or treatment with blood products that were derived from infected donor. The high frequency of disease (Kuru) on those who took part in the ritual eating of the body also suggests that transmission occurs via oral route (3). Once the host is inoculated with the prion disease they first infect tissues that are normally involved in body’s defense mechanism such as spleen and lymph nodes.
In vCJD, prions first infect lymphoreticular tissues and then infect brain causing disease symptoms. Unlike other pathogens such as bacteria, fungi or viruses the prion proteins are formed by one of the body’s own proteins and hence cannot be recognized as foreign antigen by host immune system. Lack of nucleic acid along with immune tolerance and size makes prions extremely resistance to protease, heat, radiation making it almost invincible to any known defense mechanism. The incubation period of disease may vary from several years up to 70 years. Evolution of prion disease.
It was believed that Bovine Spongiform Encephalopathy (BSE) in cattle and Creutzfeld-Jakob disease (CJD) in humans originated from prion disease called scrapie found in the sheep. Later it was found that the humans seem unable to get prion diseases from sheep infected with scrapie CJD but by consuming contaminated beef. This strongly suggests that prions in humans did not evolve from the sheep scrapie. The study of fossil of a transmembrane protein in the sequence of known vertebrate PrP suggests that it used to be an integral membrane protein probably expelled to the extracellular space by a mutation (4).
Another study suggests that the prion gene is descended from the more ancient ZIP family of metal ion transporters. Members of the ZIP protein family are well known for their ability to transport zinc and other metals across cell membranes (5). This study is consistent with the study of fossils that prion used to be an integral membrane protein as ion transporters belongs to integral membrane proteins. Epidemiology: Human TSEs (Transmissible spongiform encephalopathies) can be divided into infectious (5%) sporadic (80%) and inherited (15%).
Sporadic CJD occurs at the rate of 0. 5 to 1 million populations per year and thought to arise as a result of a conformational change in a single prion protein which then infects other normal PrPc protein. About 15% of human TSEs occur due to inherited mutations in the prion gene located on chromosome 20p (5). Over 30 pathogenic mutations have now being identified causing autosomal dominantly inherited disease. The occurance of bovine spongiform encephalopathy (BSE) in UK cow heard in 1990 led to transmission to humans (hill et al, 1997;Collinge et al,1996).
The origin of BSE still remains unclear but it is assumed that a case of sporadic BSE arose in a single cow by conformational change in single prion protein by mutation of a single prion gene in a similar manner how CJD is thought to occur in humans. The transmission of BSE to humans as vCJD gave rise to major public health problem with the death of 119 in 2002 only. Structure The normal form of the protein PrPc contains highly conserved cell surface protein attached via a glycophosphatidyl inosotol anchor.
It is expressed in a wide range of cell types particularly in neuronal cell. The normal PrPc is a sialoglycoprotein of molecular weight 33 to 35 kDa and contains high content of ? helices secondary structure that is sensitive to protease treatment and soluble in detergent. The disease associated isoforms on other hand is found only in infected brains as aggregated materials and is resistant to protease treatment and in soluble in detergent and contains high content of ? sheet secondary structure. Prion disease and complement
It is now well established that prion disease such as CJD in humans, scrapie in sheep, and BSE in cattle are disease of amyloid formation. Amyloid formation is a neurodenegenerative disorder characterized by extracellular accumulations of protease resistant isoforms on the cell surface. Previous study on amyloid plaque in human brain has revealed the deposition of complement protein but not immunoglobulins (Ishii et al. , 1984). These studies strongly suggest the role of complement in prion disease. The prion infected brain also show activation and recruitment of microglia around the plaques.
Infection of prion disease usually occurs via oral ingestion of contaminated beef during which PrPsc which penetrates the intestinal epithelium and propagates in lymphoid tissues and migrates to CNS where most of the damage occurs. Several studies had shown that the mice deficient in complement proteins are resistant to scrapie when administered intraperitonelly. Wide range of knockout mice depleting circulating immunoglobulin or individual Fc-gamma receptor showed no effect on scrapie pathogenesis. (6).
However, mice deficient in C3, Clq or complement receptor CR1/CR2 were partially or fully protected against spongiform encephalopathy when exposed to limited amounts of prions. These results clearly suggest the role of complement component during onset of prion disease. However the exact mechanism still remains elusive on how prions activates the specific complement proteins but it is clear that these components of complement is involved in initial trapping of prions in lymphoreticular organs soon after infection. Several reports have also indicated that humoral immune response to the PrPsc can prevent prion infection.
Monoclonal antibodies and F(ab) fragments recognizing PrP were also shown to abolish scrapie as well as infectivity in neuroblastoma cells (7). Immune response to prion disease For more than two decades it has been shown that prion infection does not elicit immune responses. TSE do not elicit any inflammatory responses and the antibodies to the prion protein are typically undetectable. The challenges for the researchers were to determine why prions accumulate in lymphoid organs and why various states of immune deficiency prevent peripheral prion infection.
The answers for these questions still remains unclear but the investigation of these questions had led to different detours in the aspect of research including neurophysiology and protein functions. Currently, all prion disease lack effective treatment and are universally fatal. It has now clearly established that scrapie infection fails to induce an immune system. The possible reason for this lack of immune response is due to normal scrapie protein which is modified to scrapie and the host fails to recognize scrapie as a foreign antigen. Also anti scrapie antibody generated in one host may not be effective on another host.
Therapeutic approach Several approaches have been employed to overcome the infection of prion disease. The immunomodulatory approach to immunize mucosal layer is one of the potential attractive method to produce a local immune response which might partially or completely prevent prion entry across gut barrier. However any immunomodulation approach aimed at self antigen has to be finely balanced as it may enhance potential autoimmune toxicity. Several studies using attenuated Salmonella vaccine strain expressing the prion protein showed that mucosal vaccination can protect against prion infection from a peripheral source.
This study clearly showed increased survival times of orally exposed mice and opened the door for developing possible active or passive immunization of gut mucosa to fight prion disease that more specifically target PrPsc. Other studies revealed that immunization of prion knockout mice with phosphotungstic acid purified mouse prion protein resulted in PrP specific monoclonal antibodies with binding specificities selective for both infectious PrPsc and normal PrPc proteins suggesting the possible vaccine for prion disease. However it could only delay the incubation time and only effective when administered intra peritoneally.
Another approach to fight against prion is to understand the pattern of TLR (Toll like Receptor) signaling in the glial cell. TLR are also known to be expressed in innate immune response and plays a crucial role in their activation against foreign pathogens. It was recently discovered that TLR signaling was linked to neurodegenerative diseases found in Alzheimer’s disease (AD). Both AD and PrPsc forms a plaque on the brain which led researchers to investigate more on amyloid plaque formed during AD. The study did provide some insight in caring plaque formation but failed to eliminate prions completely.
Some tradition medicins such as curcumin from curry turmeric has shown some effectiveness in preventing brain damage in mouse module due to its anti-inflammatory properties. Medical marijuana also appeared to be effective in delaying plaque formation due to its active ingredient THC. THC prevents the formation of deposits in the brain associated with amylogenesis by inhibiting acetylcholinesterase. Although researchers were able to elicit immune response against prion in all inoculated PrP knockout mice but the amount titre obtained is usually low in concentration to be used as effective anti prion antibodies.
The use of adjuvant with these anti prion antibody may provide another possible vaccine option to boost the immune system against these prion diseases. Another research on anti prion antibody suggests that anti PrP antibodies detected at the terminal stage of prion- affected mouse generates high titers of these antibodies. The problem using these types of antibody is they might evoke autoimmune disease. To understand the mechanism of prion pathogenesis scientists have directed their attention towards finding the normal functioning of prion proteins.
The normal prion protein is expressed early in embryogenesis and in adult it is present at highest levels in neurons of the brain and spinal cord. Most of the normal prion molecules are localized on the cell surface and attached to lipid bilayer at a C terminal. Some function of normal prion protein include cellular uptake or binding of copper ions, transmembrane signaling of some neuronal growth, formation and maintenance of synapses, adhesion to the extracellular matrix, protection against apoptosis and response to oxidative stress (8).
Loss of putative function of normal prion protein has little phenotypic effect. However the loss of PrPc partially impairs the cytoprotective activity of the protein in both yeast and mouse modal. Mouse lacking normal prion gene exhibit complete tolerance from PrPsc . This strongly suggests that the normal prion protein is required for onset of disease. Furthermore despite having knockout gene for normal prion protein the transgenic mouse remained healthy. The result clearly suggests that normal prion protein is not essential for normal functioning of the cell.
However the experiment is limited only to the mouse model only and exploring the redundancy in the cell for normal prion protein is essential. Several other studies were also conducted to investigate CD4 T cell mediate immune response against PrPc from different species. In one of the study carried out in 2003 by Stolze and his colleague mention about functional CD4+ T cell response against foreign normal PrPc molecules in wild type mice but they were not able to get any response against infective PrPsc (9).
The recent advances in prions research have gone a long way but disappointingly active immunization is yet to be discovered. It is evident that the main obstacle to vaccine therapy comes from the tolerance of the immune system to prions and more specifically to its own PrP protein. Some recent publication thus suggests that this may not be a problem if adequate peptides of PrP are identified. With recent advances in technology and medicine may be in future scientist will be able to develop proper vaccine and cure for prion disease. References: 1. PamKM, Baldwin M, Nguyen J et al.
Conversion of alpha helices into beta sheets features in the formation of the scrapie prion protein. Proceedings of the National Acedemics of science of the United State of America 90 (23) : 10962-6. (December 1993). 2. Paul Erlich, Chantal Dumestre-perard, Wai LI Ling, Jean Gagnon and Jean-Yves Cesbron. Complement Protein C1q Forms a Complex with Cytotoxic Prion Protein Oligomers. The journal of biological chemistry vol 285,no 25, pp 19276-19276,June 18; 2010. 3. Kitzman RL, Alpers MP, Gajdusek DC. The natural incubation period of Kuru and the episodes of transmission in three clusters of patients.
Neuroepidemiology 1984; 3; 3-20. 4. A report on Evolutionary origins of Prions disease; Science daily sept 28; 2009. 5. Pierr Aucoturier and Claude Carnaud. The ommune system and prion disease: a relationship of complicity and blindness. Journel of leukocytebiology. vol 72,dec 2002. 6. Robert B. Sim, Uday Kishore, Christian L. Villers, Pratrics N. Daniel A. Mitchell. Clq binding and complement activation by prions and amyloids. 2007; immunobiology 212; page 355-362. 7. Magdalini Polymenidou, Frank L. Heppner, Erica C. Pellicioli, Eduard Urich, Gino Miele, Nathalie Braun and Adriano Aguzzi.
Humoral immune response to native eukaryotic prion protein correlates with anti- prion protection. ; 14670-14676;PNAS’oct5,2004;vol 101. 8. Laura Westergard, Heather M. Christensen and David A. Harris. The cellular prion protein:its physiological function and rolein disease. Biochem biophys Acta 2007 juna; 1772(6):629-644. 9. L. Stolze, H. Rezaei, G. Jung, J. Grosclaude, P. Debey, H. schild and H. G Rammensee. CD4+T cell mediated immunity against prion proteins. Research article; Birkhauser verlag, 2003, Cell Mol. Life sci. 60; 629-638. 10.
Ulrich Kalinke . Vaccination against prion disease. July 27,2007. The normal prion found in cattle shares two biochemical features with the prion found in humans and apes. But neither of these features is found in the prion of sheep which when mutated causes the disease called scrapie. The discovery is a shocking for the evolutionary argument that cattles and sheep though are extremely closely related bu humans are unable to contract prion diseases from sheep infected with scrapie, they would be unlikely to contract CJD from the contaminated beef.